Use of adipose stem cells and polylactide discs for tissue engineering of the temporomandibular joint disc

Abstract

There is currently no suitable replacement for damaged temporomandibular joint (TMJ) discs after discectomy. In the present study, we fabricated bilayer biodegradable polylactide (PLA) discs comprising a non-woven mat of poly(L/D)lactide (P(L/D)LA) 96/4 and a P(L/DL)LA 70/30 membrane plate. The PLA disc was examined in combination with adipose stem cells (ASCs) for tissue engineering of the fibrocartilaginous TMJ disc in vitro. ASCs were cultured in parallel in control and chondrogenic medium for a maximum of six weeks. Relative expression of the genes, aggrecan, type I collagen and type II collagen present in the TMJ disc extracellular matrix increased in the ASC-seeded PLA discs in the chondrogenic medium. The hypertrophic marker, type X collagen, was moderately induced. Alcian blue staining showed accumulation of sulphated glycosaminoglycans. ASC differentiation in the PLA discs was close to that observed in pellet cultures. Comparison of the mRNA levels revealed that the degree of ASC differentiation was lower than that in TMJ disc-derived cells and tissue. The pellet format supported the phenotype of the TMJ disc-derived cells under chondrogenic conditions and also enhanced their hyalinization potential, which is considered part of the TMJ disc degeneration process. Accordingly, the combination of ASCs and PLA discs has potential for the development of a tissue-engineered TMJ disc replacement.

Figure 1.

A structure of the PLA disc. A non-woven mat (P(L/D)LA 96/4) closed from one side with a (P(L/DL)LA 70/30) membrane plate. Thickness of the disc is approximately 1.8 mm.

Figure 2.

A micromass attached on the fibre crossing point in a PLA disc under chondrogenic conditions. The condensation of the micromass after 3 days (a), 4 days (b) and two weeks (c) of culturing.

Figure 3.

The ASC discs stained with Alcian blue. The control ASC discs (a,d,g) are represented on the left column and the ASC discs under differentiation conditions (b,c,e,f,h,i) in two columns on the right. Boxes in (d) and (f) represent areas shown at higher magnifications in (j) and (k), respectively. Arrows indicate locations of the P(L/D)LA 96/4 fibres. The ASC discs are chosen from the different experiments, and the ASC discs 1 and 2 are not separated in the panel owing to general similarity in their behaviour.

Figure 4.

Alcian blue-stained ASC pellets from control (a–c) and differentiation (d–f) at the time points of 1.5 (a,d), 3 (b,e) and 6 (c,f) weeks of culture. The higher magnification insets are represented in (b) and (e). Pictures represent samples from different experiments.

Figure 5.

The relative mRNA levels of the ASC discs 1 and 2 for aggrecan (a), type I (b), type II (c) and type X (d) collagen in the scatter charts. The results of the ASC discs 1 and 2 are compared side by side in the same chart. The controls are presented on the left side and the differentiations on the right side. The significantly changed mRNA levels were noticed between controls, and differentiation conditions for the disc are marked with ** (p < 0.01), *** (p < 0.001) and ns (not significant). Differences between the ASC discs 1 and 2 were not found to be statistically significant. (Filled circle, control; filled upside down triangle, differentiation; line, median of the results at a time point.)

Figure 6.

The scatter charts of the relative mRNA levels of aggrecan (a), type I (b), type II (c) and type X (d) collagen in ASC pellets. An expression point for type II collagen at 1.5 weeks was defined as an outlier and thus discarded. The relative changes in gene expression were statistically significant (p < 0.01) for all the detected genes. (Filled circle, control; filled upside down triangle, differentiation; line, median of the results at a time point.)

Figure 7.

The bar charts of qRT–PCR results comparing control and differentiated samples between ASC pellets and ASC discs, and TMJ disc cell pellets at weeks 3 and 6; in addition, an mRNA sample of the TMJ disc served as a positive control. The results of one PLA disc type were included because of the general similarity between ASC discs 1 and 2. All material was derived from the same rabbit.