This study aimed at guiding osteoblast cells on biodegradable polymer carriers with well-defined surface microtopography and chemistry, and investigating the effect of cell alignment on osteoblast phenotype expression. A blend of two different polyesters, one being natural in origin (PHBV) and the other synthetic (P(L/DL)LA), was used to form a film with parallel macro- (250 &micro / m wide) or microgrooves (27 &micro / m wide) on its surface, by solvent casting on patterned templates. The micropatterned Si template was produced by photolithography, while the Teflo macropatterned template was lathe cut. Fibrinogen (Fb) was adsorbed or immobilized via epichlorohydrin spacer/crosslinker on the film surfaces to enhance cell attachment by increasing the surface hydrophilicity and by providing RGD amino acid sequence for integrin binding. Surface hydrophilicity was assessed by water contact angle measurements. Adsorption of Fb caused an increase in hydrophilicity, while the opposite was achieved with its covalent immobilization. Fb was homogeneously immobilized throughout the whole micropatterned film surface with amount of 153.1 &plusmn / 42.4 g Fb/cm2, determined with the Bradford assay, while it was adsorbed within the grooves of the micropattern. Surface characteristics of the films were studied with Scanning Electron (SEM) and Light microscopy. Osteoblast cells derived from rat bone marrow were seeded on the polymeric films with different surface topography and chemistry and were grown for one and three weeks. Osteoblast proliferation on the films was determined with Cell Titer 96 TM Non-Radioactive Cell Proliferation (MTS) test. Alkaline Phosphatase (ALP) assay and tetracycline labelling of mineralized matrix were carried out to determine osteoblast phenotype expression on different surfaces. SEM and fluorescence microscopy were used to evaluate the cell alignment. Osteoblasts on the micropatterned films with adsorbed Fb aligned along the groove axis with a mean deviation angle of 13.1o, while on the unpatterned films deviation from horizontal axis was 63.2o and cells were randomly distributed. Cell alignment did not affect cell proliferation. However, the highest ALP specific activity and the most homogeneous mineral distribution were obtained on the Fb adsorbed micropatterned films.

Various members of the insulin-like growth factor (IGF) family of growth factors are highly expressed in bone tissue and are vitally important for the normal development and function of bone. Recent studies have shown that IGF-I can associate with the extra-cellular matrix proteins vitronectin (VN) and fibronectin (FN) via IGF binding protein-5 (IGFBP-5). Furthermore, when these complexes are pre-bound to a tissue culture surface they can stimulate enhanced responses in epithelial cell types in vitro. More recently, transforming growth factor-beta 1 (TGF-β1), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have also been shown to interact with VN and to elicit functional responses in various cell types. Taken together, these findings indicate that exploitation of the adhesive properties of these ECM proteins might allow immobilisation of various growth factors at the culture surface. This may provide a novel means of coating engineered biomaterial constructs with agents which can elicit specific functional effects in therapeutically important cells, such as those used in cell-based therapeutics for the replacement and / or regeneration of damaged bone tissue. Since both VN and FN are also important matrix components of bone, this study sought to investigate the hypothesis that select pre-bound combinations of these matrix proteins and growth factors could also stimulate functional responses in bone cells and the therapeutically important so called mesenchymal stem cells. Thus it is reported here that pre-bound combinations of VN, IGFBP-5 and IGF-I or FN IGFBP-5 and IGF-I significantly stimulate cell migration in the osteoblast-like SaOS-2 cells. While, VN, IGFBP-5 and IGF-I stimulated cell proliferation over 72 hr, FN, IGFBP-5 and IGF-I did not. Moreover, I found that VN, IGFBP-5 and IGF-I could facilitate alkaline phosphatase (ALP) expression in SaOS-2 cells. VN, FN and EGF on the other hand could sustain SaOS-2 cells for up to 12 days in culture, but could not sustain ALP expression; hence it is possible that these cells may have entered a state of quiescence in response to this treatment. Extending these studies to cells derived from clinical samples, pre-bound combinations of VN / IGFBP-5 / IGF-I were not able to support initiation of human mesenchymal stem cell (hMSC) cultures. Nevertheless, VN alone in serum free media stimulated substantial metabolic activity and protein synthesis in hMSCs once the cultures were established. Moreover, the addition of IGFBP-3 or -5 together with IGF-I can enhance the response to levels equivalent to that observed with 10% FCS. I also report that the responses to VN and TGF-β1 are synergistic and stimulate greater hMSC metabolic activity than 10% FCS. Interestingly, hMSCs cultured in IGF-I or TGF-β1 and low concentrations of VN aggregated, an effect that was not observed when higher concentrations of VN were used. I hypothesise that this aggregation effect was due to endogenous protease activity, and therefore examined MMP-2 and 9 activity in hMSC conditioned media. Both pro-MMP-2 and pro-MMP-9 were constitutively expressed by hMSCs but there was no evidence of the active forms in the conditioned media, indicating that neither IGF-I nor TGF-β1 affect MMP-2 or -9 expression or activation in serum-free media. However, hMSC conditioned media could degrade IGFBP-5, suggesting that there is proteolytic activity within the conditioned media which may impact on the function of ECM / growth factor components in serum-free media settings. Thus, while ECM and growth factors may stimulate desirable responses in therapeutically important cells in serum-free culture, the role that endogenously expressed proteases have on the efficacy of such media supplements needs to be examined closely. Taken together, the studies reported in this thesis provide proof of principle data indicating that select combinations of ECM proteins and growth factors could be utilised in bone tissue engineering applications. This may be achieved for example, as a biomaterial coating, or could form the basis of a viable alternative media supplement for the serum-free culture of hMSCs.

En chirurgie reconstructive osseuse les biomatériaux remplacent le tissu osseux manquant et dans le cas de pathologies ils peuvent également délivrer des molécules actives. L’élément semi-métallique, gallium (Ga), est utilisé dans le traitement de différentes pathologies liées à la résorption accélérée de l’os dû à son effet inhibiteur sur les ostéoclastes (cellules résorbantes de l’os). Le Ga peut être incorporé dans la structure des biomatériaux osseux et nous nous sommes intéressés aux propriétés biologiques de ces derniers. In vitro, en présence de Ga nous avons mis en évidence une diminution de la différentiation des ostéoclastes, ainsi qu’une sur-expression de plusieurs marqueurs des ostéoblastes (cellules formatrices d’os). In vivo, le modèle murin de comblement du défaut osseux a montré une augmentation de la quantité de tissu osseux néoformé avec un biomatériau chargé en Ga vs. contrôle. Ces données démontrent que les biomatériaux chargés en Ga sont compatibles avec la survie et la prolifération des cellules osseuses et que le Ga peut améliorer la reconstruction osseuse. D’autre part, étant donné que des effets anti-tumoraux du Ga sont largement décrits, nous avons étudié ces effets sur une lignée cellulaire cancéreuse, choisie pour son affinité pour le tissu osseux. Nous avons montré que le Ga réduit la prolifération et probablement le potentiel tumoral de cette lignée, mais aussi la différentiation ostéoclastique induite par les cellules cancéreuses. Ces effets inhibiteurs observés dans un contexte tumoral indiquent que le Ga est un candidat intéressant pour le couplage avec des biomatériaux destinés au comblement osseux après une résection tumorale. / In bone reconstructive surgery biomaterials commonly replace the missing tissue and in case of pathologies can also serve as vectors for drug delivery. The semi-metallic element gallium (Ga) is used for the treatment of several disorders associated with accelerated bone resorption, due to its inhibitory action on bone-resorbing cells (osteoclasts). Since Ga can be incorporated into the structure of bone biomaterials, we embarked on characterising the biological properties of novel Ga-loaded materials. In vitro, we observed a decrease in osteoclast differentiation and the upregulated expression of several osteoblastic markers (bone-forming cells) in the presence of Ga-loaded biomaterial. In vivo, using a rat bone defect model, we showed an increase in newly formed bone tissue in implants filled with Ga-loaded biomaterial vs. control. Taken together, our data indicate that Ga-loaded biomaterials provide biocompatible substrates allowing bone cells survival and improved bone reconstruction in vivo. Taking into account antitumoral effects of Ga, largely described in literature, we also investigated its impact on a bone metastatic model. Using an aggressive human cancer cell line selected for its ability to invade bone tissue, we showed that Ga could reduce cancer cell proliferation and viability and reverse excessive osteoclastogenesis in bone metastatic environment. Moreover, we demonstrated that Ga modulated the expression of several marker genes hindering the tumour-propagating potential of cancer cells. Thus, due to its inhibitory action on cancer cells, Ga could represent an attractive additive to biomaterials used for tissue reconstruction after tumour resection.

Fabrication of scaffold is the key for bone tissue engineering, which is commonly regarded as the most potential route for repairing bone defects. Previously, porous ceramic scaffolds were fabricated through a variety of traditional methods, like moulding and casting, but most of them cannot produce customised tissue-engineered scaffolds. Therefore, 3D printing methods are gaining more attention and are currently being explored and developed to make scaffolds with acceptable biocompatibility. With the considerable development of bone tissue engineering, the bioactivity of scaffolds is becoming increasingly demanded, which leads to new methods and techniques to produce highly biomimetic bone scaffolds. In this study, a new fabrication process to optimise the structures of scaffolds was developed, and intensive researches were performed on the porous scaffolds to confirm their advantages in biological performance. Specifically, by combination of motor assisted extrusion deposition and gas-foaming (graphite as the porogen) technique, hierarchically porous scaffolds with improved microstructures, i.e. multi-scaled pores from nanometre to millimetre (nm-μm-mm), was successfully developed. In this thesis, the optimal content of porogen for scaffolds was studied in terms of compressive strength and in-rod porosities. The most concerned physicochemical properties of scaffolds were carefully examined and the results revealed that such scaffolds exhibit excellent physicochemical properties owing to hierarchically porous structures. Due to additional in-rod micropores and increased specific surface area, along with better hydrophilicity, hierarchically porous scaffolds exerted complete superiority in biological activity, including promoting cellular proliferation of osteoblasts, adhesion and spreading status, as well as the ability to induce cellular differentiation.

Introduction: Bone defects and non-unions caused by trauma, tumor resection, pathological degeneration, or congenital deformity pose a great challenge in the field of orthopedics. Traditionally, these defects have been repaired by using autografts and allografts. Autografts have set the gold standard for clinical bone repair because of their osteoconductivity, osteoinductivity and osteogenicity. Nevertheless, the application of autografts is limited because of donor availability and donor site morbidity. Allografts have the advantage that the tissues are readily available and can be easily applied, especially when large segments of bone are to be reconstructed. However, their use is also limited by the risk of disease transfer and immune rejection. To circumvent these limitations tissue engineering has evolved as a means to develop viable bone grafts. An ideal bone graft should be both osteoconductive and osteoinductive, biomechanically strong, minimally antigenic, and eliminates donor site morbidity and quantity issues. The biodegradable polymer, Poly lactic-co-glycolic acid (PLAGA) was chosen because of its commercial availability, biocompatibility, non-immunogenicity, controlled degradation rate, and its ability to promote optimal cell growth. To improve the mechanical properties of PLAGA, Single Walled Carbon Nanotubes (SWCNT) were used as a reinforcing material to fabricate composite scaffolds. The overall goal of this project is to develop a Single Walled Carbon Nanotube composite (SWCNT/PLAGA) for bone regeneration and to examine the interaction of MC3T3-E1 cells (mouse fibroblasts) and hBMSCs (human bone marrow derived stem cells) with the SWCNT/PLAGA composite via focusing on extracellular matrix production and mineralization; and to evaluate its toxicity and bio-compatibility in-vivo in a rat subcutaneous implant model. We hypothesize that reinforcement of PLAGA with SWCNT to fabricate SWCNT/PLAGA composites increases both the mechanical strength of the composites as well as the cell proliferation rate on the surface of the composites while expressing osteoblasts phenotypic, differentiation and mineralization markers; and SWCNT/PLAGA composites are biocompatible and non-toxic, and are ideal candidates for bone tissue engineering. Methods: PLAGA and SWCNT/PLAGA composites were fabricated with various amounts of SWCNT (5, 10, 20, 40 and 100mg), characterized and degradation studies were performed. PLAGA (poly lactic-co-glycolic acid) and SWCNT/PLAGA microspheres and composites were fabricated; characterized and mechanical testing was performed. Cells were seeded and cell adhesion/morphology, growth/survival, proliferation and gene expression analysis were performed to evaluate biocompatibility. Sprague-Dawley rats were implanted subcutaneously with Sham, poly lactic-co-glycolic acid (PLAGA) and SWCNT/PLAGA composites, and sacrificed at 2, 4, 8 and 12 week post-implantation. The animals were observed for signs of morbidity, overt toxicity, weight gain, food consumption, hematological and urinalysis parameters, and histopathology. Results: Imaging studies demonstrated uniform incorporation of SWCNT into the PLAGA matrix and addition of SWCNT did not affect the degradation rate. Composites with 10mg SWCNT resulted in highest rate of cell proliferation (p<0.05) among all composites. Imaging studies demonstrated microspheres with uniform shape and smooth surfaces, and uniform incorporation of SWCNT into PLAGA matrix. The microspheres bonded in a random packing manner while maintaining spacing, thus resembling trabeculae of cancellous bone. Addition of 10mg SWCNT led to greater compressive modulus and ultimate compressive strength. Imaging studies revealed that MC3T3-E1 cells adhered, grew/survived, and exhibited normal, non-stressed morphology on the composites. SWCNT/PLAGA composites exhibited higher cell proliferation rate and gene expression compared to PLAGA. No mortality and clinical signs were observed. All the groups showed consistent weight gain and rate-of-gain for each group was similar. All the groups exhibited similar pattern for food consumption. No difference in urinalysis parameters, hematological parameters; and absolute and relative organ weight was observed. A mild to moderate summary toxicity (sumtox) score was observed for animals treated with the PLAGA and SWCNT/PLAGA whereas the sham animals did not show any response. At all the time intervals both PLAGA and SWCNT/PLAGA showed a significantly higher sumtox score compared to the Sham group. However, there was no significant difference between PLAGA and SWCNT/PLAGA groups. Conclusion: Our SWCNT/PLAGA composites, which possess high mechanical strength and mimic the microstructure of human trabecular bone, displayed tissue compatibility similar to PLAGA, a well known biocompatible polymer over the 12 week study. Thus, the results obtained demonstrate the potential of SWCNT/PLAGA composites for application in BTE and musculoskeletal regeneration. Future studies will be designed to evaluate the efficacy of SWCNT/PLAGA composites in bone regeneration in a non-union ulnar bone defect rabbit model. As interest in carbon nanotube technology increases, studies must be performed to fully evaluate these novel materials at a nonclinical level to assess their safety. The ability to produce composites capable of promoting bone growth will have a significant impact on tissue regeneration and will allow greater functional recovery in injured patients.

La reconstruction de lésions osseuses complexes reste un défi dans le domaine de la chirurgie orthopédique et maxillo-faciale. A ce jour, la technique de référence reste la greffe d’os autologue. Cependant cette technique présente de nombreuses limites (risque d’infection, morbidité au site de prélèvement). Dans ce contexte, l’ingénierie tissulaire peut apporter des solutions de reconstruction innovantes. En effet l’utilisation de matrices ostéoconductrices et ostéoinductrices permettrait de remplacer l’autogreffe. Le premier objectif de ce travail a été de mettre au point une matrice de polysaccharides, contenant des particules d’hydroxyapatite (HA) dopées avec du strontium (Sr), afin de stimuler à la fois la régénération osseuse, mais également l’angiogenèse. Les résultats obtenus in vitro et in vivo nous ont permis d’optimiser la formulation de cette matrice, en termes de quantités de particules d’hydroxyapatite dopées par différents taux de substitution en strontium, dispersées au sein de la matrice. La deuxième partie de ce travail a été consacrée à l’application de cette matrice à la technique de Masquelet afin de remplacer l’utilisation de l’autogreffe. Cette procédure chirurgicale en deux temps, basée sur la formation d’une membrane induite, est utilisée fréquemment en chirurgie orthopédique et maxillo-faciale. Le premier temps opératoire utilise un ciment chirurgical, le (poly(méthyl)méthalcrylate, PMMA) qui entraîne la formation d’une membrane induite vascularisée. Notre travail a été de remplacer ce ciment par du silicone et d’étudier l’influence de la radiothérapie sur la qualité et la fonction de la membrane ainsi formée. Les premiers essais d’évaluation de cette matrice ont été réalisés chez le rat après résection segmentaire du fémur, suivie d’une procédure de radiothérapie. Les perspectives de ce travail sont d’évaluer la performance de cette matrice dans une lésion mandibulaire de grand volume, après irradiation, chez le gros animal. / Reconstruction of large and complex bone defects remains a challenge for orthopaedic and maxillo-facial surgery. The gold standard strategy for bone reconstruction is the autologous bone graft. However, this approach still exhibits some limitations (infection risks, morbidity at the donor site). In this context, tissue engineering can provide innovative solutions for bone reconstruction. Indeed, the use of osteoconductives and osteoinductives matrices could replace autograft. Based on previous data obtained by our laboratory, the first objective of this work was to develop a composite matrix of polysaccharides containing hydroxyapatite (HA) particles doped with strontium (Sr), to stimulate both bone formation and angiogenesis. In vitro and in vivo results allow us to optimize the amount of HA particules and the ratio of Sr-substitution within the polysaccharide-based matrix. The second part of this work was to apply this biomaterial in the context of Masquelet approach. These two time procedure surgery, based on the formation of an induced membrane, is commonly used in orthopaedic and maxillo-facial surgery. The first chirurgical step uses a surgical cement (poly(methyl)methalcrylate, PMMA) to promote around it the formation of a vascularized membrane. Our work was to replace this cement by silicone and to study the influence of radiotherapy treatment on the quality and the function of this induced membrane. The first preclinical evaluation of this matrix has been performed on a rat femoral segmental bone defect, followed by a radiotherapy procedure. The perspectives of this work are to evaluate the performances of this matrix on irradiated segmental mandibular bone defect in large animal.

Plusieurs études rapportent l’intérêt de structures poreuses synthétiques reproduisant la micro-architecture osseuse pour obtenir une régénération des pertes de substance osseuses. La fusion laser sélective de titane permet de fabriquer des implants poreux aux propriétés mécaniques très proches de celles de l’os et au potentiel d’ostéointégration élevé. Néanmoins, la recolonisation osseuse des pores de ces implants peut être limitée par leurs propriétés élastiques que nous considérons surdimensionnées. Nous avons mis au point une étude expérimentale chez la brebis afin d’évaluer l’influence des propriétés élastiques de ces implants, utilisés dans des pertes de substance mandibulaires, sur leur recolonisation osseuse. Des implants poreux et contrôles permettant une reprise intégrale de la sollicitation mécanique ont été développés. Deux groupes de six brebis ont été équipés d’implants poreux et d’implants contrôles controlatéraux de raideur variable. La régénération osseuse au sein des implants a été évaluée par caractérisation mécanique des interfaces os–implant et par la mesure du volume osseux néoformé à partir d’acquisitions micro-CT. Les implants poreux ont permis une meilleure régénération osseuse que les implants contrôles. Les implants poreux à la raideur la plus basse ont montré une régénération osseuse significativement plus élevée que les autres implants poreux. Un modèle en éléments finis a été développé afin d’optimiser la fixation des implants et la transmission des contraintes aux interfaces os-implant. / Several articles report on the regeneration of bone defects using synthetic porous structures mimicking bone micro-architecture. Porous implants exhibiting mechanical properties close to that of bone tissue with enhanced osseointegration ability can be manufactured by means of selective laser melting of titanium. However, bone growth into the pores of such implant could be limited due to oversized elastic properties. We implemented an experimental study with ewes to assess the influence of the overall stiffness of these implants on bone ingrowth in critical-size mandibular defects. Fully load-bearing porous and control implants of varying overall stiffness were developed and implanted in two groups of six ewes. Bone ingrowth was assessed by mechanical characterization of bone-implant interfaces and by the measurement of the newly formed bone volume using micro-CT imaging. Higher bone ingrowth was identified in porous implants compared to control implants. Low-stiffness porous implants exhibited significantly higher bone ingrowth as compared to porous implants with stiffness closer to that of the missing bone. A finite elements model was developed to improve bone fixation of the implant and load transfer through the bone-implant interfaces.

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