skeletal muscles are always regarded as the source of satellite stem cells or muscle precursor cells . these kinds of stem cells are at the quiescent condition under normal circumstances and activated when the repairing of muscle tissue is needed . they will be differentiated and blended together into new muscle fibers , reaching the purpose of repairing defected muscles as a result . in recent years , studies have shown that there is also another kind of stem cells in skeletal muscle , called muscle - derived stem cells ( mdscs ) . they have mesenchymal stem cells - like differentiation potential , and the ability of being differentiated into several types of terminal cells is maintained . instead of being differentiated into muscle cells , mdscs can also be differentiated into other type of cells , such as hematopoietic cells , osteoblasts , and chondroblasts   under particular conditions . therefore , the potential of mdscs as candidate seed cells in osteogenetic tissue engineering has been paid much more attention than before . bone morphogenetic proteins ( bmps ) belong to one of the -superfamily members of transforming growth factors ( tgfs ) ; bmps are a kind of multifunctional growth factors and are one of the most potent osteogenetic growth factors [ 68 ] . bmp9 possible roles include inducing and maintaining the cholinergic phenotype of embryonic basal forebrain cholinergic neurons , inhibiting hepatic glucose production and inducing the expression of key enzymes of lipid metabolism , and stimulating murine hepcidin 1 expression [ 1012 ] . although the functional role of bmp9 in the skeletal system remains to be fully understood , the potent osteogenic activity of bmp9 suggests that it may be used as one of the most effective bone regeneration agents compared with other bmps , such as bmp2 , bmp4 , or bmp7 [ 13 , 14 ] . presently , among the overwhelming majority of many osteogenetic studies of bmps , mouse mesenchymal stem cells ( such as c3h10 ) , mouse embryonic fibroblasts ( mefs ) , and bone marrow stromal cells ( bmscs ) are subjected as studying objects . however , there have been no literature reports on whether bpm9 can induce osteogenetic differentiation of muscle - derived stem cells . therefore , in this paper , we have demonstrated the potential osteogenetic ability of mdscs induced by bmp9 in vitro and in vivo testing . bmp9 , bmp2 , and gfp expression adenoviruses ( adbmp9 , adbmp2 , and ad - gfp ) were provided by dr . he ( molecular oncology laboratory , medical center , the university of chicago , usa ) and amplified in our laboratory . hank 's solution , dmem culture medium , and high - quality fetal calf serum ( fbs ) were used in cells culture ( hyclone company ) . alkaline phosphatase ( alp ) staining kit and quantitative testing kit were purchased from bd company . alizarin red s staining kit , type i collagenase , trypsin , polylysine , and vitamin c were purchased from sigma company . anti - sca-1 antibody ( wuhan boster biological technology co. , ltd . ) and nanohydroxyapatite / polyamide bone cement were provided by research center of nano - biomaterials , sichuan university . healthy rabbits with an average age of 68 weeks , as the experimental animal , were provided by the experimental animal center , chongqing medical university . after anesthesia , muscle strips ( 2  cm  0.8  cm  0.5  cm , approximately 5  g ) were cut from the rabbits and then placed into a sterilized bottle . carry out sequential digestion by two - step method ( type i collagenase and trypsin methods ) , then filter through no . 100 , 200 , and 400 stainless steel screens , and aspirate the obtained cells into a 100  ml culture bottle ( pp1 ) . 9  ml of dmem culture solution ( contained 100  ml / l fetal calf serum ) was added . 1  h later , transfer the cell suspension into another culture dish ( pp2 ) by the differential - rate wall - adhering growth method . hereafter , repeat the procedure above to obtain pp3 , pp4 , pp5 , and pp6 every 24  h. mdscs were fixed with acetone and prepared for immunohistochemical staining analysis at 48 hours after subculture . alp activity was assessed by a modified great escape seap chemiluminescence assay ( bd clontech , mountain view , ca , usa ) and/or histochemical staining assay ( using a mixture of 0.1  mg / ml of naphthol as - mx phosphate and 0.6  mg / ml of fast blue bb salt ) [ 13 , 14 ] . cultured mdscs were seeded in 24-well plate with subconfluent of 30% and infected with adbpm9 ( experimental group ) , adbpm2 ( positive control group ) , and ad - gfp ( negative control group ) , respectively . at 5 , 7 , and 9 days after infection , alp activity mdscs were seeded into a 24-well plate and infected with adbpm9 , adbmp2 , and ad - gfp . cells were fixed with 0.05% ( v / v ) glutaraldehyde at room temperature for 10 minutes . after being washed with distilled water , fixed cells were incubated with 0.4% alizarin red s ( sigma - aldrich ) for 5 minutes , followed by extensive washing with distilled water . anesthetize 36 new zealand white rabbits , then dissection was cut ( longitudinally approximately 30  mm along the inner side of the forearm ) to adequately expose the middle segment of the radius , and prepare a bone defect model of approximately 12  mm by an electric saw , in accordance with the length of the implant . randomly assign the rabbits into 3 groups and implant adv - hbmp9 + nanohydroxyapatite / polyamide bone cement , adv - hbmp2 + nanohydroxyapatite / polyamide bone cement , and adv - gfp + hydroxyapatite / polyamide bone cement at the left and right bone defect sites , respectively . suture the wound layer by layer and wrap with sterile dressings . after the surgery , each rabbit in each group is injected with gentamicin sulfate 10,000  u , for 3  d ; the rabbits of each group are fed in different cages . carry out x - ray radiography in weeks 2 , 4 , 8 , 12 , and 16 after the surgery , respectively . in weeks 2 , 4 , 6 , 8 , 12 , and 16 after the surgery , sacrifice 2 rabbits of each group , respectively , macroscopically observe the healing condition of the implant and surrounding tissues , and carry out a histological examination of the tissue in the stent . the cells of early wall - adhering growth ( pp1 ) are mostly fibroblasts , with large body and with rapid growth , without significant directional growth . the cells of late - stage wall - adhering growth ( pp6 ) are mostly short , fusiform , polygonal cells ( figure 1(a ) ) , with obviously directional growth that may form into a long chain ; however , their growth is slower than that of fibroblasts . in 710  d later , intercellular lamellar fusion occurs ; hereafter , cell growth rate is slower than before . sca-1 is one of the most characteristic phenotypes of mdscs ; when the in vitro culture density of cells reached 70%80% , sca-1 immunohistochemical staining was carried . the results showed that 80%90% of cells were positive in sca-1 staining ( figure 1(b ) ) . alp staining of mdscs transfected with bmp9 , bmp2 , and gfp at day 9 was shown ( figure 2(a ) ) . it suggested that alp expression induced by bmp9 in mdscs was higher than other groups . and also , alp activities of mdscs after infected by bmp9 , bmp2 , and gfp expression adenovirus were assessed at days 5 , 7 , and 9 ( figure 2(b ) ) . alizarin red s staining was carried out at day 14 after infection ; it was found that , under the induction of bmp9 , obvious calcium salt sedimentation occurred in mdscs , the density of calcification was higher than other groups , and at the same time , the size of calcification in bmp9 group was larger ( figure 3 ) . week 2 after the surgery : no obvious callus formation was seen around the implant in each group . week 4 after the surgery : the boundary between the broken ends of bone and the implant was veiled in the bmp9 group , and the transmittancy of the implant was decreased . week 8 after the surgery : callus grew into the implant from the two broken ends of bone and from the side of the ulna ( figure 4 ) . the amount of formed callus in the bmp2 group was less than that in the phase in the bmp9 group ( figure 4 ) . a small amount of callus was seen in the side of ulna in the gfp group ( figure 4 ) . week 12 after the surgery : in the bmp9 group , the two broken ends of bone were completely connected , and the majority of bone marrow cavities were recanalized . however , the two broken ends of bone were not completely connected in the bmp2 group . week 16 after the surgery : in the bmp9 group , the two broken ends of bone were completely connected , and the majority of bone marrow cavities were recanalized ( figure 4 ) . however , the ends of bone broken were not completely connected in the bmp2 group ( figure 4 ) . in the gfp group , there was only a certain amount of formed callus in the side of ulna and in the proximal end of bone broken ( figure 4 ) . week 2 after the surgery : in the bmp9 group , the implant was wrapped by noncohesive fibrous tissues , the two ends of the implant were connected with the recipient bone fibers , a small amount of soft tissues with blood vessels grew into the lateral aperture of the implant , no obvious new bone formation was seen , and the adhesion between the implant and the recipient bone was not firm and the implant could be shaken . in the bmp2 group , it was seen that the implant was wrapped by noncohesive fibrous tissues , no obvious new bone formation was seen , and the implant could be shaken . in the gfp group , it was seen that the implant was wrapped by a small amount of fibrous tissues . week 4 after the surgery : in the bmp9 group , the implant was wrapped by fibrous tissues , the proximal and distal ends of the implant were connected with the recipient bone and could not be shaken , a large amount of soft tissues with blood vessels grew into the lateral aperture of the implant , and there was a small amount of formed new callus . in the bmp2 group , a small amount of soft tissues with blood vessels grew into the lateral aperture of the implant , and no new bone formation was seen . in the gfp group , a small amount of soft tissues with blood vessels grew into the lateral aperture of the implant , and no new bone formation was seen . week 6 after the surgery : in the bmp9 group , the implant was wrapped completely by fibrous membranous tissues , and there were hard new bone and callus formation . in the bmp2 group , there was a small amount of formed new callus . in the gfp group , week 8 after the surgery : in the bmp9 group , there was a large amount of formed new callus . it was seen that hard calluses grew into the implant , and a bone - wedge - like structure was formed in the lateral aperture of the implant . in the bmp2 group , the lumen was full of connective tissues , and there was new callus formation . in the gfp group , weeks 12 and 16 after the surgery : in the bmp9 group , callus was increased . in the bmp2 group , it was seen that hard calluses grew into the implant . in the gfp group , a large amount of new callus was formed .   in the bmp9 group : in week 2 after the surgery , it was seen that connective tissues grew into the lumen of the implant , and there were much inflammatory cell infiltration and immature capillary formation . in week 4 after the surgery , the lumen was full of connective tissues , inflammatory cells were reduced , a mature vascular net was formed , and it was seen that a small amount of cartilage grew . in week 6 after the surgery , a mature vascular net was formed , and there was fibrous callus formation in the stent . in week 8 after the surgery , the lumen was full of mature vascular net and cartilages , and there were much fibrous callus and osteoid formations , and a bone wedge - like structure was formed in the lateral aperture of the implant . some cartilages were directly adhered to the surface of the implant ( figure 5 ) . in week 12 after the surgery , the lumen was full of cartilages ; after bone formation and moulding , mature bone trabecular formation was seen , similar to peripheral bones , observed under microscope , and was connected with the recipient bone mutually . in week 16 after the surgery , the bone cavity was filled with mature trabecular bone , and immature trabecular bone between meshes were connected into mesh - like and plate - like shapes ( figure 5 ) . in the bpm2 group : vasoformation and bone formation were similar to those in the bmp9 group ; however , the times for vasoformation and bone formation were approximately 2 weeks later than those in the bmp9 group ( figure 5 ) . in the gfp group : the times for vasoformation and bone formation were much later than those in the bmp2 group ( figure 5 ) . there are several classes of cell populations in the skeletal muscle , of which the muscle - derived stem cells ( mdscs ) have multidirectional differentiation ability [ 4 , 1517 ] . in the experiment , mdscs were extracted by the preplate technique ; the principle of this technique was based on different times for wall - adhering growth of different cells : the majority of cells of initial wall - adhering growth are fibroblasts ( pp1 ) , the main portion of cells of wall - adhering growth in 2448  h are satellite cells ( pp2-pp4 ) , and cells of last wall - adhering growth were sca-1 staining - positive mdscs . prior to wall - adhering growth , mdscs were of small and round spherical shape , with strong refractivity , and were also of round shape at the time of initial wall - adhering growth ; after spontaneous growth , they are fused into mature polynuclear myotubes . after mdscs transfected with bmp9 and bmp2 , it is found by alp staining and quantitative analysis that bmp9 induced alp activities of muscle - derived stem cells , and the activities were increased with time , reaching peak in day 9 , suggesting that mdscs had the osteogenous differentiation ability in vitro . meanwhile , the alp activities induced by bmp9 were obviously higher than those in the bmp2 group , suggesting that bmp9 had higher potential ability of osteogenesis stimulating than bmp2 . the results of the later - stage marker , that is , calcium salt sedimentation , for osteogenous differentiation of cells showed that there was obvious calcium salt sedimentation in the bmp9 group ; moreover , compared with that in the bmp2 group , bmp9 has stronger ability of stimulating calcium salt sedimentation in mdscs . these demonstrated that bmp9 was probably a more efficient osteoinductive growth factor compared with bmp2 . the result was consistent with that from the studies conducted by wada et al . . previously , the studies by d. s. musgrave found that , under the action of bmp2 , mdscs resulted in ectopic osteogenesis in upper limbs . in the studies by v. wright , mdscs transfected with bmp4-induced ectopic osteogenesis and promoted bone healing . therefore , during our study , the effects of bmp9/2/gfp in repairing radius defect were observed by using the method in which , after the middle segment defect model of radius was prepared , adv - hbmp9/2/gfp + nanohydroxyapatite / polyamide bone cement was implanted . as a result , new bone was initially emerged in week 4 in the bmp9 group ; in the bmp9 group , by week 8 , large mounts of blood vessels , soft tissues , and bone calluses were formed , and hard calluses grew into the implant . in week 16 after the surgery , the two broken ends of bone were completely connected , and the majority of bone marrow cavities were recanalized ; by histological examination , mature bone trabecular was seen , and new bone trabecular between meshes was connected into mesh - like and plate - like shapes . the repairing duration of bone defects in the bmp2 group was approximately 2 weeks longer than that in the bmp9 group . bone defects repairing in gfp group is much more slow than that in the bmp2 group . this result is consistent with the prior reports submitted by cheng et al .   and kang et al . they demonstrated that bmp9 ( in addition to bmp-2 , bmp4 , or bmp-7 ) is identified as the most potent bmps to induce orthotopic bone formation in vivo . from what have been discussed above , bmp9 has strong effects of inducing directional osteogenous differentiation of muscle - derived stem cells ; compared with other stem cells in tissue engineering , the in vitro cultivation of mdscs has the advantages of good material availability , as well as strong proliferation and differentiation abilities . meanwhile , it has also demonstrated that it is a research direction of vast potential to carry out research on artificial bone in tissue engineering , by using highly efficient osteogenous induction factor and readily available , adequate tissue stem cells in combination .
efficient osteogenetic differentiation and bone formation from muscle - derived stem cells ( mdscs ) should have potential clinical applications in treating nonunion fracture healing or bone defects . here , we investigate osteogenetic differentiation ability of mdscs induced by bone morphogenetic protein 9 ( bmp9 ) in vitro and bone formation ability in rabbit radius defects repairing model . 
 rabbit 's mdscs were extracted by type i collagenase and trypsin methods , and bmp9 was introduced into mdscs by infection with recombinant adenovirus . 
 effects of bmp9-induced osteogenetic differentiation of mdscs were identified with alkaline phosphatase ( alp ) activity and expression of later marker . in stem - cell implantation assay , mdscs have also shown valuable potential bone formation ability induced by bmp9 in rabbit radius defects repairing test . taken together , our findings suggest that mdscs are potentiated osteogenetic stem cells which can be induced by bmp9 to treat large segmental bone defects , nonunion fracture , and/or osteoporotic fracture .