Optimization of Corneal Epithelial Progenitor Cell Growth on <i>Bombyx mori</i> Silk Fibroin Membranes

Hogerheyde, Thomas A.; Suzuki, Shuko; Walshe, Jennifer; Bray, Laura J.; Stephenson, Sally‐Anne; Harkin, Damien G.; Richardson, Neil

doi:10.1155/2016/8310127

Cited by 9 publications

(8 citation statements)

References 43 publications

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“…It can therefore be assumed that surface patterning of RHC I and CLP DMTMM hydrogels may be unnecessary to attain successful LESC-enriched cultures in vitro . Our results confirm the findings of Hogerheyde et al [59], in which fibronectin coating of silk fibroin membranes did not significantly improve cell outgrowth of iHCECs or of primary LESC. Conversely, our results are in contradiction to previous research that showed the benefit of fibronectin patterning of carrier membranes on cell proliferation of iHCECs [21, 60].…”

Section: Discussionsupporting

confidence: 92%

“…When compared to our study, aforementioned reports [21, 59–62] lacked (i) extensive phenotyping and genotyping of cultured primary cells, (ii) comparison to HAM, the carrier of choice in CLET, and/or (iii) implementation of a xeno-free, standardized cultivation protocol; all of which indicate further need of optimization-described techniques.…”

Section: Discussionmentioning

confidence: 75%

“…Even though our results did not indicate a short-term benefit for in vitro cultivation of LESC, surface patterning might potentially result in an ideal microenvironment for long-term LESC proliferation and preservation. In addition to F- μ CP and 3D fabrication, other groups have suggested surface tethering and bulk incorporation of laminin, collagen type III, Coll-IV, IKVAV, YIGSR, RGD, and vitronectin [46, 59, 78, 79]. All of these possibilities support the promise of collagen hydrogels in tissue engineering, not only in ophthalmology but also in other disciplines such orthopedics [80], dermatology [81], and cardiology [82].…”

Section: Discussionmentioning

confidence: 99%

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In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds

Haagdorens

Cėpla

Melsbach

et al. 2019

Stem Cells International

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Purpose. To investigate the efficacy of recombinant human collagen type I (RHC I) and collagen-like peptide (CLP) hydrogels as alternative carrier substrates for the cultivation of limbal epithelial stem cells (LESC) under xeno-free culture conditions. Methods. Human LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified RHC I, (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked CLP (CLP-12 EDC), (4) DMTMM- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium-) crosslinked CLP (CLP-12), (5) fibronectin-patterned CLP-12, (6) “3D limbal niche-mimicking” CLP-12, and (7) DMTMM-crosslinked CLP made from higher CLP concentration solution. Cell proliferation, cell morphology, and expression of LESC markers were analyzed. All data were compared to cultures on human amniotic membrane (HAM). Results. Human LESC were successfully cultivated on six out of seven hydrogel formulations, with primary cell cultures on CLP-12 EDC being deemed unsuccessful since the area of outgrowth did not meet quality standards (i.e., inconsistence in outgrowth and confluence) after 14 days of culture. Upon confluence, primary LESC showed high expression of the stem cell marker ΔNp63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-β4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-α1, and collagen type IV. Cells showed low expression of differentiation markers KRT3 and desmoglein 3 (DSG3). Significantly higher gene expression of KRT3 was observed for cells cultured on CLP hydrogels compared to RHC I and HAM. Surface patterning of hydrogels influenced the pattern of proliferation but had no significant effect on the phenotype or genotype of cultures. Overall, the performance of RHC I and DMTMM-crosslinked CLP hydrogels was equivalent to that of HAM. Conclusion. RHC I and DMTMM-crosslinked CLP hydrogels, irrespective of surface modification, support successful cultivation of primary human LESC using a xeno-free cultivation protocol. The regenerated epithelium maintained similar characteristics to HAM-based cultures.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

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In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds

Haagdorens

Cėpla

Melsbach

et al. 2019

Stem Cells International

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“…Then washed with 1 ml PBS for 3 times. These coated silk films were prepared freshly before seeding cells 34 – 36 .…”

Section: Methodsmentioning

confidence: 99%

Silk films with nanotopography and extracellular proteins enhance corneal epithelial wound healing

Luo

Kang

Sartaj

et al. 2021

Sci Rep

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Corneal wound healing depends on extracellular matrix (ECM) and topographical cues that modulate migration and proliferation of regenerating cells. In our study, silk films with either flat or nanotopography patterned parallel ridge widths of 2000, 1000, 800 nm surfaces were combined with ECMs which include collagen type I (collagen I), fibronectin, laminin, and Poly-d-Lysine to accelerate corneal wound healing. Silk films with 800 nm ridge width provided better cell spreading and wound recovery than other size topographies. Coating 800 nm patterned silk films with collagen I proves to optimally further increased mouse and rabbit corneal epithelial cells growth and wound recovery. This enhanced cellular response correlated with redistribution and increase in size and total amount of focal adhesion. Transcriptomics and signaling pathway analysis suggested that silk topography regulates cell behaviors via actin nucleation ARP-WASP complex pathway, which regulate filopodia formation. This mechanism was further explored and inhibition of Cdc42, a key protein in this pathway, delayed wound healing and decreased the length, density, and alignment of filopodia. Inhibition of Cdc42 in vivo resulted in delayed re-epithelization of injured corneas. We conclude that silk film nanotopography in combination with collagen I constitutes a better substrate for corneal wound repair than either nanotopography or ECM alone.

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“…Finally, our studies made use of animal-component-free CnT-Prime1 culture medium for explant culture. Conventional keratinocyte growth medium contains supplements including bovine pituitary extract [30], which may not be safe for clinical use; we found that we could carry out these experiments with CnT-Prime1 medium [31] that did not require supplementation with animal products. This switch was also due in large part to a shortage of keratinocyte growth medium (Lonza) that arose during our experiments.…”

Section: Plos Onementioning

confidence: 99%

Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture

et al. 2020

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Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven techniques for treating limbal stem cell deficiency (LSCD). However, the precise regions that are most suitable for preparing explants for transplantation have not been identified conclusively. Accordingly, this in vitro study aimed at determining ideal sites to be selected for tissue harvest for limbal stem cell culture and transplantation. We evaluated cell outgrowth potential and the expression of stem cell markers in cultures from 48 limbal explants from five cadaveric donors. The limbal explants were generated from the three specific sites: Lcor (located innermost and adjacent to the cornea), Lm (middle limbus), and Lconj (located outermost adjacent to the conjunctiva). We found that explants from the Lconj and Lm sites exhibited higher growth potential than those from the Lcor site. Transcript encoding the stem cell marker and p63 isoform, ΔNp63, was detected in cells from Lm and Lconj explants; expression levels were slightly, though significantly (p-value < 0.05), higher in Lm than in Lconj, although expression of ΔNp63α protein was similar in cells from all explants. Differential expression of ATP-Binding Cassette Subfamily G Member 2 (ABCG2) did not reach statistical significance. Immunohistochemistry by indirect immunofluorescence analysis of limbus tissue revealed that the basal layer in explant tissue from Lconj and Lm contained markedly more stem cells than found in Lcor explant tissue; these findings correlate with a higher capacity for growth. Collectively, our findings suggest that explants from the Lconj and Lm sites should be selected for limbal cell expansion for both CLET and SLET procedures. These new insights may guide surgeons toward specific limbal sites that are most suitable for stem cell culture and transplantation and may ultimately improve treatment outcomes in the patients with LSCD.

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Optimization of Corneal Epithelial Progenitor Cell Growth on Bombyx mori Silk Fibroin Membranes

Cited by 9 publications

References 43 publications

In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds

In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds

Silk films with nanotopography and extracellular proteins enhance corneal epithelial wound healing

Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture

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