Critical-size Rat Femoral Defects Research Articles

A bioinspired mineral-organic composite hydrogel as a self-healable and mechanically robust bone graft for promoting bone regeneration

Despite advances in the development of osteo-regenerative biomaterials, current products are vulnerable to stress-induced formation of cracks, resulting in the loss of functionality and a limited lifespan. In the present study, a strategy based on host-guest assembly was developed to fabricate a silk fibroin-based inorganic-organic hybrid hydrogel (termed SF@HG@HA) in which silk fibroin was used as a polymer template to tether host (β-cyclodextrin) and guest (cholesterol) monomers, respectively. Due to dynamic host-guest interactions, the prepared hydrogel could repair itself spontaneously when damaged, without the assistance of any external stimuli, mimicking the self-healing characteristics of native bone tissue. Furthermore, the efficient energy dissipation mechanism provided by the host-guest crosslinking strategy endowed the hydrogel with robust mechanical properties to bear substantial mechanical loading. SF@HG@HA was shown to support cell proliferation and osteogenic differentiation in vitro and accelerate bone regeneration in critical-size rat femoral defects in vivo. Together, the silk fibroin-based self-healing hydrogel with robust mechanical properties shows potential applications in the reconstruction of bone defects, which may provide new directions for the design of functional biomaterials for tissue regeneration.

Regional gene therapy with 3D printed scaffolds to heal critical sized bone defects in a rat model.

The objective of the present study was to assess the ability of transduced rat bone marrow cells (RBMCs) that overexpress BMP-2 loaded on a three-dimensionally (3D) printed scaffold to heal a critical sized rat femoral defect. Tricalcium phosphate (TCP) scaffolds were 3D printed to fit a critical sized rat femoral defect. The RBMCs were transduced with a lentiviral (LV) vector expressing BMP-2 or GFP. The rats were randomized into the following treatment groups: (1) RBMC/LV-BMP-2 + TCP, (2) RBMC/LV-GFP + TCP, (3) nontransduced RBMCs + TCP, (4) TCP scaffold alone. The animals were euthanized at 12 weeks and evaluated with plain radiographs, microcomputed tomography (micro-CT), histology, histomorphometry, and biomechanically. Each LV-BMP-2 + TCP treated specimen demonstrated complete healing of the femoral defect on plain radiographs and micro-CT. No femurs healed in the control groups. Micro-CT demonstrated that LV-BMP-2 + TCP treated femoral defects formed 197% more bone volume compared to control groups (p < 0.05). Histologic analysis demonstrated bone formation across the TCP scaffold, uniting the femoral defect on both ends in the LV-BMP-2 + TCP treated specimens. Biomechanical assessment demonstrated similar stiffness (p = 0.863), but lower total energy to failure, peak torque, and peak displacement (p < 0.001) of the femurs treated with LV-BMP-2 + TCP when compared to the contralateral control femur. Regional gene therapy induced overexpression of BMP-2 via transduced RBMCs combined with an osteoconductive 3D printed TCP scaffold can heal a critically sized femoral defect in an animal model. The combination of regional gene therapy and 3D printed osteoconductive scaffolds has significant clinical potential to enhance bone regeneration.

Bisphosphonates reduce biomaterial turnover in healing of critical-size rat femoral defects.

Treatment of osteoporotic patients with bisphosphonates (BPs) preserves bone mass and microarchitecture. The high prescription rate of the drugs brings about increases in the numbers of fractures and bone defects requiring surgical interventions in these patients. Currently, critical-size defects are filled with biomaterials and healing is supported with bone morphogenetic proteins (BMP). It is hypothesized that BPs interfere with biomaterial turnover during BMP-supported repair of defects filled with β-tricalcium phosphate (βTCP) ceramics. To test this hypothesis, retired breeder rats were ovariectomized ( OVX). After 8 weeks, treatment with alendronate (ALN) commenced. Five weeks later, 6 mm diaphyseal femoral defects were applied and stabilized with locking plates. βTCP cylinders loaded with 1 μg and 10 μg BMP2, 10 μg L51P, an inhibitor of BMP antagonists and 1 μg BMP2/10 μg L51P were fitted into the defects. Femora were collected 16 weeks post-implantation. In groups receiving calcium phosphate implants loaded with 10 μg BMP2 and 1 μg BMP2/10 μg L51P, the volume of bone was increased and βTCP was decreased compared to groups receiving implants with 1 μg BMP2 and 10 μg L51P. Treatment of animals with ALN caused a decrease in βTCP turnover. The results corroborate the synergistic effects of BMP2 and L51P on bone augmentation. Administration of ALN caused a reduction in implant turnover, demonstrating the dependence of βTCP removal on osteoclast activity, rather than on chemical solubility. Based on these data, it is suggested that in patients treated with BPs, healing of biomaterial-filled bone defects may be impaired because of the failure to remove the implant and its replacement by authentic bone.

Effect of sonic hedgehog/β-TCP composites on bone healing within the critical-sized rat femoral defect

The creation of entirely synthetically derived bone substitute materials which are as effective as autologous bone grafts is desirable. Osteogenesis involves the concerted action of several proteins within a signaling cascade. Hedgehog proteins act upstream of this cascade, inducing the expression of various bone morphogenetic proteins (BMPs) and promoting physiological bone healing. Therefore, the hypothesis that hedgehog signaling in bone defects improves bone healing more than BMP signaling alone was tested. Recombinant N-terminal sonic hedgehog protein (N-SHh), BMP-2 or a combination of the two was added to β-tricalcium phosphate (β-TCP) and 5-mm femoral midshaft defects in nude rats were filled with these composites. The defects were stabilized with mini-plates. After eight weeks, the animals were sacrificed and the femora were explanted. The radiological evaluation was followed by a three-point bending test and histological examination. BMP-2/β-TCP composites showed a trend of increased stiffness compared with the controls (β-TCP without protein). N-SHh/β-TCP composites had lower stiffness compared with the control group and the N-SHh/BMP-2/β-TCP composites also had lower average stiffness compared with the controls (all not significant). Histomorphometry, however, revealed abundant cartilage and bone core formation in the N-SHh-composite groups. The sum of the new cartilage and bone was highest in the combination group N-SHh/BMP-2 (not significant). The addition of N-SHh to bone substitute materials appears to delay bone healing at the applied concentration and observation time but also showed a trend for higher amounts of ossifying cartilage.

Mesenchymal Stem Cell Concentration and Bone Repair: Potential Pitfalls from Bench to Bedside

Mesenchymal stem cells are multipotent and have the ability to differentiate into bone. We conducted a preclinical trial comparing the osteogenic potential of human bone marrow aspirate with that of mesenchymal stem cell-enriched bone marrow aspirate (both mixed with demineralized bone matrix) in a critical-sized rat femoral defect model. The buffy coat was extracted from human bone marrow aspirate to obtain mesenchymal stem cell-enriched bone marrow aspirate. Fifty-nine athymic rats, each with a 6-mm femoral defect, were divided into six treatment groups: defect only (Group I), demineralized bone matrix and saline solution (Group II), demineralized bone matrix and bone marrow aspirate (Group III), demineralized bone matrix and mesenchymal stem cell-enriched bone marrow aspirate (Group IV), demineralized bone matrix and recombinant human bone morphogenetic protein-2 (rhBMP-2) (Group V [positive control]), and absorbable collagen sponge and rhBMP-2 (Group VI [positive control]). All animals were killed at twelve weeks for radiographic, micro-computed tomography, histomorphometric, and histologic analysis. There was wide variability in the mesenchymal stem cell concentrations obtained from the human donors. All ten defects healed in the positive control groups (Groups V and VI). Only one defect healed in each experimental group (Groups II, III, and IV) (i.e., three of forty-four defects healed). There was no significant difference among the radiographic scores of Groups II, III, and IV (p = 0.59), and the score for each of those groups was significantly higher than that for Group I (p <or= 0.005) and significantly lower than those for Groups V and VI (p <or= 0.001). The bone volume and mineral density did not differ among Groups III, IV, and V (p = 0.53). The percent total bone volume and percent normal bone volume in Group VI were significantly higher than those values in Groups I, III, and IV (p < 0.0001) and those in Group II (p = 0.048). In Groups II through V, the cortical bone was more dense than the lace-like bone in Group VI. Neither bone marrow aspirate nor mesenchymal stem cell-enriched bone marrow aspirate mixed with demineralized bone matrix resulted in reliable healing of critical-sized bone defects. It is possible that a greater number of mesenchymal stem cells or an enhanced osteoinductive signal is required for adequate bone-healing. Mesenchymal stem cell and/or carrier variability may also contribute to differences in bone formation.