Global gene profiling reveals a downregulation of BMP gene expression in experimental atrophic nonunions compared to standard healing fractures.

Nonunion is a challenging problem that may occur following certain bone fractures. However, there has been little investigation of the molecular basis of nonunions. Bone morphogenetic proteins (BMPs) play a significant role in osteogenesis. However, little is known about the expression patterns of BMPs in abnormal bone healing that results in nonunion formation. These facts prompted us to investigate and compare the gene expression patterns of BMPs and their antagonists in standard healing fractures and nonunions using rat experimental models. Standard closed healing fractures and experimental atrophic nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, 10, 14, 21, and 28, total RNA was extracted from the callus of standard healing fracture and fibrous tissue of nonunion (n=4 per each time point and each group). Gene expression of BMPs, BMP antagonists, and other regulatory molecules were studied by methods including Genechip microarray and real-time quantitative RT-PCR. Gene expression of BMP-2, 3, 3B, 4, 6, 7, GDF-5, 7, and BMP antagonists noggin, drm, screlostin, and BAMBI were significantly lower in nonunions compared to standard healing fractures at several time points. Downregulation in expression of osteogenic BMPs may account for the nonunions of fracture. The balance between BMPs and their endogenous antagonists is critical for optimal fracture healing.     

Altered relative expression of BMPs and BMP inhibitors in cartilaginous areas of human fractures progressing towards nonunion

The present study was conducted to evaluate the hypothesis that an imbalance in the local production of bone morphogenetic proteins (BMPs) and BMP inhibitors exists within the cartilaginous intermediate of nonhealing fractures. Biopsies were recovered intraoperatively from human fractures that, upon follow-up, were found to heal normally or become nonunions. The samples were examined by immunohistochemistry to determine the expression of BMP-2, BMP-14, and the BMP inhibitors noggin and chordin. Expression was determined semiquantitatively based on the area of positive staining per area of cartilage and by determining the number of positively staining cells and the intensity of staining. There was a significant reduction in BMP-2 and BMP-14 expression in cartilaginous areas of nonhealing fractures compared to healing fractures. However, there was no difference in the expression of the BMP inhibitors between the two groups of fractures. This imbalance in the expression of BMPs and BMP inhibitors within cartilaginous areas of developing nonunions may account for their reduced bone forming ability. These data suggest strategies for preventing the development of nonunions by altering levels of BMPs and their inhibitors within fracture sites. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 752–757, 2009     

Expression and activation of the BMP-signaling components in human fracture nonunions

BACKGROUND: The potential use of bone morphogenetic proteins (BMPs) to promote bone-healing is of great interest to orthopaedic surgeons. Although the complex mechanism leading from the local presence of BMP (whether endogenous or exogenous) to bone formation is increasingly understood, limited information is available as to whether endogenous BMPs, their receptors, or other molecules involved in their signal transduction, such as Smad1, are present or disappear during the development of fracture nonunions. The purpose of the present study was to determine, by immunohistochemical analysis, whether BMPs, BMP receptors, or Smad1 disappear from tissues during the development of a fracture nonunion. METHODS: Twenty-one patients (average age, sixty-one years; range, thirty to eighty-five years) with a delayed union (four patients) or a nonunion (seventeen patients) were included. The average duration of the delayed union or nonunion was twenty-two months (range, 3.5 to 120 months). With use of immunohistochemical analysis, we studied the localization of BMP-2, BMP-4, and BMP-7 and their receptors BMPR-IA, BMPR-IB, and BMPR-II as well as pSmad1. With use of a pSmad1 antibody, we also studied whether the BMP receptors that were expressed were activated. RESULTS: The immunohistochemical localization of all seven BMP-signaling components was demonstrated in seventeen (81%) of the twenty-one patients. The remaining four patients lacked one or more of the components. Areas of newly formed bone had the highest percentage of positively staining cells, with the staining generally decreasing in areas remote from bone formation. However, even in areas of dense fibrous tissue and in specimens that lacked newly formed bone, immunostaining was still present. The staining patterns showed co-localization of the BMP-2, BMP-4, and BMP-7 proteins with the BMP receptors. The presence of pSmad1 signified the activated state of the BMP receptors, which implies that the BMP signal is transduced inside the cell.     

Coordinated regulation of mesenchymal stem cell differentiation on microstructured titanium surfaces by endogenous bone morphogenetic proteins

Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra < 0.4 μm); sandblasted/acid-etched Ti (SLA, Ra = 3.2 μm); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6 days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment.     

BMP signaling components are expressed in human fracture callus

Of the various growth factors involved in the healing response after a fracture, bone morphogenetic proteins (BMPs) are emerging as key modulators. BMPs exert their effects by binding to a complex of type I and type II receptors leading to the phosphorylation of specific downstream effector proteins called Smads. The current study examined the presence of BMP signaling components in human callus obtained from five nascent malunions undergoing fracture fixation. These callus samples represented various stages of bone healing and a mixture of endochondral and intramembraneous bone healing. We performed immunohistochemistry on the callus, using antibodies for BMP (BMP-2,-3,-4,-7), their receptors (BMPR-IA, -IB, -II), and phosphorylated BMP receptor-regulated Smads (pBMP-R-Smads). Active osteoblasts showed fairly consistent positive staining for all BMPs that were examined, with the immunoreactivity most intense for BMP-7 and BMP-3. Immunostaining for BMPs in osteoblasts appeared to colocalize with the expression of BMPR-IA, -IB, and -II. Positive immunostaining for pBMP-R-Smads suggests that the BMP receptors expressed in these cells are activated. Staining for BMPs in cartilage cells was variable. The immunostaining appeared stronger in more mature cells, whereas staining for BMP receptors in cartilage cells was less ubiquitous. However, the expression of pBMP-R-Smads in cartilage cells suggests active signal transduction. Fibroblast-like cells also had a variable staining pattern. Overall, our findings indicate the presence of BMPs, their various receptors, and activated forms of receptor-regulated Smads in human fracture callus. To the best of our knowledge, this is the first study that documents the expression of these proteins in human fracture tissue. Complete elucidation of the roles of BMP in bone formation will hopefully lead to improved fracture healing care.     

Circulating bone morphogenetic protein levels and delayed fracture healing

Objective

Despite adequate treatment 5–30 % of bone fracture patients experience delayed union. During normal fracture union, bone morphogenetic proteins (BMPs) induce healing through a sequential cascade of events. Improved fracture healing after BMP-2 or -7 supplementation in patients with impaired fracture union suggests a deficiency of one or more of these factors. We postulated that low levels of circulating BMPs may result in delayed bone healing. The aim of this study was to quantify differences in levels of circulating BMP-2, -4, -6, -7, and −9 in patients that have demonstrated normal or delayed fracture healing.

Patients and methods

Blood samples were collected from an unselected cohort of 65 patients that had been treated for a diaphyseal tibia or femur fracture. Patients were divided into a group with fracture healing within nine months after injury and a group with delayed fracture union. BMP plasma concentrations were quantified using ELISAs and compared between these two groups.

Results

Circulating plasma levels of BMP-2, -4, -6, and -7 did not differ between 34 patients with normal fracture healing and 31 patients with delayed fracture healing. Also the median BMP-9 plasma levels were not statistically different between the two groups of patients. However, the distribution in the patients with normal union showed a wider range (72–2496 pg/ml) compared with the delayed union group (120–816 pg/ml).

Conclusion

In general, circulating BMP concentrations are not statistically different between patients who demonstrated normal or delayed fracture healing. High circulating BMP-9 levels seem to be associated with faster fracture healing, but are apparently not decisive.     

Mechanical Load and BMP Signaling During Tendon Repair: A Role for Follistatin?

Healing of the rat Achilles tendon is sensitive to mechanical loading, and the callus strength is reduced by 3/4 after 14 days, if loading is prevented. Exogenous GDFs stimulate tendon healing. This response is influenced by loading: without loading, cartilage and bone formation is initiated. This implies BMP signaling is crucial during tendon healing and influenced by mechanical loading. We therefore asked if mechanical loading influences the gene expression of the BMP signaling system in intact and healing tendons, and how the BMP signaling system changes during healing. The genes were four BMPs (OP-1/BMP-7, GDF-5/CDMP-1/BMP-14, GDF-6/CDMP-2/BMP-13, and GDF-7/CDMP-3/BMP-12), two receptors (BMPR1b and BMPR2), and the antagonists follistatin and noggin. The Achilles tendon was transected in rats and left to heal. Half of the rats had one Achilles tendon unloaded by injection of Botox in the calf muscles. Ten tendons were analyzed before transection and for each of four time points. All genes except noggin were expressed at all time points, but followed different patterns during healing. Loading strongly decreased the expression of follistatin, which could lead to increased signaling. The BMP system appears involved in tendon maintenance and healing, and may respond to mechanical loading.     

Distinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.     

Expression of bone morphogenetic proteins and rat distal-less homolog genes following rat femoral fracture

Expression of the genes encoding bone morphogenetic proteins (BMPs), BMP type IA receptor (BMPR-1A), and rat distal-less homolog (rDlx) was studied in bone, callus, and the surrounding soft tissue following rat femoral closed fracture, using RT-PCR-based techniques. Before fracture, the genes encoding BMP-5, BMP-6, and BMPR-1A were found to be expressed in both bone and the surrounding soft tissue, whereas the BMP-2 gene was expressed only in bone and BMP-7 was not expressed in either tissue. Expression of these genes was unaffected by fracture. The gene encoding BMP-4 was also expressed in both bone and the surrounding soft tissue before fracture. Moreover, although unchanged in bone, 6 h after fracture BMP-4 expression was increased tenfold in the surrounding soft tissue. The increased BMP-4 expression was transient and returned to prefracture levels within 72 h. Expression of rDlx was also increased in bone after fracture, but at later times than were observed with BMP-4: elevated rDlx expression was detected after 48 h and persisted for 30 days or more. No expression of rDlx was observed in the surrounding soft tissue before or after fracture. These findings indicate that BMP-4 and rDlx are selectively expressed following femoral fracture in the rat, and also suggest that they are involved in the formation of the callus at an early point during the postfracture healing of bone. Received: March 26, 1999 / Accepted: Sept. 20, 1999     

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.

     

Expression of bone morphogenetic proteins, receptors, and tissue inhibitors in human fetal, adult, and osteoarthritic articular cartilage.

Coordinate expression of BMPs and their receptors and inhibitors is likely necessary for physiologic BMP regulation and activity. To characterize the expression of such factors in fetal, normal adult, and end-stage osteoarthritic articular cartilage, samples from these sources were analyzed. PCR-amplified sequences (BMPs 1-11), receptors (IA, IB, II), TGF-beta1, TGF-beta2, inhibitors noggin and follistatin, CDMP-1, COMP, and GAPDH from cDNAs generated from extracted total RNA were resolved by gel electrophoresis. Protein levels of BMPs 3, 7, and 8 were also analyzed by SDS-PAGE and Western blotting. RT-PCR revealed that BMPs 1, 2, 4-6, and 11, BMPR-IA and II, noggin, follistatin, CDMP-1, COMP, and GAPDH mRNAs were expressed in similar fashion in both fetal and adult (normal or osteoarthritic) cartilage. BMPs 9 and 10 mRNAs were not expressed in either group. BMPs 7, 8, and BMPR-IB mRNAs were consistently expressed in fetal but not in adult cartilage. BMP-3 mRNA was expressed in fetal and normal adult, but not in osteoarthritic samples. TGF-beta1 was expressed in both adult normal and osteoarthritic, but not fetal, samples. Similarly, Western blotting demonstrated BMPs 7 and 8 to be present in fetal but not in adult samples. BMP-3 protein was present in fetal and adult normal samples, to a lesser extent, but absent in osteoarthritic cartilage.     

Expression profiles of BMP-related molecules induced by BMP-2 or -4 in muscle-derived primary culture cells

The formation of ectopic bone in muscle following the implantation of decalcified bone matrix led to the search and eventual discovery of bone morphogenetic proteins (BMPs) in bone matrix. The precise sequence of molecular events that underpin the cellular transformation of undifferentiated mesenchymal cells into bone has not been established, and is the subject of this study. Northern and Western blot analyses were used to examine changes in gene expression of cells treated with BMP-2 or -4. The molecules, which included BMP receptors (BMPRs), Noggin (a BMP-specific antagonist), osteocalcin (OC), Smad-4, and MyoD, were examined at messenger RNA (mRNA) and protein levels. The changes in expression of these molecules were followed in mouse muscle-derived primary culture cells, and osteoblastic or nonosteoblastic embryonic cell lines. We show the early up-regulation of BMPR-1A, -2, Noggin, OC, and Smad-4 in muscle-derived primary culture cells in a dose-dependent manner in response to BMP-2 or -4. MyoD expression was not detected after BMP stimulation. The differential expression of these positive and negative regulators of BMP signaling points to a potential regulatory mechanism for bone induction in mesenchymal cells.     

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.     

脊柱后外侧融合过程中BMP-2、BMP-4基因的动态表达

目的：研究脊柱后外侧融合过程中骨形态发生蛋白(BMP)的表达情况，探讨BMP对融合过程的作用。方法：36只成年雄性新西兰白兔，制作L4、L5双侧横突间自体骨植骨模型，按术后处死时间(0d、2d、4d、1周、2周、3周、4周、5周、6周、10周、6个月、10个月)随机平均分为12组。将融合组织平均分为3等份，与横突交界的上、下区域定为边缘区，中间区域定为中央区。以RT-PCR法检测不同时间段、不同融合区域BMP-2 mRNA、BMP-4 mRNA的表达水平。结果：在脊柱融合术后1～6周，BMP-2和BMP-4表达量明显增高，并各自出现相应的峰值。中央区BMP表达水平的增幅及峰值明显低于边缘区，其开始增高的时间及峰值出现的时间滞后于边缘区1～3周，表现出明显的时间和空间差别：结论：脊柱融合中央区BMP低表达及时间滞后可能是不融合发生的重要原因。在融合早期补充外源性BMP可能有助于提高融合率。     

Colocalization of noggin and bone morphogenetic protein-4 during fracture healing.

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to bone morphogenetic protein (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, 10, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.     

Co-expression of BMPs and BMP-inhibitors in human fractures and non-unions

Bone morphogenetic proteins (BMPs) are increasingly being used clinically to enhance fracture repair and healing of non-unions. However, the potential efficacy of supraphysiological dosing for clinical results warrants further clarification of the BMP signaling pathway in human fracture healing. As BMP signaling can be fine-tuned at numerous levels, the role of BMP-inhibitors has become a major focus. The aim of the present study was to document co-expression of BMPs, pSmad 1/5/8, and BMP-inhibitors in human fracture callus and human non-unions. Using human tissue of fracture callus (n=14) and non-unions (n=4) we documented expression of BMPs (BMP2, BMP3 and BMP7), pSmad 1/5/8 and the BMP-inhibitors noggin, gremlin, chordin, Smad-6, Smad-7 and BAMBI. Co-expression of pSmad 1/5/8, BMPs and BMP-inhibitors was noted in the osteoblasts of fracture callus as well as of non-unions. Expression of BMP-inhibitors was generally stronger in non-unions than in fracture callus. The most pertinent differences were noted in the cartilaginous tissue components. Expression of BMP2 in chondrocytes was markedly decreased in non-unions compared to fracture callus and that of BMP7 was almost completely absent. Expression of BMP-inhibitors was almost the same in osteoblasts, chondrocytes and fibroblasts of fracture callus and well as in non-unions. Interestingly, although BMP ligands were present in the chondrocytes and fibroblasts of non-unions, they did not co-express pSmad 1/5/8 suggesting that BMP signaling may have been inhibited at some point before Smad 1/5/8 phosphorylation. These results suggest co-expression of BMP, pSmad 1/5/8 and BMP-inhibitors occurs in human fracture callus as well as non-unions but the relative expression of BMPs vs. BMP-inhibitors was different between these two tissue types. In contrast to our expectations, the expression of BMP inhibitors was comparable between fracture callus and non-unions, whereas the expression of BMPs was notably lower in the cartilaginous component of the non-unions in comparison to fracture callus. Based on these results, we believe that aberrations in the BMP-signaling pathway in the cartilaginous component of fracture healing could influence clinical fracture healing. An imbalance between the local presence of BMP and BMP-inhibitors may switch the direction towards healing or non-healing of a fracture.     

基于BMP-2/SMAD/Runx2通路探究骨化三醇胶囊对大鼠胫骨骨折愈合的影响

目的 探讨骨化三醇胶囊(Cal)通过骨形态发生蛋白2(BMP-2)/SMAD/Runt相关转录因子2(Runx2)通路对大鼠胫骨骨折愈合的影响及相关机制.方法 将52只SD大鼠随机分为对照组(Cont)、胫骨骨折组、Cal组(灌胃10 ng/mL Cal,连续4周)、Cal+Noggin(BMP特异抑制剂)组(灌胃10...     

BMP2 Differentially Regulates the Expression of Gremlin1 and Gremlin2, the Negative Regulators of BMP Function, During Osteoblast Differentiation

Bone morphogenetic proteins (BMPs) control the expressions of many genes involved in bone formation. On the basis of our hypothesis that BMP2 stimulation-regulated gene expression plays a critical role in osteoblast differentiation, we performed genome-wide screening of messenger RNA from BMP2-treated and -untreated C2C12 cells using a DNA microarray technique. We found that the expressions of Gremlin1 and Gremlin2, which are known BMP antagonists, were bidirectionally regulated by BMP2. Gremlin1 was down-regulated by BMP2, while Gremlin2 was up-regulated in both time- and dose-dependent manners. Ablation of Gremlin1 or Gremlin2 enhanced osteoblast differentiation induced by BMP2. On the other hand, treatment with recombinant Gremlin1 inhibited BMP2-induced osteoblast differentiation. Furthermore, treatment with Smad4 siRNA and the p38 MAPK inhibitor SB203580 suppressed BMP2-induced Gremlin2 gene expression. The differential regulation of Gremlin1 and Gremlin2 gene expressions by BMP2 may explain the critical function of these genes during osteoblast differentiation.     

L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin

Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.