Isolation, culture and characterization of endothelial cells in infantile hemangioma / 中华整形外科杂志

<p><b>OBJECTIVE</b>To establish a method for the reliable isolation and culture of infantile hemangioma endothelial cells (HemECs) in vitro.</p><p><b>METHODS</b>Proliferative hemangioma specimens were digested by collagenase to form a single cell suspension. The HemECs were isolated using anti-CD31 coated dynabeads. The CD31+ cells were grown in fibronectin coated dishes. HemECs were identified by morphological characteristics and immunocytochemistry. The cells were also examined for their ability to intake LDL.</p><p><b>RESULTS</b>The method enabled the rapid isolation of HemECs that demonstrated typical endothelial cobblestone morphology in culture. The cells were positively stained for CD31, vWF. They also were labeled with DiI-Ac-LDL.</p><p><b>CONCLUSIONS</b>This technique can effectively isolate endothelial cells from the proliferative hemangiomas. These cells could be further used to research the mechanism of proliferation and degeneration of infantile hemangioma.</p>

Constructing of smooth muscle layers of tissue engineered blood vessel in a bioreactor / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of in vitro constructing tissue engineered blood vessels in bioreactor.</p><p><b>METHODS</b>Cell-PGA (polyglycolic acid) complex was constructed by seeding smooth muscle cells isolated from canine carotid artery on PGA unwoven fibers. The cell-PGA complex was cultured in a bioreactor (pulse rate = 75/min, radical distension < 5%). After three or six weeks in vitro culture, engineered tissues were harvested and tested.</p><p><b>RESULTS</b>Grossly,the experimental groups showed a tubular structure with a round lumen and good elasticity. Histological staining revealed smooth muscle fibers layers and dense elastic fibers presented in the engineered vessel wall. Bands of smooth muscle fibers and continuous endothelial cells layer were detected by the immuno-histological staining. In contrast, the control group took on poor elasticity, collapsed lumen and pale surface in the gross observation. In addition, its arrangement of smooth muscle fibers and elastic fibers was random and disorganized by histological observation, which was also confirmed by the immunohistological staining. The structure of 6 weeks TEBVs was more mature than that of 3 weeks and the biomechanical property of dynamic ones was as much as 60% of the normal one.</p><p><b>CONCLUSIONS</b>Blood vessel with good elasticity can be constructed in a bioreactor by tissue engineering approach.</p>

The isolation, subculture and identification of human adipose derived endothelial progenitor cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the methods of isolating and identifying human adipose derived EPCs.</p><p><b>METHODS</b>The cells obtained from human lipoaspirates were plated on culture dishes coated with human fibronectin and were cultured in DMEM containing 2% FBS. Cells of passage 2 cultured in EGM-2 (2% FBS) served as the induced cells (experimental group), with cells cultured in DMEM (2% FBS) as the non-induced cells (control group) . Immunofluorescence was used to detect the expression of cell markers, including CD34, vWF and PECAM-1. FACS (fluorescence activated cell sorter) was used to quantitatively analyze the expression rate of cell markers (CD34, CD45, CD133 and PECAM-1). Fluorescence microscope was used to observe the function of taking up DiI-ac-LDL by the induced cells. To determine the ability of forming capillary-like structure in three-dimensional matrices, the induced cells were also cultured in methylcellulose.</p><p><b>RESULTS</b>The induced cells of passage 2 exhibited cobblestone morphology, similar to that of the endothelial cells. In contrast, these morphological changes were not observed in non-induced cells. Immunofluorescence detected expression of vWF, PECAM-1 in induced cells and CD34 in non-induced cells. FACS analysis showed (67.41 +/- 13.35)% of the induced cells expressed PECAM-1 and (6.73 +/- 2.21)% of the non-induced cells expressed PECAM-1 (P < 0.01), while (72.39 +/- 13.45)% of the non-induced cells expressed CD34 and (16.06 +/- 3.86)% of the induced cells expressed CD34 (P < 0.01). Fluorescence microscopy observed the induced cells took up low-density lipoprotein (LDL). The formation of "branch-like" structure confirmed their functional activity.</p><p><b>CONCLUSION</b>EPCs derived from human adipose may serve as another source of seeding cells for vascular tissue engineering.</p>

Experimental study on constructing small-caliber artery by tissue engineering approach / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the possibility of constructing small-caliber artery by means of tissue engineering.</p><p><b>METHODS</b>Cell-PGA mixtures were made by separately seeding 1 x 10(7) smooth muscle cells and 5 x 10(6) endothelial cells isolated from neonate umbilicus onto PGA scaffold, the cell-PGA constructs were wrapped around a silicone tube before its implantation subcutaneously to nude mice and the mice were sacrificed in 2 and 6 weeks. The tissue engineered artery (TEA) were examined both grossly and immunohistochemically.</p><p><b>RESULTS</b>The gross appearance of TEA was similar to that of the natural counterparts; histologic and immunohistochemical analyses of the neoformed tissues revealed a typical artery structure, including the presence of EC at the luminal surface and the presence of SMC and collagen in the wall.</p><p><b>CONCLUSION</b>TEA with histology similar to natural vessel can be constructed by tissue engineering.</p>