Synthetic vs natural scaffolds for human limbal stem cells.

246 FORENSIC SCIENCE Croat Med J. 2015;56:246-56 doi: 10.3325/cmj.2015.56.246 Synthetic vs natural scaffolds for human limbal stem cells Aim To investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane. Mirna Tominac Trcin1, Iva Dekaris2, Budimir Mijović3, Marina Bujić1, Emilija Zdraveva3, Tamara Dolenec1, Maja Pauk-Gulić2, Dragan Primorac4,5,6,7,8, Josip Crnjac8, Branimira Špoljarić9, Gordan Mršić10, Krunoslav Kuna11, Daniel Špoljarić9, Maja Popović9 University Hospital Centre Sestre Milosrdnice, Tissue bank at University Department of Traumatology, Zagreb, Croatia 1 Specialty Eye Hospital Svjetlost, Zagreb and Department of Ophthalmology Medical Faculty, University of Rijeka, Rijeka, Croatia 2 Methods Human placenta was taken at elective cesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were amniotic membrane, fibrin, PU, and PCL nanoscaffolds, with or without prior NaOH treatment. Results Scanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL, and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin. Conclusion Natural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purposes. Department of Basic Natural and Technical Sciences, Faculty of Textile Technology, University of Zagreb, Zagreb, Croatia 3 Eberly College of Science, The Pennsylvania State University, University Park, PA, USA 4 The Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA 5 Medical School, University of Osijek, Osijek, Croatia 6 Medical School, University of Split, Split, Croatia 7 University Department for Forensic Sciences, University of Split, Split, Croatia 8 Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia 9 10 Forensic Science Centre “Ivan Vučetić”, Zagreb, Croatia University Hospital Centre Sestre Milosrdnice, Gynecology and Obstetrics Department, Zagreb, Croatia 11 Received: April 28, 2015 Accepted: June 9, 2015 Correspondence to: Maja Popović Department of Biology Faculty of Veterinary Medicine University of Zagreb Heinzelova 55, Zagreb, Croatia www.cmj.hr 247 Tominac Trcin et al: Synthetic vs natural scaffolds for human limbal stem cells Like other adult stem cells, limbal stem cells are of high proliferative capacity, small in size (6-7 µm), have high nucleus to cytoplasm ratio and rarely undergo cell division. They do not express markers of terminally differentiated cells like cytokeratin (CK) 3, cytokeratin 12, and involucrin. Although specific markers for limbal stem cells are yet to be defined, commonly used are putative markers of progenitor, limbal basal cells like p63, p63 gene splice variant ΔNp63α, β1– integrin, and ABC-G2, a member of ATP-Binding Cassette (ABC) family (1-4). On the other hand, cytokeratin CK19 is known as a marker of the conjunctival epithelium, although more specific ones, like cytokeratin CK13 and S100 calcium binding protein family: S100A8 and S100A9, have recently been identified (5). Importance of limbal stem cells for homeostasis in normal corneal epithelium becomes particularly evident in patients with Limbal Stem Cell Deficiency (LSCD), where this process is seriously disrupted. LSCD can be of congenital origin (like aniridia) or acquired through events like trauma, repeated surgeries of ocular surface, inflammation of ocular surface (Stevens-Johnson syndrome) (6). Either way, stem cells from basal limbal region are depleted or dysfunctional. The corneal epithelium loses ability for renewal, which leads to chronic epithelial defects, scarring, neovascularization, conjunctivalization, and inflammation of the cornea. Symptoms may include pain, photophobia, blepharospasm, tearing and even blindness (7). For total LSCD, conventional treatment includes transplantation of limbal tissue from autologous healthy eye or from the eye of allogenic donor. Unfortunately, there is certain risk after autologous transplantation for healthy eye to develop LSCD; and transplantation of allogenic stem cells requires systemic immunosuppression of the recipient causing various side-effects of such treatment. Almost 16 years ago cultured limbal epithelial cell therapy was introduced as a treatment option for LSCD (8). Up till now several hundred patients have been treated with ex vivo cultivated cells. Long term follow up studies reported satisfying outcomes, with up to 76.6% of success defined as a permanent restoration of a transparent, avascular, and renewing cornea (9-13). be expanded in vitro with or without feeder cells, in culture media with fetal bovine serum, autologous serum, or serum free (14). The correct selection of the cell scaffold is of fundamental importance for clinical application. The primary aim of this research was to investigate the impact of different types of scaffolds on the viability and differentiation of in vitro cultured limbal epithelial cells. In this respect natural scaffolds (amniotic membrane, fibrin) were compared to electrospun ones made from two widely used synthetic polymers in tissue engineering: polyurethane and polycaprolactone. Considering hydrophobic properties of their surfaces that could attenuate cell attachment, we tested their more hydrophilic versions in parallel – the electrospun scaffolds after the NaOH treatment. Material and methods Scaffolds preparation and cell culture All aseptic procedures regarding preparation of scaffolds were respected and cell cultures were prepared in a clean room facility of Tissue Bank, University Hospital Sestre Milosrdnice (Zagreb, Croatia, 2013/2014). Amniotic membrane preparation Human placenta was collected at the Gynecology and Obstetrics Department, University Hospital Center Sestre Milosrdnice, from a healthy woman during cesarean section. The amnion was isolated from the chorion, washed in sterile physiological solution, put on nitrocellulose membrane fragments, and cryopreserved. Thawed amnion was washed (...truncated)