Three-dimensional-printed collagen scaffold with limbal stem cells derived from adipose-derived mesenchymal stem cells for the treatment of limbal stem cell deficiency

Abstract

When limbal stem cell deficiency (LSCD) is partial, the standard treatment involves covering the corneal surface with amniotic membrane (AM), which supports the proliferation of the remaining limbal stem cells (LSCs). In cases of complete LSCD, the most common treatment is cultured limbal epithelial transplantation (CLET), although there is a risk of rejection. Studies have shown that mesenchymal stem cell transplantation is equally safe and effective as CLET. Recent research has demonstrated successful differentiation of adipose-derived adult mesenchymal stem cells (ADSCs) into LSCs. Combining AM transplantation with LSCs improves treatment efficacy. However, a limitation of AM use is donor variability and the associated risk of immune rejection. We propose the use of 3D-printed collagen as a scaffold seeded with LSCs derived from ADSCs for the treatment of LSCD in a rat model. The 3D-printed collagen scaffolds exhibited good transparency. In vitro differentiation of ADSCs into LSCs showed morphological changes that were more pronounced and occurred more rapidly on 3D-printed collagen. Among the tested substrates, 3D-printed collagen was the most efficient for differentiation, yielding the highest expression of LSC-specific markers (p63α and BMI-1) and the corneal epithelial marker (SSEA-4). LSCs differentiated in either AM or 3D-printed collagen I scaffolds were transplanted into a rat model of LSCD and compared with the standard, cell-free AM treatment. In all treatment groups, the induced epithelial wound was closed; however, integration of the 3D-printed collagen scaffold was statistically superior to that of AM. However, markers for different corneal structures (PAS, BMI-1, p63α, and cytokeratins 12 and 13) indicated that the generated epithelium was conjunctival rather than corneal, suggesting that the contribution of ADSC-derived LSCs was insufficient for complete corneal re-epithelization.  

Keywords

Amniotic membrane / Bioprinting / Cornea / Limbal stem cell deficiency / Mesenchymal stem cells / Regenerative medicine / Tissue engineering / Vision loss

Funding

The authors received grants from: the Ministry of Science and Innovation, Strategic Health Action, Instituto de Salud Carlos III (ISCIII), Proyectos de I+D+I en Salud (grant number PI23/00207) and co-funded by the European Union; as well as the Foundation for Biomedical Research of La Paz University Hospital (Luis Álvarez Projects; grant number 07/252898.9/24). The study was also supported by StemVision and Carl Zeiss AG. M. Cadenas-Martín and M. Stokking were financed by the European Social Fund (PI_SEPE_MPDM PROGRAMA INVESTIGO 1785344099 and PI_SEPE_PROGRAMA INVESTIGO 26-23, respectively).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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