Rabbit Femoral Defect Research Articles

Osseointegration of titanium scaffolds manufactured by selective laser melting in rabbit femur defect model.

The aim of this study was to assess the osseointegration of two series of titanium (Ti) scaffolds with 0.8 and 1 mm cell size obtained by Selective Laser Melting (SLM) technique. One of the series had the Ti surface unmodified, while the other had the Ti surface coated with silicon-substituted nano-hydroxyapatite (nano-HapSi). The scaffolds were implanted in the femur bone defects of 6 White Californian male rabbits: three animals were implanted with 0.8 mm cell size scaffolds and three animals with 1 mm cell size scaffolds, respectively. The bone fragments and scaffolds harvested at 2, 4 and 6 months were histologically analyzed using conventional light microscopy (LM) and scanning electron microscopy (SEM) for the qualitative evaluation of the bone tissue formed in contact with the scaffold. Both LM and SEM images indicated a better osseointegration for nano-HapSi coated Ti scaffolds. LM revealed that the compact bone formed in the proximity of nano-HapSi-coated scaffolds was better organized than spongy bone associated with unmodified scaffolds. Moreover, Ti scaffolds with meshes of 0.8 mm showed higher osseointegration compared with 1 mm. SEM images at 6 months revealed that the bone developed not only in contact with the scaffolds, but also proliferated inside the meshes. Nano-HapSi-coated Ti implants with 0.8 mm meshes were completely covered and filled with new bone. Ti scaffolds osseointegration depended on the mesh size and the surface properties. Due to the biocompatibility and favorable osseointegration in bone defects, nano-HapSi-coated Ti scaffolds could be useful for anatomical reconstructions.

Fabrication of injectable bone substitute loading porous simvastatin-loaded poly(lactic-co-glycolic acid) microspheres

An injectable bone substitute (IBS) with well-controlled release mechanism of simvastatin (SIM) was fabricated from SIM loaded-porous poly(lactic-co-glycolic acid) microspheres (PLGA MSs), biphasic calcium phosphates (BCP) and CaCO3 nano powder as the solid phase and gelatin, citric acid and hydroxypropyl methylcellulose as the liquid phase. The ratios of PLGA MSs and BCP were varied to obtain IBS with optimal mechanical properties, biocompatibility and bioactivity. In vitro test with MG-63 pre-osteoblast cells showed that IBS with higher amount of PLGA MSs yield better cell adhesion and proliferation. In vivo study on rabbit femoral defect also suggested that the IBS promoted osteogenesis.

Artificial Membrane Induced by Novel Biodegradable Nanofibers in the Masquelet Procedure for Treatment of Segmental Bone Defects

The Masquelet induced-membrane technique for the treatment of segmental bone defects includes a two-stage surgical procedure, and polymethylmethacrylate (PMMA) plays a major role in the treatment. However, the PMMA spacer must be surgically removed. Here, we investigated the potential of poly(lactic-co-glycolic acid) (PLGA) nanofibers, a biodegradable material to replace the PMMA spacer, allowing the bioactive membrane to be induced and the spacer to degrade without the additional surgery on a rabbit femoral segmental bone defect model. PLGA nanofibers were shown to degrade completely six weeks after implantation in the investigated animals, and a thick membrane was found to circumferentially fold around the segmental bone defects. Results from image studies demonstrated that, in the group without the bone graft, all studied femurs exhibited either nonunion or considerable malunion. In contrast, the femurs in the bone graft group had a high union rate without considerable deformities. Histological examinations suggested that the membranous tissue in this group was rich in small blood vessels and the expression of BMP2 and VEGF increased. Our results demonstrate that the biodegradable PLGA nanofibers may be useful for replacing the PMMA spacer as the bioactive-membrane inducer, facilitating the process of healing and removing the need for repeated surgeries.

Expression of osteoprotegerin and receptor activator for the nuclear factor-κB ligand in XACB/LV-bFGF/MSCs transplantation for repair of rabbit femoral head defect necrosis.

The aim of this study is to observe the changes in osteoprotegerin (OPG) and receptor activator for the nuclear factor-κB ligand (RANKL) in a rabbit model, and to explore the therapeutic effect of tissue engineering bone on femoral head necrosis. A total of 60 rabbits were randomly divided into 5 groups. The necrosis model of femoral head defects was created by dexamethasone combined with a horse serum injection. The model of femoral head necrosis was reconstructed by tissue engineering bone. The protein expressions of OPG and RANKL were detected by Western blot analysis. The expression of OPG and the RANKL protein in group E was higher than that in the other 4 groups (P < .05); there was no significant difference between groups C and D ( P > .05). The expression of OPG protein in the rabbit femoral head necrosis group was improved by xenogeneic antigens of cancellous bone/lentiviral-basic fibroblast growth factor/mesenchymal stem cells, which were expected to be used as an effective tissue engineering material to repair the necrosis of the femoral head.

Incorporation of chitosan-alginate complex into injectable calcium phosphate cement system as a bone graft material

Calcium phosphate brushite type of cements have been used to replace bone graft materials because of their biocompatibility and other attractive features. Especially, injectability of cement allows easy handling of minimally invasive surgical techniques. New calcium phosphate cement (CPC) system, brushite based cement incorporated into polyelectrolyte complex, was developed in this study. Chitosan-alginate complex produced by an interaction between a cationic polymer (chitosan) and an anionic polymer (alginate) was loaded in the cement. This improved the functional properties and biocompatibility of the final cement. We optimized the liquid/solid (L/S) ratio of the cement components and investigated the compressive strength, setting time, pH change of CPC0 (with only citric acid) and CPC0.5, 1, and 1.5 (0.5, 1, and 1.5 v/v % chitosan-alginate complex in citric acid solution, respectively). The L/S ratio did not affect structural formation, while the addition of polymer complex showed new formation of macro-pores within CPC. However, a lower L/S ratio and higher amount of added polymer complex shortened the setting time and improved the compressive strength. The appropriate conditions for the injectable bone substitute were CPC1 with an L/S ratio of 0.45. To investigate the effect of the chitosan-alginate complex on CPC system in physiological conditions, CPC0 and CPC1 were implanted in a rabbit femoral head defect model for 1 and 3 months. Micro-computed tomography revealed improved bone formation in CPC1 compared to CPC0 3 months after implantation. Histological analysis revealed newly formed bone tissues around the peripheral sides of CPC0 and CPC1. The results indicate the potential value of the CPC system containing polymer complex as an injectable bone substitute. The study of the CPC-polymer complex system incorporating drugs or cells can be further developed into a controlled release system for faster bone regeneration.

Fabrication and evaluation of carbonate apatite-coated calcium carbonate bone substitutes for bone tissue engineering.

Carbonate apatite-coated calcium carbonate (CO3 Ap/CaCO3 ) was fabricated through a dissolution-precipitation reaction using CaCO3 granules as a precursor to accelerate bone replacement based on superior osteoconductivity of the CO3 Ap shell, along with Ca2+ release from the CaCO3 core and quicker resorption of the CaCO3 core. In the present study, CaCO3 , 10% CO3 Ap/CaCO3 , 30% CO3 Ap/CaCO3 , and CO3 Ap granules were fabricated and examined histologically to evaluate their potential as bone substitutes. Larger contents of CaCO3 in the granules resulted in higher Ca2+ release and promoted cell proliferation of murine preosteoblasts at 6days compared with CO3 Ap. Interestingly, in a rabbit femur defect model, 10% CO3 Ap/CaCO3 induced significantly higher new bone formation and higher material resorption compared with CO3 Ap at 8weeks. Nevertheless, CO3 Ap showed a superior osteoconductive potential compared with 10% CO3 Ap/CaCO3 at 8weeks. All tested granules were most likely resorbed by cell mediation including multinucleated giant cell functions. Therefore, we conclude that CO3 Ap/CaCO3 has a positive potential for bone tissue engineering based on well-controlled calcium release, bone formation, and material resorption.

Demineralized Bone Scaffolds with Tunable Matrix Stiffness for Efficient Bone Integration.

As a biophysical cue, matrix stiffness can decide the stem cell fate. However, most methods to construct three-dimensional (3D) scaffolds may change the 3D microstructure while altering their mechanical properties. In this study, demineralized bone matrix scaffolds with different compressive modulus (66.06 ± 27.83 MPa (high), 26.90 ± 13.16 MPa (medium), and 0.67 ± 0.14 MPa (low)) were constructed by controlling the decalcification duration (1 h, 12 h, and 5 days), respectively. The pore size and porosity have no significant difference between the scaffolds before and after decalcification. Cell experiments indicated that the low scaffolds could promote the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro. Rat subcutaneous implantation experiments further demonstrated that the low scaffolds could efficiently improve the cell infiltration, deposition of collagen fibers, and positive osteocalcin and osteopontin expression of endogenous cells as well as angiogenesis. Finally, rabbit femoral condylar defect experiments proved that the low scaffolds could significantly promote the bone repair and integration and stromal cell derived factor-1α/CXC chemokine receptor signal pathway was essential for the stiffness-mediated bone repair. These investigations provided a novel method for fabricating 3D bone grafts with different stiffness, which is also of great significance for studying the effect of stiffness on the biological behavior of MSCs in three dimensions.

Pilot implantation study of a borate-glass filled hydrophilic bone cement

Hydrophilic composite-resin cements reinforced with highly degradable borate-glass may provide sustained strontium release to improve local bone quality. An 8 week pilot implantation study in a rabbit femoral defect model was conducted to evaluate the local response to new bone cement materials. The materials demonstrated favorable tissue responses with evidence of direct new bone formation. Counter to the literature, there were no beneficial effects associated with strontium releasing biomaterials. Nevertheless, highly hydrophilic glass filled cements may present benefit through rapid surface mineralization, and in vivo swelling, providing a press fit coupling to bone.

Segmental Additive Tissue Engineering

Segmental bone defects caused by trauma and disease represent a major clinical problem worldwide. Current treatment options are limited and often associated with poor outcomes and severe complications. Bone engineering is a promising alternative solution, but a number of technical challenges must be addressed to allow for effective and reproducible construction of segmental grafts that meet the size and geometrical requirements needed for individual patients and routine clinical applications. It is important to devise engineering strategies and standard operating procedures that make it possible to scale up the size of bone-engineered grafts, minimize process and product variability, and facilitate technology transfer and implementation. To address these issues, we have combined traditional and modular tissue engineering approaches in a strategy referred to as Segmental Additive Tissue Engineering (SATE). To demonstrate this approach, a digital reconstruction of a rabbit femoral defect was partitioned transversally to the longitudinal axis into segments (modules) with discoidal geometry and defined thickness to enable protocol standardization and effective tissue formation in vitro. Bone grafts corresponding to each segment were then engineered using biomimetic scaffolds seeded with human induced pluripotent stem cell-derived mesodermal progenitors (iPSC-MPs) and a novel perfusion bioreactor with universal design. The SATE strategy enables the effective and reproducible engineering of segmental bone grafts for personalized skeletal reconstruction, and will facilitate technology transfer and implementation of a tissue engineering approach to segmental bone defect therapy.

Role of TGF‑β1 expressed in bone marrow‑derived mesenchymal stem cells in promoting bone formation in a rabbit femoral defect model.

Bone defects represent a major clinical and socioeconomic problem without suitable treatment options. Previous studies have shown that transforming growth factor β1 (TGF-β1) is important in the development of various diseases. The present study aimed to investigate the therapeutic potential of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) expressing TGF-β1 in the treatment of rabbit femoral defects. First, rabbit BMSCs were identified and cultured. TGF-β1 was then stably overexpressed in the rabbit BMSCs by lentivirus transfection, which was expressed at a high level in the femoral defects treated with TGF-β1-overexpressing BMSCs, compared with PBS-treated controls. In addition, the TGF-β1-overexpressing BMSCs promoted new bone formation in the rabbit femoral defect model, and increased the expression of bone-related markers at week 2 and week 6. Therefore, the study demonstrated that BMSCs overexpressing TGF-β1 may provide a novel therapeutic option for femoral defects.

In vitro and in vivo evaluation of polylactic acid-based composite with tricalcium phosphate microsphere for enhanced biodegradability and osseointegration.

A biodegradable polylactic acid composite containing tricalcium phosphate microsphere was fabricated. The composite exhibited enhanced biocompatibility and a well-interconnected porous structure that enabled tissue ingrowth after degradation. The tricalcium phosphate microspheres had an average size of 106 ± 43μm and were incorporated into the polylactic acid matrix using a high-shear mixer. The resulting bioactivity and hydrophilicity were enhanced to levels comparable to those of a polylactic acid composite containing tricalcium phosphate powder, which is a well-known material used in the medical field. An accelerated 30-day degradation test in HCl revealed successful generation of an open porous structure with ∼98% interconnectivity in the polylactic acid-tricalcium phosphate microsphere composite, demonstrating the potential of this material to induce enhanced osseointegration in the later stage of bone regeneration. The early stage osseointegration was also evaluated by implanting the composite in vivo using a rabbit femoral defect model. After 16 weeks of implantation, the bone-to-implant contact ratio of the polylactic acid-tricalcium phosphate microsphere composite was enhanced owing to tissue ingrowth through the generated pores near the surface.

Self-fitting shape memory polymer foam inducing bone regeneration: A rabbit femoral defect study

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.

Repair of Large Segmental Bone Defect using Vascularized Small Corticocancellous Bone in Rabbit Femur.

Background Although free-vascularized long-bone transfer is useful for reconstruction of a large segmental bone defect, it is limited by availability of transplantable bone, meticulous microsurgical technique, and donor-site morbidities. Hence, easier, readily available, and safer surgical procedures are warranted. This study evaluated the effects of vascularized small corticocancellous bone grafting for reconstruction of a large segmental rabbit femur defect. Methods A 1.5 cm defect was created in the femurs of 40 New Zealand white rabbits and divided into a control group (n = 10, bone defect without graft), group A (n = 10, bone defect filled with morselized autogenous bone), group B (n = 10, bone defect grafted with a vascularized small corticocancellous bone and morselized autogenous bone), and group C (n = 10, bone defect grafted with a vascularized small corticocancellous bone). Simple radiographs were taken postoperatively, and bone healing ability was scored using Taira's radiologic scale. Histologic examinations were scored using Emery's histologic scale. The expression of osteogenesis-related growth factors (BMP-2, -4, and -7, VEGF, and RANKL) was analyzed. Results Radiologically, group B showed superior biological efficacy in bone formation and consolidation over the other groups. Histologically, the defect in group B was filled with more abundant mature bone than the other groups. Group B showed higher gene expression of BMP-2, -4, and -7, and VEGF. Conclusions The grafting of the morselized autogenous bone (MSB) combined with the vascularized small corticocancellous bone is more effective than that of the MSB alone for repairing a large segmental bone defect.

Preparation and invivo evaluation of a silicate-based composite bone cement.

Silicate-based cements have been developed as a class of bioactive and biodegradable bone cements owing to their good invitro bioactivity and ability to dissolve in a simulated body fluid. Until recently, however, the invivo evidence of their ability to support bone regeneration is still scarce. In the present study, a pilot invivo evaluation of a silicate-based composite bone cement (CSC) was carried out in a rabbit femur defect model. The cement was composed of tricalcium silicate, 45S5 bioglass and calcium sulfate, and the self-setting properties of the material were established. The invivo bone integration and biodegradability of CSC were investigated and compared with those of bioactive glass particulates, and a calcium phosphate cement. The results showed that CSC underwent a relatively slower invivo degradation as compared with bioactive glass and calcium phosphate cement. Histological observation demonstrated that bone contact area at the interface between the surrounding bone and CSC gradually increased with time proceeding. CSC kept its structural integrity during implantation invivo because of its acceptable mechanical strength. These results provide evidence of effectiveness invivo and suggest potential clinical applications of the silicate-based composite bone cements.

Systematic evaluation of the osteogenic capacity of low-melting bioactive glass-reinforced 45S5 Bioglass porous scaffolds in rabbit femoral defects

Due to the low strength and high brittleness of 45S5 Bioglass®, it is still a great challenge for the three-dimensional porous 45S5 Bioglass® to treat mechanically required loaded bone defects. Therefore, 45S5 Bioglass®-derived bioactive glass-ceramic (BGC) porous scaffolds were fabricated at a low temperature sintering condition with and without the addition of 4% low-melting ZnO/B2O3 (ZB) bioactive glass as a reinforcing agent and using 350- or 500 μm paraffin microspheres as a porogen. The pore structure characterization for the scaffolds indicated that the scaffolds containing 4% ZB had very good macroporous structures of ∼313 and ∼448 μm in pore size and over 70% porosity with appreciable strength (>15 MPa), which was about four times higher than that those manufactured without ZB and with 350 μm porogen scaffolds. The open porosity was decreased with the addition of 4% ZB but the interconnected pore percentage (>50 μm) was increased with increasing the porogen size from 350 to 500 μm. In vivo investigations revealed that the stronger scaffolds containing 4% ZB and 500 μm porogen were particularly beneficial for osteogenic capacity in critical size femoral bone defects, accompanied with an accelerated bone ingrowth (6–18 weeks) and the material itself experiencing mild resorption. In contrast, both the scaffolds with smaller pore sizes exhibited a low level of new bone ingrowth (<32%) after 6–12 weeks implantation. These results suggest a promising application of such 45S5 Bioglass®-derived BGC scaffolds in a clinical setting, especially for mechanically loaded bone defects.

Evaluation of carbonate apatite blocks fabricated from dicalcium phosphate dihydrate blocks for reconstruction of rabbit femoral and tibial defects.

This study aimed to evaluate in vivo behavior of a carbonate apatite (CO3Ap) block fabricated by compositional transformation via a dissolution-precipitation reaction using a calcium hydrogen phosphate dihydrate [DCPD: CaHPO4·2H2O] block as a precursor. These blocks were used to reconstruct defects in the femur and tibia of rabbits, using sintered dense hydroxyapatite (HAp) blocks as the control. Both the CO3Ap and HAp blocks showed excellent tissue response and good osteoconductivity. HAp block maintained its structure even after 24 weeks of implantation, so no bone replacement of the implant was observed throughout the post-implantation period in either femoral or tibial bone defects. In contrast, CO3Ap was resorbed with increasing time after implantation and replaced with new bone. The CO3Ap block was resorbed approximately twice as fast at the metaphysis of the proximal tibia than at the epiphysis of the distal femur. The CO3Ap block was resorbed at an approximately linear change over time, with complete resorption was estimated by extrapolation of data at approximately 1-1.5 years. Hence, the CO3Ap block fabricated in this study has potential value as an ideal artificial bone substitute because of its resorption and subsequent replacement by bone.

Lightweight Open-Cell Scaffolds from Sea Urchin Spines with Superior Material Properties for Bone Defect Repair.

Sea urchin spines (Heterocentrotus mammillatus), with a hierarchical open-cell structure similar to that of human trabecular bone and superior mechanical property (compressive strength ∼43.4 MPa) suitable for machining to shape, were explored for potential applications of bone defect repair. Finite element analyses reveal that the compressive stress concentrates along the dense growth rings and dissipates through strut structures of the stereoms, indicating that the exquisite mesostructures play an important role in high strength-to-weight ratios. The fracture strength of magnesium-substituted tricalcium phosphate (β-TCMP) scaffolds produced by hydrothermal conversion of urchin spines is about 9.3 MPa, comparable to that of human trabecular bone. New bone forms along outer surfaces of β-TCMP scaffolds after implantation in rabbit femoral defects for one month and grows into the majority of the inner open-cell spaces postoperation in three months, showing tight interface between the scaffold and regenerative bone tissue. Fusion of beagle lumbar facet joints using a Ti-6Al-4V cage and β-TCMP scaffold can be completed within seven months with obvious biodegradation of the β-TCMP scaffold, which is nearly completely degraded and replaced by newly formed bone ten months after implantation. Thus, sea urchin spines suitable for machining to shape have advantages for production of biodegradable artificial grafts for bone defect repair.

RhBMP-2 loaded 3D-printed mesoporous silica/calcium phosphate cement porous scaffolds with enhanced vascularization and osteogenesis properties

A major limitation in the development of effective scaffolds for bone regeneration has been the limited vascularization of the regenerating tissue. Here, we propose the development of a novel calcium phosphate cement (CPC)-based scaffold combining the properties of mesoporous silica (MS) with recombinant human bone morphogenic protein-2 (rhBMP-2) to facilitate vascularization and osteogenesis. Specifically, the development of a custom MS/CPC paste allowed the three-dimensional (3D) printing of scaffolds with a defined macroporous structure and optimized silicon (Si) ions release profile to promote the ingrowth of vascular tissue at an early stage after implantation in support of tissue viability and osteogenesis. In addition, the scaffold microstructure allowed the prolonged release of rhBMP-2, which in turn significantly stimulated the osteogenesis of human bone marrow stromal cells in vitro and of bone regeneration in vivo as shown in a rabbit femur defect repair model. Thus, the combination MS/CPC/rhBMP-2 scaffolds might provide a solution to issues of tissue necrosis during the regeneration process and therefore might be able to be readily developed into a useful tool for bone repair in the clinic.

Novel Bio-functional Magnesium Coating on Porous Ti6Al4V Orthopaedic Implants: In vitro and In vivo Study

Titanium and its alloys with various porous structures are one of the most important metals used in orthopaedic implants due to favourable properties as replacement for hard tissues. However, surface modification is critical to improve the osteointegration of titanium and its alloys. In this study, a bioactive magnesium coating was successfully fabricated on porous Ti6Al4V by means of arc ion plating, which was proved with fine grain size and high film/substrate adhesion. The surface composition and morphology were characterized by X-ray diffraction and SEM equipped with energy dispersive spectroscopy. Furthermore, the in vitro study of cytotoxicity and proliferation of MC3T3-E1 cells showed that magnesium coated porous Ti6Al4V had suitable degradation and biocompatibility. Moreover, the in vivo studies including fluorescent labelling, micro-computed tomography analysis scan and Van-Gieson staining of histological sections indicated that magnesium coated porous Ti6Al4V could significantly promote bone regeneration in rabbit femoral condylar defects after implantation for 4 and 8 weeks, and has better osteogenesis and osteointegration than the bare porous Ti6Al4V. Therefore, it is expected that this bioactive magnesium coating on porous Ti6Al4V scaffolds with improved osteointegration and osteogenesis functions can be used for orthopedic applications.

3D printed poly(ε-caprolactone) scaffolds modified with hydroxyapatite and poly(propylene fumarate) and their effects on the healing of rabbit femur defects.

A large variety of approaches have been used to treat large and irregular shaped bone defects with less than optimal success due to material or design issues. In recent years patient specific constructs prepared by additive manufacturing provided a solution to the need for shaping implants to fit irregular defects in the surgery theater. In this study, cylindrical disks of poly(ε-caprolactone) (PCL) were printed by fused deposition modeling and modified with nanohydroxyapatite (HAp) and poly(propylene fumarate) (PPF) to create a mechanically strong implant with well-defined pore size and porosity, controllable surface hydrophilicity (with PPF) and osteoconductivity (with HAp). Cytotoxicity, irritation and inflammation tests demonstrated that the scaffolds were biocompatible. PCL/HAp and PCL/HAp/PPF scaffolds were implanted in the femurs of rabbits with and without seeding with rabbit Bone Marrow Stem Cells (BMSC) and examined after 4 and 8 weeks with micro-CT, mechanically and histologically. BMSC seeded PCL/HAp/PPF scaffolds showed improved tissue regeneration as determined by bone mineral density and micro-CT. Compressive and tension stiffness values (394 and 463 N mm-1) were significantly higher than those of the healthy rabbit femur (316 and 392 N mm-1, respectively) after 8 weeks of implantation. These 3D implants have great potential for patient-specific bone defect treatments.