![[ Visit Client Website ]](images/banner.gif)
Saturday, October 17, 2015: 8:45 AM
Introduction/Purpose:
Decellularization of xenologous tissues provides inductive extra-cellular matrices (ECM) for effective organ reconstruction: this promising approach has not been translated to breast reconstruction yet. We investigated effectiveness of different decellularization protocols of porcine mammary glands with the purpose of prospective breast tissue engineering.
Material and Methods:
Porcine mammary glands underwent preliminary macroscopic (anatomical) and microscopic (histological) comparative analysis to assess suitability for in vivo application. Frozen glands were cut in homogeneous samples (10x10x2cm) and processed according to three different decellularization protocols (A, B, C) via 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). Obtained specimens were analyzed by macroscopic (morphologic) and microscopic methods (hematoxylin and eosin-H&E, immunofluorescent labeling with 4′,6-diamidino-2-phenylindole-DAPI, quantitative measurement of DNA and DNA fragment size).
Results:
Histological structure of porcine glands resembled human glands. Glands could be molded to required shape and adjacent glands could be harvested together (up to 700grams). Size varied (average: 20x40cm in length and 3cm in height). Blood supply was based on reliable vascular pedicles (caliber:1,5-2mm). Decellularization protocols had variable effectiveness: all samples showed macroscopic evidence of decellularization preserving original morphology. 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 (H&E) protocol A preserved a morphology more closely resembling native architecture of ECM and preserving vascular/ductal networks. Protocols B and C slightly damaged and altered histological structure.
Conclusions:
Decellularization of porcine mammary tissue represents a novel and reliable preliminary approach for breast tissue engineering by prospective combined recellularization and in vivo implant.
Decellularization of xenologous tissues provides inductive extra-cellular matrices (ECM) for effective organ reconstruction: this promising approach has not been translated to breast reconstruction yet. We investigated effectiveness of different decellularization protocols of porcine mammary glands with the purpose of prospective breast tissue engineering.
Material and Methods:
Porcine mammary glands underwent preliminary macroscopic (anatomical) and microscopic (histological) comparative analysis to assess suitability for in vivo application. Frozen glands were cut in homogeneous samples (10x10x2cm) and processed according to three different decellularization protocols (A, B, C) via 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). Obtained specimens were analyzed by macroscopic (morphologic) and microscopic methods (hematoxylin and eosin-H&E, immunofluorescent labeling with 4′,6-diamidino-2-phenylindole-DAPI, quantitative measurement of DNA and DNA fragment size).
Results:
Histological structure of porcine glands resembled human glands. Glands could be molded to required shape and adjacent glands could be harvested together (up to 700grams). Size varied (average: 20x40cm in length and 3cm in height). Blood supply was based on reliable vascular pedicles (caliber:1,5-2mm). Decellularization protocols had variable effectiveness: all samples showed macroscopic evidence of decellularization preserving original morphology. 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 (H&E) protocol A preserved a morphology more closely resembling native architecture of ECM and preserving vascular/ductal networks. Protocols B and C slightly damaged and altered histological structure.
Conclusions:
Decellularization of porcine mammary tissue represents a novel and reliable preliminary approach for breast tissue engineering by prospective combined recellularization and in vivo implant.