Improvements Of Biological Properties Of Bio-artificial Muscle (BAM) And The Applied Study Of PLG Biodegradable Scaffold As An In Vivo Vehicle For Skeletal Muscle Cells

PartⅠGeneration of Bio-artificial muscle (BAM) andimprovement for innervationObjective To generate bio-artificial muscle (BAM) in vitro and analyze isoform typesof myosin heavy chain (MHC) in BAM.To improve sensitivity of BAM to neurotrans-mitters,we aimed to upregulate the number of AchR on BAM and induce formation ofmature clusters of AchR.Methods The C3H murine primary myoblasts that expressed GFP was mixed with120μl Fibrin hydrogel in order to generate BAM in vitro.QPCR was used to analyzeisoform types of MHC.Agrin was employed to increase the number of AchR in BAM.Laminin was used to induce mature clusters of AchR.The morphology of AchR wasobserved by confocal microscope.Results The isoform of MHC expressed in BAM without any treatment was atperinatal stage.The isoform of MHC expressed in cultured cells was at embryonic stage.Agrin treatment increased the number of AchR in BAM.Laminin treatment improvedmorphology and structure of AchR and induced mature clusters of AchR.Conclusion From perspective of development,the isoforms of MHC that wereexpressed by BAM were closer to those in mature muscle tissue.Agrin treatmentupregulated the number of AchR.Meanwhile,the addition of Laminin into BAM improvedmorphological features of BAM and induced clusters of mature AchR.It improved thestructural advantages of BAM and provided the experimental foundation for innervations. PartⅡVEGF improves formation and distribution of vascular networksurrounding bio-artificial muscle (BAM)Objective To improve the physiological properties of bio-artificial muscle by inducingneovascularization and blood vessel formation into bio-artificial muscle (BAM) andincreasing angiogensis to allow BAM long-term survival and to exert its function in host.Methods We used lenti-virus to transduce mouse myoblasts allowing them to expressVEGF and GFP,respectively.Then we fabricated bio-artificial muscle from these cells invitro by culturing them together in order to allow them to form fused cells.The resultingBAMs containing fused cells were transplanted subcutaneously into the dorsal region of7-week-old C57 mice.The confocal microscope was used to analyze structure andmorphology of those BAMs as well as cell survival rate both prior to and aftertransplantation.ELISA was employed to determine the amount of VEGF produced byBAMs.The vascular network was evaluated by Evan's staining.Results The lenti-virus transduced myoblasts were capable of differentiating intomature muscle fiber under differentiation culture conditions,indicating transduction did notinfluence differentiation of the myoblasts.We successfully fabricated BAMs from theseVEGF expressing myoblasts,these BAMs resemble physiological muscle in terms ofphysical and physiological properties.But the type of BAMs had no blood vessels and wasnot innervated before implantation.These VEGF-expressing BAMs,when implanted invivo,were capable of inducing vascular network formation around the implants,improvingthe survival rate of the muscle fibers in vivo.Conclusion VEGF expressed by myofibers were capable of strongly inducing localangiogenesis around the implant site,which could last over a few months and providedphysical and physiological basis for incorporation of BAMs to host and improved thesurvival rate of muscle cells after transplantation.These bio-artificial muscles can also beused as potential reversible gene therapy approach for treating a number of vasculardiseases. PartⅢThe study for testing PLG biodegradable scaffold as a vehiclefor skeletal muscle cells and the effects of NK cell depletion on viability ofmuscle cells seeded on PLG scaffoldObjective To use degradable PLG scaffold as a vehicle for cell delivery for the purposeof regenerating muscle injuries and test its ability to maintain the cell viability in vivo.Tocharacterize the effects resulted from depletion of NK cells in nude mice on the viability ofcells in PLG scaffolds.Methods We prepared degradable PLG scaffold using gas foaming technique.Humanmyoblasts were seeded onto the scaffold for testing if they can differentiate into maturemuscle fibers by staining tropomyosin.The scaffolds with seeded cells were thentransplanted into the subcutaneous region of the SCID mice.In vivo cell viability on thePLG scaffold was evaluated by live/dead staining and quantified by confocal microscopy.Results We successfully generated PLG scaffold.Myoblasts were able to differentiateinto mature muscle cells.Compared to no-tension control,the viability of muscle cells onPLG scaffold was increased.Four weeks post transplantation,the cell density on thescaffold decreased by 78%.This might be due to the effect of other immune cells like NKcells or macrophages.etc.After depleting NK cells we confirmed that cells can survive onthe PLG scaffold for as long as four weeks.The cell viability reached 34.72%,much higherthan that (22.72%) in SCID mice without depletion of NK cells.Conclusion PLG scaffold can be used as a vehicle for delivering myoblasts.It providestension for myoblast differentiation.Depletion of NK cells increased cell viability.Thisstudy provided basis for using PLG scaffold for the purpose of delivering cells in vivo.Webelieve that PLG scaffold will play an important role in clinical gene and cell therapy.