Dissertations / Theses on the topic 'BONE CEMENTS/therapeutic use'

　　　Strontium (Sr) is not only widely studied for its compound as a drug for treating osteoporosis, but there is also a growing interest of its addition in orthopaedic biomaterials. Over the years, the development of orthopaedic biomaterials has already advanced to a new era in the search of resorbable and/or bioactive materials. Due to its anabolic and anti-resportive properties of Sr on bone regeneration as a drug, strontium has been extensively investigated for its potential in other orthopaedic applications. The purposes of this study were to investigate strontium containing hydroxyapatite (Sr-HA) bone cement for the delivery of gentamicin and the effects of Sr incorporation in coral and borosilicate glass as bone engineering scaffolds. 　　　 　　　Three types of Sr incorporated materials are reported here, in which these include an applied study of the drug elution property of previously published bone cement and two initial studies of the biological properties of newly developed coral and borosilicate scaffolds. Firstly, the gentamicin release, bioactivity and mechanical property of bioactive bone cement filler based on Sr-HA were compared to a commercially available gentamicin-loaded poly(methyl methacrylate) (PMMA). Over the study period of 30 days, the cumulative gentamicin release from Sr-HA bone cement was much greater than PMMA bone cement (+ 34%); better bioactivity of Sr-HA was also confirmed with the apatite formation after simulated body fluid immersion. Goniopora, a highly interconnected porous coral, was hydrothermally converted to coralline hydroxyapatite (CHA) or coated with hydroxyapatite and incorporated with Sr. As the first report of incorporating Sr into coral with the structure remained, about 4-16% Sr was detected on CHA. Sr-HA coated coral was studied in vitro and in vivo (ovariectomized rat model) resulting in better cell proliferation and higher scaffold volume retention (+40%). Finally, the development of Sr incorporated borosilicate (SrB) glass scaffold explored a new material for bone tissue engineering, but more importantly, it introduced a phenomenal idea of the stimulatory effect of a local alkaline microenvironment on bone regeneration. Detections of an exceedingly high pH (~ pH 8.6) condition on the material surface and release of Sr, Si and B ions during the degradation of scaffold SrB were confirmed to stimulate osteoblasts and facilitate apatite formation. Although new bone was observed on both scaffolds, higher bone area/tissue area (B.Ar/T.Ar) on scaffold SrB indicated more new bone formation over borosilicate scaffold without Sr addition. 　　　 　　　The significance of this study is to explore and develop three orthopaedic biomaterials advancing the stimulatory effects of Sr on bone regeneration. The drug elution properties of Sr-HA bone cement provides a fascinating alternative for treating osteomyelitis. Furthermore, by incorporating Sr into CHA and borosilicate scaffold, it brings out the importance on the readiness of the Sr release of the materials in order to deliver the stimulatory effects. Subsequently, a localized pH micro-environment arisen by material degradation is emphasized as a controlling factor in bone regeneration on biomaterials.
published_or_final_version
Orthopaedics and Traumatology
Doctoral
Doctor of Philosophy

Em um estudo prospectivo sobre o tratamento das artroplastias de quadril infectadas, com perdas ósseas e fístulas ativas, 25 pacientes foram tratados em dois tempos e 36 pacientes foram tratados em dois tempos com espaçador de cimento impregnado com vancomicina. O acompanhamento médio foi de dois anos e onze meses. A taxa de recidiva infecciosa foi de 29,2% nos tratados em dois tempos e de 8,8% nos tratados com espaçador. O Escore de Harris para Quadril médio passou de 19,3 para 69,0 pontos nos casos tratados em dois tempos e de 19,7 para 72,2 pontos nos pacientes tratados com espaçador. Ao final do estudo, 86,1% dos tratados com espaçador e em 33,3% dos tratados em dois tempos tinham próteses em bom funcionamento e sem infecção. O espaçador de cimento com antibiótico é o tratamento de escolha nas próteses infectadas de quadril
We report a prospective study of 61 patients with chronically deep infected hip replacements with actively discharging sinuses, treated with a two-stage revision protocol, with and without a cement spacer impregnated with vancomycin. The average follow-up was two years and eleven months. Twenty-five patients were treated without a spacer and seven had recurrence of infection. Thirty-three patients were treated with a spacer and three had recurrence of infection. The average Harris Hip score increased from 19,3 to 69,0 on the non-spacer patients and from 19,7 to 75,2 on the spacer group. At the end of the study, the success rate was 86,1% for the spacer group and 33,3% for the non-spacer group. The use of the spacer increased the results of the two-stage chronic infected hip replacements

Mestrado em Materiais e Dispositivos Biomédicos
In recent years, the development and innovation of new bone substitutes has revolutionized the lives of millions of patients. The aim of this work is the development and characterization of a bioactive, injectable and ready-to-use system (also called putty or premixed cement) for bone regeneration. The solid phase is constituted by beta-tricalcium phosphate (β-TCP), FastOs® bioglass (FastOs® BG) and monocalcium phosphate monohydrate (MCPM) powders, while the liquid phase comprises glycerol (G). The synthesis of β-TCP powder was obtained by precipitation reactions followed by heat-treatment; FastOs® BG was obtained by melt-quenching. The characterization of the obtained powders was made through X-ray diffraction (XRD) and measurement of the mean particle sizes and particle size distribution. The putty was prepared by mixing the solid and liquid phases and placed in syringes with a screw cap. Regarding clinical application, injectability, setting time (ST) and mechanical strength were investigated to characterize the putty. Structural analyses of the putty were also performed by XRD, Fourier Tranform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The putty has a solid/liquid weight ratio (S/L) of 3.3, mean ST of ~25 min, ~96% of injectability and a maximum compressive strength of 6 MPa. Therefore, the putty exhibited excellent injectability results, absence of filter pressing effect and acceptable mechanical properties. The structural analysis of the hardened cements revealed the formation of monetite crystals covered by an amorphous apatitic layer after immersion in PBS and water. The results are encouraging and support the conclusion that ready-to-use injectable bone substitutes have excellent handling properties to be used clinically. In accordance with the Directive 93/42/EEC the putty is considered a class III medical device. In order to pave the way towards its commercial release and in order to meet the essential requirements set out in Annex I of the Directive 93/42/EEC, a clinical evaluation has been carried out.
Nos últimos anos, o desenvolvimento e a inovação de novos substitutos ósseos tem revolucionado a vida de milhões de doentes. O objetivo deste trabalho é o desenvolvimento e caracterização de um sistema bioativo, injectável e pronto-a-usar (putty) para regeneração óssea. A fase sólida é constituída por pós de fosfato tricálcico beta (β-TCP), biovidro FastOs (FastOs®BG) e fosfato monocálcico monohidratado (MCPM), enquanto a fase líquida é o glicerol (G). A síntese dos pós de β-TCP foi obtida por reações de precipitação seguida de tratamento térmico; os pós de FastOs®BG foram obtidos por fusão e arrefecimento em água fria (fritagem) (melt-quenching). A caracterização dos pós foi feita por difracção de raios-X (XRD) e medição dos tamanhos de partícula. O sistema injectável pronto-a-usar foi preparado através da mistura das fases sólida e líquida e colocado em seringas seladas com tampa roscada. Do ponto de vista de aplicação clínica, o sistema foi caracterizado tendo em conta a sua injectabilidade, tempo de presa (setting time, ST) e resistência mecânica. A análise estrutural do sistema também foi realizada, através de XRD, espectroscopia de infravermelho com transformada de Fourier (FTIR) e microscopia eletrónica de varrimento (SEM). O sistema injectável pronto-a-usar tem uma razão em peso sólido/líquido (S/L) de 3,3, um ST médio de ~25 min, ~96% de injectabilidade, e 6 MPa de resistência máxima à compressão. Deste modo, o sistema injetável demonstrou excelentes resultados de injectabilidade, tendo-se verificado ainda a ausência do efeito de filter pressing e propriedades mecânicas aceitáveis. A análise estrutural dos cimentos endurecidos revelou a formação de cristais de monetite recobertos por uma camada apatítica amorfa após imersão em PBS e em água. Os resultados obtidos são promissores e permitem concluir que o sistema injetável pronto-a-usar possui excelentes propriedades de manipulação do ponto de vista clínico. De acordo com a Directiva 93/42/CEE o sistema injetável é considerado um dispositivo médico de classe III. Com o objectivo de contribuir para o seu processo de lançamento comercial e seguindo os requisitos essenciais estabelecidos no anexo I da Directiva 93/42/CEE foi elaborado um relatório tendo em conta a avaliação clínica do sistema injectável.

In a 6-week rat model it was demonstrated that a small dose of peri-implant zoledronic acid (ZA) increased local bone formation 3-fold compared with controls. Ancillary in vitro studies using 14C-labeled ZA implant doses demonstrated biphasic elution profiles for implants coated with hydroxyapatite; complete ZA release occurred within one to three weeks in serum compared with only 60% ZA release after 12 weeks in water. Implants without hydroxyapatite coating showed more burst-type release profiles and full ZA elution within 24 hours of hydration in serum or water. Canine studies at 6 weeks using implants with 14C-labeled ZA showed that the compound remained localized, with the greatest ZA concentration immediately adjacent to the implant. Although there was evidence of skeletal ZA distribution via diffusion into the circulation, the levels were two orders of magnitude less than at the implant site.

The high performance of bone implants depends on the positive response of osteoblasts to the surface of the materials manufactured for the implant. Cell response in turn strongly depends on the nature of the initial interaction of macromolecules involved in cell adhesion and proliferation with the atomic structure of the surface of the material used for the implant. The initial interaction between bone specific extracellular matrix proteins and the solid substrate influences cell response at the cell-implant interface. This interaction is crucial for implant stability, long-term durability, and osseointegration. Despite extensive research undertaken to develop high-quality material for implants in order to improve the cell-substrate interaction, little is known about the significance of the atomic structure of the substrate and the role of molecular machinery involved in cell-substrate interaction. Using a combined approach involving material sciences and cell and molecular biology, the objectives of this research are to evaluate the response of pre-osteoblast and fibroblast cell lines to novel bulk polycrystalline and single crystal titanium based material and assess the role of crystal size and orientation.
Novel bulk nano-structured titanium substrates were produced by the process of high-pressure torsion (HPT). These materials have a significant advantage compared to conventional titanium-based materials by having higher surface wettablity, mechanical properties as well as a distinct surface oxide layer and atomic structure. A co-culture system was adapted to investigate the differential response of pre-osteoblast and fibroblast cell lines to titanium and titanium dioxide single-crystal substrates.
The results of this study provide clear evidence that crystal size and specific crystallographic orientation can be used to improve cell adhesion and proliferation. The nanostructured titanium substrates show strong interaction with pre-osteoblast cells as evident by the higher expression of fibronectin and the formation of extensive focal adhesion. Differential cell behaviour of pre-osteoblasts and fibroblasts are observed in cultures grown on the substrates with specific crystallographic orientations. The degree of cell attachment of the pre-osteoblasts is considerably higher on Ti-(1120) crystal face compared with the fibroblasts. These findings have profound implications for the improved osseointegration and inhibition of fibrosis leading to long-term implant consolidation and stability.

Background and purpose: Rhizoma Chuanxiong (CX), the dry rhizome of Ligusticum chuanxiong Hort., is a commonly used Chinese herbal medicine to treat gynecological diseases. So far, more than 60 chemical components have been identified from CX such as volatile oils (ligustilide, etc.), phenolic acids (ferulic acid, etc.) and alkaloids (chuanxiongzine, etc.). These components in CX are the basis of its wide pharmacodynamic actions including estrogen-like, progesterone-like and anti-coagulant/anti- platelet effects. In our recent survey based on previous published clinical trials, CX was ranked as one of the top 20 herbs commonly used for anti-miscarriages amongst Chinese pregnant women. However, CX should be used with caution during pregnancy as its property of 2invigorating blood circulation and removing blood stagnation3. Despite its wide applications, the safe dosage of CX in pregnant women remains unclear with no records found in the current Chinese Pharmacopoeia or other guidelines. Thus, verification regarding the impacts of CX preparations and its components in embryonic development is urgently required. In view of the limited experimental evidence that is currently available to assess the safety of CX, this project aims to (1) identify the general impacts of CX aqueous extract in maternal function and fetal development with an in vivo mouse model; and to (2) investigate the adverse impacts and underlying mechanisms of CX aqueous extract in fetal bone development with a biomarker assay and metabolomics analysis.;Concusion: CX aqueous extract at a low dosage of 2 g/kg/day (equals to the daily dosage of human adults) did not cause adverse effect in pregnant mice, and it suggested that this dosage of CX preparations should be safe for pregnant women. Our data demonstrated that high dosage and long-term use of CX aqueous extract might result in embryonic toxicities including fetal bone malformations for the first time. As the CX aqueous extract in this study was not contaminated by pesticide residues and heavy metals, the adverse impacts of CX aqueous extract should be considered as a result of its intrinsic components in the herb. Furthermore, CX aqueous extract might significantly down-regulate biomarkers related to bone formation and metabolism during osteogenesis. It is therefore valuable to establish a practical approach to systematically assess the safety of CX and other herbal medicines.;Method: Referred to the guidelines of WHO, the Chinese Pharmacopoeia and the Hong Kong Chinese Materia Medica Standards, CX aqueous extract was prepared, and its reference marker (ligustilide and ferulic acid) were quantitatively authenticated by HPLC analysis. LC/MS fingerprint analysis was performed for the quality control purposes. In addition, pesticide residues and heavy metals found in CX aqueous extract were examined using GC-MS and ICP-MS analysis. In the Segment II study as per FDA and OECD guidelines, pregnant mice were randomly assigned into 6 groups (n=18 per group): i.e. mice were orally administrated with distilled water as the negative controls (Group 1); or CX aqueous extract of 2, 16, 24 and 32 g/kg/day respectively from the gestation day (GD)6 to 16(Group 2, 3, 4 and 5); or vitamin A (200,000 IU) on GD7, 9 and 11 as the positive controls (Group 6). All mice were sacrificed to assess maternal and fetal parameters on the GD18. In the mechanistic study, the expressions of biomarkers related to fetal bone development including PICP, ICTP, B-ALP, BGP, Gdf-5, BMPs, BMP-6, BMP-8, BMP-11, IL-4, IL-4r, IL-10 and IL-10r in fetal tissue samples of the Group 1 and 5 (32 g/kg/day, n=18) were measured using ELISA analyses on GD16. Meanwhile, the metabolites of two-group samples were also analyzed by the UHD Accurate-Mass Q- TOF LC/MS, and profiling data was further analyzed by specific software. During statistical analysis, measurement data from G1, 2, 3 4 and 5 groups were analyzed using one-way ANOVA(SPSS software, version 16.0). LSD test in Post hoc method was applied to compare differences between every two groups. Pearsons x 2 - test was used to analyze category data from G1, 2, 3, 4 and 5 groups, and Fishers exact test was applied to compare differences between different groups. The student t-test was also used to compare differences between G1 and G6 groups in animal studies as well as G1 and G5 groups using ELISA or metabolomics results. An intragroup difference with a p-value less than 0.05 was considered as statistical significant.;Resutt:(1) There was no statistical significant difference in maternal and fetal parameters found between the Group 1 and 2 (p> 0.05). However, the maternal body weight (BW), gravid uterine weight, corrected BW change, live fetus/litter, mean fetal BW in the Group 4 and 5 were significantly lower than those in the Group 1(p

When human mesenchymal stem cells (hMSCs) are cultured inside a 3D collagen meshwork, they become a potential tissue engineering bone graft alternative. However, the in vitro osteogenesis rate of hMSCs is slow, leading to a low mineral deposition. To enhance the osteogenic differentiation of hMSCs, low intensity pulsed ultrasound (LIPUS) was employed as an external stumulus. The present study demonstrated the feasibility of employing daily LIPUS exposure for enhancing osteogenesis in vitro. Exposure of seven consecutive days LIPUS, each of 30 minutes duration, did not affect the cell viability, and the organization of hMSCs within the collagen meshwork was not disturbed. The calcium deposition within the collagen meshwork was enhanced after seven days of exposure. The osteoinductivity was also upregulated at the early period of culture. In order to optimizing the enhancement effects of LIPUS, various ultrasound parameters, including intensity, exposure duration and exposure repetition were investigated. Results showed the LIPUS enhancement effects are dose dependent, LIPUS exposure should be longer than 10 minutes/day in order to elicit a significant effect. Calcium deposition was higher when LIPUS exposure was done twice per day instead of one. Although individual variation exists, optimal LIPUS intensity range was between 60-120 mW/cm2 ISATA (Spatial Average Temporal Average Intensity). The interaction mechanism between LIPUS and cells was also investigated. Microbubbles were added to the culture during LIPUS exposure to find out whether cavitation is involved in the interaction. Flow sensor primary cilium was also studied in order to verify that ultrasound is transduced through fluid flow. Results showed cavitation may not be a contributing factor to osteogenesis, and primary may be involved in the transduction of LIPUS stimulation. This study demonstrated that osteogenesis of hMSCs encapsulated in collagen constructs could be enhanced by LIPUS. The LIPUS parameters were also optimized. The LIPUS interaction pathways were also being better understood. This thesis study will be a paradigm for cellular mechanotransduction studies and put an important step forward for therapeutic ultrasound.
published_or_final_version
Electrical and Electronic Engineering
Master
Master of Philosophy

Prostate cancer remains one of the most commonly diagnosed cancers in men and is a leading cause of cancer death. While great success has been achieved at curing early stage prostate cancer, limited success has been obtained when treating late-stage hormone independent prostate cancer. This is due to the increased propensity for skeletal and non-skeletal metastases. Thus development of novel effective therapeutic modalities against late stage prostate cancer is of critical importance.
Towards these objectives, I have focused my attention on the role of prostate secretory protein (PSP-94) which is expressed in normal individuals and in patients with early stage prostate cancer. Using our well established in vivo models of prostate cancer, I have evaluated the ability of PSP-94 and its amino acids 31-45 required (PCK3145) to decrease tumor growth and skeletal metastases in vivo and evaluated the potential mechanism(s) associated with PCK3145 anti-cancer actions.
Prostatic cancer can also develop as a result of epigenetic activation of tumor promoting genes. To evaluate the role of methylation in prostate cancer, late stage prostate cancer cells were treated with the universal methylating agent S-adenosylmethionine (SAM) and an anti-sense oligonucleotide directed against MBD2 (AS). Scrambled oligonucleotide was included as a control (S). Both SAM and MBD2-AS resulted in inhibition in uPA, MMP-2 and VEGF production leading to decreased tumor cell invasive capacity. However, SAM and MBD2-AS were not able to either further repress partially methylated genes (GSTP1) or reactivate already methylated genes (AR). Furthermore, SAM and MBD2-AS treatment resulted in significant reduction in tumor growth in vivo . Immunohistochemical and RT-PCR analyses carried out on SAM and MBD2-AS tumors revealed decreased protein and mRNA expression of uPA and MMP-2 which was partially due to increased methylation of the respective promoters even after 10 weeks post in vitro treatment as analyzed by bisulfate sequencing. In addition decreased levels of angiogenesis and tumor survival markers were observed.
Collectively, these studies are aimed at the development of novel reliable approached to diagnose and treat advanced, hormone refractory prostate cancer to reduce tumor associated morbidity and mortality.

Bone loss from the cranio-maxillofacial region is a major clinical problem affecting patients worldwide. Conventional treatment strategy includes the use of autogenous or allogeneic bone, biomaterials, and osteogenic growth factors. However, there has been no effective therapy for most cases so far. Stem cell-based gene therapy is the latest research method with possible applications in humans. The present study aims to (1) characterize rabbit mesenchymal stem cells (MSCs) relating to growth pattern, surface antigens, and the potential for multi-differentiation; (2) determine the transduction efficiency and duration of recombinant adeno-associated virus2 carrying enhanced green fluorescent protein (rAAV2EGFP) reporter gene in rabbit MSCs and study the effects of rAAV2EGFP transduction on stem cells’ phenotype and capacity of multi-differentiation; (3) evaluate the differentiation characteristics of rabbit MSCs following recombinant adeno-associated virus 2 carrying bone morphogenetic protein 2 gene (rAAV2BMP2) transduction; (4) investigate whether MSCs transduced by rAAV2BMP2 could successfully induce bone regeneration in rabbit critical-size cranial defects. MSCs were isolated from bone marrows of rabbit tibias and cultured. Cell counting and colony-forming assays demonstrated that growth rates of MSCs dropped substantially with increasing passages. Flow cytometry on MSCs at passage 1 showed that cells expressed high level of CD49a and low level of CD44 as well as stage-specific embryonic antigen 4 (SSEA4). Multi-differentiation and reverse transcriptase-polymerase chain reaction (RTPCR) tests demonstrated that rabbit MSCs were capable to differentiate into osteocytes, chondrocytes and adipocytes. Immunofluorescence microscopy showed that rabbit MSCs produced a series of hematopoietic growth factors, including stem cell factor (SCF), vascular endothelial growth factor-A (VEGFA) and granulocyte macrophage colony-stimulating factor (GMCSF). Subsequently, rabbit MSCs were transduced with rAAV2EGFP in vitro. By comparing the transduction efficiency with different doses of rAAV2EGFP particles, multiplicity of infection (MOI) of 1 x 10 4 was identified as an optimal parameter for the transduction of rAAV2 in rabbit MSCs. Fluorescent microscopy demonstrated long-term expression of EGFP in rabbit MSCs after transduction both in vitro and in vivo. In addition, cell proliferation assay, adipogenic induction test and flow cytometry showed that rAAV2EGFP transduced MSCs exhibited a similar pattern with non-transduced cells on the cell growth, capacity of adipogenic differentiation and expression of surface antigens, indicating that rabbit MSCs maintain their stem cell properties after rAAV2EGFP transduction.
published_or_final_version
Dentistry
Doctoral
Doctor of Philosophy

Introduction: Intervertebral disc (IVD) degeneration is suggested to begin from the nucleus pulposus (NP). Evidence from various studies highlights mesenchymal stem cells (MSC), in most cases using bone marrow derived MSC, as a potential stem cell source for NP regeneration. However MSC can be isolated from many sources with various characteristics. There are indications that fetal or close to fetal tissue sources contain MSC with relatively undifferentiated phenotype with respect to MSC from adult sources. Moreover, umbilical cord (C)-MSC may have better chondrogenic differentiation potential than bone marrow (B)-MSC. We hypothesize CMSC are different from BMSC, and more efficient than BMSC in stimulating NP regeneration. Methods: MSC were isolated from human bone marrow and umbilical cord with corresponding ethical approval. BMSC and CMSC were characterized for cell surface marker expression profile and differentiation potential.. RT-PCR of interest genes in NP cells isolated from scoliosis and degenerate discs was performed to search for NP degeneration indicators. Conditioned media (CM) was collected from confluent MSC monolayer, and used for stimulation of four batches of degenerated NP cells isolated from human degenerative intervertebral discs. Cell proliferation and cytotoxicity were assessed by MTT assay. Proteoglycan content were measured by DMMB assay. Gene expression of a series of degeneration related molecules including ACAN, SOX9, CDH2, CD55, KRT19, KRT18, FBLN1 and MGP, and fibrosis related molecules, including MMP12, HSP47, COL1A1, COL3A1 and FN1, of NP cells in MSC-CM were determined by real- time RT-PCR. All results were normalized to the control cells in basal medium. The expression of discogenic, chondrogenic and osteogenic markers on BMSC and CMSC were compared by RT-PCR. Results and Conclusion: CMSC were similar to BMSC and fulfilled the minimum criteria of MSC, however the expression of CD146, CD106 and Stro-1 was different, and BMSC had a spontaneous osteogenesis tendency while CMSC expressed chondrogenic marker even without TGF-beta stimulation. BMSC demonstrated a paracrine effect on modulating human degenerated NP cells towards a non-degenerative phenotype in stimulating cell proliferation, slightly enhancing proteoglycan production, upregulating KRT19 while downregulating MMP12. Compared with BMSC, a higher paracrine effect of CMSC was disclosed in modulating the phenotype of NP cells in all aspects tested, and an intrinsic higher expression on CMSC of ‘potential NP markers’, including KRT19, KRT18 and CD55, but lower expression of osteogenic markers, including RUNX2 and ALPL, was revealed, which indicate a higher potential of CMSC for future clinical application to treat IVD degeneration diseases. KRT19 and MMP12 were also confirmed to be the highest differentially expressed candidate genes between cultured scoliosis and degenerated human NP cells, indicating a high indicator potential of NP degeneration. Furthermore, a subpopulation was detected in the degenerated NP cells that possessed macrophage-like phenotype and activities, which may play a role in the pathogenesis of IVD degeneration. In conclusion, studies in this thesis highlighted CMSC as a superior source than BMSC for IVD repair. Further investigations into the active agents in the conditioned media and the signalling pathway may help to elucidate the mechanism of the effect.
published_or_final_version
Orthopaedics and Traumatology
Doctoral
Doctor of Philosophy

Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp8) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp8 peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp8)-liposome-antagomir-148a. Our results demonstrated that D-Asp8 could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeting delivery depended on the interaction between bone resorption surfaces and D-Asp8. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp8)-liposome-antagomir-148a. These results indicated that (D-Asp8)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.

Breast cancer is the most commonly diagnosed cancer in women. Despite recent advances in screening and early detection, breast cancer continues to result in a high incidence of morbidity and mortality. In its late stage the majority of patients exhibit signs of destructive skeletal metastasis. This complication is promoted by the production of growth factors by tumor cells which can induce tumor cell proliferation via their interaction with their respective receptors to initiate the vicious cycle of bone resorption. Inhibition of growth factors signaling through their receptors can therefore serve as a useful therapeutic approach to block bone metastasis.
The biological characteristics of cancer cells along with the targeting properties of immune system offer a novel approach in the treatment of breast cancer. Directed against HER-2/nue oncogene, the recombinant humanized monoclonal antibody, Trastuzumab (Herceptin), has shown significant clinical benefits for the treatment of HER-2 positive metastatic breast cancer.
In the present study, the effects of Herceptin and its molecular mechanism of action in abrogating the development and progression of osteolytic bone metastasis is investigated in an experimental mouse model of skeletal metastasis using human breast cancer cells BT-474 which are known to express high levels of HER-2. Treatment of BT-474 cells with Herceptin caused a dose dependent decrease in cell proliferation. In in vivo studies BT-474 cells were injected by into the left ventricle of female BALB/c nu/nu mice. Intraperitoneal infusion of Herceptin from the day of tumor cell inoculation or at the time of radiologically detectable skeletal metastasis either slowed the development or prevented the progression of skeletal metastasis as compared to control groups of animals receiving non-specific IgG. Bone histological analysis of long bones showed the ability of Herceptin to reduce the ratio of tumor volume to bone volume as well as mitotic index when Herceptin treatment was initiated from the day of tumor cell inoculation. Immunohistochemical analysis of long bones showed a significantly lower level of activated (phosphorylated) MAPK in bones of Herceptin treated animals. These studies demonstrate the ability of Herceptin to inhibit the development and abrogate the progression of skeletal metastasis associated with breast cancer by blocking the HER-2 mediated signaling pathways.

Anterior cruciate ligament (ACL) rupture is a major clinical problem in sports medicine. The current mainstay of treatment is arthroscopic-assisted ACL reconstruction with a soft tissue tendon graft. However, the affected patients are required to abstain from any pivoting activity for at least six to nine months after the operation to protect the graft-host bone interface in order to allow the graft to heal. In this study, a method to accelerate the graft healing within the bone tunnel is proposed by using a local application of an osteoconductive bone cement (Strontium enriched calcium phosphate cement, Sr-CPC) at the graft-host bone interface. It is postulated that Sr-CPC can induce earlier new bone formation in the gap between the graft and host bone tunnel and hence can result in an accelerated healing of the graft within the bone tunnel in ACL reconstruction. Preparation of Sr-CPCs using the conventional setting method (a dissolution/precipitation process) leads to a delay in setting. This study adopted a new setting reaction, a chelate reaction, to manufacture a Sr-CPC system. The Sr-CPC system was fast-setting, injectable and cohesive, and it was suitable for use in minimally invasive orthopaedics surgeries (e.g. arthroscopic-assisted ACL reconstruction). In order to investigate the biocompatibility and osteoconductivity of the Sr-CPC, in vitro cell experiments and an in vivo animal study were carried out. The in vitro experiments showed that the Sr-CPC was biocompatible with no local toxicity. In addition, a higher proliferation rate of osteoblastic-like MG-63 cells, accompanying higher alkaline phosphatase activity, was found in the Sr-CPC group. The in vivo study using a rat femur metaphyseal bone defect model showed evidence of earlier endochondral ossification which was noted at 2 weeks post operation. Moreover, a higher peri-cement bone formation rate, accompanied by a higher cement resorption rate, was found in the Sr-CPC group at 32 weeks after the operation compared with the convention calcium phosphate cement group. To study the effect of the Sr-CPC on the graft healing within the bone tunnel, a one-stage bilateral ACL reconstruction using an Achilles tendon allograft was performed in 30 rabbits. One study (15 rabbits) was to investigate the effect of the Sr-CPC on the healing of a soft tissue tendon graft within the bone tunnel, and the other study (15 rabbits) was to study the difference between the Sr-CPC and the conventional CPC in the healing of a soft tissue tendon graft within the bone tunnel. The Sr-CPC treated graft showed an accelerated healing at all of the time points when compared with the non-treated graft; and at time points of 3 to 12 weeks when compared with the CPC treated graft. In conclusion, a strontium enriched calcium phosphate cement, which is suitable for the arthroscopic use, was manufactured. It is biocompatible, osteoconductive and degradable. It accelerates the graft healing within the bone tunnels in a rabbit ACL reconstruction model using an Achilles tendon allograft when compared with both of the non-treated group and the conventional CPC-treated group.
published_or_final_version
Orthopaedics and Traumatology
Doctoral
Doctor of Philosophy

Twenty-eight college female volunteers, 18 to 24 years of age, were studied to determine their bone mineral density and hormonal status following 10 months of supplementation with boron. Seventeen of the subjects were college athletes (A) who participated in either varsity basketball, tennis, track, triathlons, or volleyball. Eleven eumenorrheic subjects were placed in the sedentary group (S). All athletes were eumenorrheic, except for two, who were amenorrheic. The subjects were randomly assigned to either a placebo (cornstarch) or 3 mg of Tri-Boron (TWin Labs, Ronkonkoma, NY) per day. The study was single blind; the placebo and boron supplements were identical in appearance. The subjects' maximal oxygen consumption (V02max ) was assessed at month 0 only, in order to compare the athletes' aerobic capacity to that of the sedentary group. Subjects were measured at time 0 and 10 months for the following: average daily food intake, bone mineral density, plasma 1,2 5-dihydroxyvi tamin D3 , and plasma alkaline phosphatase. The following parameters were determined at 0, 6, and 10 months: serum 17-Beta estradiol progesterone, and testosterone, and percent body fat, and body weight. The athletes had a significantly greater V02MX (p < 0.05) than the sedentary group. There were no differences between the A and the S groups' food intake as measured in kcals, protein, fat, carbohydrate and fiber content. Although average body weight did not differ between activity groups, the athletes had a significantly lower (p < 0.05) percent body fat than the sedentary group. The athletes showed a slight increase in bone mineral density, whereas the sedentary group showed a slight decrease. The difference between these changes was significant (p < 0.05) between the activity groups. Furthermore, the athletes showed a slight increase in plasma 1,25-dihydroxyvitamin D3 , while the sedentary group showed a decrease in this measure; the difference between these changes also proved to be significant (p < 0.05) between activity groups. There were no significant differences in plasma alkaline phosphatase values. Serum 17-B estradiol and testosterone values exhibited a significant (p < 0.05) increase for all groups combined at 6 months. There were no significant changes observed in serum progesterone. Although significant changes were observed in some of the variables measured, none of the changes were a result of the boron supplementation.
Ph. D.

Since vitamin D deficiency is common at birth, the objective of this study was to test if postnatal vitamin D supplementation would normalize bone mineralization. Forty guinea pigs were randomized to receive a diet with or without vitamin D3 during pregnancy. Newborn pups were randomized to receive 10 IU of vitamin D3 or a placebo daily until d28. Measurements at birth and d28 included whole body and regional bone mass, osteocalcin and deoxypyridinoline, plus biomechanical testing of excised tibias and femurs. Offspring from deficient sows had lower body weight, whole body and tibia bone mineral content (BMC) and lower osteocalcin and biomechanical integrity. By d28 this group had lower whole body bone density and femur BMC, unless supplemented. Interactions with gender showed males continued to have low 25(OH)D despite supplementation. Therefore, neonates born to sows with dietary vitamin D deficiency require supplemental vitamin D to support normal bone mineral accretion.

Osteoporosis is a common condition characterized by bone fragility and fractures. Hip fracture, leads to disability, morbidity, excess mortality and growing costs to health care systems.
Antiresorptive agents are used to treat osteoporosis and fractures; it is unknown if these agents are effective in preventing recurrent fractures in individuals who have sustained a hip fracture.
Using health services administrative databases, we ascertained the incidence of hip fractures and associated-mortality rates in the elderly population in Quebec, from 1996 to 2002 and, evaluated the effectiveness of antiresorptive agents for the prevention of recurrent hip fractures.
We identified 33,243 hip fractures. Age-adjusted annual rates of hip fractures decreased in women by 11% from 1996 to 2002 while they did not change in men. Overall one-year mortality rates were higher in men than in women (37% versus 24%), and remained stable over time. Patients exposed to antiresorptives had a 26% reduction in the rate of recurrent fractures (95% CI, 0.64--0.86) compared to patients who were not exposed to these agents.
Hip fractures remain a prevalent disease with serious complications. Further research is essential to confirm our results and, to clarify the association between increasing use of antiresorptive agents and the trend reversal in the incidence of hip fractures.

Uvod: Hepatička osteodistrofija je termin koji obuhvata metaboličke bolesti kosti udružene sa hroničnim bolestima jetre. U alkoholnoj cirozi (AC) jetre postoji visoka zastupljenost deficijencije vitamina D proporcionalna stepenu disfunkcije jetre, ali njena uloga u patogenezi hepatičke osteodistrofije nije dovoljno objašnjena. Nivo 25(OH)D odražava status vitamina D. Kod AC jetre izmenjena je metabolička aktivnost kosti i suprimirano je formiranje kosti što dovodi do smanjenja koštane mase. U centru interesovanja je postizanje optimalnog statusa vitamina D. Stavovi o suplementaciji vitaminom D kod AC jetre nisu jasno definisani. Cilj rada: Utvrditi nivo vitamina D, ispitati metaboličku aktivnost kosti i mineralnu gustinu kosti kod bolesnika sa AC jetre. Utvrditi efekte suplementacije sa 1000 IU vitamina D3 na dan tokom godinu dana u odnosu na metaboličku aktivnost kosti i mineralnu gustinu kosti kod ispitivanih bolesnika. Bolesnici i metode: Istraživanje je sprovedeno na Klinici za gastroenterologiju i hepatologiju Kliničkog centra Vojvodine u Novom Sadu kao prospektivna intervencijska studija sa primenom suplementacije sa 1000 IU vitamina D3 na dan kod bolesnika sa AC jetre. Grupu bolesnika koja je uključena u istraživanje (1) činilo je 70 bolesnika muškog pola sa dijagnozom AC jetre. Bolesnici su imali četiri pregleda (P), odnosno tačke studije: P1-uključivanje bolesnika i započinjanje suplementacije vitaminom D; P2, P3 i P4 posle tri, šest i dvanaest meseci suplementacije vitaminom D, redom. Prilikom svakog pregleda rađene su analize funkcije jetre, metabolizma kosti i statusa vitamina D. Na početku (P1) i na kraju istraživanja (P4) vršeno je merenje mineralne gustine kosti (BMD) DXA metodom. Gubitak bolesnika od P1 do P4 bio je dvadeset, na različitim tačkama studije. Prvi deo istraživanja odnosi se na Grupu bolesnika koja je uključena u istraživanje (1) i završila prvi pregled (P1). Pedeset bolesnika je završilo kompletno istraživanje po predviđenom protokolu i oni se zbog realizacije svih pregleda i ponovljenih merenja posmatraju kao: Grupa bolesnika koja je završila istraživanje (2). Rezultati: (1): Kod bolesnika sa AC jetre utvrđena je deficijencija vitamina D, snižen nivo osteokalcina, normalni nivoi CrossLapsa, PTH, ukupnog i jonizovanog kalcijuma, fosfora i magnezijuma. Osteopeniju je imalo 42,65% a osteoporozu 14,71% ispitanika. Kod svih ispitanika najniži BMD izmeren je na vratu femura. (2): Suplementacija vitaminom D dovela je do značajnog porasta 25(OH)D. U odnosu na osteokalcin konstatovana je pozitivna razlika vrednosti P1/P4, iako je nivo ostao ispod donje granice normale. Kod nivoa CrossLapsa i PTH razlika P1/P4 je negativna, ali su nivoi u sva četiri merenja u okviru referentnih vrednosti. Na lumbalnoj kičmi došlo je do poboljšanja BMD za 0.87%, a pogoršanja su na vratu femura -1.87 % i kuku -1.65%. Konstatovano je i poboljšanje funkcije jetre. Zaključci: Kod bolesnika sa AC jetre poboljšanje statusa vitamina D dovodi do povećanja formiranja kosti i poboljšanja koštane mase na lumbalnoj kičmi. Neophodno je određivanje statusa vitamina D kod svih bolesnika sa AC jetre i uvođenje suplementacije vitaminom D kod bolesnika koji imaju nivo 25(OH)D < 80 nmol/l, uz tromesečne kontrole efekta. Kod postavljanja dijagnoze AC jetre potrebno je inicijalno određivanje BMD. Kod suplementacije vitaminom D nakon inicijalnog DXA pregleda sledeći se preporučuje nakon jedne do dve godine.
Introduction: The term Hepatic osteodystrophy defines a group of metabolic bone diseases associated with underlying chronic liver disease. Alcoholic liver cirrhosis (ALC) is characterized by high incidence of vitamin D deficiency that is proportional to the level of liver failure; however, its role in the pathogenesis of hepatic osteodystrophy has not yet been fully elucidated. The level of 25(OH)D best reflects the vitamin D status. ALC is characterized by changed bone metabolic activity and suppressed bone formation, resulting in the decrease in bone mass. The key topic of interest is the achievement of optimal vitamin D status. The attitude of health professionals towards vitamin D supplementation in alcoholic liver cirrhosis has not yet been clearly defined. The aim of the research: Determining of vitamin D levels, investigating the metabolic activity of the bone and bone mass in patients with alcoholic liver cirrhosis (ALC); Determining the effects of vitamin D3 supplementation at the dose 1000 IU/day during a one-year period in relation to metabolic activity of the bone and bone mineral density (BMD) in the investigated patient population. Patients and methods: The research was conducted at the Clinic for Gastroenterology and Hepatology of the Clinical Centre of Vojvodina in Novi Sad. The research was designed as a prospective interventional study implicating vitamin D3 supplementation at the dose 1000 IU/day to patients with ALC. The investigated patient population (1) encompassed 70 male patients diagnosed with ALC. The patients underwent four examinations (P), that is, research phases: P1 – inclusion of the patient into the study and introduction of vitamin D supplementation; P2, P3 and P4 after 3, 6 and 12 months of vitamin D supplementation treatment, respectively. Each examination included the analysis of liver function, bone metabolism and vitamin D status. At the beginning (P1) and at the end (P4) of the investigation period, bone mineral density (BMD) was measured by means of dual-energy x-ray absorptiometry (DXA) method. Twenty patients dropped out from the research at different stages throughout the investigation period (P1 to P4). The first part of the investigation pertains to the Group of patients who were included into the study (1) and completed the first examination (P1). Fifty patients have completed the entire research according to the foreseen protocol encompassing all examinations and repeated measurements. These patients are considered a Group of patients who completed the research (2) Results: (1): In ALC patients, vitamin D deficiency and decreased osteocalcin levels were established, as well as normal levels of CrossLaps, PTH, total and ionized calcium, phosphorus and magnesium. Osteopenia and osteoporosis were established in 42.65% and 14.71% of patients, respectively. The lowest BMD was measured in the femoral neck in all patients. (2): Vitamin D supplementation resulted in significant increase in 25(OH)D. Analysis of osteocalcin level revealed positive P1/P4 difference, even though the level remained below the lower normal limit. The levels of CrossLaps and PTH revealed negative P1/P4 difference; however, the levels determined at all four measurements were within the reference values. An improvement of BMD for 0.87% was established in lumbar spine, whereas a decrease was noticed in femoral neck (1.87%) and hip (1.65%). Furthermore, an improvement of liver function was established. Conclusions: Improvement of vitamin D status in ALC patients results in an increase of bone formation and improvement of body mass in lumbar spine. Determining the vitamin D status in all patients with ALC is of outmost importance, as well as the vitamin D supplementation of patients with levels of 25(OH)D < 80 nmol/l along with the monitoring of treatment outcome at three-month intervals. Establishment of the diagnosis of alcoholic liver cirrhosis should encompass initial measurement of BMD. In case of vitamin D supplementation treatment, the initial DXA examination should be repeated after the period of one to two years.

Orientador: Elcio Marcantonio Junior
Banca: Joni Augusto Cirelli
Banca: Enilson Antonio Sallum
Banca: Luis Carlos Spolidorio
Banca: Paulo Tambasco de Oliveira
Resumo: Os objetivos deste estudo foram avaliar o efeito de biomateriais associados ao PRP na formação óssea em defeitos padronizados de rádio e avaliar o efeito de PRP+RTG+enxerto ósseo autógeno para tratamento de lesões de furca grau II em cães. Para tal, em um primeiro estudo, foram confeccionados 5 defeitos de 5mm em cada rádio (direito e esquerdo) em 05 cães, constituindo assim espaços para preenchimento com os enxertos ou substitutos ósseos avaliados, totalizando 50 cavidades. Os materiais testados foram DFDBA, vidro bioativo, osso autógeno e osso mineral bovino, associados ou não ao PRP. Estes grupos foram avaliados e comparados com os grupos representados por coágulo sangüíneo e PRP. Ao final de 60 dias, foi realizada biópsia e preparo laboratorial para avaliação histológica e histomorfométrica. Os grupos que apresentaram melhores resultados foram coágulo, PRP e osso autógeno associado ou não a PRP; e a utilização do PRP não promoveu maior formação óssea em relação aos demais grupos, com exceção do vidro bioativo+PRP que apresentou os melhores resultados. No segundo estudo, foram criados cirurgicamente defeitos periodontais de furca nos quartos pré-molares mandibulares, bilateralmente em 5 cães, cronificados por um período de três meses e tratados por PRP/RTG/enxerto ósseo autógeno (grupo experimental) ou RTG/enxerto ósseo autógeno (grupo controle). Quatro meses após o tratamento, os cães foram sacrificados. Na área de furca, houve maior preenchimento ósseo e extensão linear de novo cemento, nova adaptação conjuntiva e regeneração periodontal no grupo experimental (p<0.005) e maior extensão linear de epitélio no grupo controle (p<0.005). A associação do PRP a RTG e enxerto ósseo autógeno proporcionou maior regeneração dos tecidos periodontais. Portanto, o PRP promoveu melhores resultados na formação óssea somente... (Resumo completo, clicar acesso eletrônico abaixo).
Abstract: The aims of this study were to evaluate the effect of biomaterials in association to PRP on bone formation, in padronized defects of radius, and evaluate the effect of PRP/GTR/autogenous bone graft in the treatment of Class II furcation lesions in dogs. For that, in a first study, five defects of 5mm each were produced in each radius (left and right) in 5 dogs, so creating spaces to be filled with grafts or the bone substitutes under study, totalling 50 cavities. The biomaterials tested were DFDBA, bioglass, autogenous bone and bovine mineral bone grafts, associated, or not, with PRP. These biomaterials were compared with coagulum and PRP. At the end of 60 days biopsy was done and histological laminas were prepared. Under the experimental condictions, coagulum, PRP and autogenous bone associated or not to PRP presented the best results, with more new bone formation; and no difference was observed between the groups with biomaterials, associated or not with PRP, excepting bioactive glass/PRP which showded the best results. In the second study, periodontal furcation defects were surgically produced bilaterally in the fourth mandibular premolars in 5 dogs, cronified for 3 months and treated with PRP/GTR/autogenous bone graft (experimental group) or GTR/autogenous bone graft (control group). Four months after the treatment, the dogs were sacrified. In the furcation area, more bone filling and linear extension of new cement, new conjunctive adaptation and periodontal regeneration were observed to occur in the experimental group (p<0.005) and more linear extension of epitelium in the control group (p<0.005). The association PRP/GTR/autogenous bone graft was observed to produce more regeneration of periodontal tissues. Therefore, the PRP association showed the best results in bone formation only at bioactive glass group; and... (Complete abstract, click electronic address below).
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Os objetivos deste estudo foram avaliar o efeito de biomateriais associados ao PRP na formação óssea em defeitos padronizados de rádio e avaliar o efeito de PRP+RTG+enxerto ósseo autógeno para tratamento de lesões de furca grau II em cães. Para tal, em um primeiro estudo, foram confeccionados 5 defeitos de 5mm em cada rádio (direito e esquerdo) em 05 cães, constituindo assim espaços para preenchimento com os enxertos ou substitutos ósseos avaliados, totalizando 50 cavidades. Os materiais testados foram DFDBA, vidro bioativo, osso autógeno e osso mineral bovino, associados ou não ao PRP. Estes grupos foram avaliados e comparados com os grupos representados por coágulo sangüíneo e PRP. Ao final de 60 dias, foi realizada biópsia e preparo laboratorial para avaliação histológica e histomorfométrica. Os grupos que apresentaram melhores resultados foram coágulo, PRP e osso autógeno associado ou não a PRP; e a utilização do PRP não promoveu maior formação óssea em relação aos demais grupos, com exceção do vidro bioativo+PRP que apresentou os melhores resultados. No segundo estudo, foram criados cirurgicamente defeitos periodontais de furca nos quartos pré-molares mandibulares, bilateralmente em 5 cães, cronificados por um período de três meses e tratados por PRP/RTG/enxerto ósseo autógeno (grupo experimental) ou RTG/enxerto ósseo autógeno (grupo controle). Quatro meses após o tratamento, os cães foram sacrificados. Na área de furca, houve maior preenchimento ósseo e extensão linear de novo cemento, nova adaptação conjuntiva e regeneração periodontal no grupo experimental (p<0.005) e maior extensão linear de epitélio no grupo controle (p<0.005). A associação do PRP a RTG e enxerto ósseo autógeno proporcionou maior regeneração dos tecidos periodontais. Portanto, o PRP promoveu melhores resultados na formação óssea somente... .
The aims of this study were to evaluate the effect of biomaterials in association to PRP on bone formation, in padronized defects of radius, and evaluate the effect of PRP/GTR/autogenous bone graft in the treatment of Class II furcation lesions in dogs. For that, in a first study, five defects of 5mm each were produced in each radius (left and right) in 5 dogs, so creating spaces to be filled with grafts or the bone substitutes under study, totalling 50 cavities. The biomaterials tested were DFDBA, bioglass, autogenous bone and bovine mineral bone grafts, associated, or not, with PRP. These biomaterials were compared with coagulum and PRP. At the end of 60 days biopsy was done and histological laminas were prepared. Under the experimental condictions, coagulum, PRP and autogenous bone associated or not to PRP presented the best results, with more new bone formation; and no difference was observed between the groups with biomaterials, associated or not with PRP, excepting bioactive glass/PRP which showded the best results. In the second study, periodontal furcation defects were surgically produced bilaterally in the fourth mandibular premolars in 5 dogs, cronified for 3 months and treated with PRP/GTR/autogenous bone graft (experimental group) or GTR/autogenous bone graft (control group). Four months after the treatment, the dogs were sacrified. In the furcation area, more bone filling and linear extension of new cement, new conjunctive adaptation and periodontal regeneration were observed to occur in the experimental group (p<0.005) and more linear extension of epitelium in the control group (p<0.005). The association PRP/GTR/autogenous bone graft was observed to produce more regeneration of periodontal tissues. Therefore, the PRP association showed the best results in bone formation only at bioactive glass group; and... (Complete abstract, click electronic address below).

O efeito da terapia por ondas de choque (TOC) na consolidação óssea, após osteotomias bilaterais dos fêmures e osteossínteses com hastes bloqueadas, foi estudado em 8 cães. Os fêmures direitos constituíram o grupo controle e os esquerdos, o grupo tratado, que recebeu 2000 pulsos de ondas de choque de 18 kV, energia de 5mJ (-6dB), na linha da fratura. Radiografias após 4, 8 e 12 semanas, revelaram maior proliferação periosteal no grupo tratado. Os resultados dos exames cintilográficos (razão tratado/controle), realizados na 2a, 4a, 6a, 8a, 10a e 12a semanas, foram estatisticamente superiores no grupo tratado. A TOC provocou aumento da atividade osteogênica na consolidação de fraturas agudas
The effect of extracorporeal shock wave therapy (ESWT) on bone healing, after bilateral femoral osteotomies and osteosynthesis with interlocking nails, was studied in 8 dogs. The right femurs composed the control group, and the left femurs, the treated group, which received 2000 pulses of shock wave, with 18 kV, 5 mJ (-6dB) energy on the fracture line. Radiographs after 4, 8 and 12 weeks, revealed increased periosteal proliferation in the treated group. The results (treated/control ratio) of the scintigraphic exams, performed on weeks 2, 4, 6, 8, 10 and 12, were statistically higher for the treated group. The ESWT led to increased osteogenic activity on acute fracture's bone healing.

研究背景：常规骨科临床在治疗大段骨缺损时需要移植骨和（或）支架材料，尤其复合有治疗性生物活性成分的复合材料尤为理想。本研究的策略在于发展开发一种具有生物活性和生物降解特性的的合并有植物小分子icaritin（外源性生长因子）或者骨形态发生蛋白2（BMP-2, 内源性生长因子）的复合骨支架用于骨再生。基于聚乳酸乙交酯共聚物和磷酸三钙，我们利用先进的快速成型技术编制了新型的符合有BMP-2 或者icaritin 的支架材料， 命名为PLGA/TCP （ 对照材料组） ，PLGA/TCP/BMP-2（BMP-2 编织复合治疗材料组）， PLGA/TCP/icaritin （低，中，高剂量icaritin 编织复合治疗材料组）。
研究目标：本研究的总体目标是通过系统的体外实验和兔骨缺损的体内实验，建立和评估一种优化的复合递送系统，用于骨再生的应用。体内效果的研究体现在终点关于合并有外源性生长因子icaritin 和内源性生长因子BMP-2 的复合材料之间的比较研究。
材料和方法：低温快速成型机器用于复合材料的编制。PLGA 和TCP 作为基本载体材料，icaritin 和BMP-2 作为具有生物活性的外源性和内源性生长因子，分别进行编织复合。最终编织复合的支架材料命名为P/T 对照组，P/T/BMP-2 和低，中，高剂量P/T/icaritin 治疗组。另外，我们通过液体完全浸泡并在真空橱内干燥24 小时的方法制备了BMP-2 和icaritin 浸泡复合支架材料，分别是P/T+BMP-2(阳性对照组)和中剂量P/T+icaritin(比较组)。体外成骨潜能是通过兔骨髓干细胞和支架材料共培养的方法检测细胞接种，增殖效率，碱性磷酸酶活性，钙沉积以及成骨基因定量mRNA 表达检测。兔尺骨双侧阶段性缺损并植入复合支架材料的模型用于探讨支架材料体内成骨和成血管功效，影像学和活体检测CT 技术用于评估骨再生；借助CT的血管造影术和组织学检测新生血管；动态核磁共振技术用于检测骨缺损局部血液灌注功能，以及宿主组织和支架材料之间的相互作用。
研究结果： 对编织的支架材料的体外特性和成骨潜能进行鉴定和评估。显微CT 定量结果显示此支架材料具有互联大孔隙，平均孔隙率75±3.27%，平均孔径458±25.6μm。和对照组，icaritin 浸泡复合组，BMP-2 编织复合组比较，在icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组（n=6, p<0.01）中，与材料共培养的兔骨髓干细胞（BMSCs）表现了较高的细胞接种效率，碱性磷酸酶活性和上调的胶原酶I，骨桥蛋白mRNA 表达，以及较多的钙结节沉积。同时，BMP-2 浸泡复合组表现了最佳的效果（n=6, p<0.01）。兔尺骨缺损模型体内试验结果显示，术后2，4，8周影像学和显微CT 显示，和对照组，icaritin 浸泡复合组，BMP-2 编织复合组比较，icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组材料（n=6, p<0.01）植入的骨缺损区域有更多新生成骨。BMP-2 浸泡复合组表现了最多的新骨形成（n=6,p<0.01）。组织学结果同样也验证了在icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组（n=6, p<0.01）中，存在较多的骨样组织和典型的板层骨。BMP-2 浸泡复合组也具有最多的新骨组织生成（n=6, p<0.01）。此外， 在icaritin 编织复合支架材料（n=6, p<0.05）尤其中剂量组（n=6, p<0.01）中，借助显微CT 的血管造影术检测发现，骨缺损区域出现较大的新生血管体积，动态核磁共振检查发现较好的局部血液灌注功能。在三种icaritin 剂量浓度的编织复合材料组之间比较，我们发现中浓度icaritin 复合比例的编织复合材料组显示了最佳的成骨潜能。
研究结论： 编织复合有外源性植物分子icaritin 的PLGA/TCP 支架材料在体内体外试验中均表现了预期的成骨分化潜能和骨再生能力，尤其是中剂量icaritin 编织复合材料。传统的应用前做体外复合的BMP-2 浸泡复合支架材料和更具吸引力和方便应用的植物分子icaritin 编织复合支架材料，都可以较好的增强骨修复，这很可能为新型生物复合材料潜在的临床有效性验证提供很好的基础。
Background: Treatment of large bone defect in routine orthopaedic clinics requires bonegrafting and/or scaffold materials, especially desirable with composite material combined with therapeutic and bioactive agents for achieving better treatment outcome. The strategy of this study was to develop such a bioactive biodegradable composite bone scaffold incorporating a phytomolecule icaritin as an exogenous growth factor or bone morphogenetic protein-2 (BMP-2) as a known endogenous growth factor for bone regeneration. Based on polylactide-co-glycolide (PLGA) and Tricalcium Phosphate (TCP), we fabricated innovative BMP-2 or icaritin incorporated scaffold materials, namely PLGA/TCP (Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and high-icaritin with three different dosages of icaritin (Treatment groups) by an advanced prototyping technology.
Aims: The overall aim of the study was to establish and evaluate a local delivery system with slow release of bioactive agents for acceleration of bone regeneration in a bone defect model in rabbits. In vivo efficacy study served as end-point of this comparative study between composite scaffold incorporating exogenous growth factor icaritin and endogenous growth factor BMP-2.
Materials & Methods: Composite scaffolds were fabricated at -28ºC by a lowtemperature rapid-prototyping machine. PLGA and TCP were used as basic carrier materials, and icaritin or BMP-2 was incorporated as exogenous or endogenous bioactive growth factors, respectively. The incorporated scaffolds were named by PLGA/TCP (P/T, Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and high-icaritin (Treatment groups). In addition, we prepared BMP-2 and icaritin loading scaffolds, namely PLGA/TCP+BMP-2 as positive control group and PLGA/TCP+middle-icaritin as comparative group by entire immersion in the solution and dry in vacuum cabinet for 24 hours. In vitro osteogenic potentials of the designed bioactive composite scaffolds were tested in scaffold-co-cultured rabbit bone marrow stem cells (BMSCs) for measurement of cell seeding and proliferation efficiency, alkaline phosphatase (ALP) activity, calcium deposition, and quantitative mRNA expression of relative osteogenic genes. In vivo efficacy investigation was designed to evaluate osteogenesis and angiogenesis in a bilateral ulna bone segmental defect model implanted with composite scaffold in rabbits, with radiography and in vivo micro-CT for studying new bone regeneration and micro-CT-based angiography and histology for neovascularization, dynamic MRI for local blood perfusion function, as well as host tissue and scaffold material interactions.
Results: The in vitro characterization and osteogenic potential of the fabricated scaffolds were performed and confirmed, respectively. Micro-CT quantitation showed that the scaffolds had interconnected macropores with an average porosity of 75±3.27 % and pore size or diameter of 458±25.6 μm. Compared to P/T, P/T+icaritin and P/T/BMP-2 scaffolds, P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin (n=6, p<0.01) presented higher cell seeding efficiency, ALP activity and calcium nodules and up-regulated mRNA expressions of Collagen type I and Osteopontin of co-cultured BMSCs. P/T+BMP-2 showed the best osteogenic effects among all groups (n=6, p<0.01). In vivo measurement of x-ray and micro-CT in rabbit ulna bone defect model at week 2, 4 and 8 post-surgery showed more newly formed bone in the defects treated with P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin scaffold (n=6, p<0.01) compared with that of P/T, P/T+icaritin and P/T/BMP-2 groups. P/T+BMP-2 also showed the best bone formation among all groups (n=6, p<0.01). Histological results also demonstrated that there were more osteoid tissues and typical lamellar bone in surface and internal of the implants, as well as along the adjacent host bone in P/T/icaritin groups (n=5, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01). P/T+BMP-2 group showed the most newly formed bone (n=6, p<0.01). In addition, newly formed vessels in the defects were identified with micro-CT-based angiography and functionally supported by dynamic MRI for reflecting blood perfusion. The results showed more ingrowing new vessels in P/T/icaritin groups (n=6, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01), compared to P/T and P/T/BMP-2 groups. For comparing dose effects among three scaffolds incorporating different concentration of icaritin, we found that middle dose PLGA/TCP/icaritin composite scaffold showed the best osteogenic potential.
Conclusion: PLGA/TCP scaffolds incorporating exogenous phytomolecule icaritin demonstrated the desired osteogenic differentiation potential and bone regeneration capability as investigated in vitro and in vivo, where the middle dose of icaritin incorporating PLGA/TCP composite scaffold showed the best effects. These findings may form a good foundation for potential clinical validation of this innovative bioactive composite scaffold with either conventional endogenous BMP-2 for in vitro loading before application or more attractively and user-friendly incorporated with exogenous phytomolecule icaritin as a ready product for enhancing bone defect repair.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Chen, Shihui.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 173-198).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Acknowledgements --- p.viii
Abstract --- p.x
中文摘要 --- p.xiii
List of Abbreviations --- p.xvi
List of Tables --- p.xix
List of Figures --- p.xx
Journal Publications --- p.xxv
Journal Supplements --- p.xxv
Conference Abstracts --- p.xxvi
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- Bone Defect in Orthopaedics --- p.2
Chapter 1.2 --- Human Skeletons --- p.2
Chapter 1.2.1 --- Bone Types and Function --- p.2
Chapter 1.2.2 --- Bone Development --- p.4
Chapter 1.2.3 --- Bone Physiology and Structure --- p.6
Chapter 1.2.4 --- Bone Specific Markers --- p.7
Chapter 1.2.5 --- Bone Cells --- p.9
Chapter 1.2.6 --- Bone Marrow Stromal Cells --- p.12
Chapter 1.3 --- Bone Regeneration and Remodeling --- p.13
Chapter 1.3.1 --- Bone Defect Healing --- p.13
Chapter 1.3.2 --- Non-union and Segmental Defect --- p.15
Chapter 1.3.3 --- Bone Defect Treatment --- p.16
Chapter 1.4 --- Angiogenesis in Bone Healing --- p.19
Chapter 1.4.1 --- Blood Vessels Formation Process --- p.20
Chapter 1.4.2 --- Growth Factor in Angiogenesis --- p.21
Chapter 1.5 --- Biomaterials in Bone Tissue Engineering --- p.22
Chapter 1.6 --- Scaffold-Based Therapy --- p.23
Chapter 1.6.1 --- Bone Grafts --- p.23
Chapter 1.6.1.1 --- Autografts --- p.23
Chapter 1.6.1.2 --- Allografts --- p.25
Chapter 1.6.2 --- Bone Graft Substitutes --- p.25
Chapter 1.6.2.1 --- Bone Formation in Porous Scaffolds --- p.25
Chapter 1.6.2.2 --- Degradable Polymers --- p.27
Chapter 1.6.2.3 --- Non-Degradable Polymers --- p.29
Chapter 1.6.2.4 --- Ceramics --- p.29
Chapter 1.6.2.5 --- Bioactive Composite Materials --- p.30
Chapter 1.7 --- Growth Factor-Based Therapy --- p.31
Chapter 1.7.1 --- Endogenous Growth Factor--Bone Morphogenetic Proteins --- p.31
Chapter 1.7.2 --- Exogenous phytomoleculeIcaritin--Icaritin --- p.31
Chapter 1.7.3 --- Delivery of Growth Factor in Tissue Engineering --- p.34
Chapter 1.8 --- Fabrication of Porous Composite Scaffolds --- p.37
Chapter 1.8.1 --- Architectural Parameters of Bone Scaffolds --- p.37
Chapter 1.8.2 --- Three-Dimensional Scaffold Fabrication --- p.37
Chapter 1.9 --- Animal Models for Testing Bone Defects Healing --- p.39
Chapter Chapter 2 --- Research Rationale and Study Objectives
Chapter 2.1 --- Research Rationale --- p.42
Chapter 2.2 --- Study Objectives --- p.46
Chapter Chapter 3 --- Bioactive Composite Scaffolds: Preparation, Morphology and Release Assay
Chapter 3.1 --- Introduction --- p.49
Chapter 3.2 --- Materials and Methods --- p.50
Chapter 3.2.1 --- Materials --- p.50
Chapter 3.2.2 --- Fabrication of PLGA/TCP Incorporating BMP-2 or Icaritin --- p.51
Chapter 3.2.3 --- Morphological Analysis of Composite Scaffolds --- p.53
Chapter 3.2.3.1 --- Analysis of Porosity and Macropores Diameter Using High-resolution Micro-CT --- p.53
Chapter 3.2.3.2 --- Analysis of Surface Morphology and Elements Composition Using Scanning Electron Microscopy --- p.54
Chapter 3.2.4 --- Icaritin Content Assay in PLGA/TCP Scaffolds Incorporating Icaritin --- p.54
Chapter 3.2.5 --- Preparation of PLGA/TCP Scaffold Coating BMP-2 or Icaritin --- p.55
Chapter 3.2.6 --- In vitro Release Assay --- p.55
Chapter 3.2.6.1 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.55
Chapter 3.2.6.2 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.56
Chapter 3.2.7 --- Mechanical Properties of Composite Scaffolds --- p.56
Chapter 3.2.8 --- Statistical Analysis --- p.57
Chapter 3.3 --- Results --- p.57
Chapter 3.3.1 --- Morphological Analysis of Composite Scaffolds --- p.57
Chapter 3.3.1.1 --- Porosity and Macroscopic Diameter --- p.57
Chapter 3.3.1.2 --- Surface Morphology and Elements Composition --- p.58
Chapter 3.3.2 --- Icaritin Content in Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60
Chapter 3.3.3 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60
Chapter 3.3.4 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.61
Chapter 3.3.5 --- Mechanical Properties of Composite Scaffolds --- p.63
Chapter 3.4 --- Discussion --- p.64
Chapter 3.5 --- Summary --- p.71
Chapter Chapter 4 --- Bioactive Composite Scaffolds: In vitro Degradation and Characterization Studies
Chapter 4.1 --- Introduction --- p.73
Chapter 4.2 --- Materials and Methods --- p.74
Chapter 4.2.1 --- Preparation of Composite Scaffolds for in vitro Degradation Assay --- p.74
Chapter 4.2.2 --- Characterizations --- p.75
Chapter 4.2.2.1 --- Scaffold Volume Changes --- p.75
Chapter 4.2.2.2 --- Scaffold Weight Changes --- p.75
Chapter 4.2.2.3 --- pH Value Changes --- p.75
Chapter 4.2.2.4 --- Calcium Ion Release from Scaffolds --- p.76
Chapter 4.2.3 --- Mechanical Properties Changes --- p.76
Chapter 4.2.4 --- Statistical Analysis --- p.77
Chapter 4.3 --- Results --- p.77
Chapter 4.3.1 --- Volume Decrease --- p.78
Chapter 4.3.2 --- Weight Loss --- p.78
Chapter 4.3.3 --- pH Value Reduction --- p.79
Chapter 4.3.4 --- Calcium Ion Release --- p.79
Chapter 4.3.5 --- Mechanical Properties --- p.80
Chapter 4.4 --- Discussion --- p.81
Chapter 4.5 --- Summary --- p.84
Chapter Chapter 5 --- In vitro Evaluation of Bone Marrow Stem Cells (BMSCs) Growing on Bioactive Composite Scaffolds
Chapter 5.1 --- Introduction --- p.87
Chapter 5.2 --- Materials and Methods --- p.90
Chapter 5.2.1 --- Preparation of Composite Scaffolds for in vitro Evaluation --- p.90
Chapter 5.2.2 --- BMSCs Seeding Rate and Proliferation on Composite Scaffolds --- p.90
Chapter 5.2.3 --- Alkaline Phosphate (ALP) Activity Assay --- p.92
Chapter 5.2.4 --- Osteogenic Gene Expression Assay Using Quantitative Real-time PCR --- p.92
Chapter 5.2.5 --- Calcium Deposition Assay Using Alizarin Red Staining --- p.93
Chapter 5.2.6 --- Statistical Analysis --- p.94
Chapter 5.3 --- Results --- p.94
Chapter 5.3.1 --- Cells Seeding Efficiency and Proliferation --- p.94
Chapter 5.3.2 --- ALP Activity --- p.97
Chapter 5.3.3 --- Osteogenic Gene mRNA Expression --- p.97
Chapter 5.3.4 --- Calcium Deposition --- p.98
Chapter 5.4 --- Discussion --- p.99
Chapter 5.5 --- Summary --- p.102
Chapter Chapter 6 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds
Chapter 6.1 --- Introduction --- p.105
Chapter 6.2 --- Materials and Methods --- p.106
Chapter 6.2.1 --- Preparation of Composite Scaffolds for Implantation --- p.106
Chapter 6.2.2 --- Establishment of Ulna Bone Segmental Defect in Rabbits --- p.107
Chapter 6.2.3 --- Radiographic Evaluation of New Bone Area Fraction --- p.109
Chapter 6.2.4 --- XtremeCT Evaluation of New Bone Formation and Bone Mineral Density (BMD) --- p.110
Chapter 6.2.5 --- Histological Evaluation of New Bone Formation --- p.111
Chapter 6.2.6 --- Evaluation of Rate of New Bone Formation and Mineral Apposition Rate (MAR) --- p.114
Chapter 6.2.7 --- Evaluation of Neovascularization Using Micro-CT-based Microangiography --- p.116
Chapter 6.2.8 --- Blood Perfusion Function Using Dynamic Magnetic Resonance Imaging (MRI) --- p.119
Chapter 6.2.9 --- Statistical Analysis --- p.120
Chapter 6.3 --- Results --- p.121
Chapter 6.3.1 --- Radiographic Area Fraction of New Bone Formation --- p.123
Chapter 6.3.2 --- XtremeCT New Bone Volume Fraction and BMD --- p.128
Chapter 6.3.3 --- Histological New Bone Fraction --- p.133
Chapter 6.3.4 --- Rate of New Bone Formation and MAR --- p.136
Chapter 6.3.5 --- New Vessels Volume Evaluated Using Micro-CT-Based Microangiography --- p.140
Chapter 6.3.6 --- Dynamic Blood Perfusion Function --- p.144
Chapter 6.4 --- Discussion --- p.146
Chapter 6.5 --- Summary --- p.151
Chapter Chapter 7 --- Summaries, Conclusions, Limitations and Future Studies
Chapter 7.1 --- Introduction --- p.153
Chapter 7.2 --- Bioactive Composite Scaffolds: Preparation, Morphology and in vitro Release Evaluation --- p.155
Chapter 7.3 --- Bioactive Composite Scaffolds: in vitro Degradation and Characterization Studies --- p.159
Chapter 7.4 --- In vitro Evaluation of the Response of Bone Marrow Stem Cells Growing on Bioactive Composite Scaffolds --- p.160
Chapter 7.5 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds --- p.162
Chapter 7.6 --- Evaluation of Dose-dependent Effects of Icaritin Mechanical Property, Degradation, and Osteogenic Potentials --- p.164
Chapter 7.7 --- Conclusions --- p.170
Chapter 7.8 --- Limitations and Future Studies --- p.171
Chapter 7.9 --- References --- p.173
Chapter 7.10 --- Appendix --- p.199
Chapter 7.10.1 --- Animal Licence and Ethics --- p.199
Chapter 7.10.2 --- Safety Approval --- p.201
Chapter 7.10.3 --- Journal Supplements --- p.202
Chapter 7.10.4 --- Conference Abstracts--Posters --- p.205
Chapter 7.10.5 --- Conformation of Paper Submission --- p.208
Chapter 7.10.6 --- Published Paper --- p.209

by Chak Chi Wai.
Thesis (M.Phil)--Chinese University of Hong Kong, 1992.
Includes bibliographical references (leaves 142-155).
ACKNOWLEDGMENTS --- p.i
TABLE OF CONTENT --- p.ii
LIST OF ABBREVIATION --- p.viii
ABSTRACT --- p.x
Chapter CHAPTER ONE: --- INTRODUCTION
Chapter 1.1 --- INTRODUCTION TO ALKALINE PHOSPHATASE --- p.2
Chapter 1.1.1 --- The Alkaline Phosphatase Isoenzymes --- p.2
Chapter 1.1.2 --- The Properties of Alkaline Phosphatases --- p.5
Chapter 1.1.3 --- Serum Alkaline Phosphatases --- p.7
Chapter 1.1.3.1 --- Intestinal Alkaline Phosphatase --- p.8
Chapter 1.1.3.2 --- Placental Alkaline Phosphatase --- p.8
Chapter 1.1.3.3 --- Renal Alkaline Phosphatase --- p.9
Chapter 1.1.3.4 --- Skeletal Alkaline Phosphatase --- p.9
Chapter 1.1.3.5 --- Hepatic Alkaline Phosphatase --- p.10
Chapter 1.1.3.6 --- Miscellaneous Alkaline Phosphatases --- p.10
Chapter 1.1.4 --- Problems in Discriminating the Skeletal and Hepatic Alkaline Phosphatase in Serum --- p.12
Chapter 1.1.5 --- Wheat Germ Lectin Precipitation of the Bone- Specific Alkaline Phosphatase --- p.13
Chapter 1.2 --- STRUCTURE OF BONE AND MECHANISMS OF CALCIFICATION --- p.15
Chapter 1.2.1 --- Gross Structure of Bone --- p.15
Chapter 1.2.2 --- The Elements of Bone --- p.17
Chapter 1.2.2.1 --- Bone Cells --- p.17
Chapter 1.2.2.2 --- Organic Substances of Bone --- p.19
Chapter 1.2.2.3 --- Inorganic Substances of Bone --- p.21
Chapter 1.2.3 --- Mechanisms of Calcification --- p.22
Chapter 1.3 --- BONE FRACTURE HEALING --- p.24
Chapter 1.3.1 --- Types of Fracture --- p.24
Chapter 1.3.2 --- The Process of Bone Fracture Healing --- p.26
Chapter 1.3.2.1 --- Stage of Hematoma --- p.26
Chapter 1.3.2.2 --- Stage of Subperiosteal and Endosteal Cellular Proliferation --- p.28
Chapter 1.3.2.3 --- Stage of Fibrocartilaginous Callus --- p.28
Chapter 1.3.2.4 --- Stage of Bony Callus --- p.30
Chapter 1.3.2.5 --- Stage of Remodeling --- p.31
Chapter 1.4 --- THE OSTEOBLASTIC CHARACTERS OF UMR-106 OSTEOSARCOMA CELL LINE --- p.32
Chapter 1.4.1 --- Classification of Osteosarcoma --- p.32
Chapter 1.4.2 --- Derivation of UMR-106 Osteosarcoma Cell Line --- p.33
Chapter 1.4.3 --- Osteoblastic Characters of UMR-106 --- p.34
Chapter 1.4.3.1 --- ALP Expression --- p.34
Chapter 1.4.3.2 --- Hormone Responsive Adenylate Cyclase System --- p.35
Chapter 1.4.3.3 --- "Cytosolic Receptors for 1,25-Dihydroxy- cholecalciferol" --- p.35
Chapter 1.5 --- IN VITRO CULTURE OF FETAL RAT CALVARIAL OSTEOBLASTS --- p.37
Chapter 1.6 --- AIM AND SCOPE OF THIS DISSERTATION --- p.39
Chapter CHAPTER TWO: --- MATERIALS AND METHODS
Chapter 2.1 --- BONE FRACTURE OPERATION --- p.42
Chapter 2.1.1 --- Animals --- p.42
Chapter 2.1.2 --- Blood Sampling and Preparation of Plasma Samples --- p.42
Chapter 2.1.3 --- Bone Fracture Operation --- p.43
Chapter 2.1.3.1 --- Reagents and Apparatus --- p.43
Chapter 2.1.3.2 --- Procedures --- p.44
Chapter 2.1.4 --- Radiography --- p.50
Chapter 2.1.5 --- Removal of Tibiae --- p.51
Chapter 2.1.6 --- Extraction of Callus ALP --- p.51
Chapter 2.1.6.1 --- Reagent --- p.51
Chapter 2.1.6.2 --- Homogenization of the Callus --- p.51
Chapter 2.1.6.3 --- Extraction of ALP --- p.52
Chapter 2.1.7 --- Assay for Bone-Specific ALP --- p.53
Chapter 2.1.7.1 --- Reagents --- p.53
Chapter 2.1.7.2 --- Procedures --- p.54
Chapter 2.1.8 --- Normal Curve for Plasma Bone-Specific ALP in Rabbits --- p.56
Chapter 2.1.9 --- The Effects of Tibial Fracture on the Plasma Level of Bone-Specific ALP in Rabbits --- p.56
Chapter 2.1.10 --- Profile of Plasma Bone-Specific ALP upon a Fracture Healing --- p.57
Chapter 2.1.11 --- Profile of Callus Bone-Specific ALP at Different Stages of Fracture Healing --- p.57
Chapter 2.2 --- CLINICAL STUDIES OF PLASMA BONE-SPECIFIC ALP --- p.58
Chapter 2.2.1 --- Patient Groups --- p.58
Chapter 2.2.1.1 --- Normal Adults --- p.58
Chapter 2.2.1.2 --- Fracture Group --- p.58
Chapter 2.2.1.3 --- Tumor Group --- p.59
Chapter 2.2.2 --- Assays for Plasma Bone-Specific ALP --- p.59
Chapter 2.3 --- "IN VITRO CULTURES OF FETAL, RAT OSTEOBLASTS AND UMR-106 OSTEOSARCOMA cell line" --- p.60
Chapter 2.3.1 --- Animals --- p.60
Chapter 2.3.2 --- UMR-106 Cell Line --- p.60
Chapter 2.3.3 --- General Reagents Used for Cell Culture --- p.60
Chapter 2.3.4 --- Isolation of Calvarial Osteoblasts --- p.64
Chapter 2.3.4.1 --- Tools and Reagents --- p.64
Chapter 2.3.4.2 --- Procedures --- p.65
Chapter 2.3.5 --- Storage of UMR-106 Cell Line --- p.67
Chapter 2.3.6 --- Subculture of Confluent Monolayer --- p.68
Chapter 2.3.6.1 --- Reagents --- p.68
Chapter 2.3.6.2 --- Procedures --- p.69
Chapter 2.3.7 --- Staining for Calcium Deposits --- p.69
Chapter 2.3.7.1 --- Reagents --- p.70
Chapter 2.3.7.2 --- Procedures --- p.70
Chapter 2.3.8 --- Protein Determination --- p.71
Chapter 2.3.8.1 --- Reagents --- p.71
Chapter 2.3.8.2 --- Procedures --- p.71
Chapter 2.3.9 --- Microdetermination of Inorganic Phosphate --- p.72
Chapter 2.3.9.1 --- Reagents --- p.72
Chapter 2.3.9.2 --- Procedures --- p.73
Chapter 2.3.10 --- Determination of Calcium --- p.73
Chapter 2.3.10.1 --- Reagent --- p.73
Chapter 2.3.10.2 --- Procedures --- p.73
Chapter 2.3.11 --- Extraction and Assay for Cellular ALP --- p.74
Chapter 2.3.11.1 --- Reagents --- p.74
Chapter 2.3.11.2 --- Procedures --- p.75
Chapter 2.3.12 --- Cell Surface ALP Assay --- p.75
Chapter 2.3.12.1 --- Reagents --- p.75
Chapter 2.3.12.2 --- Procedures --- p.76
Chapter 2.3.13 --- Extraction of Calcium Phosphate Deposits --- p.76
Chapter 2.3.13.1 --- Reagent --- p.76
Chapter 2.3.13.2 --- Procedures --- p.76
Chapter 2.3.14 --- Collagen Synthesis Assay --- p.77
Chapter 2.3.14.1 --- Reagents --- p.77
Chapter 2.3.14.2 --- Procedures --- p.78
Chapter CHAPTER THREE: --- EFFECTS OF TIBIAL FRACTURE ON THE LEVEL OF BONE-SPECIFIC ALKALINE PHOSPHATASE IN RABBITS
INTRODUCTION --- p.81
results:
Chapter 3.1 --- normal curve for plasma bone-specific alp in rabbits --- p.82
Chapter 3.2 --- THE EFFECTS OF TIBIAL FRACTURE ON THE PLASMA LEVEL OF BONE-SPECIFIC ALP IN RABBITS --- p.84
Chapter 3.3 --- PROFILE OF THE PLASMA ALP LEVEL UPON HEALING OF TIBIAL FRACTURE --- p.86
Chapter 3.4 --- RADIOGRAPHY --- p.89
Chapter 3.5 --- PROFILE OF CALLUS BONE-SPECIFIC ALP ACTIVITY UPON HEALING OF TIBIAL FRACTURE --- p.93
DISCUSSION --- p.95
Chapter CHAPTER FOUR: --- CLINICAL STUDIES OF PLASMA BONE-SPECIFIC ALKALINE PHOSPHATASE
INTRODUCTION --- p.100
RESULTS:
Chapter 4.1 --- NORMAL VALUES --- p.100
Chapter 4.2 --- FRACTURE GROUP --- p.101
Chapter 4.3 --- BONE TUMOR GROUP --- p.102
DISCUSSION --- p.102
Chapter CHAPTER FIVE: --- IN VITRO CULTURE OF FETAL RAT OSTEOBLASTS AND UMR-106 CELL LINE
INTRODUCTION --- p.105
RESULTS:
Chapter 5.1 --- IN VITRO MINERALIZATION OF UMR-106 CELLS AND PRIMARY RC CELLS --- p.107
Chapter 5.2 --- STUDY OF BONE-SPECIFIC ALP RELEASED INTO MEDIUM BY UMR-106 CELLS AND PRIMARY RC CELLS --- p.113
Chapter 5.3 --- STUDY OF CELLULAR ALP ACTIVITIES AND CALCIUM PHOSPHATE DEPOSITS --- p.116
Chapter 5.4 --- STUDIES OF CELLULAR ALP ACTIVITIES AND RELATIVE RATES OF COLLAGEN SYNTHESIS --- p.125
DISCUSSION --- p.128
Chapter CHAPTER SIX: --- GENERAL DISCUSSION --- p.136
BIBLIOGRAPHY --- p.142
APPENDIX --- p.156

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library.
The growth plate is a cartilaginous structure located at the proximal and distal ends of immature long bones, which contributes to longitudinal growth through the process of endochondral ossification. Cartilage has a limited ability to regenerate and in children, injury to the the growth plate can result in limb length discrepancies and angular deformity, due to formation of a bone bridge at the damaged site which disturbs structure and function of the growth plate. Current treatments of the abnormalities arising from growth plate arrest involve surgical correction once the deformities have manifested. To date, there is no biological based therapy for the repair of injured/damaged growth plate cartilage. Mesenchymal stem cells (MSC) are self renewable mulitpotential progenitor cells with the capacity to differentiate toward the chondrogenic lineage. Since their discovery, significant interest has been generated in the potential application of these cells for cartilage regeneration. In this study, the ability of autologous bone marrow mesenchymal stem cells to regenerate growth plate cartilage in a sheep model was examined.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330837
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

by Paul, Liu Po Lung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1994.
Includes bibliographical references (leaves 86-94).
ACKNOWLEDGMENT --- p.i
TABLE OF CONTENT --- p.ii
"LIST OF TABLE, FIGURE & PHOTO" --- p.viii
ABSTRACT --- p.x
Chapter CHAPTER ONE : --- INTRODUCTION --- p.1
Chapter 1.1 --- ALKALINE PHOSPHATASE
Chapter 1.1.1 --- Alkaline Phosphatase Isoenzyme --- p.2
Chapter 1.1.2 --- The Properties of Alkaline Phosphatases --- p.4
Chapter 1.1.3 --- Serum Alkaline Phosphatases --- p.6
Chapter 1.1.3.1 --- Placental Alkaline Phosphatase --- p.7
Chapter 1.1.3.2 --- Intestinal Alkaline Phosphatase --- p.7
Chapter 1.1.3.4 --- Skeletal Alkalne Phosphatase --- p.8
Chapter 1.1.3.5 --- Hepatic Alkaline Phosphatase --- p.8
Chapter 1.1.3.3 --- Renal Alkaline Phosphatase --- p.9
Chapter 1.1.3.6 --- Miscellaneous Alkaline Phosphatase --- p.9
Chapter 1.1.4 --- Problems in Discriminating the Skeletal and Hepatic Alkaline Phosphatase in Serum --- p.11
Chapter 1.1.5 --- Quantitative measure of the Bone-Specific Alkaline Phosphatase --- p.12
Chapter 1.1.6 --- Qualitative Detection of ALP isoenzymes --- p.14
Chapter 1.2 --- OSTEOSARCOMA --- p.17
Chapter 1.2.1 --- Definition --- p.17
Chapter 1.2.2 --- Epidemiology and Statistics --- p.17
Chapter 1.2.3 --- Clinical Presentation --- p.18
Chapter 1.2.4 --- Radiographic finding --- p.19
Chapter 1.2.5 --- Staging of Musculoskeletal Neoplasms --- p.20
Chapter 1.2.6 --- Treatment of osteosarcoma --- p.21
Chapter 1.2.6.1. --- Chemotherapy in Prince of Wales Hospital --- p.21
Chapter 1.3 --- PLASMA AND TISSUE ALKALINE PHOSPHATASE IN NORMAL AND NEOPLASTIC CONDITION --- p.23
Chapter 1.3.1 --- Normal values of plasma alkaline phosphatase --- p.23
Chapter 1.3.2 --- Clinical use of elevated plasma & tissue alkaline phosphatase level in neoplastic conditions --- p.25
Chapter 1.3.2.1 --- Helping the Diagnosis of the Osteosarcoma --- p.25
Chapter 1.3.2.2 --- Monitoring the effect of chemotherapy --- p.26
Chapter 1.3.2.3 --- Predicting the clinical course --- p.26
Chapter 1.3.3 --- Qualitative measurement of ALP in plasma and tissue extract of osteosarcoma patient --- p.29
Chapter 1.4 --- AIM AND SCOPE OF THE PRESENT DISSERATION --- p.30
Chapter CHAPTER TWO : --- MATERIALS AND METHODS --- p.32
Chapter 2.1 --- DIFERENT GROUPS OF PATIENTS --- p.33
Chapter 2.1.1 --- Monitering the plasma bone specific ALP --- p.33
Chapter 2.1.1.1 --- Osteosarcoma group --- p.33
Chapter 2.1.1.2 --- Benign bone tumour group --- p.34
Chapter 2.1.1.3 --- Metastasis group --- p.34
Chapter 2.1.2 --- Collection of plasma samples preserve of tumor tissue --- p.34
Chapter 2.2 --- QUANTITATIVE ANALYSIS OF THE PLASMA AND TISSUE BONE SPECIFIC ALKALINE PHOSPHATASE --- p.36
Chapter 2.2.1 --- Extraction of tissue ALP --- p.36
Chapter 2.2.1.1. --- Reagent --- p.36
Chapter 2.2.1.2. --- Homogenization of the bone tissue --- p.36
Chapter 2.2.1.3. --- Extraction of ALP --- p.37
Chapter 2.2.2 --- Assay for Bone-specific ALP --- p.38
Chapter 2.2.2.1. --- Reagents --- p.38
Chapter 2.2.2.2. --- Procedures --- p.38
Chapter 2.3 --- QUALITATIVE MEASUREMENT OF ALP ISOENZYME --- p.40
Chapter 2.3.1 --- Equipment required --- p.40
Chapter 2.2.2 --- Practical procedure --- p.40
Chapter 2.3.3.1 --- Gel casting --- p.40
Chapter 2.3.3.2 --- Sample preparation and application --- p.42
Chapter 2.3.3.3 --- Electrofocusing --- p.42
Chapter 2.3.3.4 --- Western blotting of the protein --- p.43
Chapter 2.3.3.5 --- Detection methods --- p.45
Chapter 2.4 --- METHOD OF STATISTICAL ANALYSIS --- p.48
Chapter CHAPTER THREE : --- RESULTS --- p.49
Chapter 3.1 --- QUANTITATIVE MEASUREMENT OF PLASMA AND TISSUE BONE SPECIFIC ALKALINE PHOSPHATASE --- p.50
Chapter 3.1.1 --- General Information of the patients monitoring --- p.50
Chapter 3.1.2 --- Pretreatment evaluation --- p.52
Chapter 3.1.3 --- Correlation between the pretreatment plasma ALP levels and prognosis in the osteosarcoma patient group --- p.57
Chapter 3.1.4 --- "Correlation between the pre-operational, post- operational plasma ALP levels and the prognosis of osteosarcoma" --- p.59
Chapter 3.1.5 --- Analysis of plasma ALP levels at the time of relapse in osteosarcoma patients --- p.61
Chapter 3.1.6 --- Usefulness of the plasma ALP levels for monitoring the effectiveness of chemotherapy --- p.62
Chapter 3.1.7 --- Correlation between the ALP levels in the tumor extract and the prognosis of the osteosarcoma --- p.64
Chapter 3.2 --- QUALITATIVE ANALYSIS OF THE PLASMA AND TISSUE ALKALINE PHOSPHATASE LEVEL --- p.67
Chapter 3.2.1 --- Comparison of the result of Isoelectric focusing of the plasma ALP of the osteosarcoma patients and the normal subjects --- p.67
Chapter 3.2.1 --- Result of Isoelectric focusing of the ALP isoenzymes in the tissue extract of the osteosarcoma and normal bone --- p.70
Chapter CHAPTER FOUR : --- DISCUSSION --- p.72
Chapter 4.1 --- USE OF QUANTITATIVE MONITORING OF PLASMA ALP AND MEASURING TISSUE ALP IN OSTEOSARCOMA PATIENTS --- p.73
Chapter 4.2 --- ISOELECTRIC FORCUSING AS A TECNIQUE FOR QUALITATIVE MEASUREMENT OF PLASMA AND TISSUE ALKALINE PHOSPHATASE --- p.80
Chapter CHAPTER FIVE : --- CONCLUSION --- p.83
Chapter CHAPTER SIX : --- BIBILOGRAPHY --- p.85

Despite numerous studies have demonstrated an association of type 2 diabetes mellitus (T2DM) and osteoporosis, the underlying mechanism connecting these two conditions remains elusive. Clinically, combined calcium and vitamin D supplement is the commonest osteoporosis therapy; however, recent studies have suggested an increase in cardiovascular risks associated with calcium plus vitamin D supplementation. Therefore, an alternative strategy in treating osteoporosis patients with T2DM is urgently needed. In this study, we hypothesized that combined administration of menaquinone-4 (vitamin K₂, biologically active form of vitamin K) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃, biologically active form of vitamin D) as a novel therapy in treating osteoporosis of T2DM patients. Anabolic effect of vitamin K₂ and vitamin D₃, alone or in combination, was assessed on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (db⁺/m⁺) and obese/diabetic (db⁺/db⁺, leptin receptor-deficient) mice. Furthermore, the underlying cellular mechanism was also investigated. Serum undercarboxylated osteocalcin (an indication of vitamin K₂ level) level was higher whereas vitamin D₃ level was lower in db⁺/db⁺ mice, and sections of the iliac crests of db⁺/db⁺ mice illustrated extensive porous structures filled with enlarged adipocytes compared with db⁺/m⁺ mice. Lower levels of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4, type I collagen, OSX, alkaline phosphatase (ALP) activity, p-Smad1/5/8 and p-ERK1/2) were observed in the osteoblasts of db⁺/db⁺ mice. Acute vitamin D₃ (10 nM) application elicited a more sustained and greater magnitude of increase of [Ca²⁺]ᵢ in osteoblasts of db⁺/m⁺ mice when compared with db⁺/db⁺ mice. A significantly higher level of calcium deposits in osteoblasts was observed in db⁺/m⁺ mice when compared to db⁺/db⁺ mice. Co-administration of vitamin K₂ (10 nM) and vitamin D₃ (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K₂ and D₃ co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and transcription factors for bone formation, with a greater magnitude of increase observed in osteoblasts of db⁺/db⁺ mice. Suppressed expression of calcium-sensing receptor (CaSR), F-actin, V-ATPase, vitamin D receptor (VDR) and pregnane X receptor (PXR) observed in osteoblasts of db⁺/db⁺ mice were partially reversed by combined vitamins treatment. Moreover, combined vitamins K₂ plus D₃ treatment significantly enhanced migration and the appearance of surface microvilli and ruffles of osteoblasts of db⁺/db⁺ mice. Effects of combined vitamins K₂ plus D₃ treatment observed in osteoblasts of db⁺/db⁺ and db⁺/m⁺ mice were eradicated by warfarin (20 µM, a vitamin K epoxide reductase inhibitor). Thus, our results illustrate that vitamins K₂ plus D₃ supplementation is a novel therapeutic strategy in treating osteoporosis of T2DM patients.
儘管大量研究已證明第二類型糖尿病和骨質疏鬆症的關聯，連接這兩個病症的基本機制仍然是難以捉摸的。在臨床上，鈣和維生素D的綜合補充劑是最常見的骨質疏鬆症治療，然而最近的研究卻表明服用鈣和維生素D的綜合補充劑會增加患者的心血管風險，因此急切需要尋找可以給予同時患有骨質疏鬆症和第二類型糖尿病患者的替代治療。在本研究中，我們假設甲萘醌-4（維生素K₂，維生素K生物活性形式）和1α，25 - 二羥基維生素D₃（維生素D₃，維生素D的生物活性形式）可以嘗試在同時患有骨質疏鬆症和第二類型糖尿病患者身上作為一種革新的療法。本研究從C57BL/KsJ瘦削/非糖尿病 (db⁺/m⁺) 的小鼠和肥胖/帶有第二類型糖尿病基因 (db⁺/db⁺) 兼有瘦素受體缺陷的小鼠的髂嵴原始成骨細胞上對維生素K₂和維生素D₃單獨或組合使用的合成代謝作用進行了評估。此外，我們也對該成骨細胞的底層機制進行了一系列的研究。
在肥胖/帶有第二類型糖尿病基因的小鼠血清內低羧骨鈣素水平（維生素K₂水平的指標）較高而維生素D水平較低，另外，它們的髂嵴的部分與瘦削/非糖尿病的小鼠相比，呈現出比較廣泛的多孔結構並填滿了擴大的脂肪細胞。從肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞中，可以觀察到它們的骨合成代謝的標誌物和骨骼形成的轉錄因子 (骨鈣蛋白，Runx2，Dlx5，ATF4，第一類型骨膠原，OSX，鹼性磷酸酶 (ALP) 活性，p-Smad1/5/8和p-ERK1/2) 的水平比較低。急性維生素D₃ (10 nM) 的應用在瘦削/非糖尿病小鼠的成骨細胞比起在肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞引起更持續和更大幅度的細胞內鈣變化增加。在瘦削/非糖尿病小鼠的成骨細胞中比起在肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞有顯著較高的鈣沉積形成。維生素K₂ (10 nM) 和維生素D₃ (10 nM) 的綜合藥在兩種小鼠的成骨細胞中可以有效地增強鈣沉積的形成。維生素K₂和維生素D₃的綜合藥對增加骨合成代謝的標誌物和骨形成轉錄因子的水平有時間依賴性 (7，14和21日)，療程越長至21日，在肥胖/帶有第二類型糖尿病基因小鼠的成骨細胞中有更大的幅度的增加。合併維生素治療能部分有效地逆轉在肥胖/帶有第二類型糖尿病基因小鼠的成骨細胞中被抑制表達的鈣敏感受體 (CASR)，F-肌動蛋白，V-ATP酶，維生素D受體 (VDR) 和孕烷X受體 (PXR)。此外，結合維生素K₂加維生素D₃治療顯著增強了肥胖/帶有第二類型糖尿病基因小鼠的成骨細胞的細胞遷移和增加了成骨細胞表面外觀的微絨毛和褶皺。在瘦削/非糖尿病小鼠的成骨細胞及肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞上結合維生素K₂加維生素D₃的治療效果被華法林 (20 μM，維生素K環氧化物還原酶抑製劑) 根除。因此，我們的結果証明了維生素K₂加維生素D₃補充劑的結合使用可有效地作為治療第二類型糖尿病患者並患有骨質疏鬆症的一種新的治療策略。
Poon, Chui Wa Christina.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2014.n5203
Includes bibliographical references (leaves 135-151).
Abstracts also in Chinese.
Title from PDF title page (viewed on 26, October, 2016).
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.

激素性骨壞死是由於經常使用脈衝性激素處理非骨科性問題引起的一種常見的骨科疾病。在組織病理學上，激素性骨壞死指骨死亡，血管內血栓閉塞和血管外骨髓脂肪沉積會引起缺血導致骨修復不足。上游的分子細胞病理學機制研究表明間充質幹細胞細胞池活性下降，成骨細胞凋亡和骨小梁基質退變導致的不充分修復是激素性骨壞死發生的重要因素。
間充質幹細胞是骨髓的基質組成部分，具有分化成多種細胞的潛能。最近的研究表明，激素性骨壞死可能是骨細胞和/或間質幹細胞病變引起的一種疾病。研究發現，在接受類固醇治療而發生骨壞死的病人中，骨髓間充質幹細胞活性下降和分化潛能發生改變。在骨髓細胞中，激素能夠誘導脂肪發生。盛輝等發現來源於激素性骨壞死兔子中的間充質幹細胞成脂分化增強，謝新薈等進一步發現發生激素性骨壞死的兔子，骨缺損修復延遲，這可能是由激素導致的間充質幹細胞潛能發生改變引起的。綜合以上研究表明，間充質幹細胞在骨壞死發生和修復過程中起著重要作用。我們之前報導過淫羊藿黃酮（EFs）的腸代謝產物淫羊藿素Icaritin通過抑制血栓的形成和脂肪沉澱預防激素性骨壞死。最近，我們把Icaritin整合到聚乳酸聚乙醇酸共聚物/磷酸三鈣（PLGA/TCP）支架材料中，形成PLGA/TCP/Icaritin複合支架材料。我們發現PLGA/TCP/Icaritin複合材料可以促進激素性骨壞死骨缺損的修復，肌肉移植發現PLGA/TCP/Icaritin也能促進新生血管的發生。我們也發現單純PLGA/TCP複合材料也能夠促進激素性骨壞死骨缺損的修復，但是潛在的機制尚不清楚。
骨是一個高度血管化的組織，依賴於血管和骨細胞密切的時空連結維持骨骼的完整性。因此，血管生成在骨骼發育和骨折修復過程中發揮著舉足輕重的作用。血管為骨的發育和再生提供氧氣，為基質輸送刺激間充質細胞特異性成骨的重要信號，另一方面，骨為血管生成輸送生長因數和細胞。
本論文分為以下四個主要部分：
第一部分: 研究Icaritin對人源間充質幹細胞分化的作用及其機制。流式細胞分選鑒定結果表明我們使用的人源間充質幹細胞能夠特異表達間充質幹細胞表面標誌物。MTT實驗結果顯示Icaritin不影響間充質幹細胞的增殖；分化實驗表明Icaritin在沒有成骨誘導試劑存在的情況下無法影響間充質幹細胞的分化。在成骨誘導試劑存在的情況下，Icaritin促進間充質幹細胞成骨分化，抑制其成脂分化；即時螢光實時定量聚合酶鏈式擴增（RT-PCR）結果顯示Icaritin在間充質幹細胞分化過程中上調成骨基因的表達，下調成脂基因表達。進一步研發發現在成骨分化過程中，Icaritin能夠促進BMP2和beta-catenin 蛋白的表達，而BMP2抑制劑Noggin能夠能夠逆轉Icaritin促進的成骨發生。這些發現表明Icaritin能夠促進而非誘導間充質幹細胞的成骨分化，Icaritin調解間充質幹細胞成骨分化具有BMP2信號通路依賴性。
第二部分: 評估激素性骨壞死兔源間充質幹細胞的分化潛能及Icaritin 對異常分化的間充質幹細胞分化潛能的影響。結果表明Icaritin促進正常兔源間充質幹細胞的成骨分化，抑制其成脂分化。激素性骨壞死兔源間充質幹細胞的成骨分化潛能降低，成脂分化升高；而Icaritin能夠劑量依賴性地部分恢復降低的成骨分化潛能，抑制升高的成脂分化活性。激素性骨壞死兔源間充質幹細胞的增殖活性也下降但是不能被Icaritin恢復。Icaritin對激素性骨壞死兔源間充質幹細胞中下降的VEGF的表達無影響。這些發現顯示間充質幹細胞的分化潛能在激素性骨壞死發生過程中遭到破壞，但是能夠被Icaritin部分恢復。
第三部分: 評估Icaritin對體外成血管的影響。我們對Icaritin對人臍帶靜脈內皮細胞（HUVECs）的增殖、遷移、管狀結構形成及成血管相關基因的表達的影響進行了檢測。結果表明Icaritin不影響HUVECs的增殖、遷移和管狀結構的形成；RT-PCR結果顯示Icaritin對HUVECs中的VEGF, HIF1a, FGF2 and TGF-beta表達也沒有影響。這些發現表明Icaritin在體外並不能直接作用于血管生成。結果謝新薈和陳詩慧等人的體內研究結果可以推測在骨缺損修復過程中，Icaritin通過促進成骨間接促進血管生成。
第四部分: 主要研究Icaritin及複合生物材料在體外體內對間充質幹細胞歸巢的影響。結果表明Iaritin能夠促進間充質幹細胞的遷移並上調血管細胞黏附分子1（VCAM1）的表達。複合材料PLGA/TCP和PLGA/TCP/Icaritin在體外培養的條件下能夠募集間充質幹細胞到材料周圍及進入材料。間充質幹細胞體外用修飾性超順磁性氧化鐵（SPIO@SiO₂-NH₂）納米顆粒標記後，其分化潛能依然保留，增殖和潛能能力稍微下降。兔激素性骨壞死造模完成後，股骨遠端髓芯減壓壞死骨缺損手術，PLGA/TCP和PLGA/TCP/Icaritin複合材料植入缺損孔道，同時把SPIO@SiO₂-NH₂標記的間充質幹細胞注射到距離缺損區20毫米的骨髓腔內。結果顯示只有標記的間充質幹細胞植入而沒有材料植入時，缺損區被脂肪細胞充滿，並沒有標記的間充質幹細胞出現，而在缺損區附近和遠離缺損區的部位有標記的間充質幹細胞出現。同時植入PLGA/TCP複合材料和標記的間充質幹細胞時，標記的間充質幹細胞出現在缺損區的材料中，在缺損區附近沒有標記的間充質幹細胞出現，而在遠離缺損區的部位，有標記的間充質幹細胞出現。同時植入PLGA/TCP/Icaritin和標記的間充質幹細胞時，得到跟植入PLGA/TCP複合材料和標記的間充質幹細胞相似的結果，但是在缺損區域，SPIO陽性的間充質幹細胞數目在PLGA/TCP和PLGA/TCP/Icaritin組別中並未發現有顯著性差異。以上發現表明Icaritin和PLGA/TCP複合材料能夠在體外和體內促進間充質幹細胞的歸巢。
綜上所述，複合支架材料PLGA/TCP/Icaritin通過調節間充質幹細胞的歸巢和分化促進激素性骨壞死骨缺損的修復。Icaritin通過BMP2和Wnt/beta-catenin通路調解間充質幹細胞的成骨分化。這是首次研究發現Icaritin及PLGA/TCP支架材料影響骨缺損修復過程中幹細胞歸巢，但是分子細胞生物學機制還需要進一步的研究。
Steroid-associated osteonecrosis (SAON) is a common orthopaedic problem as the pulsed steroids are frequently prescribed for the treatment of non-orthopaedic medical conditions. Histopathologically, SAON refers to death of bone. Intravascular thrombus occlusion and extravascular marrow lipid deposition cause ischemia, which leads to an inadequate repair of the bone. Recent study revealed upstream pathological mechanism at cellular and molecular level. The decrease in activity of mesenchymal stem cell (MSC) pool, apoptosis of osteocytes, and trabecular bone matrix degeneration may cause bone inadequate repair, a key pathological feature found in SAON.
MSCs are the stromal component of bone marrow (BM) and have the potential to differentiate into several cell types. Recent studies have suggested that SAON may be a disease of bone cells and/or MSCs. With corticosteroid therapy in patients, the MSCs activity decreased and differentiation potential changed. Steroids have been also shown to produce adipogenesis in bone-marrow cells. It has been found adipogenesis of MSCs from SAON rabbits elevated (Sheng et al., 2007a) and bone defect repair was delayed in rabbits with SAON (Xie et al., 2011), this may be caused by altered MSCs potentials. All these findings imply MSCs play a vital role in SAON development and bone defect repair. It had been reported that Icaritin, an intestinal metabolite of Epimedium-derived avonoids (EF) reduced SAON incidence with inhibition of both thrombosis and lipid deposition (Zhang et al., 2009a). More recently, we found integrating Icaritin into PLGA/TCP to form PLGA/TCP/Icaritin composite scaffold could promote SAON bone defect repair and more neovascularization formed in an intramuscular implantation model, and further found PLGA/TCP scaffold only also could promote SAON bone defect repair in rabbits (Wang et al., 2012a). But the underlying mechanism remains unclear.
Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate MSC specification to promote bone formation and repair. On the other hand, bone also supplies growth factors and cells for angiogenesis. The content of this thesis is divided into the following four major parts:
Part I: to study the effect and molecular mechanism of Icaritin on the differentiation of human bone marrow-derived MSCs. Human MSC was identified first by flow cytometery and result showed our cultured human MSC expressed standard surface markers of MSCs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation ability of MSCs was not affected by Icaritin. Differentiation assay showed that without oseteogenic supplements (OS), Icaritin had no effect on osteogenic differentiation of MSCs. With presence of OS, Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs. Real- time polymerase chain reaction (RT-PCR) showed that Icaritin up-regulated osteoblastic marker genes expression during osteogenic differentiation of MSCs and inhibited adipogenic gene expression. Further studies showed that Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis. All these findings indicated Icaritin possessed osteopromotive but not osteoinductive potentials during the differentiation of MSCs. Icaritin regulated osteogenic differentiation of MSCs in BMP2 pathway dependent manner.
Part II: to evaluate the differentiation potential of MSCs derived from rabbit with SAON and the effect of Icaritin on the altered differentiation of MSCs. The results showed that Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs derived from normal rabbit. Osteogenic differentiation potential of mesenchymal stem cells derived from rabbit with SAON declined and Icaritin partly rescued the declined osteogenic differentiation potential in dose-dependent manner. Adipogenic differentiation potential of MSCs derived from rabbit with SAON enhanced while the enhanced adipogenesis could be depressed by Icaritin. The proliferation ability of MSCs derived from rabbit with SAON declined while could not be rescued by Icaritin. VEGF expression decreased in MSCs derived from rabbit with SAON but its expression could not be influenced by Icaritin. These findings showed that the differentiation potential of MSCs destroyed during SAON development and this potential could be partially restored by Icaritin.
Part III: to evaluate the in vitro angiogenic effect of Icaritin. The proliferation, migration and tube formation ability of human umbilical vein cells (HUVECs) were detected. The results showed that Icaritin did not affect HUVECs proliferation, migration and tube-like structure formation of HUVECs. Real time PCR showed that VEGF, HIF1a, FGF2 and TGF-beta expression in HUVECs was not changed when HUVECs were treated by Icaritin. These data indicated Icaritin did not directly impact angiogenesis in vitro. Combined with in vivo findings, we supposed Icaritin promoted angiogenesis through its enhanced osteogenesis during bone defect repair.
Part IV: to study Icaritin and scaffold impact on stem cell homing in vitro and in vivo. It was found Icaritin promoted the migration of rabbit MSCs and increased vascular cell adhesion molecule 1 (VCAM1) expression. Composite scaffolds PLGA/TCP and PLGA/TCP/Icaritin could recruit rabbit MSCs under in vitro culture condition. When labeled with SPIO@SiO₂-NH₂, the differentiation potential of rabbit MSCs retained while proliferation and migration ability of rabbit MSCs declined. Two weeks after SAON establishment, PLGA/TCP and PLGA/TCP/Icaritin scaffolds were implanted into the bone tunnel after core-decompression in initial necrotic bone defect in rabbits with SAON, immediately with SPIO@SiO₂-NH₂ labeled MSCs injected into bone marrow cavity locally. The results showed that without scaffold implantation, the tunnel was filled with fat cells and fibrotic tissues and there was no label MSC in the tunnel while there were more labeled cells appeared in bone marrow near the tunnel than far away the tunnel, with both PLGA/TCP and PLGA/TCP/Icaritin implantation, the labeled MSCs migrated into scaffold after its implantation into the bone tunnel while there was no labeled cell next to the tunnel but some were shown away from the tunnel. No significant difference was found in SPIO positive MSCs in bone tunnel between PLGA/TCP and PLGA/TCP/Icaritin group. The findings indicated that at least PLGA/TCP scaffold itself promoted MSCs homing in vitro and in vivo where the released icaritin could execute its osteopromotive effects.
In summary, the composite scaffold PLGA/TCP/Icaritin enhanced bone defect repair in rabbit with SAON by promoting homing and osteogenesis of MSCs. Icaritin promoted osteogenic differentiation of MSCs through BMP2 mediated signal pathway, such as Wnt/beta-catenin signal pathway. It is first time to report that PLGA/TCP scaffold promoted MSCs homing during bone defect repair, but underlying molecular and cellular mechanism need to be further studied.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Yao, Dong.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 137-158).
Abstract also in Chinese; some appendixes also in Chinese.
ACKNOWLEDGEMENTS --- p.i
TABLE OF CONTENTS --- p.iii
ABSTRACT (IN ENGLISH) --- p.x
ABSTRACT (IN CHINESE) --- p.xiv
FLOWCHART --- p.xviii
LIST OF PUBLICATIONS --- p.xix
LIST OF ABBREVIATIONS --- p.xxi
LIST OF FIGURES --- p.xxiv
Chapter CHAPTER 1: --- Introduction --- p.1
Chapter 1 --- Osteonecrosis --- p.2
Chapter 1.1. --- Etiology --- p.2
Chapter 1.2. --- Anatomy of femoral head --- p.3
Chapter 1.3. --- Pathogenesis --- p.4
Chapter 1.3.1. --- Intraosseous hypertension (Compartment Syndrome of Bone --- p.4
Chapter 1.3.2. --- Intraosseous hypertension (Compartment Syndrome of Bone) --- p.4
Chapter 1.3.3. --- Coagulation --- p.5
Chapter 1.4. --- Development stages of osteonecrosis --- p.5
Chapter 2. --- Steroids-associated osteonecrosis --- p.11
Chapter 2.1. --- Epidemiology --- p.12
Chapter 2.2. --- Histopathology --- p.12
Chapter 2.3. --- Etiopathogenesis --- p.13
Chapter 2.3.1. --- Steroid and fat metabolism --- p.14
Chapter 2.3.2. --- Steroid and endothelial cells --- p.15
Chapter 2.3.3. --- Steroid and coagulation --- p.16
Chapter 2.3.4. --- Steroid and angiogenesis --- p.17
Chapter 2.4. --- Steroid and mesenchymal stem cells (MSCs) --- p.18
Chapter 2.5. --- Treatment strategies for SAON --- p.18
Chapter 2.5.1. --- Prevention --- p.19
Chapter 2.5.2. --- Nonoperative treatment --- p.19
Chapter 2.5.3. --- Operative treatment --- p.19
Chapter 2.5.3.1. --- Core decompression strategy --- p.20
Chapter 2.5.3.2. --- Tissue engineering approach --- p.22
Chapter 3. --- Epimedium-derived flavonoids (EFs) --- p.22
Chapter 3.1. --- Icaritin -Intestinal metabolism of EFs --- p.24
Chapter 3.1.1. --- Anti-tumor activity --- p..25
Chapter 3.1.2. --- Neuroprotective effects --- p.25
Chapter 3.1.3. --- Embryonic stem cells differentiation --- p.25
Chapter 3.1.4. --- Osteogenic differentiation --- p.26
Chapter 4. --- Poly lactic-co-glycolic acid / tricalcium phosphate (PLGA/TCP) scaffold --- p.26
Chapter 5. --- PLGA/TCP/Icaritin --- p.28
Chapter 6. --- Hypothesis of this study --- p.28
Chapter 7. --- Objective --- p.29
Chapter CHAPTER 2: --- The effect of phytomolecule Icaritin on differentiation of human mesenchymal stem cells in vitro --- p.30
Chapter 1. --- Introduction --- p.31
Chapter 2. --- Material and Methods --- p.33
Chapter 2.1. --- Ethics --- p.33
Chapter 2.2. --- Reagents and cell culture --- p.33
Chapter 2.3. --- Surface phenotypes of human BM-MSCs --- p.33
Chapter 2.4. --- Osteogenic and adipogenic differentiation of human BM-MSCs treated with Icaritin --- p.34
Chapter 2.5. --- MTT assay for proliferation of BM-MSCs --- p.34
Chapter 2.6. --- ALP staining --- p.35
Chapter 2.7. --- ALP activity assay --- p.35
Chapter 2.8. --- Alizarin Red S staining --- p.35
Chapter 2.9. --- Oil Red O staining --- p.35
Chapter 2.10. --- Ribonucleic acid (RNA) isolation --- p.36
Chapter 2.11. --- Reverse transcription --- p.36
Chapter 2.12. --- Real time polymerase chain reaction (RT-PCR) --- p.37
Chapter 2.13. --- Western blotting --- p.37
Chapter 2.14. --- Osteogenetic analysis of human MSCs after the addition of BMP2 inhibitor Noggin --- p.39
Chapter 2.15. --- Statistical analysis --- p.39
Chapter 3. --- Results --- p.40
Chapter 3.1. --- Characterization of surface phenotypes of human BM-MSCs --- p.40
Chapter 3.2. --- Icaritin had no effect on human mesenchymal stem cells (MSCs) proliferation --- p..41
Chapter 3.3. --- Icaritin promoted osteogenic differentiation of MSCs in presence of osteogenic supplement --- p.42
Chapter 3.4. --- Icaritin enhanced mineralization in osteogenic differentiation of MSCs only in presence of osteogenic supplement --- p.44
Chapter 3.5. --- Icaritin upregulated mRNA expression of osteoblastic marker genes during osteogenic differentiation of MSCs --- p.45
Chapter 3.6. --- Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis --- p..48
Chapter 3.7. --- Icaritin inhibited fat droplets formation during adipogenic differentiation of MSCs --- p.50
Chapter 4. --- Discussion --- p.52
Chapter 5. --- Conclusion --- p.56
Chapter CHAPTER 3: --- Icaritin rescued abnormal differentiation potential of MSCs derived from rabbit with SAON --- p.57
Chapter 1. --- Introduction --- p.58
Chapter 2. --- Methods and materials --- p.59
Chapter 2.1. --- SAON model establishment --- p.59
Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.60
Chapter 2.3. --- Osteogenic and adipogenic differentiation of rabbit BM-MSCs treated with Icaritin --- p.61
Chapter 2.4. --- MTT Assay for Proliferation of BM-MSCs --- p.62
Chapter 2.5. --- ALP Staining --- p.62
Chapter 2.6. --- ALP Activity Assay --- p.62
Chapter 2.7. --- Alizarin Red S Staining --- p.62
Chapter 2.8. --- Oil Red O Staining --- p.63
Chapter 2.9. --- RNA Isolation --- p.63
Chapter 2.10. --- Reverse transcription --- p.64
Chapter 2.11. --- Real time Polymerase chain reaction (RT-PCR) --- p.64
Chapter 2.12. --- Western blotting performance --- p.65
Chapter 2.13. --- Statistical analysis --- p.65
Chapter 3. --- Results --- p.66
Chapter 3.1. --- The osteogenic differentiation potential declined while adipogenic differentiation ability elevated of MSCs derived from SAON rabbits --- p.66
Chapter 3.2. --- The dose-dependent effect of Icaritin on osteogenic differentiation enhancement of MSCs from normal and SAON rabbits --- p.68
Chapter 3.3. --- Icaritin inhibited adipogenic differentiation of MSCs both derived from normal and SAON rabbits --- p..71
Chapter 3.4. --- PPAR-γ and aP2 proteins expression increased in SAON rabbit while inhibited by Icaritin both in normal and SAON rabbit --- p.74
Chapter 3.5. --- Proliferation ability of MSCs derived from SAON rabbit declined and Icaritin had no effect on proliferation both derived from normal and SAON rabbit --- p.75
Chapter 3.6. --- Icaritin had no effect on the expression of VEGF which decreased in MSCs derived SAON --- p.76
Chapter 4. --- Discussion --- p.76
Chapter 5. --- Conclusion --- p.81
Chapter CHAPTER 4: --- The effect of Icaritin on angiogenesis in vitro --- p.82
Chapter 1. --- Introduction --- p.83
Chapter 2. --- Material and Methods --- p.85
Chapter 2.1. --- Cell culture --- p.85
Chapter 2.2. --- Proliferation assay --- p.85
Chapter 2.3. --- Scratch-wound healing assay --- p..86
Chapter 2.4. --- Migration Assay --- p.86
Chapter 2.5. --- In vitro Angiogenesis Assay --- p.87
Chapter 2.6. --- RNA Isolation and Real-time PCR Performance --- p.87
Chapter 2.7. --- Statistical Analysis --- p.88
Chapter 3. --- Results --- p.88
Chapter 3.1. --- Icaritin did not affect HUVECs migration --- p.88
Chapter 3.2. --- Icaritin had no effect on tube formation on growth factors reduced Matrigel --- p.92
Chapter 3.3. --- Icaritin had no effect on HUVECs proliferation --- p.94
Chapter 3.4. --- Icaritin did not change the angiogenesis related gene expression --- p.95
Chapter 4. --- Discussion --- p.96
Chapter 5. --- Conclusion --- p.100
Chapter CHAPTER 5: --- Effect of PLGA/TCP and PLGA/TCP/Icaritin composite scaffolds on stem cell homing during bone defect repair with SAON --- p.101
Chapter 1. --- Introduction --- p.102
Chapter 2. --- Material and Methods --- p.106
Chapter 2.1. --- Preparation of porous PLGA/TCP/Icaritin composite scaffolds --- p.106
Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.106
Chapter 2.3. --- Wound healing assay --- p.107
Chapter 2.4. --- In vitro MSCs recruitment assay of scaffolds --- p.107
Chapter 2.5. --- MSCs labeling with SPIO@SiO2-NH2 nanoparticle --- p.108
Chapter 2.6. --- Prussian blue staining --- p.108
Chapter 2.7. --- MTT assay for SPIO@SiO2-NH2 labeled MSCs --- p.108
Chapter 2.8. --- Osteogenic and adipogenic differentiation of SPIO@SiO2-NH2 labeled MSCs --- p.109
Chapter 2.9. --- Real time PCR --- p.109
Chapter 2.10. --- Animal model establishment --- p.109
Chapter 2.11. --- Descriptive histology and histomorphometry --- p.110
Chapter 2.12. --- In vivo magnetic resonance imaging (MRI) of nanoparticle-labeled MSCs --- p.112
Chapter 2.13. --- Statistical analysis --- p.112
Chapter 3. --- Results --- p.112
Chapter 3.1. --- Icaritin promoted MSCs migration in vitro --- p.112
Chapter 3.2. --- PLGA/TCP and PLGA/TCP/Icaritin recruited MSCs when incubated in vitro --- p.114
Chapter 3.3. --- Stem cell potentials of MSC after SPIO@SiO2-NH2 labeling --- p.118
Chapter 3.4. --- PLGA/TCP and PLGA/TCP/Icaritin promoted MSCs homing in vivo --- p.122
Chapter 4. --- Discussion --- p.126
Chapter 5. --- Conclusion --- p.136
Chapter CHAPTER 6: --- Summary of the study and future research --- p.137
Chapter 1. --- Summary of the study --- p.138
Chapter 2. --- Limitations and further studies --- p.139
APPENDIXES --- p.142
REFERENCES --- p.147

成人骨量的更新与维持通过骨重塑来实现。骨重塑包括骨吸收与骨形成两个偶联的过程，其中成骨细胞介导骨形成，破骨细胞介导骨吸收，当偶联的骨吸收超过骨吸收就会导致骨量丢失，进而导致发生骨质疏松症的发生。目前，临床治疗骨质疏松的药物如阿仑膦酸盐、雌激素受体调节剂、活性维生素D、雌激素替代治疗、降钙素、骨化三醇等都是基于针对破骨细胞的调控来抑制骨吸收，但是对于已经丢失的骨量无法恢复。唯一被美国FDA批准用来通过刺激新骨形成来恢复丢失的骨量的治疗药物就是甲状旁腺激素。然而，这种药物在刺激新骨形成的同时也刺激了骨吸收，即：在使用18个月后有明显促进骨吸收的副作用。
酪蛋白激酶相互作用蛋白-1（CKIP-1）基因是一个新发现的骨形成的负调控基因，CKIP-1基因敲除小鼠在骨发育和正常骨代谢过程中均未发现激活骨吸收。CKIP-1敲除导致小鼠胫骨近端骨量与胫骨皮质骨形成速率显著高于野生型，且这一差异随着小鼠的增龄而显著，而骨外器官没有发现异常表型，提示CKIP-1是潜在相对安全的治疗骨质疏松的靶向基因。特别是我们最近研发的一种天门冬氨酸－丝氨酸－丝氨酸重复三肽修饰的脂质体递送（(Asp-Ser-Ser)₆-liposome）系统能够实现靶向骨形成表面的小干扰核酸的递送，并明显减少了小干扰核酸在非骨组织的分布。因此，提出本课题的研究假设：特异性静默骨内CKIP-1可以促进骨形成而不刺激骨吸收，从而为骨质疏松的临床治疗提供安全有效的治疗手段。
为了确定CKIP-1基因表达在老年绝经后妇女骨骼中与骨形成内在联系，首先，我们通过对发生骨折的老年绝经后妇女的骨痂标本中CKIP-1 mRNA和蛋白表达的测定，发现CKIP-1基因mRNA和蛋白表达水平与骨形成能力负相关。并且，这种相关性在骨质疏松动物模型中进一步得到证实。其次，针对我们研究假设，从一组针对大鼠、小鼠、猴和人类的成骨样细胞的CKIP-1 mRNA的跨种属siRNA序列中筛选出体外静默效率最高CKIP-1小干扰核酸序列si-3。接着，体内外实验证实si-3序列在健康动物体内的静默效率和促进成骨的功能。同时，确定尾静脉注射（Asp-Ser-Ser）₆-liposome 包裹的CKIP-1小干扰核酸在 大鼠和小鼠为的最佳剂量分别为3.75mg/kg和7.5mg/kg以及注射周期为每两周一次。最后，为了检验CKIP-1 小干扰核酸是否可通过促进骨形成从而逆转绝经后骨质疏松症中的骨丢失，我们分别以绝经后骨质疏松大鼠和小鼠为实验动物模型，通过测定骨形态计量学参数、骨量和骨结构等来评价骨靶向递送系统（(Asp-Ser-Ser)₆-liposome）递送的CKIP-1 siRNA对老年绝经后骨质疏松症的治疗效果。动态活体CT分析结果表明，与0周未治疗的基础值相比，经6周治疗骨密度（BMD）, 相对骨体积分数（BV/TV）和骨小梁厚度（Tb.Th）在小干扰核酸治疗组显著增加。此外，在治疗6周后小干扰核酸治疗组骨密度，相对骨体积和骨小梁厚度显示较高于模型对照组。0周与其它检测时间点之间的对比分析较显示，小干扰核酸治疗组的新生骨显著高于模型组或假手术组。组织形态学分析结果表明在治疗6周后，无论是股骨远端或中段的矿化沉积率（MAR）、骨形成速率（BFR） 和组的骨形成表面（Ob.S/ BS）在OVX组和siRNA组均显著高于模型对照组，而模型对照组和小干扰核酸治疗组的骨吸收表面（Oc.S/ BS）之间无显著性差异。
结论：CKIP-1基因小核酸干扰治疗在老年绝经后骨质疏松中能够显著促进骨形成并不会加剧骨吸收，该药物具有显著逆转骨丢失的作用。
Osteoporosis is characterized by an imbalance between bone formation and bone resorption. Therefore, promoting bone formation and inhibiting bone resorption are the two major therapeutic strategies in the treatment of osteoporosis. Currently, the only Food and Drug Administration (FDA)-approved anabolic agent capable of stimulating bone formation is parathyroid hormone (PTH). However, dominant bone resorption after 18-month treatment with PTH is a great concern (Rubin and Bilezikian 2003). Thus, development of alternative bone anabolic agents is highly desirable.
Casein kinase-2 interacting protein-1 (CKIP-1), which is encoded by Plekho1, and thus also known as Plekho1, is a newly discovered negative regulator of bone formation during bone development and subsequent bone maintenance that does not activate bone resorption (Lu, Yin et al. 2008). Specifically, CKIP-1 protein functions as the auxiliary factor of ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1) to interrupt the bone anabolic BMP-signalling pathway, which has been demonstrated to be a specific suppressor of bone formation (Yamashita, Ying et al. 2005). In a previous study, we found that CKIP-1 expression in female rat bone increases with aging, whereas bone formation decreases with aging (Guo, Zhang et al. 2010). Systemic examination of the tissue distribution of CKIP-1 expression has revealed that is abundantly expressed in the musculoskeletal system but sparingly expressed in the liver, lungs, kidneys, pancreas, and other organs (Zhang, Tang et al. 2007). In addition, an abnormal tissue phenotype in heart, liver, spleen, lung, and kidney tissue has not been observed in CKIP-1 gene knockout mice (KO), even at an advanced age (Lu, Yin et al. 2008). Thus, CKIP-1 gene silencing might be a potential strategy for promoting bone anabolic action in reversing bone loss.
RNA interference (RNAi), a natural cellular process that regulates gene expression by a highly precise mechanism of sequence-directed gene silencing at the stage of translation by degrading specific messenger RNA and then blocking translation of the specific gene, has been employed for gene silencing in vivo (Frank-Kamenetsky, Grefhorst et al. 2008). Accordingly, RNAi should be an appropriate target for CKIP-1 gene silencing in vivo.
We raised the hypothesis that therapeutic RNAi targeting of CKIP-1 might promote bone formation for reversing postmenopausal bone loss. To test the hypothesis, we performed several studies to achieve the following specific aims: (1) To explore the relationship between CKIP-1 expression and bone formation in aged postmenopausal osteoporosis; (2) To Identify a cross-species CKIP-1 siRNA sequence with high knockdown efficiency; (3) To validate of the identified CKIP-1 siRNA in healthy rodents in vivo; (4) To examine the anabolic effect of the identified CKIP-1 siRNA on bone in osteoporotic animal models.
The relationship between CKIP-1 gene expression and bone formation in bone specimens from aged postmenopausal women: To explore the association between CKIP-1 gene expression and bone formation in bone specimens from aged postmenopausal women, the gene expression of CKIP-1 and ALP in the bone specimens from aged female patients were examined. We found the protein expression of CKIP-1 increased during aging and negatively correlate to bone formation as indicated by the mRNA expression of ALP (Guo., Zhang. et al. 2011). Further, we also found the decreased bone formation during aging was partly rescued in Ckip-1 KO mice during aging.
A cross-species CKIP-1 siRNA sequence: Recently, we identified a specific CKIP-1 siRNA sequence (CKIP-1 siRNA si-3) with high knockdown efficiency across rat, mouse, rhesus, and human osteoblast-like cells that does not induce immunostimulatory activity and promotes osteoblast differentiation across the species in vitro and bone formation in rats in vivo (Guo, Zheng et al. 2012).
Validation of the CKIP-1 siRNA si-3 capsulated by bone-targeted siRNA delivery system in healthy rodents in vivo: We developed a bone-targeting siRNA delivery system (tripeptide aspartate-serine-serine linked with liposome, i.e. (Asp-Ser-Ser)₆-liposome) that can remarkably reduce the exposure of non-bone tissue to CKIP-1 siRNA (Zhang, Guo et al. 2012). To validate the identified CKIP-1 siRNA in healthy rodents in vivo, the established continuous CKIP-1 gene silencing protocol is optimized in adult rats and mice in vivo by hydrodynamic tail vein injection of 3.75mg/kg for rats and 7.5 mg/kg for mice every 2 weeks (Guo, Zhang et al. 2010). The osteogenic effects of CKIP-1 siRNA in both rats and mice were further validated in vivo.
Anabolic effect of CKIP-1 siRNA si-3 on bone in aged postmenopausal osteoporosis: For evaluation of the anabolic effect of CKIP-1 siRNA si-3 on reversing bone loss due to osteoporosis in an animal model, we intravenously injected ovariectomized (OVX) rats and mice with CKIP-1 siRNA delivered by the (Asp-Ser-Ser)₆-liposome, a liposome linked with six repeated aspartate-serine-serine moiety, every 2 weeks for 6 weeks. In vivo and ex vivo microCT analysis demonstrated a change over time in the variables examined and different change patterns over time among the groups examined after administration. We found that the siRNA group had experienced a significant increase in bone mineral density (BMD), relative bone volume (BV/TV), and trabecular thickness (Tb.Th) between weeks 0 and 6; had a higher BMD, BV/TV, and Tb.Th compared to the OVX group at week 6; and had a similar Tb.Th to that of the SHAM group at week 6. Registration analysis between week 0 and other time points revealed that the siRNA had a greater number of newly formed bone than the OVX and SHAM groups. Histomorphometric analysis showed that the siRNA group had a significantly higher mineralization rate (MAR), a significantly higher bone-formation rate (BFR), a significantly larger osteoblast surface (Ob.S/BS) at both the distal and mid-shaft femur compared to the OVX group after 6 weeks of treatment but not a significantly different Oc.S/BS.
Significance: Confirmation of our hypothesis by our results helps establish CKIP-1’s role as a pivotal negative regulator of bone formation in the aging skeleton and provides evidence that inhibiting CKIP-1 is a novel anabolic treatment for osteoporosis, indicating great potential for the use of therapeutic RNAi in orthopaedics and traumatology.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Guo, Baosheng.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves [132-150]).
Abstract also in Chinese.
Declaration --- p.i
Acknowledgements --- p.ii
Abstract --- p.iii
论文摘要 --- p.vii
Table of Content --- p.ix
Abbreviations --- p.xvii
List of Figures --- p.xix
List of Tables --- p.xxii
Chapter CHAPTER 1 --- Review of recent anabolic therapy for osteoporosis --- p.1
Chapter 1.1. --- Epidemiology of postmenopausal osteoporosis --- p.1
Chapter 1.1.1. --- Definition of osteoporosis --- p.1
Chapter 1.1.2. --- Epidemiology and health challenge of postmenopausal osteoporosis --- p.2
Chapter 1.2. --- General pathophysiological understanding of osteoporosis and current challenge for osteoporosis treatment --- p.3
Chapter 1.2.1. --- Bone modeling and remodeling --- p.3
Chapter 1.2.2. --- Pathophysiological process of osteoporosis --- p.4
Chapter 1.2.3. --- Systemic risk factors in the pathophysiology of osteoporosis --- p.5
Chapter 1.2.4. --- Local risk factors in the osteoporosis pathophysiology --- p.6
Chapter 1.2.5. --- Two therapeutic strategies for osteoporosis treatment --- p.7
Chapter 1.3. --- Current and potential anabolic agents for osteoporosis treatment --- p.8
Chapter 1.3.1. --- PTH analogues --- p.8
Chapter 1.3.2. --- Potential concerns regarding PTH administration --- p.9
Chapter 1.3.3. --- Potential PTH alternatives --- p.10
Chapter 1.3.4. --- Modulation of Wnt/β-cateinin pathway --- p.10
Chapter 1.3.5. --- Aptamer-based technology in osteoporosis treatment --- p.14
Chapter 1.4. --- CKIP-1: A novel negative regulator of bone formation --- p.15
Chapter 1.4.1. --- TGF-β/BMP signaling pathways involved in regulating bone formation --- p.15
Chapter 1.4.2. --- CKIP-1 interrupts BMP signaling pathway --- p.16
Chapter 1.4.3. --- CKIP-1 negatively regulates bone formation without activating bone resorption --- p.17
Chapter 1.5. --- RNA interference strategy in anabolic therapy of osteoporosis --- p.18
Chapter 1.5.1. --- siRNA-mediated gene silencing in osteoporosis treatment --- p.18
Chapter 1.5.2. --- MicroRNAs as potential therapeutic targets in the anabolic treatment of osteoporosis --- p.20
Chapter 1.5.3. --- Bone targeted RNAi-based anabolic-agents delivery --- p.23
Chapter 1.6. --- Summary --- p.24
Chapter CHAPTER 2 --- The relationship between CKIP-1 expression and bone formation in aged postmenopausal osteoporosis --- p.26
Chapter 2.1 --- Introduction --- p.26
Chapter 2.2 --- Materials and methods --- p.28
Chapter 2.2.1 --- Bone specimen collection from aged postmenopausal women --- p.28
Chapter 2.2.2 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.28
Chapter 2.2.3 --- Total protein extraction and western blot analysis --- p.30
Chapter 2.2.4 --- CKIP-1 expression in bone and other tissues --- p.31
Chapter 2.2.5 --- Relationship between CKIP-1 expression and bone formation in aged ovariectomized rats --- p.31
Chapter 2.2.6 --- Role of CKIP-1 in regulating bone formation in aged ovariectomized mice --- p.32
Chapter 2.2.7 --- Statistics --- p.32
Chapter 2.3 --- Results --- p.33
Chapter 2.3.1 --- Correlation analysis between CKIP-1 expression and bone formation-related gene expression in bone specimens from agedd postmenopausal women across age --- p.33
Chapter 2.3.2 --- CKIP-1 gene expression pattern in bone and other tissues --- p.37
Chapter 2.3.3 --- Correlation between CKIP-1 expression and bone formation in rat bone --- p.38
Chapter 2.3.4 --- CKIP-1 negatively regulates bone formation in aged ovariectomized mice by using CKIP-1 knockout mice --- p.39
Chapter 2.4 --- Summary --- p.41
Chapter CHAPTER 3 --- Identification of a cross-species CKIP-1 siRNA sequence --- p.43
Chapter 3.1 --- Introduction --- p.43
Chapter 3.2 --- Materials and methods --- p.44
Chapter 3.2.1 --- Design rationale and modification for cross-species CKIP-1 siRNA --- p.44
Chapter 3.2.2 --- In vitro screening for cross-species CKIP-1 siRNA sequences --- p.45
Chapter 3.2.3 --- Investigation of the effects of the identified CKIP-1 siRNA on the expression of osteoblast phenotype genes --- p.47
Chapter 3.2.4 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.47
Chapter 3.2.5 --- Western blot analysis --- p.51
Chapter 3.2.6 --- Evaluation of calcium deposition --- p.51
Chapter 3.2.7 --- BMP-2 reporter activity assay in MC3T3-E1 cells --- p.52
Chapter 3.2.8 --- Isolation of the primary human blood monocytes and IFN-α and TNF-α measurement --- p.53
Chapter 3.2.9 --- Statistics --- p.54
Chapter 3.3 --- Results --- p.54
Chapter 3.3.1 --- Bio-informatic analysis of the designed CKIP-1 siRNA sequences --- p.54
Chapter 3.3.2 --- Identified the cross-species CKIP-1 siRNA sequences by In vitro screening --- p.56
Chapter 3.3.3 --- Effects of the identified CKIP-1 siRNA on the expression of osteoblast phenotype genes --- p.60
Chapter 3.3.4 --- Effects of the identified CKIP-1 siRNA on matrix mineralization --- p.65
Chapter 3.3.5 --- Effect of the identified CKIP-1 siRNA on BMP signaling --- p.67
Chapter 3.3.6 --- Effects of the identified CKIP-1 siRNA on the ratio of RANKL/OPG --- p.67
Chapter 3.3.7 --- Effects of the identified CKIP-1 siRNA on immunostimulatory activity --- p.68
Chapter 3.4 --- Summary --- p.71
Chapter 3.4.1 --- CKIP-1 siRNA si-3 as the identified sequence --- p.71
Chapter 3.4.2 --- CKIP-1 siRNA si-3 promoted osteoblast differentiation in vitro --- p.72
Chapter CHAPTER 4 --- Validation of the identified CKIP-1 siRNA in healthy rodents in vivo --- p.74
Chapter 4.1 --- Introduction --- p.74
Chapter 4.2 --- Materials and methods --- p.74
Chapter 4.2.1 --- Localization of intraosseous siRNA delivered by (Asp-Ser-Ser)₆-liposome --- p.75
Chapter 4.2.2 --- Cell-selective delivery in vivo of CKIP-1 siRNA --- p.76
Chapter 4.2.3 --- Dose-response study of CKIP-1 siRNA --- p.77
Chapter 4.2.4 --- Time-course study of CKIP-1 siRNA --- p.77
Chapter 4.2.5 --- Examination of the effect of the identified siRNA on the expression of osteoblast phenotype genes --- p.78
Chapter 4.2.6 --- Measurement for serum PINP and urinary DPD --- p.80
Chapter 4.2.7 --- 5’-RACE Analysis --- p.81
Chapter 4.2.8 --- Laser captured micro-dissection (LCM) --- p.82
Chapter 4.2.9 --- Evaluation the anabolic effect of the identified siRNA on healthy rat bone --- p.82
Chapter 4.2.10 --- Evaluation the anabolic effect of the identified siRNA on healthy mouse bone --- p.84
Chapter 4.2.11 --- Micro CT analysis --- p.84
Chapter 4.2.12 --- Dynamic bone histomorphometric analysis --- p.85
Chapter 4.2.13 --- Statistics --- p.86
Chapter 4.3 --- Results --- p.87
Chapter 4.3.1 --- Rationale of bone targeted delivery of CKIP-1 siRNA by (Asp-Ser-Ser)₆-liposome --- p.87
Chapter 4.3.2 --- Intraosseous distribution of siRNA delivered by (Asp-Ser-Ser)₆-liposome --- p.89
Chapter 4.3.3 --- Optimal dosage and duration for CKIP-1 siRNA administration in vivo --- p.92
Chapter 4.3.4 --- Knockdown efficiency of CKIP-1 siRNA in osteoblasts by LCM in combination with Q-PCR --- p.94
Chapter 4.3.5 --- Examination of the effect of the identified siRNA on the expression of osteoblast phenotype genes --- p.96
Chapter 4.3.6 --- RNAi mechanism of CKIP-1 siRNA action in vivo --- p.99
Chapter 4.3.7 --- Anabolic effect of the identified siRNA on healthy rat bone --- p.101
Chapter 4.3.8 --- Anabolic effect of the identified siRNA on healthy mouse bone . --- p.104
Chapter 4.4 --- Summary --- p.107
Chapter 4.4.1 --- Intraosseous localization of CKIP-1 siRNA after systemic administration --- p.107
Chapter 4.4.2 --- Evidence of RNAi in bone tissue from systemic administration of CKIP-I siRNA --- p.107
Chapter 4.4.3 --- CKIP-1 siRNA si-3 promots bone formation in rats and mice in vivo --- p.108
Chapter CHAPTER 5 --- Anabolic effect of the identified CKIP-1 siRNA on bone in postmenopausal osteoporostic animal models --- p.110
Chapter 5.1. --- Introduction --- p.110
Chapter 5.2. --- Materials and Methods --- p.110
Chapter 5.2.1. --- Evaluation of anabolic effect of CKIP-1 siRNA on osteoporotic mouse bone --- p.111
Chapter 5.2.2. --- Evaluation of anabolic effect of CKIP-1 siRNA on osteoporotic rat bone --- p.112
Chapter 5.2.3. --- In vivo micro-CT analysis and registration of proximal tibia from osteoporotic rats --- p.112
Chapter 5.2.4. --- Ex vivo micro-CT analysis of the distal femur and 5th lumbar vertebrae body of osteoporotic rats --- p.115
Chapter 5.2.5. --- Ex vivo micro-CT analysis of distal femur from osteoporotic mice --- p.115
Chapter 5.2.6. --- Bone histomorphometric analysis --- p.116
Chapter 5.2.7. --- Mechanical testing --- p.117
Chapter 5.2.8. --- Statistics --- p.118
Chapter 5.3. --- Results --- p.116
Chapter 5.3.1. --- Anabolic effect of CKIP-1 siRNA si-3 on osteoporotic mouse bone --- p.118
Chapter 5.3.2. --- In vivo microCT data of proximal tibia from aged osteoporotic rats --- p.121
Chapter 5.3.3. --- Ex vivo microCT data of distal femur from aged osteoporotic rats --- p.124
Chapter 5.3.4. --- Ex vivo microCT data of 5th LV body from aged osteoporotic rats --- p.126
Chapter 5.3.5. --- Bone histomorphometric analysis of aged osteoporotic rats --- p.129
Chapter 5.3.6. --- Mechanical testing of the mid-shaft femur of aged osteoporotic rats --- p.132
Chapter 5.4. --- Summary --- p.134
Chapter CHAPTER 6 --- Discussions --- p.134
Chapter 6.1 --- CKIP-1 siRNA design rationale and further modification --- p.135
Chapter 6.1.1 --- Specificity design rationale of the CKIP-1 siRNA --- p.135
Chapter 6.1.2 --- Stability enhancing modification of CKIP-1 siRNA --- p.136
Chapter 6.1.3 --- Safety concerns with CKIP-1 siRNA therapy --- p.136
Chapter 6.2 --- Development of bone-targeted siRNA delivery --- p.136
Chapter 6.3 --- Prospects for and limitation of application of study findings to clinical therapeutics --- p.137
References --- p.139
Publications --- p.159

Chi Keung Cheung.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1993.
Includes bibliographical references (leaves 219-251).
Chapter CHAPTER I --- LITERATURE REVIEW
Chapter 1 --- The structure of bone --- p.2
Chapter 1.1. --- The cortical bone --- p.3
Chapter 1.2. --- The cancellous bone --- p.3
Chapter 2 --- The composition of bone --- p.3
Chapter 2.1. --- Bone minerals --- p.4
Chapter 2.2. --- The organic matrix --- p.4
Chapter 2.3. --- The bone cells --- p.9
Chapter 2.3.1. --- The osteoblast and the osteocyte --- p.9
Chapter 2.3.2. --- The osteoclast --- p.11
Chapter 3 --- Bone turnover - modelling and remodelling of bone --- p.13
Chapter 3.1. --- Postulated sequence of bone remodelling --- p.14
Chapter 4 --- Regulation of bone resorption --- p.16
Chapter 4.1. --- Role of osteoblast and the lining cell on bone resorption --- p.17
Chapter 5 --- Regulation of bone formation --- p.19
Chapter 6 --- Effects of systemic hormones and local factors on bone metabolism --- p.20
Chapter 6.1. --- Parathyroid hormone --- p.20
Chapter 6.2. --- "1,25-dihydroxyvitamin D3" --- p.22
Chapter 6.3. --- Calcitonin --- p.23
Chapter 6.4. --- Prostaglandins --- p.23
Chapter 6.5. --- Sex hormones --- p.24
Chapter 6.6. --- Glucocorticoid --- p.26
Chapter 6.7. --- Growth hormone --- p.27
Chapter 6.8. --- Insulin --- p.28
Chapter 6.9. --- Thyroid hormones --- p.29
Chapter 6.10. --- Other systemic and local factors --- p.30
Chapter 7 --- Indices of bone turnover --- p.34
Chapter 8 --- Non-biochemical indices of bone metabolism --- p.34
Chapter 8.1. --- Radionuclide bone scan --- p.34
Chapter 8.2. --- Radiokinetic assessment --- p.35
Chapter 8.3. --- Bone biopsy --- p.35
Chapter 8.4. --- Bone densitometry --- p.36
Chapter 9 --- Biochemical indices of bone metabolism --- p.37
Chapter 10 --- Biochemical markers of bone formation --- p.38
Chapter 10.1. --- Alkaline phosphatase --- p.38
Chapter 10.1.1. --- Role and origin of bone alkaline phosphatase isoenzyme --- p.39
Chapter 10.1.2. --- Measurement of bone alkaline phosphatase --- p.41
Chapter 10.1.2.1. --- Heat inactivation --- p.42
Chapter 10.1.2.2. --- Chemical inactivation --- p.43
Chapter 10.1.2.3. --- Immunological methods --- p.44
Chapter 10.1.2.4. --- High performance liquid chromatography --- p.45
Chapter 10.1.2.5. --- Gel electrophoresis --- p.45
Chapter 10.1.2.6. --- Isoelectric focusing --- p.47
Chapter 10.2. --- Osteocalcin --- p.48
Chapter 10.3. --- Osteonectin --- p.51
Chapter 10.4. --- Matrix Gla-protein --- p.51
Chapter 10.5. --- Other non-collagenous proteins --- p.52
Chapter 10.6. --- Urinary Gla concentration --- p.52
Chapter 10.7. --- Collagen peptides and extension peptides --- p.54
Chapter 11 --- Biochemical markers of bone resorption --- p.55
Chapter 11.1. --- Urine hydroxyproline --- p.55
Chapter 11.2. --- Pyridinium cross-links --- p.58
Chapter 11.3. --- Acid phosphatase --- p.60
Chapter 11.3.1. --- Acid phosphatase isoenzymes --- p.60
Chapter 11.3.2. --- The band 5 acid phosphatase isoenzyme genetics and characteristics --- p.62
Chapter 11.3.3. --- Band 5 acid phosphatase as marker of osteoclastic function --- p.64
Chapter 11.3.4. --- Measurement of osteoclastic acid phosphatase --- p.67
Chapter 11.3.4.1. --- Specific chemical inhibitor --- p.67
Chapter 11.3.4.2. --- Electrophoresis --- p.67
Chapter 11.3.4.3. --- Immunological methods --- p.68
Chapter 12 --- Problems with current biochemical markers of bone metabolism --- p.68
Chapter 13 --- Aims of this study --- p.70
Chapter CHAPTER II --- PURIFICATION OF TARTRATE-RESISTANT ACID PHOSPHATASE AND THE DEVELOPMENT OF AN IMMUNOASSAY FOR IT'S MEASUREMENT
Chapter 1 --- Introduction --- p.72
Chapter 2 --- Materials and methods --- p.75
Chapter 2.1. --- Chemicals and reagents --- p.75
Chapter 2.1.1. --- Apparatus --- p.76
Chapter 2.2. --- Methods --- p.77
Chapter 2.2.1. --- Cord serum --- p.77
Chapter 2.2.2. --- Measurement of tartrate-resistant acid phosphatase activity --- p.77
Chapter 2.2.3. --- Measurement of protein concentration --- p.80
Chapter 2.2.4. --- Purification of TRACP from cord plasma --- p.82
Chapter 2.2.4.1. --- Cation-exchange column chromatography --- p.83
Chapter 2.2.4.2. --- Gel filtration column chromatography --- p.84
Chapter 2.2.4.3. --- Concanavalin A-affinity column chromatography --- p.85
Chapter 2.2.4.4. --- Preparative isoelectric focusing (IEF) --- p.86
Chapter 2.3. --- Characterisation of purified TRACP --- p.90
Chapter 2.3.1. --- Polyacrylamide gel electrophoresis (PAGE) --- p.91
Chapter 2.3.2. --- "Optimum pH, substrate specificity and the effects of potential activators and inhibitors on TRACP activity" --- p.99
Chapter 2.3.3. --- Amino acid composition of purified TRACP --- p.101
Chapter 2.4. --- Methods for raising anti-human TRACP antibody and characterisation of the antiserum --- p.102
Chapter 2.4.1. --- Production of rabbit anti-human TRACP antibody --- p.102
Chapter 2.4.2. --- Determination of the titre of rabbit anti-human TRACP antibody --- p.103
Chapter 2.4.3. --- Immunoblotting analyses for cross reactivity study --- p.103
Chapter 2.4.4. --- Immunohistochemical study for antibody specificity --- p.105
Chapter 2.4.5. --- Cross reactivity study of the rabbit anti-human TRACP antibody to some tissue preparations --- p.107
Chapter 2.5. --- Enzyme linked immunosorbent assay for TRACP --- p.109
Chapter 2.5.1. --- Optimisation and evaluation of the new ELISA method for TRACP --- p.111
Chapter 3 --- RESULTS --- p.113
Chapter 3.1. --- "Precision of methods for the determination of protein, TRACP and phosphate." --- p.113
Chapter 3.2. --- Isolation and purification of TRACP --- p.113
Chapter 3.2.1. --- Concanavalin A affinity chromatography --- p.120
Chapter 3.2.2. --- Isoelectric focusing (IEF) --- p.120
Chapter 3.3. --- Characterisation and homogeneity of purified TRACP --- p.128
Chapter 3.3.1. --- Characterisation of purified TRACP --- p.128
Chapter 3.3.2. --- Homogeneity of purified TRACP --- p.132
Chapter 3.3.3. --- Amino acid composition --- p.136
Chapter 3.4. --- Characterisation of the rabbit anti-human TRACP antibody --- p.136
Chapter 3.4.1. --- Antibody specificity - immunoblotting study --- p.139
Chapter 3.4.2. --- Antibody specificity - cross reactivity with partially purified non-cord plasma TRACP --- p.142
Chapter 3.4.3. --- Antibody specificity - immunohistochemical study --- p.145
Chapter 3.5. --- Enzyme linked immunosorbent assay for TRACP --- p.145
Chapter 3.5.1. --- Optimal concentration of antigen for coating of microtitre plate --- p.145
Chapter 3.5.2. --- Kinetics of reaction with the primary rabbit anti-human TRACP antibody --- p.149
Chapter 3.5.3. --- "Precision, recovery and assay range" --- p.149
Chapter 4 --- DISCUSSION --- p.155
Chapter 4.1. --- Purification of cord plasma TRACP --- p.155
Chapter 4.2. --- Characterisation of cord plasma TRACP --- p.158
Chapter 4.3. --- Characterisation of rabbit anti-human TRACP antibody --- p.163
Chapter 4.4. --- Enzyme immunoassay for TRACP --- p.165
Chapter CHAPTER III --- STUDY OF SERUM TRACP IN HEALTHY SUBJECTS AND IN PATIENTS WITH BONE RELATED DISEASES
Chapter 1 --- Introduction --- p.168
Chapter 2 --- Materials and methods --- p.171
Chapter 2.1. --- Subjects --- p.171
Chapter 2.1.1. --- Healthy subjects --- p.171
Chapter 2.1.2. --- Patients --- p.172
Chapter 2.1.2.1. --- Post-menopausal women on hormone replacement therapy --- p.172
Chapter 2.1.2.2. --- Hip fracture patients --- p.173
Chapter 2.1.2.3. --- Other patients --- p.174
Chapter 2.3. --- Measurement of other biochemical parameters --- p.175
Chapter 2.3.1. --- Bone alkaline phosphatase --- p.175
Chapter 2.3.2. --- "Measurement of urine hydroxyproline, creatinine, calcium, osteocalcin, thyroid hormones and parathyroid hormone" --- p.176
Chapter 2.4. --- Statistics --- p.178
Chapter 3 --- RESULTS --- p.179
Chapter 3.1. --- Healthy subjects --- p.179
Chapter 3.2. --- Serum TRACP concentration in post-menopausal women before and after hormone replacement therapy --- p.185
Chapter 3.3. --- TRACP concentration in elderly subjects with hip fractures --- p.189
Chapter 3.4. --- Serum TRACP concentrations in patients with other bone related diseases --- p.190
Chapter 3.4.1. --- Hyperthyroidism --- p.194
Chapter 3.4.2. --- Hyperparathyroidism --- p.198
Chapter 3.4.3. --- Haemodialysis --- p.201
Chapter 4 --- DISCUSSION --- p.204
GENERAL DISCUSSION --- p.216
REFERENCES --- p.219

Indiana University-Purdue University Indianapolis (IUPUI)
Enhancing osteoblast proliferation, survival, and extracellular matrix protein secretion are potential therapeutic approaches to treat bone fractures and diseases such as osteoporosis. BENS is a traditional medicine used in many countries such as India for thousands of years to treat many diseases including bone diseases. In this study, molecular, cell-based and in vivo approaches were utilized to investigate the effects of BENS on bone and cartilage regeneration. An osteosarcoma cell line (MG63) was incubated in serum free media with and without 0.8 mg/ml of BENS. BENS significantly increased cell survival up to 30 days and these cells retained their ability to proliferate in fresh media with serum. After adding BENS, there were statistically significant decreases in the expression of both anti-apoptotic and pro-apoptotic proteins. An in vivo non-critical size segmental bone defect Xenopus system was used to evaluate the ability of BENS to enhance cartilage formation. After a small segment of the anterior hemisection of the tarsus bone was excised, the frogs were divided into three groups and given subcutaneous injections of either phosphate-buffered saline or BENS once daily for 30 days and then bone/cartilage formation evaluated. The total cartilage area/total section area was significantly increased (2.6 fold) in the BENS treated samples. In an osteoporotic rat model, the anabolic properties of BENS on bone mass were assessed by histomorphometric analyses. Ovariectomized (OVX) rats received daily intraperitoneal injections for 4 weeks. Bone formation rates (BFRs) for the cortical periosteal bone surface of the midshaft tibia were 383.2, 223.9, 308.8, 304.9, and 370.9 µm3/µm2/year, and for the trabecular surface were 82.2, 113, 212.1, 157, and 165 µm3/µm2/year for the sham, OVX, PTH, 3 mg/kg BENS, and 30 mg/kg BENS groups, respectively. BENS increased both trabecular and cortical BFRs. It generated better results on cortical periosteal bone surface than did PTH. Taken together, these findings suggest that BENS promotes osteoblast survival due to its effects on altering the balance between pro-apoptotic and anti-apoptotic proteins. In addition, in vivo studies revealed that BENS enhanced cartilage formation in Xenopus and BFRs in rats. Therefore, BENS may possess anabolic bone/cartilage properties.

傳統中醫中藥理論遵從"腎主骨"概念。因此，中醫在治療與骨有關的疾病時一般都處方"補腎"類中藥。
ELP是一例中藥草本 "補腎" 複方。其包含三種中藥，包括淫羊藿（E）、女貞子（L）和補骨脂（P）。動物體內實驗和臨床研究已證明ELP有效治療絶經後骨質疏鬆症。可是，經口服吸收後的血清中的ELP有效物質對細胞的成骨影響從未進行過相關研究。ELP對預防在缺乏體力活動下所引起的骨質疏鬆症的療效也屬未知。此外，基於其"補腎"的特性，ELP可能潛在著能促進骨折癒合的功能。本研究的目的包括研究血清中ELP的有效物質在細胞和分子水平上的護骨能力，並測試其對預防於失重狀態下引起的骨質疏鬆症（慢性骨紊亂）的效能。本研究還旨在考察 ELP在促進骨癒合 （急性骨紊亂）上的作用。本研究分為三部分。
第一部分 -- 骨代謝的體外研究:健康大鼠分別口服草本配方ELP、EL、及單味中草藥提取物E或L、並以蒸餾水作為對照（H2O），口服給藥二小時後收集其血清作體外血清藥理學研究。分別考察含藥血清對各細胞系包括UMR106、RAW264.7、和從大鼠骨中分離出的骨髓間充質幹細胞（MSC）的增殖和分化屬性的影響，並以液質聯用技術（LC-MS）來分析血清內所含中藥的化學成份。
第二部分 -- 骨質疏鬆症的體內研究:以尾吊雄性大鼠作為卸荷狀態骨質疏鬆症的動物模型。在不同的給藥組中，大鼠口服高中低三種劑量的ELP（ELP-H、ELP-M和ELP-L），或三個不同抗骨質疏鬆藥物，包括雷洛昔芬（Ral），阿侖膦酸鈉（Aln）和雷奈酸鍶（Strn）作為陽性對照組，並以蒸餾水為安慰劑對照（TS）。另一組大鼠則沒有尾吊，作為正常對照（Non-TS）。本部分分析在吊尾期間大鼠體內生化指標和骨密度（BMD）的變化，及其後各組在骨小梁微結構和骨骼生物力學上的差異。
第三部分 -- 骨缺損癒合的體內研究:兩個鑽孔性骨缺損模型分別建立於老年雌性大鼠的左股骨骨幹和右脛骨近端骺端。其後動物分成4組：（1）ELP 口服給藥（ELP）；（2）CDNR外敷治療（CDNR為另一中藥複方，包含紅花（C）、續斷（D）、三七（N）和大黃（R））；（3）ELP口服給藥結合CDNR外敷治療（ELP+CDNR）；（4）和蒸餾水餵養（Control）。通過監測骨缺損癒合的過程、檢測大鼠血液中生化標誌物的變化、骨骼生物力學測試和形態計量學分析，考察ELP及其與CDNR在骨缺損癒合上的協同作用。
第一部分的結果顯示，口服給藥二小時後，大鼠血清中淫羊藿的標記化合物淫羊藿苷（icariin）無被檢出。在EL或E的給藥大鼠血清中，檢出淫羊藿苷的其中一個代謝產物icariside I；而其另一個代謝產物icariside II，則在ELP的給藥大鼠血清中檢測到。L和P的常見標記化合物則能從相應餵飼L和P的大鼠血清中檢出。體外血清藥理學研究結果表明含藥（ELP）大鼠血清對細胞無毒性作用，且能促進 UMR106 細胞增殖和上調其Runx2 基因表達。然而，含藥血清無增加UMR106細胞的鹼性磷酸酶活性和鈣沉積。它抑制 RAW264.7細胞的分化及其基質金屬蛋白酶9（MMP-9）和組織蛋白酶 K的基因表達。它亦能促進MSC細胞的增殖，增強其鹼性磷酸酶活性和Runx2與ALP基因的表達。
第二部分的結果指出ELP-H能減少吊尾大鼠股骨遠端及腰椎骨密度的百分比損失，抵抗股骨遠端骨小梁微結構惡化和加強股骨骨幹骨缺損部位的生物力學特性。此外，ELP-H還能降低血液骨鈣素和抗酒石酸酸性磷酸酶5b（TRAP5b）的濃度。研究亦發現ELP對骨密度、結構參數和生化指標的影響存在劑量依賴性。整體上而言，ELP在預防卸荷骨質疏鬆症的影響類似於Ral和Aln，而非Strn。
第三部分的結果表明，從顯微電腦掃描或形態計量學上分析，所有實驗組跟對照組間均沒有顯著性差異。但值得注意的是，ELP+CDNR大大提高了股骨骨幹骨缺損在癒合過程中的歸一化生物力學屬性。而ELP單獨用藥則減少了TRAP5b的濃度。
總之，這項研究結論出血清藥理學研究加上LC-MS的應用能作為找出中藥中有效成分的有效途徑。本研究還展示ELP的含藥血清對骨細胞有護骨作用。ELP可防預在卸荷狀態下形成的骨質疏鬆症，它還有助於提升外敷中藥複方CDNR在骨缺損癒合過程中的療效。從這項研究的三個部分中歸納出的共同點說明，儘管ELP擁有刺激成骨的能力，它的護骨作用主要是透過它的抗骨吸收效果。ELP在慢性（防止骨質疏鬆症）和急性（促進骨癒合）骨紊亂上均有療效。
Traditional Chinese Medicine (TCM) claims that bone health lies in the functioning of the "Kidneys". When the "Kidney" is strong, our body can stimulate growth and transformation of the bone marrow, which nourishes and strengthens the skeleton. Therefore, "Kindey-tonifying" herbs are usually used to cure bone diseases.
ELP is a "Kidney-tonifying" Chinese herbal formula containing three Chinese herbs including Herba Epimedii (E), Fructus Ligustri Lucidi (L) and Fructus Psoraleae (P). It has been proven effective to treat postmenopausal osteoporosis through in vivo and clinical studies. However, ELP is for oral administration. The osteogenic properties of its post-absorption metabolites have never been studied. The efficacy of ELP on prevention of osteoporosis development due to physical inactivity is also unknown. With its "Kindey-tonifying" property, ELP is also considered as a potential agent to facilitate fracture healing.
The aims of this study included to investigate the osteoprotective effects of ELP metabolites at cellular and molecular levels and to prove the efficacy of ELP on prevention of osteoporosis development in unloading condition - a chronic bone disorder. It also aimed to study the effect of ELP on promotion of bone defect healing - an acute bone disorder. This study was divided into three parts.
Part 1 - in vitro study of bone metabolism: Healthy rats were fed with herbal formula ELP or EL, single herbal extracts of E or L or distilled water as control (H₂O). Sera were then collected for in vitro seropharmacological study. Cell lines including UMR106 and RAW264.7, as well as mesenchymal stem cell (MSC) isolated from rats, were cultured with the sera. Their proliferation and differentiation properties of the cells were analyzed. In addition, the chemical profiles of the herbal extracts within the sera were analyzed using liquid chromatography-mass spectrometry (LC-MS).
Part 2 - in vivo study of osteoporosis: Tail-suspension male rats were used as the unloading osteoporotic animal model. The rats in different groups were fed with three different doses of ELP (ELP-H, ELP-M and ELP-L), or three different anti-osteoporosis drugs including raloxifene (Ral), alendronate (Aln) and strontium ranelate (Strn) as positive controls or distilled water as placebo control (TS). One group of rats was non-tail-suspended as normal control (Non-TS). Changes in bone mineral density (BMD), microarchitecture of trabeculae and biomechanical properties of the bone of the rats were analyzed. Changes in biochemical markers within the tail-suspension period were also studied.
Part 3 - in vivo study of bone defect healing: two drilled-hole bone defects were created in the diaphysis of left femur and proximal metaphysis of right tibia, respectively, of aged female rats. Animals were divided into 4 groups: (1) administered with ELP orally (ELP); (2) treated with another herbal formula CDNR containing Carthami Flos (C), Dipsaci Radix (D), Notoginseng Rhizoma (N) and Rhei Rhizoma (R) topically (CDNR); (3) treated with oral ELP and topical CDNR at the same time (ELP+CDNR); and (4) fed with distilled water (Control). The effects of ELP and the synergistic effects of ELP+CDNR on facilitation of the bone defect healing were monitored in vivo using viva-CT and through measurement of biochemical markers biweekly. After euthanasia of the rats, the bones were harvested for biomechanical test and histomorphometrical analysis.
Results: Part 1 revealed that the common marker compound, icariin, had not been detected in the sera of all the rats. Instead, one of the metabolites of E, icariside I, was found in the sera of the rats fed with EL or E, while another metabolite, icariside II, was detected in the serum of the rats fed with ELP. Common marker compounds of L and P were observed in the sera of the rats fed with the herbal items accordingly. The in vitro studies in this Part showed that there was no cytotoxic effect of the rat sera on the cells. The post-absorbed ELP metabolites in rat serum promoted UMR106 proliferation by 25.7%, (p < 0.05) and upregulated the Runx2 gene expression by 1.18 fold (p < 0.05) after cultured for 2 and 3 days, respectively. However, they could not increase the ALP activity and calcium deposition of UMR106. They also inhibited RAW264.7 differentiation by 29.2 % (p < 0.05) and downregulated the MMP9 and Cathepsin K gene expression of RAW264.7 by 0.46 (p < 0.05) and 0.36 (p < 0.01) fold, respectively. The ELP metabolites promoted the proliferation of MSC by 14.4 % (p < 0.001) and resulted in 42.6 % higher ALP activity than the control serum (p < 0.05). They also upregulated the Runx2 and ALP gene expression at both Day 4 and Day 7 of culture significantly.
Part 2 showed that compared with the tail-suspension control (TS), ELP in high dose (ELP-H) reduced the percentage loss of total and trabecular BMD by 5.46 and 8.52 %, respectively (p < 0.05 both) in distal femur, and by 4.67 % (p < 0.05) in trabecular region of lumbar spine of the tail-suspended rats. Analysis from micro-CT showed that microarchitectural parameters BV/TV, Tb.Th and TV density of the distal femur of ELP-H were 17.62, 11.90 and 8.09 % higher than those of the TS (p < 0.05, for all). 3-point bending test on mid-shaft femur of the rats revealed that the yield load, ultimate load and stiffness of the drill-defect of ELP-H were higher than those of TS significantly. All of the biochemical markers decreased significantly from baseline (Day 0) to Day 28 in ELP-H. In addition, osteocalcin and TRAP5b concentrations of ELP-H were lower than those of TS significantly at Day 28. The effect of ELP on BMD, microarchitectural parameters and biochemical markers were in dose-dependent manner. In general, the osteoprotective effect of ELP-H on unloading bone was similar to Ral and Aln, but not Strn.
Part 3 indicated no significant difference in BV/TV and BMD among all groups at each time point. Histomorphometrical analysis from fluorescent labeling and Goldner’s trichrome staining showed no statistical difference in new bone formation between the Control and other treatment groups. Notably, the normalized yield load, ultimate load and failure of ELP+CDNR were significantly higher than those of Control by 20.38 % (p < 0.05), 23.17 % (p< 0.001) and 25.55 % (p< 0.001), respectively. Analysis on the change of biochemical markers showed that the bone formation marker BALP increased while bone resorption markers Dpd and TRAP5b decreased within the 42-day monitoring period. BALP activity of both Control and ELP increased significantly but only ELP reduced the TRAP5b concentrations starting from Day 14 post-op. There was no statistical difference when the concentrations of the biochemical markers were compared horizontally among the 4 groups at the same time point.
In conclusion, the current study demonstrated that seropharmacological study incorporating with the application of LC-MS can be a potential efficient approach to find out active ingredients of medicine herbs. Post-absorbed metabolites of ELP also showed their osteoprotective effects on bone cells. Aqueous extract of ELP could prevent the development of osteoporosis in unloading condition and such effect was dose-dependent. It also helped elevating the efficacy of a topical applied herbal formula CDNR on improving the bone strength of healing bone defects. A common finding from the 3 parts of this study illustrated that the osteoprotective effect of ELP was mainly achieved by its anti-resorptive efficacy on bone, although it possess an ability to stimulate osteoblastogenesis. ELP was found effective for both chronic (prevent osteoporosis development) and acute (facilitate bone healing) bone disorders.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Siu, Wing Sum.
"November 2012."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 201-227).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts also in Chinese.
ABSTRACT --- p.i
摘要 --- p.vi
ACKNOWLEDGEMENTS --- p.ix
TABLE OF CONTENTS --- p.xi
LIST OF FIGURES --- p.xvii
LIST OF TABLES --- p.xxiii
PUBLICATIONS --- p.xxiv
ABBREVIATION --- p.xxv
Chapter CHAPTER 1: --- INTRODUCTION --- p.1
Chapter 1.1 --- TRADITIONAL CHINESE MEDICINE (TCM) AND BONE DISEASES --- p.1
Chapter 1.2 --- CELLULAR AND MOLECULAR MECHANISMS ON BONE METABOLISM --- p.2
Chapter 1.2.1 --- Bone formation by osteoblast --- p.3
Chapter 1.2.2 --- Bone resorption by osteoclasts --- p.4
Chapter 1.3 --- OSTEOPOROSIS --- p.5
Chapter 1.3.1 --- Postmenopausal osteoporosis --- p.6
Chapter 1.3.2 --- Disuse osteoporosis --- p.8
Chapter 1.3.3 --- Basic principle of TCM on osteoporosis --- p.10
Chapter 1.3.4 --- Common Chinese herbal medicine reported to have anti-osteoporotic effects --- p.11
Chapter 1.4 --- BONE FRACTURE --- p.11
Chapter 1.4.1 --- Biology and repair of bone fracture --- p.12
Chapter 1.4.2 --- TCM on promotion of fracture healing --- p.13
Chapter 1.4.3 --- Theories of TCM on fracture healing --- p.15
Chapter CHAPTER 2: --- OSTEOPOROSIS AND HERBS --- p.16
Chapter 2.1 --- CHINESE HERBAL MEDICINE SELECTED IN THIS PART --- p.16
Chapter 2.2 --- DESIGN OF STUDY --- p.19
Chapter 2.3 --- HYPOTHESES AND OBJECTIVES --- p.19
Chapter 2.4 --- BACKGROUND OF THE STUDY --- p.23
Chapter 2.4.1 --- In vitro study of ELP on bone cells --- p.23
Chapter 2.4.2 --- In vivo study of ELP on postmenopausal osteoporosis --- p.23
Chapter 2.4.3 --- Clinical study of ELP on postmenopausal osteoporosis --- p.24
Chapter CHAPTER 3: --- PART 1 IN VITRO SEROPHARMACOLOGICAL STUDY ON OSTEOPOROSIS --- p.26
Chapter 3.1 --- OBJECTIVES --- p.26
Chapter 3.2 --- SEROPHARMACOLOGICAL APPROACH TO STUDY ELP --- p.26
Chapter 3.3 --- TYPES OF CELLS INVOLVED IN THE CURRENT STUDY --- p.27
Chapter 3.3.1 --- UMR106 --- p.28
Chapter 3.3.2 --- RAW264.7 --- p.28
Chapter 3.3.3 --- Mesenchymal stem cell (MSC) --- p.28
Chapter 3.4 --- IN VITRO ASSESSMENTS ON BONE METABOLISM --- p.29
Chapter 3.4.1 --- Bone formation --- p.29
Chapter 3.4.1.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay --- p.29
Chapter 3.4.1.2 --- Bromodeoxyuridine (BrdU) assay --- p.30
Chapter 3.4.1.3 --- Total alkaline phosphatase (ALP) activity measurement --- p.30
Chapter 3.4.1.4 --- Calcium deposition analysis --- p.30
Chapter 3.4.2 --- Bone degradation --- p.31
Chapter 3.4.2.1 --- Tartrate-resistant acid phosphatase (TRAP) staining --- p.31
Chapter 3.4.3 --- Phenotypic markers of cells involved in bone remodeling using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) --- p.31
Chapter 3.5 --- MATERIAL AND METHODS --- p.37
Chapter 3.5.1 --- Preparation of herbal extracts --- p.37
Chapter 3.5.2 --- Serum preparation for seropharmacological study --- p.38
Chapter 3.5.2.1 --- Administration of herbal extracts and blood collection --- p.38
Chapter 3.5.2.2 --- Serum preparation --- p.38
Chapter 3.5.3 --- Analysis of marker compounds in serum using liquid chromatographymass spectrometry (LC-MS) --- p.39
Chapter 3.5.3.1 --- Serum preparation --- p.39
Chapter 3.5.3.2 --- Operation of LC-MS --- p.39
Chapter 3.5.4 --- Isolation and characterization of MSC from bone marrow --- p.40
Chapter 3.5.5 --- Cell culture --- p.42
Chapter 3.5.5.1 --- General materials --- p.42
Chapter 3.5.5.2 --- UMR106 --- p.43
Chapter 3.5.5.3 --- RAW264.7 --- p.44
Chapter 3.5.5.4 --- Bone Marrow MSC --- p.45
Chapter 3.5.6 --- Assays analyzing the responses of cells on the effect of metabolites of herbs in serum --- p.46
Chapter 3.5.6.1 --- General materials --- p.46
Chapter 3.5.6.2 --- Assays for bone formation --- p.50
Chapter 3.5.6.3 --- Assays for bone degradation --- p.55
Chapter 3.5.7 --- Statistical analysis --- p.56
Chapter 3.6 --- RESULTS --- p.57
Chapter 3.6.1 --- Chemical characterization of ELP extract --- p.57
Chapter 3.6.2 --- Marker compounds found in rat serum using LC-MS --- p.58
Chapter 3.6.3 --- Effects of herbal metabolites on UMR106 --- p.61
Chapter 3.6.3.1 --- Effect on cell viability --- p.61
Chapter 3.6.3.2 --- Effects on cell proliferation and differentiation --- p.61
Chapter 3.6.3.3 --- Regulation on osteogenesis through gene expression --- p.63
Chapter 3.6.4 --- Effects of herbal metabolites on RAW264.7 --- p.67
Chapter 3.6.4.1 --- Effect on cell viability --- p.67
Chapter 3.6.4.2 --- Inhibitory effect on RAW264.7 --- p.67
Chapter 3.6.4.3 --- Regulation on osteoclastogenesis through gene expression --- p.67
Chapter 3.6.5 --- Effects of herbal metabolites on bone marrow mesenchyma stem cell (MSC) --- p.70
Chapter 3.6.5.1 --- Confirmation of MSC isolated from bone marrow of rat using flow cytometry --- p.70
Chapter 3.6.5.2 --- Effect on cell viability --- p.70
Chapter 3.6.5.3 --- Effects on cell proliferation and differentiation --- p.71
Chapter 3.6.5.4 --- Regulation on osteogenesis through gene expression --- p.71
Chapter 3.7 --- DISCUSSION --- p.75
Chapter CHAPTER 4: --- PART 2 IN VIVO STUDY ON DISUSE OSTEOPOROSIS . --- p.83
Chapter 4.1 --- OBJECTIVES --- p.83
Chapter 4.2 --- POTENTIAL EFFECT OF ELP ON DISUSE OSTEOPOROSIS --- p.83
Chapter 4.3 --- ANIMAL MODELS FOR OSTEOPOROSIS STUDY --- p.84
Chapter 4.3.1 --- Conventional ovariectomized animal model for the studies of osteoporosis --- p.85
Chapter 4.3.2 --- Animal models for study of disuse osteoporosis --- p.85
Chapter 4.3.2.1 --- Bandaging or casting --- p.86
Chapter 4.3.2.2 --- Tail-suspension (TS) --- p.86
Chapter 4.4 --- ASSESSMENTS ON DISUSE OSTEOPOROSIS DEVELOPMENT --- p.87
Chapter 4.4.1 --- Bone mineral density (BMD) measurement --- p.87
Chapter 4.4.2 --- Micro-architecture analysis --- p.87
Chapter 4.4.3 --- Bone strength assessment --- p.88
Chapter 4.4.4 --- Bone turnover monitoring by measuring biochemical markers --- p.89
Chapter 4.4.4.1 --- Bone formation markers --- p.89
Chapter 4.4.4.2 --- Bone resorption markers --- p.91
Chapter 4.5 --- MATERIAL AND METHODS --- p.95
Chapter 4.5.1 --- Preparation of herbal extracts --- p.95
Chapter 4.5.2 --- Tail-suspension rat model --- p.95
Chapter 4.5.3 --- Animal arrangement and grouping --- p.97
Chapter 4.5.4 --- Administration of herbal extracts and drugs --- p.97
Chapter 4.5.5 --- Assessments on disuse osteoporosis development --- p.98
Chapter 4.5.5.1 --- Bone mineral density measurement using Peripheral Quantitative Computed Tomography (pQCT) --- p.98
Chapter 4.5.5.2 --- Bone micro-architecture analysis using Micro-computed Tomography (μCT) --- p.99
Chapter 4.5.5.3 --- Bone strength assessment through biomechanical bending test --- p.100
Chapter 4.5.5.4 --- Bone turnover monitoring by measuring biochemical markers --- p.100
Chapter 4.5.5.4.1 --- Serum collection --- p.100
Chapter 4.5.5.4.2 --- Measurements of biochemical markers --- p.101
Chapter 4.5.6 --- Statistical analysis --- p.105
Chapter 4.6 --- RESULTS --- p.106
Chapter 4.6.1 --- Effects of ELP on bone mineral density (BMD) --- p.106
Chapter 4.6.2 --- Effects of ELP on bone micro-architecture --- p.118
Chapter 4.6.3 --- Effects of ELP on biomechanics of bone --- p.122
Chapter 4.6.4 --- Effects of ELP on bone turnover --- p.125
Chapter 4.7 --- DISCUSSION --- p.132
Chapter CHAPTER 5: --- PART 3 IN VIVO STUDY ON BONE DEFECT HEALING --- p.140
Chapter 5.1 --- HERBAL ITEMS SELECTED IN THIS PART --- p.140
Chapter 5.2 --- DESIGN OF STUDY --- p.143
Chapter 5.3 --- HYPOTHESES AND OBJECTIVES --- p.144
Chapter 5.4 --- SPECIFIC STRATEGY ON PROMOTION OF FRACTURE HEALING OF TCM --- p.144
Chapter 5.5 --- POTENTIAL EFFECT OF ELP ON BONE HEALING --- p.144
Chapter 5.6 --- ANIMAL MODELS --- p.146
Chapter 5.6.1 --- Bone fracture model --- p.147
Chapter 5.6.2 --- Drill-hole bone defect model --- p.147
Chapter 5.7 --- ASSESSMENTS ON BONE HEALING --- p.149
Chapter 5.7.1 --- Micro-architecture analysis --- p.149
Chapter 5.7.2 --- Bone strength assessment --- p.150
Chapter 5.7.3 --- Bone turnover monitoring by measuring biochemical markers --- p.151
Chapter 5.7.4 --- Histomorphometry --- p.151
Chapter 5.8 --- MATERIALS AND METHODS --- p.153
Chapter 5.8.1 --- Preparation of herbal extracts --- p.153
Chapter 5.8.1.1 --- ELP --- p.153
Chapter 5.8.1.2 --- CDNR --- p.153
Chapter 5.8.2 --- Production of drill-hole bone defect --- p.154
Chapter 5.8.2.1 --- Femur --- p.155
Chapter 5.8.2.2 --- Tibia --- p.155
Chapter 5.8.2.3 --- Animal arrangement and grouping --- p.157
Chapter 5.8.3 --- Herbal formulae administration and application --- p.157
Chapter 5.8.3.1 --- Oral administration --- p.157
Chapter 5.8.3.2 --- Topical application --- p.157
Chapter 5.8.4 --- Assessments on bone healing --- p.158
Chapter 5.8.4.1 --- Bone micro-architecture and bone density measurement using in vivo micro-computed tomography (vivaCT) --- p.158
Chapter 5.8.4.2 --- Bone strength assessment through biomechanical bending test --- p.159
Chapter 5.8.4.3 --- Bone turnover monitoring by measuring biochemical markers --- p.160
Chapter 5.8.4.4 --- Histomorphometry --- p.160
Chapter 5.8.4.4.1 --- Fluorochrome double labeling --- p.160
Chapter 5.8.4.4.2 --- Tissue processing and sectioning --- p.161
Chapter 5.8.4.4.3 --- Staining of sections --- p.162
Chapter 5.8.4.4.4 --- Image analysis --- p.164
Chapter 5.8.5 --- Statistical analysis --- p.165
Chapter 5.9 --- RESULTS --- p.166
Chapter 5.9.1 --- Effect of ELP and CDNR on bone micro-architecture --- p.and
Chapter bone --- density at the bone defect site --- p.166
Chapter 5.9.2 --- Histomorphometrical findings in treatment of bone healing --- p.172
Chapter 5.9.3 --- Effect of ELP and CDNR on biomechanics of bone --- p.175
Chapter 5.9.4 --- Effect of ELP and CDNR on bone turnover --- p.178
Chapter 5.10 --- DISCUSSION --- p.184
Chapter CHAPTER 6: --- GENERAL DISCUSSION AND CONCLUSION --- p.193
Chapter 6.1 --- UNKNOWN AREAS FOR THE STUDY OF ELP --- p.193
Chapter 6.2 --- SUMMARY OF CRUCIAL FINDINGS OF THE OSTEOGENIC EFFECTS OF ELP IN EACH PART OF THIS STUDY --- p.194
Chapter 6.2.1 --- Part 1: in vitro seropharmacological study on osteoporosis --- p.194
Chapter 6.2.2 --- Part 2: in vivo study on disuse osteoporosis --- p.195
Chapter 6.2.3 --- Part 3: in vivo study on bone healing --- p.196
Chapter 6.3 --- COMMON OSTEOGENIC EFFECT OF ELP IN THE THREE PARTS OF THE WHOLE STUDY --- p.197
Chapter 6.4 --- LIMITATIONS OF THE PRESENT STUDY --- p.197
Chapter 6.5 --- SIGNIFICANCES OF THIS STUDY --- p.199
Chapter 6.6 --- FUTURE STUDIES --- p.199
BIBLIOGRAPHY --- p.201

Although Hormone Therapy (HT) is the most effective treatment for alleviating menopausal vasomotor symptoms and reducing bone loss, many women are reluctant to take this treatment due to side effects and concerns about safety. Epidemiological studies suggest that a significant proportion of women use Complementary and Alternative Medicine (CAM) therapies to alleviate vasomotor symptoms and improve quality of life. Anecdotal and clinical evidence indicate a number of CAM therapies, such as herbal medicine, may be effective in alleviating symptoms and modulating bone metabolism. Hence, in the context of concerns over the safety of HT and the extensive history of the clinical use of herbal medicine, this thesis investigated issues pertinent to CAM use and the menopausal transition. The aims of this thesis were to: • Examine the nature and extent of CAM use by women transitioning through menopause • Evaluate the effectiveness of a herbal formula containing Chinese herbs and Cimicifuga racemosa for alleviating vasomotor symptoms, improving quality of life and modulating bone turnover markers. From July 2003 until July 2004 the Women’s Health during Midlife Survey recruited 1,296 women aged 45-65 who were symptomatic when transitioning through menopause or asymptomatic but taking menopause specific treatments. A validated 19-item survey instrument assessed the use of CAM modalities and menopause specific products. The instrument was completed voluntarily and anonymously by women recruited from three strata; menopause clinics, clinics of general practice and government agencies. Approximately 54% of respondents had visited a CAM practitioner and/or used a CAM product during the previous 12 months. The most popular practitioners were the naturopath (7.2%) and acupuncturist (4.8%), while soy (25.4%) and evening primrose oil (EPO, 18.4%) were the most popular products. Massage and chiropractic were considered the most effective therapies, while phytoestrogen tablets and EPO were the most efficacious products. Although 26.4% of respondents indicated their doctor asked about CAM use, 71% of CAM users said they informed their physician about using CAM. Of the 60% of women using pharmaceutical medicines, 62.5% reported using a CAM product during the preceding 12 months. The survey results confirm the continued popularity of CAM use amongst women transitioning through menopause. A number of treatments were perceived to be effective in relieving symptoms. However, communication between medical practitioners and patients about CAM use is inadequate, and given the high use of pharmaceutical medicines this oversight may unnecessarily expose women to drugherb interactions. A randomised, double-blind, placebo controlled clinical trial was conducted to evaluate the effectiveness of a herbal formula derived from two traditional Chinese herbal formulae with the addition of Cimicifuga in alleviating vasomotor symptoms. The trial recruited 93 healthy women who reported at least six vasomotor symptoms per day. After a four week baseline period, women were randomly allocated to receive either herbal treatment or identical looking placebo tablets for 16 weeks. During the trial women recorded the number and severity of their flushes on a Daily Flush Diary, and at each monthly consultation two quality of life scales were completed. Forty nine eligible trial women were entered into a pilot study to assess the effect of the formula on bone turnover markers; bone specific alkaline phosphatase and deoxypyridinoline (corrected for creatinine). The herbal formula was found to be no more effective than placebo in reducing the frequency of flushing and the composite hot flush score or in improving quality of life. The pilot study found the formula had no effect on bone turnover markers after 16 weeks of treatment. Therefore, this formula cannot be recommended as a treatment for vasomotor symptoms and is unlikely to have any long term effect on bone. The use of CAM during the menopausal transition is very popular. Although this thesis found the current herbal formula to be an ineffective treatment, the scientific evaluation of potential CAM therapies is imperative so that health care professionals and consumers can make informed decisions concerning treatment options for the alleviation of symptoms.
Doctor of Philosophy (PhD)

by Cheng Yuen Yee.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves [106]-113).
Abstracts in English and Chinese.
Abstract --- p.i
Acknowledgements --- p.iv
Research out puts --- p.v
Abbreviations --- p.vii
List of Figures --- p.viii
List of Tables --- p.xiii
Table of contents --- p.xiv
Chapter Chapter 1 --- Introduction & Hypothesis
Chapter 1.1. --- General Introduction --- p.1
Chapter 1.2. --- Hypothesis --- p.4
Chapter 1.3. --- Objectives --- p.4
Chapter Chapter 2 --- An Overview of Giant Cell Tumour of Bone
Chapter 2.1. --- Introduction --- p.5
Chapter 2.2. --- Pathobiological features of GCT --- p.6
Chapter 2.2.1. --- Radiological appearances and clinical classifications of GCT --- p.7
Chapter 2.2.2. --- Histological characteristics --- p.10
Chapter 2.2.3. --- Metastatic GCT --- p.13
Chapter 2.3. --- Histogenesis of GCT --- p.14
Chapter 2.4. --- Treatment --- p.19
Chapter 2.5. --- Summary --- p.22
Chapter Chapter 3 --- Pharmacological aspect of bisphosphonates
Chapter 3.1. --- Introduction --- p.23
Chapter 3.2. --- Chemical structures of bisphosphonates --- p.28
Chapter 3.3. --- Mechanisms and actions --- p.28
Chapter 3.3.1. --- Bisphosphonates induce osteoclast apoptosis --- p.30
Chapter 3.3.2. --- Bisphosphonates induce cell apoptosis --- p.32
Chapter 3.3.3. --- Apoptosis --- p.33
Chapter 3.3.3.1. --- Morphological characteristic of apoptosis --- p.35
Chapter 3.4. --- Clinical applications of bisphosphonates --- p.36
Chapter 3.5. --- Bisphosphonates used in this study --- p.38
Chapter 3.6. --- Summary --- p.43
Chapter Chapter 4 --- Materials and methods
Chapter 4.1. --- Introduction --- p.44
Chapter 4.2. --- Primary GCT cell culture and maintenance --- p.46
Chapter 4.3. --- Drug preparation --- p.46
Chapter 4.4. --- MTT assay --- p.47
Chapter 4.5. --- Annexin-V-flous staining assay --- p.48
Chapter 4.6. --- Haematoxyline and Eosin staining --- p.51
Chapter 4.7. --- TUNEL assay (Terminal deoxynucleotidyltrasferase - mediated dUTP-biotin nick end labelling) --- p.52
Chapter 4.8. --- TEM (Transmission Electron Microscopy) --- p.54
Chapter 4.9. --- Statistical analysis --- p.54
Chapter Chapter 5 --- Bisphosphonates induce apoptosis in giant cell tumour of bone -in vitro study
Chapter 5.1. --- Introduction --- p.56
Chapter 5.2. --- Experimental design --- p.57
Chapter 5.3. --- Results
Chapter 5.3.1. --- Bisphosphonates reduce cell viability of GCT stromal tumour cell --- p.59
Chapter 5.3.2. --- Bisphosphonates induce morphological changesin GCT primary culture --- p.59
Chapter 5.3.3. --- Bisphosphonate significantly induce apoptosis in GCT stromal cells in a dose dependent manner --- p.62
Chapter 5.4. --- Discussions and Summary --- p.68
Chapter Chapter 6 --- Bisphosphonates induce apoptosis in giant cell tumour of bone -in vivo study
Chapter 6.1. --- Introduction --- p.73
Chapter 6.2. --- Experiment design --- p.74
Chapter 6.3. --- Results
Chapter 6.3.1. --- H & E observations
Chapter 6.3.2. --- Pamidronate significantly induce apoptosis in both osteoclast-like giant cells and stromal tumour cells by TUNEL labelling assay --- p.79
Chapter 6.3.3. --- Pamidronate induced cellular ultrastructural changes of GCT by TEM examination --- p.83
Chapter 6.3.4. --- Pamidronate reduce the recurrent characteristic of GCT --- p.95
Chapter 6.4. --- Discussions and Summary --- p.97
Chapter Chapter 7 --- Summary and Future Study
Chapter 7.1. --- Summary --- p.101
Chapter 7.2. --- Future directions --- p.103
Chapter Chapter 8 --- Reference --- p.105
Chapter Chapter 9 --- Appendix - solution preparation

Indiana University-Purdue University Indianapolis (IUPUI)
THE EFFECT OF FULL-CONTOUR Y-TZP CERAMIC SURFACE ROUGHNESS ON THE WEAR OF BOVINE ENAMEL AND SYNTHETIC HYDROXYAPATITE: AN IN-VITRO STUDY by Alaa Hussein Aref Sabrah Indiana University School of Dentistry Indianapolis, Indiana Full-contour yttrium-stabilized tetragonal zirconia polycrystal (Y-TZP) restorations have been advocated recently in clinical situations where occlusal/palatal space is limited, or to withstand parafunctional activities. The objectives of this in-vitro study were to investigate the effects of different polishing techniques on the surface roughness of Y-TZP (Ardent Dental, Inc.) and to investigate the effects of different polishing techniques on the wear behavior of synthetic hydroxyapatite (HA) and bovine enamel. An in-vitro study was conducted by fabrication of 48 Y-TZP sliders (diameter = 2 mm × 1.5 mm in height) using CAD/CAM technique; then the samples were embedded in acrylic resin using brass holders. Samples were then randomly allocated into four groups according to the finishing/polishing procedure: G1-as-machined (n = 8), G2- glazed (n = 16), G3-diamond bur-finishing (Brasseler, USA) (n = 8) and G4- G3+OptraFine polishing kit (Ivoclar-Vivadent) (n = 16). Thirty-two sintered HA disks (diameter = 11 mm × 2.9 mm in height) and 16 bovine enamel samples with a minimum surface area of 64 mm2 were mounted in brass holders. Baseline surface roughness (Ra and Rq, in μm) were recorded using a non-contact profilometer (Proscan 2000) for all the samples. A two-body pin-on-disk wear test was performed for 25,000 cycles at 1.2 Hz in which the four zirconia groups were tested against HA, and only G2-glazed and G4- G3+OptraFine polishing kit (Ivoclar-Vivadent) were tested against bovine enamel. Vertical substance loss (μm) and volume loss (mm3) of HA were measured (Proscan). Zirconia height loss was measured using a digital micrometer. One-way ANOVA was used for statistical analysis. The results indicated that surface roughness measurements showed significant differences among the surface treatments with G1 (Ra = 0.84, Rq = 1.13 μm) and G3 (Ra = 0.89, Rq = 1.2 μm) being the roughest, and G2 (Ra = 0.42, Rq = 0.63 μm) the smoothest. The glazed group showed the highest vertical loss (35.39 μm) suggesting wear of the glaze layer, while the polished group showed the least vertical loss (6.61 μm). HA antagonist volume loss and vertical height loss for groups (G1, G2 and G3) were similar, while polished group (1.3 mm3, 14.7 μm) showed significant lower (p = 0.0001) values. Antagonist height loss and antagonist volume loss were significantly higher for bovine antagonist than for HA antagonist (197.6 μm/116.2 μm, and 28.5 mm3/17.7 mm3 for bovine against glazed/polished zirconia sliders, respectively) (p < 0.0001). From the results it can be concluded that glazed zirconia provided an initially smooth surface, but a significant increased antagonist wear compared with the polished surface was seen. Bovine enamel showed higher wear compared with HA, which suggested that more studies should be performed to validate the use of bovine enamel as a substitute for human enamel in wear studies.