Optimal condition of heparin-conjugated fibrin with bone morphogenetic protein-2 for spinal fusion in a rabbit model

Background aimsHeparin-conjugated fibrin (HCF) is a carrier for long-term release of bone morphogenetic protein-2 (BMP-2) and has been shown to promote bone formation in animal models. We performed an experimental study to determine the optimal dose of BMP-2 with an HCF carrier that promotes bone formation comparable to that of autograft while minimizing complications in spinal fusion.MethodsTwenty-four rabbits underwent posterolateral fusion of the L5–6 spinal segments. Different concentrations of HCF BMP-2 (1/10, 1/20, 1/30 or 1/40) were implanted in the spines of experimental rabbits, and autograft or INFUSE was implanted in the spines of control animals. Eight weeks after treatment, spinal fusion efficacy was evaluated by plain radiography, micro-computed tomography (micro-CT), mechanical testing and histomorphometry.ResultsSimilar to autograft, the 1/40 HCF BMP-2 showed significant bone formation on micro-CT and histomorphometry with mechanical stability. However, the other HCF BMP-2 concentrations did not show significant bone formation compared with autograft. Although conventional BMP-2 (INFUSE) led to higher bone formation and stability, it also led to excessive ectopic bone and fibrous tissue formation.ConclusionsThis study suggests the optimal concentration of BMP-2 using HCF for spinal fusion, which may decrease the complications of high-dose conventional BMP-2.     

Synthetic extracellular matrices for in situ tissue engineering

Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed.     

In silico Mechano-Chemical Model of Bone Healing for the Regeneration of Critical Defects: The Effect of BMP-2

The healing of bone defects is a challenge for both tissue engineering and modern orthopaedics. This problem has been addressed through the study of scaffold constructs combined with mechanoregulatory theories, disregarding the influence of chemical factors and their respective delivery devices. Of the chemical factors involved in the bone healing process, bone morphogenetic protein-2 (BMP-2) has been identified as one of the most powerful osteoinductive proteins. The aim of this work is to develop and validate a mechano-chemical regulatory model to study the effect of BMP-2 on the healing of large bone defects in silico. We first collected a range of quantitative experimental data from the literature concerning the effects of BMP-2 on cellular activity, specifically proliferation, migration, differentiation, maturation and extracellular matrix production. These data were then used to define a model governed by mechano-chemical stimuli to simulate the healing of large bone defects under the following conditions: natural healing, an empty hydrogel implanted in the defect and a hydrogel soaked with BMP-2 implanted in the defect. For the latter condition, successful defect healing was predicted, in agreement with previous in vivo experiments. Further in vivo comparisons showed the potential of the model, which accurately predicted bone tissue formation during healing, bone tissue distribution across the defect and the quantity of bone inside the defect. The proposed mechano-chemical model also estimated the effect of BMP-2 on cells and the evolution of healing in large bone defects. This novel in silico tool provides valuable insight for bone tissue regeneration strategies.     

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.     

Bone morphogenetic protein 2-induced cellular chemotaxis drives tissue patterning during critical-sized bone defect healing: an in silico study

Critical-sized bone defects are critical healing conditions that, if left untreated, often lead to non-unions. To reduce the risk, critical-sized bone defects are often treated with recombinant human BMP-2. Although enhanced bone tissue formation is observed when BMP-2 is administered locally to the defect, spatial and temporal distribution of callus tissue often differs from that found during regular bone healing or in defects treated differently. How this altered tissue patterning due to BMP-2 treatment is linked to mechano-biological principles at the cellular scale remains largely unknown. In this study, the mechano-biological regulation of BMP-2-treated critical-sized bone defect healing was investigated using a multiphysics multiscale in silico approach. Finite element and agent-based modeling techniques were combined to simulate healing within a critical-sized bone defect (5 mm) in a rat femur. Computer model predictions were compared to in vivo microCT data outcome of bone tissue patterning at 2, 4, and 6 weeks postoperation. In vivo, BMP-2 treatment led to complete healing through periosteal bone bridging already after 2 weeks postoperation. Computer model simulations showed that the BMP-2 specific tissue patterning can be explained by the migration of mesenchymal stromal cells to regions with a specific concentration of BMP-2 (chemotaxis). This study shows how computational modeling can help us to further understand the mechanisms behind treatment effects on compromised healing conditions as well as to optimize future treatment strategies.

     

Optimized bone regeneration based on sustained release from three-dimensional fibrous PLGA/HAp composite scaffolds loaded with BMP-2

Contemporary treatment of critical bone defect remains a significant challenge in the field of orthopedic surgery. Engineered biomaterials combined with growth factors have emerged as a new treatment alternative in bone repair and regeneration. Our approach is to encapsulate bone morphogenetic protein-2 (BMP-2) into a polymeric matrix in different ways and characterize their individual performance in a nude mouse model. The main objective of this study is to examine whether the PLGA/HAp composite fibrous scaffolds loaded with BMP-2 through electrospinning can improve bone regeneration. The hypothesis is that different loading methods of BMP-2 and different HAp contents in scaffolds can alternate the release profiles of BMP-2 in vivo, therefore modify the performance of scaffolds in bone regeneration. Firstly, mechanical strength of scaffolds and HAp nanoparticles distribution in scaffolds were investigated. Secondly, nude mice experiments extended to 6 weeks were carried out to test the in vivo performance of these scaffolds, in which measurements, like serum BMP-2 concentration, ALP activity, X-ray qualification, and H&E/IHC tissue staining were utilized to monitor the growth of new bone and the changes of the corresponding biochemical parameters. The results showed that the PLGA/HAp composite scaffolds developed in this study exhibited good morphology/mechanical strength and HAp nanoparticles were homogeneously dispersed inside PLGA matrix. Results from the animal experiments indicate that the bioactivity of BMP-2 released from the fibrous PLGA/HAp composite scaffolds is well maintained, which further improves the formation of new bone and the healing of segmental defects in vivo. It is concluded that BMP-2 loaded PLGA/HAp composite scaffolds are promising for bone healing.     

Structure and function of plasminogen/plasmin system

The main physiological function of plasmin is blood clot fibrinolysis and restoration of normal blood flow. To date, however, it became apparent that in addition to thrombolysis, the plasminogen/plasmin system plays an important physiological and pathological role in a number of other essential processes: degradation of the extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, and tumor cell migration. This review focuses on structural features of plasminogen, regulation of its activation by physiological plasminogen activators, inhibitors of plasmin, and plasminogen activators, and the role of plasminogen binding to fibrin, cellular receptors, and extracellular ligands in various functions performed by plasmin thus formed.     

Effect of implanting a soft tissue autograft in a central-third patellar tendon defect: biomechanical and histological comparisons

Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon autograft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n?=?10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After 12 weeks of recovery, the animals were euthanized and their limbs were dedicated to biomechanical (n?=?7) or histological (n?=?3) evaluations. Only stiffness was improved by treatment with patellar tendon autograft relative to natural healing (p?=?0.009). Additionally, neither the patellar tendon autograft nor natural healing repairs regenerated a normal zonal insertion site between the tendon and bone. Immunohistochemical staining for collagen type II demonstrated that fibrocartilage-like tissue was regenerated at the tendon-bone interface for both repairs. However, the tissue was disorganized. Insufficient tissue integration at the tendon-to-bone junction led to repair tissue failure at the insertion site during testing. It is important to re-establish the tendon-to-bone insertion site because it provides joint stability and enables force transmission from muscle to tendon and subsequent loading of the tendon. Without loading, tendon mechanical properties deteriorate. Future studies by our laboratory will investigate potential strategies to improve patellar tendon autograft integration into bone using this model.     

Bone morphogenetic protein (BMP)1-3 enhances bone repair

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) regulate morphogenesis by processing precursors to mature functional extracellular matrix (ECM) proteins and several growth factors including TGFβ, BMP2, BMP4 and GFD8. We have recently discovered that BMP1-3 isoform of the Bmp-1 gene circulates in the human plasma and is significantly increased in patients with acute bone fracture. We hypothesized that circulating BMP1-3 might have an important role in bone repair and serve as a novel bone biomarker. When administered systemically to rats with a long bone fracture and locally to rabbits with a critical size defect of the ulna, recombinant human BMP1-3 enhanced bone healing. In contrast, neutralization of the endogenous BMP1-3 by a specific polyclonal antibody delayed the bone union. Invitro BMP1-3 increased the expression of collagen type I and osteocalcin in MC3T3-E₁ osteoblast like cells, and enhanced the formation of mineralized bone nodules from bone marrow mesenchymal stem cells. We suggest that BMP1-3 is a novel systemic regulator of bone repair.     

Transforming growth factor beta superfamily members: role in cartilage modeling

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover.     

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor beta 1 and beta 2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor beta (TGF-beta) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor beta isoforms (TGF-beta 1 and TGF-beta 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGF beta was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-beta isoforms.     

The effects of gelatin,fibrin-platelet glue and their combination on healing of the experimental critical bone defect in a rat model: radiological,histological, scanning ultrastructural and biomechanical evaluation

Fibrin-platelet glue (FPG) is a blood derivative, in which platelets and fibrinogen are concentrated in a small plasma volume, by differential centrifugation and precipitation. It can form a three-dimensional and biocompatible fibrin scaffold with a myriad of growth factors and proteins that are released progressively to the local environment and contribute to the accelerated postoperative bone healing. Gelatin (Gel) is a derivative of collagen and can promote cell adhesion and proliferation due to its unique sequence of amino acids, so it is suitable for bone tissue applications. This study examined the effects of Gel, FPG and their combinations as bone scaffold on the healing of surgically created critical-size defects in rat radius. Fifty critical size defects of 5 mm long were bilaterally created in the radial diaphysis of 25 rats. The animals were randomly divided into five equal groups as empty defect, autograft, Gel, FPG and Gel–FPG groups (n = 10 in each group). Radiographs of each forelimb were taken postoperatively on the 1st day and then at the 28th and 56th days post injury to evaluate bone formation, union and remodeling of the defect. After 56 days, the rats were euthanized and their harvested healing bone samples were evaluated by histopathology, scanning electron microscopy (SEM) and biomechanical testing. The results of present study showed that the Gel alone did not significantly affect bone healing and regeneration; however, the Gel treated defects promoted healing more than those that were left untreated (negative control). Furthermore, the FPG-enhanced grafts provided a good scaffold containing numerous growth factors for proliferation of osteoinduction and was effective in improving the structural and functional properties of the newly formed bone more than that of the untreated and also the Gel treated groups. Incorporation of Gel into the FPG scaffold improved healing potential of the FPG scaffold; however, it was still inferior to the autograft (positive control). Although the Gel–FPG scaffolds had best effectiveness during bone regeneration, it still needs to be further enhanced by incorporation of the ceramic and osteoinductive biomaterials.     

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.     

Isolation of recombinant BMP receptor IA ectodomain and its 2:1 complex with BMP-2

Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily which induces bone formation and regeneration, and important steps during early embryonic development. BMP-2 signals via oligomerization of type I and type II serine/threonine kinase receptors. We report here expression of the extracellular domain of the human type IA receptor for BMP-2 (BMPR-IA) in Escherichia coli. This soluble form of BMPR-IA (sBMPR-IA) was purified employing a BMP-2 affinity column. Gel filtration experiments and analysis of gel filtration fractions by polyacrylamide electrophoresis and densitometry reveal that BMP-2 forms a defined 1:2 complex with sBMPR-IA that can be purified and hopefully used for crystallization studies.     

Recombinant protein-co-PEG networks as cell-adhesive and proteolytically degradable hydrogel matrixes. Part II: biofunctional characteristics

We present here the biological performance in supporting tissue regeneration of hybrid hydrogels consisting of genetically engineered protein polymers that carry specific features of the natural extracellular matrix, cross-linked with reactive poly(ethylene glycol) (PEG). Specifically, the protein polymers contain the cell adhesion motif RGD, which mediates integrin receptor binding, and degradation sites for plasmin and matrix-metalloproteinases, both being proteases implicated in natural matrix remodeling. Biochemical assays as well as in vitro cell culture experiments confirmed the ability of these protein-PEG hydrogels to promote specific cellular adhesion and to exhibit degradability by the target enzymes. Cell culture experiments demonstrated that proteolytic sensitivity and suitable mechanical properties were critical for three-dimensional cell migration inside these synthetic matrixes. In vivo, protein-PEG matrixes were tested as a carrier of bone morphogenetic protein (rhBMP-2) to heal critical-sized defects in a rat calvarial defect model. The results underscore the importance of fine-tuning material properties of provisional therapeutic matrixes to induce cellular responses conducive to tissue repair. In particular, a lack of rhBMP or insufficient degradability of the protein-PEG matrix prevented healing of bone defects or remodeling and replacement of the artificial matrix. This work confirms the feasibility of attaining desired biological responses in vivo by engineering material properties through the design of single components at the molecular level. The combination of polymer science and recombinant DNA technology emerges as a powerful tool for the development of novel biomaterials.     

Gender Affects Skin Wound Healing in Plasminogen Deficient Mice

The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound closure in female versus male plasmin deficient mice. Further studies showed that this gender effect could not be reversed by ovariectomy, suggesting that female sex-hormones did not mediate the accelerated skin wound healing in plasmin deficient female mice. Histological examination of healed wounds revealed larger amounts of fibrotic scars in the provisional matrix of plasmin deficient male mice compared to female mice. These fibrotic scars correlated to an obstruction of cell infiltration of the granulation tissue, which is a prerequisite for wound healing. In conclusion, the presented data show that the gender dependent effect of plasmin deficiency is tissue specific and may be secondary to already established differences between genders, such as skin thickness and composition.     

Mesenchymal stem cells from osteoporotic patients reveal reduced migration and invasion upon stimulation with BMP-2 or BMP-7

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression.     

Differential growth factor adsorption to calvarial osteoblast-secreted extracellular matrices instructs osteoblastic behavior

Craniosynostosis (CS), the premature ossification of cranial sutures, is attributed to increased osteogenic potential of resident osteoblasts, yet the contribution of the surrounding extracellular matrix (ECM) on osteogenic differentiation is unclear. The osteoblast-secreted ECM provides binding sites for cellular adhesion and regulates the transport and signaling of osteoinductive factors secreted by the underlying dura mater. The binding affinity of each osteoinductive factor for the ECM may amplify or mute its relative effect, thus contributing to the rate of suture fusion. The purpose of this paper was to examine the role of ECM composition derived from calvarial osteoblasts on protein binding and its resultant effect on cell phenotype. We hypothesized that potent osteoinductive proteins present during sutural fusion (e.g., bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-β1)) would exhibit distinct differences in binding when exposed to ECMs generated by human calvarial osteoblasts from unaffected control individuals (CI) or CS patients. Decellularized ECMs produced by osteoblasts from CI or CS patients were incubated in the presence of BMP-2 or TGF-β1, and the affinity of each protein was analyzed. The contribution of ECM composition to protein binding was interrogated by enzymatically modulating proteoglycan content within the ECM. BMP-2 had a similar binding affinity for each ECM, while TGF-β1 had a greater affinity for ECMs produced by osteoblasts from CI compared to CS patients. Enzymatic treatment of ECMs reduced protein binding. CS osteoblasts cultured on enzymatically-treated ECMs secreted by osteoblasts from CI patients in the presence of BMP-2 exhibited impaired osteogenic differentiation compared to cells on untreated ECMs. These data demonstrate the importance of protein binding to cell-secreted ECMs and confirm that protein-ECM interactions have an important role in directing osteoblastic differentiation of calvarial osteoblasts.     

The Masquelet Technique for Membrane Induction and the Healing of Ovine Critical Sized Segmental Defects

The healing of critical sized segmental defects is an ongoing clinical problem. No method has achieved pre-eminence. The Masquelet technique is a relatively new innovation involving the induction of a fibrous tissue membrane around the bone defect site taking advantage of the body’s foreign body reaction to the presence of a polymethylmethacrylate (PMMA) spacer. The aim of this study was to investigate the properties and characteristics of this induced membrane and its effectiveness when used in conjunction with allograft or an allograft/autograft mix as filler materials in an ovine critical sized defect model. The resultant induced membrane was found to be effective in containing the graft materials in situ. It was demonstrated to be an organised pseudosynovial membrane which expressed bone morphogenic protein 2 (BMP2), transforming growth factor- beta (TGFβ), vascular endothelial growth factor (VEGF), von Willerbrand factor (vWF), interleukin 6 (IL-6) and interleukin 8 (IL-8). While more new bone growth was evident in the test groups compared to the controls animals at 12 weeks, the volumes were not statistically different and no defects were fully bridged. Of the two graft material groups, the allograft/autograft mix was shown to have a more rapid graft resorption rate than the allograft only group. While the Masquelet technique proved effective in producing a membrane to enclose graft materials, its ability to assist in the healing of critical sized segmental defects when compared to empty controls remained inconclusive.