Both platelet-rich plasma (PRP) and vascular endothelial growth factor (VEGF) can promote regeneration. The aim of this study was to compare the effects of these two elements on bone formation and vascularization in combination with bone marrow stromal cells (BMSC) in a critical-size bone defect in rabbits. The critical-size defects of the radius were filled with: (1) a calcium-deficient hydroxyapatite (CDHA) scaffold + phVEGF165-transfected BMSC (VEGF group), (2) CDHA and PRP, or (3) CDHA, autogenous BMSC, and PRP. As controls served: (4) the CDHA scaffold alone and (5) the CDHA scaffold and autogenous BMSC. The volume of new bone was measured by means of micro-CT scans, and vascularization was assessed in histology after 16 weeks. Bone formation was higher in the PRP + CDHA, BMSC + CDHA, and PRP + BMSC + CDHA groups than in the VEGF group (p < 0.05). VEGF transfection significantly promoted vascularization of the scaffolds in contrast to BMSC and PRP (p < 0.05), but was similar to the result of the CDHA + PRP + BMSC group. The results show that VEGF-transfected BMSC as well as the combination of PRP and BMSC improve vascularization, but bone healing was better with the combination of BMSC and PRP than with VEGF-transfected BMSC. Expression of VEGF in BMSC as a single growth factor does not seem to be as effective for bone formation as expanded BMSC alone or PRP which contains a mixture of growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Both platelet-rich plasma (PRP) and vascular endothelial growth factor (VEGF) can promote regeneration. The aim of this study was to compare the effects of these two elements on bone formation and vascularization in combination with bone marrow stromal cells (BMSC) in a critical-size bone defect in rabbits. The critical-size defects of the radius were filled with: (1) a calcium-deficient hydroxyapatite (CDHA) scaffold + phVEGF165-transfected BMSC (VEGF group), (2) CDHA and PRP, or (3) CDHA, autogenous BMSC, and PRP. As controls served: (4) the CDHA scaffold alone and (5) the CDHA scaffold and autogenous BMSC. The volume of new bone was measured by means of micro-CT scans, and vascularization was assessed in histology after 16 weeks. Bone formation was higher in the PRP + CDHA, BMSC + CDHA, and PRP + BMSC + CDHA groups than in the VEGF group (p < 0.05). VEGF transfection significantly promoted vascularization of the scaffolds in contrast to BMSC and PRP (p < 0.05), but was similar to the result of the CDHA + PRP + BMSC group. The results show that VEGF-transfected BMSC as well as the combination of PRP and BMSC improve vascularization, but bone healing was better with the combination of BMSC and PRP than with VEGF-transfected BMSC. Expression of VEGF in BMSC as a single growth factor does not seem to be as effective for bone formation as expanded BMSC alone or PRP which contains a mixture of growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The issue of prosthetic infection leading to implant failure due to the formation of bacterial biofilms on biomaterial surfaces has been widely recognized as a major issue, often leading to revision surgery. The growing number of patients requiring synthetic biomaterials as implants is on the rise and so is the risk of infection arising from pre/peri-/post-operative surgical procedures. Traditional antibiotic treatment has led to the emergence of bacterial drug resistance. Therefore, the development of novel bactericidal methods to combat drug resistant microbial pathogens is the need of the hour. The first part of the thesis is an attempt to address prosthetic infection by the development of novel ultrasmall gold nanoparticles (AuNPs) which are cytocompatible and present a therapeutic dosage window for eliciting antimicrobial property. Towards this end, ultrasmall AuNPs with 0.8 nm and 1.4 nm gold core sizes, stabilized by monosulphonated triphenylphosphine ligand shells were synthesized. Such intricately designed AuNPs with ultrasmall gold cores and phosphine-based ligand chemistry were demonstrated to be highly potent bactericidal agents against staphylococci, the most common human pathogen causing biomaterial associated infection. The antibacterial efficacy of these AuNPs was significant even in mature staphylococcal biofilms. In another study, the application of high strength pulse magnetic fields (1-4 Tesla) was examined for bacterial growth inactivation in vitro. A magnetic field strength dependent decrease in bacterial viability with a concomitant increase in the production of reactive oxygen species (ROS) and longer doubling times were recorded. The mechanism of action was explained through an analytical model which involves ion-transport interference of essential ions like Ca2+ and Mg2+ and disruption of FeS clusters leading to inactivation of bacterial redox enzymes. On the contrary, such high magnetic fields did not pose any detrimental effects to eukaryotic cells under similar exposure. Additionally, the potency of low intensity direct current electric field (DC EF: 1V/cm) against biofilm formation by methicillin resistant Staphylococcus aureus (MRSA) was explored on antimicrobial surfaces of hydroxyapatite and Zinc oxide (HA-xZnO; x = 0, 5, 7.5 and 10 wt%). An EF exposure time dependent decline in the viability and stability of MRSA biofilms were noted. Further, EF treatment resulted in bacterial membrane depolarization and reduced biofilm formation on HA-ZnO composites, independent of the substrate composition. In summary, the above three studies were cases of the developmental methods to address prothetic infection. The second part of the thesis is focused on the development of magneto-responsive biomaterials as implants for orthopaedic applications. Under this category, the sintering/ hot pressing of hydroxyapatite-magnetite (HA-xFe3O4; x = 0, 5, 10, 20 and 40 wt%) powders in oxidizing and inert atmospheres was carried out and the resulting phases and microstructure were characterized. A detailed analysis of the phase assemblage by Rietveld refinement of the X-ray diffraction (XRD) data and Mössbauer spectroscopy revealed the major retention of Fe3O4 along with wustite (FeO) formation under reducing conditions while hematite (α-Fe2O3) was the oxidized product of conventional sintering in ambient atmosphere. A good correlation between the unit cell volume increases in HA observed from Rietveld refinements and Fe incorporation into the apatite lattice from Mössbauer spectral parameters was evident. Further, the Mössbauer data analysis indicated a preferential occupancy of Fe at the Ca(1) site under oxidizing conditions and Ca(2) site in inert atmosphere. The above phase analyses were further confirmed by X-ray photoelectron spectroscopy (XPS), Infrared spectroscopy (FT-IR) and CHN analysis. The microstructure of the hot-pressed samples observed under transmission electron microscope (TEM) divulged similar phases as deduced from XRD as well as the formation of translational Moire fringe patterns due to inference of overlapping crystal planes of HA and Fe3O4 in the HA-40 wt% composite. Such translational Moire fringes suggest a preferred arrangement and orientation of the crystallites resulting from hot-pressing, which correlated well with the room temperature magnetic measurements made with the help of a vibrating sample magnetometer (VSM). The compositional similarity of Fe doping in HA to that of the tooth enamel and bone presents these HA-Fe3O4 composites as potent dental/ orthopaedic implant materials. In the conclusive study, the hot-pressed HA-xFe3O4 composites were tested for their efficacy in supporting the osteogenesis of human mesenchymal stem cells (hMSCs) assisted by intermittent static magnetic field exposure. The magneto-responsive substrates were applied as platforms for the culture of hMSCs and the effect of static magnetic field (SMF) exposure on the viability, proliferation and differentiation of hMSCs were elucidated. With a mild compromise in viability, SMF triggered the osteogenic differentiation of hMSCs mediated by proliferative arrest in the G0/G1 phase and elevated intracellular calcium levels. The early bone marker genes - Runx2, Col IA and ALP were significantly up regulated upon SMF exposure on pure HA and HA-Fe3O4 composites. Further, the late osteogenic markers – OCN and OPN were detected exclusively in the HA-xFe3O4 (x = 10 and 40 wt%) composites. Matrix mineralization was enhanced and CaP nodules were detected on similar SMF treated HA-Fe3O4 composites. A substrate magnetization and time dependent modulation of gene expression was recorded which corroborated well with the temporal trending of osteogenic genes during bone development. In conclusion, substrate magnetization can be applied as a tool to modulate the behavior of stem cells and direct them towards osteogenic lineage. Such a pertinent combination of substrate magnetization and external magnetic field stimulation can be applied synergistically for stem cell based bone tissue engineering applications.

Cette thèse transdisciplinaire réalisée en collaboration avec le laboratoire SPCTS (Sciences des Procédés Céramiques et Traitement de Surface) et l’EA 3842 (Homéostasie cellulaire et pathologies) de l’université de Limoges est un projet de recherche à l’interface entre la biologie et la chimie et a été consacrée à l’étude de l’influence des propriétés physico-chimiques de biocéramiques de phosphate de calcium sur leur comportement biologique in vitro.L’exploration des processus d’interaction entre matériaux et cellules reste une problématique scientifique de premier plan tant d’un point de vue fondamental qu’appliqué pour la mise au point de biomatériaux performants. L’objectif final est d’optimiser l’efficacité thérapeutique des céramiques phosphocalciques comme matériaux de substitution pour la régénération osseuse. La première partie de la thèse est une revue bibliographique générale présentant la problématique actuelle abordée en lien avec les besoins cliniques et les limitations des études actuelles. Les connaissances sur la biologie du tissu osseux sain ainsi que les aspects de régulation du processus de remodelage osseux ont également été abordés dans ce chapitre. Ce chapitre se termine par une synthèse bibliographique sur les biomatériaux et la régénération osseuse. Le chapitre 2 est relatif à la synthèse puis à la caractérisation physico-chimique des matériaux céramiques. Des céramiques de trois compositions chimiques : HA (hydroxyapatite : Ca10(PO4)6(OH)2 , SiHA (hydroxyapatite silicatée : Ca10(PO4)5,6(SiO4)0,42(OH)1,6 et CHA (hydroxyapatite carbonatée : Ca9,5(PO4)5,5(CO3)0,48(OH)1,08(CO3)0,23 , chacune avec deux microstructures différentes : dense ou poreuse, ont été élaborées et rigoureusement caractérisées (porosité, topographie de surface, mouillabilité, potentiel zêta, taille des grains, distribution et taille des pores, surface spécifique). Le chapitre 3 décrit l’approche expérimentale employée pour l’évaluation biologique des interactions matériaux/cellules explorées dans ce travail. Les analyses biologiques ont été réalisées avec deux lignées cellulaires différentes. La lignée cellulaire pré-ostéoblastique MC3T3-E1 et la lignée cellulaire de monocytes/macrophages, précurseurs des ostéoclastes RAW 264.7, (très importantes pour les aspects osseux, mais moins souvent explorées que les lignées ostéoblastiques dans la littérature). Enfin, le chapitre 4 reporte et commente les résultats biologiques obtenus dans ce travail. Tous les biomatériaux évalués dans cette étude sont biocompatibles, néanmoins, le biomatériau poreux CHA s’est avéré le plus prometteur des six variantes de biomatériaux testés. / This transdisciplinary thesis, carried out in collaboration with the SPCTS laboratory (sciences of ceramic processes and surface treatment) and EA 3842 (Cellular homoeostasis and pathologies) of the University of Limoges, is a research project at the interface between biology and chemistry and was devoted to the study of the influence of the physico-chemical properties of calcium phosphate bioceramics on their biological behavior in vitro.The exploration of the processes of interaction between materials and cells remains a major scientific issue, both from a fundamental and applied point of view for the development of highperformance biomaterials. The ultimate objective is to optimize the therapeutic efficiency of phosphocalcic ceramics as substitute materials for bone regeneration.The first part of the thesis is a general bibliographic review presenting the current issues tackled with the clinical needs and limitations of current studies. Knowledge of the biology of healthy bone tissue as well as the regulatory aspects of the bone remodeling process was also discussed in this chapter. It includes also a bibliographic overview of biomaterials and bone regeneration.Chapter 2 relates to the synthesis and the physico-chemical characterization of ceramic materials. HA (hydroxyapatite: Ca10 (PO4) 6 (OH) 2, SiHA (silicated hydroxyapatite: Ca10 (PO4) 5.6 (SiO4) 0.42 (OH) 1.6 and CHA (carbonated hydroxyapatite: Ca9.5 (PO4) 5.5 (CO3) 0.48 (OH) 1.08 (CO3) 0.23, ceramics each with two different microstructures : dense or porous, have been elaborated and thoroughly characterized (porosity, surface topography, wettability, zeta potential, grain size, pore size and distribution, specific surface area). Chapter 3 describes the experimental approach used for the biological evaluation of the interactions between materials and cells. Biological analyzes were performed with two different cell lines. The pre-osteoblastic MC3T3-E1 cell line and the RAW 264.7cell line of monocytes / macrophages, precursors of the steoclasts, (very important for the bone aspects, but less often explored than the osteoblastic lines in the literature). Finally, Chapter 4 reports and comments on the biological results obtained in this work. All biomaterials evaluated are biocompatible, nevertheless, the porous CHA biomaterial was the most promising of the six variants of biomaterials tested.