Osteoinductive Bone Morphogenetic Proteins Research Articles

FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis.

Osteoinductive bone morphogenetic proteins (BMPs), including BMP-2, have a unique capability of mediating bone formation both in orthotopic and ectopic locations. Immunosuppresive macrolides have been shown to potentiate BMP-2 activity through FKBP12, but these have yet to translate to effective osteoinductive therapies. Herein, we show the osteogenic activity of FK506 as a stand-alone agent in direct comparison to BMP-2 both in vitro and in vivo. FK506 was capable of producing stand-alone alkaline phosphatase induction in C2C12 cells comparable to that seen with rhBMP-2. FK506 treatment activated the BMP receptor, as shown by increased pSmad1/5 levels, and produced significantly higher mRNA levels of the early response genes in BMP and TGF-β pathways. Additionally, the FK506 induction of alkaline phosphatase was shown to be resistant to Noggin treatment. In vivo osteogenic activity of FK506 was tested by local delivery on a collagen sponge in an ectopic subcutaneous implantation model in the rat. Dose responses of FK506 showed increasing levels of ectopic mineralization comparable to the mineral volume produced by BMP-2 delivery. These findings suggest that the use of FK506 can enhance osteoblastic differentiation in vitro and can induce mineralization when delivered locally in vivo.

Sub-clinical dose of bone morphogenetic protein-2 does not precipitate rampant, sustained inflammatory response in bone wound healing.

Large bone injuries, defects, and chronic wounds present a major problem for medicine. Several therapeutic strategies are used clinically to precipitate bone including a combination therapy delivering osteoinductive bone morphogenetic protein 2 (rhBMP-2) via an osteoconductive scaffold (absorbable collagen sponge [ACS], i.e., INFUSE). Adverse side effects reportedly associated with rhBMP2 administration include rampant inflammation and clinical failures. Although acute inflammation is necessary for proper healing in bone, inflammatory cascade dysregulation can result in sustained tissue damage and poor healing. We hypothesized that a subclinical dose of rhBMP2 modeled in the murine calvarial defect would not precipitate alterations to inflammatory markers during acute phases of bone wound healing. We utilized the 5 mm critical size calvarial defect in C57BL6 wild-type mice which were subsequently treated with ACS and a subclinical dose of rhBMP2 shown to be optimal for healing. Three and 7-day postoperative time points were used to assess the role that rhBMP-2 plays in modulating inflammation vs. ACS alone by cytokine array and histological interrogation. Data revealed that rhBMP-2 delivery resulted in substantial modulation of several markers associated with inflammation, most of which decreased to levels similar to control by the 7-day time point. Additionally, while rhBMP-2 administration increased macrophage response, this peptide had a little noticeable effect on traditional markers of macrophage polarization (M1-iNOS, M2-Arg1). These results suggest that rhBMP-2 delivered at a lower dose does not precipitate rampant inflammation. Thus, an assessment of dosing for rhBMP-2 therapies may lead to better healing outcomes and less surgical failure.

Synthesis and Evaluation of BMMSC-seeded BMP-6/nHAG/GMS Scaffolds for Bone Regeneration

Bioactive scaffolding materials and efficient osteoinductive factors are key factors for bone tissue engineering. The present study aimed to mimic the natural bone repair process using an osteoinductive bone morphogenetic protein (BMP)-6-loaded nano-hydroxyapatite (nHA)/gelatin (Gel)/gelatin microsphere (GMS) scaffold pre-seeded with bone marrow mesenchymal stem cells (BMMSCs). BMP-6-loaded GMSs were prepared by cross-linking and BMP-6/nHAG/GMS scaffolds were fabricated by a combination of blending and freeze-drying techniques. Scanning electron microscopy, confocal laser scanning microscopy, and CCK-8 assays were carried out to determine the biocompatibility of the composite scaffolds in vitro. Alkaline phosphatase (ALP) activity was measured to evaluate the osteoinductivity of the composite scaffolds. For in vivo examination, critical-sized calvarial bone defects in Sprague-Dawley rats were randomly implanted with BMMSC/nHAG/GMS and BMMSC/BMP-6/nHAG/GMS scaffolds, and compared with a control group with untreated empty defects. The BMP-6-loaded scaffolds showed cytocompatibility by favoring BMMSC attachment, proliferation, and osteogenic differentiation. In radiological and histological analyses, the BMMSC-seeded scaffolds, especially the BMMSC-seeded BMP-6/nHAG/GMS scaffolds, significantly accelerated new bone formation. It is concluded that the BMP-6/nHAG/GMS scaffold possesses excellent biocompatibility and good osteogenic induction activity in vitro and in vivo, and could be an ideal bioactive substitute for bone tissue engineering.

The GDF5 mutant BB-1 enhances the bone formation induced by an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia

Background ContextTargeted delivery of osteoinductive bone morphogenetic proteins (eg, GDF5) in bioresorbable calcium phosphate cement (CPC), potentially suitable for vertebroplasty and kyphoplasty of osteoporotic vertebral fractures, may be required to counteract augmented local bone catabolism and to support complete bone regeneration. The biologically optimized GDF5 mutant BB-1 may represent an attractive drug candidate for this purpose. PurposeThe aim of the current study was to test an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming CPC containing low-dose BB-1 in a sheep lumbar osteopenia model. Study Design/ SettingThis is a prospective experimental animal study. MethodsBone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and were filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BB-1 (L5; CPC+fibers+BB-1; 5, 100, and 500 µg BB-1; n=6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BB-1) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (BV/TV) (assessed by micro-CT and histomorphometry), trabecular thickness (Tb.Th), and trabecular number (Tb.N); (3) bone formation, that is, osteoid volume/bone volume (OV/BV), osteoid surface/bone surface (OS/BS), osteoid thickness, mineralizing surface/bone surface (MS/BS), mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. ResultsCompared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BB-1 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BB-1, additional significant effects of BB-1 were demonstrated for BMD, bone structure (BV/TV, Tb.Th, and Tb.N), and bone formation (OS/BS and MS/BS). The BB-1 effects on bone formation at 3 and 9 months were dose dependent, with 100 µg as the potentially optimal dosage. ConclusionsBB-1 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as 100 µg BB-1 was sufficient to augment middle- to long-term bone formation. A CPC containing the novel GDF5 mutant BB-1 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty and kyphoplasty.

Low-dose BMP-2 is sufficient to enhance the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia

Background contextBioresorbable calcium phosphate cement (CPC) may be suitable for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures. However, additional targeted delivery of osteoinductive bone morphogenetic proteins (BMPs) in the CPC may be required to counteract the augmented local bone catabolism and support complete bone regeneration. PurposeThis study aimed at testing an injectable, poly (l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing low-dose bone morphogenetic protein BMP-2 in a sheep lumbar osteopenia model. Study Design/ SettingThis is a prospective experimental animal study. MethodsBone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BMP-2 (L5; CPC+fibers+BMP-2; 1, 5, 100, and 500 µg BMP-2; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BMP-2) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography [micro-CT] and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralizing surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. ResultsCompared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BMP-2 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BMP-2, additional significant effects of BMP-2 were demonstrated for bone structure (bone volume/total volume, trabecular thickness, trabecular number) and formation (osteoid surface/bone surface and mineralizing surface/bone surface), as well as for the compressive strength. The BMP-2 effects on bone formation at 3 and 9 months were dose-dependent, with 5–100 µg as the optimal dosage. ConclusionsBMP-2 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as ≤100 µg BMP-2 was sufficient to augment middle to long-term bone formation. The novel CPC+BMP-2 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty/kyphoplasty.

How cells internalize a growth factor presented by a biomaterial: BMP-2 delivery to cells in a matrix-bound manner using layer-by-layer films

Event Abstract Back to Event How cells internalize a growth factor presented by a biomaterial: BMP-2 delivery to cells in a matrix-bound manner using layer-by-layer films Catherine Picart1, 2, Flora Gilde1, 2, Laure Fourel3, Raphael Guillot1, 2, Isabelle Paintrand1, 2, Takaharu Okada1, 2, 4, Thomas Boudou1, 2, Corinne Albigez-Rizo3 and Catherine Picart1, 2 1 Grenoble Institute of Technology, Department of Bioengineering, France 2 UMR5628, CNRS, France 3 Institute Albert Bonniot, France 4 National Institute for Materials Science, Japan Introduction: Bone Morphogenetic Proteins (BMPs) play an essential role in bone formation and regeneration. In the field of orthopedics, delivery of BMP-2 from the surface of implantable materials would enable faster and better bone formation around the implant. A major effort has been recently undertaken to develop biomaterials for local and precise delivery of the osteoinductive bone morphogenetic proteins (BMPs)[5] . As endocytosis and signaling are tightly coupled, a better understanding of how BMP molecules interact with the biomaterial and are effectively processed by the cells is thus crucial. We previously showed that polyelectrolyte films with matrix-bound BMP-2 are bioactive in vivo when deposited on ceramic granules[3] and on titnaium implants[4]. In this study, we investigated the effect of the delivery mode (matrix-bound versus soluble) and the endocytic processes involved in BMP2-mediated Smad and non-Smad signaling in C2C12 cells, which is the early step of bone differentiation. Materials and Methods: We used a biomimetic polyelectrolyte film made by self-assembly of poly(L-Lysine) and hyaluronan, which acts as nanoreservoir to trap BMP-2 and to present it to cells in a "matrix-bound" manner[1],[2]. This polyelectrolyte film was crosslinked to a controlled extent. We used fluorescently labeled BMP-2 to track the possible diffusion of BMP-2 in the biomaterials and its internalization by the cells. We quantified the amount of BMP-2 internalized by the cells. We then used chemical inhibitors to block the major internalization routes (including clathrin,dynamin and caveolin) and knock-down the these pathways to unravel their specific effect on BMP-2 signaling. Results and Discussion: We showed that BMP-2 partially diffused within the film and that it is internalized by C2C12 cells. We then quantified the effect of film cross-linking on the amount of BMP-2 that is internalized by the cells. The use of inhibitors of endocytosis, siRNA-mediated knock-down and immuno-staining of endocytic proteins allowed to investigate the internalization pathways adopted by the cells in comparison to the delivery of BMP-2 in solution. Finally, we investigated the role of endocytosis in the activation of the BMP signaling pathways. Conclusions: We demonstrated that soluble as well as matrix-bound BMP-2 were internalized through both clathrin- and caveolin-dependent pathways. Furthermore, we established that both the initial and final steps of endocytosis process were determinant for BMP-2-induced signaling. These results highlight that a bioactive growth factor delivered via a biomaterial enables the signaling pathways to be transduced in a very efficient manner. European Research council (European Comission, FP7, GA259370, BIOMIM)

Osteophilic Multilayer Coatings for Accelerated Bone Tissue Growth

Osteophilic modular nanostructured multilayers containing hydroxyapatite nanoparticles complexed with a natural polymer chitosan create an osteoconductive surface for mesenchymal stem cells (MSCs). Coupled with the sustained release of physiological amounts of osteoinductive bone morphogenetic protein over several days from degradable poly(β-amino ester) based multilayers, this single coating results in a synergistic accelerated and upregulated differentiation of MSCs into osteoblasts laying down new bone tissue on orthopedic implants.

Bone Regeneration by Demineralized Dentin Matrix in Skull Defects of Rats

Alveolar bone defects may result in functional and morphological abnormalities. In such cases, bone can be regenerated by grafting fresh autogenous bone into the defect. Subsequently, we focused on the dentin of third molars, which are usually discarded after extraction, or teeth removed faor orthodontic therapy. As in bone, type I collagen is the main component of the organic matrix in dentin. Also, it has been proven that the organic matrix of dentin contains osteoinductive bone morphogenetic protein (BMP). It would be safe to perform grafting with the dentin of extracted teeth used as an autogenous bone substitute. Therefore, the objective of the present study was to reuse extracted teeth as a bone substitute material. Demineralized dentin matrix (DDM), an organic material derived from the dentine of bovine teeth, was grafted in rat skull defects to determine its bone regeneration ability and the possibility of its use as a bone substitute. In the present study, new bone formed from the defect margin in the control group, but in the graft group, new bone formation occurred from individual DDM granules within the defect, and not just from the margin. At Week 8, defect repair was limited in the control group. In contrast, most of the defect was covered by osseous tissue in the graft group. In conclusion, DDM may be a useful bone substitute that serves as a scaffold for bone regeneration by inducing a high level of new bone formation soon after surgery.

Discontinuous Release of Bone Morphogenetic Protein-2 Loaded Within Interconnected Pores of Honeycomb-Like Polycaprolactone Scaffold Promotes Bone Healing in a Large Bone Defect of Rabbit Ulna

The choice of an appropriate carrier and its microarchitectural design is integral in directing bone ingrowth into the defect site and determining its subsequent rate of bone formation and remodeling. We have selected a three-dimensional polycaprolactone (PCL) scaffold with an interconnected honeycomb-like porous structure to provide a conduit for vasculature ingrowth as well as an osteoconductive pathway to guide recruited cells responding to a unique triphasic release of osteoinductive bone morphogenetic proteins (BMP) from these PCL scaffolds. We hypothesize that the use of recombinant human bone morphogenetic protein 2 (rhBMP2)-PCL constructs promotes rapid union and bone regeneration of a large defect. Results of our pilot study on a unilateral 15 mm mid-diaphyseal segmental rabbit ulna defect demonstrated enhanced bone healing with greater amount of bone formation and bridging under plain radiography and microcomputed tomography imaging when compared with an empty PCL and untreated group after 8 weeks postimplantation. Quantitative measurements showed significantly higher bone volume fraction and trabecular thickness, with lower trabecular separation in the rhBMP2-treated groups. Histology evaluation also revealed greater mature bone formation spanning across the entire scaffold region compared with other groups, which showed no bone regeneration within the central defect zone. We highlight that it is the uniqueness of the scaffold having a highly porous network of channels that promoted vascular integration and allowed for cellular infiltration, leading to a discontinuous triphasic BMP2 release profile that mimicked the release profile during natural repair mechanisms in vivo. This study serves as preclinical evidence demonstrating the potential of combining osteoinductive rhBMP2 with our PCL constructs for the repair of large defects in a large animal model.

Short-Term Application of Dexamethasone Enhances Bone Morphogenetic Protein-7-Induced Ectopic Bone Formation In Vivo

Long-term administration of glucocorticoids may lead to bone loss and osteoporosis as reported in previous experimental and clinical studies. On the other hand, several in vitro studies have demonstrated that dexamethasone treatment induces proliferation and differentiation of human and murine osteoblast precursors. Thereby, a positive interaction of dexamethasone with the osteoinductive bone morphogenetic proteins (BMPs) is reported in vitro, but in vivo studies are still missing. Thus, the aim of this study was to determine whether short-term application of dexamethasone may improve BMP-7-induced bone formation in vivo. Ectopic bone formation was induced in control and dexamethasone-treated mice by application of BMP-7 into the hamstring muscles. After 20 days of treatment, each ectopic bone nodule was analyzed by contact radiography, microcomputed tomography, and histomorphometry. Furthermore, mice were subjected to histomorphometric analyses of their lumbar vertebrae and proximal tibiae to assess the systemic effect of short-term dexamethasone treatment on bone metabolism. Dexamethasone application significantly increased the bone volume and osteoblast number of the ectopic bone nodules compared with untreated controls. Histomorphometric analyses of the lumbar vertebrae and proximal tibiae revealed no significant differences between the control and dexamethasone-treated mice. This study demonstrates that BMP-7-induced ectopic bone formation is significantly enhanced by systemic short-term application of dexamethasone. These in vivo data confirm the results of previous in vitro studies and could be of interest for further studies with the intention to improve BMP-induced bone formation by short-term application of dexamethasone.

Quantitative Assessment of the Bone Morphogenetic Protein Expression From Alternate Bone Graft Harvesting Sites

Bone morphogenetic proteins (BMPs) play important roles in the stimulation of osteogenesis and osteoinduction during bone fracture healing and their expression levels may be important for bone graft efficacy. The objective of this study was to determine if there are variations in the expression of BMPs and their receptors in various bone graft harvesting sites. We analyzed autogenous marrow aspirates obtained from three different graft sites for the mRNA levels of BMPs and their receptors. Using real-time polymerase chain reaction, we analyzed the mRNA levels of BMPs and their receptors in autogenous bone marrow aspirates obtained from three different bone graft sites of 10 different human subjects. Collection of autogenous bone marrow from the iliac crest, the proximal humerus, and the proximal tibia was performed using standard sterile techniques in the operating room as part of surgery to treat an established fracture nonunion. The mRNA levels of BMP-2 and BMP-5 were the highest in the bone marrow aspirates from the three different sites, whereas the mRNA levels of the other osteoinductive BMPs (BMP-4, -5, -6, -7, -8, and -9) were lower. The mRNA levels of BMP-3, an inhibitor of osteogenesis, were the lowest in the bone marrow aspirates of all three different sites. There were no statistical significant differences in the mRNA levels of any of the BMPs or their receptors investigated in this study in the bone marrow of the three different sites. Because no statistical significant differences in the mRNA levels of the BMPs and their receptors were detected in the bone marrow aspirates from the three different sites, our findings suggest that potential differences of various graft sites in the augmentation of bone healing does not result from different expression levels of BMPs.

Recent Developments in the Biology of Fracture Repair

Fracture repair is dependent on local and systemic molecular and cellular processes. During fracture repair, mesenchymal stem cells are systemically recruited to the fracture site, and cytokines are released from the fracture site into the vascular system. In a significant minority of fractures, healing delays result from adverse clinical factors that interfere with these processes. Extrinsic factors, such as aging and smoking, adversely affect the molecular and cellular processes occurring locally in the fracture site. Fracture fixation affects healing through local changes in the biologic signaling within the fracture callus. Current biologic treatment of fractures includes the local application of osteoinductive bone morphogenetic proteins (ie, BMP-2, BMP-7) and cell-based therapies. Although clinical results with bone morphogenetic proteins have been satisfactory, they have not been as impressive as those reported in animal studies. Further understanding of the biology of fracture repair may lead to improved treatment modalities.

Three-Dimensional Technology and Bone Morphogenetic Protein in Frontal Bone Reconstruction

Osteoinductive bone morphogenetic proteins (BMPs) may be used in humans to facilitate healing of bony defects. The effect of different BMPs is, as with many other growth factors, highly dependent on the delivery vehicle. Bovine type I collagen is currently used in the clinical setting as a carrier and has been approved in several countries for human use. Here, we report the reconstruction of a frontal bone defect using heparin together with bovine type I collagen, hyaluronic acid, and fibrin as vehicles for BMP-2. A bony structure was created on the back of the patient by treating the latissimus dorsi muscle with the growth factor. A polyamide mold was used as a template to achieve the desired shape. The bone structure was transplanted into the defect site via microsurgical techniques. Although the prefabricated bone was not large enough tocover the entire frontal defect, the reconstruction was completed by using an additional cranial implant.

BMP Stimulation of Alkaline Phosphatase Activity in Pluripotent Mouse C2C12 Cells is Inhibited by Dermatopontin, One of the Most Abundant Low Molecular Weight Proteins in Demineralized Bone Matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of γ-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.

Mechanical evaluation of mandibular defects reconstructed using osteogenic protein-1 (rhOP-1) in a sheep model: a critical analysis

Osteoinductive bone morphogenetic proteins (BMPs) have been used extensively in experimental and clinical orthopaedic research. It is a natural progression for these growth regulators to be tested in the craniofacial region. The aim of this investigation was to analyse the mechanical properties of the sheep mandibles reconstructed using recombinant human osteogenic protein type 1 (rhOP-1). A unilateral 35 mm osteoperiosteal continuity defect was created at the parasymphyseal region of the mandible in six adult sheep. The animals were sacrificed 3 months after surgery and mechanical properties of the regenerated bone at the operated sides (OS) were compared to the corresponding bone at the non-operated side (NOS). The regenerated tissue at the OS were then submitted for histological and histomorphometric analysis. Although all the animals achieved complete bony union, a wide range of mechanical properties was found. The rhOP-1-induced bone achieved a mean of 36% of the strength of the bone at the NOS ( P < 0.05). The mean value of the stiffness of the OS was 24% of the NOS ( P < 0.05). While half of the samples of the OS had ‘weak’ mechanical properties (9–25% strength compared to NOS) and a low stiffness (6–18%), the rest showed relatively higher strength (47–63%) and were stiffer (35–47%). Unlike the NOS, the operated sides failed under tensile stresses and cracks initiated at the superior border of the mandible. The wide mechanical variations suggest that further basic bone biology research is needed to provide better understanding of the cellular and molecular events which take place during the process of osteoinduction.

Full Length Article

Numerous experimental studies have been published about osteoinductive bone morphogenetic proteins (BMPs). However, to our knowledge there has been no detailed histological study of a mandibular defect in a large mammal, reconstructed using BMPs. We describe here the histological features of rhBMP-7-induced bone in mandibular defects in sheep. Methods: A 35 mm osteoperiosteal defect was created at the parasymphyseal region of the mandible in six adult sheep. The continuity of the mandible was maintained using a bony plate, and rhBMP-7 was applied on a type I collagen carrier. Bone labels were injected at selected time intervals during the follow-up period. The animals were killed after 3 months and bone samples were examined histologically, histomorphometrically, and by fluorescence microscopy. Results and conclusions: We found a mixture of woven and lamellar bone that contained many cells with large nuclei. This had not reorganised to form cortical bone and the rhBMP-7-induced bone was more porous than the native bone. The newly-formed bone restored both endosteal and periosteal layers. rhBMP-7-induced bone was biocompatible and induced no ossification of soft tissue or abnormal growth of nearby vital structures. The mineral apposition rate was 1.98 μm/day (range 0.62–5.63 μm/day), a value close to that reported in humans. This suggests that BMPs have a limited effect in accelerating the rate of mineralisation, but promote the pre-mineralisation processes, and perhaps the formation of woven bone.

Histological assessment of bioengineered new bone in repairing osteoperiosteal mandibular defects in sheep using recombinant human bone morphogenetic protein-7

Numerous experimental studies have been published about osteoinductive bone morphogenetic proteins (BMPs). However, to our knowledge there has been no detailed histological study of a mandibular defect in a large mammal, reconstructed using BMPs. We describe here the histological features of rhBMP-7-induced bone in mandibular defects in sheep. Methods: A 35 mm osteoperiosteal defect was created at the parasymphyseal region of the mandible in six adult sheep. The continuity of the mandible was maintained using a bony plate, and rhBMP-7 was applied on a type I collagen carrier. Bone labels were injected at selected time intervals during the follow-up period. The animals were killed after 3 months and bone samples were examined histologically, histomorphometrically, and by fluorescence microscopy. Results and conclusions: We found a mixture of woven and lamellar bone that contained many cells with large nuclei. This had not reorganised to form cortical bone and the rhBMP-7-induced bone was more porous than the native bone. The newly-formed bone restored both endosteal and periosteal layers. rhBMP-7-induced bone was biocompatible and induced no ossification of soft tissue or abnormal growth of nearby vital structures. The mineral apposition rate was 1.98 μm/day (range 0.62–5.63 μm/day), a value close to that reported in humans. This suggests that BMPs have a limited effect in accelerating the rate of mineralisation, but promote the pre-mineralisation processes, and perhaps the formation of woven bone.

Tissue engineering skeletal muscle for orthopaedic applications.

With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

Biomechanical and histological aspects of fracture healing, stimulated with osteogenic protein-1

Fracture healing could be stimulated with osteoinductive bone morphogenetic proteins (bmp's), such as osteogenic protein-1 (OP-1), but little is known about its effectiveness in stimulation of fracture healing. In this study, biomechanical and histological aspects of fracture healing after an injection of OP-1 in the fracture gap were investigated. In 40 goats, a closed fracture was created in the left tibia. The fractures were stabilized with an external fixator and the animals were assigned to four different groups: no injection, injection of 1 mg OP-1, injection of 1 mg OP-1 with collagenous carrier material, and injection of carrier material alone. Twenty-one animals were sacrificed after 2 weeks and 19 after 4 weeks. Biomechanical testing was perfomed on both explanted tibiae. Four longitudinal samples of the fracture were sawn, processed for histology, and examined by two observers. Biomechanical evaluation showed a higher stiffness and strength at 2 weeks after injection of OP-1. Histological evaluation showed normal fracture healing patterns in all animals without adverse effects of the given injections. These data show that fracture healing can be accelerated with a single injection of OP-1, eventually resulting in normally healed bone.