Study on the Effect of Type III Recombinant Humanized Collagen on Human Vascular Endothelial Cells.

The effect and mechanism of type III recombinant humanized collagen (hCOLIII) on human vascular endothelial EA.hy926 cells at the cellular and molecular levels were investigated. The impact of hCOLIII on the proliferation of EA.hy926 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assay, the effect of hCOLIII on cell migration was investigated by scratch assay, the impact of hCOLIII on cell cycle and apoptosis was detected by flow cytometry, the ability of hCOLIII to induce angiogenesis of EA.hy926 cells was evaluated by angiogenesis assay, and the effect of hCOLIII on vascular endothelial growth factor (VEGF) expression was detected by real-time reverse transcription-polymerase chain reaction analysis. The hCOLIII at concentrations of 0.5, 0.25, and 0.125 mg/mL all showed specific effects on the proliferation and migration of human vascular endothelial cells. It could also affect the cell cycle, increase the proliferation index, and increase the expression level of VEGF in human vascular endothelial cells. In the meantime, hCOLIII at the concentration of 0.5 mg/mL also showed a promoting effect on vessel formation. hCOLIII can potentially promote the endothelization process of blood vessels, mainly by affecting the proliferation, migration, and vascular-like structure of human endothelial cells. At the same time, hCOLIII can promote the expression of VEGF. This collagen demonstrated its potential as a raw material for cardiovascular implants.

Fabrication and performance evaluation of PLCL-hCOLIII small-diameter vascular grafts crosslinked with procyanidins.

Cardiovascular disease has become one of the main causes of death. It is the common goal of researchers worldwide to develop small-diameter vascular grafts to meet clinical needs. Collagen is a valuable biomaterial that has been used in the preparation of vascular grafts and has shown good results. Recombinant humanized collagen (RHC) has the advantages of clear chemical structure, batch stability, no virus hazard and low immunogenicity compared with animal-derived collagen, which can be developed as vascular materials. In this study, Poly (l-lactide- ε-caprolactone) with l-lactide/ε-caprolactone (PLCL) and type III recombinant humanized collagen (hCOLIII) were selected as raw materials to prepare vascular grafts, which were prepared by the same-nozzle electrospinning apparatus. Meanwhile, procyanidin (PC), a plant polyphenol, was used to cross-link the vascular grafts. The physicochemical properties and biocompatibility of the fabricated vascular grafts were investigated by comparing with glutaraldehyde (GA) crosslinked vascular grafts and pure PLCL grafts. Finally, the performance of PC cross-linked PLCL-hCOLIII vascular grafts were evaluated by rabbit carotid artery transplantation model. The results indicate that the artificial vascular grafts have good cell compatibility, blood compatibility, and anti-calcification performance, and can remain unobstructed after 30 days carotid artery transplantation in rabbits. The grafts also showed inhibitory effects on the proliferation of SMCs and intimal hyperplasia, demonstrating its excellent performance as small diameter vascular grafts.

Hydrogel-complexed small-diameter vascular graft loaded with tissue-specific vascular extracellular matrix components used for tissue engineering.

Tissue engineering is thought to the most promising strategy to develop successful small diameter vascular grafts (SDVG) to meet clinical demand. The introduction of natural substances into the SDVG made from synthetic biomaterials can improve the biocompatibility to promote the regeneration of SDVG in vivo. Due to that natural materials from different sources may have property deviation, it is vital to determine the source of natural materials to optimize SDVG fabrication for tissue engineering applications. In this study, bioactive SDVGs were prepared via coating of heparin-modified poly-(ε-caprolactone) scaffolds with a precursor solution containing vascular extracellular matrix (VECM) components and subsequent in situ gelation. The mechanical properties, degradation behaviors, and morphologies of the SDVGs were thoroughly characterized and evaluated. Cell experiments demonstrated the in vitro tissue specificity of the VECM that could promote the proliferation of endothelial cells better than skin-derived collagen. Furthermore, three types of SDVGs, SDVGs with blank hydrogel, SDVGs with skin-derived collagen, and SDVGs with vascular extracellular matrix (VECM-SDVGs), were implanted into the abdominal aorta of rats for one month. The explanted SDVGs were then comprehensively evaluated using hematoxylin and eosin, Masson, von Kossa staining, and immunohistochemical staining for CD31, α-SMA, and MHC. The results showed that the VECM-SDVGs showed the best endothelium regeneration, appropriate intima regeneration, and no calcification, indicating the in vivo specificity of the fabricated VECM-SDVGs. Thus, long-term implantation of VECM-SDVGs was performed. The results showed that a complete endothelial layer formed after 6 months of implantation, and the amount of contractile SMCs in the regenerative smooth muscle layer approached the amount of native aorta at the 12th month. Consequently, relying on vascular tissue specificity, VECM-SDVGs can modulate the regenerative behavior of the implanted SDVGs in vivo to achieve satisfactory vascular regeneration both in short- and long-term implantation.

The crescendo pulse frequency of shear stress stimulates the endothelialization of bone marrow mesenchymal stem cells on the luminal surface of decellularized scaffold in the bioreactor.

A completely confluent endothelial cell (EC) monolayer is required to maintain proper vascular function in small diameter tissue-engineered vascular graft (TEVG). However, the most effective method for EC attachment to the luminal surface and formation of an entire endothelium layer that works in vitro remains a complicated challenge that requires urgent resolution. Although pulsatile flow has been shown to be better suited for the generation of functional endothelium, the optimal frequency setting is unknown. Several pulsatile flow frequencies were used to implant rat bone mesenchymal stem cells (MSC) into the lumen of decellularized porcine carotid arteries. The endothelium's integrity and cell activity were investigated in order to determine the best pulse frequency settings. The results showed that MSC were maximally preserved and exhibited maximal morphological changes with improved endothelialization performance in response to increased pulse stimulation frequency. Increased pulse frequency stimulation stimulates the expression of mechanoreceptor markers, cytoskeleton reorganization in the direction of blood flow, denser skeletal proteins fibronectin, and stronger intercellular connections when compared to constant pulse frequency stimulation. MSC eventually develops an intact endothelial layer with anti-thrombotic properties on the inner wall of the decellularized tubular lumen. Conclusion: The decellularized vessels retain the three-dimensional structure of the vasculature, have a surface topography suitable for MSC growth, and have good mechanical properties. By increasing the frequency of pulsed stimulation, MSC endothelialize the lumen of the decellularized vasculature. It is expected to have anti-thrombotic and anti-neointimal hyperplasia properties after implantation, ultimately improving the patency of TEVG.

Design and characterization of small-diameter tissue-engineered blood vessels constructed by electrospun polyurethane-core and gelatin-shell coaxial fiber.

Substitution or bypass is the most effective treatment for vascular occlusive diseases.The demand for artificial blood vessels has seen an unprecedented rise due to the limited supply of autologous blood vessels. Tissue engineering is the best approach to provide artificial blood vessels. In this study, a new type of small-diameter artificial blood vessel with good mechanical and biological properties was designed by using electrospinning coaxial fibers. Four groups of coaxial fibers vascular membranes having polyurethane/gelatin core-shell structure were cross-linked by the EDC-NHS system and characterized. The core-shell structure of the coaxial vascular fibers was observed by transmission electron microscope. After the crosslinking, the stress and elastic modulus increased and the elongation decreased, burst pressure of 0.11 group reached the maximum (2844.55 ± 272.65 mmHg) after cross-linking, which acted as the experimental group. Masson staining identified blue-stained ring or elliptical gelatin ingredients in the vascular wall. The cell number in the vascular wall of the coaxial group was found in muscle embedding experiment significantly higher than that of the non-coaxial group at all time points(p < 0.001). Our results showed that the coaxial vascular graft with the ratio of 0.2:0.11 had better mechanical properties (burst pressure reached 2844.55 ± 272.65 mmHg); Meanwhile its biological properties were also outstanding, which was beneficial to cell entry and offered good vascular remodeling performance.Polyurethane (PU); Gelatin (Gel); Polycaprolactone (PCL); polylactic acid (PLA);1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC); N-Hydroxy succinimide (NHS); 4-Morpholine-ethane-sulfonic (MES); phosphate buffered saline (PBS); fetal calf serum (FCS); Minimum Essential Medium (MEM); Dimethyl sulfoxide (DMSO); hematoxylin-eosin (HE).