[Effects of tensile force on the vascular lumen formation in three-dimensional printed tissue].

Objective: To explore the effects of tensile force on vascular lumen formation in three-dimensional printed tissue. Methods: The experimental research method was used. Human umbilical vein endothelial cells (HUVECs) were extracted from discarded umbilical cord tissue of 3 healthy women (aged 22 to 35 years) who gave birth in the Department of Gynaecology and Obstetrics of Suzhou Ruihua Orthopaedic Hospital from September 2020 to May 2021. Human skin fibroblasts (HSFs) were extracted from discarded normal skin tissue of 10 male patients (aged 20 to 45 years) who underwent wound repair in the Department of Hand Surgery of Suzhou Ruihua Orthopaedic Hospital from September 2020 to September 2022. After identification of the two kinds of cells, the 4th to 6th passage of cells were taken for the follow-up experiments. HUVECs and HSFs were used as seed cells, and polycaprolactone, gelatin, hyaluronic acid, and fibrin were used as scaffold materials, and the three-dimensional printed vascularized tissue was created by three-dimensional bioprinting technology. The printed tissue with polycaprolactone scaffold of 6 and 10 mm spacing, and without polycaprolactone scaffold were set as 6 mm spacing polycaprolactone group, 10 mm spacing polycaprolactone group, and non-polycaprolactone group, respectively. After 4 days of culture, the printed tissue in 10 mm spacing polycaprolactone group was selected to detect the cell survival by cell viability detection kit, and the cell survival rate was calculated. After 14 days of culture, the printed tissue in three groups were taken, and the shape change of tissue was observed by naked eyes; immunofluorescence staining was performed to observe the arrangement of filamentous actin, and lumen diameter, total length, and number of branches of vessel in the tissue. The tissue with micro-spring structure in the above-mentioned three groups was designed, printed, and cultured for 9 days, and the tensile force applied in the printed tissue was measured according to the force-displacement curve. The number of samples was all 3 in the above experiments. Data were statistically analyzed with one-way analysis of variance and Tukey test. Results: After 4 days of culture, the cell survival rate in printed tissue in 10 mm spacing polycaprolactone group was (91.3±2.2)%. After 14 days of culture, the shape change of printed tissue in non-polycaprolactone group was not obvious, while the shape changes of printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were obvious. After 14 days of culture, the arrangement of filamentous actin in the printed tissue in non-polycaprolactone group had no specific direction, while the arrangement of filamentous actin in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group had a specific direction. After 14 days of culture, The vascular lumen diameters of the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were (6.0±1.3) and (10.8±1.3) µm, respectively, which were significantly larger than 0 µm in non-polycaprolactone group (P<0.05), and the vascular lumen diameter of printed tissue in 10 mm spacing polycaprolactone group was significantly larger than that in 6 mm spacing polycaprolactone group (P<0.05); the total length and number of branches of blood vessel in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were significantly shorter or less than those in non-polycaprolactone group (P<0.05), and the total length and number of branches of blood vessel in the printed tissue in 10 mm spacing polycaprolactone group were significantly shorter or less than those in 6 mm spacing polycaprolactone group. After 9 days of culture, the tensile forces applied in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were (2 340±59) and (4 284±538) µN, respectively, which were significantly higher than 0 µN in non-polycaprolactone group (P<0.05), and the tensile force applied in the printed tissue in 10 mm spacing polycaprolactone group was significantly higher than that in 6 mm spacing polycaprolactone group (P<0.05). Conclusions: The three-dimensional printed scaffold structure can exert different tensile force in the printed tissue, and the vascular lumen diameter of the printed tissue can be regulated by adjusting the tensile force.

The molecular mechanism of the action of the pharmacological doses of glucocorticoids. Studies on the low-affinity glucocorticoid receptor.

The low-affinity glucocorticoid binding sites (LAGS, kDa 1-10 mumol/L) with glucocorticoid specificity were demonstrated in hepatic cytosol of rats. The induction of tyrosine aminotransferase (TAT) activity in primary cultures of rat hepatocytes by high concentration (10 mumol/L) of hydrocortisone (F) could be completely inhibited by RU486, the competitive antagonist of glucocorticoid receptor, indicating that the induction of TAT by high concentrations of F is mediated by LAGS, therefore, LAGS may be referred to as low-affinity glucocorticoid receptor (GRL). In order to study the effect of GC on GRL, the concentration of glucocorticoids (GC) in plasma was maintained over 1 mumol/L for 3 d by subcutaneous injection of F in polyvinyl alcohol into rats. The binding capacity (Ro) of high-affinity glucocorticoid receptor (GRH) decreased significantly 1 h after injection and maintained at low level, whereas the Ro of GRL increased at 1, 24, and 48 h after injection. Thus, it may be concluded that GC can downregulate GRH but upregulate GRL. These results strongly suggest that the action of pharmacological doses of GC may be mediated by GRL.

[Low-affinity binding sites of glucocorticoid].

The low-affinity glucocorticoid binding sites (LAGS) with steroid specificity were demonstrated in hepatic cytosol, cerebral cytosol, and thymocytes of rat. The Kd of LAGS was 1-10 mumol.L-1 as estimated by Scatchard, pseudo-scatchard, and competitive analysis. The specific binding of dexamethasone 1-100 nmol.L-1 was roughly parallel with its inhibition of [3H]UdR incorporation in thymocytes of rat, and both were blocked by mifepristone (RU-486), the competitive antagonist of glucocorticoids. These results suggest that the pharmacological activity of glucocorticoid might be mediated at least partially by LAGS.