Tissue engineering with meniscus cells derived from surgical debris

Abstract

Objective: Injuries to the avascular regions of the meniscus fail to heal and so are treated by resection of the damaged tissue. This alleviates symptoms but fails to restore normal load transmission in the knee. Tissue engineering functional meniscus constructs for re-implantation may improve tissue repair. While numerous studies have developed scaffolds for meniscus repair, the most appropriate autologous cell source remains to be determined. In this study, we hypothesized that the debris generated from common meniscectomy procedures would possess cells with potential for forming replacement tissue. We also hypothesized that donor age and the disease status would influence the ability of derived cells to generate functional, fibrocartilaginous matrix.

Methods: Meniscus derived cells (MDCs) were isolated from waste tissue of 10 human donors (seven partial meniscectomies and three total knee arthroplasties) ranging in age from 18 to 84 years. MDCs were expanded in monolayer culture through passage 2 and seeded onto fiber-aligned biodegradable nanofibrous scaffolds and cultured in a chemically defined media. Mechanical properties, biochemical content, and histological features were evaluated over 10 weeks of culture.

Results: Results demonstrated that cells from every donor contributed to increasing biochemical content and mechanical properties of engineered constructs. Significant variability was observed in outcome parameters (cell infiltration, proteoglycan and collagen content, and mechanical properties) amongst donors, but these variations did not correlate with patient age or disease condition. Strong correlations were observed between the amount of collagen deposition within the construct and the tensile properties achieved. In scaffolds seeded with particularly robust cells, construct tensile moduli approached maxima of approximately 40 MPa over the 10-week culture period.

Conclusions: This study demonstrates that cells derived from surgical debris are a potent cell source for engineered meniscus constructs. Results further show that robust growth is possible in MDCs from middle-aged and elderly patients, highlighting the potential for therapeutic intervention using autologous cells.

Figure 1. Morphological appearance of passage 2 human MDCs in monolayer and on fiber-aligned nanofibrous scaffolds

(A) Passage 2 MDCs in monolayer on tissue culture polystyrene demonstrate a fibroblast-like morphology. (B) Passage 2 MDC-seeded constructs cultured for one day reveal MDCs elongating in and aligning with the predominant fiber direction of the scaffold. Red: F-actin, white: fibers, blue: nuclei. Scale bar: 50μm.

Figure 2. MDC-seeded scaffolds increase in mechanical properties with time in culture in a fibro-cartilaginous medium

(A) Force-elongation plots of five scaffolds either seeded (MDC) or maintained as unseeded controls (USC) on day 70 for Donor 8. (B) Maximum load of seeded scaffolds normalized to that of paired USC scaffolds on day 70 for all ten donors. Donor # is indicated on the x-axis. Data represent the mean and standard deviation of 5 samples per donor. (C) Normalized stiffness (indicating percentage change) of MDC-seeded scaffolds from each donor compared to their paired USC scaffolds at each time point. Donor # (and age) is indicated on the x-axis.

Figure 3. Donor-to-donor variation in time-dependent changes in biochemical composition of MDC-seeded engineered meniscus constructs

(A) Total collagen and (B) total s-GAG accumulation in engineered constructs with time in culture for each donor. Donor # (and age) is indicated on the x-axis. Data represent the mean and standard deviation of 5 samples per donor per time point. (C) Percent dry weight (% DW) collagen and (D) % DW s-GAG for samples from each of the ten donors on day 70. Gray background in (C) and (D) indicates range of collagen and s-GAG found in 5 native tissue samples. Note the interrupted scale in (C) the % collagen by DW plot. USC constructs processed similarly showed no appreciable background content of s-GAG or collagen.

Figure 4. Structure-function-composition correlations for MDC-seeded constructs with time in culture

(A) Total collagen content in constructs correlates well with change in stiffness for all donors at all time points (days 14, 42, and 70). (B) Total GAG content correlates poorly with change in stiffness for all donors at all time points (days 14, 42, and 70). (C) Donor age showed a weak correlation with change in stiffness of constructs on day 70.

Figure 5. Histological analysis of constructs from best-case (Donor 8, A,C,E) and worst-case (Donor 6, B,D,F) samples on day 70

DAPI staining of cell nuclei demonstrate infiltration into the outer two-thirds of constructs under best-case conditions (A), and limited infiltration at the periphery under worst-case conditions (B). Similar findings are observed for collagen (C,D) and proteoglycan (E,F) deposition as indicated by Picrosirius Red and Alcian Blue staining, respectively. Scale Bar: 1mm.