Biomaterial properties of cholecyst‐derived scaffold recovered by a non‐detergent/enzymatic method

Abstract

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 local immunogenic potential of scaffolds prepared from porcine cholecyst, jejunum, and urinary bladder in rat subcutaneous model

“…20 The scaffolds were implanted in a rat subcutaneous model that is frequently used for evaluating local tissue response. 20 The scaffolds were implanted in a rat subcutaneous model that is frequently used for evaluating local tissue response.…”

“…20 The procedure involved ex vivo incubation of the source organ in a stabilising agent (10% neutral buffered formalin), that also caused in situ cross linking of biomolecules, followed by mechanical delamination of the ECM beneath the mucosa from other tissue layers. 20 The procedure involved ex vivo incubation of the source organ in a stabilising agent (10% neutral buffered formalin), that also caused in situ cross linking of biomolecules, followed by mechanical delamination of the ECM beneath the mucosa from other tissue layers.…”

“…19 It has been reported that compared with scaffolds isolated from small intestine and urinary bladder, CDS prepared by a non-detergent/enzymatic method contains relatively less residual cellular components. 20 However, the immunogenic potential of this less cellular biomaterial has not been studied. It is hypothesised that when prepared by a non-detergent/enzymatic method, CDS has less immunogenic potential compared with scaffolds prepared from small intestine or urinary bladder.…”

Controlled cross‐linking of porcine cholecyst extracellular matrix for preparing tissue engineering scaffold

“…Traditionally, for adequate tissue fixation, 10% NBF has been proposed for routine histology of biological tissues. Therefore, the concentration of NBF was kept as 10% in the original nonenzymatic and nondetergent method (Anilkumar et al, ) of preparing tissue engineering scaffolds from porcine cholecyst, jejunum, and bladder. This formed the rationale for the selection of 10% as the maximum concentration of the NBF.…”

“…The presence of fewer fibrils in CDS‐10 (Figure e) compared with other preparations also reflected the extent of the cross‐linking. Considering that the collagen is the most abundant protein present in the CDS (Anilkumar et al, ), the extent of cross‐linking in the variably cross‐linked scaffolds was assessed by enzymatic degradation of the CDS with collagenase. Progressive degradation of the material on decreasing the concentration of formaldehyde (Figure ) was an indication that cross‐linking had taken place in a controlled manner.…”