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In Vitro Culture of Human Corneal Endothelium on Non-Mulberry Silk Fibroin Films for Tissue Regeneration.

ABSTRACT: Purpose:The purpose of this study was to determine if non-mulberry varieties of silk are suitable for the culture of corneal endothelium (CE). Methods:Aqueous silk fibroin derived from Philosamia ricini (PR), Antheraea assamensis (AA), and Bombyx mori (BM) were cast as approximately 15 µm films with and without pores on which human CE cells were cultured. Tensile strength, elasticity, transmittance in visible range, and degradation properties of the films were characterised. Adhesion of CE to the silk films was quantified using MTT assay in addition to quantifying the number and area of focal adhesions using paxillin. Expression of CE markers was determined at the gene and protein levels using PCR and immunostaining, respectively. Barrier integrity of the cultured cells was measured as permeability to FITC dextran (10 kDa) in the presence or absence of thrombin. Results:The films exhibited robust tensile strength, >95% transmittance and a refractive index comparable to the native cornea. BM degraded significantly faster when compared to PR and AA. A comparison between the three varieties of silk showed that significantly more cells were adhered to PR and AA than to BM. This was also reflected in the expression of stable focal adhesions on PR and AA, thus enabling the formation of intact monolayers of cells on these varieties unlike on BM. Treatment with thrombin significantly increased cellular permeability to dextran. Conclusions:Our data shows that PR and AA varieties sufficiently support the growth and function of CE cells. This could be attributed to the presence of natural cell binding motifs (RGD) in these varieties. Translational Relevance:Development of a suitable carrier for engineering the CE to address a major clinical requirement of healthy donor tissues for transplantation.

SUBMITTER: Ramachandran C

PROVIDER: S-EPMC7396167 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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In Vitro Culture of Human Corneal Endothelium on Non-Mulberry Silk Fibroin Films for Tissue Regeneration.

Ramachandran Charanya C Gupta Prerak P Hazra Swatilekha S Mandal Biman B BB

Translational vision science & technology 20200309 4

<h4>Purpose</h4>The purpose of this study was to determine if non-mulberry varieties of silk are suitable for the culture of corneal endothelium (CE).<h4>Methods</h4>Aqueous silk fibroin derived from Philosamia ricini (PR), Antheraea assamensis (AA), and Bombyx mori (BM) were cast as approximately 15 µm films with and without pores on which human CE cells were cultured. Tensile strength, elasticity, transmittance in visible range, and degradation properties of the films were characterised. Adhes ...[more]

PMID: 32818099

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Project description:The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm-1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 μmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 μmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

Project description:The antimicrobial nature of Antharaea mylitta silk-fibroin (SF) is reported but antioxidant potential and the immunomodulatory role towards the fibroblast cell repair process is not explored. Polyurethane is reported to have inflammatory potential by mononuclear cells directed cytokine release, which can guide fibroblast repair. Present study demonstrates the conjunctive effect of inflammatory PU/SF to regulate the favorable shift from pro-inflammatory to anti-inflammatory cytokine stimulation for accelerated fibroblast repair. Minimal inhibitory concentration of SF was determined against pathogenic strains and the effect of SF was investigated for fibroblast NIH3T3 cell adhesion. SF doses (8, 8.5, 9 mg mL-1) were found to be greater than both the IC50 of DPPH scavenging and the ED50 for NIH3T3 proliferation. Anti-lipid peroxidase (ALP) activity of SF doses and citric acid-treated NIH3T3 cells were compared under hydrogen peroxide (H2O2) induced oxidative stress. 9 mg mL-1 SF showed greater ALP activity than the citric acid standard. SF-driven protection to oxidative damage was measured by viable cell fraction in trypan blue dye exclusion assay where 9 mg mL-1 SF showed the highest viability (p ≤ 0.05). 9 mg mL-1 SF was blended with PU for scaffold (w/v = 2 : 5, 2 : 7, 2 : 9) fabrication. The protective effect of PU/SF (2 : 5, 2 : 7, 2 : 9) against oxidative stress was verified by damaged cell survival in MTT assay and DNA quantification. The highest number of cells survived on PU/SF (2 : 9) at all intervals (p ≤ 0.01) upon oxidative damage; PU/SF (2 : 9) was also fabricated by employing the immobilization technique. Immobilized PU/SF (2 : 9) exhibited a greater zone of microbial inhibition, a higher extent of inhibition to microbial adherence, and caused more LDH release from bacterial cell membrane due to membrane rupture, resulting in bacterial cell death (E. coli, K. pneumoniae, P. aeruginosa, S. aureus) compared to the experimental results shown by blended PU/SF (2 : 9). The protective nature of PU/SF (2 : 9) against oxidative stress was ensured through the LDH activity of damaged NIH3T3 cells. Initial raised IL-6, TNF-alpha (pro-inflammatory cytokines) and lowered IL-8, IL-10 (anti-inflammatory cytokine) profiles coupled with fallen IL-6, TNF-alpha, and elevated IL-8, IL-10 at later hours synergistically progress the inflammatory phase of in vitro scratch wound repair in mononuclear culture treated by PU/SF (2 : 9).

Dataset Information

In Vitro Culture of Human Corneal Endothelium on Non-Mulberry Silk Fibroin Films for Tissue Regeneration.

Publications

In Vitro Culture of Human Corneal Endothelium on Non-Mulberry Silk Fibroin Films for Tissue Regeneration.

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