Co-culture of Early and Late Passage Chondrocytes on Biomimetic Collagen-Alginate Sulfate Films Improves the Cartilage Phenotype of Cells Utilized in Autologous Chondrocyte Transplantation

Abstract

The limited regenerative ability of avascular articular cartilage poses challenges in treating cartilage damaged by injury or disease. Autologous chondrocyte transplantation (ACT) repairs cartilage tissues by reintroducing healthy chondrocytes from an autologous cartilage biopsy into the defect. However, a significant challenge is the tendency for chondrocytes to de-differentiate during monolayer cell expansion on tissue culture plastic (TCP), which is required to reach sufficient cell numbers for ACT procedures. The de-differentiated chondrocytes adopt a fibroblastic phenotype and produce inferior cartilage with weaker mechanical properties and altered tissue composition. This study aims to assess the effects of a co-culture system of early- and late-passage chondrocytes on biomimetic collagen-alginate sulfate (Col-AlgS) films to preserve the cartilage phenotype of monolayer-expanded articular chondrocytes as an optimized cell source for ACT. Col-AlgS films were prepared through layer–by–layer (LBL) deposition of type I collagen (Col1) and alginate sulfate (AlgS), with TCP and Col1 as control substrates. Primary bovine chondrocytes were serial passaged before Passage 1 (P1) and Passage 4 (P4) chondrocytes were seeded individually or in co–cultures on the 3 substrates as follows: P4 cells alone, a 3:1 ratio of P4 cells to P1 cells, a 1:1 ratio of P4:P1, a 1:3 ratio of P4:P1, and P1 cells alone. The viability and proliferation of cells was assessed using live/dead assay and trypan blue exclusion. Chondrocyte morphology was assessed through ImageJ processing. Gene expression was quantified using quantitative real time polymerase chain reaction (q-RT-PCR) and protein synthesis was assessed using western blotting. This work showed a 2-fold decrease in cell proliferation on Col-AlgS compared to TCP and Col1 controls (p<0.05). Conversely, there was an increasing trend in cell proliferation of the co-cultures compared to P4 on TCP (1.5-fold) and Col1 as well as Col-AlgS (2-fold) substrates (p<0.05). Col-AlgS increased the expression of relevant genes more than 3-fold, including type I collagen (Col1) and type II collagen (Col2) (p<0.01 and p<0.05 respectively) as well as lubricating superficial zone protein (SZP) (p<0.01), while decreasing pro-inflammatory interleukin 6 (IL6) (p<0.05). Similarly, the co-cultures increased gene expression of Col2 and SZP (p<0.05 for both), while decreasing IL6 (p<0.01). Moreover, microscopic images indicated that chondrocytes on Col1 and AlgS substrates were found to have a rounder morphology compared to those on TCP. Thus, we conclude that a co-culture system of early- and late-passage chondrocytes on Col-AlgS limits the de-differentiation of articular chondrocytes during monolayer-expansion. This system may therefore be used for an optimized chondrocyte source in cartilage tissue engineering applications as it addresses the issue of cell numbers obtained through serial passaging with preserved phenotype.