Critical-sized Femur Defects Research Articles

Combination of Adipose-Derived Stromal/Stem Cells and Decellularized Adipose Tissue Hydrogel for Osteogenic Applications.

Adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue (DAT) are adipose tissue products obtained from individuals undergoing fat removal procedures like liposuction, lipectomy, or breast reduction. DAT hydrogel is prepared by removing the cells from the adipose tissue and digesting it to form a liquid material that forms a gel at physiological temperature. ASCs seeded on DAT have displayed osteogenic potential in vitro and in animal models of bone defects. Herein, we describe the methods for preparing DAT hydrogel, ASC seeding in DAT hydrogel, osteogenic differentiation of ASCs, creation of critical-sized femur defect model in mice, its treatment with ASC-DAT hydrogel, and analyses.

Biomimetic Structural Protein Based Magnetic Responsive Scaffold for Enhancing Bone Regeneration by Physical Stimulation on Intracellular Calcium Homeostasis.

The bone matrix has distinct architecture and biochemistry which present a barrier to synthesizing bone-mimetic regenerative scaffolds. To mimic the natural structures and components of bone, biomimetic structural decellularized extracellular matrix (ECM)/regenerated silk fibroin (RSF) scaffolds incorporated with magnetic nanoparticles (MNP) are prepared using a facile synthetic methodology. The ECM/RSF/MNP scaffold is a hierarchically organized and interconnected porous structure with silk fibroin twined on the collagen nanofibers. The scaffold demonstrates saturation magnetization due to the presence of MNP, along with good cytocompatibility. Moreover, the β-sheet crystalline domain of RSF and the chelated MNP could mimic the deposition of hydroxyapatite and enhance compressive modulus of the scaffold by ≈20%. The results indicate that an external static magnetic field (SMF) with a magnetic responsive scaffold effectively promotes cell migration, osteogenic differentiation, neogenesis of endotheliocytes in vitro, and new bone formation in a critical-size femur defect rat model. RNA sequencing reveals that the molecular mechanisms underlying this osteogenic effect involve calsequestrin-2-mediated Ca2+ release from the endoplasmic reticulum to activate Ca2+ /calmodulin/calmodulin-dependent kinase II signaling axis. Collectively, bionic magnetic scaffolds with SMF stimulation provide a potent strategy for bone regeneration through internal structural cues, biochemical composition, and external physical stimulation on intracellular Ca2+ homeostasis.

Measuring Bone Healing: Parameters and Scores in Comparison.

(1) Background: Bone healing is a complex process that can not be replicated in its entirety in vitro. Research on bone healing still requires the animal model. The critical size femur defect (CSFD) in rats is a well-established model for fractures in humans that exceed the self-healing potential. New therapeutic approaches can be tested here in vivo. Histological, biomechanical, and radiological parameters are usually collected and interpreted. However, it is not yet clear to what extent they correlate with each other and how necessary it is to record all parameters. (2) Methods: The basis for this study was data from three animal model studies evaluating bone healing. The µCT and histological (Movat pentachrome, osteocalcin) datasets/images were reevaluated and correlation analyses were then performed. Two image processing procedures were compared in the analysis of the image data. (3) Results: There was a significant correlation between the histologically determined bone fraction (Movat pentachrome staining) and bending stiffness. Bone fraction determined by osteocalcin showed no prognostic value. (4) Conclusions: The evaluation of the image datasets using ImageJ is sufficient and simpler than the combination of both programs. Determination of the bone fraction using Movat pentachrome staining allows conclusions to be drawn about the biomechanics of the bone. A standardized procedure with the ImageJ software is recommended for determining the bone proportion.

A comparative in vitro and in vivo analysis of the biological properties of the 45S5-, 1393-, and 0106-B1-bioactive glass compositions using human bone marrow-derived stromal cells and a rodent critical size femoral defect model

Since the introduction of the 45S5-bioactive glass (BG), numerous new BG compositions have been developed. Compared to the 45S5-BG, 1393-BG shows favorable processing properties due to its low crystallization tendency and the 1393-BG-based borosilicate 0106-B1-BG exhibits improved angiogenic properties due to its boron content. Despite their close (chemical) relationship, the biological properties of the mentioned BG composition have not yet been comparatively examined. In this study, the effects of the BGs on proliferation, viability, osteogenic differentiation, and angiogenic factor production of human bone marrow-derived mesenchymal stromal cells were assessed. Scaffolds made of the BGs were introduced in a critical-sized femur defect model in rats in order to analyze their impact on bone defect regeneration. In vitro, 1393-BG and 0106-B1-BG outperformed 45S5-BG with regard to cell proliferation and viability. 1393-BG enhanced osteogenic differentiation; 0106-B1-BG promoted angiogenic factor production. In vivo, 0106-B1-BG and 45S5-BG outperformed 1393-BG in terms of angiogenic and osteoclastic response resulting in improved bone regeneration. In conclusion, the biological properties of BGs can be significantly modified by tuning their composition. Demonstrating favorable processing properties and an equally strong in vivo bone regeneration potential as 45S5-BG, 0106-B1-BG qualifies as a basis to incorporate other bioactive ions to improve its biological properties.

Osteoporotic bone recovery by a bamboo-structured bioceramic with controlled release of hydroxyapatite nanoparticles.

While most bone defects can be repaired spontaneously, the healing process can be complicated due to insufficient bone regeneration when osteoporosis occurs. Synthetic materials that intrinsically stimulate bone formation without inclusion of exogenous cells or growth factors represent a highly desirable alternative to current grafting strategies for the management of osteoporotic defects. Herein, we developed a series of hydroxyapatite bioceramics composed of a microwhiskered scaffold (wHA) reinforced with multiple layers of releasable hydroxyapatite nanoparticles (nHA). These novel bioceramics (nwHA) are tunable to optimize the loading amount of nHA for osteoporotic bone formation. The utility of nwHA bioceramics for the proliferation or differentiation of osteoporotic osteoblasts in vitro is demonstrated. A much more compelling response is seen when bioceramics are implanted in critical-sized femur defects in osteoporotic rats, as nwHA bioceramics promote significantly higher bone regeneration and delay adjacent bone loss. Moreover, the nwHA bioceramics loaded with a moderate amount of nHA can induce new bone formation with a higher degree of ossification and homogenization. Two types of osteogenesis inside the nwHA bioceramic pores were discovered for the first time, depending on the direction of growth of the new bone. The current study recommends that these tailored hybrid micro/nanostructured bioceramics represent promising candidates for osteoporotic bone repair.

Acceleration of Bone Regeneration Induced by a Soft-Callus Mimetic Material.

Clinical implementation of endochondral bone regeneration (EBR) would benefit from the engineering of devitalized cartilaginous constructs of allogeneic origins. Nevertheless, development of effective devitalization strategies that preserves extracellular matrix (ECM) is still challenging. The aim of this study is to investigate EBR induced by devitalized, soft callus‐mimetic spheroids. To challenge the translatability of this approach, the constructs are generated using an allogeneic cell source. Neo‐bone formation is evaluated in an immunocompetent rat model, subcutaneously and in a critical size femur defect. Living spheroids are used as controls. Also, the effect of spheroid maturation towards hypertrophy is evaluated. The devitalization procedure successfully induces cell death without affecting ECM composition or bioactivity. In vivo, a larger amount of neo‐bone formation is observed for the devitalized chondrogenic group both ectopically and orthotopically. In the femur defect, accelerated bone regeneration is observed in the devitalized chondrogenic group, where defect bridging is observed 4 weeks post‐implantation. The authors' results show, for the first time, a dramatic increase in the rate of bone formation induced by devitalized soft callus‐mimetics. These findings pave the way for the development of a new generation of allogeneic, “off‐the‐shelf” products for EBR, which are suitable for the treatment of every patient.

The influence of different artificial extracellular matrix implant coatings on the regeneration of a critical size femur defect in rats.

Resorbable biomaterials based on artificial extracellular matrices (aECM) represent promising scaffolds for the treatment of large bone defects. Here, we investigated various glycosaminoglycan (GAG) derivatives of varying sulfation degree with respect to their influence on in vivo bone healing. The materials used in this study consisted of GAG-coated degradable polycaprolactone-co-lactide (PCL). Critical size femur defects in rats were filled with autologous bone serving as positive control or the respective coated or uncoated PCL scaffolds. After 2 and 12 weeks, progress in the healing process was investigated by analyzing the new bone matrix formation, the collagen content and hydroxyapatite formation by using micro-computed tomography (μCT), biomechanical testing, nuclear magnetic resonance spectroscopy (NMR) and histology. The sulfated GAG coating contributed substantially to bone regeneration, increased collagen synthesis and initiated mineralization of the organic matrix. Most substantial collagen production was detected in scaffolds coated with chondroitin sulfate. Scaffolds coated with hypersulfated hyaluronan induced formation of new bone volume comparable to what was observed in the positive control. GAG differing in the sugar backbone and degree of sulfation modulate the healing process at different times, eventually leading to improved bone healing.

Bone regeneration by bone morphogenetic protein-2 from porous beads with leaf-stacked structure for critical-sized femur defect model in dogs.

The objective of this study was to investigate whether porous beads with a leaf-stacked scaffold loaded with bone morphogenetic protein-2, with sustained release for up to four weeks, were beneficial to a dog model of critical long bone defects and capable of promoting bone regeneration without side effects. Critical long bone defects were created in the femoral diaphyses of 21 dogs. Each critical defect was fixed with an intramedullary pin and an universal locking plate. Leaf-stacked scaffold beads were implanted into the defect, which was covered with a membrane. Nineteen samples were evaluated 4, 8, and 12 weeks after surgery using simple radiographs, serum chemistry measurements, histological staining, and gene expression analysis. Sustained bone morphogenetic protein-2 release from the leaf-stacked scaffold continued for four weeks and consistently affected the expression of early and late osteogenesis factors. The results suggest that sustained bone morphogenetic protein-2 release is effective for bone regeneration, with minimal side effects.

Influence of the induced membrane filled with syngeneic bone and regenerative cells on bone healing in a critical size defect model of the rat’s femur

IntroductionThe induced membrane technique for the treatment of large bone defects consists of a 2-stage procedure. In the first stage, a polymethylmethacrylate (PMMA) cement spacer is inserted into the bony defect of a rat’s femur and over a period of 2–4 weeks a membrane forms that encapsulates the defect/spacer. In a second operation the membrane is opened, the PMMA spacer is removed and the resulting cavity is filled with autologous bone.Since little effort has been made to replace the need for autologous bone this study was performed to elucidate the influence of different stem cells and the membrane itself on bone healing in a critical size femur defect model in rats.Especially the question should be addressed whether the use of stem cells seeded on a β-TCP scaffold is equivalent to syngeneic bone as defect filling in combination with the induced membrane technique. Materials and MethodsA total of 96 male Sprague-Dawley (SD) rats received a 10 mm critical size defect of the femur, which was stabilized by a plate osteosynthesis and filled with PMMA cement. In a second step the spacer was extracted and the defects were filled with syngeneic bone, β-TCP with MSC + EPC or BM-MNC. In order to elucidate the influence of the induced membrane on bone defect healing the induced membrane was removed in half of the operated femurs. The defect area was analysed 8 weeks later for bone formation (osteocalcin staining), bone mineral density (BMD) and bone strength (3-point bending test). ResultsNew bone formation, bone mineral density and bone stiffness increased significantly, if the membrane was kept. The transplantation of biologically active material (syngeneic bone, stem cells on b-TCP) into the bone defect mostly led to a further increase of bone healing. Syngeneic bone had the greatest impact on bone healing however defects treated with stem cells were oftentimes comparable. ConclusionFor the first time we demonstrated the effect of the induced membrane itself and different stem cells on critical size defect healing. This could be a promising approach to reduce the need for autologous bone transplantation with its’ limited availability and donor site morbidity.

The osteo-inductive activity of bone-marrow-derived mononuclear cells resides within the CD14+ population and is independent of the CD34+ population.

Bone marrow mononuclear cells (BMC) seeded on a scaffold of β-tricalcium phosphate (β-TCP) promote bone healing in a critical-size femur defect model. Being BMC a mixed population of predominantly mature haematopoietic cells, which cell type(s) is(are) instrumental for healing remains elusive. Although clinical therapies using BMC are often dubbed as stem cell therapies, whether stem cells are relevant for the therapeutic effects is unclear and, at least in the context of bone repair, seems dubious. Instead, in light of the critical contribution of monocytes and macrophages to tissue development, homeostasis and injury repair, in the current study it was hypothesised that BMC-mediated bone healing derived from the stem cell population. To test this hypothesis, bone remodelling studies were performed in an established athymic rats critical-size femoral defect model, with β-TCP scaffolds augmented with complete BMC or BMC immunomagnetically depleted of stem cells (CD34+) or monocytes/macrophages (CD14+). Bone healing was assessed 8 weeks after transplantation. Compared to BMC-augmented controls, when CD14- BMC, but not CD34- BMC were transplanted into the bone defect, femora possessed dramatically decreased biomechanical stability and new bone formation was markedly reduced, as measured by histology. The degree of vascularisation did not differ between the two groups. It was concluded that the monocyte fraction within the BMC provided critical osteo-inductive cues during fracture healing. Which factors were responsible at the molecular levels remained elusive. However, this study marked a significant progress towards elucidating the mechanisms by which BMC elicit their therapeutic effects, at least in bone regeneration.

Teriparatide promotes healing of critical size femur defect through accelerating angiogenesis and degradation of β-TCP in OVX osteoporotic rat model

Accumulating evidence suggests that early angiogenesis has an important effect on the healing of injury. Teriparatide (PTH) is extensively applied for its potent anabolic activity on bone, while little is known about its angiogenic ability which may facilitate new bone formation. In this study, we tested the angiogenic ability of PTH and its effect on degradation of β-tricalcium phosphate (β-TCP) in an ovariectomized (OVX) rat distal femoral metaphysis model. After successful establishment of the OVX model was confirmed, a critical size defect was drilled into each distal femur of the OVX rats. Afterwards all animals were randomly divided into three groups: control group, group β-TCP and group β-TCP+PTH, then rats of group β-TCP+PTH were injected Teriparatide (30 μg/kg) subcutaneous every other day. Four weeks after femur surgery, five specimens from each group were used for Microfil perfusion to reveal blood vessels in the bone defect. The residual rats were harvested for micro-computed tomography, histological analysis and immunochemistry. The results showed Teriparatide facilitated neovascularization, degradation of β-TCP and new bone formation in combination with β-TCP, which may be relevant to neovascularization in an early phase.

Combined delivery of FGF-2, TGF-β1, and adipose-derived stem cells from an engineered periosteum to a critical-sized mouse femur defect.

Critical-sized long bone defects suffer from complications including impaired healing and non-union due to substandard healing and integration of devitalized bone allograft. Removal of the periosteum contributes to the limited healing of bone allografts. Restoring a periosteum on bone allografts may provide improved allograft healing and integration. This article reports a polysaccharide-based tissue engineered periosteum that delivers basic fibroblast growth factor (FGF-2), transforming growth factor-β1 (TGF-β1), and adipose-derived mesenchymal stem cells (ASCs) to a critical-sized mouse femur defect. The tissue engineered periosteum was evaluated for improving bone allograft healing and incorporation by locally delivering FGF-2, TGF-β1, and supporting ASCs transplantation. ASCs were successfully delivered and longitudinally tracked at the defect site for at least 7 days post operation with delivered FGF-2 and TGF-β1 showing a mitogenic effect on the ASCs. At 6 weeks post implantation, data showed a non-significant increase in normalized bone callus volume. However, union ratio analysis showed a significant inhibition in allograft incorporation, confirmed by histological analysis, due to loosening of the nanofiber coating from the allograft surface. Ultimately, this investigation shows our tissue engineered periosteum can deliver FGF-2, TGF-β1, and ASCs to a mouse critical-sized femur defect and further optimization may yield improved bone allograft healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 900-911, 2017.

Design and Fabrication of Complex Scaffolds for Bone Defect Healing: Combined 3D Plotting of a Calcium Phosphate Cement and a Growth Factor-Loaded Hydrogel

Additive manufacturing enables the fabrication of scaffolds with defined architecture. Versatile printing technologies such as extrusion-based 3D plotting allow in addition the incorporation of biological components increasing the capability to restore functional tissues. We have recently described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of an oil-based CPC paste under mild conditions. In the present study, we have developed a strategy for growth factor loading based on multichannel plotting: a biphasic scaffold design was realised combining CPC with VEGF-laden, highly concentrated hydrogel strands. As hydrogel component, alginate and an alginate-gellan gum blend were evaluated; the blend exhibited a more favourable VEGF release profile and was chosen for biphasic scaffold fabrication. After plotting, two-step post-processing was performed for both, hydrogel crosslinking and CPC setting, which was shown to be compatible with both materials. Finally, a scaffold was designed and fabricated which can be applied for testing in a rat critical size femur defect. Optimization of CPC plotting enabled the fabrication of highly resolved structures with strand diameters of only 200µm. Micro-computed tomography revealed a precise strand arrangement and an interconnected pore space within the biphasic scaffold even in swollen state of the hydrogel strands.

Alteration of Masquelet's induced membrane characteristics by different kinds of antibiotic enriched bone cement in a critical size defect model in the rat's femur

The Masquelet technique for the treatment of large bone defects consists of a 2-stage procedure. In the first stage, a polymethylmethacrylate (PMMA) cement spacer is inserted into the bony defect of a rat's femur and over a period of 2–4 weeks a membrane forms that encapsulates the defect/spacer. In a second operation the membrane is opened, the PMMA spacer is removed and the resulting cavity is filled with autologous bone. Different kinds of bone cements are available, with or without supplemental antibiotics. Both might influence the development and the characteristics of the induced membrane which might affect the bone healing response. Hence, this comparative study was performed to elucidate the effect of different bone cements with or without supplemental antibiotics on the development of an induced membrane in a critical size femur defect model in rats.A total of 72 male SD rats received a 10mm critical size defect of the femur which was stabilised by a plate osteosynthesis and filled with either Palacos+Gentamycin, Copal Gentamycin+Vancomycin, Copal+Gentamycin+Clindamycin or Copal Spacem. The induced membranes were analysed after two, four and six weeks (wks) after insertion of the cement spacers (n=6/group). Paraffin embedded histological sections of the membrane were microscopically analysed for membrane thickness, elastic fibres, vascularisation and proliferation by an independent observer blinded to the group setup.The thickness of the induced membrane increased significantly from 2 wks (553μm) to 6 wks (774μm) in group Palacos+Gentamycin whereas membrane thickness decreased significantly in groups Copal+Gentamycin+Clindamycin (682–329μm) and Copal Spacem (916μm to 371μm). The comparison between the groups revealed significantly increased membrane thickness in group Palacos+Gentamycin and Copal Gentamycin+Vancomycin in comparison to group Copal+Gentamycin+Clindamycin six weeks after induction. However, the fraction of elastic fibres was significantly increased in groups Copal+Gentamycin+Clindamycin (71%, 80%) and Copal Spacem (82%, 81%) after 2 and 4 weeks in comparison to the groups Palacos+Gentamycin (56%, 57%) and Copal Gentamycin+Vancomycin (63%, 69%). Those differences however were partly diminished after 6 wks. The ratio of immature (vWF+) to more mature (CD31+) blood vessels increased significantly in groups Palacos+Gentamycin and Copal Gentamycin+Vancomycin whereas no significant alterations were noted in groups Copal+Gentamycin+Clindamycin and Copal Spacem.For the first time we demonstrated that thickness and proportion of elastic fibres in induced membranes were influenced by the type of cement and the kind of supplemental antibiotics being used. Whether these alterations of the induced membrane have an effect on bone healing remains to be proven in future studies.

BMP-2-transduced human bone marrow stem cells enhance neo-bone formation in a rat critical-sized femur defect.

Synthetic graft materials are considered as possible substitutes for cancellous bone, but lack osteogenic and osteoinductive properties. In this study, we investigated how composite scaffolds of βTCP containing osteogenic human bone marrow mesenchymal stem cells (hBMSCs) and osteoinductive bone morphogenetic protein-2 (BMP-2) influenced the process of fracture healing. hBMSCs were loaded into βTCP scaffolds 24 h before implantation in a rat critical-sized bone defect. hBMSCs were either stimulated with rhBMP-2 or transduced with BMP-2 by gene transfer. The effect of both protein stimulation and gene transfer was compared for osteogenic outcome. X-rays were conducted at weeks 0, 1, 3, 6, 9 and 12 post-operatively. In addition, bone-labelling fluorochromes were applied at 0, 3, 6 and 9 weeks. Histological analysis was performed for the amount of callus tissue and cartilage formation. At 6 weeks, the critical-sized defect in 33% of the rats treated with the Ad-BMP-2-transduced hBMSCs/βTCP scaffolds was radiographically bridged. In contrast, in only 10% of the rats treated with rhBMP2/hBMSCs, 12 weeks post-treatment, the bone defect was closed in all treated rats of the Ad-BMP-2 group except for one. Histology showed significantly higher amounts of callus formation in both Ad-BMP-2- and rhBMP-2-treated rats. The amount of neocartilage was less pronounced in both BMP-2-related groups. In summary, scaffolds with BMP-2-transduced hBMSCs performed better than those with the rhBMP2/hBMSCs protein. These results suggest that combinations of osteoconductive biomaterials with genetically modified MSCs capable of secreting osteoinductive proteins may represent a promising alternative for bone regeneration. Copyright © 2015 John Wiley & Sons, Ltd.

In vivo experimental study on bone regeneration in critical bone defects using PIB nanogels/boron-containing mesoporous bioactive glass composite scaffold.

PurposeIn the present study, the fabrication of novel p(N-isopropylacrylamide-co-butyl methylacrylate) (PIB) nanogels was combined with boron-containing mesoporous bioactive glass (B-MBG) scaffolds in order to improve the mechanical properties of PIB nanogels alone. Scaffolds were tested for mechanical strength and the ability to promote new bone formation in vivo.Patients and methodsTo evaluate the potential of each scaffold in bone regeneration, ovariectomized rats were chosen as a study model to determine the ability of PIB nanogels to stimulate bone formation in a complicated anatomical bone defect. PIB nanogels and PIB nanogels/B-MBG composites were respectively implanted into ovariectomized rats with critical-sized femur defects following treatment periods of 2, 4, and 8 weeks post-implantation.ResultsResults from the present study demonstrate that PIB nanogels/B-MBG composites showed greater improvement in mechanical strength when compared to PIB nanogels alone. In vivo, hematoxylin and eosin staining revealed significantly more newly formed bone in defects containing PIB nanogels/B-MBG composite scaffolds when compared to PIB nanogels alone. Tartrate-resistant acid phosphatase-positive staining demonstrated that both scaffolds were degraded over time and bone remodeling occurred in the surrounding bone defect as early as 4 weeks post-implantation.ConclusionThe results from the present study indicate that PIB nanogels are a potential bone tissue engineering biomaterial able to treat defects of irregular shapes and deformities as an injectable, thermoresponsive, biocompatible hydrogel which undergoes rapid thermal gelation once body temperature is reached. Furthermore, its combination with B-MBG scaffolds improves the mechanical properties and ability to promote new bone formation when compared to PIB nanogels alone.

<i>In Vivo</i> Osteogenic Effect of Porous β- CaSiO<sub>3</sub>/PDLGA Composite Scaffolds

The aim of the present study is to evaluate the in vivo biological behaviour of porous β-CS/PDLGA scaffolds. The scaffolds were implanted in critical-sized femur defects ( 6 ×10 mm) for 4, 12 and 20 weeks with β-TCP scaffolds as the control. The in vivo bone regeneration of the scaffolds were investigated using sequential histological evaluations and Micro-CT technology. Results showed that the β-CS/PDLGA scaffolds could stimulate bone regeneration and degrade progressively at a rate proportionate with the regeneration of new bone as compared with β-TCP scaffolds. The present study suggested the potential application of β-CS/PDLGA scaffolds in hard tissue regeneration.

Delivery of PDGF-B and BMP-7 by mesoporous bioglass/silk fibrin scaffolds for the repair of osteoporotic defects

Osteoporosis is a chronic disease affecting millions of people worldwide caused by an imbalance between bone-forming osteoblasts and bone-resorbing osteoclasts. Despite recent developments in pharmacological agents to prevent osteoporotic-related fractures, much less attention has been placed on the repair of bone defects following fracture. Critical to this process is the recruitment of mesenchymal stem cells (MSCs) to defect sites by growth factors. One method which has been effective for the sustained release of growth factors is that of gene therapy. The aim of the present study was to investigate newly developed mesoporous bioglass/silk fibrin scaffolds containing adPDGF-b and adBMP-7 into osteoporotic critical-sized femur defects in ovariectomised rats following treatment periods of 2 and 4 weeks. In vivo osteogenetic efficiency evaluated by μ-CT analysis, hematoxylin and eosin staining, and immunohistochemical (type I collagen, osteopontin and BSP) revealed significantly new bone formation in defects containing adenovirus for both PDGF-b and BMP-7 when compared to scaffolds alone and scaffolds containing BMP-7. TRAP-positive staining also demonstrated the ability for these scaffolds to be degraded over time and initiate bone turnover/remodeling. Although the use of gene therapy for clinical applications is still in its infancy, results from the present study demonstrate their potent ability to recruit mesenchymal progenitor cells through sustained release of PDGF-b and BMP-7 which may be beneficial for patients suffering from osteoporotic-related fractures.

The enhancement of bone regeneration by a combination of osteoconductivity and osteostimulation using β-CaSiO3/β-Ca3(PO4)2 composite bioceramics

β-Tricalcium phosphate (β-TCP) is osteoconductive, while β-calcium silicate (β-CS) is bioactive with osteostimulative properties. Porous β-CaSiO3/β-Ca3(PO4)2 composite bioceramic scaffolds with various β-TCP:β-CS ratios were designed to combine both osteoconductivity and osteostimulation in order to enhance bone regeneration. The composite scaffolds were implanted in critical sized femur defects (6×12mm) for 4, 12 and 26weeks with pure β-TCP and β-CS scaffolds as the controls. The in vivo biodegradation and bone regeneration of the specimens were investigated using sequential histological evaluations, immunohistochemical examination and micro-computed tomography technology. The results showed that the scaffolds with 50 and 80wt.% β-CS dramatically enhanced the amount of newly formed bone and reduced the degradation rate. In contrast, porous β-CS displayed poor new bone formation due to its rapid degradation, while porous β-TCP showed moderate bone regeneration starting on the surface of the implants, due to a lack of osteostimulation. More importantly, the scaffolds with 50 and 80wt.% β-CS not only had excellent osteoconductivity, but also stimulated rapid bone formation, and they could degrade progressively at a rate matching the regeneration of new bone. In summary, our findings indicated that the degradation rate and bioactivity of β-CS/β-TCP composite bioceramic scaffolds could be adjusted by controlling the ratio of β-CS to β-TCP, suggesting the potential application of β-CS/β-TCP composite bioceramic scaffolds with 50 and 80wt.% β-CS component in hard tissue regeneration and bone tissue engineering.

The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation

Regenerating bone tissue involves complex, temporal and coordinated signal cascades of which bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF 165) play a prominent role. The aim of this study was to determine if the delivery of human bone marrow stromal cells (HBMSC) seeded onto VEGF 165/BMP-2 releasing composite scaffolds could enhance the bone regenerative capability in a critical sized femur defect. Alginate-VEGF 165/P DLLA-BMP-2 scaffolds were fabricated using a supercritical CO 2 mixing technique and an alginate entrapment protocol. Increased release of VEGF 165 (750.4 ± 596.8 ρg/ml) compared to BMP-2 (136.9 ± 123.4 ρg/ml) was observed after 7-days in culture. Thereafter, up till 28 days, an increased rate of release of BMP-2 compared to VEGF 165 was observed. The alginate-VEGF 165/P DLLA-BMP-2 + HBMSC group showed a significant increase in the quantity of regenerated bone compared to the alginate-VEGF 165/P DLLA-BMP-2 and alginate/P DLLA groups respectively in a critical sized femur defect study as indices measured by μCT. Histological examination confirmed significant new endochondral bone matrix in the HBMSC seeded alginate-VEGF 165/P DLLA-BMP-2 defect group in comparison to the other groups. These studies demonstrate the ability to deliver a combination of HBMSC with angiogenic and osteogenic factors released from biodegradable scaffold composites enhances the repair and regeneration of critical sized bone defects.