BMP-6 promotes type 2 immune response during enhancement of rat mandibular bone defect healing.

Introduction: Bone morphogenetic proteins (BMPs) are used as key therapeutic agents for the treatment of difficult fractures. While their effects on osteoprogenitors are known, little is known about their effects on the immune system. Methods: We used permutations of BMP-6 (B), vascular endothelial growth factor (V), and Hedgehog signaling pathway activator smoothened agonist (S), to treat a rat mandibular defect and investigated healing outcomes at week 8, in correlation with the cellular landscape of the immune cells in the fracture callus at week 2. Results: Maximum recruitment of immune cells to the fracture callus is known to occur at week 2. While the control, S, V, and VS groups remained as nonunions at week 8; all BMP-6 containing groups - B, BV, BS and BVS, showed near-complete to complete healing. This healing pattern was strongly associated with significantly higher ratios of CD4 T (CD45+CD3+CD4+) to putative CD8 T cells (CD45+CD3+CD4-), in groups treated with any permutation of BMP-6. Although, the numbers of putative M1 macrophages (CD45+CD3-CD11b/c+CD38high) were significantly lower in BMP-6 containing groups in comparison with S and VS groups, percentages of putative - Th1 cells or M1 macrophages (CD45+CD4+IFN-γ+) and putative - NK, NKT or cytotoxic CD8T cells (CD45+CD4-IFN-γ+) were similar in control and all treatment groups. Further interrogation revealed that the BMP-6 treatment promoted type 2 immune response by significantly increasing the numbers of CD45+CD3-CD11b/c+CD38low putative M2 macrophages, putative - Th2 cells or M2 macrophages (CD45+CD4+IL-4+) cells and putative - mast cells, eosinophils or basophils (CD45+CD4-IL-4+ cells). CD45- non-haematopoietic fractions of cells which encompass all known osteoprogenitor stem cells populations, were similar in control and treatment groups. Discussion: This study uncovers previously unidentified regulatory functions of BMP-6 and shows that BMP-6 enhances fracture healing by not only acting on osteoprogenitor stem cells but also by promoting type 2 immune response.

Current evidence on potential of adipose derived stem cells to enhance bone regeneration and future projection.

Injuries to the postnatal skeleton are naturally repaired through successive steps involving specific cell types in a process collectively termed "bone regeneration". Although complex, bone regeneration occurs through a series of well-orchestrated stages wherein endogenous bone stem cells play a central role. In most situations, bone regeneration is successful; however, there are instances when it fails and creates non-healing injuries or fracture nonunion requiring surgical or therapeutic interventions. Transplantation of adult or mesenchymal stem cells (MSCs) defined by the International Society for Cell and Gene Therapy (ISCT) as CD105+CD90+CD73+CD45-CD34-CD14orCD11b-CD79αorCD19-HLA-DR- is being investigated as an attractive therapy for bone regeneration throughout the world. MSCs isolated from adipose tissue, adipose-derived stem cells (ADSCs), are gaining increasing attention since this is the most abundant source of adult stem cells and the isolation process for ADSCs is straightforward. Currently, there is not a single Food and Drug Administration (FDA) approved ADSCs product for bone regeneration. Although the safety of ADSCs is established from their usage in numerous clinical trials, the bone-forming potential of ADSCs and MSCs, in general, is highly controversial. Growing evidence suggests that the ISCT defined phenotype may not represent bona fide osteoprogenitors. Transplantation of both ADSCs and the CD105- sub-population of ADSCs has been reported to induce bone regeneration. Most notably, cells expressing other markers such as CD146, AlphaV, CD200, PDPN, CD164, CXCR4, and PDGFRα have been shown to represent osteogenic sub-population within ADSCs. Amongst other strategies to improve the bone-forming ability of ADSCs, modulation of VEGF, TGF-ß1 and BMP signaling pathways of ADSCs has shown promising results. The U.S. FDA reveals that 73% of Investigational New Drug applications for stem cell-based products rely on CD105 expression as the "positive" marker for adult stem cells. A concerted effort involving the scientific community, clinicians, industries, and regulatory bodies to redefine ADSCs using powerful selection markers and strategies to modulate signaling pathways of ADSCs will speed up the therapeutic use of ADSCs for bone regeneration.

Correction: BMP-Non-Responsive Sca1+CD73+CD44+ Mouse Bone Marrow Derived Osteoprogenitor Cells Respond to Combination of VEGF and BMP-6 to Display Enhanced Osteoblastic Differentiation and Ectopic Bone Formation.

[This corrects the article DOI: 10.1371/journal.pone.0103060.].

Assessment of Hedgehog Signaling Pathway Activation for Craniofacial Bone Regeneration in a Critical-Sized Rat Mandibular Defect.

IMPORTANCE: Osseous craniofacial defects are currently reconstructed with bone grafting, rigid fixation, free tissue transfer, and/or recombinant human bone morphogenetic protein 2. Although these treatment options often have good outcomes, they are associated with substantial morbidity, and many patients are not candidates for free tissue transfer. OBJECTIVE: To assess whether polysaccharide-based scaffold (PS) constructs that are cross-linked with smoothened agonist (SAG), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 6 (BMP-6) would substantially increase bone regeneration. DESIGN, SETTING, AND PARTICIPANTS: This animal model study was conducted at the University of Virginia School of Medicine Cui Laboratory from March 1, 2017, to June 30, 2017. Thirty-three 10-week-old female Lewis rats were acquired for the study. Bilateral nonsegmental critical-sized defects were created in the angle of rat mandibles. The defects were either left untreated or filled with 1 of the 9 PSs. The rats were killed after 8 weeks, and bone regeneration was evaluated using microcomputed tomographic imaging and mechanical testing. Analysis of variance testing was used to compare the treatment groups. MAIN OUTCOMES AND MEASURES: Blinded analysis and computer analysis of the microcomputed tomographic images were used to assess bone regeneration. RESULTS: In the 33 female Lewis rats, minimal healing was observed in the untreated mandibles. Addition of SAG was associated with increases in bone regeneration and bone density in all treatment groups, and maximum bone healing was seen in the group with BMP-6, VEGF, and SAG cross-linked to PS. For each of the 5 no scaffold group vs BMP-6, VEGF, and SAG cross-linked to PS group comparisons, mean defect bone regeneration was 4.14% (95% CI, 0.94%-7.33%) vs 66.19% (95% CI, 54.47%-77.90%); mean bone volume, 14.52 mm3 (95% CI, 13.07-15.97 mm3) vs 20.87 mm3 (95% CI, 14.73- 27.01 mm3); mean bone surface, 68.97 mm2 (95% CI, 60.08-77.85 mm2) vs 96.77 mm2 (95% CI, 76.11-117.43 mm2); mean ratio of bone volume to total volume, 0.11 (95% CI, 0.10-0.11) vs 0.15 (95% CI, 0.10-0.19); and mean connectivity density 0.03 (95% CI, 0.02-0.05) vs 0.32 (95% CI, 0.25-0.38). On mechanical testing, mandibles with untreated defects broke with less force than control mandibles in which no defect was made, although this force did not reach statistical significance. No significant difference in force to fracture was observed among the treatment groups. CONCLUSIONS AND RELEVANCE: In this rat model study, activation of the hedgehog signaling pathway using smoothened agonist was associated with increased craniofacial bone regeneration compared with growth factors alone, including US Food and Drug Administration-approved recombinant human bone morphogenetic protein 2. Pharmaceuticals that target this pathway may offer a new reconstructive option for bony craniofacial defects as well as nonunion and delayed healing fractures. LEVEL OF EVIDENCE: NA.

A review of UHMWPE wear-induced osteolysis: the role for early detection of the immune response.

In a world where increasing joint arthroplasties are being performed on increasingly younger patients, osteolysis as the leading cause of failure after total joint arthroplasty (TJA) has gained considerable attention. Ultra-high molecular weight polyethylene wear-induced osteolysis is the process by which prosthetic debris mechanically released from the surface of prosthetic joints induces an immune response that favors bone catabolism, resulting in loosening of prostheses with eventual failure or fracture. The immune response initiated is innate in that it is nonspecific and self-propagating, with monocytic cells and osteoclasts being the main effectors. To date, detecting disease early enough to implement effective intervention without unwanted systemic side effects has been a major barrier. These barriers can be overcome using newer in vivo imaging techniques and modules linked with fluorescence and/or chemotherapies. We discuss the pathogenesis of osteolysis, and provide discussion of the challenges with imaging and therapeutics. We describe a positron emission tomography imaging cinnamoyl-Phe-(D)-Leu-Phe-(D)-Leu-Phe-Lys module, specific to macrophages, which holds promise in early detection of disease and localization of treatment. Further research and increased collaboration among therapeutic and three-dimensional imaging researchers are essential in realizing a solution to clinical osteolysis in TJA.

Dual Inhibition of Activin/Nodal/TGF-ß and BMP Signaling Pathways by SB431542 and Dorsomorphin Induces Neuronal Differentiation of Human Adipose Derived Stem Cells.

Damage to the nervous system can cause devastating diseases or musculoskeletal dysfunctions and transplantation of progenitor stem cells can be an excellent treatment option in this regard. Preclinical studies demonstrate that untreated stem cells, unlike stem cells activated to differentiate into neuronal lineage, do not survive in the neuronal tissues. Conventional methods of inducing neuronal differentiation of stem cells are complex and expensive. We therefore sought to determine if a simple, one-step, and cost effective method, previously reported to induce neuronal differentiation of embryonic stem cells and induced-pluripotent stem cells, can be applied to adult stem cells. Indeed, dual inhibition of activin/nodal/TGF-ß and BMP pathways using SB431542 and dorsomorphin, respectively, induced neuronal differentiation of human adipose derived stem cells (hADSCs) as evidenced by formation of neurite extensions, protein expression of neuron-specific gamma enolase, and mRNA expression of neuron-specific transcription factors Sox1 and Pax6 and matured neuronal marker NF200. This process correlated with enhanced phosphorylation of p38, Erk1/2, PI3K, and Akt1/3. Additionally, in vitro subcutaneous implants of SB431542 and dorsomorphin treated hADSCs displayed significantly higher expression of active-axonal-growth-specific marker GAP43. Our data offers novel insights into cell-based therapies for the nervous system repair.

Poly(lactic-co-glycolide) polymer constructs cross-linked with human BMP-6 and VEGF protein significantly enhance rat mandible defect repair.

We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (P(VEGF)) or BMP-6 (P(BMP-6)) or both (P(VEGF+BMP-6)) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with P(VEGF+BMP-6) scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the P(VEGF+BMP-6) group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in P(VEGF), P(BMP-6) and P(VEGF+BMP-6) but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with P(VEGF), P(BMP-6), or P(VEGF+BMP-6) but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on P(VEGF+BMP-6) but not on P, P(VEGF), or P(BMP-6). Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.

Inhibition of differentiation and function of osteoclasts by dimethyl sulfoxide (DMSO).

Dimethyl sulfoxide (DMSO) is an FDA-approved organosulfur solvent that is reported to have therapeutic value in osteoarthritis and osteopenia. DMSO is used as a cryoprotectant for the cryopreservation of bone grafts and mesenchymal stem cells which are later used for bone repair. It is also used as a solvent in the preparation of various scaffolds used for bone tissue engineering purposes. DMSO has been reported to inhibit osteoclast formation in vitro but the mechanism involved has remained elusive. We investigated the effect of DMSO on osteoclast differentiation and function using a conventional model system of RAW 264.7 cells. The differentiation of RAW 264.7 cells was induced by adding 50 ng/ml RANKL and the effect of DMSO (0.01 and 1% v/v) on RANKL-induced osteoclastogenesis was investigated. Addition of 1% DMSO significantly inhibited RANKL-induced formation of TRAP+, multinucleated, mature osteoclasts and osteoclast late-stage precursors (c-Kit(-) c-Fms(+) Mac-1(+) RANK(+)). While DMSO did not inhibit proliferation per se, it did inhibit the effect of RANKL on proliferation of RAW 264.7 cells. Key genes related to osteoclast function (TRAP, Integrin αVß3, Cathepsin K and MMP9) were significantly down-regulated by DMSO. RANKL-induced expression of RANK gene was significantly reduced in the presence of DMSO. Our data, and reports from other investigators, that DMSO enhances osteoblastic differentiation of mesenchymal stem cells and also prevents bone loss in ovarietcomized rats, suggest that DMSO has tremendous potential in the treatment of osteoporosis and bone diseases arising from uncontrolled activities of the osteoclasts.

Recognition of Immune Response for the Early Diagnosis and Treatment of Osteoarthritis.

Osteoarthritis is a common and debilitating joint disease that affects up to 30 million Americans, leading to significant disability, reduction in quality of life, and costing the United States tens of billions of dollars annually. Classically, osteoarthritis has been characterized as a degenerative, wear-and-tear disease, but recent research has identified it as an immunopathological disease on a spectrum between healthy condition and rheumatoid arthritis. A systematic literature review demonstrates that the disease pathogenesis is driven by an early innate immune response which progressively catalyzes degenerative changes that ultimately lead to an altered joint microenvironment. It is feasible to detect this infiltration of cells in the early, and presumably asymptomatic, phase of the disease through noninvasive imaging techniques. This screening can serve to aid clinicians in potentially identifying high-risk patients, hopefully leading to early effective management, vast improvements in quality of life, and significant reductions in disability, morbidity, and cost related to osteoarthritis. Although the diagnosis and treatment of osteoarthritis routinely utilize both invasive and non-invasive strategies, imaging techniques specific to inflammatory cells are not commonly employed for these purposes. This review discusses this paradigm and aims to shift the focus of future osteoarthritis-related research towards early diagnosis of the disease process.

Interactions between MSCs and immune cells: implications for bone healing.

It is estimated that, of the 7.9 million fractures sustained in the United States each year, 5% to 20% result in delayed or impaired healing requiring therapeutic intervention. Following fracture injury, there is an initial inflammatory response that plays a crucial role in bone healing; however, prolonged inflammation is inhibitory for fracture repair. The precise spatial and temporal impact of immune cells and their cytokines on fracture healing remains obscure. Some cytokines are reported to be proosteogenic while others inhibit bone healing. Cell-based therapy utilizing mesenchymal stromal cells (MSCs) is an attractive option for augmenting the fracture repair process. Osteoprogenitor MSCs not only differentiate into bone, but they also exert modulatory effects on immune cells via a variety of mechanisms. In this paper, we review the current literature on both in vitro and in vivo studies on the role of the immune system in fracture repair, the use of MSCs in the enhancement of fracture healing, and interactions between MSCs and immune cells. Insight into this paradigm can provide valuable clues in identifying cellular and noncellular targets that can potentially be modulated to enhance both natural bone healing and bone repair augmented by the exogenous addition of MSCs.

Cross-Talk Between VEGF and BMP-6 Pathways Accelerates Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

Deficiency in vascular endothelial growth factor (VEGF) or bone morphogenetic proteins (BMPs) results in fracture non-unions. Therefore, it is indispensable to comprehend the combined effect of VEGF and BMPs on the osteogenic differentiation of osteoprogenitor mesenchymal stem cells (MSCs) that are either naturally occurring at the fracture repair site or exogenously added to enhance the bone repair. We found that the combination of VEGF and BMP-6 enhanced COL1A2 expression, which correlated with upregulated expression of osterix, Dlx5, and Msx2 in human adipose-derived stem cells (hADSCs). Cross-talk between VEGF and BMP-6 pathways upregulated activation of p38 mitogen-activated kinase (p38 MAPK) and inhibited activation of protein kinase B (PKB, also known as Akt), whereas phosphorylation of "mothers against decapentaplegic" homologs 1/5/8 (Smads 1/5/8) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2) was not affected. Consistent with these findings, p38 inhibitor SB203580, or siRNA knockdown of osterix, abrogated crosstalk between the VEGF and BMP-6 pathways and significantly reduced the observed upregulation of COL1A2. Nuclear translocation of the phosphorylated form of osterix was also inhibited by SB203580. Although crosstalk between the VEGF-BMP-6 pathways did not show an effect on the extent of mineralization, inhibition of any one of the three components that were upregulated through the cross-talk, i.e., osterix, Dlx5, and p38 activation, led to a complete inhibition of mineralization. Inhibition of PKB/Akt activation, which is attenuated through the cross-talk, significantly enhanced ALP gene expression. These observations imply that crosstalk between the VEGF and BMP-6 signaling pathways enhances osteogenic differentiation of MSCs.

Expression of CD105 and CD34 receptors controls BMP-induced in vitro mineralization of mouse adipose-derived stem cells but does not predict their in vivo bone-forming potential.

Adipose-derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose-derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone-forming potential. Four purified populations of mouse ADSCs: CD105(+) CD34(+), CD105(-) CD34(-), CD105(+) CD34(-) and CD105(-) CD34(+) were obtained using fluorescence-activated cell sorting (FACS) and their BMP-responsiveness was determined in vitro. CD105(+) CD34(-) population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP-responsive phenotype and larger cell size while expression of CD34 correlated with low BMP-responsive phenotype and smaller cell size. CD105(+) CD34(-) population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105(+) CD34(-) ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP-responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair.

BMP-non-responsive Sca1+ CD73+ CD44+ mouse bone marrow derived osteoprogenitor cells respond to combination of VEGF and BMP-6 to display enhanced osteoblastic differentiation and ectopic bone formation.

Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.

Adenoviral Delivery of the VEGF and BMP-6 Genes to Rat Mesenchymal Stem Cells Potentiates Osteogenesis.

The combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF), and bone morphogenetic protein (BMP) to sites of bone injury results in enhanced repair compared to the administration of a single factor or a combination of two factors. Based on these findings, we hypothesized that coexpression of VEGF and BMP-6 genes would enhance the osteoblastic differentiation of rat bone-marrow-derived stem cells (rMSCs) and osteogenesis by comparison to rMSCs that do not express VEGF and BMP-6. We prepared a GFP tagged adenovirus vector (Ad-VEGF+BMP-6) that contained DNA encoding the hVEGF and hBMP-6 genes. rMSCs were transduced with the virus, and the successful transduction was confirmed by green fluorescence and by production of VEGF and BMP-6 proteins. The cells were cultured to assess osteoblastic differentiation or administered in the Fischer 344 rats to assess bone formation. Mineralization of rMSCs transduced with Ad-VEGF+BMP-6 was significantly enhanced over the nontransduced rMSCs. Only transduced rMSCs could induce osteogenesis in vivo, whereas Ad-VEGF+BMP-6 or nontransduced rMSCs alone did not induce osteogenesis. The data suggests that the combined delivery of MSCs, VEGF, and BMP-6 is an attractive option for bone repair therapy.

Combined angiogenic and osteogenic factor delivery for bone regenerative engineering.

Both osteogenesis and angiogenesis are integrated parts of bone growth and regeneration. Combined delivery of osteogenic and angiogenic factors is a novel approach in bone regenerative engineering. Exogenous addition of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) together with an osteoconductive scaffold is a very promising method to enhance bone repair. This concept has been incorporated into the development of new strategies for bone tissue engineering and significant advancements have been made in last 10 years. In contrary to previous belief that VEGF modulates bone repair only by enhancing angiogenesis in the proximity of bone injury, recent evidence also suggests that cross-talk between VEGF and BMP signaling pathways in MSCs promotes osteoblastic differentiation of MSCs which aids in fracture repair. Future studies should focus on cross-talk between angiogenesis and osteogenesis, optimization of VEGF/BMP ratios, selection of the most potent BMPs, and optimization of delivery methods for VEGF and BMP. Recent discoveries from basic research including effective delivery of growth factors and cells to the area of interest will help bring VEGF plus BMP for bone healing from the bench to the patient's bedside.

Interferon gamma and T cells inhibit osteogenesis induced by allogeneic mesenchymal stromal cells.

The mesenchymal stromal cells (MSCs) are reported to be immunoprivileged and osteogenic. We hypothesized that the use of allogeneic MSCs for bone repair was possible if they displayed an ability to induce similar osteogenesis in syngeneic as well as in allogeneic hosts. To test this hypothesis we used a cloned bone marrow derived cell, termed D1, isolated from Balb/c mice. The D1 cells were subcutaneously injected in syngeneic Balb/c, allogeneic immunocompetent B6, allogeneic T-cell deficient NCr nude, and allogeneic B6 Pfp-/- Rag2-/- mice that lack matured T and B cells as well as NK-cell cytolytic functions. D1 cells formed ectopic bones only in syngeneic or allogeneic immunocompromised hosts but not in allogeneic B6 hosts. The lack of T cells alone in allogeneic NCr mice was sufficient to promote osteogenesis in allogeneic environment. We observed a significantly higher number of T cells, B cells, macrophages and significantly higher expression of interferon gamma (IFN-γ) in B6 allogeneic implants as compared to the syngeneic implants. These factors correlated with severe inhibition of expression of alkaline phosphatase, osteocalcin, and runx2 genes in the implants from B6 mice. Our data suggest that strategies to inhibit T cells and IFN-γ functions will be useful for bone repair mediated by allogeneic MSCs.

Osteogenic response of human adipose-derived stem cells to BMP-6, VEGF, and combined VEGF plus BMP-6 in vitro.

Exogenous addition of three factors-mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF), and bone morphogenetic proteins (BMPs)-has proven to be more beneficial than delivery of any single factor for fracture repair in animal models. We studied the osteogenic differentiation of human adipose-derived stem cells (hADSCs) in the presence of VEGF, BMP-6, or VEGF plus BMP-6 to better understand their enhancement of osteoblastic differentiation of MSCs. The VEGF plus BMP-6 group demonstrated an additive effect on the enhancement of mineralization and expression of ALP and Msx2 genes. Unlike VEGF or BMP-6 alone, the combination of VEGF and BMP-6 significantly enhanced the expression of COL1A1, osterix, and Dlx5 genes. The data indicate that a cross-talk between VEGF and BMP-6 signaling pathways enhances osteogenic differentiation of hADSCs.

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and ß-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells.

New strategies such as combined utilization of growth factors may provide a better treatment for difficult fractures. We have demonstrated enhanced angiogenesis and osteogenesis through the actions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) on the osteogenic differentiation of a cloned mouse osteoprogenitor cell in vitro and ectopic bone formation in vivo. Human VEGF and BMP-6 genes expressed together produced a significant increase in alkaline phosphatase activity, expression of the RunX2 and osteocalcin genes and mineralization. Microcomputed tomographic analysis of subcutaneous implants consisting of cells transfected with VEGF and BMP-6 cDNA and delivered on a 3D poly (lactic-co-glycolic acid) scaffold confirmed the additive effects between VEGF and BMP-6. Ectopic bone formation in the VEGF plus BMP-6 group was greatest compared to that in either VEGF or BMP-6 alone. This is the first study that demonstrates osteogenesis in vitro and in vivo through the additive effects of VEGF and BMP-6.

Comparison of 16S rRNA gene sequences of genus Methanobrevibacter.

BACKGROUND: The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter. RESULTS: The phylogenetic analysis of the genus based on 786 bp aligned region from fifty-four representative sequences of the 120 available sequences for the genus revealed seven multi-member groups namely, Ruminantium, Smithii, Woesei, Curvatus, Arboriphilicus, Filiformis, and the Termite gut symbionts along with three separate lineages represented by Mbr. wolinii, Mbr. acididurans, and termite gut flagellate symbiont LHD12. The cophenetic correlation coefficient, a test for the ultrametric properties of the 16S rRNA gene sequences used for the tree was found to be 0.913 indicating the high degree of goodness of fit of the tree topology. A significant relationship was found between the 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) for the genus with the correlation coefficient (r) for logD and logS, and for [ln(-lnD) and ln(-lnS)] being 0.73 and 0.796 respectively. Our analysis revealed that for this genus, when S = 0.984, D would be <70% at least 99% of the times, and with 70% D as the species "cutoff", any 16S rRNA gene sequence showing <98% sequence similarity can be considered as a separate species. In addition, we deduced group specific signature positions that have remained conserved in evolution of the genus. CONCLUSIONS: A very significant relationship between D and S was found to exist for the genus Methanobrevibacter, implying that it is possible to predict D from S with a known precision for the genus. We propose to include the termite gut flagellate symbiont LHD12, the methanogenic endosymbionts of the ciliate Nyctotherus ovalis, and rat feces isolate RT reported earlier, as separate species of the genus Methanobrevibacter.