Human iliac vein replacement with a tissue-engineered graft.

The modification of a previously described technique to generate venous conduits in a lamb model from a decellularised matrix and autologous cells and its application to human tissue is described. A 49-year-old woman underwent surgery for a large malignant pelvic tumour (carcinoma of unknown primary) involving the right iliac artery and vein. The right iliac artery was reconstructed with a cryopreserved human arterial allograft. For iliac vein reconstruction a tissue-engineered neo-vein was developed utilising a decellularised cryopreserved vein allograft that was reseeded in a bioreactor with autologous endothelial cells derived from the recipient's great saphenous vein. Both interposition grafts were patent initially, after 3, 6, 12, and 24 months, but the tissue-engineered neo-vein had become obstructed due to evolving disease four month postoperatively. Tissue engineered neo-veins may be a therapeutic option in selected cases with symptomatic vein stenosis or obstruction not curable with interventional methods or standard prosthetic replacement.

Preclinical development of tissue-engineered vein valves and venous substitutes using re-endothelialised human vein matrix.

OBJECTIVE: The aim of the study was to evaluate the use of a decellularised scaffold and its re-endothelialisation in vitro in order to create human vascular substitutes containing venous valves. This research is clinically relevant particularly with regard to the development of venous (valve containing) transplants to replace a diseased femoral vein valve and/or obstructed veins. This technique may enable causal treatment of venous reflux and obstructions. MATERIALS AND METHODS: Valve-bearing segments of human allogeneic great saphenous veins (GSVs) were decellularised using sodium deoxycholic acid (SD) and treated with DNase I. Human venous endothelial cells (ECs) were enzymatically harvested from the GSV, expanded up to the 3rd passage using FCS (n=20) or human AB serum (hABS; n=8) supplemented media before used for re-seeding. In special bioreactors, 3D re-seeding of 28 decellularised GSV was performed with constant perfusion (A; n=8), bidirectional perfusion (B; n=8), bidirectional perfusion/reduced flow (C; n=2), static conditions (D; n=2), and bidirectional perfusion/reduced flow using hABS (E; n=8) instead of FCS. Decellularised GSV, scaled-up EC and 3D-seeded tissue-engineered valve containing neo-veins underwent immunohistochemical and PCR characterisation. RESULTS: Intact collagen and elastin networks as well as complete acellularity were shown after GSV decellularisation. In EC culture, supplementation with hABS led to a significantly higher expression of vWF compared to FCS (p=0.025). Additional EC markers such as CD 31, FLK-1 and VE-Cadherin were not altered. EC re-seeding using hABS supplemented medium (E) led to a confluent monolayer of cells that were immunohistochemically positive for FLK-1, CD 31, vWF and VE-Cadherin and by means of PCR after RNA preparation in 7 of 8 cases but was unsuccessful if FCS was used (A-D). In A-D cells presented as conglomerates positive for CD 31 and VE-Cadherin, suggesting sufficient intercellular contact but not cell-matrix contact. CONCLUSIONS: Treatment with SD and DNase enables complete decellularisation of human valve containing veins whereas 3D matrix components such as collagen and elastin remain preserved. The lumen of the scaffold including the valves can be successfully re-seeded with a human EC monolayer in a 3D bioreactor. There is substantial evidence that hABS and not FCS is essential for the completion of cell-matrix contacts in human veins.

Acellular scaffold implantation--no alternative to tissue engineering.

OBJECTIVE: Degradation mechanisms of cardiovascular bioprostheses may play an important role in bioartificial implants. The fate of acellular implanted and cellular cardiovascular scaffolds was examined in an in vivo model. METHODS: Decellularized or native ovine carotid artery (CA, n=42) and aorta (AO, n=42) were implanted subcutaneously into rats for 2, 4 and 8 weeks. Immunohistochemical methods were used to monitor repopulation. Desmin-vimentin, CD31-, CD4- and CD18-antibodies for myocytes, endothelium, and inflammatory cell-infiltration, respectively. Calcification was detected by von-Kossa staining. Cell density was quantified by DNA-isolation. RESULTS: Acellular AO and CA matrices showed progressive calcification. Cellular AO and CA matrices trigger a strong inflammatory reaction which subsides after two weeks. CA scaffolds are revascularized progressively, whereas AO biocomposites degenerate. Calcification is less pronounced in cellular AO scaffolds and lacking in CA. CONCLUSION: Acellular bioartificial implants demonstrate degradation mechanisms similar to currently applied cardiovascular bioprostheses. Cellularized viable implants are promising clinical alternatives.

Tissue-engineered bioprosthetic venous valve: a long-term study in sheep.

OBJECTIVE: to develop a graft bearing an immunologically tolerated tissue-engineered venous valve (TE graft) that will be incorporated into a native vessel, and restore normal valve function for the treatment of chronic venous insufficiency. METHODS: twenty-four TE grafts were grown using decellularised allogeneic ovine veins as donor matrix, which was subsequently repopulated with the future recipient's myofibroblasts (MFB) and endothelial cells (EC). TE grafts were implanted into the external jugular vein. Animals were sacrificed at 1, 6, and 12 weeks (n=4, each). Autografts served as controls (1 week, n=4; 6 weeks, n=4). Specimen for histology and immunohistochemistry were taken. RESULTS: the matrix was successfully repopulated with MFB and EC (n=8). Patency on venography in the TE graft-group was44,44, and 34 at 1, 6, and 12 weeks, and44 (44) in autografts at 1 (6) weeks, respectively. Except for 2 TE grafts after 12 weeks, valves were competent (duplex ultrasound). Patent TE grafts were merely distinguishable from autografts with minor inflammatory reactions. Reflux was caused by neo-intima formation related to the basis of the TE graft. CONCLUSION: acellularisation and consecutive in vitro autogeneic re-seeding of valved venous conduits can lead to immunologically acceptable, patent, and competent implants in sheep.