Disruption of BMP Signaling in Osteoblasts Through Type IA Receptor (BMPRIA) Increases Bone Mass

Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both μCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL–OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL–OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo.     

Bone Formation During Distraction Osteogenesis Is Dependent on Both VEGFR1 and VEGFR2 Signaling

Introduction : Distraction osteogenesis (DO) is characterized by the induction of highly vascularized new bone formation through an intramembranous process largely devoid of the formation of cartilage. Materials and Methods : To test the hypothesis that DO is strictly dependent on vascualrization, we inhibited vascular endothelial growth factor (VEGF) activity by antibody blockade of both receptors VEGFR1 (Flt‐1) and VEGFR2 (Flk‐1) or only VEGFR2 (Flk‐1) in a previously developed murine tibia DO model. During normal DO, VEGFR1 (Flt‐1), VEGFR2 (Flk‐1), VEGFR3 (Flt4) and all four VEGF ligand (A, B, C, and D) mRNAs are induced. Results : The expression of mRNA for the receptors generally paralleled those of the ligands during the period of active distraction. Bone formation, as assessed by μCT, showed a significant decrease with the double antibody treatment and a smaller decrease with single antibody treatment. Vessel volume, number, and connectivity showed progressive and significant inhibition in all of these of parameters between the single and double antibody blockade. Molecular analysis showed significant inhibition in skeletal cell development with the single and double antibody blockade of both VEGFR1 and 2. Interestingly, the single antibody treatment led to selective early development of chondrogenesis, whereas the double antibody treatment led to a failure of both osteogenesis and chondrogenesis. Conclusions : Both VEGFR1 and VEGFR2 are functionally essential in blood vessel and bone formation during DO and are needed to promote osteogenic over chondrogenic lineage progression.     

自体髂骨移植联合BMP治疗四肢长骨干骨折不愈合

目的分析骨折不愈合的原因，探讨骨折不愈合的治疗方法。方法选取2006年4月至2007年10月45例长骨干骨折不愈合病例，手术取出原内固定．重新复位，选用坚强合适内固定材料固定骨折断端，植人骨形态发生蛋白复合材料及自体髂骨，对于骨质缺损较多者同时植入同种异体骨。结果术后随访5～20个月，伤口均一期愈合，至随访时43例达骨性愈合，未发现内固定钢板及髓内钉松动、折弯。其中2例患者活动时肘关节疼痛，1例活动范围在0°～120°之间，1例活动范围在0°～90°之间；1例肩关节活动受限，肩关节外展范围0°～80°；1例患者活动时膝关节疼痛，活动范围在0°～60°之间。结论内固定不牢固、骨质缺损是长骨干骨折不愈合的重要原因，提高四肢长骨干骨折愈合的关键是选择合适坚强的内固定，术中充分植骨及植入成骨活性物质，促进骨折的愈合。     

Dysregulated BMP Signaling and Enhanced Osteogenic Differentiation of Connective Tissue Progenitor Cells From Patients With Fibrodysplasia Ossificans Progressiva (FOP)

The study of FOP, a disabling genetic disorder of progressive heterotopic ossification, is hampered by the lack of readily available connective tissue progenitor cells. We isolated such cells from discarded primary teeth of patients with FOP and controls and discovered dysregulation of BMP signaling and rapid osteoblast differentiation in FOP cells compared with control cells. Introduction: Fibrodysplasia ossificans progressiva (FOP), the most disabling condition of progressive heterotopic ossification in humans, is caused by a recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor, in all classically affected individuals. A comprehensive understanding of FOP has been limited, in part, by a lack of readily available connective tissue progenitor cells in which to study the molecular pathology of this disorder. Materials and Methods: We derived connective tissue progenitor cells from discarded primary teeth (SHED cells) of patients with FOP and controls and examined BMP signaling and osteogenic differentiation in these cells. Results: SHED cells transmitted BMP signals through both the SMAD and p38 mitogen‐activated protein kinase (MAPK) pathways and responded to BMP4 treatment by inducing BMP responsive genes. FOP cells showed ligand‐independent BMP signaling and ligand‐dependent hyper‐responsiveness to BMP stimulation. Furthermore, FOP cells showed more rapid differentiation to an osteogenic phenotype than control cells. Conclusions: This is the first study of BMP signaling and osteogenic differentiation in connective tissue progenitor cells from patients with FOP. Our data strongly support both basal and ligand‐stimulated dysregulation of BMP signaling consistent with in silico studies of the mutant ACVR1 receptor in this condition. This study substantially extends our understanding of dysregulated BMP signaling in a progenitor cell population relevant to the pathogenesis of this catastrophic disorder of progressive ectopic ossification.     

Prostaglandin E2 inhibits BMP signaling and delays chondrocyte maturation

While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2–treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-β to activate the TGF-β-responsive reporter, 4XSBE. Finally, PGE2 down-regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 785–792, 2009     

骨形态发生蛋白在异体骨段移植愈合中的表达及临床意义

目的：研究骨形态发生蛋白（BMP）在异体骨段移植愈中的表达及临床意义。方法：采用新西兰大白兔股骨中段切除1．5cm骨干和骨膜实验动物模型。方法：36只兔随机分实验组和对照组。实验组植入深低温保存的异体骨,对照组植入自体新鲜骨,均用髓内针固定,分别于术后第1、2、3个月用免疫组织化学染色法观察BMP在异体骨愈合过程的表达情况,结果：深低温保存的同种异体骨移植愈合BMP表达与体骨相似,移植骨段BMP表达阴性,新生骨及其周围类基质表达阳性。新生编织骨BMP表达活跃,成熟板层骨BMP表达减弱（P＜0．01）。结论：异体骨移植愈合中骨吸收和骨诱导是同时,骨诱导在异体骨愈合早期发挥了重要作用。     

复合骨形态发生蛋白胎儿骨修复长骨缺损的实验研究

寻找满意的骨形态发生蛋白（ＢＭＰ）的载体一直是应用ＢＭＰ修复骨缺损研究中的一个重要课题，为探讨胎儿骨（ＦＢ）能否成为ＢＭＰ载体，制备了复合ＢＭＰ胎儿骨（ＢＭＰ／ＦＢ），将其植入兔桡骨中段１５ｍｍ人工缺损处，以单纯ＦＢ植入、异体骨（ＡＬＢ）植入、空白作为对照，通过Ｘ线摄片，光镜观察，电子探针钙磷元素测定等方法了解各组骨缺损修复速度和愈合程度。结果发现ＢＭＰ／ＦＢ植入侧４周时骨缺损处有大量间充质细胞聚集，８周形成骨小梁，ＦＢ基本被吸收，１２周出现部分板层骨，钙磷值达到正常桡骨皮质骨水平，１６周骨细胞成熟，髓腔再通，外观塑形较好，在不同时间点骨缺损修复程度，ＢＭＰ／ＦＢ组明显优于ＦＢ组，ＦＢ组优于ＡＬＢ组，空白组骨缺损则被纤维组织，脂肪组织等填充。实验证明ＦＢ是一种良好的ＢＭＰ载体。     

Enhanced Osteoclastogenesis Causes Osteopenia in Twisted Gastrulation‐Deficient Mice Through Increased BMP Signaling

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1‐deficient (Twsg1^?/?) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1^?/? mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1^?/? mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL‐mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1^?/? mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1^?/? mice was reversed in vitro in a dose‐dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.     

骨形态发生蛋白复合同种松质骨载体修复节段性骨缺损的实验研究

目的　本实验将探讨同种松质骨作为 BMP的载体修复节段性骨缺损。方法　将 rh BMP- 2复合同种松质骨载体 (其中含 rh BMP- 2 0 .4 mg)植入兔桡骨 15 m m人工缺损处 ,以新鲜自体松质骨植入 ,单纯同种松质骨植入作为对照 ,通过放射学骨缺损修复 L ane评分 ,Nilsson骨愈合组织学评分 ,扫描电镜观察 ,比较术后 4、8、12周各组修复骨缺损的效果。结果　 rh BMP- 2复合同种骨载体组术后 4、8、12周各项评分与同期自体骨组之间无显著性差异 (P>0 .0 5 ) ,明显优于单纯同种骨组 (P<0 .0 5 )。结论　 rh BMP- 2具有高效的骨诱导能力。同种骨是一种较理想的 BMP载体 ,rh BMP- 2复合同种松质骨载体的骨修复效果与自体骨基本一致     

CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling

We identified the neuroprotein collapsing response mediator protein‐4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1–CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain‐of‐function and loss‐of‐function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4‐deficient mice (Crmp4^–/–) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild‐type controls. Increased bone mass in Crmp4^–/– mice was associated with enhanced BMP2 signaling and BMP2‐induced osteoblast differentiation in Crmp4^–/– osteoblasts (OBs). Furthermore, Crmp4^–/– OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4^–/– OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin‐dependent kinase inhibitor 1B, p27^Kip1 and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.     

Bone morphogenetic proteins in bone tumors

Bone morphogenetic proteins (BMPs), inducers of ectopic bone formation in vivo, are present in a number of osteosarcomas. BMPs are responsible for reactive bone formation, including periosteal reactions by normal osteoblasts, rather than production of tumorous osteoid by tumor cells. Osteosarcomas producing BMPs contain less-differentiated mesenchymal cells, resulting in a poorer prognosis for those patients. BMPs are also expressed in malignant fibrous histiocytomas (MFHs) of bone and dedifferentiated chondrosarcomas exhibiting undifferentiated features. However, BMPs in MFH do not show any osteoinductive activity in vivo, suggesting that those BMPs may be inactive forms and have additional functions unrelated to bone formation. Among benign bone tumors, BMPs are expressed in osteoid osteomas or osteoblastomas and effect reactive bone formation such as a surrounding sclerosis. BMPs and a BMP receptor (BMPRIB) are also detected in the cartilage cap in osteochondroma, suggesting that BMP signaling via BMPRIB might be involved in the pathogenesis of osteochondroma. Clinically, BMPs have utility as diagnostic and prognostic markers for characterizing the stage of differentiation of mesenchymal cells and mesenchymal tumors, and they may be of value in predicting the prognosis of sarcoma patients. This article reviews the accumulated information on BMPs in bone tumors, including the most recent findings, and discusses the biological and clinical significance of BMPs in bone tumors.Presented at the 36th Annual Musculoskeletal Tumor Meeting of the Japanese Orthopaedic Association, Kobe, Japan, July 2003     

骨形态发生蛋白4（BMP4）基因表达在骨折愈合过程中的定位研究

创伤后，骨组织的再生修复十分完全，其修复常无瘢痕残留。骨组织之所以有如此完美的愈合能力，是因为骨骼组织内存在有成骨因子，即骨生长因子。骨形态发生蛋白（ｂｏｎｅｍｏｒｐｈｏｇｅｎｅｔｉｃｐｒｏｔｅｉｎ，ＢＭＰ）因其直接诱导软组织成骨而受重视。本文应用ＢＭＰ４ｃＤＮＡ探针检测骨折愈合过程中外骨痂内ＢＭＰ４基因表达的定位与分布，探讨ＢＭＰ４基因表达在闭合性骨折愈合外骨痂形成中的作用。用６４只健康ＳＤ大鼠制备闭合性胫骨骨折动物模型。分别于骨折后１２小时、１、３、５、７、９、１４及２８天取材。取材后行恒冷切片，用地高辛素标记的ＢＭＰ４ｃＤＮＡ探针进行原位核酸分子杂交。大鼠骨折后１２小时～３天，骨折周围血肿内细胞及肌肉中新出现的间充质细胞内ＢＭＰ４ｍＲＮＡ表达检测为阳性信号。表明创伤激活ＢＭＰ４ｍＲＮＡ的表达，并呈区域性参与骨折的修复，也说明骨折血肿及周围软组织在骨折愈合过程中具有非常重要的地位。     

Expression of Noggin and Gremlin1 and its implications in fine‐tuning BMP activities in mouse cartilage tissues

Increasing evidence supports the idea that bone morphogenetic proteins (BMPs) regulate cartilage maintenance in the adult skeleton. The aim of this study is to obtain insight into the regulation of BMP activities in the adult skeletal system. We analyzed expression of Noggin and Gremlin1 , BMP antagonists that are known to regulate embryonic skeletal development, in the adult skeletal system by Noggin‐LacZ and Gremlin1‐LacZ knockin reporter mouse lines. Both reporters are expressed in the adult skeleton in a largely overlapping manner with some distinct patterns. Both are detected in the articular cartilage, pubic symphysis, facet joint in the vertebrae, and intervertebral disk, suggesting that they regulate BMP activities in these tissues. In a surgically induced knee osteoarthritis model in mice, expression of Noggin mRNA was lost from the articular cartilage, which correlated with loss of BMP2/4 and pSMAD1/5/8, an indicator of active BMP signaling. Both reporters are also expressed in the sterna and rib cartilage, suggesting an extensive role of BMP antagonism in adult cartilage tissue. Moreover, Noggin‐LacZ was detected in sutures in the skull and broadly in the nasal cartilage, while Gremlin1‐LacZ exhibits a weaker and more restricted expression domain in the nasal cartilage. These results suggest broad regulation of BMP activities by Noggin and Gremlin1 in cartilage tissues in the adult skeleton, and that BMP signaling and its antagonism by NOGGIN play a role in osteoarthritis development. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1671–1682, 2017.

     

Aminobisphosphonates Cause Osteoblast Apoptosis and Inhibit Bone Nodule Formation In Vitro

Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20-100 microM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ss-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.