Poster U12 - Inducing mineralization of self-assembled neocartilage through the application of triiodothyronine and β-glycerophosphate

Introduction: Inducing mineralization of self-assembled neocartilage has applications in 1) engineering calcified cartilage at the interface of osteochondral implants and 2) developing a tissue-engineered growth plate. However, functionally mineralized self-assembled neocartilage has yet to be achieved. The objective of this study is to induce hypertrophic differentiation and mineralization of self-assembled neocartilage through the use of triiodothyronine (T3), which has previously been shown to induce collagen X deposition in self-assembled neocartilage [1], and β-glycerophosphate (βGP), which has been demonstrated to induce mineralization in tissue-engineered cartilage [2]. This study aids in the development of novel cell therapies for both articular cartilage and the growth plate by allowing zonal tissue mineralization.

Materials and

Methods: The experimental design of this study compares non-stimulated control constructs with constructs stimulated via T3, βGP, or a combination of T3/βGP for a duration of 1 or 4 weeks (Figure 1). The cells utilized in this study were costal chondrocytes obtained from 5 to 8-month-old Yucatan minipigs (two male and two female) that had been banked at P5 and expanded to P7 for the present study. At P7, the chondrogenic phenotype of cells was restored through a process called aggregate rejuvenation in which cells are subjected to aggregate culture for 14 days supplemented with 10ng/mL TGF-β1, 100ng/mL growth differentiation factor 5 (GDF-5), and 100ng/mL bone morphogenetic protein 2 (BMP-2) [3]. After rejuvenation, cells were seeded at a density of 2 million cells per construct in non-adherent wells to form self-assembled neocartilage. All constructs were stimulated via TGF-β1 (10 ng/ml through day 28), c-ABC (2 Units per mL for 4 hours on day 7), and LOXL2 (0.15 μg/mL from days 7 to 21) [4]. Additionally, experimental groups were stimulated via T3 (100 ng/mL) [1], βGP (10 mM) [2], or a combination of the two for either days 1-7 or days 1-28. After 4 weeks of culture, constructs were evaluated via mechanical testing (compressive, tensile, tribological), biochemistry, and histological staining. Analysis of variance (ANOVA) tests were used to analyze data sets with Tukey’s post hoc testing at p< 0.05 (n=9-12 per group).

Results, Conclusions, and Discussions:

Results: Morphologically, all constructs were round and flat. There were no notable differences among the constructs in terms of morphology (Figure 2A). Hematoxylin and Eosin staining (H&E) was relatively consistent among groups. Alizarin red staining for mineralization revealed that only the constructs treated with both T3 and βGP for a period of 4 weeks displayed substantial mineralization (Figure 2B). Mineral deposits in the 4-week T3/βGP were visible on a macroscopic level. Surprisingly, Alizarin red was particularly concentrated on the top third of the construct. The compressive aggregate modulus of T3-treated constructs was higher than the aggregate modulus of constructs treated by βGP alone (Figure 3). Specifically, the aggregate modulus of 1-week T3 was 1.4 times that of 4-week βGP (p=0.041), 4-week T3 was 1.4 times that of 1-week βGP (p=0.012), 4-week T3 was 1.5 times that of 4-week βGP (p=0.004), and 1-week T3/βGP was 1.4 times that of 4-week βGP (p=0.033). In terms of tribological properties, control constructs exhibited the lowest coefficient of friction, but it should be noted that orientation was not monitored (Figure 4); the combination T3/βGP 4-week group was 2.0 times that of the control (p=0.017).

Discussion and conclusions: This study demonstrated that the application of T3 and βGP to self-assembled neocartilage induces mineralization and changes in the functional properties of the constructs. Interestingly, T3 and βGP work synergistically to induce mineralization of self-assembled neocartilage, and no notable mineralization was observed if the factors were applied independently. The unexpected result of mineralization localizing to the top third of the constructs demonstrates that engineering zonal organization within self-assembled neocartilage is feasible. The aggregate modulus was higher in the constructs that were stimulated via T3 than constructs that were stimulated via βGP, which is in line with previous research that has demonstrated that T3 increases the aggregate modulus of self-assembled neocartilage [1]. Additionally, the elevated coefficient of friction in constructs that were treated with T3 and βGP indicates these biochemical factors would be more suitable for non-articular applications. Overall, this study will inform the biochemical regimens used to engineer osteochondral implants and the growth plate using self-assembled neocartilage.

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