We showed previously that 1‐ethyl‐3‐(3‐dimethylamino‐propyl)‐carbodiimide hydrochloride (EDC) cross‐linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4 years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers—bulky, aromatic N‐cyclohexyl‐N0‐(2‐morpholinoethyl)‐carbodiimide (CMC), and nonbulky EDC—in a mouse corneal graft model. Murine corneas undergoing full‐thickness implantation with these gels became opaque due to dense retro‐corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c+ dendritic cells (DC) were found, suggesting a hydrogel‐specific innate immune response. More DC infiltrated the stroma surrounding EDC‐N‐hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis‐associated tenascin c expression. The opposite was true for CMC‐NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor β1 (TGF‐β1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF‐β1 neutralisation. Furthermore, tenascin c staining was found in 40‐ to 50μm long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF‐β1 alternatively activated (tolerising) DC regulate fibroblast‐mediated tenascin c secretion, possibly via local production of TGF‐β1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.
|Number of pages||11|
|Journal||Journal of Tissue Engineering and Regenerative Medicine|
|Early online date||25 Jul 2019|
|Publication status||Published - Sept 2019|
We are grateful to the Iain Fraser Flow Cytometry Centre, the Microscopy and Histology Facility, the Quantitative PCR Facility, and the Medical Research Facility at the University of Aberdeen. This work was supported by the Royal College of Surgeons of Edinburgh, UK and Saving Sight in Grampian/Development Trust of the University of Aberdeen, UK.
Saving Sight in Grampian/Development Trust of the University of Aberdeen, UK
Royal College of Surgeons of Edinburgh, UK
- corneal transplantation
- tissue repair
- transforming growth factor beta 1
- membrane nanotubes
- tectonic graft
- transforming growth factor β1, membrane nanotubes, tectonic graft