Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs.

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.

Limbal Stem Cells on Bacterial Nanocellulose Carriers for Ocular Surface Regeneration.

Limbal stem cells (LSCs) are already used in cell-based treatments for ocular surface disorders. Clinical translation of LSCs-based therapies critically depends on the successful delivery, survival, and retention of these therapeutic cells to the desired region. Such a major bottleneck could be overcome by using an appropriate carrier to provide anchoring sites and structural support to LSC culture and transplantation. Bacterial nanocellulose (BNC) is an appealing, yet unexplored, candidate for this application because of its biocompatibility, animal-free origin and mechanical stability. Here, BNC as a vehicle for human embryonic stem cells-derived LSC (hESC-LSC) are investigated. To enhance cell-biomaterial interactions, a plasma activation followed by a Collagen IV and Laminin coating of the BNC substrates is implemented. This surface functionalization with human extracellular matrix proteins greatly improved the attachment and survival of hESC-LSC without compromising the flexible, robust and semi-transparent nature of the BNC. The surface characteristics of the BNC substrates are described and a preliminary ex vivo test in simulated transplantation scenarios is provided. Importantly, it is shown that hESC-LSC retain their self-renewal and stemness characteristics up to 21 days on BNC substrates. These results open the door for future research on hESC-LSC/BNC constructs to treat severe ocular surface pathologies.

Tissue adhesive hyaluronic acid hydrogels for sutureless stem cell delivery and regeneration of corneal epithelium and stroma.

Regeneration of a severely damaged cornea necessitates the delivery of both epithelium-renewing limbal epithelial stem cells (LESCs) and stroma-repairing cells, such as human adipose-derived stem cells (hASCs). Currently, limited strategies exist for the delivery of these therapeutic cells with tissue-like cellular organization. With the added risks related to suturing of corneal implants, there is a pressing need to develop new tissue adhesive biomaterials for corneal regeneration. To address these issues, we grafted dopamine moieties into hydrazone-crosslinked hyaluronic acid (HA-DOPA) hydrogels to impart tissue adhesive properties and facilitate covalent surface modification of the gels with basement membrane proteins or peptides. We achieved tissue-like cellular compartmentalization in the implants by encapsulating hASCs inside the hydrogels, with subsequent conjugation of thiolated collagen IV or laminin peptides and LESC seeding on the hydrogel surface. The encapsulated hASCs in HA-DOPA gels exhibited good proliferation and cell elongation, while the LESCs expressed typical limbal epithelial progenitor markers. Importantly, the compartmentalized HA-DOPA implants displayed excellent tissue adhesion upon implantation in a porcine corneal organ culture model. These results encourage sutureless implantation of functional stem cells as the next generation of corneal regeneration.

Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential.

BACKGROUND: The differentiation of corneal limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) has great power as a novel treatment for ocular surface reconstruction and for modeling corneal epithelial renewal. However, the lack of profound understanding of the true LSC population identity and the regulation of LSC homeostasis is hindering the full therapeutic potential of hPSC-derived LSCs as well as primary LSCs. METHODS: The differentiation trajectory of two distinct hPSC lines towards LSCs was characterized extensively using immunofluorescence labeling against pluripotency, putative LSC, and mature corneal epithelium markers. Cell counting, flow cytometry, and qRT-PCR were used to quantify the differences between distinct populations observed at day 11 and day 24 time points. Initial differentiation conditions were thereafter modified to support the maintenance and expansion of the earlier population expressing ABCG2. Immunofluorescence, qRT-PCR, population doubling analyses, and transplantation into an ex vivo porcine cornea model were used to analyze the phenotype and functionality of the cell populations cultured in different conditions. RESULTS: The detailed characterization of the hPSC differentiation towards LSCs revealed only transient expression of a cell population marked by the universal stemness marker and proposed LSC marker ABCG2. Within the ABCG2-positive population, we further identified two distinct subpopulations of quiescent ∆Np63α-negative and proliferative ∆Np63α-positive cells, the latter of which also expressed the acknowledged intestinal stem cell marker and suggested LSC marker LGR5. These populations that appeared early during the differentiation process had stem cell phenotypes distinct from the later arising ABCG2-negative, ∆Np63α-positive third cell population. Importantly, novel culture conditions modulating the Wnt and BMP signaling pathways allowed efficient maintenance and expansion of the ABCG2-positive populations. In comparison to ∆Np63α-positive hPSC-LSCs cultured in the initial culture conditions, ABCG2-positive hPSC-LSCs in the novel maintenance condition contained quiescent stem cells marked by p27, demonstrated notably higher population doubling capabilities and clonal growth in an in vitro colony-forming assay, and increased regenerative potential in the ex vivo transplantation model. CONCLUSIONS: The distinct cell populations identified during the hPSC-LSC differentiation and ABCG2-positive LSC maintenance may represent functionally different limbal stem/progenitor cells with implications for regenerative efficacy.

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

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-ß4 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.

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks.

There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.

Hydrazone crosslinked hyaluronan-based hydrogels for therapeutic delivery of adipose stem cells to treat corneal defects.

Corneal blindness is a worldwide problem, plagued by insufficient amount of high-quality donor tissue. Cell therapy using human adipose stem cells (hASCs) has risen as an alternative to regenerate damaged corneal stromal tissue, the main structural and refractive layer of the cornea. Herein we propose a method to deliver hASCs into corneal defects in hyaluronan (HA)-based hydrogels, which form rapidly in situ by hydrazone crosslinking. We fabricated two different HA-based hydrazone-crosslinked hydrogels (HALD1-HACDH and HALD2-HAADH), and characterized their swelling, degradation, mechanical, rheological and optical properties and their ability to support hASC survival. To promote hASC attachment and survival, we incorporated collagen I (col I) to the more stable HALD1-HACDH hydrogel, since the HALD2-HAADH hydrogel suffered swift degradation in culture conditions. We then used an organ culture model with excised porcine corneas to study the delivery of hASCs in these three hydrogels for stromal defect repair. Although all hydrogels showed good hASC survival directly after encapsulation, only the collagen-containing HALD1-HACDH-col I hydrogel showed cells with elongated morphology, and significantly higher cell metabolic activity than the HALD1-HACDH gel. The addition of col I also increased the stiffness and reduced the swelling ratio of the resulting hydrogel. Most importantly, the corneal organ culture model demonstrated these hydrogels as clinically feasible cell delivery vehicles to corneal defects, allowing efficient hASC integration to the corneal stroma and overgrowth of corneal epithelial cells.

Digital image analysis of the tissue surface areas of site-designated and bilaterally pooled prostate biopsies.

Initial reports about the length of bilaterally pooled biopsies showed alarming tissue loss compared to individual biopsies, but the current understanding of "noodle biopsies" and better embedding techniques may have improved their quality. Here, we implemented digital image analysis to study the differences in tissue surface areas between individual and pooled cores. Prostate biopsy reports from 1242 consecutive patients were reviewed. Urologist-dependent bias on the biopsy quality was eliminated by identifying four urologists who submitted equally individual and bilaterally pooled biopsies. Digital image analysis was applied to the tissue surface areas of 936 virtual slides containing 1440 biopsy cores (12 cores per patient x 120 patients) taken by the four urologists. The median (range) surface areas were 73.8 mm² (40.1-102.5) for the site-designated (n=57) and 77.1 mm² (49.5-119.2) for the bilaterally pooled biopsies (n=63) (p=0.19). For three urologists, the median surface areas were 69.5 mm² (60.4-93.2), 75.5 mm² (48.2-98.7) and 78.2 mm² (47.1-92.7) for the site-designated and 79.2 mm² (49.5-116.4), 69.3mm² (49.6-119.2) and 79.2 mm² (55.1-96.7) for the pooled biopsies, respectively (p=0.58-0.75). For one urologist, the median surface area was marginally higher for the pooled biopsies, 68.1 mm² (40.1-102.5) vs. 81.6 mm² (62.7-108.8) (p=0.03). In conclusion, the histological yields of individual and pooled prostate biopsies were practically equal. The results should not be considered as a recommendation to increasingly submit unspecified bilateral cores but to encourage pathology laboratories to embed and cut all received prostate biopsies with special attention, regardless of submission type.

Length of prostate biopsies is not necessarily compromised by pooling multiple cores in one paraffin block: an observational study.

BACKGROUND: Individually submitted prostatic needle biopsies are recommended by most guidelines because of their potential advantage in terms of core quality. However, unspecified bilateral biopsies are commonly submitted in many centers. The length of the core is the key quality indicator of prostate biopsies. Because there are few recent publications comparing the quality of 12 site-designated biopsies versus pooled biopsies, we compared the lengths of the biopsies obtained by both methods. METHODS: The material was obtained from 471 consecutive subjects who underwent prostatic needle biopsy in the Tampere University Hospital district between January and June 2013. Biopsies from 344 subjects fulfilled the inclusion criteria. The total number of cores obtained was 4047. The core lengths were measured on microscope slides. Extraprostatic tissue was subtracted from the core length. RESULTS: The aggregate lengths observed were 129.5 ± 21.8 mm (mean ± SD) for site-designated cores and 136.9 ± 26.4 mm for pooled cores (p = 0.09). The length of the core was 10.8 ± 1.8 mm for site-designated cores and 11.4 ± 2.2 mm for pooled cores (p = 0.87). The median length for pooled cores was 11 mm (range 5 mm - 18 mm). For individual site-designated cores, the median length was 11 mm (range 7 mm -15 mm). The core length was not correlated with the number of cores embedded into one paraffin block (r = 0.015). There was no significant difference in cancer detection rate (p = 0.62). CONCLUSIONS: Our results suggest that unspecified bilateral biopsies do not automatically lead to reduced core length. We conclude that carefully embedded multiple (three to nine) cores per block may yield cores of equal quality in a more cost-efficient way and that current guidelines favoring individually submitted cores may be too strict.