Objective Human acellular amniotic membrane (HAAM) contains collagens, glucoproteins, proteinpolysaccharide,integrin, and lamellar, which can supply rich nutrition to cell prol iferation and differentiation. To explore the possibil ity of HAAM with adi pose-derived stem cells (ADSCs) as a good engineered skin substitute for repairing skin defect. Methods Primary ADSCs were obtained from inguinal fat of 30 healthy 4-month-old SD rats, male or female, weighing 250-300 g, and cultured in vitro and purified. The 3rd passage ADSCs were used to detect CD44, CD49d and CD34 by immunocytochemistry staining. After physical and trypsin preparation, the HAAM was observed by HE staining and scanning electron microscope(SEM) respectively. ADSCs were seeded on epithel ial side of HAAM at the density of 2 × 105/cm2, cocultured, and observed by SEM at different time. MTT test was used to detect viabil ity of cells that seeded on HAAM, the group without HAAM was used as control. Thirty SD rats were made models of full-thickness skin wound and randomly divided into three groups (A, B, and C). Wound was repaired with HAAM/ADSCs composites in group A, with HAAM in group B, and with gauze as control in group C. The rats underwent postoperative assessment of wound heal ing rate and histological observation at the 1st, 2nd, and 4th weeks. Results HE staining showed that the 3rd passage ADSCs was spindle-shaped with an ovoid nucleus which located in the middle of cell; the immunocytochemistry staining showed positive result for CD44 and CD49d and negative result for CD34. There were no residues of cells in the HAAM by HE staining. SEM showed that there were different structures at the two sides of HAAM；one side had compact reticular structure and the other side had fibrous structure. After 3 days of co-culture, ADSCs showed good growth on HAAM; the cells were closely packed onto the HAAM, attached firmly and prol iferated to confluence on the stromal surface of HAAM. MTT test showed that the cells on the HAAM grew well and had b prol iferation vital ity. There was no significant difference between ADSCs cultured in the HAAM and control group (P gt; 0.05). One, 2, 4 weeks after graft, there were significant differences in wound heal ing rate between group A and groups B, C (P lt; 0.05), between group B and group C (P lt; 0.05). HE staining showed that wound healed faster in group A than in groups B, C. Cytokeratin 19 (CK19) immunohistochemical statining showed that there were more CK19 positive cells in group A than in groups B, C. Conclusion The graft of HAAM with ADSCs plays an effective role in promoting the repair of full-thickness skin wound
Objective To explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The label ing efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell’ s activity. Rabbits were randomly divided into experimental group (n=8) and control group (n=4), the labeled BMSCs and non-labeled BMSCs (5 × 105/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl’s fluid to observe cell survival and distribution. Results The label ing efficiency of BMSCs with SPIO was 95.65% ± 1.06%, the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell prol iferation within 7 days between non-labeled and labeled cells (P gt; 0.05). After 8 weeks of operation, the injected cells was al ive. ConclusionLabeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.