An Efficient Method for Decellularization of Human Prepuce as a Biologic Scaffold for Skin Regeneration

Background and Aim: The use of ECM derived from decellularizedtissues and PAM (preputial acellular matrix) are widely used in bothpreclinical animal studies and clinical applications to repair tissues inregenerative medicine. Depending on a number of factors including theefficacy of decellularization, the use of chemical crosslinking agents, andthe age of the source tissue from which ECM scaffolds are harvested,the host response to implanted ECM-derived biomaterials may vary fromunacceptable to excellent. In this research, using ionic detergent, TritonX-100and Hank’s buffer, we optimized the preparation procedure for thedecellularized Prepuce.Methods: Prepuce tissues were prepared from circumcision of children (aninformed consent was obtained from parents) under the sterile condition.The outer-layer fat was removed mechanically by microdissection. Then,cellular components of prepuces were removed by using 5% (SDS)at room temperature for 4 h, by shaking. In the next step, the treatedprepuces were washed with distilled water. Afterward, trypsin (0.05%) (EDTA) (0.01%) was added and incubated at 4C for 4h. Fragmentswere rinsed with Hank’s balanced salt solution (HBSS), digested with 1%Triton X-100 for 1 h, and then washed in HBSS at 4°C for 48 h. In order toevaluate the efficacy of our acellularization procedure, acellular sampleswere fixed in 10% formalin, the samples were paraffin embedded andsectioned to 5 thickness. Hematoxylin and eosin (H&E) were appliedto visualize extracellular matrices and fibers. The DNA content in tissuesamples was assessed by Hoechst staining and then observed under alight microscope.Results: H&E and Hoechst staining of PAM showed an acellular collagenbasedmatrix with fibers orientation similar to the natural prepuces tissue.No cellular or nuclear remnants were preserved in scaffolds, while ECMwas satisfactorily preservedConclusion: Many variables including cell and matrix density,thickness, and morphology can alter the efficacy of tissue and organdecellularization as well as the integrity and physical properties of theresulting ECM scaffold. The current method can be used efficiently forthe decellularization of the prepuces biologic scaffold with satisfyingbiocompatibility for both in vivo and in vitro applications

Keywords:

Decellularization , Human prepuce , Skin regeneration