Chronic wounds affect millions of patients worldwide, and it is estimated that this number will increase steadily in the future due to population ageing. The research of new therapeutic approaches to wound healing includes the development of nanofibrous meshes and the use of platelet lysate (PL) to stimulate skin regeneration. This study considers a combination of a degradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) membranes (NF) and fibrin loaded with various concentrations of PL aimed at the development of bioactive skin wound healing dressings. The cytocompatibility of the NF membranes, as well as the effect of PL, was evaluated in both monocultures and co-cultures of human keratinocytes and human endothelial cells. We determined that the keratinocytes were able to adhere on all the membranes, and their increased proliferation and differentiation was observed on the membranes that contained fibrin with at least 50% of PL (Fbg + PL) after 14 days. With respect to the co-culture experiments, the membranes with fibrin with 20% of PL were observed to enhance the metabolic activity of endothelial cells and their migration, and the proliferation and differentiation of keratinocytes. The results suggest that the newly developed NF combined with fibrin and PL, described in the study, provides a promising dressing for chronic wound healing purposes.

Faculty of Health Technical University of Liberec Studentska 1402 2 461 17 Liberec 1 Czech Republic

Faculty of Science Humanities and Education Technical University of Liberec Studentska 1402 2 461 17 Liberec 1 Czech Republic

Faculty of Textile Engineering Technical University of Liberec Studentska 1402 2 461 17 Liberec 1 Czech Republic

Institute of Macromolecular Chemistry of the Czech Academy of Sciences Heyrovskeho nam 2 162 06 Prague 6 Czech Republic

Institute of Nanomaterials Advanced Technologies and Innovation Bendlova 1409 7 460 01 Liberec 1 Czech Republic

Institute of Physiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

Regional Hospital Liberec Husova 357 28 460 01 Liberec 1 Czech Republic

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Martinez-Zapata M., Martí-Carvajal A., Solà I., Expósito J., Bolíbar I., Rodríguez L., Garcia J., Zaror C. Autologous platelet-rich plasma for treating chronic wounds (Review) Cochrane Database Syst. Rev. 2016 doi: 10.1002/14651858.CD006899.pub3. PubMed DOI PMC

Sen C.K., Gordillo G.M., Roy S., Kirsner R., Lambert L., Hunt T.K., Gottrup F., Gurtner G.C., Longaker M.T. Human skin wounds: A major and snowballing threat to public health and the economy: PERSPECTIVE ARTICLE. Wound Repair Regen. 2009;17:763–771. doi: 10.1111/j.1524-475X.2009.00543.x. PubMed DOI PMC

Bacakova L., Zikmundova M., Pajorova J., Broz A., Filova E., Blanquer A., Matejka R., Stepanovska J., Mikes P., Jencova V., et al. Applications of Nanobiotechnology. IntechOpen; London, UK: 2020. Nanofibrous Scaffolds for Skin Tissue Engineering and Wound Healing Based on Synthetic Polymers.

Jeong S.I., Jun I.D., Choi M.J., Nho Y.C., Lee Y.M., Shin H. Development of electroactive and elastic nanofibers that contain polyaniline and poly(L-lactide-co-ε-caprolactone) for the control of cell adhesion. Macromol. Biosci. 2008;8:627–637. doi: 10.1002/mabi.200800005. PubMed DOI

Losi P., Barsotti M.C., Foffa I., Buscemi M., De Almeida C.V., Fabbri M., Gabbriellini S., Nocchi F., Ursino S., Urciuoli P., et al. In vitro human cord blood platelet lysate characterisation with potential application in wound healing. Int. Wound J. 2020;17:65–72. doi: 10.1111/iwj.13233. PubMed DOI PMC

Robson M.C. The role of growth factors in the healing of chronic wounds. Wound Repair Regen. 1997;5:12–17. doi: 10.1046/j.1524-475X.1997.50106.x. PubMed DOI

Nardini M., Perteghella S., Mastracci L., Grillo F., Marrubini G., Bari E., Formica M., Gentili C., Cancedda R., Torre M.L., et al. Growth factors delivery system for skin regeneration: An advanced wound dressing. Pharmaceutics. 2020;12:120. doi: 10.3390/pharmaceutics12020120. PubMed DOI PMC

Lai H.-J., Kuan C.-H., Wu H.-C., Tsai J.-C., Chen T.-M., Hsieh D.-J., Wang T.-W. Tailored design of electrospun composite nanofibers with staged release of multiple angiogenic growth factors for chronic wound healing. Acta Biomater. 2014;10:4156–4166. doi: 10.1016/j.actbio.2014.05.001. PubMed DOI

Chong D.L.W., Trinder S., Labelle M., Rodriguez-Justo M., Hughes S., Holmes A.M., Scotton C.J., Porter J.C. Platelet-derived transforming growth factor-β1 promotes keratinocyte proliferation in cutaneous wound healing. J. Tissue Eng. Regen. Med. 2020;14:645–649. doi: 10.1002/term.3022. PubMed DOI PMC

Barrientos S., Stojadinovic O., Golinko M.S., Brem H., Tomic-Canic M. PERSPECTIVE ARTICLE: Growth factors and cytokines in wound healing. Wound Repair Regen. 2008;16:585–601. doi: 10.1111/j.1524-475X.2008.00410.x. PubMed DOI

Martin P. Wound healing—Aiming for perfect skin regeneration. Science. 1997;276:75–81. doi: 10.1126/science.276.5309.75. PubMed DOI

Borzini P., Mazzucco L. Platelet gels and releasates. Curr. Opin. Hematol. 2005;12:473–479. doi: 10.1097/01.moh.0000177831.70657.e8. PubMed DOI

Tenci M., Rossi S., Bonferoni M.C., Sandri G., Boselli C., Di Lorenzo A., Daglia M., Icaro Cornaglia A., Gioglio L., Perotti C., et al. Particulate systems based on pectin/chitosan association for the delivery of manuka honey components and platelet lysate in chronic skin ulcers. Int. J. Pharm. 2016;509:59–70. doi: 10.1016/j.ijpharm.2016.05.035. PubMed DOI

Ranzato E., Patrone M., Mazzucco L., Burlando B. Platelet lysate stimulates wound repair of HaCaT keratinocytes. Br. J. Dermatol. 2008;159:537–545. doi: 10.1111/j.1365-2133.2008.08699.x. PubMed DOI

Baik S.Y., Lim Y.A., Kang S.J., Ahn S.H., Lee W.G., Kim C.H. Effects of platelet lysate preparations on the proliferation of hacat cells. Ann. Lab. Med. 2014;34:43–50. doi: 10.3343/alm.2014.34.1.43. PubMed DOI PMC

Pedram Rad Z., Mokhtari J., Abbasi M. Calendula officinalis extract/PCL/Zein/Gum arabic nanofibrous bio-composite scaffolds via suspension, two-nozzle and multilayer electrospinning for skin tissue engineering. Int. J. Biol. Macromol. 2019;135:530–543. doi: 10.1016/j.ijbiomac.2019.05.204. PubMed DOI

Ahmed S.M., Ahmed H., Tian C., Tu Q., Guo Y., Wang J. Whey protein concentrate doped electrospun poly(epsilon-caprolactone) fibers for antibiotic release improvement. Colloids Surf. B Biointerfaces. 2016;143:371–381. doi: 10.1016/j.colsurfb.2016.03.059. PubMed DOI

Mikes P., Horakova J., Saman A., Vejsadova L., Topham P., Punyodom W., Dumklang M., Jencova V. Comparison and characterization of different polyester nano/micro fibres for use in tissue engineering applications. J. Ind. Text. 2021;50:870–890. doi: 10.1177/1528083719848155. DOI

Horakova J., Mikes P., Saman A., Jencova V., Klapstova A., Svarcova T., Ackermann M., Novotny V., Suchy T., Lukas D. The effect of ethylene oxide sterilization on electrospun vascular grafts made from biodegradable polyesters. Mater. Sci. Eng. C. 2018;92:132–142. doi: 10.1016/j.msec.2018.06.041. PubMed DOI

Riedel T., Brynda E., Dyr J.E., Houska M. Controlled preparation of thin fibrin films immobilized at solid surfaces. J. Biomed. Mater. Res. Part A. 2009;88:437–447. doi: 10.1002/jbm.a.31755. PubMed DOI

Riedelová-Reicheltová Z., Brynda E., Riedel T. Fibrin Nanostructures for Biomedical Applications. Physiol. Res. 2016:S263–S272. doi: 10.33549/physiolres.933428. PubMed DOI

Jackson M., Mantsch H.H. The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure. Crit. Rev. Biochem. Mol. Biol. 1995;30:95–120. doi: 10.3109/10409239509085140. PubMed DOI

Jing X., Mi H.-Y., Peng J., Peng X.-F., Turng L.-S. Electrospun aligned poly(propylene carbonate) microfibers with chitosan nanofibers as tissue engineering scaffolds. Carbohydr. Polym. 2015;117:941–949. doi: 10.1016/j.carbpol.2014.10.025. PubMed DOI

Szewczyk P., Ura D., Metwally S., Knapczyk-Korczak J., Gajek M., Marzec M., Bernasik A., Stachewicz U. Roughness and Fiber Fraction Dominated Wetting of Electrospun Fiber-Based Porous Meshes. Polymers. 2018;11:34. doi: 10.3390/polym11010034. PubMed DOI PMC

Horakova J., Mikes P., Saman A., Svarcova T., Jencova V., Suchy T., Heczkova B., Jakubkova S., Jirousova J., Prochazkova R. Comprehensive assessment of electrospun scaffolds hemocompatibility. Mater. Sci. Eng. C. 2018;82:330–335. doi: 10.1016/j.msec.2017.05.011. PubMed DOI

Lukášek J., Hauzerová Š., Havlíčková K., Strnadová K., Mašek K., Stuchlík M., Stibor I., Jenčová V., Řezanka M. Cyclodextrin-Polypyrrole Coatings of Scaffolds for Tissue Engineering. Polymers. 2019;11:459. doi: 10.3390/polym11030459. PubMed DOI PMC

Sovkova V., Vocetkova K., Rampichova M., Mickova A., Buzgo M., Lukasova V., Dankova J., Filova E., Necas A., Amler E. Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers. Platelets. 2018;29:395–405. doi: 10.1080/09537104.2017.1316838. PubMed DOI

Zamani M., Yaghoubi Y., Movassaghpour A., Shakouri K., Mehdizadeh A., Pishgahi A., Yousefi M. Novel therapeutic approaches in utilizing platelet lysate in regenerative medicine: Are we ready for clinical use? J. Cell. Physiol. 2019:1–15. doi: 10.1002/jcp.28496. PubMed DOI

Mosesson M.W. Fibrinogen and fibrin structure and functions. J. Thromb. Haemost. 2005;3:1894–1904. doi: 10.1111/j.1538-7836.2005.01365.x. PubMed DOI

Safari M., Ghahari L., Babak Hoss M.D. Effects of Epidermal Growth Factor, Platelet Derived Growth Factor and Growth Hormone on Cultured Rat Keratinocytes Cells in vitro. Pak. J. Biol. Sci. 2014;17:931–936. doi: 10.3923/pjbs.2014.931.936. PubMed DOI

Pluemsakunthai W., Kuroda S., Shimokawa H., Kasugai S. A basic analysis of platelet-rich fibrin: Distribution and release of platelet-derived growth factor-BB. Inflamm. Regen. 2013;33:164–172. doi: 10.2492/inflammregen.33.164. DOI

Reinartz J., Batrla R., Boukamp P., Fusenig N., Kramer M.D. Binding and Activation of Plasminogen at the Surface of Human Keratinocytes. Exp. Cell Res. 1993;208:197–208. doi: 10.1006/excr.1993.1238. PubMed DOI

Klicova M., Klapstova A., Chvojka J., Koprivova B., Jencova V., Horakova J. Novel double-layered planar scaffold combining electrospun PCL fibers and PVA hydrogels with high shape integrity and water stability. Mater. Lett. 2020;263:127281. doi: 10.1016/j.matlet.2019.127281. DOI

Schuhladen K., Raghu S.N.V., Liverani L., Neščáková Z., Boccaccini A.R. Production of a novel poly(ɛ-caprolactone)-methylcellulose electrospun wound dressing by incorporating bioactive glass and Manuka honey. J. Biomed. Mater. Res. Part B Appl. Biomater. 2020:1–13. doi: 10.1002/jbm.b.34690. PubMed DOI

Lorden E.R., Miller K.J., Bashirov L., Ibrahim M.M., Hammett E., Jung Y., Medina M.A., Rastegarpour A., Selim M.A., Leong K.W., et al. Mitigation of hypertrophic scar contraction via an elastomeric biodegradable scaffold. Biomaterials. 2015;43:61–70. doi: 10.1016/j.biomaterials.2014.12.003. PubMed DOI

Pal P., Dadhich P., Srivas P.K., Das B., Maulik D., Dhara S. Bilayered nanofibrous 3D hierarchy as skin rudiment by emulsion electrospinning for burn wound management. Biomater. Sci. 2017;5:1786–1799. doi: 10.1039/C7BM00174F. PubMed DOI

Croisier F., Atanasova G., Poumay Y., Jérôme C. Polysaccharide-Coated PCL Nanofibers for Wound Dressing Applications. Adv. Healthc. Mater. 2014;3:2032–2039. doi: 10.1002/adhm.201400380. PubMed DOI

Filová E., Brynda E., Riedel T., Bacáková L., Chlupác J., Lisá V., Houska M., Dyr J.E. Vascular endothelial cells on two-and three-dimensional fibrin assemblies for biomaterial coatings. J. Biomed. Mater. Res. A. 2009;90:55–69. doi: 10.1002/jbm.a.32065. PubMed DOI

Filová E., Suchý T., Sucharda Z., Supová M., Zaloudková M., Balík K., Lisá V., Slouf M., Bačáková L. Support for the initial attachment, growth and differentiation of MG-63 cells: A comparison between nano-size hydroxyapatite and micro-size hydroxyapatite in composites. Int. J. Nanomed. 2014;9:3687–3706. doi: 10.2147/IJN.S56661. PubMed DOI PMC

Pajorova J., Bacakova M., Musilkova J., Broz A., Hadraba D., Lopot F., Bacakova L. Morphology of a fibrin nanocoating influences dermal fibroblast behavior. Int. J. Nanomed. 2018;13:3367–3380. doi: 10.2147/IJN.S162644. PubMed DOI PMC

Barsotti M.C., Losi P., Briganti E., Sanguinetti E., Magera A., Al Kayal T., Feriani R., Di Stefano R., Soldani G. Effect of platelet lysate on human cells involved in different phases of wound healing. PLoS ONE. 2013;8:e84753. doi: 10.1371/journal.pone.0084753. PubMed DOI PMC

Schoop V.M., Fusenig N.E., Mirancea N. Epidermal Organization and Differentiation of HaCaT Keratinocytes in Organotypic Coculture with Human Dermal Fibroblasts. J. Invest. Dermatol. 2003;112:343–353. doi: 10.1046/j.1523-1747.1999.00524.x. PubMed DOI

Tan J., Zhao C., Zhou J., Duan K., Wang J., Lu X., Weng J., Feng B. Co-culturing epidermal keratinocytes and dermal fibroblasts on nano-structured titanium surfaces. Mater. Sci. Eng. C. 2017;78:288–295. doi: 10.1016/j.msec.2017.04.036. PubMed DOI

Werner S., Krieg T., Smola H. Keratinocyte-fibroblast interactions in wound healing. J. Invest. Dermatol. 2007;127:998–1008. doi: 10.1038/sj.jid.5700786. PubMed DOI

Baltazar T., Merola J., Catarino C., Xie C.B., Kirkiles-Smith N.C., Lee V., Hotta S., Dai G., Xu X., Ferreira F.C., et al. Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells. Tissue Eng. Part A. 2019:1–29. doi: 10.1089/ten.tea.2019.0201. PubMed DOI PMC

Castaño O., Pérez-Amodio S., Navarro-Requena C., Mateos-Timoneda M.Á., Engel E. Instructive microenvironments in skin wound healing: Biomaterials as signal releasing platforms. Adv. Drug Deliv. Rev. 2018;129:95–117. doi: 10.1016/j.addr.2018.03.012. PubMed DOI

Feliciani C., Gupta A.K., Sauder D.N. Keratinocytes and cytokine/growth factors. Crit. Rev. Oral Biol. Med. 1996;7:300–318. doi: 10.1177/10454411960070040101. PubMed DOI

Zhang X., Yin M., Zhang L.J. Keratin 6, 16 and 17-Critical Barrier Alarmin Molecules in Skin Wounds and Psoriasis. Cells. 2019;8:807. doi: 10.3390/cells8080807. PubMed DOI PMC

Stankova L., Musilkova J., Broz A., Potocky S., Kromka A., Kozak H., Izak T., Artemenko A., Stranska D., Bacakova L. Alterations to the adhesion, growth and osteogenic differentiation of human osteoblast-like cells on nanofibrous polylactide scaffolds with diamond nanoparticles. Diam. Relat. Mater. 2019;97:107421. doi: 10.1016/j.diamond.2019.05.007. DOI

Sakariassen K.S., Bolhuis P.A., Sixma J.J. Human blood platelet adhesion to artery subendothelium is mediated by factor VIII–Von Willebrand factor bound to the subendothelium. Nature. 1979;279:636–638. doi: 10.1038/279636a0. PubMed DOI

Romaldini A., Ulivi V., Nardini M., Mastrogiacomo M., Cancedda R., Descalzi F. Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability. Cells. 2019;8:331. doi: 10.3390/cells8040331. PubMed DOI PMC

Cui T., Kirsner R.S., Li J. Advances in Wound Care. Mary Ann Liebert, Inc.; New Rochelle, NY, USA: 2010. Angiogenesis in Chronic Wounds; pp. 347–352.

PLCL/PCL Dressings with Platelet Lysate and Growth Factors Embedded in Fibrin for Chronic Wound Regeneration

The Effect of the Controlled Release of Platelet Lysate from PVA Nanomats on Keratinocytes, Endothelial Cells and Fibroblasts