The effect of the carbodiimide cross-linker on the structural and biocompatibility properties of collagen-chondroitin sulfate electrospun mat

Sheyda Akhshabi,1 Esmaeil Biazar,2 Vivek Singh,3 Saeed Heidari Keshel,4 Nagaraja Geetha1 1Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysuru, Karnataka, India; 2Department of Biomaterials Engineering, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran; 3Prof Brien Holden Eye Research Center, Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, L. V. Prasad Eye Institute, Hyderabad, Telangana, India; 4Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Background: Collagen and chondroitin sulfate (CS) are an essential component of the natural extracellular matrix (ECM) of most tissues. They provide the mechanical stability to cone the compressive forces in ECM. In tissue engineering, electrospun nanofibrous scaffolds prepared by electrospinning technique have emerged as a suitable candidate to imitate natural ECM functions. Cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxy succinimide can overcome the weak mechanical integrity of the engineered scaffolds in addition to the increased degradation stability under physiological conditions. Materials and methods: This study has synthesized nanofibrous collagen–CS scaffolds by using the electrospinning method. Results: The results have shown that incorporation of CS in higher concentration, along with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxy succinimide, enhanced mechanical stability. Scaffolds showed more resistance to collagenase digestion. Fabricated scaffolds showed biocompatibility in corneal epithelial cell attachment. Conclusion: These results demonstrate that cross-linked electrospun CO–CS mats exhibited a uniform nanofibrous and porous structure, especially for lower concentration of the cross-linker and may be utilized as an alternative effective substrate in tissue engineering. Keywords: collagen, chondroitin sulfate, CS, extracellular matrix, ECM, cross linker, electrospinning, nanofibe

3D-Printed membrane as an alternative to amniotic membrane for ocular surface/conjunctival defect reconstruction: An in vitro and in vivo study

Background The aim of this study was to evaluate the surgical handling and clinical applicability of a specific 3D-printed membrane design fabricated using a gelatin, elastin and sodium hyaluronate blend for conjunctival reconstruction and compare it with amniotic membrane (AM), which is normally used in such surgeries. Methods 3D printing technique was employed to fabricate the membrane based on gradient design. Prior to printing, rheometry was employed to optimize the ink composition. The printed membranes were then fully characterized in terms of physical and mechanical properties. In vitro viability, proliferation and adhesion of human limbal epithelial cells were assessed using MTT assay and scanning electron microscopy (SEM), respectively. Prior to in vivo experiment, surgical handling of each membrane was evaluated by three surgeons. In vivo evaluation was conducted through implanting the gelatin-based membranes and AM on induced conjunctival defects in rabbits (n = 8). Clinical observations, including epithelialization, inflammation severity, scar tissue formation and presence of granulation tissue, were recorded from day 1 through day 28. Histological examination was performed on all enucleated eyes on day 28. In addition to H&amp;E staining, specific stains including Periodic Acid Schiff staining, Masson's Trichrome staining and immuno-histochemical staining for α-SMA were further used to assess goblet cell proliferation, healed sub-epithelial stroma and scar tissue formation and the presence of myofibroblasts, respectively. Results Among all the examined compositions, a blend of 8% w/v gelatin, 2% w/v elastin and 0.5% w/v sodium hyaluronate was found to be appropriate for printing. The printed membranes had favorable optical characteristics (colorless and transparent), and the surgical handling was significantly easier compared to AM. Epithelial cells cultivated on the membranes indicated suitable viability and proliferation, and SEM images presented appropriate cell adhesion on the surface of the membranes. Clinical observations suggested similar epithelialization time (approximately 3 weeks) for both the membrane and AM grafted eyes but significantly lower levels of clinical inflammation in the membrane group from day 1 through day 28 (p = 0.01), which is a key advantage of using the printed membranes over the AM. Histological examination showed similar qualities in the healed epithelium in terms of cell morphology and cell layers. However, twice the density of goblet cells per 100 cells was observed in the gelatin-based membrane grafted group. Remnant of the degraded implant was seen in only 3 of the membranes, but in 7 of the AM grafted eyes. Inflammation and granulomatous reaction was significantly higher in sections containing the AM compared to membrane (p ≺ 0.01 and p = 0.01, respectively). α-SMA staining was more evident, but not significantly different from the gelatin-based membrane, for the AM group (p = 0.25). Conclusion The designed gelatin-based membrane offers the necessary physical and mechanical characteristics needed for successful ocular surface/conjunctival defect construction and may be considered a promising alternative to AM due to a more predictable degradation pattern, higher goblet cell density on the healed epithelium, less inflammation and reduced scar tissue formation.</p