Effect of rhBMP‐2 on tibial plateau fractures in a canine model

This study was to determine the efficacy of recombinant human bone morphogenetic protien‐2 (rhBMP‐2)/calcium phosphate matrix (CPX) paste to accelerate healing in a canine articular fracture model with associated subchondral defect. rhBMP‐2/CPX (BMP), CPX alone (CPX) or autogenous bone graft (ABG) was administered to a canine articular tibial plateau osteotomy with a subchondral defect in each of 21 female dogs. The unoperated contralateral limbs served as controls. Ground reaction forces, synovial fluid, radiographic changes, mechanical testing, bone density, and histology of bone and synovium were analyzed at 6 weeks after surgery. Radiographic analysis demonstrated that the BMP and CPX groups showed improved bony healing compared to the ABG group at week 6. Histomorphometric analysis demonstrated that the BMP group had significantly increased trabecular bone volume compared to the CPX and ABG groups. Mechanical testing revealed that the BMP group had significantly greater maximum failure loads than the ABG group. Histological analysis demonstrated that the BMP group had significantly less sub‐synovial inflammation than CPX group. This study demonstrated that rhBMP‐2/CPX accelerated healing of articular fractures with subchondral defect compared to ABG in most of the parameters evaluated, and had less subsynovial inflammation than the CPX alone in a canine model. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 466–471, 2009     

Enhancement of posterolateral lumbar spine fusion using low‐dose rhBMP‐2 and cultured marrow stromal cells

We tested the hypothesis that the dose of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) required to induce spine fusion can be reduced by combination with mesenchymal stem cells (MSCs). Twenty‐four adult rabbits underwent posterolateral intertransverse fusion at the L4–L5 level. The animals were divided into four groups based on the implant material: autologous iliac graft, Alginate‐MSCs composite, Alginate‐BMP‐2‐MSCs composite, and Alginate‐BMP‐2 composite. After 16 weeks, the rabbits were euthanized for radiographic examination, manual palpation, biomechanical testing, and histology. Radiographic union of 12 intertransverse fusion areas for the autogenous iliac graft, Alginate‐MSCs, Alginate‐BMP‐2‐MSCs, and Alginate‐BMP‐2 groups was 11, 8, 11, and 0, respectively. Moreover, manual palpation of six fusion segments in each subgroup found solid union to be 6, 1, 5, and 0, respectively. The average torques at failure of the first three groups were 2278 ± 135, 1943 ± 140, and 2334 ± 187 N‐mm, respectively. The failure torque did not differ significantly between the autograft and Alginate‐BMP‐2‐MSCs groups; both groups were significantly higher than the Alginate‐MSCs group. The results indicate that MSCs delivered with in vitro cellular doses of rhBMP‐2 are more osteoinductive than MSCs without rhBMP‐2. In combination with MSCs, a low dose (2.5 µg) of rh‐BMP‐2 could enhance bone formation and posterolateral spine fusion success in the rabbit model. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:380–384, 2009     

Recombinant human bone morphogenetic protein‐type 2 (rhBMP‐2) enhances local bone formation in the lumbar spine of osteoporotic sheep

The failure of orthopedic implants in osteoporotic patients is attributed to the lack of sufficient bone stock and regenerative capacity but most treatments for osteoporosis fail to address this issue. rhBMP‐2 is known to promote bone formation under normal conditions but has not been used clinically in the osteoporotic condition. Osteoporosis was induced in 19 ewes using ovariectomy, low calcium diet, and steroid injection. After induction, the steroid was withdrawn and pellets containing inert carrier with rhBMP‐2 in either slow or fast‐release formulation were implanted into the lumbar vertebrae of each animal. After 2, 3, and 6 months the spines were harvested and assessed for changes in BMD and histomorphometric indices. BMD did not change after cessation of steroid treatment. After 2 months BV/TV increased in the vicinity of the pellets containing the fast‐release rhBMP‐2 and was sustained for the duration of the study. Focal voids surrounding all implants, particularly the slow‐release formulation, were observed initially but resolved with time. Increased BV/TV adjacent to rhBMP‐2 pellets suggests it could be used for localized treatment of osteoporosis. Refinement of the delivery system and supplementary treatments may be necessary to overcome the initial catabolic effects of rhBMP‐2. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1390–1397, 2013.     

Enhanced healing of goat femur‐defect using BMP7 gene‐modified BMSCs and load‐bearing tissue‐engineered bone

Segmental defect regeneration is still a clinical challenge. In this study, we investigated the feasibility of bone marrow stromal cells (BMSCs) infected with adenoviral vector containing the bone morphogenetic protein 7 gene (AdBMP7) and load‐bearing to enhance bone regeneration in a critically sized femoral defect in the goat model. The defects were implanted with AdBMP7‐infected BMSCs/coral (BMP7 group) or noninfected BMSCs/coral (control group), respectively, stabilized with an internal fixation rod and interlocking nails. Bridging of the segmental defects was evaluated by radiographs monthly, and confirmed by biomechanical tests. Much callus was found in the BMP7 group, and nails were taken off after 3 months of implantation, indicating that regenerated bone in the defect can be remodeled by load‐bearing, whereas after 6 months in control group. After load‐bearing, it is about 5 months; the mechanical property of newly formed bone in the BMP7 group was restored, but 8 months in control group. Our data suggested that the BMP7 gene‐modified BMSCs and load‐bearing can promote bone regeneration in segmental defects. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:412–418, 2010     

ACL reconstruction using bone‐tendon‐bone graft engineered from the semitendinosus tendon by injection of recombinant BMP‐2 in a rabbit model

We attempted to generate a bone‐tendon‐bone structure by injecting human‐type recombinant human bone morphogenetic protein‐2 (rhBMP‐2) into the semitendinosus tendon, and an anterior cruciate ligament (ACL) defect was reconstructed by grafting the engineered bone‐tendon‐bone graft. Two ossicles with a separation distance of 1 cm were generated within the left semitendinosus tendon of a rabbit 6 weeks after the injection of rhBMP‐2 (15 µg at each site). The engineered bone‐tendon‐bone graft was transplanted in order to reconstruct the ACL by passing the graft through the bone tunnels. In the control group, the ACL was reconstructed with the semitendinosus tendon without BMP‐2 using the same methods as those used in the experimental group. The animals were harvested at 4 or 8 weeks after surgery and examined by radiographic, histological, and biomechanical methods. In the experimental group, ossicles in the bone‐tendon‐bone graft were successfully integrated into the host bone of the femur and tibia. Histological analysis revealed that characteristic features identical to the normal direct insertion morphology had been restored. Biomechanical pull‐out testing showed that the ultimate failure load and stiffness of the reconstructed ACL in the experimental group were significantly higher than those in the control group at both 4 and 8 weeks (p < 0.05). These results indicate the potential of regenerative reconstruction of the ACL, and the reconstruction resulted in the restoration of morphology and function equivalent to those of the normal ACL. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1923–1930, 2011     

Enhancement of recombinant human BMP‐7 bone formation with bmp binding peptide in a rodent femoral defect model

Bone morphogenetic binding peptide (BBP) is an 18.5 kDa fragment of a bone matrix protein peptide. A rat femoral defect model was used to test the effect of BBP combined with recombinant human bone morphogenetic protein‐7 (rhBMP‐7) to induced bone healing. Two doses of BBP (500 and 1000 µg) were tested with two doses of rhBMP‐7 (2 and 5 µg), and the results were compared with a positive control (10 µg rhBMP‐7). Bone healing was evaluated by radiology, manual palpation, microcomputed tomography, and histology. The high dose of 10 µg of rhBMP‐7 resulted in a consistent 100% bone union rate and a mature histological appearance on histology, and was used as a positive control. When 1000 µg of BBP was combined with lower doses of BMP‐7 (2 µg rhBMP‐7 or 5 µg rhBMP‐7) significant differences were seen in radiographic scores, manual palpation, and bone volume, when compared to 2 µg rhBMP‐7 or 5 µg rhBMP‐7 alone. The combination of 1000 µg of BBP and 5 µg rhBMP‐7 also achieved 100% fusion rate, induced a larger amount of bone formation, and yielded similar maturity of bone marrow when compared with the high dosage 10 µg rhBMP‐7 group. This study demonstrated that when combined together, BBP can enhance the bone healing of rhBMP‐7. Improved healing imparted by the addition of BBP may result in lesser amounts of rhBMP‐7 needed to achieve union in the clinical setting. © 2010 OrthopaedicResearchSociety.PublishedbyWileyPeriodicals, Inc.JOrthopRes29:753–759,2011     

Repair of rat cranial bone defects with nHAC/PLLA and BMP‐2‐related peptide or rhBMP‐2

An ideal artificial substitute has good biocompatibility properties and is able to provide for rapid bone formation. Bone morphogenetic protein‐2 (BMP‐2) is considered as one of the most important growth factors for bone regeneration. In this study, a synthetic BMP‐2‐related peptide (designated P24) corresponding to residues of the knuckle epitope of BMP‐2 was introduced into a bioactive scaffold based on nano‐hydroxyapatite/collagen/poly(L ‐lactic acid) (nHAC/PLLA); its in vitro release kinetics was then measured. A 5 mm diameter cranial bone defect was created in the calvariae of 30 rats and randomly implanted with three groups of biomaterials: Group A (nHAC/PLLA alone); Group B (P24/nHAC/PLLA composite); and Group C (recombinant human BMP‐2 (rhBMP‐2)/nHAC/PLLA composite). The P24/nHAC/PLLA implants significantly stimulated bone growth similarly to the rhBMP‐2/nHAC/PLLA implants based on the radiographic and three‐dimensional CT evaluation and histological examination, thereby confirming the enhanced bone healing rate of these compounds compared with the stand‐alone nHAC/PLLA scaffold material. The osteoinductive ability of 3 mg P24 was similar to that of 1 µg rhBMP‐2. P24/nHAC/PLLA is a promising scaffold biomaterial for bone tissue regeneration. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1745–1752, 2011     

Hexa‐D‐arginine treatment increases 7B2•PC2 activity in hyp‐mouse osteoblasts and rescues the HYP phenotype

Inactivating mutations of the “phosphate regulating gene with homologies to endopeptidases on the X chromosome” (PHEX/Phex) underlie disease in patients with X‐linked hypophosphatemia (XLH) and the hyp‐mouse, a murine homologue of the human disorder. Although increased serum fibroblast growth factor 23 (FGF‐23) underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF‐23 degradation and/or enhance production remains unknown. Here we show that treatment of wild‐type mice with the proprotein convertase (PC) inhibitor, decanoyl‐Arg‐Val‐Lys‐Arg‐chloromethyl ketone (Dec), increases serum FGF‐23 and produces the HYP phenotype. Because PC2 is uniquely colocalized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX‐dependent FGF‐23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF‐23, whereas Signe1 (7B2) RNA interference (RNAi) transfection, which limited 7B2 protein production, decreased FGF‐23 degradation and increased Fgf‐23 mRNA and protein. The mechanism by which decreased 7B2?PC2 activity influences Fgf‐23 mRNA was linked to reduced conversion of the precursor to bone morphogenetic protein 1 (proBMP1) to active BMP1, which resulted in limited cleavage of dentin matrix acidic phosphoprotein 1 (DMP1), and consequent increased Fgf‐23 mRNA. The significance of decreased 7B2?PC2 activity in XLH was confirmed by studies of hyp‐mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF‐23 cleavage and increased FGF‐23 synthesis, secondary to decreased BMP1‐mediated degradation of DMP1. Subsequent Hexa‐D‐Arginine treatment of hyp‐mice enhanced bone 7B2?PC2 activity, normalized FGF‐23 degradation and production, and rescued the HYP phenotype. These data suggest that decreased PHEX‐dependent 7B2?PC2 activity is central to the pathogenesis of XLH. © 2013 American Society for Bone and Mineral Research     

Open tibial fractures grade IIIC treated successfully with external fixation,negative‐pressure wound therapy and recombinant human bone morphogenetic protein 7

The aim of the therapy in open tibial fractures grade III was to cover the bone with soft tissue and achieve healed fracture without persistent infection. Open tibial fractures grade IIIC with massive soft tissue damage require combined orthopaedic, vascular and plastic–reconstructive procedures. Negative‐pressure wound therapy (NPWT), used in two consecutive cases with open fracture grade IIIC of the tibia diaphysis, healed extensive soft tissue defect with exposure of the bone. NPWT eventually allowed for wound closure by split skin graft within 21–25 days. Ilizarov external fixator combined with application of recombinant human bone morphogenetic protein‐7 at the site of delayed union enhanced definitive bone healing within 16–18 months.     

Local delivery of mutant CCL2 protein‐reduced orthopaedic implant wear particle‐induced osteolysis and inflammation in vivo

Total joint replacement (TJR) has been widely used as a standard treatment for late‐stage arthritis. One challenge for long‐term efficacy of TJR is the generation of ultra‐high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade which may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here, we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle‐induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle‐induced osteolysis and the effects of 7ND treatment were evaluated using micro‐CT, histology, and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle‐induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle‐induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:58–64, 2016.     

Regulation of BMP2‐induced intracellular calcium increases in osteoblasts

Although bone morphogenetic protein‐2 (BMP2) is a well‐characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca²⁺ signaling. In this study, intracellular Ca²⁺ concentration ([Ca²⁺]_i) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3‐E1 osteoblast cell line using fluorescence resonance energy transfer‐based biosensors. Increase in [Ca²⁺]_i, FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF‐573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2‐stimulated [Ca²⁺]_i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2‐induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca²⁺]_i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725–1733, 2016.     

Chd4 and associated proteins function as corepressors of Sox9 expression during BMP‐2–induced chondrogenesis

Mouse embryonic fibroblasts (MEFs) differentiate into fully functional chondrocytes in response to bone morphogenetic protein‐2 (BMP‐2). However, the comprehensive proteomic aspect of BMP‐2–induced chondrogenesis remains unknown. We took advantage of quantitative proteomic analysis based on isobaric tag for relative and absolute quantitation (iTRAQ) and on‐line 2D nano‐liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify proteins differentially expressed during BMP‐2–induced chondrogenic differentiation of MEFs. We found 85 downregulated proteins, and ingenuity pathways analysis (IPA) revealed a protein‐protein network with chromodomain‐helicase‐DNA‐binding protein 4 (Chd4) in the center. Chromatin immunoprecipitation (ChIP) and nuclease hypersensitivity assays showed that Chd4, interacting with Hdac1/2, cooperates with its related proteins Kap1 and Cbx1 to bind at ?207/?148 of the Sox9 promoter. We also provided evidence that let‐7a targets the 3'UTR of Chd4 to promote chondrogenesis of MEFs. Together, our findings indicate that BMP‐2 induced the upregulation of let‐7a, targeting Chd4 and positively controlling the chondrogenic differentiation of MEFs. These findings illustrate epigenetic regulation of the chondrogenic differentiation process and also expand the understanding of the involved intracellular mechanisms.     

糖尿病肾病患者血清BMP2、BMP7水平与钙磷代谢的关系

目的探讨糖尿病肾病患者血清骨形态蛋白-2(BMP2)、骨形态蛋白-7(BMP7)水平与钙磷代谢的关系。方法选取2016年11月至2018年11月西电集团医院收治的126例糖尿病肾病患者作为研究组,采用系统性回顾性分析法分析研究组临床资料,根据不同分期阶段慢性肾脏病(CKD)将患者分为5组,分别为CKD 1组22例、CKD 2组21例、CKD 3组27例、CKD 4组31例、CKD 5组25例,另选取36例同期于西电集团医院门诊体检的健康人群作为对照组,测定患者血清BMP2、BMP7、血磷、血钙、肌酐(Scr)、甲状旁腺激素(iPTH),分析血清BMP2、BMP7与钙磷代谢的关系。结果①对照组与CKD 1~2期在血磷、血钙、Scr、iPTH、BMP2、BMP7水平之间差异无明显统计学意义(P>0.05);与对照组比较,CKD 4期以后血钙及血清BMP7水平明显降低(P<0.05);CKD 3期以后血清Scr、iPTH、BMP2水平明显开始升高、血清BMP7水平明显开始降低(P<0.05);CKD 4期以后血磷、Scr、iPTH、BMP2水平明显升高,血钙、BMP7水平明显降低;CKD 5期的变化情况同CKD 4期;②经Pearson积差相关分析,血清BMP2与血磷、Scr及iPTH呈正相关,与血钙呈负相关(P<0.05);BMP7与血钙呈正相关,与血磷、Scr及iPTH呈负相关(P<0.05);③经多因素多元逐步回归分析,血磷、血钙、Scr及iPTH均为影响糖尿病肾病患者血清BMP2、BMP7水平的相关因素(P<0.05)。结论CKD 3期以后血清BMP2开始升高、BMP7开始降低,且血清BMP2与血钙呈明显负相关,BMP7与血磷、Scr及iPTH呈明显负相关;钙磷代谢情况可能是糖尿病肾病患者血清BMP2升高、BMP7降低的重要相关因素。     

NUCB2/nesfatin‐1: A new adipokine expressed in human and murine chondrocytes with pro‐inflammatory properties,an in vitro study

Nesfatin‐1 is a recently discovered satiety‐inducing adipokine identified in hypothalamic regions that regulates energy balance. So far, no data exist on NUCB2/nesfatin‐1 localization in human and murine chondrocytes. Here, we therefore investigated NUCB2/nesfatin‐1 gene and protein expression in human and murine chondrocytes and the effect of nesfatin‐1 on pro‐inflammatory cytokines expression. Peptide localization was performed by laser confocal microscopy, NUCB2 mRNA expression was studied by RT‐PCR and protein secretion was measured by XMap technology and Western blot analysis. First, we demonstrated cytoplasmic localization of NUCB2/nesfatin‐1 peptide in both human and murine chondrocytes. We present evidence that both mRNA and protein expression of NUCB2 were increased during the differentiation of ATDC5 murine chondrocyte cell line. Furthermore, we demonstrated that nesfatin‐1 induces IL‐6 and MIP‐1α mRNA expression and protein secretion in ATDC‐5 cells challenged with IL‐1, and also increases COX‐2 mRNA expression in these cells. Finally, nesfatin‐1 provoked a clear induction of pro‐inflammatory agents, such as COX‐2, IL‐8, IL‐6, and MIP‐1α in human primary chondrocytes from OA patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:653–660, 2014.