Repair Of Critical Bone Defects Research Articles

Histological Evaluation of a New Beta-Tricalcium Phosphate/Hydroxyapatite/Poly (1-Lactide-Co-Caprolactone) Composite Biomaterial in the Inflammatory Process and Repair of Critical Bone Defects

Background: The use of biomaterials is commonplace in dentistry for bone regeneration. The aim of this study was to evaluate the performance of a new alloplastic material for bone repair in critical defects and to evaluate the extent of the inflammatory process. Methods: Forty-five New Zealand rabbits were divided into five groups according to evaluation time (7, 14, 30, 60, 120 days), totaling 180 sites with six-millimeter diameter defects in their tibiae. The defects were filled with alloplastic material consisting of poly (lactide-co-caprolactone), beta-tricalcium phosphate, hydroxyapatite and nano-hydroxyapatite (BTPHP) in three different presentations: paste, block, and membrane. Comparisons were established with reference materials, such as Bio-ossTM, Bio-oss CollagenTM, and Bio-gideTM, respectively. The samples were HE-stained and evaluated for inflammatory infiltrate (scored for intensity from 0 to 3) and the presence of newly formed bone at the periphery of the defects. Results: Greater bone formation was observed for the alloplastic material and equivalent inflammatory intensity for both materials, regardless of evaluation time. At 30 days, part of the synthetic biomaterial, regardless of the presentation, was resorbed. Conclusions: We concluded that this novel alloplastic material showed osteoconductive potential, biocompatibility, low inflammatory response, and gradual resorption, thus an alternative strategy for guided bone regeneration.

Vascularized Periosteal Flaps Accelerate Osteointegration and Revascularization of Allografts in Rats.

Surgical reconstruction of large bone defects with structural bone allografts can restore bone stock but is associated with complications such as nonunion, fracture, and infection. Vascularized reconstructive techniques may provide an alternative in the repair of critical bone defects; however, no studies specifically addressing the role of vascularized periosteal flaps in stimulating bone allograft revascularization and osseointegration have been reported. (1) Does a vascularized periosteal flap increase the likelihood of union at the allograft-host junction in a critical-size defect femoral model in rats? (2) Does a vascularized periosteal flap promote revascularization of a critical-size defect structural bone allograft in a rat model? (3) What type of ossification occurs in connection with a vascularized periosteal flap? Sixty-four rats were assigned to two equal groups. In both the control and experimental groups, a 5-cm critical size femoral defect was created in the left femur and then reconstructed with a cryopreserved structural bone allograft and intramedullary nail. In the experimental group, a vascularized periosteal flap from the medial femoral condyle, with a pedicle based on the descending genicular vessels, was associated with the allograft. The 32 rats of each group were divided into subgroups of 4-week (eight rats), 6-week (eight rats), and 10-week (16 rats) followup. At the end of their assigned followup periods, the animals were euthanized and their femurs were harvested for semiquantitative and quantitative analysis using micro-CT (all followup groups), quantitative biomechanical evaluation (eight rats from each 10-week followup group), qualitative confocal microscopic, backscattered electron microscopic, and histology analysis (4-week and 6-week groups and eight rats from each 10-week followup group). When making their analyses, all the examiners were blinded to the treatment groups from which the samples came. There was an improvement in allograft-host bone union in the 10-week experimental group (odds ratio [OR], 19.29 [3.63-184.50], p < 0.05). In contrast to control specimens, greater bone neoformation in the allograft segment was observed in the experimental group (OR [4-week] 63.3 [39.6-87.0], p < 0.05; OR [6-week] 43.4 [20.5-66.3], p < 0.05; OR [10-week] 62.9 [40.1-85.7], p < 0.05). In our biomechanical testing, control samples were not evaluable as a result of premature breakage during the embedding and assembly processes. Therefore, experimental samples were compared with untreated contralateral femurs. No difference in torsion resistance pattern was observed between both groups. Both backscattered electron microscopy and histology showed newly formed bone tissue and osteoclast lacunae, indicating a regulated process of bone regeneration of the initial allograft in evaluated samples from the experimental group. They also showed intramembranous ossification produced by the vascularized periosteal flap in evaluated samples from the experimental group, whereas samples from the control group showed an attempted endochondral ossification in the allograft-host bone junctions. A vascularized periosteal flap promotes and accelerates allograft-host bone union and revascularization of cryopreserved structural bone allografts through intramembranous ossification in a preclinical rat model. If large-animal models substantiate the findings made here, this approach might be used in allograft reconstructions for critical defects using fibular or tibial periosteal flaps as previously described.

Bone repair with raloxifene and bioglass nanoceramic composite in animal experiment

ABSTRACTOBJECTIVE: This study proposes to evaluate, by means of microtomographic analysis, the topical potential of raloxifene combined with BioGran® through the sonochemical method in the repair of critical bone defects in the calvaria of rats. The hypothesis was that the homogenization of Raloxifene to Biogran at the 20% concentration would improve the bone repair at the grafted site.MATERIALS AND METHODS: A 5-mm calvaria bone defect was induced in three groups: CTR (100% BioGran®); RAL10 (90% BioGran® and 10% raloxifene), and RAL20 (80% BioGran® and 20% raloxifene). The animals were euthanized after 30 days and the microCT analysis was then performed to evaluate the parameters bone volume (BV), bone volume percentage (BV/TV), trabecular bone thickness (Tb.Th), and the separation and number of trabeculae (Tb.Sp and Tb.N). The obtained results were compared using ANOVA and Tukey test (p < 0.05).RESULTS: The best results were found for the CTR and RAL20 groups, in which the BV, BV/TV, Tb.Sp, and Tb.N parameters were statistically significant in comparison with RAL10 (p < 0.05).CONCLUSIONS: In view of the results obtained in this experiment, we can conclude that BioGran® alone or in an 80/20 mass concentration with raloxifene can lead to favorable bone formation.

Bone repair of critical-sized defects in Wistar rats treated with autogenic, allogenic or xenogenic bone grafts alone or in combination with natural latex fraction F1

Bone grafts are used in the medical-surgical field for anatomical and functional reconstruction of lost bone areas, aiding the bone repair process by osteogenesis, osteinduction and osteoconduction. New materials such as F1 (fraction 1) protein extracted from the rubber tree Hevea brasiliensis have been investigated and currently present important properties for tissue repair, and are associated with neoangiogenesis, promoting cell adhesion and extracellular matrix formation. The main objective of this study was to investigate the association of F1 protein to different bone grafts in the repair of critical bone defects in the calvaria of Wistar rats. A total of 112 Wistar rats were divided as follows: autograft (AuG), allograft (AlG), xenograft (XeG), autograft/F1 (AuG–F1), allograft/F1 (AlG–F1), xenograft/F1 (XeG–F1), F1 (F1), control (CTL), with a waiting period of 4 and 6 weeks (w). The stereological AuG, AlG, AuG–F1 and AlG–F1 results had greater bone neoformation (p < 0.05). For immunohistochemistry, the angiogenic and osteogenic factors were higher for AuG–F1 and AlG–F1. TRAP-positive cells were higher in XeG–F1 and AlG (37 ± 9.53, 13.3 ± 4.16) (4 w) and XeG, AlG–F1 and XeG–F1 (20.33 ± 7.37; 15.25 ± 6.02, 19.33 ± 3.21) (6 w). For zymography, F1 showed increased gelatinolytic activity of MMP-2 and -9. It was concluded that the bone graft associated or not with F1 increases the angiogenic and osteogenic, biochemical and stereological factors.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy&lt;em&gt; In Vivo&lt;/em&gt; in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Enhanced bone repair induced by human adipose-derived stem cells on osteogenic extracellular matrix ornamented small intestinal submucosa.

Our aim was to design an osteogenic extracellular matrix (ECM) coated bioscaffold and to apply it to critical bone defect repair with adipose-derived stem cells (ADSCs). Morphology of scaffolds was scanned by scanning electron microscope. Cell adhesion, proliferation and osteogenic differentiation of ADSCs on ECM-small intestinal submucosa (SIS) were evaluated by immunofluorescences staining, cell counting kit-8 and real-time qPCR, respectively. A mouse calvarial defect model was used to assess effects on bone regeneration in vivo. Abundant ECM was coated on SIS, which facilitated cell adhesion and proliferation of ADSCs. ECM-SIS induced osteogenic differentiation of ADSCs even without osteogenic inductive factors. Bone regeneration in vivo was enhanced by ECM-SIS + ADSCs via BMP/SMAD pathway. This work suggested a biofabricated SIS scaffold coated with osteogenic ECM-facilitated bone regeneration with ADSCs synergistically.

Osteoblast-seeded bioglass/gelatin nanocomposite: a promising bone substitute in critical-size calvarial defect repair in rat.

Amid the plethora of methods to repair critical bone defects, there is no one perfect approach. In this study, we sought to evaluate a potent 3-dimensional (3D) bioactive SiO2-CaO-P2O5 glasses (bioglass)/gelatin (gel) scaffold for its biocompatibility by seeding cells as well as for its regenerative properties by animal implantation. Osteoblast cells were seeded onto nanocomposite scaffolds to investigate the process of critical-size calvarial defect via new bone formation. Scanning electron microscopy (SEM) was used to validate topography of the scaffolds, its homogeneity and ideal cellular attachment. Proliferation assay and confocal microscopy were used to evaluate its biocompatibility. To validate osteogenesis of the bioactive nanocomposite scaffolds, they were first implanted into rats and later removed and analyzed at different time points post mortem using histological, immunohistochemical and histomorphometric methods. Based on in vitro results, we showed that our nanocomposite is highly cell-compatible material and allows for osteoblasts to adhere, spread and proliferate. In vivo results indicate that our nanocomposite provides a significant contribution to bone regeneration and is highly biodegradable and biocompatible. So, seeded scaffolds with osteoblasts enhanced repair of critical bone defects via osteogenesis. We demonstrate the feasibility of engineering a nanocomposite scaffold with an architecture resembling the human bone, and provide proof-of-concept validation for our scaffold using a rat animal model.

Controlling Arteriogenesis and Mast Cells Are Central to Bioengineering Solutions for Critical Bone Defect Repair Using Allografts.

Although most fractures heal, critical defects in bone fail due to aberrant differentiation of mesenchymal stem cells towards fibrosis rather than osteogenesis. While conventional bioengineering solutions to this problem have focused on enhancing angiogenesis, which is required for bone formation, recent studies have shown that fibrotic non-unions are associated with arteriogenesis in the center of the defect and accumulation of mast cells around large blood vessels. Recently, recombinant parathyroid hormone (rPTH; teriparatide; Forteo) therapy have shown to have anti-fibrotic effects on non-unions and critical bone defects due to inhibition of arteriogenesis and mast cell numbers within the healing bone. As this new direction holds great promise towards a solution for significant clinical hurdles in craniofacial reconstruction and limb salvage procedures, this work reviews the current state of the field, and provides insights as to how teriparatide therapy could be used as an adjuvant for healing critical defects in bone. Finally, as teriparatide therapy is contraindicated in the setting of cancer, which constitutes a large subset of these patients, we describe early findings of adjuvant therapies that may present future promise by directly inhibiting arteriogenesis and mast cell accumulation at the defect site.

Fabrication of mesoporous calcium silicate/calcium phosphate cement scaffolds with high mechanical strength by freeform fabrication system with micro-droplet jetting

In this study, we explored the feasibility of fabrication bioactive mesoporous calcium silicate/calcium phosphate cements (MCS/CPC) scaffolds with high mechanical strength by Freeform Fabrication System with Micro-Droplet Jetting. After preparation of ordered mesoporous calcium silicate (MCS) powder, ready-to-use MCS/CPC paste was formed by mixing calcium phosphate cement (CPC) powder and MCS powder with the binder polyvinyl alcohol (PVA) aqueous solution at a certain ratio of powder to liquid. MCS/CPC scaffolds with various architectures, pore sizes, and interconnectivity were then directly printed at room temperature using MCS/CPC paste. The mechanical strength, apatite formation, degradation rate, and cytocompatibility of the composite scaffolds were systematically investigated. The results showed that MCS/CPC paste exhibited outstanding printability to form MCS/CPC scaffolds. The hybrid MCS/CPC scaffolds with predefined pore size of 350 μm showed fast degradation rate, high mechanical strength, and good cytocompatibility. It was indicated that the hybrid MCS/CPC scaffolds might be a promising candidate for critical bone defect repair.

Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability.

Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects.

Avaliação histomorfométrica da associação entre biovidro e osso bovino liofilizado no tratamento de defeitos ósseos críticos criados em calvárias de ratos. Estudo piloto

OBJETIVO: Avaliar histomorfometricamente o efeito de biovidro (B), osso bovino liofilizado (OB) ou da mistura desses dois biomateriais (B/OB - 1:1) no reparo de defeitos ósseos críticos em calvária de ratos. MATERIAL E MÉTODO: Defeitos ósseos (8 mm Ø) foram criados cirurgicamente na calvária de 24 ratos, distribuídos em 4 grupos com 6 animais, de acordo com o tipo de biomaterial: coágulo sanguíneo (GC), biovidro (GB), osso bovino liofilizado (GOB) e a mistura desses dois biomateriais (GB/OB). Os animais foram eutanasiados após 15 e 60 dias do procedimento cirúrgico (3 animais por período). A avaliação histológica foi baseada na descrição da morfologia dos tecidos neoformados, enquanto para a avaliação histomorfométrica foi realizada quantificação da porcentagem de tecido ósseo, de tecido conjuntivo fibroso neoformados e de biomaterial remanescente no defeito ósseo. RESULTADO: Nos dois períodos experimentais, a análise histológica apresentou neoformação óssea, principalmente nas bordas dos defeitos, e ao redor de partículas de biomateriais remanescentes. A avaliação histomorfométrica demonstrou que no período de 15 dias o grupo GC apresentou maior percentagem de tecido ósseo em relação aos demais grupos estudados, enquanto que aos 60 dias o grupo GOB apresentou maior porcentagem de tecido ósseo em relação ao grupo GB. CONCLUSÃO: O osso bovino liofilizado apresentou maior formação óssea em relação ao biovidro, mas nenhum dos biomateriais foi superior ao coágulo. A associação do biovidro e osso bovino liofilizado não adicionou vantagem à formação óssea.

Thyroid Hormone-Induced Hypertrophy in Mesenchymal Stem Cell Chondrogenesis Is Mediated by Bone Morphogenetic Protein-4

Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair.

Hydrogel of polysaccharide of sugarcane molasses as carrier of bone morphogenetic protein in the reconstruction of critical bone defects in rats

To evaluate the benefit of using carriers such as the biopolymer gel (hidrogel of polysaccharide of sugarcane molasses) associated with the bone morphogenetic proteins (BMP's) in the repair of critical bone defects in calvaria of Wistar rats. Forty-two rats were submitted to a surgical calvaria bone defects. These animals were divided into two experimental groups, positive control group and negative control group. The Group I the calvaria defect was filled up with biopolymer gel, biological membrane, BMP and lyophilized graft. The Group II was treated with biopolymer gel, BMP and lyophilized graft. And the group III (positive control group) was treated with BMP, lyophilized graft and biological membrane. In the negative control group (Group IV) a defect was made in the rat calvaria and the animals were sacrificed immediately after the surgery. The animals of experimental groups and positive control group were slaughtered after subsequent periods of 90 and 180 days. In these periods, the histological analysis and image assessment by cone bean tomographic imaging were obtained. There was highest bone tissue formation with statistically significant results in the groups that associated biopolymer gel and membrane (Group I), followed by the group III (BMP, lyophilized graft and biological membrane). The lower bone formation occurred in the group not using the sugarcane biopolymer gel (Group II). The radiolucent areas of the analyzes of 180 days among the groups studied were respectively, 14.98 mm(2), 26.65 mm(2) and 35.81 mm(2). The biopolymer gel showed to be an excellent bone morphogenetic protein carrier, probably by facilitating the controlled release of these proteins in the process of bone repair.

Effects of Hydroxyapatite-Containing Composite Nanofibers on Osteogenesis of Mesenchymal Stem Cells In vitro and Bone Regeneration In vivo

Among a variety of polymers, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a microbial polyester, with biodegradable, nonantigenic, and biocompatible properties, is attracting more and more attention in tissue engineering. Hydroxyapatite (HA), similar to the mineral component of natural bone, is known to be osteoconductive, nontoxic, and noninflammatory. In this study, aligned and random-oriented PHBV nanofibrous scaffolds loaded with HA nanoparticles were fabricated through electrospinning technique. Mesenchymal stem cells (MSCs) derived from rat bone marrow were used to investigate the effects of HA and orientation of fibers on cell proliferation and differentiation in vitro. Cell proliferation tested with CCK-8 assay indicated that the MSCs attached and proliferated more favorably on random-oriented PHBV nanofibrous meshes without HA. After one, two and four weeks of cell seeding, osteogenic markers including alkaline phosphate (ALP), osteocalcin (OCN), and mineralized matrix deposits were detected, respectively. The results indicated that the introduction of HA could induce MSCs to differentiate into osteoblasts. Moreover, 3D PHBV/HA scaffolds made from aligned and random-oriented nanofibers were implanted into critical-sized rabbit radius defects and exhibited significant effects on the repair of critical bone defects, implying their promising applications in bone tissue engineering.

In Situ Collagen Polymerization of Layered Cell-Seeded Electrospun Scaffolds for Bone Tissue Engineering Applications

Electrospun scaffolds have been studied extensively for their potential use in bone tissue engineering applications. However, inherent issues with the electrospinning approach limit the thickness of these scaffolds and constrain their use for repair of critical-sized bone defects. One method to increase overall scaffold thickness is to bond multiple electrospun scaffolds together with a biocompatible gel. The objective of this study was to determine whether multiple human adipose-derived stem cell (hASC-seeded electrospun, nanofibrous scaffolds could be layered via in situ collagen assembly and whether the addition of laser-ablated micron-sized pores within the electrospun scaffold layers was beneficial to the bonding process. Pores were created by a laser ablation technique. We hypothesized that the addition of micron-sized pores within the electrospun scaffolds would encourage collagen integration between scaffold layers, and promote osteogenic differentiation of hASCs seeded within the layered electrospun scaffolds. To evaluate the benefit of assembled scaffolds with and without engineered pores, hASCs were seeded on individual electrospun scaffolds, hASC-seeded scaffolds were bonded with type I collagen, and the assembled ∼3-mm-thick constructs were cultured for 3 weeks to examine their potential as bone tissue engineering scaffolds. Assembled electrospun scaffolds/collagen gel constructs using electrospun scaffolds with pores resulted in enhanced hASC viability, proliferation, and mineralization of the scaffolds after 3 weeks in vitro compared to constructs using electrospun scaffolds without pores. Scanning electron microscopy and histological examination revealed that the assembled constructs that included laser-ablated electrospun scaffolds were able to maintain a contracted structure and were not delaminated, unlike assembled constructs containing nonablated electrospun scaffolds. This is the first study to show that the introduction of engineered pores in electrospun scaffolds assists with multilayered scaffold integration, resulting in thick constructs potentially suitable for use as scaffolds for bone tissue engineering or repair of critical bone defects.

Isolation, characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models

One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.

A biodegradable porous composite scaffold of PGA/β-TCP for bone tissue engineering

Polyglycolic acid (PGA) and beta-tricalcium phosphate (β-TCP) each have many applications as tissue repair materials. In this study, three-dimensional (3D) porous composite scaffolds of PGA/β-TCP (in 1:1 and 1:3 weight ratios) were fabricated using the solvent casting and particulate leaching method. PGA/β-TCP scaffolds with high porosity, interconnected 3D pores and rough surfaces were obtained and were observed using scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). The PGA/β-TCP scaffolds were investigated during the repair of critical bone defects (3 mm diameter, 2 mm depth) in rat femoral medial-epicondyles, compared with hydroxylapatite (HAP) and no implant as controls. Quantitative imageology analysis (volume and density of new bone) and qualitative histological evaluations (hematoxylin and eosin staining; tartrate-resistant acid phosphatase-hematoxylin counterstaining) were characterized using in vivo micro-CT images and histological sections at 0, 14, 30 and 90 days after surgery. Significant differences of all variables were tested by multivariate analysis (p<0.05). The results showed that the bone reformation by using the PGA/β-TCP scaffolds began within 14 days of surgery, and were healing well at 30 days after surgery. By 90 days after surgery, the bone replacement was almost completed and presented a healthy bone appearance. The new bone mineral densities (mg/cm3) with HAP, PGA/β-TCP (1:1) and PGA/β-TCP (1:3) at 90 days after surgery were: 390.4±18.1, 563.8±26.9 and 606.3±26.9, respectively. The new bone mineral density with the PGA/β-TCP scaffold was higher than with HAP (p<0.001), and with the PGA/β-TCP (1:3) scaffold was higher than with the PGA/β-TCP (1:1) scaffold at each time examined (p<0.05). The biodegradation percents (%) of HAP, PGA/β-TCP (1:1) and PGA/β-TCP (1:3) at 90 days after surgery were: 35.1±5.5, 99.0±1.0 and 96.2±3.3, respectively. The biodegradation percents of the PGA/β-TCP scaffolds were higher than HAP at each time examined (p<0.01), and matched the osteogenesis rates. The PGA/β-TCP scaffolds were almost replaced by new growing bone within 90 days after surgery. Thus the PGA/β-TCP composite scaffold, especially weight ratio 1:3, exhibited a strong ability for osteogenesis, mineralization and biodegradation for bone replacement.

Bone grafts cultured with bone marrow stromal cells for the repair of critical bone defects: An experimental study in mice

Tissue engineering of autologous bone combined with osteoprogenitor cells is a suitable strategy for filling large bone defects. The aim of this study was to evaluate the osteogenicity of a xenogenic bone graft cultured with allogenic bone marrow stromal cells (BMSC) in a mouse critical size craniotomy. Bovine trabecular bone grafts were made free of bone marrow cells or debris and were delipidated. BMSC were harvested from C57BL/6-Tg(ACTbEGFP)1Osb/J mice (GFP+ cells) and were cultured 14 days on bone grafts in control or osteogenic medium. Engineered grafts were implanted in calvarial defect in C57BL/6 mice. Four groups were studied: graft with BMSC differentiated in osteoblasts (G-Ob), graft with BMSC (G-BMSC), graft without cells (G) and no graft. Calvariae were studied 2 and 8 weeks after implantation by radiographic and histomorphometric analyses. G group: the bone ingrowth was limited to the edges of the defect. The center of the graft was filled by a fibrovascular connective tissue. G-BMSC or G-Ob groups: bone formation occurred early in the center of the defect and did not increase between 2 and 8 weeks; the newly formed woven bone was partially replaced by lamellar bone. The preoperative osteoblastic differentiation of BMSC did not allow faster and better bone regeneration. After 2 weeks, GFP+ cells were observed around the grafted bone but no GFP+ osteocyte was present in the newly formed bone. No GFP+ cell was noted after 8 weeks. However, pre-implantation culture of the biomaterial with allogenic BMSC greatly enhanced the bone regeneration.

The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model

The efficacy and safety of a material derived from human bones in the repair of critical segmental bone defects are evaluated in a rhesus monkey model. Frozen human bones were chemically and physically processed into a partially demineralized and deproteinized material in blocks. The complete tissue-engineered (TE) bone was constructed of the material preseeded with allogeneic bone marrow mesenchymal stem cells (MSCs). The material alone and the TE bone were, respectively, implanted to bridge 2.5 cm-long critical defects in right and left radii of 15 monkeys. At weeks 1, 2, 3, 6 and 12 post -implantation, the grafts were collected from three animals and assessed for the local expression of osteogenic markers, histological and roentgenographic features, and immune reactions. It was shown that defects were well repaired with both treatments whereas the bone defects in 2 additional untreated animals remained the same size after 12 weeks. In radii implanted with the TE bones, the repair processes were approximately 3 weeks faster and new bones were formed in a multipoint way. There was neither observable toxic effect nor overt immune rejection in any animals. Taken together, these observations suggest that the TE bone blocks composited of the allogeneic or xenogeneic bone-derived scaffold and allogeneic MSCs may provide an ideal method for repairing large segmental bone defects.