Regional effects of enzymatic digestion on knee meniscus cell yield and phenotype for tissue engineering

Abstract

An abundant cell source is the cornerstone of most tissue engineering strategies, but extracting cells from the knee meniscus is hindered by its dense fibrocartilaginous matrix. Identifying a method to efficiently isolate meniscus cells is important, as it can reduce the cost and effort required to perform meniscus engineering research. In this study, six enzymatic digestion regimens used for cartilaginous cell isolation were used to isolate cells from the outer, middle, and inner regions of the bovine knee meniscus. Each regimen in each region was assessed in terms of cell yield, impact on cell phenotype, and cytotoxicity. All digestion regimens caused an overall upregulation of cartilage-specific genes Sox9, collagen type I (Col 1), collagen type II (Col 2), cartilage oligomeric matrix protein, and aggrecan (AGC) in cells from all meniscus regions, but was highest for cells isolated using 1075 U/mL of collagenase for 3 h (high collagenase). In response to isolation, outer meniscus cells showed highest upregulation of Sox9 and Col 1 genes, whereas greatest upregulation for middle meniscus cells was seen in Col 1 expression, and Col 2 expression for inner cells. Cell yield was highest in all regions when subjected to 45 min of 61 U/mL pronase followed by 3 h of 1075 U/mL collagenase (pronase/collagenase [P/C]) digestion regimen (outer: 6.57±0.37, middle: 12.77±1.41, inner: 22.17±1.47×10(6) cells/g tissue). The second highest cell yield was achieved using the low collagenase (LC) digestion regimen that applied 433 U/mL of collagenase for 18 h (outer: 1.95±0.54, middle: 3.3±4.4, inner: 6.06±2.44×10(6) cells/g tissue). Cytotoxicity analysis showed higher cell death in the LC group compared with the P/C group. Self-assembled constructs formed from LC-isolated cells were less dense than constructs formed from P/C-isolated cells, and P/C constructs showed higher glycosaminoglycan content and compressive moduli than LC constructs. All isolation methods tested resulted in similar phenotypic changes in meniscus cells from each region. These results indicate that, compared with other common isolation protocols, the P/C isolation method is able to more efficiently isolate meniscus cells from all regions that can produce tissue engineered constructs.

FIG. 1.

Cell yield from different meniscus regions using six isolation methods. The number of cells per gram of tissue was determined for each isolation regimen in each radial region of the meniscus. For comparison, native tissue average cellularity is depicted for each meniscus region (dashed lines). Overall, cell yield from the inner region was highest, followed by the middle and outer regions, respectively. Among the isolation regimens, the pronase/collagenase (P/C) treatment resulted in the highest overall cell yield from all regions. Results were analyzed using a two-way analysis of variance (ANOVA), with significance set at p<0.05. Groups not connected by the same letter are statistically different. LC, low collagenase; HC, high collagenase; T/C, trypsin/collagenase; H/C, hyaluronidase/collagenase; H/T/C, hyaluronidase/trypsin/collagenase.

FIG. 2.

Gene expression of meniscus cells in response to isolation. Outer (A), middle (B), and inner (C) meniscus cells were isolated from NT, or subjected to one of six isolation regimens: LC, HC, P/C, T/C, H/C, or H/T/C. Gene expression levels were normalized to native tissue values for Sox9, Col 1, Col 2, COMP, and AGC. Results for each cell type were analyzed using a two-way ANOVA, p<0.05. Groups not connected by the same letter are statistically different. NT, native tissue; Col1, collagen type 1; Col2, collagen type 2; COMP, cartilage oligomeric matrix protein; AGC, aggrecan.

FIG. 3.

Phase 2 cell yield and live-dead analysis. (A) Cells from whole medial menisci were isolated using the LC and P/C protocols from phase 1. Cell yield was higher for the P/C method. (B) Live-dead staining of the resultant isolated cells showed more dead cells relative to live cells in the LC-isolated population compared with the P/C-isolated cells. Quantitative data were analyzed using a Student's t-test, with significance set at p<0.05. Groups not connected by the same letter are statistically different. Color images available online at www.liebertonline.com/tec

FIG. 4.

Phase 2 gross morphology, biochemistry, and compressive mechanics. Self-assembled constructs were formed using cells isolated by the LC or P/C method (A). LC constructs were significantly thicker than P/C constructs; however, construct diameter was not different between groups. Biochemical analysis of GAG and collagen content (B and D, respectively) showed increased GAG content per cell in the P/C constructs, but no difference in collagen content between groups. P/C constructs showed higher modulus of relaxation (E_r) and instantaneous modulus (E_i) compared with LC constructs when subjected to unconfined compression stress-relaxation at 20% strain (C). Student's t-tests were performed on each data set, with significance set at p<0.05. Groups not connected by the same letter are statistically different. GAG, glycosaminoglycan. Color images available online at www.liebertonline.com/tec