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 a-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.

Development and Characterization of Acellular Extracellular Matrix Scaffolds from Porcine Menisci for Use in Cartilage Tissue Engineering

“…The acellular menisci demonstrated in vitro biocompatibility for murine NIH 3T3 embryonic fibroblast. Stapleton et al 49,50 decellularized menisci by exposing them to several freeze-thaw cycles and incubating them in hypotonic Tris buffer and SDS in hypotonic buffer plus protease inhibitors, nucleases, and hypertonic buffer followed by disinfection with peracetic acid. The menisci scaffold showed immunobiocompatibility, no evidence of the expression of the major xenogeneic epitope (galactose-a-1,3-galactose), and was capable of supporting the attachment and infiltration of human fibroblasts and porcine meniscal cells.…”

An injectable extracellular matrix derived hydrogel for meniscus repair and regeneration

“…79 The decellularized meniscal materials have been produced as 80 cell-free scaffolds for meniscus repair and regeneration [19,20]. 81 These scaffolds also were combined with primary cells, such as 82 human primary chondrocytes, murine fibroblasts and porcine 83 meniscal cells, and stem cells such as rat mesenchymal stromal 84 cells (rMSC) to promote meniscus regeneration [21][22][23][24][25] fill an irregularly shaped defect, and provide a stable microenviron-98 ment for cell growth to facilitate new tissue formation [27]. Inject-99 able hydrogels derived from the heart [9, 17,28,29], dermis [27], 100 bone [30], urinary bladder [31], liver [32], nervous system [33], 101 blood vessel [34], adipose [35], and brain [36], have been reported.…”

“…A rat http://dx.doi.org/10.1016/j.actbio.2015.01.027 1742-7061/Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.meniscus-derived scaffold seeded with rMSCs was implanted into 86 a rat defected meniscus, that showed better chondroprotective 87 effect than the conventional meniscectomy treatment[22]. However, the decellularized meniscal scaffold partially failed to be 89 recellularized for full thickness meniscus regeneration because of 90 the dense structure[21,23,26]. If the solid decellularized ECM scaf-91 fold can be processed into an injectable hydrogel, the cells can be 92 easily encapsulated into the hydrogel with uniform distribution.Furthermore, the injectable hydrogel is compatible with popular 94 minimally-invasive arthroscopic meniscectomy surgery in clinics95 [5], while the ECM scaffold commonly requires surgery with inci-96 sion.…”

A novel surgical technique for a rat subcutaneous implantation of a tissue engineered scaffold

“…Despite time and costs, it is crucial to have a reliable method for implantation. Subcutaneous implantation allows for the observation of an inflammatory response, cellularization of the extracellular matrix, and angiogenesis validation (Modulevsky et al, 2016; Guarnieri et al, 2014; Stapleton et al, 2010; Meagher et al, 2016; Mazza et al, 2015). …”