Basic Research On Application Of Cell Captured By Porous Materials In Tissue Engineering

Stem cells are classically defined by their multipotent long-term proliferation and self-renewal capabilities. They are used to treat a wide range of diseases. Candidates for such strategies include embryonic stem cells, induced pluripotent stem cells, and adult stem cells (ASC). Because ASC are, easy to harvest, and no ethical and security issues, they may become the most important cell for clinical application, particularly hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC). Some studies have indicated that HSC and MSC exist in bone marrow (BM), skeletal muscle (SM), and fat. However, there are very low frequencies of stem cells in tissues, which makes the isolation of cells difficult. Developing an alternative method that could give rise to a better source or harvest of stem cells is a hot area of research.In this study, we developed a new method to harvest HSC and MSC by implanting biomaterials into the spatium intermusculare of mice hind limbs. Our previous studies have shown that HSC could be found in captured cells (CC), and the adherent cells in CC expressed surface markers of MSC. However, whether MSC existed in CC needs to be further confirmed, and the proportion and the application value of HSC and MSC also need further exploration.Objectives:1. Comparing the proportion of HSC in CC and BM, to explore application value of HSC.2. Confirming MSC in the CC and comparing the biological characteristics of MSC in CC, BM and SM, to explore application value of MSC. 3. To investigate the treatment effects of captured MSC transplantation on the skeletal muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD).Methods:1. Gelatin sponges were implanted into the spatium intermusculare of mice hind limbs, and removed at 3, 6, 9, 12, 15, and 18 days after implanting. The sponges were weighed, and CC were counted with a hemocytometer. The viability of CC was determined by trypan blue exclusion assay and compared with BM, and the proportion of HSC in CC was detected and compared with that in BM by CFC assay.2. Gelatin sponges were implanted into the spatium intermusculare of mice hind limbs, and removed at 12 days after implanting. MSC were isolated from CC, BM, and muscle by differential adhesion method. The cell morphology, colony forming units-fibroblastics (CFU-F), proliferation capacity, and multi-differentiation potentials of CC-MSC were determined and compared with those of SM-MSC and BM-MSC by invert microscopy, CFU-F assay, cell counting assay, in vitro differentiation medium, cell staining and so on. In additional, RT-PCR was used to analyze expression of " Pluripotent " surface markers in captured MSC, and the source of MSC in CC was detected by sex chromosome tracking experiment.3. The BM-MSC and captured MSC of normal male C57BL/6 mice were injected through multiple injection points per muscle into the female mdx mice, with normal C57BL/6 mice and untreated mdx mice as the controls. At 12 weeks after transplantation, the change of motor function of the mice was observed by forced swimming test, the pathological phenotype of skeletal muscle was detected by HE staining, and dystrophin expression in gastrocnemius was detected by immunofluorescence, RT-PCR and Western blot. In additional, chimerism in female mdx mice after transplantation was confirmed by PCR analysis with primers specific for the sex-determinant Y chromosome (Sry).Results:1. Sponge weights were slightly reduced within 12 days after implanting, but deeply decreased after 12 days. However, CC number significantly increased with the implanted time prolonging, and reached the maximum on 12 days. The viability of CC was weaker than that of BM (p<0.05). The frequency of hematopoietic colonies and each colonies in CC was much higher than that in BM (p<0.05). However, the proportion of each colony type from the CC (BFU-E:GFU-GM:CFU-GEMM) was similar to that of BM.2. The characteristics of adherent cells were similar to that of SM-MSC and BM-MSC, including morphology, proliferation potential and multilineage differentiation capacity. The frequency of CFU-F in CC was much higher than in BM (p<0.05) and muscle (p<0.05). In additional, the result of RT-PCR indicated that the captured MSC expressed "Pluripotent" surface marker rex-1 and nanog, but did not express oct-4, sex chromosome tracking experiment demonstrated that MSC in the CC originated from the peripheral blood.3. The forced swimming time of the recipient mdx mice at 12 weeks after transplantation increased, but no significant increase was observed (P>0.05). The central nuclei in myofibers and the pathological phenotypes of skeletal muscle improved obviously(P<0.05). Expression of dystrophin was detected in the recipient mice by immunofluorescence, RT-PCR and Western blot. There was no significant difference in frequency of CNF and dystrophin-positive cells between CC-MSC and BM-MSC transplantation (p>0.05). In additional, sry gene was present in the recipient mice.Conclusions:1. The 12th day after implanting is a suitable time to retrieve the sponge. The frequency of HSC in the CC was much higher than in the BM, this shows that captured HSC may have a promising clinical application.2. MSC existed in CC, and the frequency of MSC in the CC was much higher than in the BM and SM, this shows that captured MSC may have a promising clinical application.3. Captured MSC can be used to treat DMD, but further research is needed to improve the effect.