OBJECTIVE

To assess the odontogenic potential of bone marrow mesenchymal stem cells (BMMSCs) to differentiate into odontoblast-like cells under the morphogenetic influence of dentine matrix as a possible basis for new stem cell-mediated therapeutic approaches to pulp diseases.

METHODS

BMMSCs were harvested from the whole bone marrow and cells at passages 3-5 were used for subsequent experiments. For in vitro studies, 1×10(4) cells were seeded on the surface of dentine slabs and co-cultured for 2 weeks in 24-well plates, then fixed, decalcified, embedded in paraffin and serial sections were processed for analyses. Haematoxylin-eosin (HE) staining was used for the morphological analysis of BMMSCs on the dentine slabs. The protein expression of dentine sialoprotein (DSP) in co-cultured BMMSCs was detected by immunohistochemical (IHC) staining. For in vivo studies, 5×10(6) cells were collected as cell pellets, seeded onto dentine slices and transplanted into renal capsules for 6 weeks. Histological analyses of harvested tissues were performed as described for the in vitro studies. Total RNA and protein were extracted from harvested tissues and Dspp/DSP expression was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively.

RESULTS

After 2 weeks of co-culture with dentine slabs, BMMSCs demonstrated good viability in terms of morphological appearance and some showed polarization and extension of their cytoplasmic processes into dentine tubules with DSP expression. In vivo study demonstrated similar morphological changes and DSP expression in cells adjacent to dentine. RT-PCR and Western blot also demonstrated that the expression of Dspp/DSP in the co-cultured BMMSCs groups was higher than in the control groups.

CONCLUSIONS

Dentine matrix can signal morphogenic induction of differentiation of BMMSCs into odontoblast-like cells in vivo and in vitro.

The influence of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and transforming growth factor beta (TGF-beta) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24 h with graded concentrations of TGF-beta1 (0, 0.1, 1.0, 5.0, and <em>10</em> ng/ml). TGF-beta1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-beta1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-beta1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.

The purpose of the research was to explore the therapeutic action of simvastatin-loaded poly(ethylene glycol)-b-poly(gamma-benzyl l-glutamate) (PEG-b-PBLG50) on intestinal ischemia/reperfusion injury (II/RI) through downregulating <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4)/cyclooxygenase-2 (COX-2) pathway as compared to free simvastatin (Sim). Sprague Dawley rats were preconditioned with 20 mg/kg Sim or simvastatin/PEG-b-PBLG50 (Sim/P) compounds, and then subjected to 45 min of ischemia and 1 h of reperfusion. The blood and small intestines were collected, serum levels of interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-<em>10</em> (IL-<em>10</em>), tumor necrosis factor-α, and nitric oxide (NO) were checked, and the dry/wet intestine ratios, superoxide dismutase activity, myeloperoxidase content, reactive oxygen species, endothelial nitric oxide synthase, <em>protein</em> 47 kDa phagocyte oxidase (p47phox), BMP4, COX-2, and p38 mitogen-activated <em>protein</em> kinase (p38MAPK) expressions were measured in intestinal tissues. Both Sim and Sim/P pretreatment reduced intestinal oxidative damnification, restricted inflammatory harm, and downregulated the BMP4 and COX-2 expressions as compared to II/RI groups, while Sim/P remarkably improved this effect.

Clinical trials using <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP2) for <em>bone</em> reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect of an alternative treatment strategy in which human <em>bone</em> marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with <em>10</em> and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured in osteogenic medium. The effects of preconditioning were analyzed with regard to proliferation and expression of osteogenic markers at both gene and <em>protein</em> level. The results were compared to those from cultures with continuous BMP2 stimulation. A significant increase in proliferation was seen with both precondition and continuous stimulation with BMP2, with no difference between the treatments. Preconditioning with BMP2 significantly increased gene expression of RUNX2, COLI, ALP, and OC, and <em>protein</em> levels of COLI and ALP. This was not found with continuous stimulation. The role of preconditioning with BMP2 in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of hMSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided.

To study the relationship between the expression of <em>10</em> selected genes in cumulus cells and the corresponding oocyte development competence, and the effect of patient age and body mass index on gene expression of cumulus cells, we collected 354 cumulus cell masses associated with individual oocyte from 48 women. The expression levels of the genes involved in glucose metabolism (<i>PFKP, PKM2, LDHA</i> and <i>GFPT</i>) and expansion (<i>HAS2, VCAN, TNFAIP6, PTGS2, PTX3 and SDC4</i>) in cumulus cells were detected by reverse transcription polymerase chain reaction. These were compared among oocyte maturity, fertilization, embryo morphology and implantation, and analyzed the effect of the subject's age and body mass index. Cumulus cell <i>PFKP</i> expression from mature oocytes was higher than those from immature oocytes (P = 0.014), and <i>VCAN</i> expression was higher from oocytes that developed into high-quality embryos (P = 0.024). <i>TNFAIP6</i> expression in cumulus cells from fertilized oocytes was lower than that from unfertilized oocytes (P = 0.044). The levels of <i>VCAN, TNFAIP6, PTX3</i> and <i>SDC4</i> were changed significantly as a function of the subject's age and body mass index. In conclusion, the level of <i>VCAN</i> expression in cumulus cells is positively correlated with the early embryo morphology score, and with further development could perhaps be used to evaluate oocyte developmental competence to complement embryonic morphological assessment.<b>Abbreviations:</b> CCs: cumulus cells; GDF9: growth differentiation factor 9; BMP15: <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15; PTGS2: prostaglandin synthase 2; HAS2: hyaluronic acid synthase 2; VCAN: versican; GREM1: gremlin 1; PFKP: phosphofructokinase, platelet; PKM2: pyruvate kinase isozyme type M2; LDHA: lactic dehydrogenase; GFPT: glucosaminefructo-6-phosphate transaminase; TNFAIP6: tumor necrosis factor 6 <em>protein</em>; PTX3: penetrin 3; SDC4: syndecan-4; BMI: body mass index; MD: median values; IQR: interquartile range; FSH: follicle-stimulating hormone; LH: luteinizing hormone; HCG: human chorionic gonadotropin; ICSI: intracytoplasmic sperm injection; GnRH: gonadotropin-releasing hormone; hMG: human menopausal gonadotropin; GV: germinal vesicle; M I: metaphase I; M II: metaphase II; cDNA: complementary DNA; SD: standard deviation.

Fibroblast growth factor 7 (FGF7) plays an important role in regulating the proliferation, migration, and differentiation of cells. However, the role of FGF7 in <em>bone</em> formation is not yet fully understood. We examined the effect of FGF7 on <em>bone</em> formation using a rat model of mandible defects. Rats underwent mandible defect surgery and then either scaffold treatment alone (control group) or FGF7-impregnated scaffold treatment (FGF7 group). Micro-CT and histological analyses revealed that the FGF7 group exhibited greater <em>bone</em> formation than did the control group <em>10</em> weeks after surgery. With the exception of total porosity (%), all <em>bone</em> parameters had higher values in the FGF7 group than in the control group at each follow-up after surgery. The FGF7 group showed greater expression of osteogenic markers, such as runt-related transcription factor 2, osterix, osteocalcin, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2, osteopontin, and type I collagen in newly formed <em>bone</em> than did the control group. The delivery of FGF7 also increased the messenger RNA expression of stromal-cell-derived factor 1 (SDF-1) and CXCR4 in newly formed <em>bone</em> in the FGF7 group compared with the control group. Further, addition of exogenous FGF7 induced migration of rat <em>bone</em> marrow stromal cells and increased the expression of SDF-1 and CXCR4 in the cells. Furthermore, the addition of FGF7 augmented mineralization in the cells with increased expression of osteogenic markers, and this augmentation was significantly suppressed by an inhibitor specific for c-Jun N-terminal kinase (SP600125) or extracellular-signal-regulated kinase (PD98059). Collectively, these results suggest that local delivery of FGF7 increases <em>bone</em> formation in a mandible defect with enhanced osteogenesis and chemoattraction.

UNASSIGNED

Bone morphogenetic protein (BMP)15 is an oocyte-specific growth factor, which, together with growth differentiation factor (GDF) 9, regulates folliculogenesis and ovulation rate. Multiple mutations in BMP15 have been identified in women with primary ovarian insufficiency (POI), supporting a pathogenic role; however, the underlying biological mechanism of many of these mutants remains unresolved.

UNASSIGNED

To determine how mutations associated with ovarian dysfunction alter the biological activity of human BMP15.

UNASSIGNED

The effects of 10 mutations in BMP15 on protein production, activation of granulosa cells, and synergy with GDF9 were assessed.

UNASSIGNED

Sequencing of 35 patients with POI identified both an unrecognized BMP15 variant (c.986G>A, R329H) and a variant (c.581T>C, F194S) previously associated with the condition. Assessing expression and activity of these and 8 other BMP15 mutants identified: (1) multiple variants, including L148P, F194S, and Y235C, with reduced mature protein production; (2) three variants (R138H, A180T, and R329H) with ∼fourfold lower activity than wild-type BMP15; and (3) 3 variants (R68W, F194S, and N196K) with a significantly reduced ability to synergize with GDF9.

UNASSIGNED

Mutations in BMP15 associated with POI reduce mature protein production, activity, or synergy with GDF9. The latter effect is perhaps most interesting given that interactions with GDF9 most likely underlie the physiology of BMP15 in the human ovary.

OBJECTIVE

To prepare new pharmaceutical forms with sustained delivery properties of recombinant human bone morphogenetic protein-2 (rhBMP2) for tissue engineering and guided tissue regeneration (GTR) use.

METHODS

rhBMP2-loaded dextran-based hydrogel microspheres (rhBMP2-MPs), which aimed to keep rhBMP2 bioactivity and to achieve long-term sustained release of rhBMP2, were prepared by double-phase emulsified condensation polymerization. The physical, chemical performances and biological characteristics of those microspheres were studied both in vitro and in vivo.

RESULTS

The microspheres' average diameter was 30.33+/-4.32 microm with 75.4% ranging from 20 microm to 40 microm and the drug loading and encapsulation efficiency were 7.82% and 82.25%, respectively. The rhBMP2-releasing profiles in vitro showed that rhBMP2 release could be maintained more than 10 d. The rhBMP2-MPs, with good swelling and biodegradation behavior, could be kept for 6 months at below 4 degree without significant characteristic change or bioactivity loss. Cytology studies showed that rhBMP2-MPs could promote the proliferation of periodontal ligament cells (PDLCs) approximately 10 d, while the bioactivity of concentrated rhBMP2 solution could keep no more than 3 d. Scanning electron microscope showed that rhBMP2-MPs could be enchased into the porous structure of calcium phosphate ceremic (CPC) and the eugonic growth of PDLCs in CPC/rhBMP2-MPs scaffolds. Animal experiments indicated that using CPC/rhBMP2-MPs scaffolds could gain more periodontal tissue regeneration than using rhBMP2 compound firsthand with CPC (CPC/rhBMP2).

CONCLUSIONS

By encapsulating rhBMP2 into dextran-based microspheres, a small quantity of rhBMP2 could achieve equivalent effects to the concentrated rhBMP2 solution and at the same time, could prolong rhBMP2 retention both in vitro and in vivo.

BACKGROUND

Recombinant human bone morphogenetic protein (rhBMP)-2 is a potent osteoinductive agent; however, its clinical use has been reduced because of safety and efficacy concerns. In preclinical studies involving a critical-sized defect in a rat model, sclerostin antibody (Scl-Ab) treatment increased bone formation within the defect but did not result in reliable healing. The purpose of the current study was to evaluate bone repair of a critical-sized femoral defect in a rat model with use of local implantation of rhBMP-2 combined with systemic administration of Scl-Ab.

METHODS

A critical-sized femoral defect was created in rats randomized into three treatment groups: local rhBMP-2 and systemic Scl-Ab (Scl + BMP), local rhBMP-2 alone, and collagen sponge alone (operative control). The Scl + BMP group received local rhBMP-2 (10 μg) on a collagen sponge placed within the defect intraoperatively and then twice weekly injections of Scl-Ab (25 mg/kg) administered postoperatively. The femora were evaluated at twelve weeks with use of radiography, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing.

RESULTS

At twelve weeks, all Scl + BMP and rhBMP-2 only samples were healed. No femora healed in the operative control group. Histomorphometric analysis demonstrated more bone in the Scl + BMP samples than in the samples treated with rhBMP-2 alone (p = 0.029) and the control samples (p = 0.003). MicroCT revealed that the Scl + BMP group had a 90% greater bone volume within the defect region compared with the rhBMP-2 group and a 350% greater bone volume compared with the operative control group (p < 0.001). Biomechanical testing showed that the group treated with Scl + BMP had greater torsional strength and rigidity compared with the rhBMP-2 group (p < 0.001 and p = 0.047) and the intact femoral control group (p < 0.001). Torque to failure was lower in the rhBMP-2 group compared with the intact femoral control group (p < 0.002). Mean energy to failure was higher in the Scl + BMP samples compared with the rhBMP-2 only samples (p = 0.001).

CONCLUSIONS

In a critical-sized femoral defect in a rat model, local rhBMP-2 combined with systemic administration of Scl-Ab resulted in more robust healing that was stronger and more rigid than results for rhBMP-2 alone and intact nonoperative femora.

CONCLUSIONS

Our study demonstrated that combining an osteoinductive agent with a systemically administered antibody that promotes bone formation can enhance bone repair and has potential as a therapeutic regimen in humans.

OBJECTIVE

To evaluate bone invasion, survival, and expression of bone morphogenetic protein-6 (BMP-6) in oral cavity cancer in the context of known biomarkers indicative of poor prognosis.

METHODS

Molecular expression study combined with retrospective chart review of corresponding patients at a tertiary care center.

METHODS

Between 2000 and 2009, a total of 197 patients underwent resection for oral cavity squamous cell carcinoma. Of these, 30 pathologic specimens were chosen for further molecular analysis. These 30 patients were separated into three groups (10 per group) based on American Joint Committee on Cancer (AJCC) staging and staging based on size alone (TAJCC /SIZE ). The first group consisted of tumors staged as T2 /2 based on size less than 4 cm and that had no evidence of bone invasion. The T2 /4 group consisted of tumors that were upstaged from T2 based on bone invasion. The T4 /4 group consisted of tumors that were large with and without bone invasion. The expression of extracellular matrix metalloproteinase inducer (EMMPRIN), BMP-6, and epidermal growth factor receptor (EGFR) was examined using immunohistochemistry techniques. Patient demographics, tumor characteristics, survival, and recurrence were compared.

RESULTS

Average follow-up was 21 months. Expression of BMP-6 was significantly higher in the T2 /4 cohort (tumor less than 4 cm with bony invasion) than the larger tumors without bone invasion (T4 /4 cohort, P = .05). In addition, increased BMP-6 expression correlated with aggressive behavior in the smaller tumors. Furthermore, increased EGFR expression positively correlated with increased levels of BMP-6.

CONCLUSIONS

Increased expression of BMP-6 in oral cavity cancer may affect bone invasion.

Autologous vascularized <em>bone</em> grafts, allografts, and biocompatible artificial <em>bone</em> substitutes each have their shortcomings. <em>Bones</em> regenerated using recombinant human <em>bone</em> <em>morphogenetic</em> <em>proteins</em>, demineralized <em>bone</em> powder, or combinations of these are generally small and do not meet the need. The current trend is to use tissue engineering approaches with <em>bone</em> marrow mesenchymal stem cells (MSCs) to generate <em>bones</em> of a desired size and shape. A suspension of osteogenically induced MSCs (CD11a-, CD29+, CD44+) was added to 2% alginate, gelled by mixing this combination with calcium sulfate (CaSO(4) 0.2 g/mL), and injected into the subcutaneous pocket in the dorsal aspect of nude mice. Cells of various concentrations (0, <em>10</em>, 50, and 70 million/mL) were used. These implanted constructs were harvested at predetermined times up to 30 weeks for histology. The doubling time of bovine MSCs is 3.75 +/- 1.96 days and the proliferation is rapid. Histological evaluation revealed signs of endochondrosis with woven <em>bone</em> deposition. The equilibrium modulus increased with time in vivo, though less than that of normal tissue. Implants seeded with 70 million cells/mL for 6 months resulted in the best formation of equilibrium modulus. This approach has several advantages: (i) obtaining MSCs is associated with low donor morbidity; (ii) MSCs proliferate rapidly in vitro, and a large number of viable cells can be obtained; and (iii) the MSC/alginate constructs can develop into <em>bone</em>-like nodules with high cell viability. Such a system may be useful in large-scale production of bony implants or in the repair of bony defects. The fact that endochondral <em>bone</em> formation led to woven <em>bone</em> suggests its potential feasibility in regional cell therapy.

Endothelial cell (EC) activation and vascular injury are hallmark features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Caveolin-1 (Cav-1) is highly expressed in pulmonary microvascular ECs and plays a key role in maintaining vascular homeostasis. The aim of this study was to determine if the lung inflammatory response to Escherichia coli lipopolysaccharide (LPS) promotes priming of ECs via Cav-1 depletion and if this contributes to the onset of pulmonary vascular remodeling. To test the hypothesis that depletion of Cav-1 primes ECs to respond to profibrotic signals, C57BL6 wild-type (WT) mice (Tie2.Cre-;Cav1fl/fl ) were exposed to nebulized LPS (<em>10</em> mg; 1 h daily for 4 days) and compared with EC-specific Cav1-/- (Tie2.Cre+;Cav1fl/fl ). After 96 h of LPS exposure, total lung Cav-1 and <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type II (BMPRII) expression were reduced in WT mice. Moreover, plasma albumin leakage, infiltration of immune cells, and levels of IL-6/IL-6R and transforming growth factor-β (TGF-β) were elevated in both LPS-treated WT and EC-Cav1-/- mice. Finally, EC-Cav1-/- mice exhibited a modest increase in microvascular thickness basally and even more so on exposure to LPS (96 h). EC-Cav1-/- mice and LPS-treated WT mice exhibited reduced BMPRII expression and endothelial nitric oxide synthase uncoupling, which along with increased TGF-β promoted TGFβRI-dependent SMAD-2/3 phosphorylation. Finally, human lung sections from patients with ARDS displayed reduced EC Cav-1 expression, elevated TGF-β levels, and severe pulmonary vascular remodeling. Thus EC Cav-1 depletion, oxidative stress-mediated reduction in BMPRII expression, and enhanced TGF-β-driven SMAD-2/3 signaling promote pulmonary vascular remodeling in inflamed lungs.

Ectopic tendon mineralization can develop following tendon rupture or trauma surgery. The pathogenesis of ectopic tendon mineralization and its clinical impact have not been fully elucidated yet. In this study, we utilized a mouse Achilles tendon injury model to determine whether ectopic tendon mineralization alters the biomechanical properties of the tendon and whether BMP signaling is involved in this condition. A complete transverse incision was made at the midpoint of the right Achilles tendon in 8-week-old CD1 mice and the gap was left open. Ectopic cartilaginous mass formation was found in the injured tendon by 4weeks post-surgery and ectopic mineralization was detected at 8 to <em>10</em>weeks post-surgery. Ectopic mineralization grew over time and volume of the mineralized materials of 25-weeks samples was about 2.5 fold bigger than that of <em>10</em>-weeks samples, indicating that injury-induced ectopic tendon mineralization is progressive. In vitro mechanical testing showed that max force, max stress and mid-substance modulus in the 25-weeks samples were significantly lower than the <em>10</em>-weeks samples. We observed substantial increases in expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> family genes in injured tendons 1week post-surgery. Immunohistochemical analysis showed that phosphorylation of both Smad1 and Smad3 was highly increased in injured tendons as early as 1week post-injury and remained high in ectopic chondrogenic lesions 4-weeks post-injury. Treatment with the BMP receptor kinase inhibitor (LDN193189) significantly inhibited injury-induced tendon mineralization. These findings indicate that injury-induced ectopic tendon mineralization is progressive, involves BMP signaling and associated with deterioration of tendon biomechanical properties.

Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced <em>bone</em> formation. Since IL-17 is known to induce osteoclastogenesis, we studied the interactions between IL-17 and BMP-2 in ceramic scaffolds either or not carrying a coating with the bisphosphonate zoledronic acid (ZOL). Histological evaluation revealed that IL-17 alone did not induce any osteoclasts at day <em>10</em>. On the other hand, BMP-2 clearly stimulated early tissue ingrowth and osteoclastogenesis. Both of these processes were blocked in presence of ZOL. IL-17 signaling restored early vascularized connective tissue formation and osteoclastogenesis induced by BMP-2 in ZOL-coated scaffolds. After 12 weeks, the <em>bone</em> volume induced by co-delivery of BMP-2 and IL-17 was doubled as compared to that induced by BMP-2 alone. We conclude that IL-17 has osteo-stimulatory effects through a synergy with <em>bone</em>-inductive BMP-2. Although local and single application of IL-17 does not mediate osteoclast formation, it could promote other processes involved in <em>bone</em> formation such as connective tissue ingrowth. The use of IL-17 may contribute to the development of improved <em>bone</em> graft substitutes.

The alveolar epithelium consists of (ATI) and type II (ATII) cells. ATI cells cover the majority of the alveolar surface due to their thin, elongated shape and are largely responsible for barrier function and gas exchange. During lung injury, ATI cells are susceptible to injury, including cell death. Under some circumstances, ATII cells also die. To regenerate lost epithelial cells, ATII cells serve as progenitor cells. They proliferate to create new ATII cells and then differentiate into ATI cells [¹,²,³]. Regeneration of ATI cells is critical to restore normal barrier and gas exchange function. Although the signaling pathways by which ATII cells proliferate have been explored [⁴,⁵,⁶,⁷,⁸,⁹,^<em>10</em>,¹¹,¹²], the mechanisms of ATII-to-ATI cell differentiation have not been well studied until recently. New studies have uncovered signaling pathways that mediate ATII-to-ATI differentiation. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signaling inhibits ATII proliferation and promotes differentiation. Wnt/β-catenin and ETS variant transcription factor 5 (Etv5) signaling promote proliferation and inhibit differentiation. Delta-like 1 homolog (Dlk1) leads to a precisely timed inhibition of Notch signaling in later stages of alveolar repair, activating differentiation. Yes-associated <em>protein</em>/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) signaling appears to promote both proliferation and differentiation. We recently identified a novel transitional cell state through which ATII cells pass as they differentiate into ATI cells, and this has been validated by others in various models of lung injury. This intermediate cell state is characterized by the activation of Transforming growth factor beta (TGFβ) and other pathways, and some evidence suggests that TGFβ signaling induces and maintains this state. While the abovementioned signaling pathways have all been shown to be involved in ATII-to-ATI cell differentiation during lung regeneration, there is much that remains to be understood. The up- and down-stream signaling events by which these pathways are activated and by which they induce ATI cell differentiation are unknown. In addition, it is still unknown how the various mechanistic steps from each pathway interact with one another to control differentiation. Based on these recent studies that identified major signaling pathways driving ATII-to-ATI differentiation during alveolar regeneration, additional studies can be devised to understand the interaction between these pathways as they work in a coordinated manner to regulate differentiation. Moreover, the knowledge from these studies may eventually be used to develop new clinical treatments that accelerate epithelial cell regeneration in individuals with excessive lung damage, such as patients with the Acute Respiratory Distress Syndrome (ARDS), pulmonary fibrosis, and emphysema.

This study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with <em>10</em>-9 M melatonin (DOs + MT), cumulus-oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.

Transforming growth factor β isoforms (TGF-β) are among the most recently evolved members of a signaling superfamily with more than 30 members. TGF-β play vital roles in regulating cellular growth and differentiation, and they signal through a highly restricted subset of receptors known as TGF-β type I receptor (TβR-I) and TGF-β type II receptor (TβR-II). TGF-β's specificity for TβR-I has been proposed to arise from its pre-helix extension, a five-residue loop that binds in the cleft between TGF-β and TβR-II. The structure and back<em>bone</em> dynamics of the unbound form of the TβR-I extracellular domain were determined using NMR to investigate the extension's role in binding. This showed that the unbound form is highly similar to the bound form in terms of both the β-strand framework that defines the three-finger toxin fold and the extension and its characteristic cis-Ile54-Pro55 peptide bond. The NMR data further showed that the extension and two flanking 3(<em>10</em>) helices are rigid on the nanosecond-to-picosecond timescale. The functional significance of several residues within the extension was investigated by binding studies and reporter gene assays in cultured epithelial cells. These demonstrated that the pre-helix extension is essential for binding, with Pro55 and Pro59 each playing a major role. These findings suggest that the pre-helix extension and its flanking prolines evolved to endow the TGF-β signaling complex with its unique specificity, departing from the ancestral promiscuity of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> subfamily, where the binding interface of the type I receptor is highly flexible.

Periodontal ligament stem cells (PDLSCs) are considered as potential mesenchymal stem cell sources for future clinical applications in periodontal regeneration therapy. Simvastation, widely used for lowering serum cholesterol, is known to have a <em>bone</em> stimulatory effect. However, it is not clear whether simvastation affects the differentiation of PDLSCs. This study examined the effects of simvastatin on human PDLSCs in vitro and in vivo. Using the limiting dilution technique, human PDLSCs were isolated and expanded. PDLSCs were cultured with simvastatin (0.01-<em>10</em> μM), and the proliferation was measured. The osteogenic differentiation was characterized by alkaline phosphatase (ALP) activity and Alizarin Red-S staining for calcium deposition. The gene expression levels of osteogenic markers were evaluated by RT-PCR. In addition, PDLSCs were transplanted into nude mice with ceramic bovine <em>bone</em> powders as carriers to observe the capacity of mineralized tissue formation in vivo. Simvastatin at concentrations <1 μM did not suppress the proliferation of PDLSCs. After the administration of 0.1 μM simvastatin, the expression of ALP, <em>bone</em> sialo<em>protein</em>, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 genes were significantly upregulated, and the ALP activity and mineralized nodule formation were significantly higher in the simvastatin-treated cells than the control cells. In addition, the in vivo transplantation results showed that simvastatin treatment promoted the degree of mineralized tissue formation. Collectively, simvastatin has positive effects on osteogenic differentiation of human PDLSCs in vitro and in vivo. This suggests that simvastatin might be a useful osteogenic induction agent for periodontal <em>bone</em> regeneration.

Good bioactivity and osteogenesis of three-dimensional porous alginate scaffolds (PAS) are critical for <em>bone</em> tissue engineering. In this work, alginate and <em>bone</em>-forming peptide-1 (BFP-1), derived from <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7), have been combined together (without carbodiimide chemistry treatment) to develop peptide-incorporated PAS (p-PAS) for promoting <em>bone</em> repairing ability. The mechanical properties and SEM images show no difference between pure PAS and p-PAS. The release kinetics of the labeled peptide with 6-carboxy tetramethyl rhodamine from the PAS matrix suggests that the peptide is released in a relatively sustained manner. In the cell experiment, p-PAS show higher cell adhesion, spreading, proliferation and alkaline phosphatase (ALP) activity than the pristine PAS group, indicating that the BFP-1 released from p-PAS could significantly promote the aggregation and differentiation of osteoblasts, especially at <em>10</em>μg/mL of trapped peptide concentration (p-PAS-<em>10</em>). Furthermore, p-PAS-<em>10</em> was implanted into Beagle calvarial defects and <em>bone</em> regeneration was analyzed after 4 weeks. New <em>bone</em> formation was assessed by calcein and Masson's trichrome staining. The data reveal that p-PAS group exhibits significantly enhanced oseto-regenerative capability in vivo. The peptide-modified PAS with promoted bioactivity and osteogenic differentiation in vitro as well as <em>bone</em> formation ability in vivo could be promising tissue engineering materials for repairing and regeneration of <em>bone</em> defects.

<AbstractText>Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease with high heritability. Although several predisposing genes have been linked to IPAH, the genetic aetiology remains unknown for a large number of IPAH cases.</AbstractText><AbstractText>We conducted an exome-wide gene-based burden analysis on two independent case-control studies, including a total of 331 IPAH cases and <em>10</em> 508 controls. Functional assessments were conducted to analyse the effects of genetic mutations on <em>protein</em> biosynthesis and function.</AbstractText><p><div><b>RESULTS</b></div>The gene encoding human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 9 (<i>BMP9</i>) was identified as a novel genetic locus displaying exome-wide association with IPAH in the discovery cohort (OR 18.8; p=1.9×<em>10</em>^-11). This association was authenticated in the independent replication cohort (p=1.0×<em>10</em>^-5). Collectively, the rare coding mutations in <i>BMP9</i> occurred in 6.7% of cases, ranking this gene second to <i>BMPR2</i>, comprising a combined significance of 2.7×<em>10</em>^-19 (OR 21.2). Intriguingly, the patients with <i>BMP9</i> mutations had lower plasma levels of BMP9 than those without. Functional studies showed that the <i>BMP9</i> mutations led to reduced BMP9 secretion and impaired anti-apoptosis ability in pulmonary arterial endothelial cells.</p><p><div><b>CONCLUSION</b></div>We identify <i>BMP9</i> as an IPAH culprit gene.</p>

The <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) pathway is essential for the morphogenesis of multiple organs in the digestive system. Abnormal BMP signaling has also been associated with disease initiation and progression in the gastrointestinal (GI) tract and associated organs. Recent studies using animal models, tissue organoids, and human pluripotent stem cells have significantly expanded our understanding of the roles played by BMPs in the development and homeostasis of GI organs. It is clear that BMP signaling regulates GI function and disease progression that involve stem/progenitor cells and inflammation in a tissue-specific manner. In this review we discuss these new findings with a focus on the esophagus, stomach, and intestine. Expected final online publication date for the <i>Annual Review of Physiology</i>, Volume 82 is February <em>10</em>, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Demineralized <em>bone</em> matrix (DBM) reproducibly induces extraskeletal <em>bone</em> formation in rodents, but its effects in dogs and primates are negative or uncertain. In previous studies on the squirrel monkey, DBM did not induce <em>bone</em>, although the same implants were effective in nude rats. In the present study, the DBM was augmented with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). <em>Bone</em> was formed in <em>10</em> of 12 monkeys, as verified by histology and calcium content. However, in 4 monkeys, the induced <em>bone</em> mass appeared smaller than the original implant. DBM controls induced microscopic amounts of <em>bone</em> in 2 out of <em>10</em> monkeys. In the nude rats, all DBM controls and augmented implants induced <em>bone</em>. The difficulties in achieving <em>bone</em> induction in higher animals may be overcome, at least partially, by using a higher concentration of the inductive <em>protein</em> than is present in DBM.

Previous studies have suggested that <em>bone</em> <em>morphogenetic</em> <em>protein</em>-6 (BMP-6) has a pronounced upregulation in rat brains subjected to traumatic brain injury. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-<em>10</em> (BMP-<em>10</em>) is a newly identified cardiac-specific peptide growth factor that belongs to the TGF-β superfamily. To elucidate the dynamic expression changes and cellular localization of BMP-<em>10</em> during traumatic brain injury (TBI), we performed an acute traumatic brain injury model in adult rats. Western blot analysis, immunohistochemistry and RTPCR revealed that BMP-<em>10</em> expression in impaired cerebral cortex was more strongly induced not only at <em>protein</em> level but also at mRNA level compared to that in normal group. Double immunofluorescence labeling suggested that BMP-<em>10</em> was localized mainly in the cytoplasm of neurons, microglias, and astrocytes within 3 mm from the lesion site at day 3 post-injury. And there was a specific upregulation of BMP-<em>10</em> in astrocytes following brain injury. Besides, co-localization of BMP-<em>10</em> and proliferating cell nuclear antigen (PCNA) was detected in Glial fibrillary acidic <em>protein</em> (GFAP) (+) cells. We also examined the expression profiles of PCNA and GFAP whose change was correlated with the expression profiles of BMP-<em>10</em> in the incised injury model used here. Another experiment in which astrocytes were treated with BMP-<em>10</em> was also performed to confirm the relationship between the upregulation of BMP-<em>10</em> and proliferation of astrocytes following TBI. Taken together, this is the first description of BMP-<em>10</em> expression during the central nervous system (CNS) lesion and repair. Thus, the present data suggested that BMP-<em>10</em> may be implicated in CNS pathophysiology after TBI. But, further studies are needed to understand the cell signal pathway which can direct the exact role of BMP-<em>10</em> following traumatic brain injury.

According to 5- and <em>10</em>-year clinical follow-up studies large-scale <em>bone</em> allografts have a high failure rate, largely due to poor allograft incorporation with adjacent <em>bone</em> and subsequent poor remodeling. The goal of this study was to develop a methodology to deliver growth factors from large-scale <em>bone</em> allografts in a temporally controlled manner. Intact long <em>bone</em> allografts were coated with a micron-scale thick layer of degradable polymer that maintained inherent pore structures and acted as a delivery vehicle for <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and vascular endothelial growth factor. VEGF was loaded onto the surface of the polymer to produce rapid release, to encourage initial vascularization at the defect site, while BMP-2 was encapsulated within the polymer layer to promote a more sustained release, to encourage <em>bone</em> formation over time. Release kinetics from factor-loaded polymer-coated allografts show an early burst release of VEGF over the first 7 days followed by a more sustained release of BMP-2 over the second and third week. In vitro cell studies using human mesenchymal stem cells confirm the bioactivity of the released BMP-2. In-vivo results show robust <em>bone</em> formation over the first 8 weeks of healing in femoral segmental defects in rats implanted with BMP-2 loaded polymer-coated allografts. A microscale thin coating of degradable polymer on a large-scale <em>bone</em> allograft provides temporal control over the delivery of growth factor loaded onto one allograft, while maintaining its microscale pore structure. Enhancing the incorporation and subsequent remodeling of allografts would reduce the incidence of allograft failure over time, and potentially speed healing at the earliest stages after implantation.