Investigation of the regenerative capacity of an acellular porcine medial meniscus for tissue engineering applications

Abstract

Previously, we have described the development of an acellular porcine meniscal scaffold. The aims of this study were to determine the immunocompatibility of the scaffold and capacity for cellular attachment and infiltration to gain insight into its potential for meniscal repair and replacement. Porcine menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic, and final washing in phosphate-buffered saline. In vivo immunocompatibility was assessed after implantation of the acellular meniscal scaffold subcutaneously into galactosyltransferase knockout mice for 3 months in comparison to fresh and acellular tissue treated with α-galactosidase (negative control). The cellular infiltrates in the explants were assessed by histology and characterized using monoclonal antibodies against: CD3, CD4, CD34, F4/80, and C3c. Static culture was used to assess the potential of acellular porcine meniscal scaffold to support the attachment and infiltration of primary human dermal fibroblasts and primary porcine meniscal cells in vitro. The explants were surrounded by capsules that were more pronounced for the fresh meniscal tissue compared to the acellular tissues. Cellular infiltrates compromised mononuclear phagocytes, CD34-positive cells, and nonlabeled fibroblastic cells. T-lymphocytes were sparse in all explanted tissue types and there was no evidence of C3c deposition. The analysis revealed an absence of a specific immune response to all of the implanted tissues. Acellular porcine meniscus was shown to be capable of supporting the attachment and infiltration of primary human fibroblasts and primary porcine meniscal cells. In conclusion, acellular porcine meniscal tissue exhibits excellent immunocompatibility and potential for cellular regeneration in the longer term.

FIG. 1.

Diagrammatic overview of sectioning/observation of mouse explants for histological assessment.

FIG. 2.

Histological images of fresh/decellularized porcine medial meniscal tissue after 3-month subcutaneous implantation in GTKO mice vaccinated with porcine red blood cells. (A) H&E-stained fresh porcine medial meniscal tissue surrounded by fibrous capsule, 40× mag; (B) H&E-stained acellular porcine medial meniscal tissue surrounded by fibrous capsule, 40 × mag; (C) H&E-stained acellular porcine medial meniscal tissue showing cellular infiltration, 200× mag. CI, capsular interface; GTKO, galactosyltransferase knock-out; H&E, hematoxylin and eosin; I, meniscal implant; mag, magnification; ST, murine subcutaneous mouse tissue. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Capsule thickness of fresh, decellularized, α-galactosidase-treated porcine medial meniscal explants from nonvaccinated/vaccinated GTKO mice. The error bars represent 95% confidence limits (n = 3). *A significant difference as determined by analysis of variance (p < 0.05).

FIG. 4.

Phenotype analysis of cellular infiltrate of fresh, decellularized, and α-galactosidase-treated porcine medial meniscal samples after 3-month implantation in nonvaccinated and vaccinated GTKO mice. Error bars have been removed for presentation purposes; however, data represent the mean (n = 3).

FIG. 5.

Histology and SEM of acellular porcine medial meniscal tissue after 24 h culture with (A1, B2) human dermal fibroblasts and (C1, D2) porcine medial meniscal cells at 37°C in an atmosphere of 5% (v/v) CO₂ in air. Cells were seeded at the following concentrations: (A1, B2) 6 × 10⁵ cells/cm² and (C1, D2) 4 × 10⁵ cells/cm². Analysis were performed using (1) H&E staining, 200× mag and (2) SEM. Scale bar represents 30 μm. FC, flattened cells; SEM, scanning electron microscopy. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Histology and SEM of acellular porcine medial meniscal tissue after 7-day culture with primary human dermal fibroblasts at 37°C (continues overleaf ) in an atmosphere of 5% (v/v) CO₂ in air. Cells were seeded at 6 × 10⁵ cells/cm². (A) Image of stacked cells on the acellular scaffold, stained with H&E, 200× mag; (B) image of stacked cells on the acellular scaffold, stained with Hoechst 33258 dye, 200× mag; (C) image of cells infiltrated into the acellular scaffold, stained H&E, 200× mag; (D) image of cells infiltrated into the acellular scaffold, stained with Hoechst 33258 dye, 200× mag; (E) SEM image of cell monolayer viewed from above, scale bar represents 100 μm; (F) SEM image of cell monolayer viewed from above, scale bar represents 30 μm. Color images available online at www.liebertonline.com/ten.

FIG. 7.

Histology and SEM of acellular porcine medial meniscal tissue after 7 day culture with primary porcine meniscal cells at 37°C in an atmosphere of 5% (v/v) CO₂ in air. Cells were seeded at 4 × 10⁵ cells/cm². (A) Image of cellular monolayer on the acellular scaffold, stained with H&E, 200× mag; (B) image of cellular monolayer on the acellular scaffold, stained with Hoechst 33258 dye, 200× mag; (C) SEM image of cell monolayer viewed from above, scale bar represents 100 μm; (D) SEM image of cell monolayer viewed from above, scale bar represents 30 μm. Color images available online at www.liebertonline.com/ten.