The Inhibiting Effect of Bone Protein Hydrolysates on Lipid Oxidation in Pork Patties

Bone protein hydrolysates were prepared by limited alcalase hydrolysis （5 h）. The hydrolysates were formulated （0-3%, w/w） into pork patties to determine the antioxidant efficacy. 0.02% BHA （butylated hydroxyanisole） was used as a positive control. Lipid oxidation in patties during storage was analyzed by measuring the TBARS and protein carbonyl content. The results showed that bone protein hydrolysates possessed significant antioxidant activity, and antioxidant activity increased with the increasing hydrolysates concentration. Sensory evaluation indicated that bone protein hydrolysates improved the color and decreased lipid oxidation flavor of pork patties. The 2% bone hydrolysates possessed the highest antioxidant activity and better sensory quality, and its effect was closed to 0.02% BHA.

Crosstalk between Wnt and bone morphogenetic protein signaling during osteogenic differentiati

Mesenchymal stem cells(MSCs)originate from many sources,including the bone marrow and adipose tissue,and differentiate into various cell types,such as osteoblasts and adipocytes.Recent studies on MSCs have revealed that many transcription factors and signaling pathways control osteogenic development.Osteogenesis is the process by which new bones are formed;it also aids in bone remodeling.Wnt/β-catenin and bone morphogenetic protein(BMP)signaling pathways are involved in many cellular processes and considered to be essential for life.Wnt/β-catenin and BMPs are important for bone formation in mammalian development and various regulatory activities in the body.Recent studies have indicated that these two signaling pathways contribute to osteogenic differen-tiation.Active Wnt signaling pathway promotes osteogenesis by activating the downstream targets of the BMP signaling pathway.Here,we briefly review the molecular processes underlying the crosstalk between these two pathways and explain their participation in osteogenic differentiation,emphasizing the canonical pathways.This review also discusses the crosstalk mechanisms of Wnt/BMP signaling with Notch-and extracellular-regulated kinases in osteogenic differentiation and bone development.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Cloning of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes

Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.

Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft

The precise mechanism of bone regeneration in different bone graft substitutes has been well studied in recent researches. However, miRNAs regulation of the bone formation has been always mysterious. We developed the anterior lumbar interbody fusion （ALIF） model in pigs using equine bone protein extract （BPE）, recombinant human bone morphogenetic protein-2 （rhBMP-2） on an absorbable collagen sponge （ACS）, and autograft as bone graft substitute, respectively. The miRNA and gene expression profiles of different bone graft materials were examined using microarray technology and data analysis, including self-organizing maps, KEGG pathway and Biological process GO analyses. We then jointly analyzed miRNA and mRNA profiles of the bone fusion tissue at different time points respectively. Results showed that miRNAs, including let-7, miR-129, m iR-21, miR-133, miR-140, miR-146, miR-184, and miR-224, were involved in the regulation of the immune and inflammation response, which provided suitable inflammatory microenvironment for bone formation. At late stage, several miRNAs directly regulate SMAD4, Estrogen receptor 1 and 5-hydroxytryptamine （serotonin） receptor 2C for bone formation. It can be concluded that miRNAs play important roles in balancing the inflammation and bone formation.

Extraction, Purification and Identification of Bone Morphogenetic Protein in Conditioned Medium of Osteosarcoma Cell (MG-63)

Objective: To find out a method of extraction and purification of bone morphogenetic protein (BMP) from osteosarcoma cell conditioned medium, and evaluate the biological activity of BMP.Methods: Conditioned medium of osteosarcoma cell lines (MG-63) was collected, concentrated and dialyzed. The concentrated protein was purified through gel chromatography on Sephcryl-S-100. The purified protein was tested by BMP monoclonal antibody (McAb), its molecular weight (MW) was determined by SDS-PAGE and its biological activity was demonstrated by heterotopic ossification.Results: The purified protein was proved to be BMP by BMP McAb, had a satisfactory heterotopic ossification, and its MW was about 21 kD.Conclusion: BMP existed in the conditioned medium of osteosarcoma cell and had a satisfactory biological activity after purification. Because osteosarcoma cell can be cultured and grew for a long timein vitro, this method will be helpful to a vast extraction of BMP and clinical application. Key words osteosarcoma cell - conditioned medium - bone morphogenetic protein - protein purification This project was a key scientific and technological program of Hubei Provicial Scientific and Technological Committee (No. 002p1503).

An overview on bone protein extract as the new generation of demineralized bone matrix

Bone protein extract is regarded as the new generation of demineralized bone matrix. The aim of this paper is to describe and characterize the properties of demineralized bone matrix and its new generation product in addition to its application in animal and human studies. Bone protein extract has features of osteoconductivity, osteoinductivity and osteogenicity, which originate from its unique and precise processing. It has exhibited powerful bone formation capacity both in animal experiments and in clinical trials by providing an optimal microenvironment for osteogenesis. Furthermore, not only does it have excellent bio- compatibility, it also has good compatibility with other implant materials, helping it bridge the host and implanted materials. Bone protein extract could be a promising alternative for demineralized bone matrix as a bone graft substitute.

Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats

AIM: To observe the effect of Danshao Huaxian capsule (DHC) on the expression of Gremlin and bone morphogenetic protein-7 (BMP-7) in the liver of hepatic fibrosis rats.

The biological function of type I receptors of bone morphogenetic protein in bone

Bone morphogenetic proteins （BMPs） have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors （BMPRI and BMPRII）. In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 （ALK2, also called type IA activin receptor）, activin- llke kinase 3 （ALK3, also called BMPRIA）, and activin-like kinase 6 （ALK6, also called BMPRIB）. The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future.

Expression of Vascular Endothelial Growth Factor in Bone Morphogenetic Protein-2 Induced Osteogenesis

An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in bone morphogenetic protein- 2 induced osteogenesis . The experimental results demonstrated that the expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenehymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenehymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14 th day, accompanied by numerons hypertrophic chondrocytes. When mature cartilage calcified and neu, bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. Signals of VEGF mRNA and VEGF can not be detected in the control groups.

Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

RETINOIC ACID DOWN-REGULATES BONE MORPHOGENETIC PROTEIN 7 EXPRESSION IN RAT WITH CLEFT PALATE

Objective To evaluate the effects of retinoic acid （RA） on expression of bone morphogenetic protein 7 （ BMP-7 ） in rat fetus with cleft palate, and the effects of RA on proliferation and apoptosis of osteoblasts. Methods All-trans RA （ATRA） was used to induce congenital cleft palate in Wistar rat. BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis. Flow cytometry and MTF assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells. BMP-7 mRNA and protein expressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis. Remilts ATRA could induce cleft palate of rat fetus. The incidence rate of cleft palate induced by 100 mg/kg AT-RA （45.5%） was significantly higher than 50 mg/kg ATRA （ 12.5%, P 〈 0. 05 ）. BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate. MC-3T3-E1 cells proliferation treated with 1 × 10^-6 mol/L ATRA decreased by 60%, the cell apoptosis increased by 2 times. BMP-7 mRNA and protein levels in MC-3T3-E1 cells treated with 1 × 10^-6 mol/L ATRA decreased by 60% and 80%, respectively, compared with ATRA-untreated cells （ P 〈 0.05 ）. Conclusions BMP-7 may play an important role in embryonic palate development. RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.

Use of bone morphogenetic proteins in mesenchymal stemcell stimulation of cartilage and bone repair

The extracellular matrix-associated bone morphogenetic proteins(BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell(MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted celltype lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair.

Influence of bone morphogenetic protein type IA receptor conditional knockout in lens on expression of bone morphogenetic protein 4 in lens

AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the vertebrate eye.METHODS: Cre-positive mice were mated with Crenegative mice to generate 50% Cre-positive(conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring(wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm.Removal of paraffin wax and dehydrating for sections,and then the procedure of in situ hybridization was processed, BMP4 MK1784-m(BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P【0.05 showed that the difference was statistically significant.· RESULTS: Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of BMP4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P 【0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5.CONCLUSION: ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.

AN IMMUNOCYTOCHEMICAL STUDY OF BONE MORPHOGENETIC PROTEIN IN EXPERIMENTAL FRACTURE HEALING OF THE RABBIT MANDIBLE

A monoclonal antibody raised against bone morphogenetic protein (BMP-McAb) has been used to demonstrate the presence of bone morphogenetic protein(BMP) in experimental fracture healing. Rabbit mandibles were fractured using standardized methods and left to heal for 3, 7, 14, 21 and 24 d, respectively. The avidin-biotin complex (ABC) method demonstrated an accumulation of positively stained primitive mesenchymal cells at the fracture site in the hematoma stage of bone repair. These cells appeared to undergo differentiation into positively-stained chondroblasts and osteoblasts during the phase of callus formation. Undifferentiated mesenchymal cells showed a high positive reactivity in the early post-fracture stages but a much lower reactivity during the remodelling phase.The results of our study suggest that bone inductive processes are accompanied by the presence of BMP in osteoprogenitor cells during fracture healing of the mandible and that BMP may play a significant role in osteogenesis during bone healing.

Bone morphogenetic proteins:Relationship between molecular structure and their osteogenic activity

Bone morphogenetic proteins(BMPs)are a family of potent,multifunctional growth factors belonging to transforming growth factor-(TGF-).They are highly conservative in structures.Over 20 members of BMPs with varying functions such as embryogenesis,skeletal formation,hematopoiesis and neurogenesis have been identified in human body.BMPs are unique growth factors that can induce the formation of bone tissue individually.BMPs can induce the differentiation of bone marrow mesenchymal stem cells into osteoblastic lineage and promote the proliferation of osteoblasts and chondrocytes.BMPs stimulate the target cells by specific membrane-bound receptors and signal transduced through mothers against decapentaplegic(Smads)and mitogen activated protein kinase(MAPK)pathways.It has been demonstrated that BMP-2,BMP-4,BMP-6,BMP-7,and BMP-9 play an important role in bone formation.This article focuses on the molecular characterization of BMPs family members,mechanism of osteogenesis promotion,related signal pathways of osteogenic function,relationships between structure and osteogenetic activity,and the interactions among family members at bone formation.

Bone morphogenetic protein-7 represses hepatic stellate cell activation and liver fibrosis via regulation of TGF-β/Smad signaling pathway

BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.

Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis inSchistosoma japonicum-infected micevia transforming growth factor-β/Smad signaling

AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95±6.66vs 2.02±0.76; week 15: 12.84±4.36 vs 1.74±0.80; P<0.05), but significantly lower than that in group B (week 9: 22.95±6.66 vs 34.43±6.96; week 15: 12.84±4.36 vs 18.90±5.07;P<0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24±5.73 vs 0.33±0.20; week 15: 12.42±4.88 vs 0.34±0.27; TGF-β1: week 9: 37.00±13.74 vs 3.73±2.14; week 15: 16.71±9.80 vs 3.08±2.35; pSmad2/3: week 9: 12.92±4.81 vs 0.83±0.48; week 15: 7.87±4.09 vs 0.90±0.45; P<0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24±5.73 vs 34.39±5.74; week 15: 12.42±4.88 vs 25.90±7.01; TGF-β1: week 9: 37.00±13.74 vs 55.66±14.88; week 15: 16.71±9.80 vs 37.10±12.51; pSmad2/3: week 9: 12.92±4.81 vs 19.41±6.87; week 15: 7.87±4.09vs 13.00±4.98;P<0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46±3.95 vs 1.00±0.40 and 8.46±3.95 vs 0.77±0.42; P<0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09±0.38 vs 0.97±0.42 vs 0.89±0.39; P>0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistentwith the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-β/Smad signaling pathway.

Drilling Combined with Adipose-derived Stem Cells and Bone Morphogenetic Protein-2 to Treat Femoral Head Epiphyseal Necrosis in Juvenile Rabbits

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells （ADSCs） and bone morphogenetic protein-2 （BMP-2） to treat femoral head epiphyseal ischemic necrosis, which can be done in juvenile rabbits. Passagefour bromodeoxyuridine （BrdU）-labeled ADSCs were cultured, assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability. Twomonth-old, healthy male rabbits （1.2 to 1.4 kg, n=45） underwent ischemic induction and were randomly divided into five groups （group A： animal model control; group B： drilling; group C： drilling ＆ ADSCs; group D： drilling ＆ BMP-2; and group E： drilling ＆ ADSCs ＆ BMP-2）. Magnetic resonance imaging （MRI）, X-ray imaging, hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4, 6 and 10 weeks after treatment. Approximately 90% of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability. Similar results were observed in the rabbits in groups C and E at weeks 6 and 10. The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B （P〈0.01）. Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B （P〈0.05）. In summary, drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

Induction of Bone Matrix Protein Expression by Native Bone Matrix Proteins in C2C12 Culture

Objective To study the expression of bone matrix protein （BMP） induced by bovine bone morphogenetic proteins （BMPs） in vitro. Methods Type 1 collagen, osteopontin （OPN）, osteonectin （ON）, osteocalcin （OC）, and bone sialoprotein （BSP） were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. Results The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the lkaline phosphatase （ALP） activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblsts in vivo and in differentiating osteoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. Conclusion Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type I collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C 12 in vitro.