In skeletal muscle tissue engineering, innervation and vascularization play an essential role in the establishment of functional skeletal muscle. For adequate three-dimensional assembly, biocompatible aligned nanofibers are beneficial as matrices for cell seeding. The aim of this study was to analyze the impact of Schwann cells (SC) on myoblast (Mb) and adipogenic mesenchymal stromal cell (ADSC) cocultures on poly-ɛ-caprolactone (PCL)-collagen I-nanofibers in vivo. Human Mb/ADSC cocultures, as well as Mb/ADSC/SC cocultures, were seeded onto PCL-collagen I-nanofiber scaffolds and implanted into the innervated arteriovenous loop model (EPI loop model) of immunodeficient rats for 4 weeks. Histological staining and gene expression were used to compare their capacity for vascularization, immunological response, myogenic differentiation, and innervation. After 4 weeks, both Mb/ADSC and Mb/ADSC/SC coculture systems showed similar amounts and distribution of vascularization, as well as immunological activity. Myogenic differentiation could be observed in both groups through histological staining (desmin, myosin heavy chain) and gene expression (MYOD, MYH3, ACTA1) without significant difference between groups. Expression of CHRNB and LAMB2 also implied neuromuscular junction formation. Our study suggests that the addition of SC did not significantly impact myogenesis and innervation in this model. The implanted motor nerve branch may have played a more significant role than the presence of SC.
Impact statement
Innervation and vascularization play an essential role in skeletal muscle tissue engineering (TE). The presented research investigated the impact of Schwann cells (SC) on cocultures of human myoblasts and adipogenic mesenchymal stromal cells on nanofiber scaffolds in the innervated arteriovenous loop model. The study concludes that the addition of SC did not significantly affect myogenesis and innervation in the model used. With the establishment of this in vivo model, enabling vascularization and innervation of the cell-scaffold-construct, this study contributes substantially to translational skeletal muscle TE and regeneration.
Get full access to this article
View all access options for this article.
References
1.
AguilarCA, GreisingSM, WattsA, et al.Multiscale analysis of a regenerative therapy for treatment of volumetric muscle loss injury. Cell Death Discov, 2018; 4:33; doi: 10.1038/s41420-018-0027-8
KohGY, KlugMG, SoonpaaMH, et al.Differentiation and long-term survival of C2C12 myoblast grafts in heart. J Clin Invest, 1993; 92(3):1548–1554; doi: 10.1172/JCI116734
4.
KhademhosseiniA, LangerR. A decade of progress in tissue engineering. Nat Protoc, 2016; 11(10):1775–1781; doi: 10.1038/nprot.2016.123
5.
RosenbaumAJ, GrandeDA, DinesJS. The use of mesenchymal stem cells in tissue engineering: A global assessment. Organogenesis, 2008; 4(1):23–27; doi: 10.4161/org.6048
6.
Torres-GuzmanRA, AvilaFR, MaitaKC, et al.Bone morphogenic protein and mesenchymal stem cells to regenerate bone in calvarial defects: A systematic review. J Clin Med, 2023; 12(12):4064; doi: 10.3390/jcm12124064
7.
ZukPA, ZhuM, AshjianP, et al.Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002; 13(12):4279–4295; doi: 10.1091/mbc.E02-02-0105
8.
Stern-StraeterJ, BonaterraGA, JuritzS, et al.Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells. Int J Mol Med, 2014; 33(1):160–170; doi: 10.3892/ijmm.2013.1555
9.
TrevorLV, Riches-SumanK, MahajanAL, et al.Adipose tissue: A source of stem cells with potential for regenerative therapies for wound healing. J Clin Med, 2020; 9(7):2161; doi: 10.3390/jcm9072161
10.
KuleszaA, BurdzinskaA, SzczepanskaI, et al.The mutual interactions between mesenchymal stem cells and myoblasts in an autologous co-culture model. PLoS One, 2016; 11(8):e0161693; doi: 10.1371/journal.pone.0161693
11.
VacantiCA. The history of tissue engineering. J Cell Mol Med, 2006; 10(3):569–576; doi: 10.1111/j.1582-4934.2006.tb00421.x
12.
ObedD, DastagirN, LiebschC, et al.In vitro differentiation of myoblast cell lines on spider silk scaffolds in a rotating bioreactor for vascular tissue engineering. J Pers Med, 2022; 12(12):1986; doi: 10.3390/jpm12121986
13.
SörgelCA, SchmidR, Kengelbach-WeigandA, et al.Air-pressure-supported application of cultured human keratinocytes in a fibrin sealant suspension as a potential clinical tool for large-scale wounds. J Clin Med, 2022; 11(17):5032; doi: 10.3390/jcm11175032
14.
MalikmammadovE, TanirTE, KiziltayA, et al.PCL and PCL-based materials in biomedical applications. J Biomater Sci Polym Ed, 2018; 29(7–9):863–893; doi: 10.1080/09205063.2017.1394711
15.
DippoldD, CaiA, HardtM, et al.Novel approach towards aligned PCL-Collagen nanofibrous constructs from a benign solvent system. Mater Sci Eng C Mater Biol Appl, 2017; 72:278–283; doi: 10.1016/j.msec.2016.11.045
16.
ChoiJS, LeeSJ, ChristGJ, et al.The influence of electrospun aligned poly(epsilon-caprolactone)/collagen nanofiber meshes on the formation of self-aligned skeletal muscle myotubes. Biomaterials, 2008; 29(19):2899–2906; doi: 10.1016/j.biomaterials.2008.03.031
17.
Simon Kratzer. Vascularization of poly-ɛ-caprolactone-collagen I-nanofibers with or without sacrificial fibers in the neurotized arteriovenous loop model. Cells, 2022; 11(23):3774; doi: 10.3390/cells11233774
18.
BittoFF, KlumppD, LangeC, et al.Myogenic differentiation of mesenchymal stem cells in a newly developed neurotised AV-loop model. Biomed Res Int, 2013; 2013:935046; doi: 10.1155/2013/935046
19.
WilsonSJ, HarrisAJ. Formation of myotubes in aneural rat muscles. Dev Biol, 1993; 156(2):509–518; doi: 10.1006/dbio.1993.1097
20.
Alvarez-SuarezP, GaworM, PrószyńskiTJ. Perisynaptic schwann cells—The multitasking cells at the developing neuromuscular junctions. Semin Cell Dev Biol, 2020; 104:31–38; doi: 10.1016/j.semcdb.2020.02.011.
21.
CaiA, ZhengZ-M, HimmlerM, et al.Schwann cells promote myogenic differentiation of myoblasts and adipogenic mesenchymal stromal cells on poly-ɛ-caprolactone-collagen I-nanofibers. Cells, 2022; 11(9):1436; doi: 10.3390/cells11091436
22.
CasellaGTB, BungeRP, WoodPM. Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro. Glia, 1996; 17(4):327–338; doi: 10.1002/(SICI)1098-1136(199608)17:4<327::AID-GLIA7>3.0.CO;2-W
23.
StrattonJA, KumarR, SinhaS, et al.Purification and characterization of Schwann cells from adult human skin and nerve. eNeuro, 2017; 4(3); doi: 10.1523/ENEURO.0307-16.2017
24.
ZhengZ. Myogenic Differentiation of Human Myoblasts, ADSC, and Schwann Cells on PCL-Collagen I-Nanofibers. 2022.
25.
WeigandA, BoosAM, TasbihiK, et al.Selective isolation and characterization of primary cells from normal breast and tumors reveal plasticity of adipose derived stem cells. Breast Cancer Res, 2016; 18(1):32; doi: 10.1186/s13058-016-0688-2
26.
DippoldD, CaiA, HardtM, et al.Investigation of the batch-to-batch inconsistencies of Collagen in PCL-Collagen nanofibers. Mater Sci Eng C Mater Biol Appl, 2019; 95:217–225; doi: 10.1016/j.msec.2018.10.057
27.
Heltmann-MeyerS, SteinerD, MüllerC, et al.Gelatin methacryloyl is a slow degrading material allowing vascularization and long-term use in vivo. Biomed Mater, 2021; 16(6).
28.
AnR, StrisselPL, Al-AbboodiM, et al.An innovative arteriovenous (AV) loop breast cancer model tailored for cancer research. Bioengineering (Basel), 2022; 9(7):280; doi: 10.3390/bioengineering9070280
29.
SteinerD, LangG, FischerL, et al.Intrinsic vascularization of recombinant eADF4(C16) spider silk matrices in the arteriovenous loop model. Tissue Eng Part A, 2019; 25(21–22):1504–1513; doi: 10.1089/ten.TEA.2018.0360
30.
WinklerS, MutschallH, BiggemannJ, et al.Human umbilical vein endothelial cell support bone formation of adipose-derived stem cell-loaded and 3D-printed osteogenic matrices in the arteriovenous loop model. Tissue Eng Part A, 2021; 27(5–6):413–423; doi: 10.1089/ten.TEA.2020.0087
31.
CaiA, ZhengZ, Müller-SeubertW, et al.Microsurgical transplantation of pedicled muscles in an isolation chamber—A novel approach to engineering muscle constructs via perfusion-decellularization. J Pers Med, 2022; 12(3):442; doi: 10.3390/jpm12030442
32.
SteinerD, WinklerS, Heltmann-MeyerS, et al.Enhanced vascularization andde novotissue formation in hydrogels made of engineered RGD-tagged spider silk proteins in the arteriovenous loop model. Biofabrication, 2021; 13(4); doi: 10.1088/1758-5090/ac0d9b.
33.
Heltmann-MeyerS, SteinerD, MüllerC, et al.Gelatin methacryloyl is a slow degrading material allowing vascularization and long-term usein vivo. Biomed Mater, 2021; 16(6); doi: 10.1088/1748-605X/ac1e9d
34.
ShuJ, QiuG, MohammadI. A Semi-automatic Image Analysis Tool for Biomarker Detection in Immunohistochemistry Analysis. In: 2013 Seventh International Conference on Image and Graphics. IEEE; 2013, pp. 937–942; doi: 10.1109/ICIG.2013.197
35.
MinamiK, HiwatashiK, UenoS, et al.Prognostic significance of CD68, CD163 and Folate receptor-β positive macrophages in hepatocellular carcinoma. Exp Ther Med, 2018; 15(5):4465–4476; doi: 10.3892/etm.2018.5959
36.
BirkR, SommerJU, HaasD, et al.Influence of static magnetic fields combined with human insulin-like growth factor 1 on human satellite cell cultures. In Vivo, 2014; 28(5):795–802.
37.
EntezariM, MozafariM, BakhtiyariM, et al.Three-dimensional-printed polycaprolactone/polypyrrole conducting scaffolds for differentiation of human olfactory ecto-mesenchymal stem cells into Schwann cell-like phenotypes and promotion of neurite outgrowth. J Biomed Mater Res A, 2022; 110(5):1134–1146; doi: 10.1002/jbm.a.37361
38.
GriffithsIR, MitchellLS, McPhilemyK, et al.Expression of myelin protein genes in Schwann cells. J Neurocytol, 1989; 18(3):345–352; doi: 10.1007/BF01190837
39.
Afshar BakooshliM, LippmannES, MulcahyB, et al.A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction. eLife, 2019; 8; doi: 10.7554/eLife.44530
KaufmanT, KaplanB, PerryL, et al.Innervation of an engineered muscle graft for reconstruction of muscle defects. Am J Transplant, 2019; 19(1):37–47; doi: 10.1111/ajt.14957
42.
KangH, LichtmanJW. Motor axon regeneration and muscle reinnervation in young adult and aged animals. J Neurosci, 2013; 33(50):19480–19491; doi: 10.1523/JNEUROSCI.4067-13.2013
43.
AsakuraA, HiraiH, KablarB, et al.Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle. Proc Natl Acad Sci U S A, 2007; 104(42):16552–16557; doi: 10.1073/pnas.0708145104
44.
Stern-StraeterJ, BranG, RiedelF, et al.Characterization of human myoblast cultures for tissue engineering. Int J Mol Med, 2008; 21(1):49–56.
45.
FerriP, BarbieriE, BurattiniS, et al.Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J Cell Biochem, 2009; 108(6):1302–1317; doi: 10.1002/jcb.22360
46.
SchiaffinoS, RossiAC, SmerduV, et al.Developmental myosins: Expression patterns and functional significance. Skelet Muscle, 2015; 5:22; doi: 10.1186/s13395-015-0046-6
47.
Stern-StraeterJ, BonaterraGA, KassnerSS, et al.Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis. J Tissue Eng Regen Med, 2011; 5(8):e197–e206; doi: 10.1002/term.417
48.
BeedleAM. Distribution of myosin heavy chain isoforms in muscular dystrophy: Insights into disease pathology. Musculoskelet Regen, 2016; 2:e1365.
LaingNG, DyeDE, Wallgren-PetterssonC, et al.Mutations and polymorphisms of the skeletal muscle alpha-actin gene (ACTA1). Hum Mutat, 2009; 30(9):1267–1277; doi: 10.1002/humu.21059
51.
SacconeNL, SacconeSF, HinrichsAL, et al.Multiple distinct risk loci for nicotine dependence identified by dense coverage of the complete family of nicotinic receptor subunit (CHRN) genes. Am J Med Genet B Neuropsychiatr Genet, 2009; 150B(4):453–466; doi: 10.1002/ajmg.b.30828
52.
VogtJ, HarrisonBJ, SpearmanH, et al.Mutation analysis of CHRNA1, CHRNB1, CHRND, and RAPSN genes in multiple pterygium syndrome/fetal akinesia patients. Am J Hum Genet, 2008; 82(1):222–227; doi: 10.1016/j.ajhg.2007.09.016
53.
KimJH, KimI, SeolY-J, et al.Neural cell integration into 3D bioprinted skeletal muscle constructs accelerates restoration of muscle function. Nat Commun, 2020; 11(1):1025; doi: 10.1038/s41467-020-14930-9
54.
HorchRE, BeierJP, KneserU, et al.Successful human long-term application of in situ bone tissue engineering. J Cell Mol Med, 2014; 18(7):1478–1485; doi: 10.1111/jcmm.12296
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
