Experimental Study On Gene-modified Myoblasts Combined With Injectable Thermosensitive Hydrogel Scaffold To Fabricate Tissue Engineered Bone

Background and objectives: The repairing of bone defect is one of the toughest tasksin clinical orthopedics. The emergent bone tissue engineering provides a new method tosolve this problem. Muscle satellite cells (MSCs) serve as the main source of stem cells inskeletal muscles. MSCs and their cultivating progenies---myoblasts, possess the plasticityto give rise to all the mesodermic lineages, especially a high potential of osteogenicdifferentiation. MSCs have remarkable advantages such as: rich source, harvest withminimal invasion, easy to culture and identify. Moreover, they are frequently used ascarriers for gene delivery. Therefore, MSCs become an attractive candidate for seed cells inbone tissue engineering with broad prospect. However, there remain a couple of problemsin myoblasts’ application in bone tissue engineering:1. There are very limited studies onscaffold materials suitable for myoblasts, especially lack of studies on chitosan derivedthermosensitive hydrogels as injectable scaffolds.2. During the cell expansion in vitro,myoblasts lose their stemness rapidly, resulting in severe impairment in their potential ofosteogenic differentiation and transplantation efficacy. Based on these facts, the objectivesof this study include:1. Fabricating a novel injectable thermosensitive hydrogel scaffold－chitosan/β-glycerophosphate/collagen (C/GP/Co), to observe its effect on myoblasts’growth and proliferation, to investigate the feasibility of its application as a scaffoldmaterial for myoblasts.2. By modifying myoblasts with msx1(muscle segmenthomeobox-1) gene via retroviral transfection, to study the effect of msx1on maintaining thestemness of myoblasts during the in vitro expansion process.3. Through experiment ofosteogenic induction in vitro and ectopic bone formation in vivo, to evaluate the osteogenicefficacy of myoblasts with msx1gene modification.Methods:1. Myoblasts were isolated and cultured in vitro, then the influences offreshly fabricated C/GP/Co scaffold on myoblasts’ growth and differentiation were investigated.(1) Through differential adhesion method combined with clones isolating,myoblasts from green fluorescent protein (GFP) transgenic mouse (C57BL/Ka-βactin-EGFP) were obtained then cultured and passaged. They were identified byimmuno-staining of desmin, myogenic induction and myosin heavy chain (MHC)immuno-staining;(2) C/GP/Co hydrogel was fabricated by mixing2.0wt%chitosan (C)solution,50wt%β-glycerophosphate (GP) and2mg/mL collagen (Co) solution at avolume ratio of5:1:6, then its ultrastructure was observed under a scanning electronmicroscopy (SEM);(3) C/GP/Co hydrogel extraction fluids with various concentrationswere prepared and then used as medium for myoblasts’ culture. To assess the cytotoxicityof this scaffold, cell viability was determined by cell counting kit-8(CCK-8), the relativegrowth rate (RGR,%) was calculated;(4) The growth of myoblasts in two-dimensionalculture was examined by cell viability assay and that in three-dimensional culture wasexamined by laser scanning confocal microscopy (LSCM) and SEM, respectively;(5)Myoblasts were encapsulated in liquid C/GP/Co compound, then injected into thesubcutaneous dorsum of nude mice. The change of GFP signal was monitored by smallanimal in vivo living fluorescent imaging system. The mice were sacrificed4weeks aftercell transplantation and the survival of myoblasts was verified by histological examination;(6) The influences of hydrogel substrate on capability of myoblasts toward myogenic,osteogenic and adipogenic differentiation were evaluated. In control group myoblasts werecultured on collagen-coated dishes while in C/GP/Co group myoblasts were cultured onC/GP/Co gel-coated dishes. Myogenic differentiation was assessed by fusion index (FI) andmyotube size. Osteogenic differentiation was assessed by Alizarin Red staining andadipogenic differentiation by Oil Red O staining.2. Msx1gene was transfected intomyoblasts via retrovirus vector, then whether msx1-modified myoblasts can help maintaintheir stemness was investigated:(1) Myoblasts were transfected by msx1gene viaretrovirus, and then identified by western blot.(2) The following features betweenmsx1-modified myoblasts (experimental group) and non-modified myoblasts (control group)were compared: cell morphology, the ratio of senescent cells, the capability of proliferation,the expression level of differentiation-related markers and migration capability. Cellmorphology was observed under a transmission electron microscopy (TEM), the ratio ofsenescent cells was calculated in myoblasts stained by β-D galaetosidase, the proliferation capability was assessed by cell proliferation assay, proliferation index(PI), Ki67index andcolony forming frequency, the differentiation-related markers include myoD(myogenicdifferentiation factor), Pax7(paired box transcription factor-7) and SSEA-1(stage specificembryonic antigen-1), the cell migration capability was assessed by transwell experiment;(3) The transplantation efficacy of myoblasts in the same generation between control groupand experimental group, and myoblasts in different generation from experimental groupwas compared in scid/mdx mice through transplantated into tibilias anterior muscles;3. Theosteogenic efficacy of msx1-modified myoblasts was evaluated through the followingaspects:(1) After osteogenic induction in vitro, the following indicators were employed toassess the osteogenic efficacy: ALP activity, area of mineralization and mRNA expressionlevel of osteogenesis-related genes;(2) Myoblasts were embedded with C/GP/Co hydrogelto fabricate an injectable tissue engineered bone(TEB), then TEB was transplanted intonude mice to perform an experiment of in vivo ectopic bone formation. Bone quality wasassessed by the following methods: gross observation of dissected implants, bone mineraldensity, H&E staining and immunohistochemical staining for osteocalcin etc.Results:1. GFP-labeled myoblasts were isolated from GFP-transgenic murine skeletalmuscles, then cultured and passaged. C/GP/Co hydrogel scaffold was successfullyfabricated and its ultrastructure was observed. This scaffold material was non-cytotoxic andexhibited good biocompatibility to myoblasts. Moreover, it demonstrated a remarkablecapability to enhance the potential of multilineage differentiation during the period of cellexpansion in vitro.(1) The fluorescent staining for desmin was positive in culturedmyoblasts. In myogenic assay, the cultured myoblasts differentiated into myotubes and thelatter was positive in immuno-staining of myosin heavy chain (MHC);(2)The preparedC/GP/Co solution gelated into hydrogel around10min. Under SEM, it exhibited a porouscobweb-like homogenous structure;(3) In the culture with extraction fluids, cytotoxic gradeof the scaffold material was0～1, indicating it was non-cytotoxic;(4) Myoblasts grew wellboth in two-dimensinal and three-dimensional hydrogel culture;(5) Hydrogel embeddedwith GFP-labeled myoblasts was transplanted into subcutaneous dorsum of nude mice.Green fluorescence was visible4weeks after implantation and histological examinationrevealed that a large number of myoblasts still survived in the scaffold;(6) The myoblastsexhibited an enhanced myogenic, osteogenic and adipogenic differentiation on the hydrogel surface. In myogenic assay, both the fusion index (FI) and size of myotube increasedsignificantly in C/GP/Co group (p<0.05). In osteogenic assay, the ALP activity at everytime point and the percentage of mineralization at day21were significantly higher inC/GP/Co group than that of control (p<0.05). In adipogenic assay, the area of positive OilRed O staining within cytoplasm was significantly larger in C/GP/Co group (p<0.05).2.Msx-1modified myoblasts maintained their stemness better in culture.(1) Myoblasts weresuccessfully transfected with msx1gene via retrovirus vector containing a doxycline tet-offsystem and the expression of msx1was verified by western blot analysis.(2) Compared tocontrol group, the myoblasts in experimental group: showing a smaller, more roundedcontour and more rejuvenized ultrastructue under TEM; the percentage of senescent cellsdecreased significantly (p<0.05); the proliferation index (PI), Ki67index and colonyforming frequency was significantly higher (p<0.05); the expression level of SSEA-1andPax-7increased significantly (p<0.05) while the expression level of myoD decreasedsignificantly (p<0.05), measured by either percentage of positive staining and mean opticaldensity; they exhibited a stronger capability of migration in transwell experiment (p<0.05)and this capability was proved to be correlated to SDF-1/CXCR4(stromal cell-derivedfactor-1/C-X-C chemokine receptor type4) axis.(3) Myoblasts in experimental groupexhibited a higher engraftment efficiency in scid/mdx mice. Much more myoblasts survivedand new myofibers expressing dystrophin formed in the host tibilias anterior muscle(p<0.05). Moreover, the high transplantation efficiency could be effectively maintained incell passaging process.3. The myoblasts in experimental group exhibited a higherosteogenic efficacy compared to myoblasts in control group:(1) In osteogenic assay in vitro,ALP activity, percentage of mineralization area and mRNA expression level of someosteogenesis-related genes were higher in experimental group at examined time point(p<0.05).(2) In the process of ectopic bone formation in vivo, the new-forming tissueengineered bones exhibited a better quality in experimental group: they were larger andstiffer in gross examination, they possessed greater bone mineral density (p<0.05), higherpercentage of new-formed trabecula area (p<0.05) and larger area of positive staining forosteocalcin.Conclusions:1. As an injectable thermosensitive hydrogel scaffold, C/GP/Co is easyto fabricate and biocompatible to myoblasts both in vitro and in vivo. It demonstrated a remarkable capability to maintain the plasticity of myoblasts during the period of in vitrocell expansion. Therefore, it could serve as an ideal scaffold to carry myoblasts.2. Msx1modification is helpful to maintain the stemness of myoblasts during cell culture, and themodified myoblasts acquire a high engrafting efficiency which can be preserved during theprocess of cell passaging.3. Msx1-modified myoblasts possess an improved osteogenicefficacy. It is feasible to combine them with C/GP/Co hydrogel to fabricate an injectablethermosensitive scaffold which can be used in bone tissue engineering.