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. 2018 Mar 15;69:120-130.
doi: 10.1016/j.actbio.2018.01.011. Epub 2018 Jan 31.

Short Peptide Analogs as Alternatives to Collagen in Pro-Regenerative Corneal Implants

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Free PMC article

Short Peptide Analogs as Alternatives to Collagen in Pro-Regenerative Corneal Implants

Jaganmohan R Jangamreddy et al. Acta Biomater. .
Free PMC article

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Abstract

Short collagen-like peptides (CLPs) are being proposed as alternatives to full-length collagen for use in tissue engineering, on their own as soft hydrogels, or conjugated to synthetic polymer for mechanical strength. However, despite intended clinical use, little is known about their safety and efficacy, mechanism of action or degree of similarity to the full-length counterparts they mimic. Here, we show the functional equivalence of a CLP conjugated to polyethylene glycol (CLP-PEG) to full-length recombinant human collagen in vitro and in promoting stable regeneration of corneal tissue and nerves in a pre-clinical mini-pig model. We also show that these peptide analogs exerted their pro-regeneration effects through stimulating extracellular vesicle production by host cells. Our results support future use of CLP-PEG implants for corneal regeneration, suggesting the feasibility of these or similar peptide analogs in clinical application in the eye and other tissues.

Statement of significance: Although biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable analogs that are functionally equivalent, and can be readily scaled-up are therefore very desirable for pre-clinical to clinical translation. Here, we demonstrate, using cornea regeneration as our test bed, that collagen-like-peptides conjugated to multifunctional polyethylene glycol (CLP-PEG) when grafted into mini-pigs as corneal implants were functionally equivalent to recombinant human collagen-based implants that were successfully tested in patients. We also show for the first time that these materials affected regeneration through stimulation of extracellular vesicle production by endogenous host cells that have migrated into the CLP-PEG scaffolds.

Keywords: Collagen-like peptide; Cornea; Exosomes; Recombinant human collagen; Regeneration.

Figures

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Fig. 1
Fig. 1
Implants, structure and mechanical stability. Control RHCIII-MPC implants (a) and CLP-PEG implants (b) are both optically clear as 500 μm thick corneal shaped implants. Similarly, mimicking RHCIII-MPC implants (c) CLP-PEG implants (d) display a nanofibrillar structure when observed using high pressure freezing TEM imaging. Scale bars, 200 nm. Oscillatory rheology of CLP-hydrogels pre- and post-grafting into rabbit corneas compared to healthy, unoperated rabbit corneas. (e) Storage modulus, (f) Loss modulus and (g) Loss tangent as a function of oscillation frequency at 0.27% shear strain amplitude.
Fig. 2
Fig. 2
Biocompatibility of Implants. Neither RHCIII-MPC (a) nor CLP-PEG (b) homogenized hydrogels tested at dilutions of 10% (1/10 w/v) and 1% (1/100 w/v) showed any noticeable trace of genotoxicity or cytotoxicity unlike the respective positive controls, BaP and 4NQO. A PrestoBlue assay (c) showed that proliferation rates of human corneal epithelial cells on RHCIII-MPC and CLP-PEG hydrogels were similar. Live/dead assay further shows minimal cell death among the RHCIII-MPC and CLP-PEG like TCP control (d). Scale bars, 100 µm. For a positive staining of Live/dead assay the TCP cultured cells are treated with 0.1% saponin for 30 min at 20 °C.
Fig. 3
Fig. 3
RHCIII-MPC and CLP-PEG corneal implants and their performance in mini-pigs. Examples of optically clear RHCIII-MPC and CLP-PEG hydrogel implants (a). After 12 months of implantation in corneas of mini-pigs, the implants remain clear like the unoperated cornea. Arrows indicate the boundaries of the implants. In vivo confocal microscopy shows that both RHCIII-MPC and CLP-PEG implanted corneas have regenerated their epithelium (b), stroma (c) and sub-epithelial nerve plexus (d) to resemble their counterparts in the normal, healthy cornea. Scale bars, 150 µm. Aesthesiometry measures the pressure needed to obtain a blink reaction, i.e. touch sensitivity, which is correlated to nerve function. Pre-operatively, all healthy corneas showed a response to light touch. At 5 weeks post-operation, the implanted corneas were non-responsive, even with maximal pressure exerted. At 3 months, touch sensitivity is returning so less pressure was needed for a response. By 6 months, sensitivity was back to normal levels. * – p < .05 as compared to un-operated eyes (Kruskal-Wallis test with Bonferroni post hoc test).
Fig. 4
Fig. 4
Characterization of regenerated neo-corneas. (a) H&E sections through a healthy, unoperated cornea, and regenerated neo-corneas at 12 months after implantation with control RHCIII-MPC and CLP-PEG scaffolds. Epithelial hyperplasia was noted in the implanted corneas, which is normal in post-grafting tissues (b). Scale bars, 100 µm. The regenerated corneal epithelium shows staining with cytokeratin 3/12, a marker of differentiated cells in all three samples. Stromal collagens types III (c) and V (d) are present in the implanted as well as unoperated corneas, showing in particular that remodeling and extracellular matrix production is occurring in CLP-PEG implants that contained no collagen. Sub-epithelial nerve plexus stained with b-tubulin (e) showing nerve regeneration in CLP-PEG hydrogels as in control RHCIII-MPC and unoperated contralateral eyes. Scale bars, 100 µm. At the ultrastructural level, TEM micrographs of all three samples show an epithelium with distinct layers of elongated, cuboidal and flattened cells, characteristic of healthy corneas (f). Scale bar, 50 µm. TEM images show a regular, lamellar arrangement in both unoperated and regenerated stromas (g). Scale bar, 20 µm. The CLP-PEG neo-corneas have slightly less regular stromas as seen in 3D reconstructed SBF-SEM images (epithelium rendered in blue) (h) suggesting an on-going process. Both neo-corneas contained collagen fibrils decorated with proteoglycans (i), similar to the matrix of the healthy, unoperated cornea. Scale bar, 200 nm.
Fig. 5
Fig. 5
Extracellular vesicle release from regenerating neo-corneas stimulated by biomimetic implants. (a) 3D reconstructed serial block face scanning electron micrographs of an unoperated healthy cornea and regenerating neo-corneas at 12 months after implantation with control RHCIII-MPC or CLP-PEG, showing the release of extracellular vesicles (rendered in gold) from the lower epithelial layers into the stroma. (b) Immunofluorescent micrographs of exosome marker CD-9 stained cornea samples. (c) Corresponding 3D reconstructions of CD9-stained sections confirm that the extracellular vesicles released by the epithelium include positively-stained exosomes. (d) Rab7 stained endosomes-exosomes in both unoperated and implanted corneas. Cell nuclei are counterstained blue with DAPI. Scale bars, 25 µm.

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