Breast Tissue Engineering

Abstract

INTRODUCTION: Decellularization of tissues provides inductive extra-cellular matrices (ECM) for effective organ reconstruction: this promising approach has not been translated to breast reconstruction yet.1,2 We investigated the effectiveness of different decellularization protocols applied to porcine mammary glands with the purpose of developing an inductive biological matrix for prospective breast tissue engineering. METHODS: We cut porcine mammary glands into homogeneous samples (10x10x2cm) and processed these according to three different decellularization protocols (A, B, C). All protocols combined multiple chemical treatments (A: 0.02% trypsin, 0.05% ethylenediaminetetraacetic acid-EDTA, 3% Triton X-100, 4% deoxycholic acid; B: collagenase 3mg/g, 0.02% trypsin, 0.05% EDTA, 10U/mL, 10U/mL lipase; C: collagenase 3mg/g, 0.05% EDTA, 4% sodium deoxycholate, 1% sodium dodecyl sulfate, 0.9% NaCl in TRIS-HCl containing protease inhibitors). We analyzed processed specimens by macroscopic (morphologic) and microscopic methods (Hematoxylin and Eosin staining, Immuno-fluorescent labeling with 4′,6-diamidino-2-phenylindole-DAPI, quantitative measurement of DNA and DNA fragment size) to assess quality of decellularization and structural preservation of the matrix. RESULTS: Harvested mammary glands could be molded to required shape; adjacent glands could be harvested together (up to 700grams). Maximal size of obtainable glands based on a single blood supply varied (average: 20x40x3cm). Blood supply was based on a single reliable vascular pedicle, potentially suitable for microsurgical anastomosis in vivo. Decellularization protocols had variable effectiveness: all samples showed macroscopic evidence of decellularization preserving original morphology at gross examination. DAPI, quantitative measurement of DNA (below 50 ng/mg dry tissue weight) and of DNA fragment size (below 200 base-pairs) showed effective reduction of immunogenic components in each protocol. At histological analysis (Hematoxylin and Eosin) protocol A provided a morphology more closely resembling native architecture of ECM and a more effective preservation of the vascular and ductal networks. Protocols B and C, instead, slightly damaged and altered histological structure of the matrix. CONCLUSIONS: Decellularization of porcine mammary tissue represents a novel and reliable preliminary approach to develop a bio-inductive matrix for potential subsequent breast tissue engineering.