Dataset Information

Proteomic, mechanical, and biochemical development of tissue-engineered neocartilage.

ABSTRACT:

Background

The self-assembling process of cartilage tissue engineering is a promising technique to heal cartilage defects, preventing osteoarthritic changes. Given that chondrocytes dedifferentiate when expanded, it is not known if cellular expansion affects the development of self-assembled neocartilage. The objective of this study was to use proteomic, mechanical, and biochemical analyses to quantitatively investigate the development of self-assembled neocartilage derived from passaged, rejuvenated costal chondrocytes.

Methods

Yucatan minipig costal chondrocytes were used to create self-assembled neocartilage constructs. After 1, 4, 7, 14, 28, 56, or 84 days of self-assembly, constructs were analyzed through a variety of histological, biomechanical, biochemical, and proteomic techniques.

Results

It was found that temporal trends in neocartilage formation are similar to those seen in native hyaline articular cartilage development. For example, between days 7 and 84 of culture, tensile Young's modulus increased 4.4-times, total collagen increased 2.7-times, DNA content decreased 69.3%, collagen type II increased 1.5-times, and aggrecan dropped 55.3%, mirroring trends shown in native knee cartilage. Importantly, collagen type X, which is associated with cartilage calcification, remained at low levels (≤ 0.05%) at all neocartilage developmental time points, similar to knee cartilage (< 0.01%) and unlike donor rib cartilage (0.98%).

Conclusions

In this work, bottom-up proteomics, a powerful tool to interrogate tissue composition, was used for the first time to quantify and compare the proteome of a developing engineered tissue to a recipient tissue. Furthermore, it was shown that self-assembled, costal chondrocyte-derived neocartilage is suitable for a non-homologous approach in the knee.

SUBMITTER: Bielajew BJ

PROVIDER: S-EPMC9308280 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM). Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC) immobilized in alginate hydrogels, and used immunohistochemistry (IHC) and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG) microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml) showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide meaningful insight into ECM assembly of neocartilage, which will lead to better understanding of cartilage formation and development of new tissue engineering strategies.