Floating Plastic Co-cultured In Smooth Muscle Cells To Endothelial Cells In Blood Vessel Formation And Function

The ability to grow new blood vessels is very important for virtually any strategy for tissue repair. Many ischemia patients are not amenable to conventional surgical revascularization and new biological revascularization strategies have been developed to address this problem. These approaches rely on the recapitulation of the developmental processes of blood vessel formation (including vasculogenesis and angiogenesis). Therefore, successful production of fully functional tissue-engineered blood vessels (TEBVs) can provide an alternated approach to treat complications from cardiovascular diseases. A fully functional TEBV must have mechanical properties that mimic those of healthy native vessels as well as a confluent endothelium that can resist thrombosis. In normal vascular wall exists two important type of cells, endothelial cells (ECs) and smooth muscle cells (SMCs). ECs lining the lumen of blood vessels have many important functions, such as vasodilation, antithrombosis, clot formation, and immune response. To obtain long-term potency of TEBVs, a confluent and functional endothelium must be present. In a healthy, mature blood vessel, SMCs exhibit a latent contractile phenotype, characterized by expression of several smooth muscle specific contractile proteins, the lack of migration, proliferation, and extracellular matrix (ECM) protein synthesis. Many researches on ECs and SMCs interactions within TEBVs showed that the co-culture of ECs/SMCs is crucial to better understand ECs/SMCs cross-talk and model vascular healing.HUVECs were isolated by perfusing human umbilical vein with 0.1% collagenase and cultured in endothelial cell growth medium. hSMCs were isolated from human umbilical cord artery by using tissue section culture after collagenase removing endothelial cells. The cells were serially passaged and expanded in humidified incubator at 37℃with 5%CO₂. HUVECs and hSMCs were assessed to confirm their cell phenotype by phase contrast light microscopic and immunofluorescent microscopic. The proliferation of co-culture cells and monoculture cells was tested by MTS assay; the growth of HUVECs on floating gels was observed by scanning electron microscopy (SEM).The rat tail collagen type I was diluted to a concentration of 1.5 mg/ml and the pH was neutralized to around 7.2 just before use. The floating gel co-culture model consisted of a subconfluent HUVECs (5x10⁴/well) attaching on the downside floating gel, when oppositely culturing a single layer of hSMCs (1x10⁴/well) on cell culture plate. The vessel-like structure visualization and quantification was observed and analyzed by Leica TCS SP2 confocal microscope and Image Measurement (version1.0) software. The expression of TM, MMP-1, MMP-9, lamininγ2 and TFPI-2 was analyzed by RT-PCR. The immunofluorescence and quantification of protein expression was analyzed by Leica TCS SP2 confocal microscope and Leica Image Quantification software.Results showed that isolated HUVECs kept their cobblestone-like shape, while the hSMCs displayed the spindle shape and specific "hill and valley" morphology. The immunofluorescence staining also confirmed the CD31 of endothelial cell specific marker and theα-SMA of SMCs. MTS assay showed that in a simplified co-culture system where HUVECs and hSMCs directly co-cultured together without collagen gel, the cell proliferation declined rapidly from day 2 to day 4. After 4 days, the cell proliferation maintained a low level without significant decreasing, compared with monocultured HUVECs and monocultured hSMCs. This indicated that interaction of HUVECs and hSMCs mutually influenced the viability of cell proliferation one another, and the low proliferation level of hSMCs in co-culture system showed a relative quiescent statethat is desired in normal blood vessels.In the floating gel co-culture system, data showed that there was higher density of vessel-like structures than monoculture group; in co-culture group, average density of vessel-like structure was 62%, 121%, 163% higher compared to monoculture group at day 3, 5, 7 respectively. There was a statistically significant difference due to the co-culture (P<0.001) and the time of co-culture (P<0.05). The gene expression of TM, MMP-1, and MMP-9 was significantly higher in co-culture group than in monoculture according to RT-PCR. The protein expression of TM was also higher in co-culture than in monoculture control. The interaction between HUVECs and hSMCs in co-culture results in a low thrombogenicity phenotype of HUVECs, and this co-culture system consisted of HUVECs, hSMCs and Collage type I can improve HUVECs function and ability of network formation. Moreover such a novel co-culture system is of great importance to better understand the EC/SMC interaction between these major cell types in the TEBVs.