Rozprawy doktorskie na temat „Scaffold Bone Defect”

Bone graft substitute is a continuously developing field in orthopedics. When compared to tradition biomaterial in the field such as PLA or PCL, elastomer like polyurethane offers advantages in its high elasticity and flexibility, which establish an intimate contact with surrounding bones. This tight contact can provide a stable bone-material interface for cell proliferation and ingrowth of bone. The aim of this study is to evaluate the osteogenesis capabilities of a porous polyurethane scaffold in a critical size bone defect. In this study, a porous scaffold synthesized from segmented polyurethane is put under in vitro and in vivo tests to evaluate its potential in acting as a bone graft substitute for critical size bone defects. In vitro results indicate osteoblast-like cells are proliferating on the polyurethane scaffold during the 21-days experiment. Cells express their normal morphology when seeded on polyurethane under fluorescent staining. Although cells show a relatively lower cell activity then that seeded on culture plate, they share a similar alkaline phosphatase activity profile with the controls during the experiment period. In the in vivo animal model, reconstructed images from micro CT scanning indicates there are bone ingrowth inside the scaffold. Histology also indicates a tight interface has formed between bone and polyurethane, with osteogenic cells proliferating on the surface. The result has indicates polyurethane is a potential material for orthopedics in acting as a bone graft substitute.<br>published_or_final_version<br>Orthopaedics and Traumatology<br>Master<br>Master of Philosophy

Non-healing bone defects have a significant socioeconomic impact in the U.S. with approximately 600,000 bone grafting procedures performed annually. Autografts and allografts are clinically the most common treatments; however, autologous donor bone is in limited supply, and allografts often have poor mechanical properties. Therefore, tissue engineering and regenerative medicine strategies are being developed to address issues with clinical bone grafting. The overall objective of this work was to develop bone tissue engineering strategies that enhance healing of orthotopic defects by targeting specific osteogenic cell signaling pathways. The general approach included the investigation of two different tissue engineering strategies, which both focused on directed osteoblastic differentiation to promote bone formation. In the first cell-based strategy, we hypothesized that constitutive overexpression of the osteoblast-specific transcription factor, Runx2, in bone marrow stromal cells (BMSCs) would promote orthotopic bone formation in vivo. We tested this hypothesis by delivering Runx2-modified BMSCs on synthetic scaffolds to critically-sized defects in rats. We found that Runx2-modified BMSCs significantly increased orthotopic bone formation compared to empty defects, cell-free scaffolds and unmodified BMSCs. This gene therapy approach to bone regeneration provides a mineralizing cell source which has clinical relevance. In the second biomaterial-based strategy, we hypothesized that incorporation of the collagen-mimetic peptide, GFOGER, into synthetic bone scaffolds would promote orthotopic bone formation in vivo without the use of cells or growth factors. We tested this hypothesis by passively adsorbing GFOGER onto poly-caprolactone (PCL) scaffolds and implanting them into critically-sized orthotopic defects in rats. We found that GFOGER-coated scaffolds significantly increased bone formation compared to uncoated scaffolds in a dose dependent manner. Development of this cell-free strategy for bone tissue engineering provides an inexpensive therapeutic alternative to clinical bone defect healing, which could be implemented as a point of care application. Both strategies developed in this work take advantage of specific osteoblastic signaling pathways involved in bone healing. Further development of these tissue engineering strategies for bone regeneration will provide clinically-relevant treatment options for healing large bone defects in humans by employing well-controlled signals to promote bone formation and eliminating the need for donor bone.

Repair of bone defects is a major challenge in orthopaedic surgery. Current bone graft treatments, including autografts, allografts and xenografts, have many limitations making it necessary to develop a biomaterial to be a bone graft substitute. One such biomaterial is bioactive resorbable silica-calcium phosphate nanocomposite (SCPC). SCPC was processed using a 3D rapid prototyping technique and sintered at different temperatures to create porous scaffolds. SEM analyses and mercury intrusion porosimetry showed SCPC to be highly porous with micro- and nanopores. BET analysis indicated that SCPC had high surface area. Mechanical testing demonstrated that SCPC had a compressive strength similar to trabecular bone. Analysis of different thermal treatment temperatures indicated as the temperature was increased, the porosity decreased and the mechanical strength increased. When loaded with rhBMP-2 (SCPC-rhBMP-2), SCPC provided a sustained release profile of rhBMP-2 for 14 days. This was shown to be a greater release than hydroxyapatite (HA)-rhBMP-2. After immersion in SBF, ICP analyses showed the calcium concentration of SBF dropped drastically after one day of immersion. In conjunction, FTIR showed the formation of a hydroxyapatite layer on the SCPC surface and was confirmed by SEM. SCPC thermally treated at 850 ??C demonstrated the greatest dissolution/precipitation reactions when immersed in SBF. Processing the SCPC-rhBMP-2 hybrid using a rapid prototyping technique allowed for an exact replica of the rabbit ulna to be fabricated. This was implanted into a 10 mm segmental defect in the rabbit ulna. CT scans during the healing of the defect showed intimate union between SCPC-rhBMP-2 and the bone and about 65% healing of the defect after 4 weeks. Rabbits were euthanized after 12 and 16 weeks. Digital images show almost complete healing of the defect after 16 weeks. Torsional testing of the ulna after 12 weeks demonstrated restoration of maximum torque and angle at failure. Histological evaluation after 12 weeks showed the regenerated bone has all the morphological characteristics of mature bone. Through in-vitro and in-vivo testing, it can be recommended that the porous bioactive SCPC can serve as a successful delivery system for biological growth factors and serve as an alternative to autologous bone grafting.

Over the last decade, technological development has led to a revolution in the treatment of bone injuries. Few technologies hold more promise than 3D printing of biological material, which includes the field of bone science. This dissertation focused on 3D printed biodegradable scaffolds by using a novel Voronoy structured scaffold for critical-size bone defects that follows the definitions defined by mathematician Georgy Voronoy. These offer innovative healing opportunities for patients experiencing large bone defects, induced either by accidental or pathological causes, to regenerate the damaged tissues by using a scaffold as a structural skeleton for cell interaction and mechanical stability.

Currently, well established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, their application, however, is associated with disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration. As a result, recent research focuses on the development of complementary therapeutic concepts. The field of tissue engineering has emerged as an important alternative approach to bone regeneration. Tissue engineering unites aspects of cellular biology, biomechanical engineering, biomaterial sciences and trauma and orthopaedic surgery. To obtain approval by regulatory bodies for these novel therapeutic concepts the level of therapeutic benefit must be demonstrated rigorously in well characterized, clinically relevant animal models. Therefore, in this PhD project, a reproducible and clinically relevant, ovine, critically sized, high load bearing, tibial defect model was established and characterized as a prerequisite to assess the regenerative potential of a novel treatment concept in vivo involving a medical grade polycaprolactone and tricalciumphosphate based composite scaffold and recombinant human bone morphogenetic proteins.

This PhD-research was focused on the development and evaluation of innovative scaffold-based bone tissue engineering concepts for the treatment of large volume bone defects, which still represent a major challenge in orthopaedic and reconstructive surgery. Two different types of bone tissue engineering constructs were investigated and successfully applied to regenerate critically-sized segmental bone defects in ovine animal models. The results outlined in the PhD thesis represent a significant contribution to potential future clinical translations of bone tissue engineering concepts from bench to bedside.

This thesis explores the feasibility of donor-receiver concept for joint replacement where cartilage-bone tissues can be taken from either human or other mammals and prepared scientifically for repairing focal joint defects in knees, hips and shoulders. The manufactured construct is immunologically inert and is capable of acting as a scaffold for engineering new cartilage-bone laminates when placed in the joint. Innovative manufacturing procedures and assessment techniques were developed for appraising this tissue-based scaffold. This research has demonstrated that tissue replacement technology can be applied in situations where blood vessels are absent such as in articular cartilage.

Made available in DSpace on 2016-08-17T18:39:36Z (GMT). No. of bitstreams: 1 3621.pdf: 12488772 bytes, checksum: 3b893af6e3b0ea869fd5eda12f270c8a (MD5) Previous issue date: 2011-02-24<br>Financiadora de Estudos e Projetos<br>Several resources have been studied in order to accelerate the process of bone repair. Among these resources, bioactive materials and low level laser therapy (LLLT) have gained prominence. Several studies suggest that both resources are able of stimulating osteoblast proliferation and osteogenesis at the fracture site, promoting a greater deposition of bone mass, which is fundamental for the consolidation process. Within this context, this project aimed to assess the effects of LLLT (_ = 830nm), with the fluencies of 120J/cm ² and scaffold Biosilicate®, used associated or not, on consolidation of induced tibial bone defects in the rats. In this study it was used 40 male Wistar rats (3 months ± 250g) divided into four groups (with 10 animals each): group control bone defect without any treatment (GC), group bone defect irradiated with LLLT 830nm (GL); group bone defect treated with implantation of scaffolds Biosilicate ® (GB); group bone defect treated with implantation of scaffolds Biosilicate ® and LLLT 830nm (GBL). The animals were submitted to laser irradiation (Ga-As-Al, 830nm, 100mW) at a single point on the bone defect for eight sessions, on alternate days. The euthanasia of animals occurred at day 15 after surgery, 24 hours after the last laser treatment session. Morphological analysis revealed that the laser group, showed better tissue organization in relation to other groups. Furthermore, morphometric analysis revealed that the irradiated animals showed a higher amount of newly formed bone compared to the other groups. The expression of COX-2 and RUNX-2/CBFA-1 were higher in GB and GBL groups. Also, biomechanical analysis revealed no statistical differences among experimental groups. From the results obtained in this study, it is possible to suggest that both treatments had osteogenic potential 15 days after surgery, but the LLLT was more effective in bone repair when compared to the biomaterials, or even when the two treatment modalities were associated.<br>Vários recursos têm sido estudados com o intuito de acelerar o processo de reparação óssea. Dentre esses recursos, os materiais bioativos e a Terapia Laser de Baixa Intensidade (LLLT) vêm se destacando, vários estudos sugerem que ambos os recursos são capazes de estimular a proliferação de osteoblastos e a osteogênese no local da fratura, promovendo maior deposição de massa óssea, fundamental para o processo de consolidação. Dentro deste contexto, esse projeto teve como objetivo verificar os efeitos da LLLT (_ = 830nm), com fluência de 120J/cm² e do scaffold de Biosilicato®, utilizados independentemente ou associados na consolidação de defeitos ósseos induzidos em tíbias de ratos. Foram utilizados 40 ratos machos da linhagem Wistar (3 meses de idade ± 250 gramas) distribuídos em 4 grupos experimentais com 10 animais cada: grupo controle com defeito ósseo e sem tratamento (GC); grupo defeito ósseo tratado com Laser 830nm (GL); grupo defeito ósseo tratado com implante de scaffolds de Biosilicato® (GB); grupo defeito ósseo tratado com implante de scaffolds de Biosilicato® e Laser 830nm (GBL). Os animais foram submetidos a irradiação Laser (Ga-As-Al, 830nm, 100mW) em um único ponto sobre o defeito ósseo por oito sessões de tratamento, em dias alternados. A eutanásia dos animais aconteceu no 15º dia após a cirurgia, 24 horas após a última sessão de tratamento Laser. A análise morfológica revelou que o grupo Laser, apresentou melhor organização tecidual em relação os demais grupos experimentais. Além disso, a análise morfométrica evidenciou uma maior quantidade de osso neoformado no grupo tratado com Laser comparado aos animais dos demais grupos. A expressão à COX-2 e a RUNX-2/CBFA-1 mostrou-se mais intensa nos grupos GB e GBL e na análise biomecânica não houve diferença estatística entre os grupos experimentais. A partir dos resultados obtidos neste estudo, pode-se sugerir que ambos os tratamentos apresentaram potencial osteogênico 15 dias após a cirurgia, porém a Terapia Laser de Baixa Intensidade foi mais eficaz no processo de reparo ósseo, quando comparado ao biomaterial, ou mesmo quando as duas modalidades de tratamento foram associadas.

Establishing the sheep model for translational research of mandible (jaw) segmental defect regeneration. Providing a framework from which additional experimentation and evaluation of novel tissue engineered constructs may be undertaken, compared and collated. For current and future novel approaches to mandible segmental defect reconstruction that may be transferable to the human condition and, ultimately, the operative table.

The aim of this pilot study was to evaluate the bioactive, surface-coated polycaprolactone-co-lactide scaffolds as bone implants in a tibia critical size defect model. Polycaprolactone-co-lactide scaffolds were coated with collagen type I and chondroitin sulfate and 30 piled up polycaprolactone-co-lactide scaffolds were implanted into a 3 cm sheep tibia critical size defect for 3 or 12 months (n¼5 each). Bone healing was estimated by quantification of bone volume in the defects on computer tomography and microcomputer tomography scans, plain radiographs, biomechanical testing as well as by histological evaluations. New bone formation occurred at the proximal and distal ends of the tibia in both groups. The current pilot study revealed a mean new bone formation of 63% and 172% after 3 and 12 months, respectively. The bioactive, surface coated, highly porous three-dimensional polycaprolactone-co-lactide scaffold stack itself acted as a guide rail for new bone formation along and into the implant. These preliminary data are encouraging for future experiments with a larger group of animals.

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Transplanting bone graft from one site in the patient to the site of fracture or bone void, i.e. autologous bone grafting is commonly used throughout the world. The transplanted bone not only fills voids, but is also bone inductive, housing the particular cells that are needed for bone regeneration. Nevertheless, a regenerative complement to autograft is of great interest and importance because the benefits from an off-the-shelf product with as good of healing capacity as autograft will circumvent most of the drawbacks with autograft. With a regenerative-medicine approach, the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of limited volume of material. Two such regenerative products that utilize bone morphogenetic protein 7 and 2 have been used for more than a decade in the clinic. However, some severe side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery, use of supra physiological doses of the BMPs due to poor localization and retention of the growth factors. The purpose of this thesis was to harness the strong inductive capability of the BMP-2 by optimizing the carrier of this bioactive protein, thereby minimizing the side effects that are associated with the clinical products and facilitating safe and localized bone regeneration at the desired site. We focused on an injectable hyaluronan-based carrier. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident hyaluronidase enzymes. Earlier studies have shown a more controlled release and improved mechanical properties when adding a weight of 25 percent of hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that the bone formation was enhanced when using nano-sized hydroxyapatite. We wished to further develop the carrier system but were lacking an animal model with high output and easy access. We also wanted to provide paired data and were committed to the 3 Rs of refinement, reduction and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper II. In Paper III, we characterized and optimized the handling properties of the carrier. In Paper IV, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. In Paper V, we sought to further optimize biomaterial properties of the hydrogel through covalently bonding of bisphosphonates to the hyaluronan hydrogel. The results demonstrated exceptional retention of the growth factor BMP-2. In Paper VI, the in vivo response related to the release of the growth factor was examined by combining a SPECT/PET/µCT imaging method to visualize both the retention of the drug, and the in-vivo response in terms of mineralization.

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Autologous bone grafting is commonly used throughout the world. Autograft both fills the void and is bone inductive, housing the particular cells that are needed for bone regeneration. However, a regenerative complement to autograft is of great interest as the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of a limited volume of material. Two such regenerative products that utilise bone morphogenetic protein (BMP)-7 and -2 have been used for more than a decade clinically. Unfortunately, several side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery and use of supra physiological doses of the BMPs due to poor localisation and retention of the growth factor. The purpose of this thesis was to harness the strong inductive capacity of the BMP-2 by optimising the carrier of this bioactive protein, thereby minimising the side effects that are associated with the clinical products and facilitating safe and localised bone regeneration. We focused on an injectable hyaluronan-based carrier developed through polymer chemistry at the University of Uppsala. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident enzymes. Earlier studies have shown improved properties when adding hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that bone formation was enhanced when using nano-sized hydroxyapatite. In Paper II, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. We wished to further develop the carrier system, but were lacking an animal model with relatively high throughput, facilitated access, paired data, and we were also committed to the 3Rs of refinement, reduction, and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper III. In Paper IV, we sought to further optimise the biomaterial properties of the hydrogel through covalent bonding of bisphosphonates to the hyaluronan hydrogel. This resulted in exceptional retention of the growth factor BMP-2. In Paper V, SPECT/PET/µCT was combined as a tri-modal imaging method to allow visualisation of the biomaterial’s in situ action, in terms of drug retention, osteoblast activity and mineralisation. Finally, in Paper VI the correlation between existing in vitro results with in vivo outcomes was observed for an array of biomaterials. The study identified a surprisingly poor correlation between in vitro and in vivo assessment of biomaterials for osteogenesis.

Large bone defects present a major challenge to orthopaedic surgeons. Tissue engineering technology may provide a potential strategy to repair such defects, which involves the implantation of scaffold constructs typically loaded with cells and growth factors. It is expected that such growth factors loaded scaffolds can produce optimal microenvironments for the differentiation of seeded cells. The purpose of this project was to construct a dual growth factor release system in vitro using gelatin hydrogel and microspheres (MSs) to carry and deliver two growth factors, which have different effects on the growth and development of bone marrow derived mesenchymal stem cells (BMSCs) as well as bone tissues. The experimental studies of this project were divided into three sections as detailed below. In the first section of this study, both the conventional double emulsion technique and the electrospraying technology were combined with the thermally induced phase separation (TIPS) approach to prepare the bovine serum albumin (BSA)-loaded poly (lactic-co-glycolic acid) (PLGA) porous MSs. The particle size, surface morphology and the internal porous structure of the MSs were characterised using scanning electron microscopy (SEM). The release profile and the encapsulation efficiency of the BSA as a model protein in the PLGA MSs were established. The results showed that the novel electrosprayed MSs had uniform particle sizes between 400-600mm, many well-connected internal pores, high efficiencies of encapsulation and loading of the model protein, and a higher porosity than those MSs derived from the conventional double-emulsion method. Thus, the combination of the electrospraying technique with a low temperature freezing had proved to be a suitable method to produce polymer MSs for the controlled-release of the loaded-protein.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Medical Science<br>Griffith Health<br>Full Text

To synthesize a polyurethane (PU) foam-like scaffold which could perform both as a resorbable membrane and as a cavity filling material in oro-maxillary bone defects. MATERIALS & METHODS: A PU foam was synthesized via a onepot reaction starting from a pre-polymerized isocyanate and a biocompatible polyester diol, using water as a foaming agent. Different foaming conditions were examined, with the aim of creating a dense/porous functional graded material. The obtained PU was characterized in terms of morphological and mechanical properties. Biocompatibility assessment was performed in combination with bonemarrow- derived human mesenchymal stromal cells (hBMSC). RESULTS: PU showed a highly porous structure, consisting of interconnected round pores with a diameter larger than 200 μm. Degradation test showed a slow degradation (ca. 1% weight loss after 6 weeks). Mechanical properties were strongly dependent upon foaming conditions, and not significantly affected by in vitro degradation process. In vitro biocompatibility assessments combined with hBMSCs proved the materials non cytotoxic, with cell viability values higher than 95% after 24 hours. CONCLUSIONS: This work demonstrates the feasibility of fabricating biphasic dense/porous polyurethane foams by a confined foaming reaction. Results support the potential application of the synthesized materials in the treatment of oro-maxillary bone defects.

Thesis (M.S.)--University of Akron, Dept. of Biomedical Engineering, 2006.<br>"December, 2006." Title from electronic thesis title page (viewed 06/27/2007) Co-Advisors, Glen O. Njus, Daniel B. Sheffer; Faculty Reader, Mary C. Verstraete; Department Chair, Daniel B. Sheffer; Dean of the College, George K. Haritos; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

La région cranio-faciale est particulièrement vulnérable aux pertes de structures. Sa localisation et sa visibilité font qu'une atteinte entraîne des troubles, aussi bien physiques (alimentation, phonation...) que psychologiques (intégrité de la personne...). Les traitements actuels (régénération osseuse guidée, autogreffe osseuse ou allogreffe) sont particulièrement invasifs et présentent un taux d'échec élevé. Tout cela affecte fortement la qualité de vie du patient. De plus, le coût direct de ces traitements est important pour les systèmes de santé et le patient. Il existe donc un réel besoin de développer des traitements innovants basés sur des approches biomimétiques d'ingénierie tissulaire pour la régénération/réparation osseuse. L'objectif de ce travail est de développer une approche d'ingénierie tissulaire pour la réparation/régénération de tissus osseux cranio-faciaux lésés. Il est basé sur l'utilisation de matrices cellularisées avec des cellules souches mésenchymateuses issues de la pulpe dentaire : les Dental Pulp Stem Cells (DPSCs). De nombreux travaux ont démontré la grande plasticité de ces cellules, qui dérivent initialement de la crête neurale, mais aussi leur rôle trophique dans la réparation de tissus lésés par leur capacité de différenciation ostéogénique et chondrocytaire. Par ailleurs, ces cellules présentent des propriétés pro-angiogéniques supérieures aux cellules mésenchymateuses de la moelle osseuse (MSCs) et l'accès à cette réserve est aisé puisqu'elles peuvent être obtenues à partir de dents extraites. Dans ce contexte, nous avons à ce jour utilisé des matrices denses de collagène contenant des cellules souches pulpaires pour régénérer un tissu osseux crânien après réalisation de défauts critiques. L'objectif est d'induire très précocement une néo-angiogenèse favorisant à court terme la survie des cellules implantées, puis de stimuler leur maintien à long terme au sein du néo-tissu implanté, pour enfin provoquer une ostéoformation. Nous avons, ainsi, pu étudier et valider différents aspects de cette thématique : .1 L'impact positif de l'utilisation de matrices denses de collagène comme support ostéoconducteur, .2 Le suivi à long terme des cellules après implantation in vivo .3 L'impact positif d'un pré-traitement à l'hypoxie sur i/ la survie des cellules après implantation in vivo ii/ la potentialisation de leur apport pour la régénération/réparation osseuse en orientant leur différenciation vers une voie ostéoblastique, .4 L'apport significatif des techniques d'imageries pour le suivi des animaux grâce à la tomographie par émission de positons (utilisation de traceurs spécifiques de la minéralisation au sein des matrices et de la néo-angiogenèse) et au microscanner à rayons X (suivi cinétique de la qualité et de la quantité de matrice osseuse régénérée), .5 La validation et la confirmation de l'ensemble de ces résultats par l'histologie. Ainsi, ces résultats nous ont permis de répondre à l'objectif de travail et de perfectionner certains aspects de la composante cellulaire. Toutefois, il reste nécessaire d'optimiser le biomatériau lui-même. Il est en effet envisageable d'améliorer les matrices de collagène compressées que nous utilisons actuellement, en y intégrant par exemple des céramiques bioactives. En perspective, potentialiser les biomatériaux des matrices et combiner les DPSCs avec un support plus adapté à leur survie et à leur croissance permettrait d'améliorer considérablement la cicatrisation osseuse. Ces dernières années, l'étude des cellules souches a progressé d'approche in vitro vers l'in vivo. Les modèles in vivo établis pour étudier ces cellules dans le domaine cranio-facial ont déjà apporté des renseignements et ce travail s'inscrit dans leur continuité en cherchant à concevoir des stratégies adaptées pour l'utilisation future des DPSCs en ingénierie tissulaire<br>The craniofacial area is particularly vulnerable to structural loss. Its location and visibility make a loss causes disorders, both physical (food, phonation...) than psychological (integrity of the person...). Current treatments (autografts, allografts or synthetic bone grafts) are particularly invasive and have a high failure rate. All this strongly affects the quality of life of the patient. In addition, the cost of these treatments is significant for the health systems and the patient. Therefore, there is a real need to develop innovative treatments based on biomimetic tissue approaches for bone repair. The purpose of this thesis is to develop a tissue engineering approach for the repair/regeneration of injured cranial-facial bone tissue. It is based on the use of cellularized scaffolds with mesenchymal stem cells derived from the dental pulp: Dental Pulp Stem Cells (DPSCs). Many studies have demonstrated the high plasticity of these cells, which initially derive from the neural crest, but also their trophic ability in the repair of damaged tissues by their osteogenic and chondrocyte differentiation capacity. Moreover, these cells have better's pro-angiogenic properties than mesenchymal cells of the bone marrow (MSCs) and access to this reserve is easy since they can be obtained from extracted teeth. In this context, we have used dense collagen scaffolds seeded with DPSCs to regenerate cranial bone tissue on critical defects model. The objective is to induce a very early neo-angiogenesis for improved short-term survival of implanted cells, then stimulate the long-term maintenance of cells in the implanted neo-tissue, finally to cause osteoformation. We were able to study and validate various aspects of this theme: 1- The positive impact of the use of dense collagen scaffold as osteoconductive support, 2- Long-term follow-up of the cells after implantation in vivo (thanks to the use of a cell line constitutively expressing an intracellular fluorescence protein), 3- The positive impact of a pre-treatment with hypoxia on i/ the survival of the cells after implantation in vivo ii/ their contribution to bone regeneration / repair by orienting their differentiation towards an osteoblastic pathway, 4- The significant contribution of imaging techniques for the monitoring of animals (less sacrifice and longitudinal follow-up...) thanks to positron emission tomography (use of specific tracers of the mineralization within the scaffolds and neo-angiogenesis) and X-ray microscanner (kinetic monitoring of the quality and quantity of regenerated bone matrix) 5- Validation and confirmation of all these results by histology. Thus, these different results allowed us to respond to the working hypothesis and optimize some aspects of the cellular component. However, it remains necessary to optimize the biomaterial itself. It is indeed possible to improve the compressed collagen scaffolds that we currently use, for example by incorporating bioactive ceramics such as bioglasses or hydroxyapatite. In recent years, the study of stem cells has progressed from in vitro to in vivo. The in vivo models established to study these cells in the craniofacial area have already provided valuable information and this work is a continuation of these previous studies by seeking to build on better strategies (right characterization, environment oriented...) for the future use of DPSCs for tissue engineering purposes. In view of this work, potentiating the biomaterials of the scaffolds and combining the DPSCs with a support more adapted to their survival and their growth would considerably improve bone healing, as well as bone regeneration / repair

博士<br>國立臺北科技大學<br>化學工程與生物科技系化學工程博士班<br>106<br>An osteoconductive scaffold can facilitate bone defect repair. Bone defects are serious complications that are most commonly caused by extensive trauma, tumors, infections, or congenital musculoskeletal disorders. If nonunion occurs, the implantation of biomaterials developed as a bone defect filler, which can promote bone regeneration, is essential. In order to evaluate biomaterials for potential development as bone substitutes for bone defect repair, it is essential to establish clinically relevant in vitro and in vivo testing models to investigate their biocompatibility, mechanical properties, degradation, and interactions with the culture medium or host tissues. In clinical practice, the treatment of bone defect would commonly use bone cement or bone graft with powder. However, the exothermic reaction would at the potential risk of injuring harm to the outside organization while filling bone cement. Instead of the use of powdered bone-filling materials also has the issue of insufficient mechanical strength which would be assisted with some bone screws or bone plates that made the clinical applications much more complexity. In this study, a novel elastic porous composite comprising poly(propylene carbonate) (PPC), poly(D-lactic acid) (PDLA), and β-tricalcium phosphate (β-TCP) was prepared as an osteoconductive scaffold. A salt-leaching method is a nonsolvent and easily operated method used to mold up the porous scaffolds. 　　The novel malleable composite scaffolds composed of PPC/PDLA/TCP were evaluated as bone substitutes for bone defect repair. The animal model results demonstrated that a PPTE porous scaffold made with a PPC/PDLA/TCP weight ratio of 90/8/2 is (1) biocompatible, yielding a positive cell culture study and minimal inflammatory response in vivo; (2) malleable, such that the scaffold can be molded into the bone defect easily without fracturing; and (3) biodegradable and osteoconductive, promoting the progressive formation of new bone into the bone defect. These results indicated that the combination of this scaffold with osteoinductive agents, such as bone morphogenetic protein, demineralized bone matrix, or mesenchymal cells, might generate new biomaterial for bone defect repair.

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。<br>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。<br>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。<br>本論文分為以下四個主要部分：<br>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。<br>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。<br>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。<br>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。<br>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。<br>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.<br>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.<br>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:<br>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.<br>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.<br>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.<br>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.<br>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.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Yao, Dong.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 137-158).<br>Abstract also in Chinese; some appendixes also in Chinese.<br>ACKNOWLEDGEMENTS --- p.i<br>TABLE OF CONTENTS --- p.iii<br>ABSTRACT (IN ENGLISH) --- p.x<br>ABSTRACT (IN CHINESE) --- p.xiv<br>FLOWCHART --- p.xviii<br>LIST OF PUBLICATIONS --- p.xix<br>LIST OF ABBREVIATIONS --- p.xxi<br>LIST OF FIGURES --- p.xxiv<br>Chapter CHAPTER 1: --- Introduction --- p.1<br>Chapter 1 --- Osteonecrosis --- p.2<br>Chapter 1.1. --- Etiology --- p.2<br>Chapter 1.2. --- Anatomy of femoral head --- p.3<br>Chapter 1.3. --- Pathogenesis --- p.4<br>Chapter 1.3.1. --- Intraosseous hypertension (Compartment Syndrome of Bone --- p.4<br>Chapter 1.3.2. --- Intraosseous hypertension (Compartment Syndrome of Bone) --- p.4<br>Chapter 1.3.3. --- Coagulation --- p.5<br>Chapter 1.4. --- Development stages of osteonecrosis --- p.5<br>Chapter 2. --- Steroids-associated osteonecrosis --- p.11<br>Chapter 2.1. --- Epidemiology --- p.12<br>Chapter 2.2. --- Histopathology --- p.12<br>Chapter 2.3. --- Etiopathogenesis --- p.13<br>Chapter 2.3.1. --- Steroid and fat metabolism --- p.14<br>Chapter 2.3.2. --- Steroid and endothelial cells --- p.15<br>Chapter 2.3.3. --- Steroid and coagulation --- p.16<br>Chapter 2.3.4. --- Steroid and angiogenesis --- p.17<br>Chapter 2.4. --- Steroid and mesenchymal stem cells (MSCs) --- p.18<br>Chapter 2.5. --- Treatment strategies for SAON --- p.18<br>Chapter 2.5.1. --- Prevention --- p.19<br>Chapter 2.5.2. --- Nonoperative treatment --- p.19<br>Chapter 2.5.3. --- Operative treatment --- p.19<br>Chapter 2.5.3.1. --- Core decompression strategy --- p.20<br>Chapter 2.5.3.2. --- Tissue engineering approach --- p.22<br>Chapter 3. --- Epimedium-derived flavonoids (EFs) --- p.22<br>Chapter 3.1. --- Icaritin -Intestinal metabolism of EFs --- p.24<br>Chapter 3.1.1. --- Anti-tumor activity --- p..25<br>Chapter 3.1.2. --- Neuroprotective effects --- p.25<br>Chapter 3.1.3. --- Embryonic stem cells differentiation --- p.25<br>Chapter 3.1.4. --- Osteogenic differentiation --- p.26<br>Chapter 4. --- Poly lactic-co-glycolic acid / tricalcium phosphate (PLGA/TCP) scaffold --- p.26<br>Chapter 5. --- PLGA/TCP/Icaritin --- p.28<br>Chapter 6. --- Hypothesis of this study --- p.28<br>Chapter 7. --- Objective --- p.29<br>Chapter CHAPTER 2: --- The effect of phytomolecule Icaritin on differentiation of human mesenchymal stem cells in vitro --- p.30<br>Chapter 1. --- Introduction --- p.31<br>Chapter 2. --- Material and Methods --- p.33<br>Chapter 2.1. --- Ethics --- p.33<br>Chapter 2.2. --- Reagents and cell culture --- p.33<br>Chapter 2.3. --- Surface phenotypes of human BM-MSCs --- p.33<br>Chapter 2.4. --- Osteogenic and adipogenic differentiation of human BM-MSCs treated with Icaritin --- p.34<br>Chapter 2.5. --- MTT assay for proliferation of BM-MSCs --- p.34<br>Chapter 2.6. --- ALP staining --- p.35<br>Chapter 2.7. --- ALP activity assay --- p.35<br>Chapter 2.8. --- Alizarin Red S staining --- p.35<br>Chapter 2.9. --- Oil Red O staining --- p.35<br>Chapter 2.10. --- Ribonucleic acid (RNA) isolation --- p.36<br>Chapter 2.11. --- Reverse transcription --- p.36<br>Chapter 2.12. --- Real time polymerase chain reaction (RT-PCR) --- p.37<br>Chapter 2.13. --- Western blotting --- p.37<br>Chapter 2.14. --- Osteogenetic analysis of human MSCs after the addition of BMP2 inhibitor Noggin --- p.39<br>Chapter 2.15. --- Statistical analysis --- p.39<br>Chapter 3. --- Results --- p.40<br>Chapter 3.1. --- Characterization of surface phenotypes of human BM-MSCs --- p.40<br>Chapter 3.2. --- Icaritin had no effect on human mesenchymal stem cells (MSCs) proliferation --- p..41<br>Chapter 3.3. --- Icaritin promoted osteogenic differentiation of MSCs in presence of osteogenic supplement --- p.42<br>Chapter 3.4. --- Icaritin enhanced mineralization in osteogenic differentiation of MSCs only in presence of osteogenic supplement --- p.44<br>Chapter 3.5. --- Icaritin upregulated mRNA expression of osteoblastic marker genes during osteogenic differentiation of MSCs --- p.45<br>Chapter 3.6. --- Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis --- p..48<br>Chapter 3.7. --- Icaritin inhibited fat droplets formation during adipogenic differentiation of MSCs --- p.50<br>Chapter 4. --- Discussion --- p.52<br>Chapter 5. --- Conclusion --- p.56<br>Chapter CHAPTER 3: --- Icaritin rescued abnormal differentiation potential of MSCs derived from rabbit with SAON --- p.57<br>Chapter 1. --- Introduction --- p.58<br>Chapter 2. --- Methods and materials --- p.59<br>Chapter 2.1. --- SAON model establishment --- p.59<br>Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.60<br>Chapter 2.3. --- Osteogenic and adipogenic differentiation of rabbit BM-MSCs treated with Icaritin --- p.61<br>Chapter 2.4. --- MTT Assay for Proliferation of BM-MSCs --- p.62<br>Chapter 2.5. --- ALP Staining --- p.62<br>Chapter 2.6. --- ALP Activity Assay --- p.62<br>Chapter 2.7. --- Alizarin Red S Staining --- p.62<br>Chapter 2.8. --- Oil Red O Staining --- p.63<br>Chapter 2.9. --- RNA Isolation --- p.63<br>Chapter 2.10. --- Reverse transcription --- p.64<br>Chapter 2.11. --- Real time Polymerase chain reaction (RT-PCR) --- p.64<br>Chapter 2.12. --- Western blotting performance --- p.65<br>Chapter 2.13. --- Statistical analysis --- p.65<br>Chapter 3. --- Results --- p.66<br>Chapter 3.1. --- The osteogenic differentiation potential declined while adipogenic differentiation ability elevated of MSCs derived from SAON rabbits --- p.66<br>Chapter 3.2. --- The dose-dependent effect of Icaritin on osteogenic differentiation enhancement of MSCs from normal and SAON rabbits --- p.68<br>Chapter 3.3. --- Icaritin inhibited adipogenic differentiation of MSCs both derived from normal and SAON rabbits --- p..71<br>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<br>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<br>Chapter 3.6. --- Icaritin had no effect on the expression of VEGF which decreased in MSCs derived SAON --- p.76<br>Chapter 4. --- Discussion --- p.76<br>Chapter 5. --- Conclusion --- p.81<br>Chapter CHAPTER 4: --- The effect of Icaritin on angiogenesis in vitro --- p.82<br>Chapter 1. --- Introduction --- p.83<br>Chapter 2. --- Material and Methods --- p.85<br>Chapter 2.1. --- Cell culture --- p.85<br>Chapter 2.2. --- Proliferation assay --- p.85<br>Chapter 2.3. --- Scratch-wound healing assay --- p..86<br>Chapter 2.4. --- Migration Assay --- p.86<br>Chapter 2.5. --- In vitro Angiogenesis Assay --- p.87<br>Chapter 2.6. --- RNA Isolation and Real-time PCR Performance --- p.87<br>Chapter 2.7. --- Statistical Analysis --- p.88<br>Chapter 3. --- Results --- p.88<br>Chapter 3.1. --- Icaritin did not affect HUVECs migration --- p.88<br>Chapter 3.2. --- Icaritin had no effect on tube formation on growth factors reduced Matrigel --- p.92<br>Chapter 3.3. --- Icaritin had no effect on HUVECs proliferation --- p.94<br>Chapter 3.4. --- Icaritin did not change the angiogenesis related gene expression --- p.95<br>Chapter 4. --- Discussion --- p.96<br>Chapter 5. --- Conclusion --- p.100<br>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<br>Chapter 1. --- Introduction --- p.102<br>Chapter 2. --- Material and Methods --- p.106<br>Chapter 2.1. --- Preparation of porous PLGA/TCP/Icaritin composite scaffolds --- p.106<br>Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.106<br>Chapter 2.3. --- Wound healing assay --- p.107<br>Chapter 2.4. --- In vitro MSCs recruitment assay of scaffolds --- p.107<br>Chapter 2.5. --- MSCs labeling with SPIO@SiO2-NH2 nanoparticle --- p.108<br>Chapter 2.6. --- Prussian blue staining --- p.108<br>Chapter 2.7. --- MTT assay for SPIO@SiO2-NH2 labeled MSCs --- p.108<br>Chapter 2.8. --- Osteogenic and adipogenic differentiation of SPIO@SiO2-NH2 labeled MSCs --- p.109<br>Chapter 2.9. --- Real time PCR --- p.109<br>Chapter 2.10. --- Animal model establishment --- p.109<br>Chapter 2.11. --- Descriptive histology and histomorphometry --- p.110<br>Chapter 2.12. --- In vivo magnetic resonance imaging (MRI) of nanoparticle-labeled MSCs --- p.112<br>Chapter 2.13. --- Statistical analysis --- p.112<br>Chapter 3. --- Results --- p.112<br>Chapter 3.1. --- Icaritin promoted MSCs migration in vitro --- p.112<br>Chapter 3.2. --- PLGA/TCP and PLGA/TCP/Icaritin recruited MSCs when incubated in vitro --- p.114<br>Chapter 3.3. --- Stem cell potentials of MSC after SPIO@SiO2-NH2 labeling --- p.118<br>Chapter 3.4. --- PLGA/TCP and PLGA/TCP/Icaritin promoted MSCs homing in vivo --- p.122<br>Chapter 4. --- Discussion --- p.126<br>Chapter 5. --- Conclusion --- p.136<br>Chapter CHAPTER 6: --- Summary of the study and future research --- p.137<br>Chapter 1. --- Summary of the study --- p.138<br>Chapter 2. --- Limitations and further studies --- p.139<br>APPENDIXES --- p.142<br>REFERENCES --- p.147

La medicina rigenerativa è oggi uno tra i campi più interessante della biotecnologia, in grado di combinare diversi aspetti della medicina della biologia cellulare e molecolare dei biomateriali e dell'ingegneria dei tessuti, finalizzata a rigenerare, riparare o sostituire tessuti. La linea di ricerca proposta, sulla base di studi che analizzano vari aspetti della rigenerazione ossea in campo medico è rivolta al miglioramento delle tecniche già utilizzate in questo ambito e si propone di realizzare un sistema integrato standardizzato. Il substrato scelto per l'analisi è costituito da osso equino in blocchi di cui è stata analizzata con osservazioni S.E.M. la superficie dopo modellazione mediante strumentazione rotativa o vibrante, la possibilità di sterilizzazione raggiungendo SAL 10–6, valutando possibili modifiche macro e microstrutturali ed infine il possibile inserimento del materiale in un workflow digitale che partendo dalla rilevazione radiologica di un difetto osseo, passa alla modellazione di un modello tridimensionale computerizzato, che porti alla produzione di un blocco mediante tecnica sottrattiva, customizzato in relazione al difetto rilevato.

碩士<br>南台科技大學<br>化學工程與材枓工程系<br>96<br>This study is to fabricate there dimensional porous scaffolds through freeze-drying method by completely mixing of either polylactic-co-glycolic acid (PLGA) or PLGA-OH, dioxane and water. It’s biocompatibility and mechanical properties could be enhanced by adding hydroxyapatite (HAP) and nano-SiO2. The different ratio of dioxane and water, dipping temperature, and the concentration of PLGA on the effect of morphology, porosity, pore size, and degradation test of scaffolds were investigated in this study. The SEM results showed that the pore size changed with different reaction conditions. The pore size of scaffolds which was dipped at 4°C for 30 minutes was uniform when the ratio of dioxane and water was 9：1. The pore size and porosity were 107.2 μm and 82.4% in 10 % PLGA(w/v), and those were 108.7 μm and 83.7% in 10 % PLGA-OH(w/v). The addition of HAP and nano-SiO2 would not change the structure of pore and synthesis procedure of the scaffolds. The XRD peak of HAP decreased when the amount of nano-SiO2 increased. The compression test results revealed that the strength of scaffolds increased with nano-SiO2, and the compression modulus was 18MPa. The degradation of scaffolds with HAP and nano size SiO2 addition could sustain up to 16 weeks.

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）。<br>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。<br>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。<br>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。<br>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。<br>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.<br>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.<br>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.<br>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.<br>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.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Chen, Shihui.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 173-198).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.<br>Acknowledgements --- p.viii<br>Abstract --- p.x<br>中文摘要 --- p.xiii<br>List of Abbreviations --- p.xvi<br>List of Tables --- p.xix<br>List of Figures --- p.xx<br>Journal Publications --- p.xxv<br>Journal Supplements --- p.xxv<br>Conference Abstracts --- p.xxvi<br>Chapter Chapter 1 --- Introduction<br>Chapter 1.1 --- Bone Defect in Orthopaedics --- p.2<br>Chapter 1.2 --- Human Skeletons --- p.2<br>Chapter 1.2.1 --- Bone Types and Function --- p.2<br>Chapter 1.2.2 --- Bone Development --- p.4<br>Chapter 1.2.3 --- Bone Physiology and Structure --- p.6<br>Chapter 1.2.4 --- Bone Specific Markers --- p.7<br>Chapter 1.2.5 --- Bone Cells --- p.9<br>Chapter 1.2.6 --- Bone Marrow Stromal Cells --- p.12<br>Chapter 1.3 --- Bone Regeneration and Remodeling --- p.13<br>Chapter 1.3.1 --- Bone Defect Healing --- p.13<br>Chapter 1.3.2 --- Non-union and Segmental Defect --- p.15<br>Chapter 1.3.3 --- Bone Defect Treatment --- p.16<br>Chapter 1.4 --- Angiogenesis in Bone Healing --- p.19<br>Chapter 1.4.1 --- Blood Vessels Formation Process --- p.20<br>Chapter 1.4.2 --- Growth Factor in Angiogenesis --- p.21<br>Chapter 1.5 --- Biomaterials in Bone Tissue Engineering --- p.22<br>Chapter 1.6 --- Scaffold-Based Therapy --- p.23<br>Chapter 1.6.1 --- Bone Grafts --- p.23<br>Chapter 1.6.1.1 --- Autografts --- p.23<br>Chapter 1.6.1.2 --- Allografts --- p.25<br>Chapter 1.6.2 --- Bone Graft Substitutes --- p.25<br>Chapter 1.6.2.1 --- Bone Formation in Porous Scaffolds --- p.25<br>Chapter 1.6.2.2 --- Degradable Polymers --- p.27<br>Chapter 1.6.2.3 --- Non-Degradable Polymers --- p.29<br>Chapter 1.6.2.4 --- Ceramics --- p.29<br>Chapter 1.6.2.5 --- Bioactive Composite Materials --- p.30<br>Chapter 1.7 --- Growth Factor-Based Therapy --- p.31<br>Chapter 1.7.1 --- Endogenous Growth Factor--Bone Morphogenetic Proteins --- p.31<br>Chapter 1.7.2 --- Exogenous phytomoleculeIcaritin--Icaritin --- p.31<br>Chapter 1.7.3 --- Delivery of Growth Factor in Tissue Engineering --- p.34<br>Chapter 1.8 --- Fabrication of Porous Composite Scaffolds --- p.37<br>Chapter 1.8.1 --- Architectural Parameters of Bone Scaffolds --- p.37<br>Chapter 1.8.2 --- Three-Dimensional Scaffold Fabrication --- p.37<br>Chapter 1.9 --- Animal Models for Testing Bone Defects Healing --- p.39<br>Chapter Chapter 2 --- Research Rationale and Study Objectives<br>Chapter 2.1 --- Research Rationale --- p.42<br>Chapter 2.2 --- Study Objectives --- p.46<br>Chapter Chapter 3 --- Bioactive Composite Scaffolds: Preparation, Morphology and Release Assay<br>Chapter 3.1 --- Introduction --- p.49<br>Chapter 3.2 --- Materials and Methods --- p.50<br>Chapter 3.2.1 --- Materials --- p.50<br>Chapter 3.2.2 --- Fabrication of PLGA/TCP Incorporating BMP-2 or Icaritin --- p.51<br>Chapter 3.2.3 --- Morphological Analysis of Composite Scaffolds --- p.53<br>Chapter 3.2.3.1 --- Analysis of Porosity and Macropores Diameter Using High-resolution Micro-CT --- p.53<br>Chapter 3.2.3.2 --- Analysis of Surface Morphology and Elements Composition Using Scanning Electron Microscopy --- p.54<br>Chapter 3.2.4 --- Icaritin Content Assay in PLGA/TCP Scaffolds Incorporating Icaritin --- p.54<br>Chapter 3.2.5 --- Preparation of PLGA/TCP Scaffold Coating BMP-2 or Icaritin --- p.55<br>Chapter 3.2.6 --- In vitro Release Assay --- p.55<br>Chapter 3.2.6.1 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.55<br>Chapter 3.2.6.2 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.56<br>Chapter 3.2.7 --- Mechanical Properties of Composite Scaffolds --- p.56<br>Chapter 3.2.8 --- Statistical Analysis --- p.57<br>Chapter 3.3 --- Results --- p.57<br>Chapter 3.3.1 --- Morphological Analysis of Composite Scaffolds --- p.57<br>Chapter 3.3.1.1 --- Porosity and Macroscopic Diameter --- p.57<br>Chapter 3.3.1.2 --- Surface Morphology and Elements Composition --- p.58<br>Chapter 3.3.2 --- Icaritin Content in Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60<br>Chapter 3.3.3 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60<br>Chapter 3.3.4 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.61<br>Chapter 3.3.5 --- Mechanical Properties of Composite Scaffolds --- p.63<br>Chapter 3.4 --- Discussion --- p.64<br>Chapter 3.5 --- Summary --- p.71<br>Chapter Chapter 4 --- Bioactive Composite Scaffolds: In vitro Degradation and Characterization Studies<br>Chapter 4.1 --- Introduction --- p.73<br>Chapter 4.2 --- Materials and Methods --- p.74<br>Chapter 4.2.1 --- Preparation of Composite Scaffolds for in vitro Degradation Assay --- p.74<br>Chapter 4.2.2 --- Characterizations --- p.75<br>Chapter 4.2.2.1 --- Scaffold Volume Changes --- p.75<br>Chapter 4.2.2.2 --- Scaffold Weight Changes --- p.75<br>Chapter 4.2.2.3 --- pH Value Changes --- p.75<br>Chapter 4.2.2.4 --- Calcium Ion Release from Scaffolds --- p.76<br>Chapter 4.2.3 --- Mechanical Properties Changes --- p.76<br>Chapter 4.2.4 --- Statistical Analysis --- p.77<br>Chapter 4.3 --- Results --- p.77<br>Chapter 4.3.1 --- Volume Decrease --- p.78<br>Chapter 4.3.2 --- Weight Loss --- p.78<br>Chapter 4.3.3 --- pH Value Reduction --- p.79<br>Chapter 4.3.4 --- Calcium Ion Release --- p.79<br>Chapter 4.3.5 --- Mechanical Properties --- p.80<br>Chapter 4.4 --- Discussion --- p.81<br>Chapter 4.5 --- Summary --- p.84<br>Chapter Chapter 5 --- In vitro Evaluation of Bone Marrow Stem Cells (BMSCs) Growing on Bioactive Composite Scaffolds<br>Chapter 5.1 --- Introduction --- p.87<br>Chapter 5.2 --- Materials and Methods --- p.90<br>Chapter 5.2.1 --- Preparation of Composite Scaffolds for in vitro Evaluation --- p.90<br>Chapter 5.2.2 --- BMSCs Seeding Rate and Proliferation on Composite Scaffolds --- p.90<br>Chapter 5.2.3 --- Alkaline Phosphate (ALP) Activity Assay --- p.92<br>Chapter 5.2.4 --- Osteogenic Gene Expression Assay Using Quantitative Real-time PCR --- p.92<br>Chapter 5.2.5 --- Calcium Deposition Assay Using Alizarin Red Staining --- p.93<br>Chapter 5.2.6 --- Statistical Analysis --- p.94<br>Chapter 5.3 --- Results --- p.94<br>Chapter 5.3.1 --- Cells Seeding Efficiency and Proliferation --- p.94<br>Chapter 5.3.2 --- ALP Activity --- p.97<br>Chapter 5.3.3 --- Osteogenic Gene mRNA Expression --- p.97<br>Chapter 5.3.4 --- Calcium Deposition --- p.98<br>Chapter 5.4 --- Discussion --- p.99<br>Chapter 5.5 --- Summary --- p.102<br>Chapter Chapter 6 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds<br>Chapter 6.1 --- Introduction --- p.105<br>Chapter 6.2 --- Materials and Methods --- p.106<br>Chapter 6.2.1 --- Preparation of Composite Scaffolds for Implantation --- p.106<br>Chapter 6.2.2 --- Establishment of Ulna Bone Segmental Defect in Rabbits --- p.107<br>Chapter 6.2.3 --- Radiographic Evaluation of New Bone Area Fraction --- p.109<br>Chapter 6.2.4 --- XtremeCT Evaluation of New Bone Formation and Bone Mineral Density (BMD) --- p.110<br>Chapter 6.2.5 --- Histological Evaluation of New Bone Formation --- p.111<br>Chapter 6.2.6 --- Evaluation of Rate of New Bone Formation and Mineral Apposition Rate (MAR) --- p.114<br>Chapter 6.2.7 --- Evaluation of Neovascularization Using Micro-CT-based Microangiography --- p.116<br>Chapter 6.2.8 --- Blood Perfusion Function Using Dynamic Magnetic Resonance Imaging (MRI) --- p.119<br>Chapter 6.2.9 --- Statistical Analysis --- p.120<br>Chapter 6.3 --- Results --- p.121<br>Chapter 6.3.1 --- Radiographic Area Fraction of New Bone Formation --- p.123<br>Chapter 6.3.2 --- XtremeCT New Bone Volume Fraction and BMD --- p.128<br>Chapter 6.3.3 --- Histological New Bone Fraction --- p.133<br>Chapter 6.3.4 --- Rate of New Bone Formation and MAR --- p.136<br>Chapter 6.3.5 --- New Vessels Volume Evaluated Using Micro-CT-Based Microangiography --- p.140<br>Chapter 6.3.6 --- Dynamic Blood Perfusion Function --- p.144<br>Chapter 6.4 --- Discussion --- p.146<br>Chapter 6.5 --- Summary --- p.151<br>Chapter Chapter 7 --- Summaries, Conclusions, Limitations and Future Studies<br>Chapter 7.1 --- Introduction --- p.153<br>Chapter 7.2 --- Bioactive Composite Scaffolds: Preparation, Morphology and in vitro Release Evaluation --- p.155<br>Chapter 7.3 --- Bioactive Composite Scaffolds: in vitro Degradation and Characterization Studies --- p.159<br>Chapter 7.4 --- In vitro Evaluation of the Response of Bone Marrow Stem Cells Growing on Bioactive Composite Scaffolds --- p.160<br>Chapter 7.5 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds --- p.162<br>Chapter 7.6 --- Evaluation of Dose-dependent Effects of Icaritin Mechanical Property, Degradation, and Osteogenic Potentials --- p.164<br>Chapter 7.7 --- Conclusions --- p.170<br>Chapter 7.8 --- Limitations and Future Studies --- p.171<br>Chapter 7.9 --- References --- p.173<br>Chapter 7.10 --- Appendix --- p.199<br>Chapter 7.10.1 --- Animal Licence and Ethics --- p.199<br>Chapter 7.10.2 --- Safety Approval --- p.201<br>Chapter 7.10.3 --- Journal Supplements --- p.202<br>Chapter 7.10.4 --- Conference Abstracts--Posters --- p.205<br>Chapter 7.10.5 --- Conformation of Paper Submission --- p.208<br>Chapter 7.10.6 --- Published Paper --- p.209

The ultimate goal of this research was to develop a “self-fitting” shape memory polymer (SMP) scaffold for the repair of craniomaxillofacial (CMF) bone defects. CMF defects may be caused by trauma, tumor removal or congenital abnormalities and represent a major class of bone defects. Their repair with autografts is limited by availability, donor site morbidity and complex surgical procedures. In addition, shaping and positioning of these rigid grafts into irregular defects is difficult. Herein, we have developed SMP scaffolds which soften at T > ~56 °C, allowing them to conformally fit into a bone defect. Upon cooling to body temperature, the scaffold becomes rigid and mechanically locks in place. This research was comprised of four major studies. In the first study, photocrosslinkable acrylated (AcO) SMP macromers containing a poly(ε-caprolactone) (PCL) segment and polydimethylsiloxane (PDMS) segments were synthesized with the general formula: AcO-PCL40-block-PDMSm-block-PCL40-OAc. By varying the PDMS segment length (m), solid SMPs with highly tunable mechanical properties and excellent shape memory abilities were prepared. In the second study, porous SMP scaffolds were fabricated based on AcO-PCL40-block-PDMS37-block-PCL40-OAc via a revised solvent casting particulate leaching (SCPL) method. By tailoring scaffold parameters including salt fusion, macromer concentration and salt size, scaffold properties (e.g. pore features, compressive modulus and shape memory behavior) were tuned. In the third study, porous SMP scaffolds were produced from macromers with variable PDMS segment lengths (m = 0 – 130) via an optimized SCPL method. The impact on pore features, thermal, mechanical, and shape memory properties as well as degradation rates were investigated. In the final study, a bioactive polydopamine coating was applied onto pore surfaces of the SMP scaffold prepared from PCL diacrylate. The thin coating did not affect intrinsic bulk properties of the scaffold. However, the coating significantly increased its bioactivity, giving rise to the formation of “bone-bonding” hydroxyapatite (HAp) when exposed to simulated body fluid (SBF). It was also shown that the coating largely enhanced the scaffold’s capacities to support osteoblasts adhesion, proliferation and osteogenesis. Thus, the polydopamine coating should enhance the performance of the “self-fitting” SMP scaffolds for the repair of bone defects.

Despite the growing number of survival cases, due to the increase in cases, the death rate from cancer-related diseases has been increasing over the years. Being less than 0.2% of all cancers, primary bone cancers are extremely unusual and often curable. However, about 50% of these tumors can metastasize, so early intervention is frequently necessary. Tumor or tumor-like resection derived from osteosarcoma usually leads to the creation of large bone defects, which constitute a reconstructive problem. The current standard procedures used to resolve this and other related issues are viable solutions. However, they are far from being ideal, still carrying many risks, and often failing. With the ongoing advances in a variety of theoretical subjects and in manufacturing (namely 3D printing), bone graft substitutes using smart biodegradable scaffolds are revolutionizing bone tissue engineering and regenerative medicine. In addition, despite the numerous options currently available, the scientific consensus is that the ideal bone graft should likely have a hybrid composition. The goal of this thesis is to provide an overall review of the current state-of-the-art on the main concepts associated with chitosan-based 3D scaffolds. Porous scaffolds produced by conventional fabrication techniques, using chemically cross-linked chitosan hydrogel are analysed. Due to its growing popularity, cross-linking agent genipin was selected as a case study. Follows an analysis on the most recent methods of scaffold fabrication reinforced by rapid prototyping techniques (PLA is further examined as printing material). The incorporation of bioactive agents and cells is evaluated in both options. The different factors that influence the properties of each material, the overall performance of the 3D structures, and the most common methods of surface modification are also discussed topics. To finalize, the key aspects that still need improvement and future perspectives for scaffold technology are highlighted.<br>Representando menos de 0,2% de todos os casos de cancro, os cancros ósseos primários são extremamente incomuns e, na maioria das vezes, curáveis, no entanto, cerca de 50% destes tumores podem metastizar e, portanto, uma intervenção precoce é frequentemente necessária. A ressecção de tumores ou de variantes destes geralmente leva à criação de grandes defeitos ósseos que constituem um problema reconstrutivo. Os procedimentos padrões usados atualmente para resolver este e outros problemas relacionados são soluções viáveis, no entanto, ainda longe de serem ideais, constatando-se a presença de diversos riscos e de um elevado rácio de insucesso. Com os contínuos avanços nos demais conteúdos teóricos e em fabricação (nomeadamente impressão 3D), os substitutos de scaffolds (“andaimes”) ósseos recorrendo a suportes biodegradáveis inteligentes têm vindo a revolucionar a engenharia de tecidos ósseos e a medicina regenerativa. Para além disso, apesar das mais diversas opções atualmente disponíveis, o consenso científico é que o scaffold ideal deverá ter uma composição híbrida. Esta tese pretende fornecer uma revisão geral do estado da arte atual acerca dos principais conceitos associados a scaffolds 3D à base de quitosano. São analisados scaffolds porosos produzidos por técnicas convencionais, à base de hidrogel de quitosano reticulado quimicamente. Devido à sua crescente popularidade, o agente de reticulação genipina foi selecionado como estudo de caso. Segue se uma análise relacionada com os métodos mais recentes de fabrico de scaffoldsreforçados por técnicas de prototipagem rápida (PLA é posteriormente examinado como material de impressão). A incorporação de agentes bioativos e células é avaliada em ambas as opções. Os diferentes fatores que influenciam as propriedades de cada material, o desempenho geral da estrutura 3D e os métodos mais comuns de preparação de superfície são também tópicos discutidos. Para finalizar, destacam-se os aspetos ainda sujeitos a aperfeiçoamento e as perspetivas futuras para a tecnologia de scaffolds.

碩士<br>國防醫學院<br>牙醫科學研究所<br>106<br>Background: In guiding bone formation, creating an adequate space is critical due to defect complexity.  In this study, a polycaprolactones (PCL) scaffold, computer designed and 3D-printed, was implanted into the critical-sized calvaria defect in rat, whereas the effect of plasma-rich fibrin (PRF) on the bone formation was also tested. Methods:  Two round defects, 6 mm in diameter, were surgically created in parietal bones of 32 rats, then 4 defect groups, including defect control, PRF, PCL, and PCL-plus-PRF (PRF+PCL), were divided according to scaffold implantation and PRF delivery.  Four and eight weeks after, half of the rats were sacrificed, respectively. Bone formation was assessed by dental radiography, micro-computed tomography (µ-CT), and histology. Results: By dental radiography and µ-CT, the radio-opaque row-by-row squares were observed in the defects of PCL and PCL+PRF groups. The opaque areas/volumes, as well as their relative values (areas/volumes), in defects were similar among 4 groups; however, significantly greater values were noted in central, but not peripheral zone in the groups with PCL (PCL and PRF+PCL groups) than those without PCL (defect control and PRF groups). By histology, the bones formed in the spaces between PCL struts; however, there was no bones-struts contact observed. Histometry showed that the tissue areas were significant greater in the groups with PCL than that without, particularly in central zone, in spite of the intervals. In week 8, similar findings were observed, including the bone and connective tissue areas. Similar areas for PCL residuals were observed in the PCL and the PRF+PCL groups. (太長) Conclusions: In the critical-sized calvarial defect of rat, our results showed the bones formed within the spaces supported by the computer-designed 3D-printed PCL scaffold but no contact with PCL struts. Strategy of using the customized scaffold for supporting spaces in augmenting bone formation is therefore suggested.

Knorpeldefekte des Kniegelenks zeichnen sich durch eine sehr begrenzte spontane Heilungstendenz aus und führen im Verlauf häufig zur Arthrose. Trotz intensiver Forschungsbemühungen konnte bisher keine neue Therapieoption eine zufrieden-stellende Alternative zu den bisherigen Therapien hervorbringen. Eine ACI in Kombination mit einem künstlich hergestellten Knorpel-Knochen-Ersatzmaterial scheint jedoch großes Potential für die Therapie von Knorpel-Knochen-Schäden zu besitzen. Im vorliegenden Langzeittierversuch mit Kaninchen wurde eine einzeitige ACI mit einem biphasischen Ersatzmaterial (TruFit®) und platelet-rich-plasma (PRP) kombiniert. Zu diesem Zweck wurde in der medialen Femurkondyle ein critical-size-Defekt mit einem Durchmesser von 4,5 mm gesetzt. In der ersten Versuchsgruppe blieb der Defekt unbehandelt (Leer). Bei der zweiten Gruppe wurde die Defekthöhle mit einem TruFit®-Zylinder aufgefüllt (TFP). Gruppe drei erhielt zusätzlich PRP (TFP+PRP) und Gruppe vier wurde darüber hinaus mit einer einzeitigen ACI kombiniert (TFP+PRP+C), bei der Chondrozyten mit Hilfe eines speziellen Kollagenase-Schnellverdaus isoliert werden konnten. Die Auswertung der Knorpel-Knochen-Regeneration erfolgte nach 12 Monaten durch eine Mikroradiographie, eine intravitale Fluoreszenzmarkierung des Knochens und durch Toluidinblau-O- und Safranin-O-Färbungen. Verwendet wurden die Scores nach Wakitani und O’Driscoll. Dabei konnte gezeigt werden, dass eine TruFit®-Therapie die Knochenregeneration positiv beeinflussen kann. Die Zugabe von PRP bewirkte die Bildung von zahlreichen dünnen Trabekeln mit einer erhöhten Anzahl trabekulärer Verbindungen, allerdings auch eine schlechtere Rekonvaleszenz der subchondralen Knochenschicht. Bezüglich der Knorpelheilung schnitt die Gruppe TFP+PRP+C am besten ab, wobei die Unterschiede nicht signifikant waren. Insgesamt zeigten alle Versuchsgruppen eine unzureichende osteochondrale Regeneration, so dass für die Therapie am Menschen zunächst weitere Studien nötig sind, die sowohl ossär als auch chondral eine verbesserte Heilungspotenz demonstrieren können. Bisher fehlen groß angelegte Studien um Therapieempfehlungen bezüglich des Ersatzmaterials, der genauen Durchführung der einzeitigen ACI und Zusätzen wie Wachstumsfaktoren zu machen.