Rat Femoral Bone Defect Research Articles

Infection Microenvironment-Responsive Coating on Titanium Surfaces for On-Demand Release of Therapeutic Gas and Antibiotic.

Aseptic loosening and bacterial infection pose significant challenges in the clinical application of titanium (Ti) orthopedic implants, which are primarily caused by insufficient osseointegration and bacterial contamination. To address these issues, a responsive coating on Ti surface is constructed, which achieves enhanced osseointegration and infection elimination by on-demand release of therapeutic gas hydrogen sulfide (H2S) and antibiotic. TiO2 nanotubes (TNT) are anodized on the Ti surface to enhance its bioactivity and serve as reservoirs for the antibiotic. An infection microenvironment-responsive macromolecular H2S donor layer is coated on top of TNT to inhibit premature leakage of antibiotic. This layer exhibits a sustained release of low-dosage H2S, which is capable of promoting the osteogenic differentiation and migration of cells. Moreover, the compactness of the macromolecular H2S donor layer could be broken by bacterial invasion, leading to rapid antibiotic release thus preventing infection. In vitro antibacterial experiments validates significant antibacterial activity of the coating against both Gram-negative (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). Crucially, this coating effectively suppresses implant-associated infection with 98.7% antibacterial efficiency in a rat femoral bone defect model, mitigates inflammation at the defect site and promotes osseointegration of the Ti orthopedic implant.

Heparin-conjugated injectable hydrogels with sustained releasing capability for promotion of H-type vessels formation and rat femoral bone defects repair

Adequate vascularization is crucial for the repair of bone defects. Platelet-derived growth factor-BB (PDGF-BB) has been shown to promote H-type vessel formation, a specialized blood vessel, strongly positive for CD31 and endomucin, coupling angiogenesis and osteogenesis. However, the lack of a controllable growth factor delivery limits its clinical application, causing certain complications associated with PDGF-BB administration. This study developed an injectable heparin-conjugated hydrogel (GHH) for controlled delivery to facilitate the effective binding of PDGF-BB and rapid vascularized bone regeneration. Herein, in vitro experiments involving the controlled release of PDGF-BB showed that the PDGF-BB-loaded GHH (GHHP) hydrogel demonstrated excellent cytocompatibility and osteogenic properties. The subcutaneous implantation model in rats revealed that the GHHP hydrogel has the potential to induce the formation of H-type blood vessels in vivo. Furthermore, the healing of distal femoral condyle defects in rat models in vivo demonstrated that GHHP could substantially promote H-type angiogenesis and effectively repair bone defects. Our injectable heparin-conjugated sustained-release PDGF-BB hydrogel has excellent angiogenic and osteogenic properties, thus providing a novel and promising solution for bone defect repair.

Efficient drug supply in stem cell cytosol via pore-forming saponin nanoparticles promotes in vivo osteogenesis and bone regeneration

Directional differentiation of stem cells is a key step in stem cell therapy. In this study, we developed saponin-based nanoparticles (Ad-SNPs) containing dexamethasone (Dex) and alpha-lipoic acid (ALA) to promote osteogenic differentiation of human mesenchymal stem cells (hMSCs) and bone regeneration. The Ad-SNPs can achieve rapid cellular uptake through a pore-forming effect without cytotoxic cationic charges. They also provide extended retention in cell cytosol due to their uptake route. These properties are advantageous in efficiently supplying drugs to the hMSCs. The combination of Dex and ALA facilitated mitochondrial fusion and prevented reactive oxygen species–induced DNA damage. It also helped to preserve mitochondrial dynamics, and the efficient supply of it provided by the Ad-SNPs induced differentiation of hMSCs into osteoblasts. The Ad-SNPs showed outstanding performance in osteoblast differentiation, maturation, and mineralization in 3D culture compared with NPs without saponin and with free drugs. When Ad-SNP-treated hMSCs were tested in a rat femoral bone defect model, they showed the fastest regeneration of bones and complete repair in the shortest period among all groups. To the best of our knowledge, this study is the first application of pore-forming saponin-based NPs with rapid cellular uptake and extended retention to stem cell therapy, and we demonstrated their promising potential in bone regeneration and efficient delivery of Dex and ALA.

ATF2-driven osteogenic activity of enoxaparin sodium-loaded polymethylmethacrylate bone cement in femoral defect regeneration

BackgroundPolymethylmethacrylate (PMMA) bone cement loaded with enoxaparin sodium (PMMA@ES) has been increasingly highlighted to affect the bone repair of bone defects, but the molecular mechanisms remain unclear. We addressed this issue by identifying possible molecular mechanisms of PMMA@ES involved in femoral defect regeneration based on bioinformatics analysis and network pharmacology analysis.MethodsThe upregulated genes affecting the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were selected through bioinformatics analysis, followed by intersection with the genes of ES-induced differentiation of BMSCs identified by network pharmacology analysis. PMMA@ES was constructed. Rat primary BMSCs were isolated and cultured in vitro in the proliferation medium (PM) and osteogenic medium (OM) to measure alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and the expression of RUNX2 and OCN using gain- or loss-of-function experiments. A rat femoral bone defect model was constructed to detect the new bone formation in rats.ResultsATF2 may be a key gene in differentiating BMSCs into osteoblasts. In vitro cell assays showed that PMMA@ES promoted the osteogenic differentiation of BMSCs by increasing ALP activity, extracellular matrix mineralization, and RUNX2 and OCN expression in PM and OM. In addition, ATF2 activated the transcription of miR-335-5p to target ERK1/2 and downregulate the expression of ERK1/2. PMMA@ES induced femoral defect regeneration and the repair of femoral defects in rats by regulating the ATF2/miR-335-5p/ERK1/2 axis.ConclusionThe evidence provided by our study highlighted the ATF2-mediated mechanism of PMMA@ES in the facilitation of the osteogenic differentiation of BMSCs and femoral defect regeneration.

Extracellular vesicles secreted by human gingival mesenchymal stem cells promote bone regeneration in rat femoral bone defects.

Extracellular vesicles (EVs), important components of paracrine secretion, are involved in various pathological and physiological processes of the body. In this study, we researched the benefits of EVs secreted by human gingival mesenchymal stem cells (hGMSC-derived EVs) in promoting bone regeneration, thereby providing new ideas for EVs-based bone regeneration therapy. Here, we successfully demonstrated that hGMSC-derived EVs could enhance the osteogenic ability of rat bone marrow mesenchymal stem cells and the angiogenic capability of human umbilical vein endothelial cells. Then, femoral defect rat models were created and treated with phosphate-buffered saline, nanohydroxyapatite/collagen (nHAC), a grouping of nHAC/hGMSCs, and a grouping of nHAC/EVs. The results of our study indicated that the combination of hGMSC-derived EVs and nHAC materials could significantly promote new bone formation and neovascularization with a similar effect to that of the nHAC/hGMSCs group. Our outcomes provide new messages on the role of hGMSC-derived EVs in tissue engineering, which exhibit great potential in bone regeneration treatment.

Influence of porosity on osteogenesis, bone growth and osteointegration in trabecular tantalum scaffolds fabricated by additive manufacturing.

Porous tantalum implants are a class of materials commonly used in clinical practice to repair bone defects. However, the cumbersome and problematic preparation procedure have limited their widespread application. Additive manufacturing has revolutionized the design and process of orthopedic implants, but the pore architecture feature of porous tantalum scaffolds prepared from additive materials for optimal osseointegration are unclear, particularly the influence of porosity. We prepared trabecular bone-mimicking tantalum scaffolds with three different porosities (60%, 70% and 80%) using the laser powder bed fusing technique to examine and compare the effects of adhesion, proliferation and osteogenic differentiation capacity of rat mesenchymal stem cells on the scaffolds in vitro. The in vivo bone ingrowth and osseointegration effects of each scaffold were analyzed in a rat femoral bone defect model. Three porous tantalum scaffolds were successfully prepared and characterized. In vitro studies showed that scaffolds with 70% and 80% porosity had a better ability to osteogenic proliferation and differentiation than scaffolds with 60% porosity. In vivo studies further confirmed that tantalum scaffolds with the 70% and 80% porosity had a better ability for bone ingrowh than the scaffold with 60% porosity. As for osseointegration, more bone was bound to the material in the scaffold with 70% porosity, suggesting that the 3D printed trabecular tantalum scaffold with 70% porosity could be the optimal choice for subsequent implant design, which we will further confirm in a large animal preclinical model for better clinical use.

Surgical Classification for Preclinical Rat Femoral Bone Defect Model: Standardization Based on Systematic Review, Anatomical Analysis and Virtual Surgery.

Though surgical techniques profoundly influence in vivo experiments, significant heterogeneity exists in current surgeries for inducing rat femoral bone defects. Such variations reduce the reproducibility and comparability of preclinical studies, and are detrimental to clinical translation. The purposes of this study were: (1) to conduct a systematic review of rat femoral defect models, summarizing and analyzing the surgical techniques; (2) to analyze surgical design and potential pitfalls via 3D anatomy and virtual surgeries for fostering future precision research; and (3) to establish a surgical classification system, for improving the reproducibility and comparability among studies, avoiding unnecessary repetitive experiments. The online database PubMed was searched to identify studies from January 2000 to June 2022 using keywords, including rat, femur, bone defect. Eligible publications were included for a review of surgical methods. Anatomical analysis and virtual surgeries were conducted based on micro-CT reconstruction of the rat femur for further investigation and establishment of a classification system. A total of 545 publications were included, revealing marked heterogeneity in surgical methods. Four major surgical designs were reported for inducing defects from the proximal to distal femur: bone tunnel, cortical window, segmental defect, and wedge-shaped defect. Anatomical analysis revealed potential pitfalls hindering efficient clinical translation. A classification system was established according to the anatomical region, surgical design, and fixation devices. This systematic review in combination with 3D analysis and virtual surgery provides a general overview of current surgical approaches to inducing femoral defects in rats, and establishes a surgical classification facilitating preclinical research of quality and translational value.

Fabrication of a bio-instructive scaffold conferred with a favorable microenvironment allowing for superior implant osseointegration and accelerated in situ vascularized bone regeneration via type H vessel formation

The potential translation of bio-inert polymer scaffolds as bone substitutes is limited by the lack of neovascularization upon implantation and subsequently diminished ingrowth of host bone, most likely resulted from the inability to replicate appropriate endogenous crosstalk between cells. Human umbilical vein endothelial cell-derived decellularized extracellular matrix (HdECM), which contains a collection of angiocrine biomolecules, has recently been demonstrated to mediate endothelial cells(ECs) – osteoprogenitors(OPs) crosstalk. We employed the HdECM to create a PCL (polycaprolactone)/fibrin/HdECM (PFE) hybrid scaffold. We hypothesized PFE scaffold could reconstitute a bio-instructive microenvironment that reintroduces the crosstalk, resulting in vascularized bone regeneration. Following implantation in a rat femoral bone defect, the PFE scaffold demonstrated early vascular infiltration and enhanced bone regeneration by microangiography (μ-AG) and micro-computational tomography (μ-CT). Based on the immunofluorescence studies, PFE mediated the endogenous angiogenesis and osteogenesis with a substantial number of type H vessels and osteoprogenitors. In addition, superior osseointegration was observed by a direct host bone-PCL interface, which was likely attributed to the formation of type H vessels. The bio-instructive microenvironment created by our innovative PFE scaffold made possible superior osseointegration and type H vessel-related bone regeneration. It could become an alternative solution of improving the osseointegration of bone substitutes with the help of induced type H vessels, which could compensate for the inherent biological inertness of synthetic polymers.

Bilayer Membrane Composed of Mineralized Collagen and Chitosan Cast Film Coated With Berberine-Loaded PCL/PVP Electrospun Nanofiber Promotes Bone Regeneration.

Bone defects are difficult to repair and reconstruct as bone regeneration remains technically challenging, with exogenous factors required to accelerate this process. Biodegradable synthetic scaffolds are promising materials for stimulating bone tissue repair. In this study, we investigated whether a bilayer membrane that includes mineralized collagen (MC) and chitosan (CS) delivering berberine (BER)—a typical Chinese herbal monomer—could promote bone healing in a rat model. An MC/CS cast film was coated with polycaprolactone (PCL)/polyvinylpyrrolidone (PVP) electrospun nanofibers loaded with BER, yielding the BER@PCL/PVP-MC/CS bilayer membrane. The 3-dimensional structure had nanofibers of uniform diameter and showed good hydrophilicity; the bilayer membrane showed favorable mechanical properties. BER@PCL/PVP-MC/CS enhanced the proliferation and attachment of MC3T3-E1 cells in vitro and induced bone regeneration when implanted into a rat femoral bone defect. These findings provide evidence that BER@PCL/PVP-MC/CS has clinical potential for effective bone repair.

Efficacy of electrical polarization on a rat femoral bone defect model with a custom-made external fixator.

We have developed a technology to electrically polarize living bone. The effects of stored electrical charge in electrical polarized bone on the facilitation of new bone formation were assayed. Stimulated depolarized current measurement was performed in electrically polarized and nonpolarized femora of SD rats. These bone specimens were implanted into bone defects of the rat femora and fixed with a custom-made external fixator. X-ray imaging of the implant was performed every week. After 3 weeks, micro-CT scanning was performed to evaluate the displacement rate. Histological observation was performed, and the occupancy ratio of the newly formed bone was calculated from tissue specimens stained with Villanueva's Goldner method. There was a tendency for the displacement rate of the implant to be smaller and the occupancy ratio of the newly formed bone to be larger, especially at the distal end, in the polarized group compared with the nonpolarized group. The time of callus appearance was significantly earlier in the polarized group than in the nonpolarized group, and bridging callus grew from the distal to the proximal end. Bone specimens can be electrically polarized, and the stored electrical charge can work effectively to facilitate new bone formation.

Effects of Bone Morphogenetic Protein-2 on Neovascularization During Large Bone Defect Regeneration.

Insufficient blood vessel supply is a primary limiting factor for regenerative approaches to large bone defect repair. Recombinant bone morphogenetic protein-2 (BMP-2) delivery induces robust bone formation and has been observed to enhance neovascularization, but whether the angiogenic effects of BMP-2 are due to direct endothelial cell stimulation or due to indirect paracrine signaling remain unclear. In this study, we evaluated the effects of BMP-2 delivery on vascularized bone regeneration and tested whether BMP-2 induces neovascularization directly or indirectly. We found that delivery of BMP-2 (5 μg) enhanced both bone formation and neovascularization in critically sized (8 mm) rat femoral bone defects; however, BMP-2 did not directly stimulate angiogenesis in vitro. In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced endothelial cell migration in vitro, suggesting a paracrine mechanism of BMP-2 action. Consistent with this inference, codelivery of BMP-2 with endothelial colony forming cells to a heterotopic site, distant from the skeletal stem cell-rich bone marrow niche, induced ossification but had no effect on neovascularization. Taken together, these data suggest that paracrine activation of osteoprogenitor cells is an important contributor to neovascularization during BMP-2-mediated bone regeneration. Impact Statement In this study, we show that bone morphogenetic protein-2 (BMP-2) robustly induces neovascularization during tissue-engineered large bone defect regeneration, and we found that BMP-2 induced angiogenesis, in part, through paracrine signaling from osteoprogenitor cells.

Multimodal porogen platforms for calcium phosphate cement degradation.

Calcium phosphate cements (CPCs) represent excellent bone substitute materials due to their biocompatibility and injectability. However, their poor degradability and lack of macroporosity limits bone regeneration. The addition of poly(d,l‐lactic‐co‐glycolic acid) (PLGA) particles improves macroporosity and therefore late stage material degradation. CPC degradation and hence, bone formation at an early stage remains challenging, due to the delayed onset of PLGA degradation (i.e., after 2–3 weeks). Consequently, we here explored multimodal porogen platforms based on sucrose porogens (for early pore formation) and PLGA porogens (for late pore formation) to enhance CPC degradation and analyzed mechanical properties, dynamic in vitro degradation and in vivo performance in a rat femoral bone defect model. Porogen addition to CPC showed to decrease compressive strength of all CPC formulations; transition of the crystal phase upon in vitro incubation increased compressive strength. Although dynamic in vitro degradation showed rapid sucrose dissolution within 1 week, no additional effects on CPC degradation or bone formation were observed upon in vivo implantation. © 2019 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1713–1722, 2019.

The comparison of biocompatibility and osteoinductivity between multi-walled and single-walled carbon nanotube/PHBV composites.

The applications of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in tissue engineering have been widely studied. This study aimed to compare the biocompatibility and osteoinductivity of single-walled carbon nanotubes (SWCNTs)/PHBV composites with multi-walled CNTs (MWCNTs)/PHBV composites. CNTs were dispersed in PHBV by ultrasonication and composites were created using thermal injection moulding. In order to test their biocompatibility and osteoinductivity. Rat osteoblasts (rOBs) were then cultured and seeded on the composites. The composites were implanted in rat femoral bone defects. Our results showed that lower weight percentages of SWCNTs and MWCNTs (2-4%) improved both their mechanical and thermal decomposition properties. However, further reduction of rOBs cell death was observed in MWCNTs/PHBV. SWCNTs were shown to upregulate the expression of Runx-2 and Bmp-2 in early stage significantly, while MWCNTs showed a stronger long-term effect on Opn and Ocn. The in vivo result was that MWCNTs/PHBV composites induced intact rounding new bone, increased integration with new bone, and earlier completed bone remodeling when compared with SWCNTs. Immunohistochemistry also detected higher expression of RUNX-2 around MWCNTs/PHBV composites. In conclusion, there were no differences observed between SWCNTs and MWCNTs in the reinforcement of PHBV, while MWCNTs/PHBV composites showed better biocompatibility and osteoinductivity both in vitro and in vivo.

Efficacy of intraoperatively prepared cell-based constructs for bone regeneration

BackgroundConventional cell-based bone regeneration suffers from the major disadvantage of limited cell supply, time-consuming in vitro expansion cultures, and limited patient-friendliness related to cell isolation and multiple visits to the clinic. Here, we utilized an alternative concept using “easy access cells” that can be obtained in an intraoperative manner to prepare cell-based constructs.MethodsWe used stromal vascular fraction (SVF) from human adipose tissue and human monocytes for intraoperative preparation of bone constructs. Conventional constructs grafted with expanded human adipose tissue mesenchymal stem cells (ADMSCs) derived from the same donor were set as positive controls. Additionally, we combined both cell types either or not with monocytes. The cellular interaction of human SVF and ADMSCs with human monocytes was evaluated in vitro. The feasibility and bone-regenerative capacity of intraoperative constructs were determined histologically and histomorphometrically in a rat femoral condyle bone defect model.ResultsSVF displayed equal in vitro osteogenic differentiation compared to donor-matched expanded ADMSCs, which for both was significantly enhanced upon co-culture with monocytes. Moreover, SVF and ADMSCs displayed different immunoregulatory effects on monocytes/macrophages. Upon implantation in rat femoral bone defects, SVF constructs demonstrated superior bone formation compared to ADMSC constructs and cell-free controls; no effects of monocyte addition were observed.ConclusionIn conclusion, we here demonstrate the feasibility of intraoperative SVF construct preparation and superior bone-regenerative capacity thereof compared to donor-matched ADMSC constructs. The superiority of SVF constructs was found to be linked to the distinct differences between immunoregulatory effects of SVF and ADMSCs.

Loading BMP-2 on nanostructured hydroxyapatite microspheres for rapid bone regeneration.

IntroductionTissue engineering is a promising strategy for bone regeneration in repairing massive bone defects. The surface morphology of implanted materials plays a key role in bone healing; these materials incorporate osteoinductive factors to improve the efficiency of bone regeneration.Materials and methodsIn the current study, nanostructured hydroxyapatite (nHAp) micro-spheres were prepared via a hydrothermal transformation method using calcium silicate (CS) microspheres as precursors; the CS microspheres were obtained by a spray-drying method. The nHAp microspheres constructed by the nano-whiskers significantly improved the ability of the microspheres to adsorb the bioactive protein (BMP-2) and reduce its initial burst release. To evaluate the in vivo bone regeneration of microspheres, both conventional hydroxyapatite (HAp) and nHAp microspheres were either loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) or not loaded with the protein; these microspheres were implanted in rat femoral bone defects for 4 and 8 weeks.Results and discussionThe results of our three-dimensional (3D) micro-computed tomography (CT) and histomorphometric observations showed that the combination of the nano-structured surface and rhBMP-2 obviously improved osteogenesis compared to conventional HAp microspheres loaded with rhBMP-2. Our results suggest that the nHAp microspheres with a nanostructured surface adsorb rhBMP-2 for rapid bone formation; they therefore show the potential to act as carriers in bone tissue regeneration.

Bioresorbable Scaffolds Based on Fibroin for Bone Tissue Regeneration

Using the tissue-engineered constructs based on scaffolds that imitate the extracellular matrix of living tissues unveils new opportunities in the treatment of various pathologies and injuries associated with tissue and organ damage. Silk fibroin of silkworm Bombyx mori is a biocompatible and bioresorbable polymer with high mechanical strength and elasticity that allows creating scaffolds on its basis for regeneration of various tissues, including bone. In the present work, fibroin scaffolds were obtained. They were designed in the form of porous sponges, films, and hybrid scaffolds of a bilayer structure in which the porous sponge threedimensional structure is limited on one side by a film. The structure of the scaffolds and their biocompatibility were studied: immortalized and primary fibroblasts, as well as the osteoblast-like cells, have been shown to successfully adhere and proliferate on the surface of the studied scaffolds. Numerous osteogenesis foci have been observed in the implant region 4 weeks after the fibroin porous scaffold implantation in the in vivo experiments in a rat femoral bone defect model indicating the osteoconduction of the scaffolds.

Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration

Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.

PLGA-linked alendronate enhances bone repair in diaphysis defect model.

Alendronate (ALN) is known as an anti-resorptive drug for the treatment of osteoporosis. Recently, ALN was found to stimulate osteogenic differentiation in mesenchymal stem cells and enhance new bone formation in calvarial bone defects. Previous in vitro and in vivo studies found that the effective concentration of ALN was approximately 1-10 μm. In the present study, a poly (lactic-co-glycolic acid) (PLGA) cross-linked ALN (PLGA-ALN) with a short-term controlled-release property for local application to enhance bone repair was developed. An in vitro drug-release kinetic test showed that PLGA-ALN microspheres released an effective concentration (50-100nm) of ALN for 9days. The effect of PLGA-ALN on bone repair was tested in a rat femoral bone defect model. The biomechanical study results showed that the maximal strength, stiffness and energy absorption were significantly increased in the PLGA-ALN group compared with the PLGA group. The microstructure of the newly formed bone at the defect site was analysed using microcomputed tomography. The PLGA-ALN group significantly improved the trabecular bone volume at the defect site compared with the PLGA group. The fibril collagen and immunolocalized bone morphogenetic protein 2 were evident in the newly formed trabecular bone in the PLGA-ALN group. Local use of newly developed PLGA-ALN-enhanced bone repair was attributable to increasing bone matrix formation, which improved the ultrastructure of the newly formed bone and thus increased the biomechanical properties of the repaired bone. It is suggested that PLGA-ALN may be a potential bone graft substitute to enhance bone repair. Copyright © 2016 John Wiley & Sons, Ltd.

Bone regeneration using injectable BMP-7 loaded chitosan microparticles in rat femoral defect

Injectable chitosan microparticles were prepared using a simple coacervation method under physiologically friendly conditions by eliminating oil or toxic chemical, and employing low temperature and pressure for growth factor stability. Amount of 200ng of bone morphogenetic protein-7 (BMP-7) was incorporated in the chitosan microparticles by two methods: encapsulating and coating techniques. These microparticles were tested in vivo to determine the biological response in a rat femoral bone defect at 6 and 12weeks. Four groups (n=10) were tested which include two groups for BMP-7 incorporated microparticles (by two techniques), microparticles without BMP-7, and defect itself (negative control). Healthy bone formation was observed around the microparticles, which were only confined to the defect site and did not disperse. Histology indicated minor inflammatory response around the microparticles at 6weeks, which reduced by 12weeks. Micro-CT analysis of bone surface density and porosity was found to be significantly more (p<0.05) for microparticles containing groups, in comparison with controls, which suggests that the new bone formed in the presence of microparticles is more interconnected and porous. Collagen fibrils analysis conducted using multiphoton microscopy showed significant improvement in the formation of bundled collagen area (%) in microparticles containing groups in comparison with controls, indicating higher cross-linking between the fibrils. Microparticles were biocompatible and did not degrade in the 12week implant period.