BMP7-Loaded Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorate Liver Fibrosis by Targeting Activated Hepatic Stellate Cells.

Purpose: Human umbilical cord mesenchymal stem cell (hucMSC)-derived small extracellular vesicles (sEVs) are natural nanocarriers with promising potential in treating liver fibrosis and have widespread applications in the fields of nanomedicine and regenerative medicine. However, the therapeutic efficacy of natural hucMSC-sEVs is currently limited owing to their non-specific distribution in vivo and partial removal by mononuclear macrophages following systemic delivery. Thus, the therapeutic efficacy can be improved through the development of engineered hucMSC-sEVs capable to overcome these limitations. Patients and Methods: To improve the anti-liver fibrosis efficacy of hucMSC-sEVs, we genetically engineered hucMSC-sEVs to overexpress the anti-fibrotic gene bone morphogenic protein 7 (BMP7) in parental cells. This was achieved using lentiviral transfection, following which BMP7-loaded hucMSC-sEVs were isolated through ultracentrifugation. First, the liver fibrosis was induced in C57BL/6J mice by intraperitoneal injection of 50% carbon tetrachloride (CCL4) twice a week for 8 weeks. These mice were subsequently treated with BMP7+sEVs via tail vein injection, and the anti-liver fibrosis effect of BMP7+sEVs was validated using small animal in vivo imaging, immunohistochemistry (IHC), tissue immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Finally, cell function studies were performed to confirm the in vivo results. Results: Liver imaging and liver histopathology confirmed that the engineered hucMSC-sEVs could reach the liver of mice and aggregate around activated hepatic stellate cells (aHSCs) with a significantly stronger anti-liver fibrosis effect of BMP7-loaded hucMSC-sEVs compared to those of blank or negative control-transfected hucMSC-sEVs. In vitro, BMP7-loaded hucMSC-sEVs promoted the phenotypic reversal of aHSCs and inhibited their proliferation to enhance the anti-fibrotic effects. Conclusion: These engineered BMP7-loaded hucMSC-sEVs offer a novel and promising strategy for the clinical treatment of liver fibrosis.

FGF-stimulated tendon cells embrace a chondrogenic fate with BMP7 in newt tissue culture.

Newts can regenerate functional elbow joints after amputation at the joint level. Previous studies have suggested the potential contribution of cells from residual tendon tissues to joint cartilage regeneration. A serum-free tissue culture system for tendons was established to explore cell dynamics during joint regeneration. Culturing isolated tendons in this system, stimulated by regeneration-related factors, such as fibroblast growth factor (FGF) and platelet-derived growth factor, led to robust cell migration and proliferation. Moreover, cells proliferating in an FGF-rich environment differentiated into Sox9-positive chondrocytes upon BMP7 introduction. These findings suggest that FGF-stimulated cells from tendons may aid in joint cartilage regeneration during functional elbow joint regeneration in newts.

The miR-3074/BMP7 axis regulates TGF-ß-caused activation of hepatic stellate cells in vitro and CCl4-caused murine liver fibrosis in vivo.

Continuously progressive hepatic fibrosis might cause chronic liver diseases, resulting in hepatic failure. The activation of hepatic stellate cells (HSCs) residing in the liver might induce and influence hepatic fibrosis. In the present study, microRNA 3074 (miR-3074) was found increased within transforming growth factor-ß (TGF-ß)-activated HSCs and enriched within the TGF-ß signaling. In activated HSCs by TGF-ß, miR-3074 overexpression aggravated TGF-ß-induced fibrotic changes, whereas miR-3074 inhibition exerted opposite effects. miR-3074 directly targeted bone morphogenetic protein 7 (BMP7) and inhibited BMP7 expression. Under TGF-ß induction, overexpressed BMP7 notably attenuated the promotive roles of miR-3074 overexpression in TGF-ß-activated HSCs. Within carbon tetrachloride (CCl4)-caused liver fibrosis murine model, miR-3074 agomir administration promoted, while LV-BMP7 administration alleviated CCl4-induced fibrotic changes; LV-BMP7 significantly attenuated the effects of miR-3074 agomir. Lastly, mmu-miR-3074 also targeted mouse BMP7 and inhibited mouse BMP7 expression. In conclusion, the miR-3074/BMP7 axis regulates TGF-ß-caused activation of HSCs in vitro and CCl4-caused murine liver fibrosis in vivo. BMP7-mediated Smad1/5/8 activation might be involved.

BMP7 expression in mammalian cortical radial glial cells increases the length of the neurogenic period.

The seat of human intelligence is the human cerebral cortex, which is responsible for our exceptional cognitive abilities. Identifying principles that lead to the development of the large-sized human cerebral cortex will shed light on what makes the human brain and species so special. The remarkable increase in the number of human cortical pyramidal neurons and the size of the human cerebral cortex is mainly because human cortical radial glial cells, primary neural stem cells in the cortex, generate cortical pyramidal neurons for more than 130 days, whereas the same process takes only about 7 days in mice. The molecular mechanisms underlying this difference are largely unknown. Here, we found that bone morphogenic protein 7 (BMP7) is expressed by increasing the number of cortical radial glial cells during mammalian evolution (mouse, ferret, monkey, and human). BMP7 expression in cortical radial glial cells promotes neurogenesis, inhibits gliogenesis, and thereby increases the length of the neurogenic period, whereas Sonic Hedgehog (SHH) signaling promotes cortical gliogenesis. We demonstrate that BMP7 signaling and SHH signaling mutually inhibit each other through regulation of GLI3 repressor formation. We propose that BMP7 drives the evolutionary expansion of the mammalian cortex by increasing the length of the neurogenic period.

Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.

Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7.

Mammalian cardiomyocytes (CMs) undergo maturation during postnatal heart development to meet the increased demands of growth. Here, we found that omentin-1, an adipokine, facilitates CM cell cycle arrest and metabolic maturation. Deletion of omentin-1 causes mouse heart enlargement and dysfunction in adulthood and CM maturation retardation in juveniles, including delayed cell cycle arrest and reduced fatty acid oxidation. Through RNA sequencing, molecular docking analysis, and proximity ligation assays, we found that omentin-1 regulates CM maturation by interacting directly with bone morphogenetic protein 7 (BMP7). Omentin-1 prevents BMP7 from binding to activin type II receptor B (ActRIIB), subsequently decreasing the downstream pathways mothers against DPP homolog 1 (SMAD1)/Yes-associated protein (YAP) and p38 mitogen-activated protein kinase (p38 MAPK). In addition, omentin-1 is required and sufficient for the maturation of human embryonic stem cell-derived CMs. Together, our findings reveal that omentin-1 is a pro-maturation factor for CMs that is essential for postnatal heart development and cardiac function maintenance.

Molecular Characterization and Function of Bone Morphogenetic Protein 7 (BMP7) in the Pacific Abalone, Haliotis discus hannai.

Bone morphogenetic proteins (BMPs) play important roles in a lot of biological processes, such as bone development, cell proliferation, cell differentiation, growth, etc. However, the functions of abalone BMP genes are still unknown. This study aimed to better understand the characterization and biological function of BMP7 of Haliotis discus hannai (hdh-BMP7) via cloning and sequencing analysis. The coding sequence (CDS) length of hdh-BMP7 is 1251 bp, which encodes 416 amino acids including a signal peptide (1-28 aa), a transforming growth factor-ß (TGF-ß) propeptide (38-272 aa), and a mature TGF-ß peptide (314-416 aa). The analysis of expression showed that hdh-BMP7 mRNA was widely expressed in all the examined tissues of H. discus hannai. Four SNPs were related to growth traits. The results of RNA interference (RNAi) showed that the mRNA expression levels of hdh-BMPR I, hdh-BMPR II, hdh-smad1, and hdh-MHC declined after hdh-BMP7 was silenced. After RNAi experiment for 30 days, the shell length, shell width, and total weight were found to be reduced in H. discus hannai (p < 0.05). The results of real-time quantitative reverse transcription PCR revealed that the hdh-BMP7 mRNA was lower in abalone of the S-DD-group than in the L-DD-group. Based on these data, we hypothesized that BMP7 gene has a positive role in the growth of H. discus hannai.

Integrated single-cell RNA-seq analysis revealed podocyte injury through activation of the BMP7/AMPK/mTOR mediated autophagy pathway.

BACKGROUND: Nephrotic syndrome (NS) is a chronic kidney disease mainly caused by impaired podocytes, ultimately resulting in massive proteinuria or even end-stage renal disease (ESRD). METHODS: The objective of this study was to explore the potential pathogenesis of NS caused by podocyte injury, and further explore the underlying mechanism through data mining, bioinformatics analysis, and experimental verification. The integrated analyses including Seurat, CellChat, gene ontology (GO), and molecular docking were performed based on the single-cell RNA-seq data (scRNA-seq). The adriamycin (ADR)-induced podocyte injury model in vitro was established to conduct the experimental verification for bioinformatics analysis results through western blot and real-time quantitative PCR (RT-qPCR). RESULTS: The results of bioinformatics analysis revealed that the bone morphogenetic protein (BMP) signaling pathway was involved in the podocyte-to-podocyte communication, which plays a crucial role in podocyte injury. The expression of BMP7 was significantly increased in ADR-induced podocytes through activating the Adenosine-monophosphate activated-protein kinase/Mammalian target of rapamycin (AMPK/mTOR) mediated autophagy pathway, and these findings were confirmed by in vitro experiments. CONCLUSION: This study first demonstrated that BMP7 participated in ADR-induced podocyte injury. The BMP7/AMPK/mTOR mediated autophagy pathway may play a crucial role in podocyte injury, which may be the potential therapeutic target for NS patients.

Gamma secretase activity modulates BMP-7-induced dendritic growth in primary rat sympathetic neurons.

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons.

BMP7 Attenuates Neuroinflammation after Spinal Cord Injury by Suppressing the Microglia Activation and Inducing Microglial Polarization Via the STAT3 Pathway.

Excessive activation of pro-inflammatory (M1) microglia phenotypes after spinal cord injury (SCI) disrupts tissue repair and increases the risk of secondary SCI. We previously reported that adeno-associated virus (AAV) mediated delivery of bone morphogenetic protein 7 (BMP7) promotes functional recovery after SCI by reducing oligodendrocyte loss and demyelination; however, little is known about the early effects of BMP7 in ameliorating neuroinflammation in the acute SCI phase. Herein, we demonstrate that treatment with recombinant human BMP7 (rhBMP7) suppresses the viability of LPS-induced HMC3 microglia cells and increases the proportion with the M2 phenotype. Consistently, in a rat SCI model, rhBMP7 decreases the activation of microglia and promotes M2 polarization. After rhBMP7 administration, the STAT3 signaling pathway was activated in LPS-induced HMC3 cells and microglia in spinal cord lesions. Furthermore, the levels of TNF-α and IL-1ß were significantly decreased in cell culture supernatants, lesion sites of injured spinal cords, and cerebrospinal fluid circulation after rhBMP7 administration, thus reducing neuron loss in the injured spinal cord and promoting functional recovery after SCI. These results provide insight into the immediate early mechanisms by which BMP7 may ameliorate the inflammation response to secondary SCI.

Assessment of BMP7, SMAD4, and CDH1 Expression Profile and Regulatory miRNA-542-3p in Eutopic and Ectopic Endometrium of Women with Endometriosis.

Alterations in the expression of numerous genes and the miRNAs that are recognized as their regulators in the endometrial cells of women with endometriosis may disrupt the intracellular signaling pathways associated with epithelial-mesenchymal transition (EMT). So far, the functional role of BMP7 in endometrial physiology has been confirmed, especially in the context of fertility, but the role of the activation of a specific mechanism operating through the BMP-SMAD-CDH1 axis in the formation of endometrial lesions remains unexplored. The aim of this study was to evaluate the expression profile of miR-542-3p and the EMT markers (BMP7, SMAD4, CDH1) in matched eutopic endometrium (EUE) and ectopic endometrium (ECE) samples from women with endometriosis in relation to healthy women. The levels of expression of the studied genes and miRNA in peripheral blood mononuclear cells (PBMCs) obtained from women diagnosed with endometriosis and those without the disease were also evaluated. Fifty-four patients (n = 54: with endometriosis-n = 29 and without endometriosis-n = 25) were included in the study. A comparative analysis of the relative mean expression values (RQ) of the studied mRNA and miRNA assessed by RT-qPCR demonstrated downregulation of BMP7, SMAD4, and CDH1 expression in ectopic lesions and upregulation in the eutopic endometrium compared with the control group. In the eutopic tissue of women with endometriosis, miR-542-3p expression was similar to that of the control but significantly lower than in endometrial lesions. We also confirmed a trend towards a negative correlation between miR-542-3p and BMP7 in ectopic tissue, and in PBMC, a significant negative correlation of miR-542-3p with further BMP signaling genes, i.e., SMAD4 and CDH1, was observed. These results indicate that the miRNA selected by us may be a potential negative regulator of BMP7-SMAD4-CDH1 signaling associated with EMT. The different patterns of BMP7, SMAD4, and CDH1 gene expression in ECE, EUE, and the control endometrium observed by us suggests the loss of the endometrial epithelium phenotype in women with endometriosis and demonstrates their involvement in the pathogenesis and pathomechanism of this disease.

Effect of crocin and losartan on biochemical parameters and genes expression of FRMD3 and BMP7 in diabetic rats.

BACKGROUND: Diabetes is a multifactorial and growing disease, one of the severe complications of which is diabetic nephropathy (DN), which is the most common cause of chronic renal failure. FERM domain containing 3 (FRMD3) is responsible for maintaining the shape and integrity of nephron cells, and bone morphogenetic protein 7 (BMP7) helps maintain function and reduce kidney damage. This study aimed to evaluate the effect of crocin and losartan on biochemical parameters and the expression of FRMD3 and BMP7 genes in streptozotocin (STZ)-induced diabetic rats. METHODS: Forty male Wistar rats were randomly divided into five experimental groups as healthy, diabetic control (D), crocin, losartan, and diabetic rats treated with losartan-crocin (n = 8). A single dose of STZ (50 mg/kg intraperitoneally injection) was used to induce diabetes. Four weeks after induction of diabetes, rats received crocin (50 mg/kg) and losartan (25 mg/kg) daily for four weeks orally. Rats were sacrificed at the end of the intervention, and blood samples were taken to determine serum levels of glucose, urea, creatinine (Cr), malondialdehyde (MDA), and thiol. Real-time polymerase chain reaction (PCR) was used to assess the expression of the FRMD3 and BMP7 genes in the kidney samples. RESULTS: Diabetes induction increased serum levels of glucose, Cr, urea, MDA, and thiol, but decreased BMP7 and FRMD3 genes expression. Treatment with crocin and losartan decreased these biochemical parameters and increased the expression of the BMP7 and FRMD3 genes. DISCUSSION: Crocin may be a promising therapeutic agent for preventing and improving diabetes-related kidney disease due to its antidiabetic and antioxidant properties.

BMP7 ameliorates intervertebral disc degeneration in type 1 diabetic rats by inhibiting pyroptosis of nucleus pulposus cells and NLRP3 inflammasome activity.

BACKGROUND: Accumulating evidence indicates that intervertebral disc degeneration (IDD) is associated with diabetes mellitus (DM), while the underlying mechanisms still remain elusive. Herein, the current study sought to explore the potential molecular mechanism of IDD in diabetic rats based on transcriptome sequencing data. METHODS: Streptozotocin (STZ)-induced diabetes mellitus type 1 (T1DM) rats were used to obtain the nucleus pulposus tissues for transcriptome sequencing. Next, differentially expressed genes (DEGs) in transcriptome sequencing data and GSE34000 microarray dataset were obtained and intersected to acquire the candidate genes. Moreover, GO and KEGG enrichment analyses were performed to analyze the cellular functions and molecular signaling pathways primarily regulated by candidate DEGs. RESULTS: A total of 35 key genes involved in IDD of T1DM rats were mainly enriched in the extracellular matrix (ECM) and cytokine adhesion binding-related pathways. NLRP3 inflammasome activation promoted the pyroptosis of nucleus pulposus cells (NPCs). Besides, BMP7 could affect the IDD of T1DM rats by regulating the inflammatory responses. Additionally, NPCs were isolated from STZ-induced T1DM rats to illustrate the effects of BMP7 on IDD of T1DM rats using the ectopic expression method. Both in vitro and in vivo experiments validated that BMP7 alleviated IDD of T1DM rats by inhibiting NLRP3 inflammasome activation and pyroptosis of NPCs. CONCLUSION: Collectively, our findings provided novel mechanistic insights for understanding of the role of BMP7 in IDD of T1DM, and further highlighted BMP7 as a potential therapeutic target for preventing IDD in T1DM.

Modulation of Osteogenic Gene Expression by Human Osteoblasts Cultured in the Presence of Bisphenols BPF, BPS, or BPAF.

Bone effects attributed to bisphenols (BPs) include the inhibition of growth and differentiation. This study analyzes the effect of BPA analogs (BPS, BPF, and BPAF) on the gene expression of the osteogenic markers RUNX2, osterix (OSX), bone morphogenetic protein-2 (BMP-2), BMP-7, alkaline phosphatase (ALP), collagen-1 (COL-1), and osteocalcin (OSC). Human osteoblasts were obtained by primary culture from bone chips harvested during routine dental work in healthy volunteers and were treated with BPF, BPS, or BPAF for 24 h at doses of 10-5, 10-6, and 10-7 M. Untreated cells were used as controls. Real-time PCR was used to determine the expression of the osteogenic marker genes RUNX2, OSX, BMP-2, BMP-7, ALP, COL-1, and OSC. The expression of all studied markers was inhibited in the presence of each analog; some markers (COL-1; OSC, BMP2) were inhibited at all three doses and others only at the highest doses (10-5 and 10-6 M). Results obtained for the gene expression of osteogenic markers reveal an adverse effect of BPA analogs (BPF, BPS, and BPAF) on the physiology of human osteoblasts. The impact on ALP, COL-1, and OSC synthesis and therefore on bone matrix formation and mineralization is similar to that observed after exposure to BPA. Further research is warranted to determine the possible contribution of BP exposure to the development of bone diseases such as osteoporosis.

Chaperone-mediated production of active homodimer human bone morphogenetic protein - 2 in E. coli.

Human bone morphogenetic protein 2 (hBMP-2) plays a leading role in the process of osteogenesis and is one of the key components of osteoplastic materials, ensuring their high osteoinduction. In order to obtain a homodimeric form hBMP-2 using the E. coli expression system, a number of problems associated with refolding in vitro and purification from monomer and oligomeric forms must be solved. The developed method for co-expression of the target protein with chaperone proteins makes it possible to obtain the biologically active homodimeric form of hBMP-2 in vivo. Purification with simple ion-exchange sorbents without the use of denaturing reagents affecting the structure of the protein molecule provides a chromatographic purity of the product of at least 97%. The expressed hBMP-2 was identified by Western blotting and the LC-ESI-TOF mass spectrometry confirmed its molecular weight of 26052.72 Da. Circular dichroism spectroscopy showed that recombinant hBMP-2 has a native secondary structure.

Conserved miR-370-3p/BMP-7 axis regulates the phenotypic change of human vascular smooth muscle cells.

Endothelial dysfunction and inflammatory immune response trigger dedifferentiation of vascular smooth muscle cells (SMCs) from contractile to synthetic phenotype and initiate arterial occlusion. However, the complex vascular remodeling process playing roles in arterial occlusion initiation is largely unknown. We performed bulk sequencing of small and messenger RNAs in a rodent arterial injury model. Bioinformatic data analyses reveal that six miRNAs are overexpressed in injured rat carotids as well as synthetic-type human vascular SMCs. In vitro cell-based assays show that four miRNAs (miR-130b-5p, miR-132-3p, miR-370-3p, and miR-410-3p) distinctly regulate the proliferation of and monocyte adhesion to the vascular SMCs. Individual inhibition of the four selected miRNAs strongly prevents the neointimal hyperplasia in the injured rat carotid arteries. Mechanistically, miR-132-3p and miR-370-3p direct the cell cycle progression, triggering SMC proliferation. Gene ontology analysis of mRNA sequencing data consistently reveal that the miRNA targets include gene clusters that direct proliferation, differentiation, and inflammation. Notably, bone morphogenic protein (BMP)-7 is a prominent target gene of miR-370-3p, and it regulates vascular SMC proliferation in cellular and animal models. Overall, this study first reports that the miR-370-3p/BMP-7 axis determines the vascular SMC phenotype in both rodent and human systems.

Differentiation of rat bone marrow mesenchymal stem cells into neurons induced by bone morphogenetic protein 7 in vitro.

OBJECTIVES: Spinal cord injury (SCI) is caused by external direct or indirect factors with high disability rate, which may even endanger the life of patients. To explore the role of bone morphogenetic protein 7 (BMP-7) in the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into neurons in vitro. METHODS: BMSCs were isolated and cultured by whole bone marrow adherence method. Adipogenic induction and osteogenic differentiation were used to test the multi⁃directional differentiation ability of BMSCs. RESULTS: After 28 days of adipogenic induction, BMSCs showed lipid droplets in the cytoplasm. After osteogenic induction, there were opaque lumps of mineral nodules in BMSCs. There were also orange-red or red mineral nodules in the extracellular matrix. The BMSCs in the 75 ng/ml BMP-7 group were morphologically similar to the neurons. After induction with BMP-7 for 2 h, the NF200 mRNA expression was higher, mRNA expression levels of SYN1, MAP2 and GFAP were higher. Positive rate of immunofluorescence staining in the BMP-7 group was notably increased. The positive rate of NSE immunofluorescence staining in the BMP-7 group was higher. CONCLUSION: BMP-7 can induce rat BMSCs to differentiate into neurons in vitro.

Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation.

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.

Regenerative Potential of Bone Morphogenetic Protein 7-Engineered Mesenchymal Stem Cells in Ligature-Induced Periodontitis.

Periodontitis is an oral disease caused by bacterial infection that has stages according to the severity of tissue destruction. The advanced stage of periodontitis presents irreversible destruction of soft and hard tissues, which finally results in loss of teeth. When conventional treatment modalities show limited results, tissue regeneration therapy is required in patients with advanced periodontitis. In the present study, we aimed to evaluate the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) delivering bone morphogenetic protein 7 (BMP7) on tissue regeneration in a periodontitis model. BMP7 is a member of the BMP family that shows bone-forming ability; however, BMPs rapid clearing and degradation and unproven efficacy make it difficult to apply it in clinical dentistry. To overcome this, we established BMP7-expressing engineered BM-MSCs (BMP7-eBMSCs) that showed superior osteogenic differentiation potential when subcutaneously transplanted with a biphasic calcium phosphate scaffold into immunocompromised mice. Furthermore, the efficacy of BMP7-eBMSC transplantation for periodontal tissue regeneration was evaluated in a rat ligature-induced periodontitis model. Upon measuring two-dimensional and three-dimensional amounts of regenerated alveolar bone using microcomputed tomography, the amounts were found to be significantly higher in the BMP7-eBMSC transplantation group than in the eBMSC transplantation group. Most importantly, fibrous periodontal ligament (PDL) tissue regeneration was also achieved upon BMP7-eBMSC transplantation, which was evaluated by calculating the modified relative connective tissue attachment. The amount of connective tissue attachment in the BMP7-eBMSC transplantation group was significantly higher than that in the ligature-induced periodontitis group, although the increase was comparable between the BMP7-eBMSC and human PDL stem cell transplantation groups. Taken together, our results suggested that sustainable release of BMP7 induces periodontal tissue regeneration and that transplantation of BMP7-eBMSCs is a feasible treatment option for periodontal regeneration. Impact Statement Periodontitis is the second most common human dental disease affecting chronic systemic diseases. Despite the tremendous efforts trying to cure the damaged periodontal tissues using tissue engineering technologies, a definitive regenerative method has not been in consensus. Researchers are seeking more feasible and abundant source of mesenchymal stem cells (MSCs), and furthermore, how to use reliable growth factors under more efficient control are the issues to be solved. In this study, we aimed to evaluate the effect of bone morphogenetic protein 7 (BMP7) gene delivering bone marrow-derived MSCs on periodontal tissue regeneration to evaluate the efficacy of BMP7 and engineered BMSCs for periodontal tissue regeneration.

Doxycycline-doped collagen membranes accelerate in vitro osteoblast proliferation and differentiation.

OBJECTIVE: The aim of the study was to evaluate the effect of doxycycline- and dexamethasone-doped collagen membranes on the proliferation and differentiation of osteoblasts. BACKGROUND: Collagen barrier membranes are frequently used to promote bone regeneration and to boost this biological activity their functionalization with antibacterial and immunomodulatory substances has been suggested. METHODS: The design included commercially available collagen membranes doped with doxycycline (Dox-Col-M) or dexamethasone (Dex-Col-M), as well as undoped membranes (Col-M) as controls, which were placed in contact with cultured MG63 osteoblast-like cells (ATCC). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and differentiation by measuring the alkaline phosphatase (ALP) activity using spectrophotometry. Real-time quantitative polymerase chain reaction was used to study the expression of the genes: Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3. Scanning electron microscopy was used to study osteoblast morphology. Data were assessed using one-way analysis of variance or Kruskal-Wallis tests, once their distribution normality was assessed by Kolmogorov-Smirnov tests (p > .05). Bonferroni for multiple comparisons were carried out (p < .05). RESULTS: Osteoblast proliferation was significantly enhanced in the functionalized membranes as follows: (Col-M < Dex-Col-M < Dox-Col-M). ALP activity was significantly higher on cultured osteoblasts on Dox-Col-M. Runx-2, OSX, ALP, OSC, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3 were overexpressed, and RANKL was down-regulated in osteoblasts cultured on Dox-Col-M. The osteoblasts cultured in contact with the functionalized membranes demonstrated an elongated spindle-shaped morphology. CONCLUSION: The functionalization of collagen membranes with Dox promoted an increase in the proliferation and differentiation of osteoblasts.