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.

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.

Therapeutic Effects of Albumin-Fused BMP7 on 2 Experimental Models of Liver Fibrosis.

Despite the fact that liver fibrosis is an intractable disease with a poor prognosis, effective therapeutic agents are not available. In this study, we focused on bone morphogenetic factor 7 (BMP7) that inhibits transforming growth factor (TGF)-ß signaling, which is involved in liver fibrosis. We prepared an albumin-fused BMP7 (HSA-BMP7) that is retained in the blood and evaluated its inhibitory effect on liver fibrosis. Bile duct ligated mice were used as an acute liver fibrosis model, and carbon tetrachloride-induced liver fibrosis mice were used as a chronic model. All mice were administered HSA-BMP7 once per week. In the mice with bile duct ligation, the administration of HSA-BMP7 significantly suppressed the infiltration of inflammatory cells, the area of fibrosis around the bile duct, and decreased in the level of hydroxyproline as compared with saline administration. The mRNA expression of TGF-ß and its downstream fibrosis-associated genes (α-SMA and Col1a2) were also suppressed by the administration of HSA-BMP7. In the carbon tetrachloride-induced liver fibrosis mice, the HSA-BMP7 administration significantly decreased the hepatic fibrosis area and the level of hydroxyproline. Based on these results, it appears that HSA-BMP7 has the potential for serving as a therapeutic agent for the treatment of liver fibrosis.

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.

Mechanical strain triggers endothelial-to-mesenchymal transition of the endocardium in the immature heart.

BACKGROUND: Endothelial-to-mesenchymal-transition (EndMT) plays a major role in cardiac fibrosis, including endocardial fibroelastosis but the stimuli are still unknown. We developed an endothelial cell (EC) culture and a whole heart model to test whether mechanical strain triggers TGF-ß-mediated EndMT. METHODS: Isolated ECs were exposed to 10% uniaxial static stretch for 8 h (stretch) and TGF-ß-mediated EndMT was determined using the TGF-ß-inhibitor SB431542 (stretch + TGF-ß-inhibitor), BMP-7 (stretch + BMP-7) or losartan (stretch + losartan), and isolated mature and immature rats were exposed to stretch through a weight on the apex of the left ventricle. Immunohistochemical staining for double-staining with endothelial markers (VE-cadherin, PECAM1) and mesenchymal markers (αSMA) or transcription factors (SLUG/SNAIL) positive nuclei was indicative of EndMT. RESULTS: Stretch-induced EndMT in ECs expressed as double-stained ECs/total ECs (cells: 46 ± 13%; heart: 15.9 ± 2%) compared to controls (cells: 7 ± 2%; heart: 3.1 ± 0.1; p < 0.05), but only immature hearts showed endocardial EndMT. Inhibition of TGF-ß decreased the number of double-stained cells significantly, comparable to controls (cells/heart: control: 7 ± 2%/3.1 ± 0.1%, stretch: 46 ± 13%/15 ± 2%, stretch + BMP-7: 7 ± 2%/2.9 ± 0.1%, stretch + TGF-ß-inhibitor (heart only): 5.2 ± 1.3%, stretch + losartan (heart only): 0.89 ± 0.1%; p < 0.001 versus stretch). CONCLUSIONS: Endocardial EndMT is an age-dependent consequence of increased strain triggered by TGF- ß activation. Local inhibition through either rebalancing TGF-ß/BMP or with losartan was effective to block EndMT. IMPACT: Mechanical strain imposed on the immature LV induces endocardial fibroelastosis (EFE) formation through TGF-ß-mediated activation of endothelial-to-mesenchymal transition (EndMT) in endocardial endothelial cells but has no effect in mature hearts. Local inhibition through either rebalancing the TGF-ß/BMP pathway or with losartan blocks EndMT. Inhibition of endocardial EndMT with clinically applicable treatments may lead to a better outcome for congenital heart defects associated with EFE.

Bone Tissue Engineering in Rat Calvarial Defects Using Induced Bone-like Tissue by rhBMPs from Immature Muscular Tissues In Vitro.

This study aimed to induce bone-like tissue from immature muscular tissue (IMT) in vitro using commercially available recombinant human bone morphogenetic protein (rhBMP)-2, rhBMP-4, and rhBMP-7, and then implanting this tissue into a calvarial defect in rats to assess healing. IMTs were extracted from 20-day-old Sprague-Dawley (SD) fetal rats, placed on expanded polytetrafluoroethylene (ePTFE) with 10 ng/µL each of rhBMP-2, BMP-4, and BMP-7, and cultured for two weeks. The specimens were implanted into calvarial defects in 3-week-old SD rats for up to three weeks. Relatively strong radiopacity was observed on micro-CT two weeks after culture, and bone-like tissue, comprising osteoblastic cells and osteoids, was partially observed by H&E staining. Calcium, phosphorus, and oxygen were detected in the extracellular matrix using an electron probe micro analyzer, and X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra of the specimen were found to have typical apatite crystal peaks and spectra, respectively. Furthermore, partial strong radiopacity and ossification were confirmed one week after implantation, and a dominant novel bone was observed after two weeks in the defect site. Thus, rhBMP-2, BMP-4, and BMP-7 differentiated IMT into bone-like tissue in vitro, and this induced bone-like tissue has ossification potential and promotes the healing of calvarial defects. Our results suggest that IMT is an effective tissue source for bone tissue engineering.

Osteoinduction within adipose tissue fragments by heterodimeric bone morphogenetic Proteins-2/6 and -2/7 versus homodimeric bone morphogenetic protein-2: Therapeutic implications for bone regeneration.

BACKGROUND: Fragments of subcutaneous adipose tissue that have been genetically modified to express bone morphogenetic protein-2 (BMP-2) regenerate large segmental osseous lesions in rodents. Gene-activated adipose tissue can be implanted into osseous defects without prior cell extraction and cell culture. The present study aimed to explore whether the heterodimers BMP-2/6 or BMP-2/7 exceed the osteoinductive effect of BMP-2 on adipose tissue. METHODS: In an in vitro tissue culture system, freshly harvested rat subcutaneous adipose tissue was cultivated in the presence of either BMP-2 or BMP-2/6 or BMP-2/7 at a high (200 ng/ml) and low (50 ng/ml) concentration. Gene expression analysis as well as histological and immunohistochemical methods were applied to test for osteoinduction. RESULTS: A concentration of 200 ng/ml of homodimeric BMP-2 induced osteogenic differentiation most potently, showing more calcification and a higher expression level of bone markers than both concentrations of BMP-2/6 or -2/7. A concentration of 50 ng/ml of BMP-2 was a significantly stronger osteogenic inducer than both concentrations of BMP-2/6 and the low concentration of BMP-2/7. The most potent heterodimeric driver of osteoinduction was BMP-2/7 at a high concentration, demonstrating effects similar to those of BMP-2 at a low concentration. CONCLUSIONS: Homodimeric BMP-2 evoked osteoinduction within adipose tissue more potently and at a lower concentration than heterodimeric BMP-2/6 or BMP-2/7. This result agrees well with the fact that it might be easier to translate adipose grafts activated by homodimeric BMP-2 clinically. Preclinical in vivo gene transfer studies are necessary to confirm the results of the present study.

Ultrasound triggered topical delivery of Bmp7 mRNA for white fat browning induction via engineered smart exosomes.

BACKGROUND: Efficient and topical delivery of drugs is essential for maximized efficacy and minimized toxicity. In this study, we aimed to design an exosome-based drug delivery platform endowed with the ability of escaping from phagocytosis at non-target organs and controllably releasing drugs at targeted location. RESULTS: The swtichable stealth coat CP05-TK-mPEG was synthesized and anchored onto exosomes through the interaction between peptide CP05 and exosomal surface marker CD63. Chlorin e6 (Ce6) was loaded into exosomes by direct incubation. Controllable removal of PEG could be achieved by breaking thioketal (TK) through reactive oxygen species (ROS), which was produced by Ce6 under ultrasound irradiation. The whole platform was called SmartExo. The stealth effects were analyzed in RAW264.7 cells and C57BL/6 mice via tracing the exosomes. To confirm the efficacy of the engineered smart exosomes, Bone morphogenetic protein 7 (Bmp7) mRNA was encapsulated into exosomes by transfection of overexpressing plasmid, followed by stealth coating, with the exosomes designated as SmartExo@Bmp7. Therapeutic advantages of SmartExo@Bmp7 were proved by targeted delivering Bmp7 mRNA to omental adipose tissue (OAT) of obese C57BL/6 mice for browning induction. SmartExo platform was successfully constructed without changing the basic characteristics of exosomes. The engineered exosomes effectively escaped from the phagocytosis by RAW264.7 and non-target organs. In addition, the SmartExo could be uptaken locally on-demand by ultrasound mediated removal of the stealth coat. Compared with control exosomes, SmartExo@Bmp7 effectively delivered Bmp7 mRNA into OAT upon ultrasound irradiation, and induced OAT browning, as evidenced by the histology of OAT and increased expression of uncoupling protein 1 (Ucp1). CONCLUSIONS: The proposed SmartExo-based delivery platform, which minimizes side effects and maximizing drug efficacy, offers a novel safe and efficient approach for targeted drug delivery. As a proof, the SmartExo@Bmp7 induced local white adipose tissue browning, and it would be a promising strategy for anti-obesity therapy.

Nerve-mediated expression of histone deacetylases regulates limb regeneration in axolotls.

Axolotls have amazing abilities to regenerate their lost limbs. Nerve and wound epidermis have great impacts on this regeneration. Histone deacetylases (HDACs) have been shown to play roles in the regeneration of amphibian tails and limbs. In this study, a bi-phasic up-regulation of HDAC1 was noted before early differentiation stage of axolotl limb regeneration. Limb regeneration was delayed in larvae incubated with an HDAC inhibitor MS-275. Local injection of MS-275 or TSA, another HDAC inhibitor, into amputation sites of the juveniles did not interfere with wound healing but more profoundly inhibited local HDAC activities and blastema formation/limb regeneration. Elevation of HDAC1 expression was more apparent in wound epidermis than in mesenchyme. Prior denervation prohibited this elevation and limb regeneration. Supplementation of nerve factors BMP7, FGF2, and FGF8 in the stump ends after amputation on denervated limbs not only enabled HDAC1 up-regulation but also led to more extent of limb regeneration. In conclusion, nerve-mediated HDAC1 expression is required for blastema formation and limb regeneration.

Generation of thalamic neurons from mouse embryonic stem cells.

The thalamus is a diencephalic structure that plays crucial roles in relaying and modulating sensory and motor information to the neocortex. The thalamus develops in the dorsal part of the neural tube at the level of the caudal forebrain. However, the molecular mechanisms that are essential for thalamic differentiation are still unknown. Here, we have succeeded in generating thalamic neurons from mouse embryonic stem cells (mESCs) by modifying the default method that induces the most-anterior neural type in self-organizing culture. A low concentration of the caudalizing factor insulin and a MAPK/ERK kinase inhibitor enhanced the expression of the caudal forebrain markers Otx2 and Pax6. BMP7 promoted an increase in thalamic precursors such as Tcf7l2+/Gbx2+ and Tcf7l2+/Olig3+ cells. mESC thalamic precursors began to express the glutamate transporter vGlut2 and the axon-specific marker VGF, similar to mature projection neurons. The mESC thalamic neurons extended their axons to cortical layers in both organotypic culture and subcortical transplantation. Thus, we have identified the minimum elements sufficient for in vitro generation of thalamic neurons. These findings expand our knowledge of thalamic development.

BMP7 antagonizes proliferative vitreoretinopathy through retinal pigment epithelial fibrosis in vivo and in vitro.

The major pathogenesis of proliferative vitreoretinopathy (PVR) is that retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transition (EMT) because of disordered growth factors, such as TGF-ß, in the vitreous humor. Bone morphogenetic proteins (BMPs) are pluripotent growth factors. In this study, we identified the antifibrotic activity of BMP7 in a PVR model both in vivo and in vitro. BMP7 expression was confirmed on the PVR proliferative membranes. BMP7 was down-regulated in the PVR vitreous humor and TGF-ß-induced RPE cell EMT. In the in vivo studies, BMP7 injection attenuated PVR progression in the eyes of the rabbit model. Additionally, BMP7 treatment maintained RPE cell phenotypes and relieved TGF-ß2-induced EMT, migration, and gel contraction in vitro. BMP7 inhibited the TGF-ß2-induced up-regulation of fibronectin and α-smooth muscle actin and the down-regulation of E-cadherin and zona occludens-1 by balancing the TGF-ß2/Smad2/3 and BMP7/Smad1/5/9 pathways. These findings provide direct evidence of the ability of BMP7 in PVR inhibition and the potential of BMP7 for use in PVR therapeutic intervention.-Yao, H., Ge, T., Zhang, Y., Li, M., Yang, S., Li, H., Wang, F. BMP7 antagonizes proliferative vitreoretinopathy through retinal pigment epithelial fibrosis in vivo and in vitro.

Biofabrication of SDF-1 Functionalized 3D-Printed Cell-Free Scaffolds for Bone Tissue Regeneration.

Large segmental bone defects occurring after trauma, bone tumors, infections or revision surgeries are a challenge for surgeons. The aim of our study was to develop a new biomaterial utilizing simple and cheap 3D-printing techniques. A porous polylactide (PLA) cylinder was printed and functionalized with stromal-derived factor 1 (SDF-1) or bone morphogenetic protein 7 (BMP-7) immobilized in collagen type I. Biomechanical testing proved biomechanical stability and the scaffolds were implanted into a 6 mm critical size defect in rat femur. Bone growth was observed via x-ray and after 8 weeks, bone regeneration was analyzed with µCT and histological staining methods. Development of non-unions was detected in the control group with no implant. Implantation of PLA cylinder alone resulted in a slight but not significant osteoconductive effect, which was more pronounced in the group where the PLA cylinder was loaded with collagen type I. Addition of SDF-1 resulted in an osteoinductive effect, with stronger new bone formation. BMP-7 treatment showed the most distinct effect on bone regeneration. However, histological analyses revealed that newly formed bone in the BMP-7 group displayed a holey structure. Our results confirm the osteoinductive character of this 3D-biofabricated cell-free new biomaterial and raise new options for its application in bone tissue regeneration.

Apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis.

The role of apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) in bone healing remains to be understood. To address this issue, we investigated the requirement of inflammasome-related genes in response to bone morphogenetic protein 7 (BMP7)-induced osteoblast differentiation in vitro. To validate the importance of ASC on osteogenesis, we subjected wild-type (WT) and ASC knockout C57BL/6 mice (ASC KO) to tibia defect to evaluate the bone healing process (up to 28 days). Our in vitro data showed that there is an involvement of ASC during BMP7-induced osteoblast differentiation, concomitant to osteogenic biomarker expression. Indeed, primary osteogenic cells from ASC KO presented a lower osteogenic profile than those obtained from WT mice. To validate this hypothesis, we evaluated the bone healing process of tibia defects on both WT and ASC KO mice genotypes and the ASC KO mice were not able to fully heal tibia defects up to 28 days, whereas WT tibia defects presented a higher bone de novo volume at this stage, evidencing ASC as an important molecule during osteogenic phenotype. In addition, we have shown a higher involvement of runt-related transcription factor 2 in WT sections during bone repair, as well as circulating bone alkaline phosphatase isoform when both were compared with ASC KO mice behavior. Altogether, our results showed for the first time the involvement of inflammasome during osteoblast differentiation and osteogenesis, which opens new avenues to understand the pathways involved in bone healing.

Bone morphogenetic protein-7 incorporated polycaprolactone scaffold has a great potential to improve survival and proliferation rate of the human embryonic kidney cells.

Renal failures treatment has been faced with several problems during the last decades. Kidney tissue engineering has been created many hopes to improve treatment procedures with scaffold fabrication that can modulate kidney cells/stem cells migration to the lesion site and increase the survival of these cells at that site with imitating the role of the kidney extracellular matrix. In this study, bone morphogenetic protein-7 (BMP7) as a vital factor for kidney development and regeneration was incorporated in the polycaprolactone (PCL) nanofibers and after morphological, mechanical, and biocompatible characterization, proliferation, and survival of the human embryonic kidney cells (HEK) were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and gene expression while cultured on scaffolds. Mechanical properties of the PCL nanofibers modulated after combining with BMP7 and hydration degree, protein adsorption and cell adhesion were enhanced in PCL-BMP7 compared to the pure PCL. Proliferation rate and growth increased significantly in HEK cells cultured on PCL-BMP7 when compared with that of PCL and tissue culture plate, whereas these data were also confirmed via significant decrease in apoptotic genes expression level in HEK cell cultured on PCL-BMP7. According to the results, PCL-BMP7 demonstrated positive effects on the survival and proliferation rate of the kidney cells and showed has also a great potential to use as a bioimplant for kidney tissue engineering applications.

IGF-1 and BMP-7 synergistically stimulate articular cartilage repairing in the rabbit knees by improving chondrogenic differentiation of bone-marrow mesenchymal stem cells.

Knee injury is known as a frequently occurred damage related to sports, which may affect the function of cartilage. This study aims to explore whether Insulin-like growth factor 1 (IGF-1) and bone morphogenetic protein-7 (BMP-7)-modified bone-marrow mesenchymal stem cells (BMSCs) affect the repair of cartilage damage found in the knee. Primarily, BMSCs were treated with a series of pEGFP-C1, IGF-1, and BMP-7, followed by determination of IGF-1 and BMP-7 expression. A rabbit cartilage defect model was also established. Afterfward, cell morphology, viability, cartilage damage repair effect, and expression of collagen I and collagen II at the 6th and the 12th week were measured. BMSCs treated with pEGFP-C1/IGF-1, pEGFP-C1/BMP-7, and pEGFP-C1/BMP-7-IGF-1 exhibited elevated expression of BMP-7 and IGF-1. Besides, BMSCs in the P10 generation displayed decreased cell proliferation. Moreover, BMSCs treated with IGF-1, BMP-7, and IGF-1-BMP-7 showed reduced histological score and collagen I expression while elevated collagen II expression, as well as better repair effect, especially in those treated with IGF-1-BMP-7. Collectively, these results demonstrated a synergistic effect of IGF-1 and BMP-7 on the BMSC chondrogenic differentiation on the articular cartilage damage repair in the rabbit knees, highlighting its therapeutic potential for the treatment of articular cartilage damage.

Tamoxifen and bone morphogenic protein-7 modulate fibrosis and inflammation in the peritoneal fibrosis model developed in uremic rats.

BACKGROUND: Peritoneal fibrosis (PF) represents a long-term complication of peritoneal dialysis (PD), affecting peritoneal membrane (PM) integrity and function. Understanding the mechanisms underlying PF development in an uremic environment aiming alternative therapeutic strategies for treating this process is of great interest. The aim of this study was to analyze the effects of tamoxifen (TAM) and recombinant BMP7 (rBMP7) in an experimental model of PF developed in uremic rats. METHODS: To mimic the clinical situation of patients on long-term PD, a combo model, characterized by the combination of PF and CKD with severe uremia, was developed in Wistar rats. PF was induced by intraperitoneal (IP) injections of chlorhexidine gluconate (CG), and CKD was induced by an adenine-rich diet. Uremia was confirmed by severe hypertension, increased blood urea nitrogen (BUN> 120 mg/dL) and serum creatinine levels (> 2 mg/dL). Uremic rats with PF were treated with TAM (10 mg/Kg by gavage) or BMP7 (30 µg/Kg, IP). Animals were followed up for 30 days. RESULTS: CG administration in uremic rats induced a striking increase in PM thickness, neoangiogenesis, demonstrated by increased capillary density, and failure of ultrafiltration capacity. These morphological and functional changes were blocked by TAM or rBMP7 treatment. In parallel, TAM and rBMP7 significantly ameliorated the PM fibrotic response by reducing α-SMA, extracellular matrix proteins and TGF-ß expression. TAM or rBMP7 administration significantly inhibited peritoneal Smad3 expression in uremic rats with PF, prevented Smad3 phosphorylation, and induced a remarkable up-regulation of Smad7, an intracellular inhibitor of TGFß/Smad signaling, contributing to a negative modulation of profibrotic genes. Both treatments were also effective in reducing local inflammation, possibly by upregulating IκB-α expression in the PM of uremic rats with PF. In vitro experiments using primary peritoneal fibroblasts activated by TGF-ß confirmed the capacity of TAM or rBMP7 in blocking inflammatory mediators, such as IL-1ß expression. CONCLUSIONS: In conclusion, these findings indicate important roles of TGF-ß/Smad signaling in PF aggravated by uremia, providing data regarding potential therapeutic approaches with TAM or rBMP7 to block this process.

Recombinant Human Bone Morphogenetic Protein 7 Exerts Osteo-Catabolic Effects on Bone Grafts That Outweigh Its Osteo-Anabolic Capacity.

This study aimed to investigate the effects of recombinant human bone morphogenetic protein (rhBMP-7) on human cancellous bone grafts (BGs) while differentiating between anabolic and catabolic events. Human BGs alone or supplemented with rhBMP-7 were harvested 14 weeks after subcutaneous implantation into NOD/Scid mice, and studied via micro-CT, histomorphometry, immunohistochemistry and flow cytometry. Immunohistochemical staining for human-specific proteins made it possible to differentiate between grafted human bone and newly formed murine bone. Only BGs implanted with rhBMP-7 formed an ossicle containing a functional hematopoietic compartment. The total ossicle volume in the BMP+ group was higher than in the BMP- group (835 mm3 vs. 365 mm3, respectively, p < 0.001). The BMP+ group showed larger BM spaces (0.47 mm vs. 0.28 mm, p = 0.002) and lower bone volume-to-total volume ratio (31% vs. 47%, p = 0.002). Immunohistochemical staining for human-specific proteins confirmed a higher ratio of newly formed bone area (murine) to total area (0.12 vs. 0.001, p < 0.001) in the BMP+ group, while the ratio of grafted bone (human) area to total area was smaller (0.14 vs. 0.34, p = 0.004). The results demonstrate that rhBMP-7 induces BG resorption at a higher rate than new bone formation while creating a haematopoietic niche. Clinicians therefore need to consider the net catabolic effect when rhBMP-7 is used with BGs. Overall, this model indicates its promising application to further decipher BMPs action on BGs and its potential in complex bone tissue regeneration.

MicroRNAs are Necessary for BMP-7-induced Dendritic Growth in Cultured Rat Sympathetic Neurons.

Neuronal connectivity is dependent on size and shape of the dendritic arbor. However, mechanisms controlling dendritic arborization, especially in the peripheral nervous system, are not completely understood. Previous studies have shown that bone morphogenetic proteins (BMPs) are important initiators of dendritic growth in peripheral neurons. In this study, we examined the hypothesis that post-transcriptional regulation mediated by microRNAs (miRNAs) is necessary for BMP-7-induced dendritic growth in these neurons. To examine the role of miRNAs in BMP-7-induced dendritic growth, microarray analyses was used to profile miRNA expression in cultured sympathetic neurons from the superior cervical ganglia of embryonic day 21 rat pups at 6 and 24 h after treatment with BMP-7 (50 ng/mL). Our data showed that BMP-7 significantly regulated the expression of 43 of the 762 miRNAs. Of the 43 miRNAs, 22 showed robust gene expression; 14 were upregulated by BMP-7 and 8 were downregulated by BMP-7. The expression profile for miR-335, miR-664-1*, miR-21, and miR-23b was confirmed using qPCR analyses. Functional studies using morphometric analyses of dendritic growth in cultured sympathetic neurons transfected with miRNA mimics and inhibitors indicated that miR-664-1*, miR-23b, and miR-21 regulated early stages of BMP-7-induced dendritic growth. In summary, our data provide evidence for miRNA-mediated post-transcriptional regulation as important downstream component of BMP-7 signaling during early stages of dendritic growth in sympathetic neurons.

Interaction of ovarian steroidogenesis and clock gene expression modulated by bone morphogenetic protein-7 in human granulosa cells.

A functional link between clock gene expression and ovarian steroidogenesis was studied using human granulosa KGN cells. Similarities between changes in the mRNA and protein expression levels of Bmal1 and Clock and those of Per2 and Cry1 were found in KGN cells after treatment with forskolin. Among the interrelationships between the expression levels of clock and steroidogenic factors, Clock mRNA had a strongly positive correlation with P450arom and a negative correlation with 3ßHSD. Knockdown of Clock gene by siRNA resulted in a significant reduction of estradiol production by inhibiting P450arom expression, while it induced a significant increase of progesterone production by upregulating 3ßHSD in KGN cells treated with forskolin. Moreover, BMP-7 had an enhancing effect on the expression of Clock mRNA and protein in KGN cells. Thus, the expression levels of Clock, being upregulated by forskolin and BMP-7, were functionally linked to estradiol production and progesterone suppression by human granulosa cells.

Initial Harm Reduction by N-Acetylcysteine Alleviates Cartilage Degeneration after Blunt Single-Impact Cartilage Trauma in Vivo.

Joint injuries are highly associated with the development of post-traumatic osteoarthritis. Previous studies revealed cell- and matrix-protective effects of N-acetylcysteine (NAC) after ex vivo cartilage trauma, while chondroanabolic stimulation with bone morphogenetic protein 7 (BMP7) enhanced type II collagen (COL2) expression. Here, as a next step, we investigated the combined and individual efficacy of intra-articular antioxidative and chondroanabolic treatment in a rabbit in vivo cartilage trauma model. Animals were randomly divided into group A (right joint: trauma (T); left joint: T+BMP7) and group B (right joint: T+NAC; left joint: T+BMP7+NAC). Condyles were impacted with the use of a spring-loaded impact device to ensure defined, single trauma administration. After 12 weeks, histopathological analysis was performed and the presence of matrix metalloproteinase 13 (MMP-13) and COL2 was assessed. Trauma-induced hypocellularity, MMP-13 expression, and cell cluster formation were reduced in NAC-treated animals. In contrast, BMP7 further increased cluster formation. Moreover, synovial concentrations of COL2 carboxy propeptide (CPII) and proteoglycan staining intensities were enhanced in NAC- and NAC+BMP7-treated joints. For the first time, the efficacy of NAC regarding early harm reduction after blunt cartilage trauma was demonstrated in vivo. However, parallel administration of BMP7 was not significantly superior compared to NAC alone.