Optimization of a Fibroin-Based Substrate for Delivering L-MSCs and Epithelial Cells to the Ocular Surface

Background and Aim: Silk fibroin membranes support the growth oflimbal mesenchymal stromal cells (L-MSCs) and limbal epithelial cells(L-ECs). Nevertheless, this biomaterial requires improvement in celladhesion, permeability (for optimal communication between the cellsseeded on opposing side of the membrane) and strength for suturing.Thus, we evaluated the properties of an advanced formulation of fibroinusing a genetically modified fibroin containing RGD (cell adhesionmotifs). It is hypothesized that this modified fibroin could be a potentialvehicle for co-application of L-ECs and L-MSCs to the ocular surface.Methods: Genetically modified RGD-fibroin (RGD-F) was obtainedfrom the National Agriculture and Food Research Organization (NARO,Tsukuba, Japan). L-MSCs and L-ECs were separately applied to eitherculture surfaces (tissue culture plastic [TCP], TCP coated with standardfibroin [TCP-F], and TCP coated with RGD-F [TCP-RGD-F]) for 90minutes. The subsequent morphology and number of attached cellswere examined under both serum-free and serum-supplemented growthconditions. The number of attached cells was quantified using a Picogreenassay (estimating dsDNA). Morphology and proliferation of L-MSCs wereassessed in long-term (6 and 10 days) cultures on TCP, TCP-F, and TCPRGD-F. A comparison was made between the co-cultures of L-MSCsand L-ECs established on RGD-F free-standing membranes fabricatedusing the regular formulation versus RGD-F prepared with poly(ethylene)Glycol (PEG as a porogen) and horseradish peroxidase (HRP as a crosslinkingagent). Finally, the feasibility of applying co-cultures of L-ECs/LMSCsgrowing on RGD/PEG/HRP fibroin membrane to the ocular surfacewas examined in the rabbit model.Results: In 90 minutes cell attachment assay for both L-MSCs and L-ECs,no significant difference was detected between culture conditions.Morphological examination of long-term (6 and 10 days) L-MSCcultures revealed that while L-MSCs cultured on TCP-F generally formedsparsely scattered clusters, those cultured on TCP-RGD-F were moreuniform in adherence. Moreover, at 6 days L-MSCs displayed evidenceof significantly (P < 0.05; n = 4) superior attachment and proliferationon TCP-RGD-F compared with TCP-F as indicated by a Picogreen assay.Confocal microscopy of the L-MSC/L-ECs co-cultures grown on theopposing surface of the RGD/HRP/PEG-fibroin membrane revealed thatL-MSCs enhanced stratification of L-ECs (2-4 fold more stratified thanthose grown in the absence of L-MSCs). The RGD/HRP/PEG-fibroinmembrane was successfully sutured to the ocular surface withouttearing. Pilot treatment of L-MSCs/L-ECs implantation using RGD/HRP/PEG-fibroin membrane (n=1) initially produced promising results (nearlycomplete re-epithelialization in 14 days), but subsequently led to aprominent epithelial defect and conjunctivalization of the ocular surface. Conclusion: Results of the current study suggest that the benefitsassociated with the inclusion of RGD in silk fibroin are most effective inthe long-term (6-10 day) cultures of L-MSCs. Moreover, the feasibility ofusing RGD/HRP/PEG-fibroin as a vehicle for delivering limbal stem cellsto the ocular surface was preliminarily confirmed. The outcomes of thisstudy provide a foundation for the use of the L-MSCs, L-ECs and the newRGD silk fibroin scaffold. This offers a stepping stone for future studies incellular therapy for limbal stem cell diseases.

کلیدواژه ها:

Limbal ، MSCs ، Epithelial ، Silk fibroin ، RGD