Growth characterization of neo porcine cartilage pellets and their use in an interactive culture model.

OBJECTIVE: The aim of this study was to evaluate the growth characteristics of freshly isolated porcine chondrocytes in high-density pellet cultures and to preliminary investigate their use in an interactive in vitro model with synovial fibroblast cell lines to study rheumatoid arthritis (RA). DESIGN: 1.8x10(6) chondrocytes/cm2 were seeded in 48-multiwell plates. Thickness, cell number and cell distribution in pellet cross sections were documented over a 22-day-long period. Alcian blue staining, type I and type II collagen staining, real-time reverse transcriptase polymerase polymerase chain reaction (RT-PCR) and high performance liquid chromatography (HPLC) were used to characterize cartilage extracellular matrix (ECM) formation, and cell proliferation was demonstrated by Ki67 staining. Furthermore, 2-week-old chondrocyte pellets were co-cultured for additional 2 weeks with two human synovial fibroblast cell lines derived from a normal donor (non-invasive cell line) and a RA patient (invasive-aggressive (IA) cell line), respectively. RESULTS: Chondrocyte pellets from 11 individual preparations showed a significant increase in pellet thickness from 44+/-19 microm (day 3) to 282+/-19 microm (day 22). Calculation of chondrocyte distribution, cell number and pellet thickness indicated that pellet growth was due to ECM formation and not cell proliferation. This was also confirmed by low numbers of Ki67 positive chondrocytes and absence of cell clusters. HPLC, messenger RNA-analysis, histochemistry and antibody staining verified the expression of ECM components such as type II collagen, whereas type I collagen expression was very low. In contrast to the non-aggressive synovial fibroblast cell line, the IA synovial fibroblast cell line clearly showed cartilage invasion. CONCLUSION: Pellet formation of freshly isolated chondrocytes followed a reproducible developmental kinetics and showed typical immature hyaline cartilage properties. Such uniform cartilage pellets are very useful as a substrate for interactive cell culture models that simulate diseases like RA.

Re-endothelialization of punctured ePTFE graft: an in vitro study under pulsed perfusion conditions.

BACKGROUND: When used as arteriovenous (AV) shunts for haemodialysis, small diameter expanded polytetrafluoroethylene (ePTFE) grafts have a high failure rate in vivo. Attempts to improve graft patency are various, and focus on either improvement of implantation techniques or graft tissue engineering. The tissue engineering approach attempts to reproduce in grafts the properties of pristine vasculature. As shown in previous experiments, it is possible to grow on ePTFE grafts under shear stress in vitro an autologous endothelial cell layer, which will withstand physiological stress under in vivo conditions of blood flow. The aim of this study was to investigate in an in vitro model the regenerative potency of a tissue-engineered prosthetic vascular graft after repeated cannulation with a haemodialysis cannula. METHODS: Pig endothelial cells were harvested from an external jugular vein. Following processing of the endothelial cells, seven ePTFE grafts were coated with an inner cell layer and were kept under pulsed perfusion. Each graft was then cannulated three times with a standard shunt needle. The endothelium was then left to regenerate for a maximum of 48 h. The grafts were stained with haematoxylin/eosin before histological study. RESULTS: All grafts were endothelialized over the puncture sites within 48 h. Histological analysis revealed a confluent endothelial cell lining at each puncture site. Cell morphology and cell pattern over puncture sites were not different from randomly picked locations over the graft lumen. CONCLUSION: Our results underline the potential of endothelial tissue engineering in vascular shunt surgery. Vascular bio-hybrids that have the properties of pristine vascular endothelium may be a key step forward in maintaining angio-access in patients who require haemodialysis.