基因修饰成肌细胞结合可注射温敏凝胶支架构建组织工程骨的实验研究
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摘要
研究背景及目的：骨缺损修复是骨科临床实践中常见难题之一，新兴的骨组织工程研究为解决该项难题提供了全新的思路。肌卫星细胞(muscle satellite cells, MSCs)是骨骼肌内最主要的干细胞成分，MSCs及其体外培养的子细胞---成肌细胞(myoblast)具有中胚层谱系内的可塑性，尤其具备较强的成骨分化能力。此外，成肌细胞还具有来源丰富、取材容易创伤小及培养方便等优势，且常被用作重要的基因载体，因而成为一种极具应用前景的骨组织工程种子细胞。但成肌细胞应用于骨组织工程尚面临一些问题：1.对成肌细胞适宜的支架材料研究不多，尤其是对以壳聚糖温敏凝胶为代表的可注射支架缺乏相应研究；2.成肌细胞体外扩增时其干性(stemness)会迅速丢失，将严重影响细胞的成骨分化潜能及移植效率。因此，本实验的研究目的包括：1.制备一种新型的可注射温敏凝胶支架---壳聚糖/β-甘油磷酸钠/胶原(C/GP/Co)，观察它对成肌细胞增殖分化的影响，探讨其作为成肌细胞运载支架的可行性；2.在成肌细胞中通过逆转录病毒转染msx1基因(肌节同源盒基因-1)，观察msx1短暂表达对成肌细胞培养过程中“干性”保持的影响；3.通过体外成骨分化诱导实验和体内异位成骨实验，检验经msx1基因修饰后成肌细胞的成骨效能。
方法：1.分离培养成肌细胞并观察制备的C/GP/Co凝胶支架对成肌细胞生长分化的影响：(1)采用差速贴壁结合克隆分选法从绿色荧光蛋白(GFP)转基因小鼠骨骼肌中分离出成肌细胞并通过结蛋白染色、成肌分化、肌球蛋白重链染色加以鉴定；(2)以5:1:6体积比将2.0wt%的壳聚糖溶液(C)、50wt%的β-甘油磷酸钠(GP)及2mg/mL的I型胶原蛋白溶液(Co)配置成C/GP/Co凝胶，通过扫描电镜观察其超微结构；(3)制备不同浓度凝胶浸提液进行细胞培养，以CCK-8试剂盒测定细胞活力，并以此计算相对增殖率(RGR)评价支架毒性；(4)成肌细胞在二维培养条件下的生长情况通过细胞活力实验进行观察，在三维培养条件下的细胞形态则通过激光共聚焦显微镜和扫描电镜进行观察；(5)用液态的C/GP/Co复合物包裹成肌细胞，注射移植到裸鼠背部皮下组织。通过小动物活体荧光成像系统观察裸鼠体内移植物荧光信号的变化情况。4w后处死裸鼠，通过组织学观察了解成肌细胞在凝胶内的存活情况；(6)评价凝胶介质对成肌细胞向成肌、成骨、成脂方向分化能力的影响。以常规胶原包被表面培养的成肌细胞为对照组，C/GP/Co凝胶表面上进行培养的成肌细胞为C/GP/Co组。成肌分化通过融合指数(FI)和肌管大小进行评价；成骨分化通过碱性磷酸酶(ALP)活性、茜素红染色进行评价，成脂分化通过油红O染色进行评价。2.利用逆转录病毒载体将msx1转染到成肌细胞，并评价经基因修饰后的成肌细胞后是否能保持“干性”：(1)逆转录病毒转染msx1基因到成肌细胞并采用免疫印迹实验(WB)予以鉴定；(2)比较经msx1基因修饰(实验组)和未经该基因修饰的成肌细胞(对照组)在细胞形态、衰老细胞比例、增殖能力、分化相关标记分子表达、迁移能力等指标上的差异。细胞形态通过透射电镜(TEM)进行观察，衰老细胞比例通过β-半乳糖苷酶染色实验获取相关数据，增殖能力通过细胞增殖实验、增殖指数(PI)、Ki67指数及集落形成率进行评价，分化相关标记包括myoD(成肌分化因子)、Pax-7(配对盒转录因子-7)和SSEA-1(阶段特异性胚胎抗原-1)，迁移能力通过Transwell实验进行评价；(3)比较同代实验组与对照组成肌细胞在scid/mdx小鼠模型中的移植效率以及不同代次的实验组细胞在该模型中的移植效率；3.评价经msx1基因修饰后成肌细胞的成骨效能：(1)通过成骨诱导分化实验比较实验组与对照组成肌细胞碱性磷酸酶(ALP)活性、钙盐沉积面积及成骨分化相关基因表达水平；(2)将成肌细胞与C/GP/Co凝胶复合构建可注射组织工程复合物，在裸鼠体内进行异位成骨实验，通过大体观察、骨密度检测、组织切片H&E染色以及骨钙素免疫组织化学染色等方法比较对照组与实验组成骨质量。
结果：1.从小鼠骨骼肌内分离出带有GFP标记的成肌细胞并进行培养、传代；成功地制备了C/GP/Co凝胶支架并观察了该材料的超微结构；该支架材料无毒，并且对成肌细胞具有良好的生物相容性，对成肌细胞体外扩增期间多向分化能力的保持具有促进作用：(1)培养的成肌细胞结蛋白染色阳性，成肌诱导条件下能形成肌管，肌球蛋白重链染色阳性；(2)制备的C/GP/Co复合物在37℃时约10min内由液体形成水凝胶，扫描电镜下该凝胶为蜂窝状均质多孔结构；(3)浸提液培养实验中支架毒性分级为0到1级，说明该材料无毒；(4)成肌细胞在凝胶二维及三维培养时均生长良好；(5)凝胶携带成肌细胞移植到裸鼠皮下4w后仍可观察到绿色荧光，组织学检查发现支架上有大量成肌细胞存活；(6)凝胶介质对成肌细胞的成肌、成骨及成脂分化均有增强作用。在成肌分化实验中，C/GP/Co组细胞分化后的融合指数及肌管大小均显著增加(p<0.05)；在成骨分化实验中，C/GP/Co组细胞在各检测时相点的ALP活性均显著增强(p<0.05)，在分化21d时的矿化面积百分比也显著增加(p<0.05)；在成脂分化实验中，C/GP/Co组细胞内油红O染色阳性区域面积显著增加(p<0.05)。2.Msx1转染成肌细胞后能有助于细胞保持“干性”：(1)逆转录病毒成功转染成肌细胞并使后者异位表达msx1蛋白；(2)实验组成肌细胞较对照组成肌细胞相比：在TEM镜下具有更小而圆的外型及更年轻化的超微结构；衰老细胞百分比显著降低(p<0.05)；具有更高的增殖指数(p<0.05)、Ki67指数(p<0.05)及集落形成率(p<0.05)；在免疫荧光阳性率和平均光密度值两项评价指标中，SSEA-1和Pax-7表达水平均上调而myoD表达水平下调(p<0.05)；Transwell实验中表现出更强的迁移能力(p<0.05)，且该能力与SDF-1/CXCR4(基质细胞衍生因子-1/细胞表面趋化因子受体4)轴有关；(3)移植到scid/mdx小鼠模型后实验组细胞存活率更高，随时间推移有更多细胞在宿主体内增殖并分化融合并表达肌营养不良蛋白(p<0.05)，并且这种高移植效率在细胞培养传代过程中能有效保持。3.实验组成肌细胞与对照组相比具有更高的成骨效能：(1)体外成骨诱导实验中，各检测时相点实验组的ALP活性、矿化面积百分比均显著高于对照组，成骨相关基因的表达水平也显著高于对照组(p<0.05)；(2)异位成骨实验中实验组可注射组织工程复合物：大体观察中形成的骨样组织体积及硬度更大，骨密度(p<0.05)、新生骨小梁面积百分比(p<0.05)以及骨钙素染色阳性区域面积均显著高于对照组。
结论：1.C/GP/Co是一种可注射的温敏凝胶支架，制备简便，在体内外实验均表现出对成肌细胞具有良好的生物相容性，且有助于体外扩增培养期间保持成肌细胞的多向分化潜能，是成肌细胞理想的支架材料。2.Msx1基因修饰成肌细胞将有助于促使成肌细胞在体外培养过程中保持“干性”，增强成肌细胞的移植效率，并且该能力在传代过程中能够有效保持。3.经msx1基因修饰的成肌细胞具有更强的成骨效能，与C/GP/Co凝胶构建可注射温敏支架用于骨组织工程确实可行。
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.
方法：1.分离培养成肌细胞并观察制备的C/GP/Co凝胶支架对成肌细胞生长分化的影响：(1)采用差速贴壁结合克隆分选法从绿色荧光蛋白(GFP)转基因小鼠骨骼肌中分离出成肌细胞并通过结蛋白染色、成肌分化、肌球蛋白重链染色加以鉴定；(2)以5:1:6体积比将2.0wt%的壳聚糖溶液(C)、50wt%的β-甘油磷酸钠(GP)及2mg/mL的I型胶原蛋白溶液(Co)配置成C/GP/Co凝胶，通过扫描电镜观察其超微结构；(3)制备不同浓度凝胶浸提液进行细胞培养，以CCK-8试剂盒测定细胞活力，并以此计算相对增殖率(RGR)评价支架毒性；(4)成肌细胞在二维培养条件下的生长情况通过细胞活力实验进行观察，在三维培养条件下的细胞形态则通过激光共聚焦显微镜和扫描电镜进行观察；(5)用液态的C/GP/Co复合物包裹成肌细胞，注射移植到裸鼠背部皮下组织。通过小动物活体荧光成像系统观察裸鼠体内移植物荧光信号的变化情况。4w后处死裸鼠，通过组织学观察了解成肌细胞在凝胶内的存活情况；(6)评价凝胶介质对成肌细胞向成肌、成骨、成脂方向分化能力的影响。以常规胶原包被表面培养的成肌细胞为对照组，C/GP/Co凝胶表面上进行培养的成肌细胞为C/GP/Co组。成肌分化通过融合指数(FI)和肌管大小进行评价；成骨分化通过碱性磷酸酶(ALP)活性、茜素红染色进行评价，成脂分化通过油红O染色进行评价。2.利用逆转录病毒载体将msx1转染到成肌细胞，并评价经基因修饰后的成肌细胞后是否能保持“干性”：(1)逆转录病毒转染msx1基因到成肌细胞并采用免疫印迹实验(WB)予以鉴定；(2)比较经msx1基因修饰(实验组)和未经该基因修饰的成肌细胞(对照组)在细胞形态、衰老细胞比例、增殖能力、分化相关标记分子表达、迁移能力等指标上的差异。细胞形态通过透射电镜(TEM)进行观察，衰老细胞比例通过β-半乳糖苷酶染色实验获取相关数据，增殖能力通过细胞增殖实验、增殖指数(PI)、Ki67指数及集落形成率进行评价，分化相关标记包括myoD(成肌分化因子)、Pax-7(配对盒转录因子-7)和SSEA-1(阶段特异性胚胎抗原-1)，迁移能力通过Transwell实验进行评价；(3)比较同代实验组与对照组成肌细胞在scid/mdx小鼠模型中的移植效率以及不同代次的实验组细胞在该模型中的移植效率；3.评价经msx1基因修饰后成肌细胞的成骨效能：(1)通过成骨诱导分化实验比较实验组与对照组成肌细胞碱性磷酸酶(ALP)活性、钙盐沉积面积及成骨分化相关基因表达水平；(2)将成肌细胞与C/GP/Co凝胶复合构建可注射组织工程复合物，在裸鼠体内进行异位成骨实验，通过大体观察、骨密度检测、组织切片H&E染色以及骨钙素免疫组织化学染色等方法比较对照组与实验组成骨质量。
结果：1.从小鼠骨骼肌内分离出带有GFP标记的成肌细胞并进行培养、传代；成功地制备了C/GP/Co凝胶支架并观察了该材料的超微结构；该支架材料无毒，并且对成肌细胞具有良好的生物相容性，对成肌细胞体外扩增期间多向分化能力的保持具有促进作用：(1)培养的成肌细胞结蛋白染色阳性，成肌诱导条件下能形成肌管，肌球蛋白重链染色阳性；(2)制备的C/GP/Co复合物在37℃时约10min内由液体形成水凝胶，扫描电镜下该凝胶为蜂窝状均质多孔结构；(3)浸提液培养实验中支架毒性分级为0到1级，说明该材料无毒；(4)成肌细胞在凝胶二维及三维培养时均生长良好；(5)凝胶携带成肌细胞移植到裸鼠皮下4w后仍可观察到绿色荧光，组织学检查发现支架上有大量成肌细胞存活；(6)凝胶介质对成肌细胞的成肌、成骨及成脂分化均有增强作用。在成肌分化实验中，C/GP/Co组细胞分化后的融合指数及肌管大小均显著增加(p<0.05)；在成骨分化实验中，C/GP/Co组细胞在各检测时相点的ALP活性均显著增强(p<0.05)，在分化21d时的矿化面积百分比也显著增加(p<0.05)；在成脂分化实验中，C/GP/Co组细胞内油红O染色阳性区域面积显著增加(p<0.05)。2.Msx1转染成肌细胞后能有助于细胞保持“干性”：(1)逆转录病毒成功转染成肌细胞并使后者异位表达msx1蛋白；(2)实验组成肌细胞较对照组成肌细胞相比：在TEM镜下具有更小而圆的外型及更年轻化的超微结构；衰老细胞百分比显著降低(p<0.05)；具有更高的增殖指数(p<0.05)、Ki67指数(p<0.05)及集落形成率(p<0.05)；在免疫荧光阳性率和平均光密度值两项评价指标中，SSEA-1和Pax-7表达水平均上调而myoD表达水平下调(p<0.05)；Transwell实验中表现出更强的迁移能力(p<0.05)，且该能力与SDF-1/CXCR4(基质细胞衍生因子-1/细胞表面趋化因子受体4)轴有关；(3)移植到scid/mdx小鼠模型后实验组细胞存活率更高，随时间推移有更多细胞在宿主体内增殖并分化融合并表达肌营养不良蛋白(p<0.05)，并且这种高移植效率在细胞培养传代过程中能有效保持。3.实验组成肌细胞与对照组相比具有更高的成骨效能：(1)体外成骨诱导实验中，各检测时相点实验组的ALP活性、矿化面积百分比均显著高于对照组，成骨相关基因的表达水平也显著高于对照组(p<0.05)；(2)异位成骨实验中实验组可注射组织工程复合物：大体观察中形成的骨样组织体积及硬度更大，骨密度(p<0.05)、新生骨小梁面积百分比(p<0.05)以及骨钙素染色阳性区域面积均显著高于对照组。
结论：1.C/GP/Co是一种可注射的温敏凝胶支架，制备简便，在体内外实验均表现出对成肌细胞具有良好的生物相容性，且有助于体外扩增培养期间保持成肌细胞的多向分化潜能，是成肌细胞理想的支架材料。2.Msx1基因修饰成肌细胞将有助于促使成肌细胞在体外培养过程中保持“干性”，增强成肌细胞的移植效率，并且该能力在传代过程中能够有效保持。3.经msx1基因修饰的成肌细胞具有更强的成骨效能，与C/GP/Co凝胶构建可注射温敏支架用于骨组织工程确实可行。
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.
引文
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