The knee meniscus contains a mixed population of cells that exhibit fibroblastic as well as chondrocytic characteristics. Tissue engineering studies and future therapies for the meniscus require a large population of cells that are seeded on scaffolds. To achieve this, monolayer expansion is often used as a technique to increase cell number. However, the phenotype of these cells may be significantly different from that of the primary population. The objective of this study was to investigate changes in meniscal fibrochondrocytes at the gene expression level over four passages using quantitative real-time reverse transcriptase polymerase chain reaction. Cells from the inner two-thirds of bovine medial menisci were used. Four extracellular matrix (ECM) molecules, commonly found in the meniscus, were investigated, namely collagen I, collagen II, aggrecan and cartilage oligomeric matrix protein (COMP). In addition, primary and passaged meniscus fibrochondrocytes were placed on surfaces coated with collagen I or aggrecan protein to investigate whether any gene expression changes resulting from passage could be reversed. Collagen I expression was found to increase with the number of passages, whereas collagen II and COMP expression decreased. Collagen I and aggrecan surface coatings were shown to downregulate and upregulate collagen I and COMP expression levels, respectively, in passaged cells. However, decreases in collagen II expression could not be reversed by either protein coating. These results indicate that although monolayer expansion results in significant changes in gene expression in meniscal fibrochondrocytes, protein coatings may be used to regain the primary cell expression of several ECM molecules.

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The combinatorial effects of TGF-β1 and hydrostatic pressure (HP) were investigated on meniscus cell-seeded PLLA constructs using a two-phase sequential study. The objective was to identify potentially synergistic effects of these stimuli toward enhancing the biomechanical and compositional characteristics of the engineered constructs. In Phase I, the effects of TGF-β1 were examined on the ability of meniscus cells to produce ECM. In Phase II, meniscus cell-seeded PLLA constructs were cultured for 4 wks with a combination of TGF-β1 and HP (10 MPa, 0 Hz or 10 MPa, 0.1 Hz). TGF-β1 was found to increase collagen and GAG deposition in the scaffolds 15-fold and 8-fold, respectively, in Phase I. In Phase II, the combination of TGF-β1 and 10 MPa, 0 Hz HP resulted in 4-fold higher collagen deposition (additive increase), 3-fold higher GAG deposition and enhanced compressive properties (additive and synergistic increases), when compared to the unpressurized no growth factor culture control. Though significant correlations were observed between the compressive properties (moduli and viscosity), and the GAG and collagen content of the constructs, the correlations were stronger with collagen. This study provides robust evidence that growth factors and HP can be used successfully in combination to enhance the functional properties of in vitro engineered knee meniscus constructs.

The knee meniscus, a fibrocartilaginous tissue located in the knee joint, is characterized by heterogeneity in extracellular matrix and biomechanical properties. To recreate these properties using a tissue engineering approach, co-cultures of meniscus cells (MCs) and articular chondrocytes (ACs) were seeded in varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) on poly-L-lactic acid (PLLA) scaffolds and cultured in serum-free medium for 4 weeks. Histological, biochemical, and biomechanical tests were used to assess constructs at the end time point. Strong staining for collagen and glycosaminoglycan (GAG) was observed in all groups. Constructs with 100% MCs were positive for collagen I and constructs cultured with 100% ACs were positive for collagen II, while a mixture of collagen I and II was observed in other co-culture groups. Total collagen and GAG per construct increased as the percentage of ACs increased (27 +/- 8 microg, 0% AC to 45 +/- 8 microg, 100% ACs for collagen and 12 +/- 4 microg, 0% ACs to 40 +/- 5 microg, 100% ACs for GAG). Compressive modulus (instantaneous and relaxation modulus) of the constructs was significantly higher in the 100% ACs group (63 +/- 12 and 22 +/- 9 kPa, respectively) when compared to groups with higher percentage of MCs. No differences in tensile properties were noted among groups. Specific co-culture ratios were identified mimicking the GAG/DW of the inner (0:100, 25:75, and 50:50) and outer regions (100:0) of the meniscus. Overall, it was demonstrated that co-culturing MCs and ACs on PLLA scaffolds results in functional tissue engineered meniscus constructs with a spectrum of biochemical and biomechanical properties.

The meniscus is a fibrocartilaginous tissue that is critically important to the loading patterns within the knee joint. If the meniscus structure is compromised, there is little chance of healing due to limited vascularity in the inner portions of the tissue. Several tissue engineering techniques to mimic the complex geometry of the meniscus have been employed. Of these, a self-assembly, scaffoldless approach employing agarose molds avoids drawbacks associated with scaffold use while still allowing formation of robust tissue. In this experiment two factors were examined, agarose percentage and mold surface roughness, in an effort to consistently obtain constructs with adequate geometric properties. Co-cultures of ACs and MCs (50:50 ratio) were cultured in smooth or rough molds composed of 1% or 2% agarose for 4 wks. Morphological results showed that constructs formed in 1% agarose molds, particularly smooth molds, were able to maintain their shape over the 4 wk culture period. Significant increases were observed for the collagen II to collagen I ratio, total collagen, GAG, and tensile and compressive properties in smooth wells. Cell number per construct was higher in the rough wells. Overall, it was observed that the topology of an agarose surface may be able to affect the phenotypic properties of cells that are on that surface, with smooth surfaces supporting a more chondrocytic phenotype. In addition, wells made from 1% agarose were able to prevent construct buckling potentially due to their higher compliance.