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(Circulation. 2002;106:I-63.)
© 2002 American Heart Association, Inc.

Surgery for Valvular Heart Disease

Construction of Autologous Human Heart Valves Based on an Acellular Allograft Matrix

Serghei Cebotari, MD; Heike Mertsching, PhD; Klaus Kallenbach, MD; Sawa Kostin, MD; Oleg Repin, MD; Aurel Batrinac, MD; Carmen Kleczka, MS; Anatol Ciubotaru, MD, PhD; Axel Haverich, MD, PhD

From the Leibniz Research Laboratory for Biotechnology and Artificial Organs, Hannover, Germany (S.C., H.M., K.K., A.H.); Division of Cardio-thoracic and Vascular Surgery, Hannover, Germany (K.K., A.H.); Hannover Medical School, ARTISS GmbH, Hannover, Germany (H.M., C.K., A.H.); Department of Experimental Cardiology, Max-Plank-Institute, Bad Nauheim, Germany (S.K.); and Republican Center of Cardiac Surgery, State Medical and Pharmaceutical University, Chisinau, Moldova (S.C., O.R., A.B., A.C.).

Correspondence to Serghei Cebotari, MD, Department of Cardio-thoracic and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail serjciub{at}yahoo.com

Objective Tissue engineered heart valves based on polymeric or xenogeneic matrices have several disadvantages, such as instability of biodegradable polymeric scaffolds, unknown transfer of animal related infectious diseases, and xenogeneic rejection patterns. To overcome these limitations we developed tissue engineered heart valves based on human matrices reseeded with autologous cells.

Methods and Results Aortic (n=5) and pulmonary (n=6) human allografts were harvested from cadavers (6.2±3.1 hours after death) under sterile conditions. Homografts stored in Earle’s Medium 199 enriched with 100 IU/mL Penicillin-Streptomycin for 2 to 28 days (mean 7.3±10.2 days) showed partially preserved cellular viability (MTT assay) and morphological integrity of the extracellular matrix (H-E staining). For decellularization, valves were treated with Trypsin/EDTA resulting in cell-free scaffolds (DNA-assay) with preserved extracellular matrix (confocal microscopy). Primary human venous endothelial cells (HEC) were cultivated and labeled with carboxy-fluorescein diacetate-succinimidyl ester in vitro. After recellularization under fluid conditions, EC were detected on the luminal surfaces of the matrix. They appeared as a monolayer of positively labeled cells for PECAM-1, VE-cadherin and Flk-1. Reseeded EC on the acellular allograft scaffold exhibited high metabolic activity (MTT assay).

Conclusions Earle’s Medium 199 enriched with low concentration of antibiotics represents an excellent medium for long time preservation of extracellular matrix. After complete acellularization with Trypsin/EDTA, recellularization under shear stress conditions of the allogeneic scaffold results in the formation of a viable confluent HEC monolayer. These results represent a promising step toward the construction of autologous heart valves based on acellular human allograft matrix.

Key Words: valves • endothelium • heart diseases • surgery