Critical-sized Femoral Segmental Defect Research Articles

Escherichia coli-derived BMP-2-absorbed β-TCP granules induce bone regeneration in rabbit critical-sized femoral segmental defects.

This study investigated whether Escherichia coli-derived bone morphogenetic protein (BMP)-2 (E-BMP-2) adsorbed onto β-tricalcium phosphate (β-TCP) granules can induce bone regeneration in critical-size femoral segmental defects in rabbits. Bone defects 20mm in size and stabilized with an external fixator were created in the femur of New Zealand white rabbits, which were divided into BMP-2 and control groups. E-BMP-2-loaded β-TCP granules were implanted into defects of the BMP-2 group, whereas defects in the controls were implanted with β-TCP granules alone. At 12 and 24weeks after surgery, radiographs were obtained of the femurs and histological and biomechanical assessments of the defect area were performed. Bone regeneration was quantified using micro-computed tomography at 24weeks. Radiographic and histologic analyses revealed bone regeneration in the BMP-2 group but not the control group; no fracturing of newly formed bone occurred when the external fixator was removed at 12weeks. At 24weeks, tissue mineral density, the ratio of bone volume to total volume, and volumetric bone mineral density of the callus were higher in the BMP-2 group than in control animals. In the former, ultimate stress, extrinsic stiffness, and failure energy measurements for the femurs were higher at 24weeks than at 12weeks. E-BMP-2-loaded β-TCP granules can effectively promote bone regeneration in long bone defects.

Hypoxic Preconditioning of Mesenchymal Stem Cells with Subsequent Spheroid Formation Accelerates Repair of Segmental Bone Defects.

Cell-based approaches for musculoskeletal tissue repair are limited by poor cell survival and engraftment. Short-term hypoxic preconditioning of mesenchymal stem cells (MSCs) can prolong cell viability in vivo, while the aggregation of MSCs into spheroids increases cell survival, trophic factor secretion, and tissue formation in vivo. We hypothesized that preconditioning MSCs in hypoxic culture before spheroid formation would increase cell viability, proangiogenic potential, and resultant bone repair compared with that of individual MSCs. Human MSCs were preconditioned in 1% O2 in monolayer culture for 3 days (PC3) or kept in ambient air (PC0), formed into spheroids of increasing cell density, and then entrapped in alginate hydrogels. Hypoxia-preconditioned MSC spheroids were more resistant to apoptosis than ambient air controls and this response correlated with duration of hypoxia exposure. Spheroids of the highest cell density exhibited the greatest osteogenic potential in vitro and vascular endothelial growth factor (VEGF) secretion was greatest in PC3 spheroids. PC3 spheroids were then transplanted into rat critical-sized femoral segmental defects to evaluate their potential for bone healing. Spheroid-containing gels induced significantly more bone healing compared with gels containing preconditioned individual MSCs or acellular gels. These data demonstrate that hypoxic preconditioning represents a simple approach for enhancing the therapeutic potential of MSC spheroids when used for bone healing. Stem Cells 2018;36:1393-1403.

Bioinspired Composite Matrix Containing Hydroxyapatite-Silica Core-Shell Nanorods for Bone Tissue Engineering.

Development of multifunctional bioinspired scaffolds that can stimulate vascularization and regeneration is necessary for the application in bone tissue engineering. Herein, we report a composite matrix containing hydroxyapatite (HA)-silica core-shell nanorods with good biocompatibility, osteogenic differentiation, vascularization, and bone regeneration potential. The biomaterial consists of a crystalline, rod-shaped nanoHA core with uniform amorphous silica sheath (Si-nHA) that retains the characteristic phases of the individual components, confirmed by high-resolution transmission electron microscopy, X-ray diffractometer, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The nanorods were blended with gelatinous matrix to develop as a porous, composite scaffold. The viability and functionality of osteogenically induced mesenchymal stem cells as well as endothelial cells have been significantly improved through the incorporation of Si-nHA within the matrix. Studies in the chicken chorioallantoic membrane and rat models demonstrated that the silica-containing scaffolds not only exhibit good biocompatibility, but also enhance vascularization in comparison to the matrix devoid of silica. Finally, when tested in a critical-sized femoral segmental defect in rats, the nanocomposite scaffolds enhanced new bone formation in par with the biomaterial degradation. In conclusion, the newly developed composite biomimetic scaffold may perform as a promising candidate for bone tissue engineering applications.

Electrospun Yarn Reinforced NanoHA Composite Matrix as a Potential Bone Substitute for Enhanced Regeneration of Segmental Defects.

Nanohydroxyapatite (nanoHA) is a well-established synthetic bone substitute with excellent osteoconduction and osteointegration. However, brittleness coupled with slow degradation curtails its load-bearing and bone regeneration potential, respectively. To address these limitations, nanoHA composite matrix reinforced with electrospun fibrous yarns was fabricated and tested in vitro and in vivo. Different weight percentages (5, 10, 15 wt%) and varying lengths (short and continuous) of poly(l-lactic acid) yarns were randomly dispersed in a gelatinous matrix containing nanoHA. This significantly improved the compressive strength as well as work of fracture, especially for continuous yarns at high weight percentages (10 and 15 wt%). Incorporation of yarns did not adversely affect the pore size (50-350 μm) or porosity of the scaffolds as well as the in vitro cellular response. Finally, when tested in a critical-sized femoral segmental defect in rat, the nanocomposite scaffolds induced osteoblast cell infiltration at 2 months that subsequently underwent increased mature lamellar bone formation at 4 months, in both the mid and peripheral defect regions. Histomorphometric analysis demonstrated that new bone formation and biomaterial degradation were significantly enhanced in the composite scaffold when compared to commercially available HA. Overall, the composite matrix reinforced with electrospun yarns proved to be a potential bone substitute having an appropriate balance between mechanical strength, porosity, biodegradation, and bone regeneration ability.

Combined Use of Low-Intensity Pulsed Ultrasound and rhBMP-2 to Enhance Bone Formation in a Rat Model of Critical Size Defect

Bone repair is regulated by biological factors and the local mechanical environment. We hypothesize that the combined use of low-intensity pulsed ultrasound (LIPUS) and recombinant human bone morphogenetic protein-2 (rhBMP-2) will synergistically or additively enhance bone regeneration in a model simulating the more difficult scenarios in orthopaedic traumatology. Femoral defects in rats were replaced with absorbable collagen sponges carrying rhBMP-2 (0, 1.2, 6, or 12 μg; n = 30). Each group was divided equally to receive daily treatment of either LIPUS or sham stimulation. At 4 weeks, new bone formation was assessed using quantitative (radiography and microcomputed tomography), qualitative (histology), and functional (biomechanical) end points. LIPUS with 1.2 μg of rhBMP-2 significantly improved the radiographic healing as compared with its sham control starting as early as 2 weeks. Quantitatively, the use of LIPUS with 6 μg of rhBMP-2 significantly increased the bone volume. However, using LIPUS with 12 μg of rhBMP-2 indicated a reduction in callus size, without compromising the bone volume, which was also observable histologically, showing organized lamellar bone and repopulated marrow in the original defect region. Histologically, 1.2 μg of rhBMP-2 alone showed the presence of uncalcified cartilage in the defect, which was reduced with LIPUS treatment. Biomechanically, LIPUS treatment significantly increased the peak torsion and stiffness in the 6- and 12 μg rhBMP-2 groups. LIPUS enhances rhBMP-2-induced bone formation at lower doses (1.2 and 6 μg) and callus maturation at 12-μg dose delivered on absorbable collagen sponge for bone repair in a rat critical-sized femoral segmental defect.

Three-dimensionally presented anti-fouling zwitterionic motifs sequester and enable high-efficiency delivery of therapeutic proteins

Zwitterions are well known for their anti-biofouling properties. Past investigations of zwitterionic materials for biomedical uses have been centered on exploiting their ability to inhibit non-specific adsorption of proteins. Here, we report that zwitterionic motifs, when presented in three dimensions (e.g. in crosslinked hydrogels), could effectively sequester osteogenic bone morphogenetic protein-2 (rhBMP-2). The ionic interactions between rhBMP-2 and the 3-D zwitterionic network enabled dynamic sequestering and sustained release of the protein with preserved bioactivity. We further demonstrated that the zwitterionic hydrogel confers high-efficiency in vivo local delivery of rhBMP-2. It can template the functional healing of critical-size femoral segmental defects in rats with rhBMP-2 at a loading dose substantially lower than those required for current natural or synthetic polymeric carriers. These findings reveal a novel function of zwitterionic materials beyond their commonly perceived anti-biofouling property, and may establish 3-D zwitterionic matrices as novel high-efficiency vehicles for protein/ionic drug therapeutic delivery.

Local injection of lovastatin in biodegradable polyurethane scaffolds enhances bone regeneration in a critical-sized segmental defect in rat femora

Statins, a class of naturally-occurring compounds that inhibit HMG-CoA reductase, are known to increase endogenous bone morphogenetic protein-2 (BMP-2) expression. Local administration of statins has been shown to stimulate fracture repair in in vivo animal experiments. However, the ability of statins to heal more challenging critical-sized defects at the mid-diaphyseal region in long bones has not been investigated. In this study, we examined the potential of injectable lovastatin microparticles combined with biodegradable polyurethane (PUR) scaffolds in preclinical animal models: metaphyseal small plug defects and diaphyseal segmental bone defects in rat femora. Sustained release of lovastatin from the lovastatin microparticles was achieved over 14 days. The released lovastatin was bioactive, as evidenced by its ability to stimulate BMP-2 gene expression in osteoblastic cells. Micro-computed tomography (CT) and histological examinations showed that lovastatin microparticles, injected into PUR scaffolds implanted in femoral plug defects, enhanced new bone formation. Furthermore, bi-weekly multiple injections of lovastatin microparticles into PUR scaffolds implanted in critical-sized femoral segmental defects resulted in increased new bone formation compared to the vehicle control. In addition, bridging of the defect with newly formed bone was observed in four of nine defects in the lovastatin microparticle treatment group, whereas none of the defects in the vehicle group showed bridging. These observations suggest that local delivery of lovastatin combined with PUR scaffold can be an effective approach for treatment of orthopaedic bone defects and that multiple injections of lovastatin may be useful for large defects.

Nell-1 Enhances Bone Regeneration in a Rat Critical-Sized Femoral Segmental Defect Model

Effective regeneration of bone is critical for fracture repair and incorporation and healing of bone grafts used during orthopedic, dental, and craniofacial reconstructions. Nel-like molecule-1 (Nell-1) is a secreted protein identified from prematurely fused cranial sutures of craniosynostosis patients that has been found to specifically stimulate osteogenic cell differentiation and bone formation. To test the in vivo osteoinductive capacity of Nell-1, a critical-sized femoral segmental defect model in athymic rats was used. A 6-mm defect, which predictably leads to nonunion if left untreated, was created in the left femur of each rat. Three treatment groups (n = 8 each) were created consisting of rats treated with (1) 1.5 mg/ml Nell-1, (2) 0.6 mg/ml Nell-1, and (3) phosphate-buffered saline only as a Nell-free control. Phosphate-buffered saline or Nell-1 was mixed with demineralized bone matrix as a carrier before implantation. All animals were euthanized 12 weeks after surgery, and bone regeneration was evaluated using radiographic, three-dimensional micro-computed tomographic, and histologic analysis. Both Nell-1-treated groups had significantly greater bone formation compared with the Nell-free group, with bone volume increasing with increasing Nell-1 concentration. Nell-1 in a demineralized bone matrix carrier can significantly improve bone regeneration in a critical-sized femoral segmental defect in a dose-dependent manner. The results of this study demonstrate that Nell-1 is a potent osteospecific growth factor that warrants further investigation. Results also support the potential application of Nell-1 as a bone graft substitute in multiple clinical scenarios involving repair of critical bone loss when autograft bone is limited or unavailable.

Silk based biomaterials to heal critical sized femur defects

Bone auto- and allografts have inherent drawbacks, therefore the treatment of non-unions and critical size defects in load bearing long bones would benefit from the use of osteopromotive biodegradable, biocompatible and mechanically durable matrices to enhance migration or delivery of cell populations and/or morphogens/cytokines. Silk fibroin biomaterial scaffolds were evaluated as osteopromotive matrices in critical sized mid-femoral segmental defects in nude rats. Four treatment groups were assessed over 8 weeks in vivo: silk scaffolds (SS) with human mesenchymal stem cells (hMSCs) that had previously been differentiated along an osteoblastic lineage in vitro (group I; pdHMSC/SS); SS with undifferentiated hMSCs (group II; udHMSC/SS); SS alone (group III; SS); and empty defects (group IV). When hMSCs were cultured in vitro in osteogenic medium for 5 weeks, bone formation was characterized with bimodal peak activities for alkaline phosphatase at 2 and 4 weeks. Calcium deposition started after 1 week and progressively increased to peak at 4 weeks, reaching cumulative levels of deposited calcium at 16 μg per mg scaffold wet weight. In vivo osteogenesis was characterized by almost bridged defects with newly formed bone after 8 weeks in group I. Significantly ( P < 0.01) greater bone volumes were observed with the pdHMSC/SS (group I) implants than with groups II, III or IV. These three groups failed to induce substantial new bone formation and resulted in the ingrowth of cells with fibroblast-like morphology into the defect zone. The implantation of pdHMSC/SS resulted in significantly ( P < 0.05) greater maximal load and torque when compared to the other treatment regimens. The pdHMSC/SS implants demonstrated osteogenic ability in vitro and capacity to thrive towards the healing of critical size femoral segmental defects in vivo. Thus, these new constructs provide an alternative protein-based biomaterial for load bearing applications.

The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.

Recombinant human bone morphogenetic proteins (rhBMPs) can induce bone formation, but the inability to identify an ideal delivery system limits their clinical application. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone-marrow cells, which allow delivery of the BMP-2 to a specific anatomical site. The autologous BMP-2-producing bone-marrow cells then were used to heal a critical-sized femoral segmental defect in syngeneic rats. Femoral defects in five groups of rats were filled with 5 x 10(6) BMP-2-producing bone-marrow cells, created through adenoviral gene transfer (twenty-four femora, Group I); twenty micrograms of rhBMP-2 (sixteen femora, Group II); 5 x 10(6) beta-galactosidase-producing rat-bone-marrow cells, created through adenoviral gene transfer of the lacZ gene (twelve femora, Group III); 5 x 10(6) uninfected rat-bone-marrow cells (ten femora, Group IV); or guanidine hydrochloride-extracted demineralized bone matrix only (ten femora, Group V). Guanidine hydrochloride-extracted demineralized bone matrix served as a substrate in all experimental groups. Specimens that were removed two months postoperatively underwent histological and histomorphometric analysis as well as biomechanical testing. Twenty-two of the twenty-four defects in Group I (BMP-2-producing bone-marrow cells) and all sixteen defects in Group II (rhBMP-2) had healed radiographically at two months postoperatively compared with only one of the thirty-two defects in the three control groups (beta-galactosidase-producing rat-bone-marrow cells, uninfected rat-bone-marrow cells, and guanidine hydrochloride-extracted demineralized bone matrix alone). Histological analysis of the specimens revealed that defects that had received BMP-2-producing bone-marrow cells (Group I) were filled with coarse trabecular bone at two months postoperatively, whereas in those that had received rhBMP-2 (Group II) the bone was thin and lace-like. Defects that had been treated with bone-marrow cells producing beta-galactosidase (Group III), uninfected bone-marrow cells (Group IV), or guanidine hydrochloride-extracted demineralized bone matrix only (Group V) demonstrated little or no bone formation. Histomorphometric analysis revealed a significantly greater total area of bone formation in the defects treated with the BMP-2-producing bone-marrow cells than in those treated with the rhBMP-2 (p = 0.036). Biomechanical testing demonstrated no significant differences, with the numbers available, between the healed femora that had received BMP-2-producing bone-marrow cells and the untreated (control) femora with respect to ultimate torque to failure or energy to failure. This study demonstrated that BMP-2-producing bone-marrow cells created by means of adenoviral gene transfer produce sufficient protein to heal a segmental femoral defect. We also established the feasibility of ex vivo gene transfer with the use of biologically acute autologous short-term cultures of bone-marrow cells.