Critical-sized Bone Defect Model Research Articles

Treatment of Large Bone Defects with a Vascularized Periosteal Flap in Combination with Biodegradable Scaffold Seeded with Bone Marrow-Derived Mononuclear Cells: An Experimental Study in Rats.

The surgical treatment of large bone defects continues to pose a major challenge in modern trauma and orthopedic surgery. In this study we test the effectiveness of a tissue engineering approach, using three-dimensional (3D) β-tricalcium phosphate (β-TCP) scaffolding plus bone marrow-derived mononuclear cells (BM-MNCs), combined with a vascularized periosteal flap, in a rat femur critical size defect model. Eighty rats were randomly allocated into four equal groups. Under general anesthesia, critical size defects were created on their femurs and were treated with (1) Vascularized periosteal flap alone, (2) Vascularized periosteal flap+β-TCP scaffolding, (3) Vascularized periosteal flap+β-TCP scaffolding+ligated vascular pedicle, and (4) Vascularized periosteal flap+β-TCP scaffolding+BM-MNCs. After 4 and 8 weeks animals were euthanized and the bone defects were harvested for analysis of new bone formation, vascularization, and strength using histology, immunohistology, micro-CT, and biomechanical testing, respectively. Group 1: (P. flap) Increase in new bone formation and vascularization. Group 2: (P. flap+scaffold) Increase in new bone formation and vascularization. Group 3: (P. flap+scaffold+ligated vascular pedicle) No new bone formation and no vascularization. Group 4: (P. flap+scaffold+BM-MNCs) A significant (p < 0.05) increase was seen in new bone formation, vascularization, and strength in bones treated with flaps, scaffold, and BM-MNCs, when compared with the other treatment groups. Combining a vascularized periosteal flap with tissue engineering approach (β-TCP scaffolding and BM-MNC) results in significantly improved bone healing in our rat femur critical size bone defect model.

Osteogenesis of peripheral blood mesenchymal stem cells in self assembling peptide nanofiber for healing critical size calvarial bony defect.

Peripheral blood mesenchymal stem cells (PBMSCs) may be easily harvested from patients, permitting autologous grafts for bone tissue engineering in the future. However, the PBMSC’s capabilities of survival, osteogenesis and production of new bone matrix in the defect area are still unclear. Herein, PBMSCs were seeded into a nanofiber scaffold of self-assembling peptide (SAP) and cultured in osteogenic medium. The results indicated SAP can serve as a promising scaffold for PBMSCs survival and osteogenic differentiation in 3D conditions. Furthermore, the SAP seeded with the induced PBMSCs was splinted by two membranes of poly(lactic)-glycolic acid (PLGA) to fabricate a composited scaffold which was then used to repair a critical-size calvarial bone defect model in rat. Twelve weeks later the defect healing and mineralization were assessed by H&E staining and microcomputerized tomography (micro-CT). The osteogenesis and new bone formation of grafted cells in the scaffold were evaluated by immunohistochemistry. To our knowledge this is the first report with solid evidence demonstrating PBMSCs can survive in the bone defect area and directly contribute to new bone formation. Moreover, the present data also indicated the tissue engineering with PBMSCs/SAP/PLGA scaffold can serve as a novel prospective strategy for healing large size cranial defects.

Remineralization of demineralized bone matrix in critical size cranial defects in rats: A 6‐month follow‐up study

The key drawback of using demineralized bone matrix (DBM) is its low initial mechanical stability due to the severe depletion of mineral content. In the present study, we investigated the long-term regeneration of DBM in a critical size bone defect model and investigated the remineralization after 6 months. Bone defects were created in the cranium of male Wistar rats which were filled with DBM or left empty as negative control. In vivo bone formation was monitored with computed tomography after 11, 19, and 26 weeks postoperatively. After 6 months, parietal bones were subjected to micro-CT. Mineral content was determined with spectrophotometric analysis. After 11 weeks the DBM-filled bone defects were completely closed, while empty defects were still open. Density of the DBM-treated group increased significantly while the controls remained unchanged. Quantitative analysis by micro-CT confirmed the in vivo results, bone volume/tissue volume was significantly lower in the controls than in the DBM group. The demineralization procedure depleted the key minerals of the bone to a very low level. Six months after implantation Ca, P, Na, Mg, Zn, and Cr contents were completely restored to the normal level, while K, Sr, and Mn were only partially restored. The remineralization process of DBM is largely complete by the 6th month after implantation in terms of bone density, structure, and key mineral levels. Although DBM does not provide sufficient sources for any of these minerals, it induces a faster and more complete regeneration process. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1336-1342, 2016.

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.

Simple evaluation method for osteoinductive capacity of cells or scaffolds using ceramic cubes.

Mesenchymal stem cells are good candidates for the clinical application of bone repair because of their osteogenic differentiation potential, but in vivo osteoinduction potential should be verified for culture expanded cells before clinical application. This study analyzed in vivo bone formation by MSCs quantitatively after implantation of MSCs planted porous biphasic ceramic cubes into athymic mice. MSCs were divided into osteogenic differentiation-induced and normal groups and also tested in vitro to evaluate the degree of differentiation into osteoblasts. The osteogenic induced group showed higher alkaline phosphatase and calcium level in vitro and corresponding higher level of bone formation in vivo compared to control group. Whereas there was no bone formation observed in fibroblast-implanted negative control group. In critical sized bone defect models, commonly used for evaluation of bone regeneration ability, it is difficult to distinguish between osteoinduction and osteoconduction, and quantitative analysis is not simple. However, this method for evaluating osteoinduction is both accurate and simple. In conclusion, the analysis of in vivo bone formation using porous ceramic cubes is a powerful and simple method for evaluating the osteoinduction ability of target cells and, furthermore, can be applied for evaluation of scaffolds for their osteoinductive properties.

Establishment of reproducible, critical-sized, femoral segmental bone defects in rats.

Development of new treatment methodologies for bone nonunion requires validated experimental models for their application into tissue engineering approaches. Critical-sized bone defect (CSBD) models identify the smallest size at which tissue defects fail to completely heal during the experimental period. The purpose of this study was to systematically determine a CSBD in rat femurs using external fixation. Thirty Fischer 344 rats were equally divided into six groups. Bone defects of 1, 2, 3, 4, 5, and 6 mm were created in the diaphysis of the femur, before external fixation. Defects were evaluated radiographically at weekly intervals and histologically at the predefined healing period of 8 weeks. Defects of 4 mm or more failed to achieve union in our model. Nonunions were characterized by the absence of a bridging callus and filling of the defect with fibrous and muscle tissue. Radiographically, bone ends in nonunions were rounded up to 4 weeks postsurgery. Our systematic approach for identification of a CSBD in rat femurs, revealed that externally fixed defects of 4 mm and larger, failed to heal within the 8-week time frame.

A comparison of bone regeneration with human mesenchymal stem cells and muscle-derived stem cells and the critical role of BMP

Adult multipotent stem cells have been isolated from a variety of human tissues including human skeletal muscle, which represent an easily accessible source of stem cells. It has been shown that human skeletal muscle-derived stem cells (hMDSCs) are muscle-derived mesenchymal stem cells capable of multipotent differentiation. Although hMDSCs can undergo osteogenic differentiation and form bone when genetically modified to express BMP2; it is still unclear whether hMDSCs are as efficient as human bone marrow mesenchymal stem cells (hBMMSCs) for bone regeneration. The current study aimed to address this question by performing a parallel comparison between hMDSCs and hBMMSCs to evaluate their osteogenic and bone regeneration capacities. Our results demonstrated that hMDSCs and hBMMSCs had similar osteogenic-related gene expression profiles and had similar osteogenic differentiation capacities in vitro when transduced to express BMP2. Both the untransduced hMDSCs and hBMMSCs formed very negligible amounts of bone in the critical sized bone defect model when using a fibrin sealant scaffold; however, when genetically modified with lenti-BMP2, both populations successfully regenerated bone in the defect area. No significant differences were found in the newly formed bone volumes and bone defect coverage between the hMDSC and hBMMSC groups. Although both cell types formed mature bone tissue by 6 weeks post-implantation, the newly formed bone in the hMDSCs group underwent quicker remodelling than the hBMMSCs group. In conclusion, our results demonstrated that hMDSCs are as efficient as hBMMSCs in terms of their bone regeneration capacity; however, both cell types required genetic modification with BMP in order to regenerate bone in vivo.

CD146+ Human Umbilical Cord Perivascular Cells Maintain Stemness under Hypoxia and as a Cell Source for Skeletal Regeneration

The human umbilical cord perivascular cells (HUCPVCs) have been considered as an alternative source of mesenchymal progenitors for cell based regenerative medicine. However, the biological properties of these cells remain to be well characterized. In the present study, HUCPVCs were isolated and sorted by CD146+ pericyte marker. The purified CD146+ HUCPVCs were induced to differentiate efficiently into osteoblast, chondrocyte and adipocyte lineages in vitro. Six weeks following subcutaneous transplantation of CD146+ HUCPVCs-Gelfoam-alginate 3D complexes in severe combined immunodeficiency (SCID) mice, newly formed bone matrix with embedded osteocytes of donor origin was observed. The functional engraftment of CD146+ HUCPVCs in the new bone regenerates was further confirmed in a critical-sized bone defect model in SCID mice. Hypoxic conditions suppressed osteogenic differentiation while increased cell proliferation and colony-forming efficiency of CD146+ HUCPVCs as compared to that under normoxic conditions. Re-oxygenation restored the multi-differentiation potential of the CD146+ HUCPVCs. Western blot analysis revealed an upregulation of HIF-1α, HIF-2α, and OCT-4 protein expression in CD146+ HUCPVCs under hypoxia, while there was no remarkable change in SOX2 and NANOG expression. The gene expression profiles of stem cell transcription factors between cells treated by normoxia and hypoxic conditions were compared by PCR array analysis. Intriguingly, PPAR-γ was dramatically downregulated (20-fold) in mRNA expression under hypoxia, and was revealed to possess a putative binding site in the Hif-2α gene promoter region. Chromatin immunoprecipitation assays confirmed the binding of PPAR-γ protein to the Hif-2α promoter and the binding was suppressed by hypoxia treatment. Luciferase reporter assay showed that the Hif-2α promoter activity was suppressed by PPAR expression. Thus, PPAR-γ may involve in the regulation of HIF-2α for stemness maintenance and promoting the expansion of CD146+ HUCPVCs in response to hypoxia. CD146+ HUCPVCs may serve as a potential autologous cell source for bone regeneration.

Repair of critical-sized bone defect by a novel bone bioengineering construct after co-transfection of recombinant human bone morphogenetic protein-2 and basic fibroblast growth factor into bone marrow stem cells and combination with nano-hydroxyapatite/recombinant human-like collagen/polylactic acid scaffold

Objective To repair a 1.5 cm critical-sized rabbit bone defect model by a novel bone bioengineering construct after co-transfection of recombinant human bone morphogenetic protein-2 （rhBMP-2） and basic fibroblast growth factor （bFGF） into bone marrow stem cells （BMSCs） and combination with nano-hydroxyapatite/recombinant human-like collagen/polylactic acid （nHA/RHLC/PLA） scaffold. Methods A 1.5 cm critical-sized bone defect model was created. BMSCs after gene transfeetion were combined with nHA/RHLC/PLA scaffolds to fabricate various biocomposites. Six groups were established： rhBMP-2- bFGF/BMSCs/nHA/RHLC/PLA （A）, rhBMP-2/BMSCs/nHA/RHLC/PLA （B）, bFGF/BMSCs/nHA/ RHLC/PLA （C） , BMSCs/nHA/RHLC/PLA （ D）, nHA/RHLC/PLA （E） and untreated bone defect group （F）. At 6th and 12th week postoperation, the samples were harvested and subjected to radiographic and histologic analyses. Results At 6th week after transplantation, obvious new bone tissue was formed in group A with bone area of （47.24 ± 4. 37 ） %, significantly greater than in group B [（28.24 ± 2. 35 ） %] and group C [（ 13.46 ± 2. 27 ） %] （ P 〈 0. 05 ）. No bone formation was observed in groups D and E. Twelve weeks later, bone integrity was restored and medullary cavity recanalized in group A with bone area of （ 96.84 ± 2. 28 ） % ; bone integrity was restored but medullary cavity was not recanalized yet in group B with bone area of （ 92. 24 ± 1.75 ） % ; slightly enhanced bone tissue formation was seen in group C [（24.73 ±2. 01 ）%] and few new bone tissue was found in group D; no bone tissue was formed in group E. The therapeutic effect for bone defect in group A was superior to other groups （ P 〈 0. 05 ）, and the bone defect in groups C, D and E was not repaired. Conclusion A new bone tissue engineering biocomposite by the combination of nHA/RHLC/PLA and BMSCs after double-transfection of rhBMP-2 and bFGF can produce apparently bone tissue in orthotopic site, which provides a new view relating to tissue-engineering repair of larze bone defect in clinical area. Key words: Recombinant bone morphogenetic protein-2; Basic fibroblast growth factor; Bonemarrow stem cells; Bone defect

BMP2 genetically engineered MSCs and EPCs promote vascularized bone regeneration in rat critical-sized calvarial bone defects.

Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.

Synthetic hydrogel scaffold is an effective vehicle for delivery of INFUSE (rhBMP2) to critical‐sized calvaria bone defects in rats

Medtronic's INFUSE Bone Graft provides surgeons with a potent tool for stimulating bone formation. Current delivery vehicles that rely on Absorbable Collagen Sponges (ACS) require excessive quantities of the active ingredient in INFUSE, recombinant human Bone Morphogenic Protein-2 (rhBMP2), to achieve physiologically relevant concentrations of the growth factor, driving up the cost of the product and increasing the likelihood of undesirable side effects in neighboring tissues. We demonstrate that a simple light-mediated, thiol-ene chemistry can be used to create an effective polymer delivery vehicle for rhBMP2, eliminating the use of xenographic materials and reducing the dose of rhBMP2 required to achieve therapeutic effects. Comprised entirely of synthetic components, this system entraps rhBMP2 within a biocompatible hydrogel scaffold that is degraded by naturally occurring remodeling enzymes, clearing the way for new tissue formation. When tested side-by-side with ACS in a critical-sized bone defect model in rats, this polymeric delivery system significantly increased bone formation over ACS controls.

Establishment of a femoral critical-size bone defect model in immunodeficient mice

BackgroundThe development of innovative therapies for bone regeneration requires the use of advanced site-specific bone defect small-animal models. The achievement of proper fixation with a murine model is challenging due to the small dimensions of the murine femur. The aim of this investigation was to find the optimal defect size for a murine critical-size bone defect model using external fixation method. MethodsAn external fixation device was attached to the right femur of 30 mice. Femoral bone defects of 1 mm (n = 10), 2 mm (n = 10), and 3 mm (n = 10) were created. Wounds were closed without any additional treatment. To investigate bone healing during the 12-wk observation period, x-ray analysis, histomorphology, immunohistochemistry, and μCT scans were performed. ResultsMicroCT analyses after 12 wk showed that 3/8 1-mm defects, 5/8 2-mm defects, and 8/8 3-mm defects remained as nonunions. The defect volumes were 0.36 ± 0.42 mm³ (1-mm group), 1.40 ± 0.88 mm³ (2-mm group), and 2.88 ± 0.28 mm³ (3-mm group; P < 0.001, between all groups). ConclusionUsing external fixation, a defect size of 3 mm is necessary to reliably create a persisting femoral bone defect in nude mice.

An Innovative Intramedullary Nail for Stabilizing Mouse Femoral Defects

The study by Garcia and colleagues, published in the Journal of Surgical Research, describes an innovative intramedullary fixator for stabilizing long bone defects in mice [1]. The authors demonstrate that their device, which they have named the Locking Mouse Nail, can stabilize mouse femoral segmental defects measuring 0, 0.25, and 2.0 mm in length. Furthermore, by showing that 0.25 mm gaps show complete healing while 2 mm gaps develop atrophic non-unions, the authors have helped confirm that critical-size femoral defects in mice are 2 mm or less in length [2]. Generally speaking, critical-sized bone defect models are distinguished from fracture healing models by their behavior following rigid fixation [2–5]. While fracture models are expected to universally achieve bone healing, segmental defect models involving a substantial gap achieve a fibrous union. Fracture models can be used to describe the cellular events surrounding normal bone healing, while segmental defect models are useful for understanding the limitations of those cellular events, as well as providing a platform for evaluating methodologies for treating the non-unions. The challenge to date has been the development of effective fixation systems for the mouse, which reproduce the types of hardware that are commonly used clinically. In this study, the authors have combined an intramedullary rod with transcortical pins to achieve multi-dimensional stabilization of individual bone segments. The rod prevents angular deformity across the fracture, while the pins prevent axial and rotational movements. They evaluated the construct’s stability, and found it is exceeded only by an external fixation system. As the authors point out, the chief advantage of this system is that it allows the study of bone healing in transgenic and knockout mouse models. For instance, the role in osteoblast function of a variety signaling pathways, such as the Wnts, as well as the efficacy of novel agents which modulate these pathways, can now be evaluated during fracture healing [6–10]. Likewise, the role of stem cells and other osteoprogenitor cells during the formation of new bone can be better defined [11]. The greatest limitations that the model offers may be availability, cost, and ease of use. It is as yet unclear whether this device will be available for widespread dissemination, and how much training an investigator will need before employing it consistently. Despite these concerns, the authors have done innovative work in creating novel mouse fixation models. They should be commended and

Establishment of a Preclinical Ovine Model for Tibial Segmental Bone Defect Repair by Applying Bone Tissue Engineering Strategies

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties; however, they are limited in access and availability and associated with donor-site morbidity, hemorrhage, risk of infection, insufficient transplant integration, graft devitalization, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench-to-bedside translations are still infrequent as the process toward approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence, commercialization, is referred to as the "Valley of Death" and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes and scalable designs and to apply these in preclinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopedic bone engineering from bench to bedside by establishing a preclinical ovine critical-sized tibial segmental bone defect model, and we discuss our preliminary data relating to this decisive step.

Multilayered chitosan scaffold for bile duct reconstruction

A new method called cloud point thermally induced phase separation (CP-TIPS) was developed to prepare poly (l-lactic acid) (PLLA) scaffolds with macropores over 300 μm in PLLA/dioxane/H2O ternary system. The concentration of PLLA was 4% (w/v), and the solvent was dioxane with 14% of deionized water. By maintaining the clear solution of the ternary system near and above the cloud point for 2 h, followed by a large enough quenching depth (−80 °C), the crystallization of the α′-form crystal became privileged, and the microstructure of the pore wall was dominated with nanofibers. In contrast, conventional TIPS process resulted in scaffolds with similar pore size but platelet-like microstructure on the pore walls. The unique macroporous nanofibrous PLLA scaffold showed well elasticity and flexibility after swelling in 70% acetone. This property further enabled a facile “mechanical squeeze and intake” of high concentration viscous chitosan solution into the porous structure to modify the composite scaffolds. The compressive modulus of macroporous nanofibrous PLLA/CS scaffolds reached about 1 MPa. The hydrophilicity of the scaffold was greatly enhanced and the pH value remained stable in the first 3 weeks after degradation in vitro. The effect of chitosan concentration in the composite scaffolds on cell affinity and the osteogenic differentiation was evaluated. Finally, a critical-size rat calvarial bone defect model was used to evaluate the osteogenesis of the scaffolds in vivo and the composite scaffold with 3% chitosan significantly enhanced bone regeneration.

Increased Bone Formation Using Calcium Sulfate-Calcium Phosphate Composite Graft

Calcium phosphates (CaPO4) and faster-resorbing calcium sulfate (CaSO4) are successfully employed as synthetic bone grafts for treatment of contained defects. We used a canine critical-sized bone defect model to study an injectable CaSO4/CaPO4 composite graft that incorporated a matrix of CaSO4 and dicalcium phosphate dihydrate into which beta-tricalcium phosphate granules were distributed. The area fraction, ultimate compressive stress, and elastic modulus of restored bone and the relative rates of material resorption were compared between the CaSO4/CaPO4 composite graft and pure CaSO4 pellets and to normal canine bone. The area fraction of bone in stained sections and the ultimate compressive stress of the regenerated bone were greater using the CaSO4/CaPO4 composite graft compared to pure CaSO4 pellets after 13 and 26 weeks and were greater than normal bone. The elastic modulus of restored bone in defects treated with CaSO4/CaPO4 composite graft was greater than in defects treated with CaSO4 pellets after 26 weeks, but similar to specimens of normal bone. A small amount of CaSO4/CaPO4 composite graft and no CaSO4 pellets remained after 13 or 26 weeks. This novel CaSO4/CaPO4 composite holds promise for clinical applications where a strong, injectable, slower-resorbing, and biocompatible bone graft substitute would be advantageous.

Growth Hormone Stimulates Bone Healing in a Critical-sized Bone Defect Model

Growth hormone plays an important role in bone metabolism. Treating bone deficits is a major topic in orthopaedic surgery. Our hypothesis was that local continuous growth hormone administration stimulates bone healing in a canine critical-sized bone defect model. Bone formation in the defects was quantified using densitometric image analysis and histomorphometry. After growth hormone treatment, expression levels of insulin-like growth factors-I and II, and growth hormone receptor were determined in the bone regenerate of the original defects. Circulating plasma concentrations of insulin-like growth factors-I and II, and insulin- like growth factor binding proteins-4, and 6 were measured during treatment. Growth hormone administration resulted in healing of bone defects but without an additional effect of local infusion. Expression of insulin-like growth factor-I in the bone regenerate was lower in the growth hormone-treated dogs, whereas insulin-like growth factor-II and growth hormone receptor expression were not increased. Growth hormone increased circulating insulin-like growth factor-I and growth factor-II plasma concentrations. Continuous infusion of growth hormone stimulated bone healing in a canine critical-sized bone defect model. Local delivery of growth hormone did not additionally enhance bone healing. Increased circulating plasma concentrations of insulin-like growth factors-I and II most likely induced bone formation.

Allogenic peripheral blood derived mesenchymal stem cells (MSCs) enhance bone regeneration in rabbit ulna critical-sized bone defect model

Mesenchymal stem cells (MSCs) were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. Whether or not the PB derived MSCs (PBMSCs) could enhance the regeneration of large bone defects have not been reported. In this study, rabbit MSCs were obtained from mononuclear cells (MNCs) cultures of both the PB and bone marrow (BM) origin. The number of PBMSCs was relatively lower, with the colony forming efficiency (CFE) ranging from 1.2 to 13 per million MNCs. Under specific inductive conditions, PBMSCs differentiated into osteoblasts, chondrocytes, and adipocytes, showing multidifferentiation ability similar to BMMSCs. Bilateral 20 mm critical-sized bone defects were created in the ulnae of 12 6-month-old New Zealand white rabbits. The defects were treated with allogenic PBMSCs/Skelite (porous calcium phosphate resorbable substitute), BMMSCs/Skelite, PBMNCs/Skelite, Skelite alone, and left empty for 12 weeks. Bone regeneration was evaluated by serial radiography, peripheral quantitative computed tomography (pQCT), and histological examinations. The X-ray scores and the pQCT total bone mineral density in the PBMSCs/Skelite and BMMSCs/Skelite treated groups were significantly greater than those of the PBMNCs/Skelite and Skelite alone groups ( p < 0.05), respectively. Histologically, newly formed bone was evident in the PBMSCs/Skelite and BMMSCs/Skelite treated groups. The findings demonstrated that the rabbit PBMSCs possessed multidifferentiation potential comparable with BMMSCs, allogenic PBMSCs seeded onto porous calcium phosphate resorbable substitutes enhanced bone regeneration in the rabbit ulna critical-sized bone defect model, suggesting allogenic PBMSCs may be a new source of circulating osteogenic stem cells for bone regeneration and tissue engineering.

Repair of an intercalated long bone defect with a synthetic biodegradable bone-inducing implant

Recombinant human bone morphogenetic protein (rhBMP)-2 in a block copolymer composed of poly- d, l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) as a carrier and porous β-tricalcium phosphate ( β-TCP) blocks were used to generate a new fully absorbable osteogenic biomaterial. The bone regenerability of the rhBMP-2/PLA-DX-PEG/ β-TCP composite was studied in a critical-sized rabbit bone defect model. In an initial study, a composite of PLA-DX-PEG (250 mg) and β-TCP (300 mg) loaded with or without rhBMP2 (50 μg) was implanted into a 1.5 cm intercalated bone defect created in a rabbit femur. Defects were assessed by biweekly radiography until 8 weeks postoperatively. The bony union of the defect was recognized only in the BMP-loaded group. To obtain further data on biomechanical and remodeling properties, another BMP-loaded composites group was made and observed up to 24 weeks. All defects were completely repaired without residual traces of implants. Anatomical and mechanical properties of the repaired bone examined by histology, 3-dimensional CT (3D-CT) and mechanical testing were essentially equivalent to the nonoperated-on femur at 24 weeks. These experimental results indicate that fully absorbable rhBMP-2/PLA-DX-PEG/ β-TCP is a promising composite having osteogenicity efficient enough for repairing large bone defects.