Driving Osteocytogenesis from Mesenchymal Stem Cells in Osteon-like Biomimetic Nanofibrous Scaffolds.

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.

Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-ß2 stimulation in retinal pigment epithelial cells through modulation of the p38 and JNK MAPK pathways.

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-ß2 (TGF-ß2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-ß2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-ß2. The treatment of RPE cells with TGF-ß2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-ß2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-ß2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-ß2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.

Bone morphogenetic protein 6 induces downregulation of pentraxin 3 expression in human granulosa lutein cells in women with polycystic ovary syndrome.

PURPOSE: To evaluate whether PTX3 is differentially expressed in the granulosa lutein cells derived from women with PCOS and whether BMP6 can regulate the expression of PTX3 in hGL cells. METHODS: The expression levels of BMP6 and PTX3 in granulosa lutein cells were evaluated by RT-qPCR. The correlation between the expression levels of BMP6 /PTX3 and oocyte quality indexes were analyzed using clinical samples. The cells were incubated with BMP6 at different concentrations and times to check the expression of PTX3 in KGN cells. TGF-ß type I inhibitors and small interfering RNA targeting ALK2/3/6,SMAD1/5/8 and SMAD4 were used to study the involvement of SMAD dependent pathways in KGN cells. RESULTS: The levels of BMP6 in hGL cells were negatively correlated with the corresponding oocyte maturation rate and high-quality embryo rate, whereas the levels of PTX3 were positively correlated with the corresponding oocyte maturation rate in PCOS. Additionally, the in vitro cell cultured results showed BMP6 significantly inhibited the expression of PTX3 in KGN cells. Furthermore, using a dual inhibition approach (kinase inhibitors and small interfering RNAs), we identified the ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors and the downstream SMAD1/SMAD5-SMAD4 signaling pathway were responsible for the BMP6-induced cellular activities in KGN cells. CONCLUSIONS: The suppressive effect of BMP6 on PTX3 was mediated by ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors in granulosa cells through the SMAD1/5-SMAD4 dependent signaling pathway in PCOS.Our findings provides new insights into the understanding of the pathogenesis of PCOS-related ovulatory disorders.

Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes.

Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron stimulates expression of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells, which in turn binds to BMP receptors on hepatocytes and induces the SMAD signaling cascade for transcriptional activation of the hepcidin-encoding HAMP mRNA. SMAD signaling is also essential for inflammatory HAMP mRNA induction by the IL-6/STAT3 pathway. Herein, we utilized human Huh7 hepatoma cells and primary murine hepatocytes to assess the effects of iron perturbations on signaling to hepcidin. Iron chelation appeared to slightly impair signaling to hepcidin. Subsequent iron supplementation not only failed to reverse these effects, but drastically reduced basal HAMP mRNA and inhibited HAMP mRNA induction by BMP6 and/or IL-6. Thus, treatment of cells with excess iron inhibited basal and BMP6-mediated SMAD5 phosphorylation and induction of HAMP, ID1 and SMAD7 mRNAs in a dose-dependent manner. Iron also inhibited IL-6-mediated STAT3 phosphorylation and induction of HAMP and SOCS3 mRNAs. These responses were accompanied by induction of GCLC and HMOX1 mRNAs, known markers of oxidative stress. We conclude that hepatocellular iron overload suppresses hepcidin by inhibiting the SMAD and STAT3 signaling pathways downstream of their respective ligands.

Fibroblastic differentiation of mesenchymal stem/stromal cells (MSCs) is enhanced by hypoxia in 3D cultures treated with bone morphogenetic protein 6 (BMP6) and growth and differentiation factor 5 (GDF5).

INTRODUCTION: Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis. METHODS: Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21 days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy. RESULTS: Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6. CONCLUSIONS: Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.

The lower in vitro chondrogenic potential of canine adipose tissue-derived mesenchymal stromal cells (MSC) compared to bone marrow-derived MSC is not improved by BMP-2 or BMP-6.

Mesenchymal stromal cells (MSC) are used for cell-based treatment for canine osteoarthritis (OA). Compared with human MSCs, detailed information on the functional characterisation of canine MSCs is limited. In particular, the chondrogenic differentiation of canine adipose tissue-derived MSCs (cAT-MSCs) is challenging. In this study, we aimed to compare cAT-MSCs with bone marrow-derived MSCs (cBM-MSCs), focusing specifically on their in vitro chondrogenic potential, with or without bone morphogenetic proteins (BMP). cBM-MSCs and cAT-MSCs were characterised using flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The chondrogenic differentiation potential of all cMSC preparations in the presence of TGF-ß1 alone or when supplemented with 10, 100, or 250 ng/mL BMP-2 or BMP-6 was investigated using RT-qPCR, and biochemical, histochemical and immunohistological analyses. Both cBM-MSCs and cAT-MSCs expressed the surface markers CD90, CD73, and CD29, and were negative for CD45 and CD34, although the expression of CD73 and CD271 varied with donor and tissue origin. Interestingly, expression of ACAN and SOX9 was higher in cBM-MSCs than cAT-MSCs. In contrast with cBM-MSCs, cAT-MSCs could not differentiate toward the chondrogenic lineage without BMP-2/-6, and their in vitro chondrogenesis was inferior to cBM-MSCs with BMP-2/-6. Thus, cAT-MSCs have lower in vitro chondrogenic capacity than cBM-MSC under the studied culture conditions with 10, 100, or 250 ng/mL BMP-2 or BMP-6. Therefore, further characterisation is necessary to explore the potential of cAT-MSCs for cell-based OA treatments.

Therapeutic Effects of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Combined with Cartilage Acellular Matrix Mediated Via Bone Morphogenic Protein 6 in a Rabbit Model of Articular Cruciate Ligament Transection.

Osteoarthritis (OA) is a general joint disease. Cartilage damage is associated with a decrease in the density of chondrocytes. Mesenchymal stem cells (MSCs) differentiate into adipocytes, osteocytes and chondrocytes, and are an excellent source of cell therapy. Cartilage-derived extracellular matrix (ECM) promotes chondrogenesis of MSCs. However, the role of MSCs stimulated by ECM is not well known in OA. The purpose of this study is to determine the role of specific factors generated by the application of ECM and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in managing OA symptoms. Cartilage acellular matrix (CAM), which is a cartilage-derived ECM, was used to promote the chondrogenesis of UCB-MSCs. Induced MSCs were analyzed using chondrogenic markers (aggrecan, collagen type 2, and SOX9) and bone morphogenic protein 6 (BMP6). BMP6 is known to be involved in early chondrogenesis of MSCs. As a result, treatment with CAM significantly increased the expression of chondrogenic markers and BMP6 in UCB-MSCs. Treatment with recombinant human BMP6 also dramatically increased the levels of chondrogenic markers in UCB-MSCs. In addition, UCB-MSCs and CAM were used to evaluate OA symptom improvement in a rabbit articular cruciate ligament transection (ACLT) model. Application of UCB-MSCs and CAM enhanced not only the structure and synthesis of proteoglycan and collagen type 2 but also anti-inflammatory effects in both rabbit joint and synovial fluid. Moreover, the detection of human cells and involvement of BMP6 were confirmed in rabbit cartilage tissues. This study indicates that therapeutic potential of UCB-MSCs with CAM is mediated via BMP6 in OA.

The BMP-SMAD pathway mediates the impaired hepatic iron metabolism associated with the ERFE-A260S variant.

The erythroferrone (ERFE) is the erythroid regulator of hepatic iron metabolism by suppressing the expression of hepcidin. Congenital dyserythropoietic anemia type II (CDAII) is an inherited hyporegenerative anemia due to biallelic mutations in the SEC23B gene. Patients with CDAII exhibit marked clinical variability, even among individuals sharing the same pathogenic variants. The ERFE expression in CDAII is increased and related to abnormal erythropoiesis. We identified a recurrent low-frequency variant, A260S, in the ERFE gene in 12.5% of CDAII patients with a severe phenotype. We demonstrated that the ERFE-A260S variant leads to increased levels of ERFE, with subsequently marked impairment of iron regulation pathways at the hepatic level. Functional characterization of ERFE-A260S in the hepatic cell system demonstrated its modifier role in iron overload by impairing the BMP/SMAD pathway. We herein described for the first time an ERFE polymorphism as a genetic modifier variant. This was with a mild effect on disease expression, under a multifactorial-like model, in a condition of iron-loading anemia due to ineffective erythropoiesis.

Hyaluronan hydrogels delivering BMP-6 for local targeting of malignant plasma cells and osteogenic differentiation of mesenchymal stromal cells.

Multiple myeloma is a malignant disease characterized by accumulation of clonal plasma cells in the bone marrow. Uncoupling of bone formation and resorption by myeloma cells leads to osteolytic lesions. These are prone to fracture and represent a possible survival space for myeloma cells under treatment causing disease relapse. Here we report on a novel approach suitable for local treatment of multiple myeloma based on hyaluronic acid (HA) hydrogels mimicking the physical properties of the bone marrow. The HA hydrogels are complexed with heparin to achieve sustained presentation and controlled release of bone morphogenetic protein 6 (BMP-6). Others and we have shown that BMP-6 induces myeloma cell apoptosis and bone formation. Using quartz crystal microbalance and enzyme-linked immunosorbent assay, we measured an initial surface density of 400 ng BMP6/cm2, corresponding to two BMP-6 per heparin molecule, with 50% release within two weeks. HA-hydrogels presenting BMP-6 enhanced the phosphorylation of Smad 1/5 while reducing the activity of BMP-6 antagonist sclerostin. These materials induced osteogenic differentiation of mesenchymal stromal cells and decreased the viability of myeloma cell lines and primary myeloma cells. BMP-6 functionalized HA-hydrogels represent a promising material for local treatment of myeloma-induced bone disease and residual myeloma cells within lesions to minimize disease relapse or fractures. STATEMENT OF SIGNIFICANCE: Multiple myeloma is a hematological cancer characterized by the accumulation of clonal plasma cells in the bone marrow and local suppression of bone formation, resulting in osteolytic lesions and fractures. Despite recent advances in systemic treatment of multiple myeloma, it is rare to achieve a targeted suppression of myeloma cells and healing of bone lesions. Here we present hydrogels which mimic the physico-chemical properties of the bone marrow, consisting of hyaluronic acid with crosslinked heparin for the controlled presentation of bioactive BMP-6. The hydrogels decrease the viability of myeloma cell lines and primary myeloma cells and induces osteogenic differentiation of mesenchymal stromal cells. The presentation of BMP-6 in the hyaluronan hydrogels enhances the phosphorylation of Smad1/5 while reducing the activity of the BMP-6 antagonist sclerostin. As such, BMP-6 functionalized hyaluronan hydrogels represent a promising material for the localized eradication of myeloma cells.

SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells†.

Connective tissue growth factor (also known as CTGF or CCN2) is a secreted matricellular protein that belongs to the CCN family. With wide-ranging biological activities and tissue expression patterns, CTGF plays a critical role in regulating various cellular functions. In the female reproductive system, CTGF is highly expressed in granulosa cells in growing ovarian follicles and is involved in the regulation of follicular development, ovulation, and luteal function. In the mammalian ovary, bone morphogenetic protein 6 (BMP6) is an important intraovarian modulator of follicular development. In this study, we demonstrated that BMP6 treatment significantly increased the expression of CTGF in both primary and immortalized human granulosa cells. Using both pharmacological inhibitors and Small interfering RNA-mediated knockdown approaches, we showed that ALK2 and ALK3 type I receptors are required for BMP6-induced cellular activities. Furthermore, this effect is most likely mediated by a Sma- and Mad-related protein (SMAD)-dependent pathway. Our studies provide novel insight into the molecular mechanisms by which an intraovarian growth factor affects the production of another factor via a paracrine effect in human granulosa cells.

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 novel role of bone morphogenetic protein 6 (BMP6) in glucose homeostasis.

AIMS: Bone morphogenetic proteins (BMPs) are involved in the development and homeostasis of multiple organs and tissues. There has been a significant focus on understanding the role of BMPs in pancreatic ß-cell dysfunction associated with type 2 diabetes (T2D). Our objective was to investigate the relationship between BMP6 and glucose homeostasis. METHODS: Ob/ob mice were treated with BMP6 for 6 days and analyzed for insulin release, body weight, lipid parameters and glucose tolerance. Quantitative real-time PCR, chromatin immunoprecipitation and glucose output assays were used to assess BMP6 effect on gluconeogenesis in rat hepatoma H4IIE cells. Specificity of BMP6 receptors was characterized by the utilization of various receptor Fc fusion proteins in luciferase reporter gene and glucose output assays in INS1 and H4IIE cells. RESULTS: Treatment of ob/ob mice with BMP6 for 6 days resulted in a reduction of circulating glucose and lipid levels, followed by a significantly elevated plasma insulin level in a dose-dependent manner. In addition, BMP6 improved the glucose excursion during an oral glucose tolerance test, lowering the total glycemic response by 21%. In rat H4IIE hepatoma cells, BMP6 inhibited gluconeogenesis and glucose output via downregulation the PepCK expression. Moreover, BMP6 inhibited glucose production regardless of the presence of cAMP, antagonizing its glycogenolytic effect. BMP6 acted on pancreatic and liver cells utilizing Alk3, Alk6 and ActRIIA serine/threonine kinase receptors. CONCLUSIONS: Collectively, we demonstrate that BMP6 improves glycaemia in T2D mice and regulates glucose metabolism in hepatocytes representing an exciting prospect for future treatments of diabetes.

Antiviral activity of bone morphogenetic proteins and activins.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

HIF-1α-mediated BMP6 down-regulation leads to hyperproliferation and abnormal differentiation of keratinocytes in vitro.

Hypoxia-inducible factor-1α (HIF-1α) has been reported to be up-regulated in psoriatic epidermis, resulting in increased proliferation and abnormal differentiation of human keratinocytes (KCs). However, the role of HIF-1α in psoriatic epidermis, which is mainly composed of KCs, is poorly understood. Here, we show that morphogenic protein 6 (BMP6) is down-regulated when HIF-1α is upregulated in patients with psoriasis skin lesions. HIF-1α overexpression in primary human KCs promoted proliferation and inhibited terminal differentiation. Furthermore, HIF1-α repressed the expression of BMP6 by binding directly to the hypoxia-response element (HRE) in the BMP6 promotor region, which shows that BMP6 is a novel target gene of HIF-1α. We also found that HIF-1α-mediated BMP6 suppression could alter the proliferation status by modulating the expression levels of cell cycle regulatory proteins and also affect the early differentiation of KCs. Therefore, we suggest that HIF-1α-dependent BMP6 suppression has a critical role in the induction of hyper-proliferation and abnormal differentiation in psoriatic KCs.

Effect of Bone Morphogenetic Protein 6 on Immunomodulatory Functions of Salivary Gland-Derived Mesenchymal Stem Cells in Sjögren's Syndrome.

Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in the salivary glands. Recent studies reported that bone morphogenetic protein 6 (BMP6), which is a member of transforming growth factor beta (TGF-ß) superfamily, was highly expressed in SS patients. To investigate the role of BMP6 in SS, we treated the salivary gland-derived mesenchymal stem cells (SGMSCs) with BMP6 and found that BMP6 could impair immunomodulatory properties of normal SGMSCs by downregulating the Prostaglandin E2 synthase through DNA-binding protein inhibitor-1. Neutralizing the BMP6 could significantly restore the SGMSC's immunoregulatory function in vitro and delay the SS disease activity in vivo. In conclusion, BMP6 could not only affect the secreting function of epithelial cells in the salivary gland but also influence the immunomodulatory properties of SGMSCs, which may trigger or enhance the autoimmune reflection in SS.

Advancing osteochondral tissue engineering: bone morphogenetic protein, transforming growth factor, and fibroblast growth factor signaling drive ordered differentiation of periosteal cells resulting in stable cartilage and bone formation in vivo.

BACKGROUND: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. METHODS: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-ß1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry. RESULTS: Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. CONCLUSION: Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.

Long-term evaluation of osteoarthritis sheep knee, treated with TGF-ß3 and BMP-6 induced multipotent stem cells.

BACKGROUND: Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography. RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance. CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-ß3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.

Synergistic effects of BMP-2, BMP-6 or BMP-7 with human plasma fibronectin onto hydroxyapatite coatings: A comparative study.

Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. STATEMENT OF SIGNIFICANCE: The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects.

Insulin-like growth factor-1 (IGF-1) enhances the osteogenic activity of bone morphogenetic protein-6 (BMP-6) in vitro and in vivo, and together have a stronger osteogenic effect than when IGF-1 is combined with BMP-2.

Bone morphogenetic protein-2 (BMP-2) is widely used in orthopedic surgery and bone tissue engineering because of its strong osteogenic activity. However, BMP-2 treatments have several drawbacks and many groups are actively exploring alternatives. Since BMP-6 has been demonstrated to be more osteoinductive, its use, either alone or together with other growth factors, might be an interesting option. In this work, we have compared the effect of BMP-2, BMP-6, or insulin-like growth factor-1 (IGF-1), either alone or in combination. Murine preosteoblasts were treated with 15 nM IGF-1 and/or 6 nM BMP-2 or -6 and the expression of osteogenic marker genes, proliferation, and alkaline phosphatase (ALP) activity in vitro were analyzed. The results showed that IGF-1 greatly enhanced the BMP-induced osteogenic differentiation of these cells in general and that the ALP activity in the cultures was higher when the combination was made with BMP-6 than with BMP-2. Furthermore, we tested the osteogenic potential of these treatments in vivo by loading 25 pmoles of IGF-1 and/or 10 pmoles of BMP-2 or -6 onto absorbable collagen sponges and implanting them into an ectopic bone formation model in rats. This study revealed that only BMP-6 was able to induce bone formation at the used dose and that the addition of IGF-1 contributed to an increase of the mineralization in the implants. Hence, the combination of BMP-6 with IGF-1 might be a better alternative than BMP-2 for orthopedic surgery or bone tissue engineering approaches. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1867-1875, 2017.

Suppression of osteoblast-related genes during osteogenic differentiation of adipose tissue derived stromal cells.

Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was evaluated by quantification of osteoblast-related marker genes using real-time polymerase chain reaction (RT-PCR). Matrix mineralization was assessed by Alizarin Red S staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. Osteogenic medium (OM) significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (p < 0.05) and led to a stable matrix mineralization. Under the influence of BMP-9+IGF-2 and BMP-2,-6,-9 the ALP expression further increased compared to ASCs cultured with OM only (p < 0.01). However, multiple osteogenic markers showed no change or decreased under the influence of OM and BMP combinations (p < 0.05). The current results indicate a restricted osteogenic differentiation potential of ASCs and suggest careful reconsideration of their use in bone tissue engineering applications.