Professor Cornell University Ithaca, New York, United States
Introduction: The meniscus-to-bone transition comprises diverse gradients in mineralization, cellular phenotype, extracellular matrix (ECM) composition, and microstructure that help bridge stress between the meniscus and bone. Recapitulating the native structure of the meniscus-to-bone transition is a complex engineering challenge. Previous research has developed different strategies to engineer collagen fiber architecture [1] and mineral matrix gradients [2]; however, recreating the complex cellular heterogeneity present at the mineralization gradient has been unexplored. Bone Morphogenetic Protein-2 (BMP-2) is a growth factor found in the knee joint during meniscus development [3] and is known to play a role in the endochondral ossification of the growth plate. In addition, BMP-2 has been shown to promote mineralization and cellular differentiation in mesenchymal condensates [4]. BMP-2 may help spatially pattern the different phenotypes present at the meniscus-to-bone transition. However, studies looking at the effect of BMP-2 in meniscal fibrochondrocytes have only focused on its proliferative and chemokinetic effects [5]. To date, no studies have evaluated the hypertrophic differentiation potential of BMP-2 on meniscal fibrochondrocytes. The objective of this study was to characterize the dose-dependent response of BMP-2 on meniscal fibrochondrocytes encapsulated in collagen gels to inform the design of tissue-engineered enthesis. We hypothesized that BMP-2 will differentiate meniscal fibrochondrocytes into a hypertrophic phenotype demonstrated by an increase in glycosaminoglycan synthesis, a larger and rounded morphology, and the expression of the hypertrophic marker, collagen X.
Materials and
Methods: To assess how meniscal fibrochondrocytes respond to BMP-2 induction, meniscal fibrochondrocytes were isolated from a total of 6 neonatal bovids, encapsulated in a collagen gel and cultured for 30 days in high glucose DMEM and BMP-2 levels ranging from 0.1 ng/ml to 800 ng/ml. To evaluate glycosaminoglycan (GAG) production, constructs were digested at the end of the culture and GAG was quantified through the dimethyl methylene blue (DMMB) assay. Resulting GAG concentrations were normalized to construct wet weight and fitted to a sigmoidal 4-parameter line fit using GraphPad Prism. To qualitatively evaluate cell morphology and GAG distribution, constructs were fixed in buffered formalin, sectioned, and stained in Alcian blue. To evaluate the extent distribution of hypertrophy throughout the gel, immunohistochemistry (IHC) of collagen X was performed on sectioned samples.
Results, Conclusions, and Discussions: Biochemical and histological analysis revealed induction of hypertrophy in meniscal fibrochondrocytes in a dose-dependent manner to BMP-2 stimulation. GAG retention in constructs had a dose-dependent response to BMP-2 stimulation (Figure 1). The half-maximal effective concentration was 87.07 ng/ml and there is close to an 8-fold increase in GAG retention between low and high doses of BMP-2 which is indicative of a change in phenotype. Alcian blue staining revealed that no BMP-2 stimulation resulted in lower GAG deposition represented by the light blue staining and no discernible cytoplasm. In contrast, a distinct hypertrophic morphology characterized by increased GAG deposition and a rounded morphology with discernible cytoplasm was present in constructs exposed to high doses of BMP-2 (Figure 2). Interestingly, the hypertrophic differentiation was not homogenous throughout the construct (data not shown). IHC of collagen X revealed faint staining throughout the construct while heavily staining the cellular body of hypertrophic cells. (Figure 3). This study shows hypertrophic differentiation in meniscal fibrochondrocytes. The findings of this study can be used to spatially pattern a hypertrophic phenotype and recreate the mineralized fibrocartilage region of the meniscus-to-bone transition.
