Although fracture healing is a well-optimized biological process that leads to healing, approximately <em>10</em>-20% of fractures result in impaired or delayed healing and these fractures may benefit from the use of biotechnologies to enhance skeletal repair. Peptide signaling molecules such as the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> have been shown to stimulate the healing of fresh fractures, nonunions, and spinal fusions and side effects from their use appear to be minimal. Other growth factors currently being studied for local application include growth and differentiation factor-5 (GDF-5), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and platelet-derived growth factor (PDGF). Molecules such as prostaglandin E receptor agonists and the thrombin-related peptide, TP508, have shown promise in animal models of fracture repair. Gene therapy using various growth factors or combinations of factors might also aid in fracture repair, particularly as new methods for delivery that do not require viral vectors are developed. Systemic therapy with agents such as parathyroid hormone (PTH), growth hormone (GH), and the HMG-CoA reductase inhibitors are also under investigation. As these and other technologies are shown to be safe and effective, their use will become a part of the standard of care in managing skeletal injuries.

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) 2 on <em>bone</em>. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfal) consensus sequence (5'-AACCACA3') at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear <em>proteins</em> to an oligonucleotide spanning the Cbfal site was inhibited or supershifted by specific antibodies to Cbfal. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long <em>bones</em>, BMP-2 induced osteoclast formation in cells from COX-2(+/+) mice but not in cells from COX-2(-/-) mice. In vivo, BMP-2 (<em>10</em> microg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (microCT), was decreased by 78% in COX-2(-/-) mice compared with COX-2(+/+) mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfal binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic <em>bone</em> formation in vivo.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of <em>bone</em> pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate <em>bone</em> formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague-Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 x <em>10</em>(5) PFU/well of BMP viral vector was 4890 x <em>10</em>(-12) U/well for ADCMVBMP-9, 302 x <em>10</em>(-12) U/well for ADCMVBMP-4, 220 x <em>10</em>(-12) U/well for ADCMVBMP-6, 45 x <em>10</em>(-12) U/well for ADCMVBMP-2, and 0.43 x <em>10</em>(-12) U/well for ADCMVBMP-7. The average volume of new <em>bone</em> induced by <em>10</em>(7) PFU of BMP vector in athymic nude rats was 0.37+/-0.03 cm(3) for ADCMVBMP-2, 0.89+/-0.07 cm(3) for ADCMVBMP-4, 1.02+/-0.07 cm(3) for ADCMVBMP-6, 0.24+/-0.05 cm(3) for ADCMVBMP-7, and 0.63+/-0.07 cm(3) for ADCMVBMP-9. In immunocompetent Sprague-Dawley rats, no <em>bone</em> formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of <em>10</em>(8) PFU induced 0.<em>10</em>+/-0.03 cm(3) of new <em>bone</em>, whereas ADCMVBMP-9 at a lower viral dose of <em>10</em>(7) PFU induced more <em>bone</em>, with an average volume of 0.29+/-0.01 cm(3).

Eschericha coli was genetically engineered to produce recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system. High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 gl(-1)) were obtained by applying a high-cell-density cultivation procedure. After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to <em>10</em>0 mgl(-1) by means of a simple dilution method with yields exceeding 50%. Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer. With the established renaturation and purification protocols, yields of more than <em>10</em> mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth. The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells.

Previously we demonstrated that <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) treatment polarizes monocytes into M2 macrophages and increases the expression of anti-inflammatory cytokines. Despite these findings, the mechanisms for the observed BMP-7 induced monocyte polarization into M2 macrophages are completely unknown. In this study, we demonstrate the mechanisms involved in the polarization of monocytes into M2 macrophages. Apoptotic conditioned media (ACM) was generated to mimic the stressed conditions, inducing monocyte polarization. Monocytes were treated with ACM along with BMP-7 and/or its inhibitor, follistatin, for 48 hours. Furthermore, an inhibitor of the PI3K pathway, LY-294002, was also studied. Our data show that BMP-7 induces polarization of monocytes into M2 macrophages while significantly increasing the expression of anti-inflammatory markers, arginase-1 and IL-<em>10</em>, and significantly (p<0.05) decreasing the expression of pro-inflammatory markers iNOS, IL-6, TNF-α and MCP-1; (p<0.05). Moreover, addition of the PI3K inhibitor, LY-294002, significantly (p<0.05) decreases upregulation of IL-<em>10</em> and arginase-1, suggesting involvement of the PI3K pathway in M2 macrophage polarization. Next, following BMP-7 treatment, a significant (p<0.05) increase in p-SMAD1/5/8 and p-PI3K expression resulting in downstream activation of p-Akt and p-mTOR was observed. Furthermore, expression of p-PTEN, an inhibitor of the PI3K pathway, was significantly (p<0.05) increased in the ACM group. However, BMP-7 treatment inhibited its expression, suggesting involvement of the PI3K-Akt-mTOR pathway. In conclusion, we demonstrate that BMP-7 polarizes monocytes into M2 macrophages and enhances anti-inflammatory cytokine expression which is mediated by the activated SMAD-PI3K-Akt-mTOR pathway.

The immature disc nucleus pulposus (NP) consists of notochordal cells (NCs). With maturation NCs disappear in humans, to be replaced by chondrocyte-like mature NP cells (MNPCs); this change in cell phenotype coincidences with early signs of disc degeneration. The reasons for NC disappearance are important to understand disc degeneration, but remain unknown, yet. This study investigated, whether loading induced a change from a notochordal nucleus phenotype to a chondrocyte-like one. An in vivo disc compression model with fixateur externe was used in 36 mature rabbits. Discs were compressed for different time periods (1, 28, 56 days), and compared with uncompressed control discs (56 days without treatment), and discs with sham compression (28 days). Nucleus cell phenotype was determined by histology and immunohistochemistry. NCs, but not MNPCs highly expressed <em>bone</em>-<em>morphogenetic</em>-<em>protein</em> 2 and cytokeratin 8, thus NC and MNPC numbers could be determined. A histologic score was used to detect structural endplate changes after compression (28 days). Control and sham compressed discs contained around 70% NCs and 30% MNPCs, to be decreased to (<em>10</em>% NCs after 28-56 days of loading. NC density fell sharply by >50% after 28-56 days of compression (P < 0.05 vs. controls). Signs of decreased endplate cellularity and increased endplate sclerosis and fibrosis were found after loading. These experiments show that NCs were less resistant to mechanical stress than MNPCs suggesting that increased intradiscal pressures after loading, and limited nutrition through structurally altered endplates could instigate the disappearance of NCs.

The aim of this study was to develop a nanosized, controlled growth factor release system to incorporate into tissue engineering scaffolds and thus activate the cells seeded in the scaffold. Nanocapsules of poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were loaded with the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> BMP-2 and BMP-7, respectively, and with bovine serum albumin (BSA), the model <em>protein</em>. BSA-loading efficiency and release kinetics were used to determine the most appropriate nanocapsule pair to achieve the delivery of growth factors in a sequential manner, as occurs in natural processes. BSA-encapsulation efficiency was highest when the polymer concentration used in the preparation of PLGA and PHBV nanocapsules was <em>10</em>% (w/v) (84.75% and 16.72%, respectively). Release of BSA was faster from PLGA than it was from PHBV. Based on the encapsulation efficiency and release data, <em>10</em>% PLGA and <em>10</em>% PHBV nanocapsules were chosen to provide the early BMP-2 and later BMP-7 release, respectively. Simultaneous, sequential delivery and individual release of the BMPs were studied for 7, 14, and 21 days, using rat <em>bone</em> marrow mesenchymal stem cells. Individual BMP-2 release suppressed cell proliferation while providing higher alkaline phosphatase activity with respect to BMP-7. The sequential delivery of BMP-2 and BMP-7 provided slightly lower proliferation than did simultaneous delivery, but the highest alkaline phosphatase activity of all indicated a synergistic effect on the osteogenic differentiation of mesenchymal stem cells caused by the use of the two growth factors in a sequential fashion.

METHODS

Rat spinal fusion model.

OBJECTIVE

To compare the efficacy of human adipose tissue-derived mesenchymal stem cells (HATDMSCs) and human bone marrow-derived mesenchymal stem cells (HBMDMSCs) transduced with an adenovirus containing the cDNA for bone morphogenetic proteins (BMP)-2 for inducing spinal fusion in an athymic rat model.

BACKGROUND

Recombinant BMPs have successfully induced spinal fusion in clinical trials. However, large doses are required for adequate bone repair. Regional gene therapy may deliver proteins to specific anatomic sites more efficiently. Gene transfer techniques using HATDMSCs have recently been tested.

METHODS

Spinal fusion was performed in rats with different treatments: Group I (n = 10) collagen sponge containing HATDMSCs transfected with adeno-BMP-2, Group II (n = 10) collagen sponge containing HBMDMSCs transfected with adeno-BMP-2, Group III (n = 10) collagen sponge containing recombinant BMP-2 (10 mug), Group IV (n = 6) collagen sponge containing HATDMSCs transfected with adeno-LacZ, Group V (n = 6) collagen sponge containing HBMDMSCs transfected with adeno-LacZ, and Group VI (n = 6) collagen sponge alone. Radiographs were obtained at 4, 6, and 8 weeks. After sacrifice, the rat spines were assessed by manual palpation, microcomputed tomography, and histologic analysis.

RESULTS

At 8 weeks, spinal fusion was observed in all Groups I, II, and III rats. 75% (15 of 20) of the gene therapy treatment animals (Groups I and II rats) had spontaneous extension of the fusion to a second level. No Groups IV, V, and VI rats developed fusion. New bone volume was significantly greater in Groups I and II than in Group VI.

CONCLUSIONS

HATDMSCs transfected with adeno-BMP-2 induce abundant bone formation and have a similar posterolateral spinal fusion in rats as similarly genetically modified HBMDMSCs. Both are potential strategies for spinal fusion and may be a more efficient method of obtaining spinal fusion over currently used grafting substances.

METHODS

A prospective randomized study investigated the radiographic progress of fusion at 6, 12, and 24 months in 42 patients who underwent a single-level anterior lumbar interbody fusion using cylindrical interbody fusion cages.

OBJECTIVE

To determine the patterns and rates of osteoinduction associated with the use of recombinant human bone morphogenetic protein type 2 (rhBMP-2) and an absorbable collagen sponge carrier in anterior lumbar interbody fusion with a tapered cylindrical fusion device.

BACKGROUND

Studies have shown that rhBMP-2 used with allograft dowels increases the rate of interbody fusion by promoting osteoinduction and enhancing incorporation of the allograft. In a small series of human patients undergoing anterior lumbar interbody fusion with a tapered cylindrical fusion cage, rhBMP-2 has been shown to promote osteoinduction and fusion.

METHODS

In this prospective nonblinded study, 42 patients were randomly divided into two groups. The investigational group underwent interbody fusion using two tapered cylindrical fusion cages (LT-CAGE) and rhBMP-2 on an absorbable collagen sponge, and a control group underwent the procedure, receiving the devices and autogenous iliac crest bone graft. Plain radiographs and computed tomographic scans were used to evaluate the pattern of osteoinduction in the interbody space and the progression of fusion 6, 12, and 24 months after surgery.

RESULTS

All the patients who received rhBMP-2 showed radiographic evidence of osteoinduction in the interbody cages 6 months after surgery. The density in the cages had increased an average of 142 Hounsfield units by 6 months. At 12 months, the increase had reached 228.7 Hounsfield units New bone formation occurred in the disc space outside the cages by 6 months in 18 of the patients in the investigational group (18/22; 82%). By 24 months, all the investigational patients showed new formation outside the cages. In the autograft control group, the density in the cages increased an average of 42 Hounsfield units, and 10 patients (10/20; 50%) showed evidence of bone formation outside the cages.

CONCLUSIONS

The use of rhBMP-2 is a promising method for facilitating anterior intervertebral spinal fusion in patients who have undergone anterior lumbar fusion surgery.

Endoglin, a type I membrane glyco<em>protein</em> expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to <em>Bone</em> <em>Morphogenetic</em> <em>Protein</em> (BMP)-9 and BMP-<em>10</em>. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known <em>protein</em>, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

Because articular cartilage has a poor regeneration capacity, numerous cell-based approaches to therapy are currently being explored. The present study involved the use of gene transfer as a means to provide sustained delivery of chondrogenic <em>proteins</em> to primary mesenchymal stem cells (MSCs). In previous work, we found that adenoviral-mediated gene transfer of transforming growth factor-beta1 (TGF-beta1) and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2), but not insulin-like growth factor 1 (IGF-1), could be used to induce chondrogenic differentiation of MSCs in an aggregate culture system. In the present study, we examined the effects on chondrogenesis of these transgenes when delivered in combination. Cultures of <em>bone</em> marrow-derived MSCs were infected with 2.5 x <em>10</em>(2) or 2.5 x <em>10</em>(3) viral particles/cell of each adenoviral vector individually, or in combination, seeded into aggregates, and cultured for 3 weeks in a defined serum-free medium. Levels of transgene product in the medium were initially high, approximately <em>10</em>0 ng/mL TGF-beta1, 120 ng/mL BMP-2, and 80 ng/mL IGF-1 at day 3, and declined thereafter. We found that co-expression of IGF-1 and TGF-beta1, BMP-2, or both at low doses resulted in larger aggregates, higher levels of glycosaminoglycan synthesis, stronger staining for proteoglycans and collagen type II and X, and greater expression of cartilage-specific marker genes than with either transgene alone. Gene-induced chondrogenesis of MSCs using multiple genes that act synergistically may enable the administration of reduced viral doses in vivo and could be of considerable benefit for the development of cell-based therapies for cartilage repair.

The purpose of this study was to evaluate the efficacy and safety of recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (rhBMP-7 or OP-1) as a <em>bone</em>-stimulating agent in the treatment of persistent fracture non-unions. Twenty-five consecutive patients [19 males, mean age 39.4 years (range: 18-79)] with 26 fracture non-unions were treated with rhBMP-7. There were <em>10</em> tibial non-unions, eight femoral, three humeral, three ulnar, one patellar, and one clavicular non-union. The mean follow-up was 15.3 months. The mean number of operations performed prior to rhBMP-7 application was 3.2, with autologous <em>bone</em> graft and <em>bone</em> marrow injection being used in <em>10</em> cases (38.5%). Both clinical and radiological union occurred in 24 (92.3%) cases, within a mean time of 4.2 months and 5.6 months, respectively. Of the remaining two cases, one patient ultimately underwent a below knee amputation, secondary to recurrence of deep sepsis. The other patient with recalcitrant ulnar non-union although the radiological union was incomplete, declined further intervention, as he was asymptomatic. No complications or adverse effects from the use of rhBMP-7 were encountered. This study supports the view that the application of rhBMP-7 as a <em>bone</em>-stimulating agent is safe and a power adjunct to be considered in the surgeon's armamentarium for the treatment of these challenging clinical conditions.

BACKGROUND

It has been previously shown that in patients with osteoporosis, mesenchymal stem cell (MSC) growth rate and osteogenic potential is decreased contributing to inferior fracture consolidation. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2), BMP-7, parathyroid hormone (PTH), and platelet-derived growth factor (PDGF) on proliferation and osteogenic differentiation of MSCs derived from patients with osteoporosis.

METHODS

Trabecular bone was obtained from 10 patients (four males, mean age 76 years) with lower extremity osteoporotic fractures. MSCs were isolated by enzymatic digestion. Functional assays of proliferation and osteogenic differentiation were performed under the influence of a wide range of concentrations of BMP-2, BMP-7, PTH, and PDGF-BB. Proliferation was assessed using CFU-F and XTT assays. Osteogenic differentiation was assessed by alkaline phosphatase activity and total calcium production.

RESULTS

MSC proliferation was found to be stimulated by supplementation with BMP-7 and PDGF-BB, whereas BMP-2 and PTH had little effect. The largest increase in proliferation rate was observed after administration 100 ng/mL of BMP-7. All four molecules induced alkaline phosphatase activity and calcium production in growing osteoblasts with a dose-dependent effect noted. BMP-2 and BMP-7 at their highest studied concentration (100 ng/mL) produced a threefold increase in the osteogenic potential of MSCs.

CONCLUSIONS

BMP-7, BMP-2, PTH, and PDGF-BB were observed to have a positive effect on osteogenic differentiation of MSCs. BMP-7 and PDGF-BB (in high doses) could be considered most potentially advantageous because they enhance both proliferation and osteogenic differentiation of MSCs derived from elderly osteoporotic bone.

In preclinical studies, human adipose stem cells (ASCs) have been shown to have therapeutic applicability, but standard expansion methods for clinical applications remain yet to be established. ASCs are typically expanded in the medium containing fetal bovine serum (FBS). However, sera and other animal-derived culture reagents stage safety issues in clinical therapy, including possible infections and severe immune reactions. By expanding ASCs in the medium containing human serum (HS), the problem can be eliminated. To define how allogeneic HS (alloHS) performs in ASC expansion compared to FBS, a comparative in vitro study in both serum supplements was performed. The choice of serum had a significant effect on ASCs. First, to reach cell proliferation levels comparable with <em>10</em>% FBS, at least 15% alloHS was required. Second, while genes of the cell cycle pathway were overexpressed in alloHS, genes of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor-mediated signaling on the transforming growth factor beta signaling pathway regulating, for example, osteoblast differentiation, were overexpressed in FBS. The result was further supported by differentiation analysis, where early osteogenic differentiation was significantly enhanced in FBS. The data presented here underscore the importance of thorough investigation of ASCs for utilization in cell therapies. This study is a step forward in the understanding of these potential cells.

Further understanding of how mechanical cues modulate skeletal tissue differentiation can identify potential means of enhancing repair following injury or disease. Prior studies examined the effects of mechanical loading on osteogenesis, chondrogenesis, and fibrogenesis in an effort to enhance bony union. However, exploring how mechanical stimuli can divert the <em>bone</em> healing process towards formation of other mesenchymal tissues, as an endpoint, may elucidate new avenues for repair and regeneration of tissues such as cartilage and fibrous tissue. This study investigated the use of mechanical stimulation to promote cartilage rather than <em>bone</em> formation within an osteotomy. Our overall goal was to define skeletal tissue distribution and molecular expression patterns induced by the stimulation. Retired breeder Sprague-Dawley rats (n = 85) underwent production of a mid-diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day <em>10</em> and continuing for 1, 2, or 4 weeks, a cyclic bending motion (+35 degrees/-25 degrees at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal <em>bone</em> healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage-related genes (COL2A1 and COL<em>10</em>A1) and downregulation of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) -4, -6 and -7. However, BMP-3 was upregulated with stimulation. These findings illustrate that mechanical cues can selectively modulate osteogenesis and chondrogenesis in vivo, and suggest a potential basis for treatment regimens for injured or diseased cartilaginous tissues.

Structural allografts used for critical <em>bone</em> defects have limited osteogenic properties for biointegration. Although ex vivo tissue-engineered constructs expressing <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP2) have demonstrated efficacy in critical defect models, similar success has not been achieved with off-the-shelf acellular approaches, including allografts coated with freeze-dried single-stranded adeno-associated virus (ssAAV-BMP2). To see whether the self-complementary AAV serotype 2.5 vector (scAAV2.5-BMP2) could overcome this, we performed side-by-side comparisons in vitro and in the murine femoral allograft model. Although ssAAV-BMP2 was unable to induce BMP2 expression and differentiation of C3H<em>10</em>T1/2 cells in culture, scAAV2.5-BMP2 transduction led to dose-dependent BMP2 expression and alkaline phosphatase activity, and displayed a 25-fold increased transduction efficiency in vivo. After 6 weeks, the ssAAV-BMP2 coating failed to demonstrate any significant effects. However, all allografts coated with <em>10</em>(<em>10</em>) scAAV2.5-BMP2 formed a new cortical shell that was indistinguishable to that formed by live autografts. Additionally, coated allografts experienced reduced resorption resulting in a threefold increase in graft <em>bone</em> volume versus autograft. This led to biomechanical superiority versus both allografts and autografts, and equivalent torsional rigidity to unfractured femur. Collectively, these results demonstrate that scAAV2.5-BMP2 coating overcomes the major limitations of structural allografts, which can be used to heal critical defects of any size.

We assessed the distribution and relative staining intensity of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five <em>10</em>-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP <em>proteins</em> were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of <em>bone</em> remodeling.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily that have been implicated in tissue growth and remodelling. Recent evidence suggests that several BMPs are expressed in the developing and adult brain. Specifically, we show that BMP 2 and BMP 6 are expressed in the developing midbrain floor of the rat. We studied potential neurotrophic effects of BMPs on the in vitro survival, transmitter uptake and protection against MPP+ toxicity of mesencephalic dopaminergic neurons cultured from the embryonic midbrain floor at embryonic day (E) 14. At <em>10</em> ng/ml and under serum-free conditions, most BMPs promoted the survival of dopaminergic neurons visualized by tyrosine hydroxylase immunocytochemistry during an 8-day culture period, but to varying extents (relative potencies: BMP 6 = 12>> 2, 4, 7). BMPs 6 and 12 were as effective as fibroblast growth factor-2 (FGF-2) and glial cell line-derived neurotrophic factor, promoting survival 1.7-fold compared with controls. BMPs 9 and 11 were not effective. Dose-response curves revealed an EC50 for BMPs 2, 6 and 12 of 2 ng/ml. BMPs 2, 4, 6, 7, 9 and 12 also promoted DNA synthesis and astroglial cell differentiation, visualized by 5-bromodeoxyuridine (BrdU) incorporation and glial fibrillary acidic <em>protein</em> (GFAP) immunocytochemistry respectively. Suppression of cell proliferation and subsequent maturation of GFAP-positive cells by 5-fluorodeoxyuridine or aminoadipic acid abolished the neuron survival-promoting effect of BMP 2. This suggests that BMPs, like other non-TGF-beta factors affecting dopaminergic neuron survival, act indirectly, probably by stimulating the synthesis and/or release of glial-derived trophic factors. BMP 6 and BMP 7 also increased the uptake of [3H]dopamine without affecting the uptake of [3H]5-hydroxytryptamine and [3H]GABA, underscoring the specificity of the trophic effect. We conclude that several BMPs share a neurotrophic capacity for dopaminergic midbrain neurons with other members of the TGF-beta superfamily, but act indirectly, possibly through glial cells.

BACKGROUND

Rotator cuff tendon-to-bone healing occurs by formation of a scar tissue interface after repair, which makes it prone to failure. Bone morphogenetic protein-13 (BMP-13) has been implicated in tendon and cartilage repair, and thus may augment rotator cuff repairs. The purpose of this study was to determine if the application of mesenchymal stem cells (MSCs) transduced with BMP-13 could improve regeneration of the tendon-bone insertion site in a rat rotator cuff repair model.

OBJECTIVE

Mesenchymal stem cells genetically modified to overexpress BMP-13 will improve rotator cuff healing based on histologic and biomechanical outcomes.

METHODS

Controlled laboratory study.

METHODS

Sixty Lewis rats underwent unilateral detachment and repair of the supraspinatus tendon and 10 rats were used for MSC harvest. Animals were randomized into 2 groups (30 animals/group). The experimental group received 10⁶ MSCs transduced with adenoviral-mediated gene transfer of human BMP-13 (Ad-BMP-13). The second group received untransduced MSCs. Fifteen animals in each group were sacrificed at 2 and 4 weeks. At each time point, 12 animals were allocated for biomechanical testing, and 3 for histomorphometric analysis.

RESULTS

There were no differences in the amount of new cartilage formation or collagen fiber organization between groups at either time point. There were also no differences in the biomechanical strength of the repairs, the cross-sectional area, peak stress at failure, or stiffness.

CONCLUSIONS

Application of MSCs genetically modified to overexpress BMP-13 did not improve healing in a rat model of rotator cuff repair.

CONCLUSIONS

Further studies are needed to evaluate various growth factors and combinations of growth factors to determine the optimal factor for the biologic augmentation of rotator cuff repairs.

L-PRF (leukocyte- and platelet-rich fibrin) is one of the four families of platelet concentrates for surgical use and is widely used in oral and maxillofacial regenerative therapies. The first objective of this article was to evaluate the mechanical vibrations appearing during centrifugation in four models of commercially available table-top centrifuges used to produce L-PRF and the impact of the centrifuge characteristics on the cell and fibrin architecture of a L-PRF clot and membrane. The second objective of this article was to evaluate how changing some parameters of the L-PRF protocol may influence its biological signature, independently from the characteristics of the centrifuge. In the first part, four different commercially available centrifuges were used to produce L-PRF, following the original L-PRF production method (glass-coated plastic tubes, 400 g force, 12 minutes). The tested systems were the original L-PRF centrifuge (Intra-Spin, Intra-Lock, the only CE and FDA cleared system for the preparation of L-PRF) and three other laboratory centrifuges (not CE/FDA cleared for L-PRF): A-PRF 12 (Advanced PRF, Process), LW-UPD8 (LW Scientific) and Salvin 13<em>10</em> (Salvin Dental). Each centrifuge was opened for inspection, two accelerometers were installed (one radial, one vertical), and data were collected with a spectrum analyzer in two configurations (full-load or half load). All clots and membranes were collected into a sterile surgical box (Xpression kit, Intra-Lock). The exact macroscopic (weights, sizes) and microscopic (photonic and scanning electron microscopy SEM) characteristics of the L-PRF produced with these four different machines were evaluated. In the second part, venous blood was taken in two groups, respectively, Intra-Spin 9 ml glass-coated plastic tubes (Intra-Lock) and A-PRF <em>10</em> ml glass tubes (Process). Tubes were immediately centrifuged at 2700 rpm (around 400 g) during 12 minutes to produce L-PRF or at 1500 rpm during 14 minutes to produce A-PRF. All centrifugations were done using the original L-PRF centrifuge (Intra-Spin), as recommended by the two manufacturers. Half of the membranes were placed individually in culture media and transferred in a new tube at seven experimental times (up to 7 days). The releases of transforming growth factor β-1 (TGFβ-1), platelet derived growth factor AB (PDGF-AB), vascular endothelial growth factor (VEGF) and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) were quantified using ELISA kits at these seven experimental times. The remaining membranes were used to evaluate the initial quantity of growth factors of the L-PRF and A-PRF membranes, through forcible extraction. Very significant differences in the level of vibrations at each rotational speed were observed between the four tested centrifuges. The original L-PRF centrifuge (Intra-Spin) was by far the most stable machine in all configurations and always remained under the threshold of resonance, unlike the three other tested machines. At the classical speed of production of L-PRF, the level of undesirable vibrations on the original centrifuge was between 4.5 and 6 times lower than with other centrifuges. Intra-Spin showed the lowest temperature of the tubes. A-PRF and Salvin were both associated with a significant increase in temperature in the tube. Intra-Spin produced the heaviest clot and quantity of exudate among the four techniques. A-PRF and LW produced much lighter, shorter and narrower clots and membranes than the two other centrifuges. Light microscopy analysis showed relatively similar features for all L-PRF types (concentration of cell bodies in the first half). However, SEM illustrated considerable differences between samples. The original Intra-Spin L-PRF showed a strongly polymerized thick fibrin matrix and all cells appeared alive with a normal shape, including the textured surface aspect of activated lymphocytes. The A-PRF, Salvin and LW PRF-like membranes presented a lightly polymerized slim fibrin gel and most of the visible cell bodies appeared destroyed (squashed or shrunk). In the second part of this study, the slow release of the three tested growth factors from original L-PRF membranes was significantly stronger (more than twice stronger, p<0.001) at all experimental times than the release from A-PRF membranes. No trace of BMP2 could be detected in the A-PRF. A slow release of BMP2 was detected during at least 7 days in the original L-PRF. Moreover, the original L-PRF clots and membranes (produced with 9 mL blood) were always significantly larger than the A-PRF (produced with <em>10</em> mL blood). The A-PRF membranes dissolved in vitro after less than 3 days, while the L-PRF membrane remained in good shape during at least 7 days. Each centrifuge has its clear own profile of vibrations depending on the rotational speed, and the centrifuge characteristics are directly impacting the architecture and cell content of a L-PRF clot. This result may reveal a considerable flaw in all the PRP/PRF literature, as this parameter was never considered. The original L-PRF clot (Intra-Spin) presented very specific characteristics, which appeared distorted when using centrifuges with a higher vibration level. A-PRF, LW and Salvin centrifuges produced PRF-like materials with a damaged and almost destroyed cell population through the standard protocol, and it is therefore impossible to classify these products in the L-PRF family. Moreover, when using the same centrifuge, the original L-PRF protocol allowed producing larger clots/membranes and a more intense release of growth factors (biological signature at least twice stronger) than the modified A-PRF protocol. Both protocols are therefore significantly different, and the clinical and experimental results from the original L-PRF shall not be extrapolated to the A-PRF. Finally, the comparison between the total released amounts and the initial content of the membrane (after forcible extraction) highlighted that the leukocytes living in the fibrin matrix are involved in the production of significant amounts of growth factors. The centrifuge characteristics and centrifugation protocols impact significantly and dramatically the cells, growth factors and fibrin architecture of L-PRF.

Although the action of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) on osteoblast differentiation has been extensively investigated, its effect on osteoclast differentiation remains unknown. In the present study, in vitro effects of BMP-2 on osteoclast-like cell formation and <em>bone</em> resorption were examined. BMP-2 (1-<em>10</em>0 ng/ml) significantly stimulated <em>bone</em> resorption by preexistent osteoclast-like cells in mouse <em>bone</em> cell cultures containing stromal cells, whereas it did not affect the <em>bone</em>-resorbing activity of isolated rabbit osteoclast-like cells. When BMP-2 was added to unfractionated <em>bone</em> cells after degeneration of preexistent osteoclast-like cells, BMP-2 dose-dependently stimulated osteoclast-like formation at a minimal effective concentration of <em>10</em> pg/ml. BMP-2 also enhanced the osteoclast-like cell formation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Moreover, osteoclast-like cells newly formed by BMP-2 from unfractionated <em>bone</em> cells possessed the ability to form pits on dentine slices. Because these results indicated that BMP-2 directly or indirectly stimulated osteoclast differentiation and activity, we next examined the direct effect of BMP-2 on osteoclast precursors in the absence of stromal cells using hemopoietic blast cells derived from spleen cells. The mRNA for BMP-2/4 receptor was detected in hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as osteoblastic MC3T3-E1 cells and MC3T3-G2/PA6 stromal cells by RNase protection assay. BMP-2 dose-dependently stimulated osteoclast-like cell formation from hemopoietic blast cells supported by GM-CSF at a minimal effective concentration of <em>10</em> pg/ml. BMP-2 also enhanced 1,25(OH)2D3-induced osteoclast-like formation from hemopoietic blast cells. The present data are the first to indicate that BMP-2 stimulates <em>bone</em> resorption through both direct stimulation of osteoclast formation and activation of mature osteoclasts, possibly via stomal cells, in vitro.

Myocardin is a muscle lineage-restricted transcriptional coactivator that has been shown to transduce extracellular signals to the nucleus required for SMC differentiation. We now report the discovery of a myocardin/BMP<em>10</em> (where BMP<em>10</em> indicates <em>bone</em> <em>morphogenetic</em> <em>protein</em> <em>10</em>) signaling pathway required for cardiac growth, chamber maturation, and embryonic survival. Myocardin-null (Myocd) embryos and embryos harboring a cardiomyocyte-restricted mutation in the Myocd gene exhibited myocardial hypoplasia, defective atrial and ventricular chamber maturation, heart failure, and embryonic lethality. Cardiac hypoplasia was caused by decreased cardiomyocyte proliferation accompanied by a dramatic increase in programmed cell death. Defective chamber maturation and the block in cardiomyocyte proliferation were caused in part by a block in BMP<em>10</em> signaling. Myocardin transactivated the Bmp<em>10</em> gene via binding of a serum response factor-myocardin <em>protein</em> complex to a nonconsensus CArG element in the Bmp<em>10</em> promoter. Expression of p57kip2, a BMP<em>10</em>-regulated cyclin-dependent kinase inhibitor, was induced in Myocd-/- hearts, while BMP<em>10</em>-activated cardiogenic transcription factors, including NKX2.5 and MEF2c, were repressed. Remarkably, when embryonic Myocd-/- hearts were cultured ex vivo in BMP<em>10</em>-conditioned medium, the defects in cardiomyocyte proliferation and p57kip2 expression were rescued. Taken together, these data identify a heretofore undescribed myocardin/BMP<em>10</em> signaling pathway that regulates cardiomyocyte proliferation and apoptosis in the embryonic heart.

Activity of voltage-gated K(+) (K(V)) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated K(V) channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on K(V) channel function and expression in normal human PASMC. BMP-2 (<em>10</em>0 nM for 18-24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA<em>10</em>, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the>><em>10</em>-fold downregulation of KCNG2 and KCNV2, two electrically silent gamma-subunits that form heterotetramers with functional K(V) channel alpha-subunits (e.g., KCNB1-2). Furthermore, the amplitude and current density of whole cell K(V) currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K(+) currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional K(V) channels. These results suggest that BMP-2 divergently regulates mRNA expression of various K(V) channel alpha-, beta-, and gamma-subunits and significantly increases whole cell K(V) currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in K(V) channel activity and regulation of K(V) channel expression. The increased K(V) channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC.

Follistatin (FS) is an important regulator of pituitary FSH secretion through its potent ability to bind and bioneutralize activin. It also represents a prototype for binding <em>proteins</em> that control bioavailability of other TGFbeta-related growth factors such as the <em>bone</em> <em>morphogenetic</em> <em>proteins</em>. The 288-residue FS molecule has a distinctive structure comprised principally of three <em>10</em>-cysteine FS domains. These are preceded by an N-terminal segment shown by us previously to contain hydrophobic residues essential for activin binding. To establish the contribution of the FS domains themselves to FS's bioactivity, we prepared mutants with deleted or exchanged domains and intradomain point mutations. Mutants were expressed from mammalian (Chinese hamster ovary) cells and evaluated for activin binding and for biological activity in assays measuring differing aspects of FS bioactivity: activin-mediated transcriptional activity and suppression of FSH secretion in primary pituitary cell cultures. The N-terminal domain (residues 1-63) alone could not bind activin or suppress activin-mediated transcription, either alone or combined in solution with the FS domain region (residues 64-288). Deletion of FS domains 1 or 2 abolished activin binding and biological activity in both assays, whereas deletion of domain 3 was tolerated. Bioactivity was also reduced or eliminated after exchange of domains (FS 2/1/3 and FS 3/1/2) or doubling of domain 1 (FS 1/1/3) or domain 2 (FS 2/2/3). Several hydrophobic residues clustered within the C-terminal region of FS domains 1 and 2 are highly conserved among all FS domains. Mutation of any of these to Asp or Ala either reduced or eliminated FS bioactivity and disrupted distant epitopes for heparin binding (FS domain 1) or antibody recognition (FS domain 2), suggesting their role in maintaining the conformational integrity of the domain and possibly the FS molecule as a whole. These results are consistent with the importance of domain conformation as well as the overall order of the domains in FS function. A continuous sequence comprising the N-terminal domain and followed by FS domains 1 and 2 fulfills the minimum structural requirement for activin binding and FS bioactivity.