Strategies to Mitigate Variability in Engineering Human Nasal Cartilage

Abstract

Skin cancer and its associated treitments can have devastating consequences for survivors; this is particularly true when cancer occurs on the nose. Recent work has applied cell-based tissue engineering (TE) strategies to develop nasal cartilage constructs for reconstruction of the nose. In this study, we have generated human nasal cartilage on a clinically approved collagen scaffold to investigate the donor-to-donor variability of TE cartilage and evaluated strategies to mitigate it. We also evaluated the gene expression of the family of fibroblast growth factor receptors (FGFR1-4) and their association with tissue quality. FGFR 1 was significantly positively correlated with GAG/DNA; a measure of chondrogenic capacity. We implemented two strategies: hypoxic culture and co-culture with mesenchymal stromal cells (MSCs) to increase tissue quality. Total glycosaminoglycan (GAG) content varied significantly between donors initially, with >10–fold difference between the best and worst donor tissue. Our co-culture strategy was able to increase TE construct quality from poor quality donor tissue while supressing hypertrophy relative to MSCs alone. However, no differences were observed with the use of hypoxic culture. Tissues generated using co-culture with MSCs became vascularized and calcified in vivo, demonstrating a non-stable cartilage phenotype in co-culture and MSCs cartilage constructs.

Table_10.DOCX

“…Most significantly, after 3 weeks of implantation in nude mice, coculture constructs treated with 200 ng/mL of PTHrP prior to implantation showed no calcification, as demonstrated by absence of Alizarin Red S staining. In contrast, control coculture constructs revealed considerable Alizarin Red S staining after the 3 weeks of implantation, which is consistent with previous reports (Andrews et al, 2017). Again, this suggests that pre-treatment of coculture constructs with PTHrP, prior to implantation, improves the phenotypic stability of the constructs.…”

“…The pellets were retrieved; their gross appearance was recorded, stored in PBS, and fixed in 10% (v/v) formalin, as previously described. The pellets were processed, embedded, and sectioned for histology, as previously described (Andrews et al, 2017).…”

“…Nasal septal cartilage is relatively easy to isolate from the septum and the NC derived from septal cartilage have been shown to proliferate rapidly in culture while maintaining their chondrogenic lineage (Fulco et al, 2014;Mumme et al, 2016). The use of fibroblast growth factor-2 (FGF2) and transforming growth factor β-1 (TGFβ-1) supplemented growth media during NC expansion has been shown to facilitate chondrogenic redifferentiation and the expression of functional cartilage extracellular matrix (ECM) in NC (Tay et al, 2004;Pelttari et al, 2014;Andrews et al, 2017). NC have been shown to produce stable cartilage constructs in vivo and have been used to repair articular cartilage of the knee and reconstruct nasal cartilage in skin cancer patients (Fulco et al, 2014;Mumme et al, 2016); however, engineered cartilage from NC suffers from large donor-to-donor variability (Fulco et al, 2014;Andrews et al, 2017).…”

“…Furthermore, coculture of NC and BM-MSC has been shown as an effective strategy to mitigate the donor-to-donor variability in NC derived engineered cartilage. However, it has been shown that the engineered cartilage from cocultured BM-MSC and NC are prone to undergo calcification when implanted in nude mice in similitude to implanted engineered cartilage microtissues from chondrogenically-induced BM-MSC (Pelttari et al, 2006;Andrews et al, 2017). In contrast, the engineered cartilage from NC alone did not undergo in vivo calcification (Acharya et al, 2012).…”

Table_11.DOCX

“…PTHrP did not affect GAG matrix production. In addition, when chondro-induction was calculated, it was found that PTHrP did not affect the synergistic effect that has been both previously reported of NC:BM-MSC cocultures (Acharya et al, 2012;Andrews et al, 2017) and observed in this study. In addition, this was further supported by ACAN gene expression, which showed no significant change with PTHrP treatments.…”