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The bilayer skin substitute based on human adipose-derived mesenchymal stem cells and neonate keratinocytes on the 3D nanofibrous PCL-platelet gel scaffold

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Abstract

In the present study, neonate keratinocytes were cocultured with human adipose-derived mesenchymal stem cells (hAMSCs) on the electrospun polycaprolactone-platelet gel (PCL–PG) scaffold. To evaluate its potential of wound healing and skin tissue engineering, neonate keratinocytes must be differentiated. The PCL scaffold prepared by the electrospinning technique was fabricated and coated by PG. Then scaffolds were fully characterized by SEM, contact angle, FTIR–ATR, and tensile test. Following seeding hAMSCs as a feeder layer, neonate keratinocytes were cocultured directly on PCL–PG scaffolds and neat PCL. The hAMSCs and neonate keratinocytes' viability was measured using MTT assay for up to 10 days (1st, 3rd, 5th, 7th, and 10th days). To examine the epidermal maturation, cytokeratin 10 and loricrin determinants detection was used through immunocytochemistry (ICC). In addition, real-time PCR was used to examine keratinocyte marker genes in keratin 10, keratin 14, and Involucrin. The MTT assay results showed higher cell viability and the proliferation of neo-keratinocytes on PCL–PG fibrillar scaffolds than the PCL scaffold. On the PCL–PG scaffolds in the presence of hAMSCs as a feeder layer, the PCR and ICC analysis had a higher cell differentiation compared to neat PCL scaffolds. Moreover, SEM images showed that the keratinocytes cocultured with hAMSCs demonstrated a better proliferation and adhesion on PCL–PG nanofiber scaffolds. Based on results, PG increased the nanofibrous PCL scaffold's biological properties, including hydrophilicity, cell attachment, cell viability, and expression of keratinocyte markers in the neat keratinocytes and hAMSCs coculture system. The findings support the potential of this engineered construct for engineering skin tissue and wound dressing.

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Abbreviations

hAMSCs:

Human adipose-derived mesenchymal stem cells

neo-K:

Neonate Keratinocytes

PCL:

Polycaprolactone

PG:

Platelet gel

SEM:

Scanning electron microscope

FTIR-ATR:

Fourier transform infrared Spectroscopy-Attenuated total reflection

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ICC:

Immunocytochemistry

PCR:

Polymerase chain reaction

3D:

Three dimensional

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Acknowledgments

The authors thank their colleagues in the Stem Cell Technology Research Center, Tehran, Iran.

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Authors and Affiliations

  1. Department of Clinical Biochemistry and Applied Cell Sciences, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran

    Parviz Ranjbarvan & Ali Golchin

  2. Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Parviz Ranjbarvan

  3. Regenerative Medicine Group (REMED), Universal Scientific Education and Research Network (USERN), Tehran, Iran

    Ali Golchin

  4. Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Arezo Azari

  5. Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Zahra Niknam

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  1. Parviz Ranjbarvan
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  4. Zahra Niknam
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PR and AG: conception, performing, collecting data, data analyzing, and interpreting. All authors cooperated in manuscript writing and final approval of the manuscript.

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Correspondence to Ali Golchin.

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The study protocol was approved by the Tehran University of Medical Sciences of Iran, and all procedures were performed following the guidelines.

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Ranjbarvan, P., Golchin, A., Azari, A. et al. The bilayer skin substitute based on human adipose-derived mesenchymal stem cells and neonate keratinocytes on the 3D nanofibrous PCL-platelet gel scaffold. Polym. Bull. 79, 4013–4030 (2022). https://doi.org/10.1007/s00289-021-03702-0

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