Objective To introduce the related issues in the clinical translational application of adipose-derived stem cells (ASCs). Methods The latest papers were extensively reviewed, concerning the issues of ASCs production, management, transportation, use, and safety during clinical application. Results ASCs, as a new member of adult stem cells family, bring to wide application prospect in the field of regenerative medicine. Over 40 clinical trials using ASCs conducted in 15 countries have been registered on the website (http://www.clinicaltrials.gov) of the National Institutes of Health (NIH), suggesting that ASCs represents a promising approach to future cell-based therapies. In the clinical translational application, the related issues included the quality control standard that management and production should follow, the prevention measures of pathogenic microorganism pollution, the requirements of enzymes and related reagent in separation process, possible effect of donor site, age, and sex in sampling, low temperature storage, product transportation, and safety. Conclusion ASCs have the advantage of clinical translational application, much attention should be paid to these issues in clinical application to accelerate the clinical translation process.
Objective To introduce types and differentiation potentials of stem cells from adipose tissue, and its applications on regenerative medicine and advantages. Methods The literature of original experimental study and clinical research about bone marrow mesenchymal stem cells (BMSCs), adipose-derived stem cells (ADSCs), and dedifferentiated fat (DFAT) cells was extensively reviewed and analyzed. Results ADSCs can be isolated from stromal vascular fraction. As ADSCs have multi-lineage potentials, such as adipogenesis, osteogenesis, chondrogenesis, angiogenesis, myogenesis, and neurogenesis, they have already been successfully used in regenerative medicine areas. Dramatically, mature fat cells can be dedifferentiated and changed into fibroblast-like cells, named DFAT cells, via ceiling culture method. DFAT cells also had the same multi-lineage potentials as ADSCs, differentiating into adipocytes, osteocytes, chondrocytes, endothelial cells, muscle cells, and nerve cells. Compared with BMSCs which are commonly used as adult stem cells, ADSCs and DFAT cells have extensive sources and can be easily acquired. While compared with ADSCs, DFAT cells have good homogeneity and b proliferation capacity. Conclusion As a potential source of stem cells, adipose tissue will provide a new promising for regenerative medicine.
Objective To find a kind of simple and effective method for purifying and label ing stromal vascular fraction cells (SVFs) so as to provide a theoretical basis for cl inical application of SVFs. Methods The subcutaneous adi pose tissue were harvested form volunteers. The adi pose tissue was digested with 0.065%, 0.125%, and 0.185% type I collagenase,respectively. SVFs were harvested after digestion and counted. After trypan blue staining, the rate of viable cells was observed. SVFs was labeled by 1, 1’-dioctadecyl-3, 3, 3’, 3’-2-tetramethy-lindocyanine perchlorate (DiI). The fluorescent label ing and growth was observed under an inverted fluorescence microscope. MTT assay was used to detect cell proliferation. Results The number of SVFs was (138.68 ± 11.64) × 104, (183.80 ± 10.16) × 104, and (293.07 ± 8.31) × 104 in 0.065% group, 0.125% group, and 0.185% group, respectively, showing significant differences among 3 groups (P lt; 0.01). The rates of viable cells were 91% ± 2%, 90% ± 2%, and 81% ± 2% in 0.065% group, 0.125% group, and 0.185% group, respectively, and it was significantly higher in 0.065% group and 0.125% group than in 0.185% group (P lt; 0.01), but no significant difference was found between 0.065% group and 0.125% group (P=0.881). Inverted fluorescence microscope showed that the cell membranes could be labeled by DiI with intact cell membrane, abundant cytoplasm, and good shape, but nucleus could not labeled. SVFs labeled by DiI could be cultured successfully and maintained a normal form. MTT assay showed that similar curves of the cell growth were observed before and after DiI labeled to SVFs. Conclusion The optimal collagenase concentration for purifying SVFs is 0.125%. DiI is a kind of ideal fluorescent dye for SVFs.
ObjectiveTo evaluate the mechanism of stromal vascular fraction (SVF) promoting angiogenesis and tissue regeneration in tissue engineering chamber. MethodsTwenty-four 6-month-old New Zealand white rabbits, male or female, weighing 2.5-2.8 kg, were selected. Thoracic dorsal arteriovenous bundle combined with collagen type I scaffold was transplanted to dorsal side, and wrapped by cylindrical hollow silicone chamber; all animals were randomly divided into the experimental group (n=12) and the control group (n=12). SVF was isolated from the back fat pads of rabbits in experimental group and labelled with DiI at 2 weeks after operation. The 1 mL cell suspension (1×106 cells/mL) and equal saline were injected into the chamber in experimental group and control group, respectively. The regenerative tissues were harvested for general observation and HE staining at 2 and 4 weeks after injection;and immunofluorescent staining was carried out in experimental group at 4 weeks. ResultsAt 2 weeks after injection, the regenerative tissue was cylindrical; obvious vessel network and incompletely degradable collagen scaffold could be seen on the surface of the new tissue in 2 groups. The volume of new tissue was (0.87±0.11) mL in experimental group, and (0.72±0.08) mL in control group at 2 weeks, showing significant difference (t=2.701, P=0.011). At 4 weeks, little collagen scaffold could be seen on the surface in control group, but no collagen scaffold in experimental group; the volume of new tissue was (0.74±0.14) mL in experimental group, and (0.64±0.10) mL in control group, showing no significant difference (t=1.424, P=0.093). HE staining showed new mature vessels at 4 weeks, but no adipose tissue or fat lobulus formed in both groups; the capillary density was significantly higher in experimental group than in control group at 2 weeks (t=6.291, P=0.000) and at 4 weeks (t=5.445, P=0.000). The immunofluorescent staining found that SVF survived and located at the edge area after 4 weeks; the expressions of CD31 and DiI were positive in some endothelial cells. ConclusionSVF can promote the angiogenesis and tissue regeneration in tissue engineering chamber, but it can not differentiate into adipocyte spontaneously without adipogenic microenvironment.
ObjectiveTo observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model.MethodsSVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson’s trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining.ResultsThe cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C (P<0.05), and in group B than groups A and C (P<0.05). HE staining, Masson’s trichrome staining, and immunohistochemistry staining for OCN staining exhibited a large number of cartilage cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and there was no effective new bone formation in groups A and C.ConclusionThe combination of sustained release of rhBMP-2 and freshly SVFs can significantly promote spinal fusion in rat model, providing a theoretical basis for further clinical applications.