组织工程人工肌肉生物学特性的改善及PLG生物支架作为骨骼肌细胞载体的应用研究
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摘要
第一部分组织工程人工肌肉的构建与评价及神经支配特性的改进
目的：体外构建组织工程人工肌肉，并分析组织工程人工肌肉中肌球蛋白重链亚型(MHC)表达情况。为进一步提高人工肌肉对神经递质的敏感性，我们尝试上调乙酰胆碱受体在人工肌肉表面的数目并诱导人工肌肉形成形态成熟的乙酰胆碱受体。
方法：将表达绿色荧光蛋白的C3H小鼠原代成肌细胞与120μl Fibrin水凝胶混合，体外构建人工肌肉。利用实时定量PCR对平面培养细胞和组织工程人工肌肉中的MHC亚型进行分析。利用Agrin蛋白上调乙酰胆碱受体在人工肌肉表面的数目。用Laminin蛋白在人工肌肉表面诱导形成形态成熟的乙酰胆碱受体。并用激光共聚焦显微镜计数乙酰胆碱受体数目，观察乙酰胆碱受体形态。
结果：未经过任何处理的组织工程人工肌肉的MHC亚型处于胚胎期和成熟期之间，相当于围周期(perinatal)的发育水平。平面培养的细胞中MHC亚型为胚胎期水平。Agrin蛋白诱导，增加了乙酰胆碱受体在人工肌肉表面的数目。Laminin蛋白处理，改善了乙酰胆碱受体的结构，形成形态成熟乙酰胆碱受体。
结论：从发育学意义上看，人工组织肌肉表达更加成熟的MHC亚型，更类似成熟肌肉组织。Agrin蛋白诱导成功增加乙酰胆碱受体数目。Laminin蛋白诱导，改善了乙酰胆碱受体的结构，使之变得更加成熟有利于更多神经突触的形成，进一步提高了人工组织肌肉在神经生物学上的结构优势，为在人工肌肉中引入神经分布奠定了基础。
第二部分VEGF改善组织工程人工肌肉周围血管网络分布的研究
目的：改善人工组织工程肌肉的生物特性，增加其周围血管生成，达到增强其与宿主循环系统营养与代谢交流的目的，使之在移植后更好与宿主整合，长期生存，在宿主体内发挥作用。
方法：逆转录病毒感染成肌细胞，使其分别表达VEGF和GFP。用分化培养液，体外融合表达VEGF和GFP的成肌细胞，以形成杂交人工肌肉。用共聚焦显微镜观察杂交人工肌肉组织形态和结构，以及移植前后细胞存活率。用免疫酶连反应检测VEGF含量。用伊文思蓝染色评价人工肌肉周围血管网络形成情况。
结果：表达VEGF的细胞可在分化条件下分化为成熟肌细胞，并形成肌纤维，肌细胞分化过程不受VEGF生长因子表达的抑制。用这种细胞在体外成功构建组织工程人工肌肉，结构形态与生理状态下的肌肉相似。VEGF的表达不影响杂交人工肌肉中肌细胞的存活率。调节杂交比例可调节VEGF的生成量。移植到小鼠皮下之后，表达VEGF的人工肌肉明显增加人工肌肉周围血管网络的生成，提高肌细胞的存活率。
结论：肌细胞分泌的VEGF可在移植区域强烈促进局部微血管生成，这种影响可持续几个月，为其移植后与宿主整合提供了生理基础，也有利于肌细胞所表达的治疗性蛋白尽可能多进入血液循环，在宿主体内发挥有效作用。
第三部分PLG生物支架作为骨骼肌细胞载体的研究及NK细胞移除对支架上肌细胞体内存活率的影响
目的：评价人工合成的可降解生物支架PLG作为成骨骼肌细胞载体的效果，以及去除裸鼠NK细胞对PLG支架上成熟肌细胞成活率的影响。方法：利用高压气泡法成功制备可降解的多孔PLG生物支架。将人类原代骨骼成肌细胞接种至PLG生物支架上，检测成肌细胞在PLG支架是否可体外分化为成熟肌细胞。将接种肌细胞的PLG生物支架移植至免疫缺陷型小鼠体内，观察肌细胞在体内存活率。利用抗NK细胞抗体去除裸鼠体内NK细胞，检测去除NK细胞后，PLG生物支架上肌细胞的存活率。
结果：成功制备PLG生物支架。成肌细胞可在PLG生物支架上分化为成熟肌细胞。与无张力组相比，PLG支架显著提供肌细胞在体存活率。PLG在移植四周后，PLG生物支架上的肌细胞密度降低了78％。去除裸鼠体内NK细胞的影响，在第4周，肌纤维中肌细胞的存活率达34．72％，远高于未去除NK细胞组的存活率22．72％。经过NK细胞抗体处理过的动物肌纤维绿色荧光强度比未经过处理组高2倍以上。
结论：PLG生物支架可作为成肌细胞的载体，为肌细胞提供张力，成肌细胞在其上可分化为成熟肌细胞，形成肌纤维，并长期存活。去除NK细胞可使肌细胞在移植之后存活率提高。该实验为肌细胞以及PLG生物支架用作基因疗法的载体提供了实验依据。
PartⅠGeneration of Bio-artificial muscle (BAM) andimprovement for innervation
Objective 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 scaffold
Objective 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.
目的：体外构建组织工程人工肌肉，并分析组织工程人工肌肉中肌球蛋白重链亚型(MHC)表达情况。为进一步提高人工肌肉对神经递质的敏感性，我们尝试上调乙酰胆碱受体在人工肌肉表面的数目并诱导人工肌肉形成形态成熟的乙酰胆碱受体。
方法：将表达绿色荧光蛋白的C3H小鼠原代成肌细胞与120μl Fibrin水凝胶混合，体外构建人工肌肉。利用实时定量PCR对平面培养细胞和组织工程人工肌肉中的MHC亚型进行分析。利用Agrin蛋白上调乙酰胆碱受体在人工肌肉表面的数目。用Laminin蛋白在人工肌肉表面诱导形成形态成熟的乙酰胆碱受体。并用激光共聚焦显微镜计数乙酰胆碱受体数目，观察乙酰胆碱受体形态。
结果：未经过任何处理的组织工程人工肌肉的MHC亚型处于胚胎期和成熟期之间，相当于围周期(perinatal)的发育水平。平面培养的细胞中MHC亚型为胚胎期水平。Agrin蛋白诱导，增加了乙酰胆碱受体在人工肌肉表面的数目。Laminin蛋白处理，改善了乙酰胆碱受体的结构，形成形态成熟乙酰胆碱受体。
结论：从发育学意义上看，人工组织肌肉表达更加成熟的MHC亚型，更类似成熟肌肉组织。Agrin蛋白诱导成功增加乙酰胆碱受体数目。Laminin蛋白诱导，改善了乙酰胆碱受体的结构，使之变得更加成熟有利于更多神经突触的形成，进一步提高了人工组织肌肉在神经生物学上的结构优势，为在人工肌肉中引入神经分布奠定了基础。
第二部分VEGF改善组织工程人工肌肉周围血管网络分布的研究
目的：改善人工组织工程肌肉的生物特性，增加其周围血管生成，达到增强其与宿主循环系统营养与代谢交流的目的，使之在移植后更好与宿主整合，长期生存，在宿主体内发挥作用。
方法：逆转录病毒感染成肌细胞，使其分别表达VEGF和GFP。用分化培养液，体外融合表达VEGF和GFP的成肌细胞，以形成杂交人工肌肉。用共聚焦显微镜观察杂交人工肌肉组织形态和结构，以及移植前后细胞存活率。用免疫酶连反应检测VEGF含量。用伊文思蓝染色评价人工肌肉周围血管网络形成情况。
结果：表达VEGF的细胞可在分化条件下分化为成熟肌细胞，并形成肌纤维，肌细胞分化过程不受VEGF生长因子表达的抑制。用这种细胞在体外成功构建组织工程人工肌肉，结构形态与生理状态下的肌肉相似。VEGF的表达不影响杂交人工肌肉中肌细胞的存活率。调节杂交比例可调节VEGF的生成量。移植到小鼠皮下之后，表达VEGF的人工肌肉明显增加人工肌肉周围血管网络的生成，提高肌细胞的存活率。
结论：肌细胞分泌的VEGF可在移植区域强烈促进局部微血管生成，这种影响可持续几个月，为其移植后与宿主整合提供了生理基础，也有利于肌细胞所表达的治疗性蛋白尽可能多进入血液循环，在宿主体内发挥有效作用。
第三部分PLG生物支架作为骨骼肌细胞载体的研究及NK细胞移除对支架上肌细胞体内存活率的影响
目的：评价人工合成的可降解生物支架PLG作为成骨骼肌细胞载体的效果，以及去除裸鼠NK细胞对PLG支架上成熟肌细胞成活率的影响。方法：利用高压气泡法成功制备可降解的多孔PLG生物支架。将人类原代骨骼成肌细胞接种至PLG生物支架上，检测成肌细胞在PLG支架是否可体外分化为成熟肌细胞。将接种肌细胞的PLG生物支架移植至免疫缺陷型小鼠体内，观察肌细胞在体内存活率。利用抗NK细胞抗体去除裸鼠体内NK细胞，检测去除NK细胞后，PLG生物支架上肌细胞的存活率。
结果：成功制备PLG生物支架。成肌细胞可在PLG生物支架上分化为成熟肌细胞。与无张力组相比，PLG支架显著提供肌细胞在体存活率。PLG在移植四周后，PLG生物支架上的肌细胞密度降低了78％。去除裸鼠体内NK细胞的影响，在第4周，肌纤维中肌细胞的存活率达34．72％，远高于未去除NK细胞组的存活率22．72％。经过NK细胞抗体处理过的动物肌纤维绿色荧光强度比未经过处理组高2倍以上。
结论：PLG生物支架可作为成肌细胞的载体，为肌细胞提供张力，成肌细胞在其上可分化为成熟肌细胞，形成肌纤维，并长期存活。去除NK细胞可使肌细胞在移植之后存活率提高。该实验为肌细胞以及PLG生物支架用作基因疗法的载体提供了实验依据。
PartⅠGeneration of Bio-artificial muscle (BAM) andimprovement for innervation
Objective 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 scaffold
Objective 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.
引文
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4. Nagy JA, Dvorak AM, Dvorak HF. VEGF-A and the Induction of Pathological Angiogenesis. Annual Review of Pathology: Mechanisms of Disease. 2006, 2:251-275.
5. De Coppi P, Delo D, Farrugia L, et al. Angiogenic gene-modified muscle cells for enhancement of tissue formation. Tissue Eng. 2005,11:1034-1044.
6. Suzuki K, Murtuza B, Smolenski RT, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation. 2001,104:1207-1212.
7. Powell CA, Shansky J, Tatto MD, et al. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy. Hum. Gene Ther. 1999,10: 565-577.
8. Powell CA, Shansky J, Tatto MD, et al. Bioartificial muscles in gene therapy. In Methods in Molecular Medicine, vol. 69: Gene Therapy Protocols (J. R. Morgan,Ed.). 2001,149 - 160. Humana Press, Totowa, NJ.
9. Powell CA, Smiley BL, Mills J, et al. Mechanical stimulation improves tissue-engineered human skeletal muscle. Am. J. Physiol. Cell Physiol. 2002, 283:1557-1565.
10. van Deutekom JC, Hoffman EP, Huard J. Muscle maturation: implications for gene therapy. Mol Med Today. 1998, 4(5):214-220.
11. Shoemaker K, Rubin J, Zumbro GL, et al. Evans blue and gentian violet: alternatives to methylene blue as a surgical marker dye. J Thorac Cardiovasc Surg.1996,112(2):542-544.
12. Gussoni E, Blau HM, Kunkel LM. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat. Med. 1997, 3: 970-977.
13. Hacein-Bey-Abina S, Kalle CV, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCED-Xl. Science. 2003, 302:415-419.
1. Skuk D, Caron NJ, Goulet M, et al. Resetting the problem of cell death following muscle-derived cell transplantation: detection, dynamics and mechanisms. J Neuropathol Exp Neurol. 2003, 62(9):951-967.
2. Beauchamp JR, Morgan JE, Pagel CN, et al. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol. 1999,144(6):1113-1122.
3. Qu Z, Balkir L, van Deutekom JC, et al. Development of approaches to improve cell survival in myoblast transfer therapy. J Cell Bio/. 1998,142(5):1257-1267.
4. Hill E, Boontheekul T, Mooney DJ. Designing scaffolds to enhance transplanted myoblast survival and migration. Tissue Eng. 2006,12(5): 1295-1304.
5. Hill E, Boontheekul T, Mooney DJ. Regulating activation of transplanted cells controls tissue regeneration. Proc Natl Acad Sci USA. 2006,103(8):2494-2499.
6. Smith MK, Peters MC, Richardson TP, et al. Locally enhanced angiogenesis promotes transplanted cell survival. Tissue Eng. 2004,10(l):63-71.
7. Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol 2001,19(11): 1029-1034.
8. Lee KY, Peters MC, Anderson KW, et al. Controlled growth factor release from synthetic extracellular matrices. Nature. 2000, 408(6815):998-1000.
9. Cohen S, Yoshioka T, Lucarelli M, et al. Controlled delivery systems for proteins based on poly (lactic/glycolic acid) microspheres. Pharm Res. 1991, 8(6):713-720.
10. Harris LD, Kim BS, Mooney DJ. Open pore biodegradable matrices formed with gas foaming. J Biomed Mater Res. 1998, 42(3):396-402.
11. Mooney DJ, Baldwin DF, Suh NP, et al. Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents.Biomaterials. 1996,17(14): 1417-1422.
12. Hennessey JV, Chromiak JA, Delia Ventura S, et al. Increase in percutaneous muscle biopsy yield with a suction-enhancement technique. J Appl Physiol.1997', 82(6): 1739-1742.
13. Powell C, Shansky J, Tatto MD. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy. Hum. Gene Ther. 1999,10: 565-577.
14. Blau HM, Springer ML. Muscle-mediated gene-therapy. N Engl J Med. 1995,333(23):1554-1556.
15. Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol. 2001,19(11):1029-1034.
1. Huard J, Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am. 2002, 84: 822-832.
2. Grefte S, Marie A, Toensma R, et al. Skeletal muscle development and regeneration.Stem cells and development. 2007,16:857-868.
3. Gates C, Huard J. Management of skeletal muscle injuries in military personnel.Operative Techniques in Sports Medicine. 2005,13(4): 247-256.
4. Alderton JM, RA Steinhardt. Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. J Bio Chem. 2000, 275:9452-9460.
5. Armstrong RB. Initial events in exercise -induced muscular injury. Med Sci Sports Exerc. 1990, 22:429-435.
6. Belcastro AN, Shewchunk LD, Raj DA. Exercise-induced muscle injury: a calpain hypothesis. Mol Cell Biochem. 1998,179:135-145.
7. Hurme T, H Kalimo, M Lehto, et al. Healing of skeletal muscle injury: an ultrastructure and immunohistochemical study. Med Sci Sports Exerc. 1991, 23:801-810.
8. Orimo S, Hiyamuta E, Arahata K, et al. Analysis of inflammatory cells and complement C3 in bupivacaine-induced myonecrosis. Muscle Nerve. 1991, 14:515-520.
9. Tidball JG. Inflammatory cell response to acute muscle injury. Med Sci Sports Exercise. 1995, 27:1022-1032.
10. Almekinders LC, Gilbert JA. Healing of experimental muscle strains and the effects of nonsteroidal anti-inflammatory medication. Am J Sports Med. 1986, 14 :303-308.
11. Lescaudron L, Peltekian E, Fontaine-Perus J, et al. Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant. Neuromuscular Disorders. 1999, 9: 72-80.
12. Merly F, Lescaudron L, Rouaud T, et al. Macrophages enhance muscle satellite cell proliferation and delay their differentiation. Muscle Nerve. 1999, 22: 724-732.
13. Robertson TA, Maley MA, Grounds MD, et al. The role of macrophages in skeletal
13. Robertson TA, Maley MA, Grounds MD, et al. The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis. Exp Cell Res. 1993,207: 321-331.
14. Peault B, Torrente Y, Cossu G, et al. Stem and Progenitor Cells in Skeletal Muscle Development, Maintenance, and Therapy. Mol Then 2007,15(5): 867-877.
15. Cossu G. Satellite cells, myoblasts and other occasional myogenic progenitors:possible origin, phenotypic features and role in muscle regeneration. Seminars in Cell & Developmental Biology. 2005,16(4-5): 623-631.
16. Hill E, Boontheekul T, Mooney DJ. Regulating activation of transplanted cells controls tissue regeneration. PNAS. 2006,103(8):2494-2499.
17. Grefte S. Skeletal muscle development and regeneration. Stem Cells Dev. 2007,16(5): 857-868.
18. Langer R, Vacanti JP. Tissue engineering. Science. 1993, 260:920-926.
19. Griffith LG, Naughton G. Tissue engineering--current challenges and expanding opportunities. Science. 2002, 295:1009-1014.
20. Smythe GM, Grounds MD. Problems and solutions in myoblast transfer therapy.J.CelLMol.Med. 2001, 5(1): 33-47.
21. Skuk D, Tremblay JP. Myoblast transplantation: the current status of a potential therapeutic tool for myopathies. Muscle Res Cell Motil. 2003, 24:285-300.
22. Skuk D. First test of a "high-density injection" protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up. Neuromuscul. Disord. 2007,17:38-46.
23. Yang SY, Goldspink G. Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Letters. 2006, 580(10):2530.
24. Saxena AK, Marler J, Benvenuto M, et al. Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers: preliminary studies. Tissue Eng. 1999, 5:525-532.
25. Chromiak JA, Shansky J, Perrone C, et al. Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids. In Vitro Cell Dev. Biol. Anim. 1998, 34:694-703.
26. Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 2001,91:534-551.
27. Wagers AJ, Conboy IM. Cellular and molecular signatures of muscle regeneration:current concepts and controversies in adult myogenesis. Cell. 2005, 122:659-667.
28. Charge SBP, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev. 2004, 84:209-238.
29. Gallagher JT, Lyon M. Molecular structure of Heparan Sulfate and interactions with growth factors and morphogens. Proteoglycans: structure, biology and molecular interactions. CRC Press. 2000.27-59.
30. Johnson SE, Allen RE. Activation of Skeletal Muscle Satellite Cells and the Role of Fibroblast Growth Factor Receptors. Exp Cell Res. 1995, 219(2): 449-453.
31. Yang S, Alnaqeeb M, Simpson H, et al. Cloning and characterization of an IGF-1 isoform expressed in skeletal muscle subjected to stretch. J Muscle Res Cell Motil.1996,17:487-495.
32. Hill M, Goldspink G. Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage. J Physiol. 2003, 549:409-418.
33. Jacquemin V, Furling D, Bigot A, et al. IGF-1 induces human myotube hypertrophy by increasing cell recruitment. Exp Cell Res. 2004, 299:148-158.
34. Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials. 2003, 24:4337-4351.
2. Vilquin JT. Myoblast transplantation: Clinical trials and perspectives. A mini review. Acta Myol. 2005, 24:119-127.
3. Mouly V, Aamiri A, Perie S, et al. Myoblast transfer therapy: Is there any light at the end of the tunnel? Acta. Myol. 2005, 24:128-133.
4. Bach AD, Beier JP, Stern-Staeter J, et al. Skeletal muscle tissue engineering. J. Cell Mol. Med. 2004, 8:413-422.
5. Bian W, Bursac N. Tissue engineering of functional skeletal muscle: challenges and recent advances. IEEE engineering in medicine and biology magazine. 2008, September/Octorber:109-113.
6. Vandenburgh HH. Functional assessment and tissue design of skeletal muscle. Ann. NY Acad. Sci. 2002, 961:201-202.
7. Levenberg S, Rouwkema J, Macdonald M, et al. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol. 2005, 23:879-884.
8. Bach AD, Beier JP, Stark GB. Expression of Trisk 51, agrin and nicotinic-acetycholine receptor epsilon-subunit during muscle development in a novel three-dimensional muscleneuronal co-culture system. Cell Tissue Res. 2003,314:263-274.
9. De Coppi P, Delo D, Farrugia L, et al. Angiogenic gene-modified muscle cells for enhancement of tissue formation. Tissue Eng. 2005,11:1034-1044.
10. Suzuki K, Murtuza B, Smolenski RT, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation. 2001,104:1207-1212.
11. Bursac N, Loo Y, Leong K, et al. Novel anisotropic engineered cardiac tissues: studies of electrical propagation. Biochem. Biophys. Res. Commun. 2007, 361:847-853.
1. Mooney DJ, Mikos AG. Growing new organs. 5c/. Am. 1999, 280: 60-65.
2. Allen DL, Leinwand LA. Postnatal Myosin Heavy Chain Isoform Expression in Normal Mice and Mice Null for IIb or IId Myosin Heavy Chains. Dev Biol. 2001,229(2), 383-395.
3. Lu BD, Allen DL, Leinwand LA,et al. Spatial and temporal changes in myosin heavy chain gene expression in skeletal muscle development.Dev Biol. 1999,216(l):312-326.
4. Berg JS, Powell BC, Cheney RE. A millennial myosin census. Mol Biol. Cell 2001,12 (4): 780-94.
5. Bach AD, Beier JP, Stern-Staeter J, et al. Skeletal muscle tissue engineering.Journal of Cellular and Molecular Medicine. 2007, 8(4), 413-422.
6. Bowe MA, Fallon JR. The role of agrin in synapse formation. Annu Rev Neurosci.1995,18:443-62.
7. Bach AD, Beier JP, Stark GB. Expression of Trisk 51, agrin and nicotinic-acetycholine receptor epsilon-subunit during muscle development in a novel three-dimensional muscleneuronal co-culture system. Cell Tissue Res. 2003, 314:263-274.
8. Sanes JR, Lichtman JW. Development of the vertebrate neuromuscular junction.Annual Review of Neuroscience. 1999, 22: 389-442.
9. Kummer TT, Misgeld T, Lichtman JW. Nerve-independent formation of a topologically complex postsynaptic apparatus. J Cell Biol. 2004,164(7):1077-1087.
10. Nishimune H, Valdez G, Jarad G, et al. Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction. J Cell Biol. 2008,182(6):1201-1215.
1. Blau HM, Springer ML. Muscle-mediated gene-therapy. N Engl J Med. 1995,333(23): 1554-1556.
2. Quenneville SP, Chapdelaine P, Rousseau J, et al. Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts. Mol Ther. 2004,10(4):679-687.
3. Vandenburgh HH, et al. Tissue-engineered skeletal muscle organoids for reversible gene therapy. Hum. Gene Ther. 1996, 7: 2195-2200.
4. Nagy JA, Dvorak AM, Dvorak HF. VEGF-A and the Induction of Pathological Angiogenesis. Annual Review of Pathology: Mechanisms of Disease. 2006, 2:251-275.
5. De Coppi P, Delo D, Farrugia L, et al. Angiogenic gene-modified muscle cells for enhancement of tissue formation. Tissue Eng. 2005,11:1034-1044.
6. Suzuki K, Murtuza B, Smolenski RT, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation. 2001,104:1207-1212.
7. Powell CA, Shansky J, Tatto MD, et al. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy. Hum. Gene Ther. 1999,10: 565-577.
8. Powell CA, Shansky J, Tatto MD, et al. Bioartificial muscles in gene therapy. In Methods in Molecular Medicine, vol. 69: Gene Therapy Protocols (J. R. Morgan,Ed.). 2001,149 - 160. Humana Press, Totowa, NJ.
9. Powell CA, Smiley BL, Mills J, et al. Mechanical stimulation improves tissue-engineered human skeletal muscle. Am. J. Physiol. Cell Physiol. 2002, 283:1557-1565.
10. van Deutekom JC, Hoffman EP, Huard J. Muscle maturation: implications for gene therapy. Mol Med Today. 1998, 4(5):214-220.
11. Shoemaker K, Rubin J, Zumbro GL, et al. Evans blue and gentian violet: alternatives to methylene blue as a surgical marker dye. J Thorac Cardiovasc Surg.1996,112(2):542-544.
12. Gussoni E, Blau HM, Kunkel LM. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat. Med. 1997, 3: 970-977.
13. Hacein-Bey-Abina S, Kalle CV, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCED-Xl. Science. 2003, 302:415-419.
1. Skuk D, Caron NJ, Goulet M, et al. Resetting the problem of cell death following muscle-derived cell transplantation: detection, dynamics and mechanisms. J Neuropathol Exp Neurol. 2003, 62(9):951-967.
2. Beauchamp JR, Morgan JE, Pagel CN, et al. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol. 1999,144(6):1113-1122.
3. Qu Z, Balkir L, van Deutekom JC, et al. Development of approaches to improve cell survival in myoblast transfer therapy. J Cell Bio/. 1998,142(5):1257-1267.
4. Hill E, Boontheekul T, Mooney DJ. Designing scaffolds to enhance transplanted myoblast survival and migration. Tissue Eng. 2006,12(5): 1295-1304.
5. Hill E, Boontheekul T, Mooney DJ. Regulating activation of transplanted cells controls tissue regeneration. Proc Natl Acad Sci USA. 2006,103(8):2494-2499.
6. Smith MK, Peters MC, Richardson TP, et al. Locally enhanced angiogenesis promotes transplanted cell survival. Tissue Eng. 2004,10(l):63-71.
7. Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol 2001,19(11): 1029-1034.
8. Lee KY, Peters MC, Anderson KW, et al. Controlled growth factor release from synthetic extracellular matrices. Nature. 2000, 408(6815):998-1000.
9. Cohen S, Yoshioka T, Lucarelli M, et al. Controlled delivery systems for proteins based on poly (lactic/glycolic acid) microspheres. Pharm Res. 1991, 8(6):713-720.
10. Harris LD, Kim BS, Mooney DJ. Open pore biodegradable matrices formed with gas foaming. J Biomed Mater Res. 1998, 42(3):396-402.
11. Mooney DJ, Baldwin DF, Suh NP, et al. Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents.Biomaterials. 1996,17(14): 1417-1422.
12. Hennessey JV, Chromiak JA, Delia Ventura S, et al. Increase in percutaneous muscle biopsy yield with a suction-enhancement technique. J Appl Physiol.1997', 82(6): 1739-1742.
13. Powell C, Shansky J, Tatto MD. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy. Hum. Gene Ther. 1999,10: 565-577.
14. Blau HM, Springer ML. Muscle-mediated gene-therapy. N Engl J Med. 1995,333(23):1554-1556.
15. Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol. 2001,19(11):1029-1034.
1. Huard J, Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am. 2002, 84: 822-832.
2. Grefte S, Marie A, Toensma R, et al. Skeletal muscle development and regeneration.Stem cells and development. 2007,16:857-868.
3. Gates C, Huard J. Management of skeletal muscle injuries in military personnel.Operative Techniques in Sports Medicine. 2005,13(4): 247-256.
4. Alderton JM, RA Steinhardt. Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. J Bio Chem. 2000, 275:9452-9460.
5. Armstrong RB. Initial events in exercise -induced muscular injury. Med Sci Sports Exerc. 1990, 22:429-435.
6. Belcastro AN, Shewchunk LD, Raj DA. Exercise-induced muscle injury: a calpain hypothesis. Mol Cell Biochem. 1998,179:135-145.
7. Hurme T, H Kalimo, M Lehto, et al. Healing of skeletal muscle injury: an ultrastructure and immunohistochemical study. Med Sci Sports Exerc. 1991, 23:801-810.
8. Orimo S, Hiyamuta E, Arahata K, et al. Analysis of inflammatory cells and complement C3 in bupivacaine-induced myonecrosis. Muscle Nerve. 1991, 14:515-520.
9. Tidball JG. Inflammatory cell response to acute muscle injury. Med Sci Sports Exercise. 1995, 27:1022-1032.
10. Almekinders LC, Gilbert JA. Healing of experimental muscle strains and the effects of nonsteroidal anti-inflammatory medication. Am J Sports Med. 1986, 14 :303-308.
11. Lescaudron L, Peltekian E, Fontaine-Perus J, et al. Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant. Neuromuscular Disorders. 1999, 9: 72-80.
12. Merly F, Lescaudron L, Rouaud T, et al. Macrophages enhance muscle satellite cell proliferation and delay their differentiation. Muscle Nerve. 1999, 22: 724-732.
13. Robertson TA, Maley MA, Grounds MD, et al. The role of macrophages in skeletal
13. Robertson TA, Maley MA, Grounds MD, et al. The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis. Exp Cell Res. 1993,207: 321-331.
14. Peault B, Torrente Y, Cossu G, et al. Stem and Progenitor Cells in Skeletal Muscle Development, Maintenance, and Therapy. Mol Then 2007,15(5): 867-877.
15. Cossu G. Satellite cells, myoblasts and other occasional myogenic progenitors:possible origin, phenotypic features and role in muscle regeneration. Seminars in Cell & Developmental Biology. 2005,16(4-5): 623-631.
16. Hill E, Boontheekul T, Mooney DJ. Regulating activation of transplanted cells controls tissue regeneration. PNAS. 2006,103(8):2494-2499.
17. Grefte S. Skeletal muscle development and regeneration. Stem Cells Dev. 2007,16(5): 857-868.
18. Langer R, Vacanti JP. Tissue engineering. Science. 1993, 260:920-926.
19. Griffith LG, Naughton G. Tissue engineering--current challenges and expanding opportunities. Science. 2002, 295:1009-1014.
20. Smythe GM, Grounds MD. Problems and solutions in myoblast transfer therapy.J.CelLMol.Med. 2001, 5(1): 33-47.
21. Skuk D, Tremblay JP. Myoblast transplantation: the current status of a potential therapeutic tool for myopathies. Muscle Res Cell Motil. 2003, 24:285-300.
22. Skuk D. First test of a "high-density injection" protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up. Neuromuscul. Disord. 2007,17:38-46.
23. Yang SY, Goldspink G. Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Letters. 2006, 580(10):2530.
24. Saxena AK, Marler J, Benvenuto M, et al. Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers: preliminary studies. Tissue Eng. 1999, 5:525-532.
25. Chromiak JA, Shansky J, Perrone C, et al. Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids. In Vitro Cell Dev. Biol. Anim. 1998, 34:694-703.
26. Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 2001,91:534-551.
27. Wagers AJ, Conboy IM. Cellular and molecular signatures of muscle regeneration:current concepts and controversies in adult myogenesis. Cell. 2005, 122:659-667.
28. Charge SBP, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev. 2004, 84:209-238.
29. Gallagher JT, Lyon M. Molecular structure of Heparan Sulfate and interactions with growth factors and morphogens. Proteoglycans: structure, biology and molecular interactions. CRC Press. 2000.27-59.
30. Johnson SE, Allen RE. Activation of Skeletal Muscle Satellite Cells and the Role of Fibroblast Growth Factor Receptors. Exp Cell Res. 1995, 219(2): 449-453.
31. Yang S, Alnaqeeb M, Simpson H, et al. Cloning and characterization of an IGF-1 isoform expressed in skeletal muscle subjected to stretch. J Muscle Res Cell Motil.1996,17:487-495.
32. Hill M, Goldspink G. Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage. J Physiol. 2003, 549:409-418.
33. Jacquemin V, Furling D, Bigot A, et al. IGF-1 induces human myotube hypertrophy by increasing cell recruitment. Exp Cell Res. 2004, 299:148-158.
34. Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials. 2003, 24:4337-4351.