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Complete Myogenic Differentiation of Adipogenic Stem Cells Requires Both Biochemical and Mechanical Stimulation

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Abstract

Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by treatment with transforming growth factor-β, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10 days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (αSMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased αSMA and calponin expression by either pulsatile (12.48-fold; p < 0.01 and 38.15-fold; p = 0.07) or static incubation plus TSB pre-treatment (8.91-fold; p < 0.05 and 37.69-fold; p < 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (p < 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.

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Acknowledgments

We thank S. Zippusch for her help with the fibrinogen isolation.

Funding

This work has been carried out as an integral part of the BIOFABRICATION FOR NIFE (2012) Initiative, which is financially supported by the Lower Saxonian Ministry for Science and Culture and the VolkswagenStiftung. (NIFE is the Lower Saxonian Center for Biomedical Engineering, Implant Research and Development—a joint translational research center of the Hannover Medical School, the Leibniz University Hannover, the Foundation University of Veterinary Medicine Hannover and the Laser Center Hannover).

Author Contributions

There were no contributions that do not justify authorship.

Conflict of interest

No conflict of interest exists.

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Correspondence to Ulrike Böer.

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Associate Editor Joel Stitzel oversaw the review of this article.

Electronic Supplementary Material

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Supplemental Fig. S1: A: Composition of the vessel mold. The vessel mold consisted of two half-shells of polyether ether ketone (PEEK), two Teflon®-cuffs with drill holes for the application of the fibrinogen/thrombin-mixture and a tubular placeholder (I). The mold was fixed using a hose clamp and adhesion tape and brought into an upright position for initial static polymerization (II). The casted tubes were 10 cm in length with a luminal diameter of 4.75 mm and a wall thickness of 1.63 mm. B: Manufacturing setup of the rotation unit. The rotation unit consisted of a computer-controlled water-cooled electric motor and a rotation chamber (I) containing a rotating metal tube. After static polymerization, the vessel mold including the fibrin segment was placed in the metal tube, which contained drill holes to allow the outflow of excessive fluid during rotation and was closed with a sealing cap (II). Supplementary material 1 (TIFF 3961 kb)

Supplemental Fig. S2: Effect of fibrin compaction on biomechanical strength. Burst pressure or tensile strength was measured of uncompacted or compacted fibrin scaffolds. Shown are means ± SD of 6 scaffolds. **p < 0.01 and #p < 0.0001 by student’s T test. Supplementary material 2 (TIFF 103 kb)

Supplemental Fig. S3: Quantification of cyclic strain. Fibrin segments (n = 12, 3 runs) were exposed to pulsatile perfusion under video recordings. Images of segments under maximal (systolic) and minimal (diastolic) pressure were subsequently analyzed using the measurement-software “MB Ruler”©. Wall strain was calculated using the formula Strain (%) = (Diameter Systolic/Diameter Diastolic)*100–100. Supplementary material 3 (TIFF 7318 kb)

Supplemental Fig. S4: Live/dead staining for analyzing Zell viability after the compaction process and determine the optimal zell count per tube. Fibrin scaffolds where seeded with 0.5, 1 and 1.5 × 107 cells/tube and live/dead staining was performed on plane sections of 0.5 cm Rings on day 1, 5 and 8 after the compaction process. Living cells are stained green (Calcein) and dead cells are stained red (Ethidium-Homodimer). Scale bar = 100 µm. Supplementary material 4 (TIFF 4965 kb)

Supplemental Fig. S5: Impact of treatment with myogenic factors on scaffold calcification. Fibrin segments containing either adipose-derived mesenchymal stem cells (ASC) or pre-differentiated smooth muscle cells (SMC) were statically incubated or exposed to pulsatile perfusion for 10 days with or without the myogenic factors TGFβ1, SPC and BMP4 (TSB). Shown are cryo sections stained by Alizarin Red. No calcium deposits in either group (A-C) were observed as shown in the positive control (D; see purple arrows pointing to calcium crystals in calcified decellularized equine carotid artery as vascular graft in sheep as published in Böer et al., Int J Art Org, 2013, 36(3) 184). Scale bar: 100 µm. Supplementary material 5 (TIFF 26182 kb)

Supplemental Fig. S6: Expression of myogenic marker proteins in myogenic factor pre-differentiated ASC. Adipose-derived mesenchymal stem cells (ASC) cultured with 1 % FCS or differentiated towards the myogenic phenotype with TGFβ1, BMP4 and SPC for 8 days (n = 3 in both groups) were analyzed by Western blot analysis for the expression of α smooth muscle actin (αSMA), calponin and smoothelin prior to embedding into the fibrin graft. FCS = fetal calf serum, TGFβ1 = Transforming growth factor β1, BMP4 = Bone morphogenetic protein 4, SPC = Sphingosylphosphorylcholine. Supplementary material 6 (TIFF 5721 kb)

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Helms, F., Lau, S., Klingenberg, M. et al. Complete Myogenic Differentiation of Adipogenic Stem Cells Requires Both Biochemical and Mechanical Stimulation. Ann Biomed Eng 48, 913–926 (2020). https://doi.org/10.1007/s10439-019-02234-z

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Keywords

  • Bioreactor technique
  • Mechanical strain
  • Contractile phenotype