Isolation procedures for the recovery of extracellular matrices (ECMs) from animal organs/tissues that are useful in regenerative medicine involve multiple sequential steps/stages including collection of the source organ at slaughter, their transportation to laboratory, decellularization, decontamination, stabilization, and sterilization. Most of these steps require extensive use of chemicals/reagents/enzymes which may also adversely affect the quality of the scaffold. With an effort to minimize the use of chemicals/reagents/enzymes, while extracting biomaterial-grade ECM from porcine cholecyst (gall bladder), we performed preisolation ex situ incubation of the organ in a stabilizing agent that also caused in situ crosslinking of tissue-components and delaminated the collagen-rich ECM from the tissue-layer beneath the mucosa. The physical, chemical, and biological properties of the isolated scaffolds were similar to that of a commercially available porcine small intestinal submucosa. The cholecyst-derived scaffold not only satisfied preclinical safety-test procedures such as cytotoxicity, local response, and endotoxin load but also showed the potential to promote healing of full-thickness skin wound in a rabbit model. The procedure was also suitable for isolating scaffolds from other hollow organs such as jejunum and urinary bladder. It was concluded that enzyme/detergent treatment may be an avoidable step while isolating biomaterial-grade scaffolds from hollow organs.

Comparative histomorphological assessment of local response to implanted reference biomaterial, also called biocompatibility testing/evaluation, in an appropriate animal model is a widely practiced safety evaluation procedure performed on biomaterials before clinical use. Standardized protocols and procedures, originally designed for testing synthetic materials, available for the testing/evaluation do not account for the immunogenic potential of a candidate biomaterial. Therefore, it is appropriate to supplement the routine biocompatibility test reports with adjunct data that may provide insight into the immunogenic potential of candidate biomaterials, especially when testing biomaterials that are derived from mammalian sources. This article presents expanded safety evaluation data of a porcine cholecyst-derived scaffold (CDS) intended as a xenogeneic graft. The biocompatibility was tested in rat subcutaneous model in comparison with a reference material and the CDS was found biocompatible. However, when studied by immunohistochemistry and real-time reverse transcription polymerase chain reaction for the number and/or polarization of M1 macrophage, M2 macrophage, cytotoxic T-cell, helper T cell, TH1 cell, and TH2 cell, the CDS appeared to induce a differential local immunopathological tissue reaction despite the similarity in biocompatibility with the reference material. The adjunct data collected were useful for objectively assessing the safety of CDS as a xenograft.

Extracellular matrices isolated from several mammalian organs/tissues have found several clinical uses as xenografts or implants. However, they may cause complications because of adverse immunologic reactions. Scaffolds that promote favorable graft-acceptance reaction are preferred for fabricating xenografts. The objective of this study was to evaluate the immunogenic potential of a porcine cholecyst-derived scaffold (CDS), prepared by a non-detergent/enzymatic method, in comparison with jejunum and urinary bladder-derived scaffolds in a rat subcutaneous model. Key graft-rejection/acceptance reaction was evaluated at the site of implantation by studying the occurrence and/or function of immunocompetent cells in the tissue reaction. There was differential occurrence of M1-macrophage, M2-macrophage, T-helper cells, T-cytotoxic cells, B-cells, and mast cells in the tissue reaction and the CDS attracted few cells compared with other scaffolds. Real-time polymerase chain reaction for evaluating mRNA of functional markers like inducible nitric oxide synthase (M1 macrophage), arginase 1 (M2 macrophage), interferon gamma (TH1 lymphocytes), and interlukin-4 (TH2 lymphocytes) suggested that the CDS, compared with the scaffolds prepared from small intestine and urinary bladder, elicited M2 macrophage and TH2 lymphocyte polarization that are congenial graft-acceptance reactions. The results indicated that CDS has less immunogenic potential compared with the scaffolds prepared from jejunum and urinary bladder when used as subcutaneous graft in rats. It was concluded that CDS is a promising animal-derived xenograft for biomedical application.

Graft-assisted healing is an important strategy for treating full-thickness skin wounds. This study evaluated the properties of porcine cholecyst–derived scaffold and its use for treating full-thickness skin wound in rabbit. The physical properties of cholecyst-derived scaffold were congenial for skin-graft application. Compared to a commercially available skin-graft substitute made of porcine small intestinal submucosa, the cholecyst-derived scaffold was rich in natural biomolecules like elastin and glycosaminoglycans. When used as a xenograft, it promoted healing with excess cell proliferation at early phases and acceptable collagen deposition in the later remodelling phases.

This study evaluated the modulatory effect of chitosan sponge co-cultured with keratinocyte and fibroblast on wound healing. Dermal fibroblasts and keratinocyte isolated from rabbit skin were co-cultured on chitosan sponge, to fabricate cell-loaded chitosan tissue engineered construct. Full thickness excision wounds created on the rabbit dorsum were treated with three types of graft materials – a noncellular chitosan graft, homologous keratinocyte fibroblast loaded chitosan, and a commercial product. Postgraft skin-wound samples were examined histomorphologically at 7th, 14th, and 28th day after staining with hematoxylin and eosin, picrosirius red and/or immunohistochemistry. Wound healing parameters considered were the extent of re-epithelialization, collagen deposition, and neoangiogenesis. The number of proliferating cells, vimentin positive cells, and alpha smooth muscle actin cells were also quantified. The histology results suggested that the grafts aided wound healing but, the cell-loaded graft induced a differential pattern of healing and had lower scarring tendency. The cell-loaded tissue construct may be useful as a therapeutic graft for treating wounds where there is a total loss of tissue and cells as in burn injury.