Application of Concentrated Growth Factors Membrane for Human Umbilical Cord Wharton’s Jelly Mesenchymal Stem Cell Differentiation towards Keratinocytes

Abstract

Concentrated growth factors are extracted from platelet-rich plasma obtained from healthy adult veins by physical gradient centrifugation, and the activated platelets release various growth factors and cytokines, which can be further converted into concentrated growth factors liquid or gel preparations by different centrifuge tubes. These preparations are widely used in clinical treatments in various fields, such as dentistry, dermatology and surgery. In this article, concentrated growth factors gel and platelet-poor plasma gel obtained from six healthy adults were pressed into a concentrated growth factors membrane and platelet-poor plasma membrane. We examined whether the 3D fibrin mesh and the various concentrated growth factors within the concentrated growth factors membrane could be used as a bioscaffold for the human Wharton’s jelly umbilical cord stem cell line or the HaCaT cell line to attach, proliferate and form epidermal-like tissue. We also aimed to implant umbilical cord stem cells on the concentrated growth factors membrane or platelet-poor plasma membrane, and further compare the characteristics of similar tissues after 4 weeks in in vitro culture. The results showed that human Wharton’s jelly umbilical cord mesenchymal stem cells, implanted on the upper surface of the concentrated growth factors membrane, showed subsequent cell attachment and proliferation. After 4 weeks of ex vivo tissue culture, a multi-layer epidermal-like tissue formed on the upper surface of the membrane containing concentrated growth factors. This tissue had a minimum thickness of 89.91 µm to a maximum of 204.19 µm, mean ± SD = 144.36 µm ± 43.14 µm. Sections of these multi-layer epidermal-like tissues were used for immunohistochemical staining. We found that 79.8% ± 7.2% of the cells expressed the pancytokeratin marker, 29.5% ± 9.4% of the cells expressed the P63 marker, and 71.7% ± 3.9% of the cells expressed the vimentin marker. After the same 4 weeks in the in vitro culture, the HaCaT cells could attach to the concentrated growth factors membrane and proliferate to form a multi-layer tissue, The tissue had a minimum thickness of 63.17 µm to a maximum of 100.26 µm, mean ± SD = 74.05 µm ± 13.44 µm. We found that 88.1% ± 4.9% of the cells expressed the pancytokeratin marker, 63.6% ± 11.4% of the cells expressed the P63 marker, and 79% ± 9.9% of the cells expressed the vimentin marker. Also, after 4 weeks in the in vitro culture, it showed that umbilical cord stem cells could attach to the platelet-poor plasma membrane, proliferate and distribute in the whole-tissue sections. We found that 9.7% ± 2.4% of the cells expressed the pancytokeratin marker, 7.45% ± 1.9% of the cells expressed the P63 maker, and 95.9% ± 3.7% of the cells expressed the vimentin marker. In terms of the percentage of umbilical cord stem cells expressing pancytokeratin, P63, or vimentin cell markers, there was a significant difference between cultivating in the concentrated growth factors membrane scaffold and the platelet-poor plasma membrane scaffolds. In terms of the percentage of umbilical cord stem cells or HaCaT cells (cultivating in the concentrated growth factors membrane) expressing pancytokeratin, P63, or vimentin cell markers, there was no significant difference. These results suggested that umbilical cord Wharton’s jelly mesenchymal stem cells can use the concentrated growth factors membrane (composed of 3D fibrin mesh, and various growth factors and cytokines) as an effective and self-contained bioscaffold to differentiate towards keratinocytes-like cells. In the future, donors’ own concentrated growth factors membrane can be applied as an auxiliary tool for autologous tissue regeneration.