| Background and ObjectivesUn-healing wound is common in diabetes, circulation obstacles in limbs, deep wounds and other diseases. How to promote the healing of such injuries has been an important research in the field of trauma. Although the researchers have got a profound understanding of the pathophysiological mechanisms of wound healing, and also have accumulated a wealth of experiences in the treatment of wounds. But there is still lack effective method in the treatment of un-healing wounds, especially in advanced diabetics with un-healing wounds. Current conventional therapy and growth factors approved by FDA in unconventional therapy have got a certain effect, but the effect is not as good as expected, even ineffective for some patients. Environmental changes in body caused by ischemia is one of the important mechanisms affecting wound repair and healing, and how to choose effective exogenous anti-injury material according to the mechanism to prevent the ischemic damage is a new measure of treatment.Although the evidence have proved that mesenchymal stem cells (MSCs) in wound healing can be differentiated to other cells so as to promote the healing, but the therapeutic effects are most likely due to the soluble factors released by MSCs that regulate local cellular responses to cutaneous injury.When the angiogenic response was impaired, MSCs could secrete stromal derived factor-1(SDF-1), vascular endothelial growth factor (VEGF), insulin-like growth factor1(IGF-1), epidermal growth factor (EGF), keratinocyte growth factor(KGF), angiopoietin(Ang)-1, macrophage inflammatory protein (MIP)-1α and β and erythropoietin(EPO), and matrix metalloproteinase-9(MMP-9), or other cytokines to stimulate recruitment, proliferation, and differentiation of EPCs, hence promoting angiogenesis and tissue regeneration. Therefore, MSCs are expected to have a positive impact in promoting unhealing wound healing.Impaired wound healing in diabetic wound is the most common in clinical, therefore, in this study we constructed the non-healing wound model based on type Ⅱ diabetes mice.To investigate the effect of bone marrow mesenchymal stem cells (BMMSCs) on angiogenesis and promoting healing in un-healing wounds, we transplanted BMMSCs in diabetic wounds locally. Diabetes mellitus (DM) is a chronic metabolic disease, many complications may occur in diabetics due to the long-term of hyperglycemia. Therefore, the biological effects of diabetics BMMSCs may also be affected. Hence, in this study we compared the differences of the biological effects in vitro environment and the angiogenesis and promoting healing in un-healing wound between the normal mice and the diabetic mice BMMSCs.Contents and Methods1. Construction of the type Ⅱ diabetic mice modelWe used C57BL/6mice and C57BL/6background GFP mice as the experimental animals, the fodder which we choose was high-fat and high-sugar, and the mice were feed with this diet in the long term. Intraperitoneal injection of Streptozotocin (STZ) was used to induce the type Ⅱ diabetes mice model, the dose of STZ was40mg/kg weight every24hours, and the intraperitoneal injection were repeated for three times. At last, we selected the mice whose fasting blood glucose were greater than or equal to16.7mmol/L and last for two weeks for the type II diabetes mice model.2. Isolation and culture of normal mice and diabetic mice GFP-BMMSCs in vitroThe normal mice and diabetic mice were killed with cervical dislocation and were disinfected with75%alcohol immediately. The process of isolation of tibia and femur were performed in super clean bench. The two ends of the bones were removed to expose bone marrow cavity, and the bone marrow cavity was washed by syringe repeatedly until the cavity was washed to white. The whole bone marrow cells were collected and cultured in complete culture medium (including penicillin-streptomycin) containing10%FBS in vitro. Non-adherent cells were removed by changed the medium. Cells were subcultured using0.25%trypsin-EDTA digestion when the cells confluence to90%in culture flask. P3generations of GFP-BMMSCs were used for experiments.3. Identification the osteogenesis and adipogenesis ability of normal mice and diabetic mice GFP-BMMSCsP3generations of GFP-BMMSCs in the logarithmic phase were selected in both groups. GFP-BMMSCs were cultured in osteogenic induction medium for mice and the medium was changed every72h. Alizarin red staining was used to identify calcium formation when GFP-BMMSCs were cultured in osteogenic induction medium for20days. There are two solutions composed of A solution and B solution in the adipogenesis induction medium for mice. First, A solution was used to induced for72h and then maintain in B solution for24h.Oil red O staining was used to evaluate the adipogenesis when GFP-BMMSCs were cultured in adipogenesis induction medium for3cycles.4. Detection of surface antigen in the GFP-BMMSCs from normal mice and diabetic miceP3generations of GFP-BMMSCs in the logarithmic phase were selected in both groups. The MSCs were diluted by PBS to a concentration of5×106cells/ml. Then,200μl of the cell suspension containing1×106cells was added to each tube, and PE-conjugated antibodies against Sca-1、CD14、CD2、CD34、CD45、CD90and CD105was added to each tube. The fluorescence markers of cells were detected by flow cytometry, and PE-conjugated mouse IgG antibody was used as the negative control.5. Groups of experimental animals 60C57BL/6type II diabetes mice were divided into four groups. Construction of6mm diameter circular wound in the same part of the left leg was done in each group. Wounds in the first group were injected with1×106P3-generation BMMSCs from GFP normal mice, and the first group was called as the normal BMMSCs group, n=20, the assay time was Od,2d,7d,14d. Wounds in second group were injected with1×106P3-generation BMMSCs from GFP diabetic mice and the group was named as the diabetic BMMSCs group, n=20, the assay time was Od,2d,7d,14d. Wounds in the third group were injected with the same dose of PBS and the group was called as the PBS group, n=10, the assay time was7d,14d. Wounds in the fourth group did not receive any treatment was called as the blank control group, n=10, the assay time was7d,14d.5samples to be detected at each time point.6. Track the location of GFP-BMMSCs in the woundDue to wounds in the PBS group and the blank control group were not injected with GFP-BMMSCs, we detected the location of GFP-BMMSCs in the wounds only in the normal BMMSCs group and the diabetic BMMSCs group. Fresh wound tissues were took from each group on Od (1h after injection) and2d. Tissues were embedded in OCT then the tissues were performed for frozen sections, and the thickness of slice was6μm. Cell nucleus were fluorescence-labeled by DAPI, each section was observed at340nm and509nm excitation light path by inverted fluorescence microscope and photographed.7. Detection of the VEGF expression level in woundsThe times we detected were7d and14d respectively. Fresh wound tissues were embedded in OCT then performed for the frozen tissues sections, and the thickness of slice was6μm. Tissues were fixed by4%paraformaldehyde for10min,0.5%Triton-X-100to permeabilize the membrane of cells for30min at room temperature, imported goat serum blocking solution for60min at room temperature, diluted mouse-derived VEGF antibodies(1:100) to incubate overnight at4℃. After balanced at room temperature for30min, then the sections were incubated with Cy3-labeled goat anti-mouse IgG (H+L) in the dark at room temperature for2h. Finally, cell nucleus was fluorescence-labeled by DAPI. Each section was observed by inverted fluorescence microscope and photographed, and we utilized Image-Pro Plus6.0software to measure the mean optical density (OD) values.8. Histopathologic examinationThe times we detected were7d and14d. After fixed in10%formalin for24h, the fresh wound tissues were embedded in paraffin. With the traditional paraffin section method, tissues were sliced and the thickness of section was5μm, then stained by HE staining. Finally, tissues was observed under an optical microscope and photographed.9. The rate of wound healingDigital cameras were used to record overall wound healing condition in each group at Od,7d,14d. We utilized Image J software to measure the wound area, and then the wound healing rates in each group7d and14d were calculated.10. Statistical analysisWe used SPSS13.0statistical software to analysis the data in this study. One-Way ANOVA was adopted to compare the values among the four groups, independent samples T-test was performed to compare the values between two groups. The description of data was used as±s, p<0.05was considered as statistically significant difference.Results1. Culture and the ability of osteogenesis and adipogenesis of GFP-BMMSCs in vitroAfter inoculated for24h, BMMSCs isolated from normal GFP mice began to grow adherent. Adherent cells began to increase on the third day, and the adherent cells were increased significantly on the seventh day. The disk-shaped colony forming could be observed and the shapes of cells were mostly like fusiform or spindle. After inoculated for10to12days, gradual integration could be observed between the large clones, and the cells were arranged radially or swirlingly, and the cells covered the entire bottom of the culture flask. Compared with normal GFP-BMMSCs group at the same growth stage, less adherent cells were found in the culture flask on the third day in the diabetic GFP-BMMSCs group, and the state of cell growth was poorer, and the colony forming was decreased.After cultured in osteogenic induction medium for mice for18days, the normal GFP-BMMSCs in the center of nodules fused gradually and lost cell structure, and the formation of calcium nodules could be apparently observed. Alizarin red staining was used to show the calcium nodules after the GFP-BMMSCs cultured in osteogenic induction medium for20days. Calcium nodules were presented as red nodules after stained by alizarin red. It suggested that the cells could differentiate into osteoblasts when cultured in osteogenic induction medium. After cultured in osteogenic induction medium for20days, diabetic GFP-BMMSCs were stained by alizarin red staining, red nodules also could be observed, but the number was less than normal GFP-BMMSCs, and the formation of colony was smaller.After cultured in adipogenic induction medium for mice for12days, the cell shape of normal GFP-BMMSCs became to round and lipid droplets filled the cytoplasm. When the cells were stained by oil red O staining, full sizes red lipid droplets were found intracellular. It indicated that the cells cultured in vitro could differentiate into adipose cells when cultured in adipogenic induction medium. After cultured in adipogenic induction medium for mice for12days, diabetic GFP-BMMSCs were stained by oil red O staining, red lipid droplets also could be observed, but the number of red lipid droplets was less than normal GFP-BMMSCs, and the size of lipid droplets were smaller.2. Detection of surface antigen in GFP-BMMSCs from normal mice and diabetic miceSpecific molecular markers of GFP-BMMSCs surface from normal mice and diabetic mice were detected by flow cytometry. The results showed that CD14, CD34, CD45which were hematopoietic stem cells and lymphocytes markers were both negative in two groups. But Sca-1, CD29, CD90, CD105which were MSCs specific molecule markers were positive in both groups. Statistical analysis of the positive rates in two groups showed that the positive rates of Sca-1, CD29, CD90, CD105in the normal BMMSCs group were94.72±4.82%,96.42±3.79%,97.77±2.86%,94.49±4.85%respectively, and were91.72±5.51%,79.14±7.41%,70.59± 7.26,92.44±4.90%in the diabetes BMMSCs group. Independent samples T-test was adopted to compare the relative expression positive rates between two groups, and the results showed that Sca-1P=0.386, CD29P=0.002, CD90P=0.000, CD105P=0.524, there were significantly difference in CD29and CD90relative expression positive rates between normal mice and diabetic mice GFP-BMMSCs.3. Track the location of GFP-BMMSCs in the woundThere were more GFP-BMMSCs tracked in the wound from the normal BMMSCs group and the diabetic BMMSCs group on Od (after injection lh). And we counted GFP-BMMSCs under the200magnification. There were46.8±8.9cells/per field and45.8±8.1cells/per field in normal BMMSCs group and diabetes BMMSCs group respectively. Independent samples T-test was adopted to analyze the cell number between two groups, and the results showed that there was no significant difference between the two groups (P=0.858). And we also counted the GFP-BMMSCs under the200magnification on2d, there was fewer GFP-BMMSCs left in the wound. The counts of cells were12.2±3.7cells/per field and5.6±2.6/per field in normal BMMSCs group and diabetes BMMSCs group respectively. Independent samples T-test was adopted to analyze the cell number between two groups, and the results showed that there were significant difference between the two groups (P=0.012).4. Detection of the expression level of VEGF in woundsFluorescence microscope was used to detect the expression levels of VEGF in wounds, and the positive expression of VEGF was presented as red fluorescence. On the seventh day, the levels of VEGF expression were both low in the control group and the PBS group, and there was no significant difference between the control group and the PBS group. But there was high expression of VEGF in the normal BMMSCs group. In addition, although the expression level in the diabetic BMMSCs group was obviously higher than the control group and the PBS group, but it was lower than the normal BMMSCs group. On the fourteenth day, the expression level of VEGF was increased in the control group and the PBS group compared with the seventh day, and the positive expression was mainly located in the lower dermis and the distribution was sparse. There was also no significant difference between the two groups. In the normal BMMSCs groups, the distribution of VEGF was the most widely and closed to the epidermis. In the diabetic group, the distribution of VEGF was sparser compared with the normal BMMSCs group, but more than the control group and the PBS group. The mean optical density (OD) values in each group at different times were analyzed with statistical method. The results showed that the OD values in normal BMMSCs group and diabetic BMMSCs group were both higher than the PBS group and the control group (P<0.05). And there were significant differences between the normal BMMSCs group and diabetic BMMSCs group, PBS group, control group (P<0.05).5. Histopathologic examination in woundsHE staining was used to perform histopathologic examination in wounds. On the seventh day, thick dried blood on the skin surface could be both observed obviously in the control group and the PBS group. Inflammatory infiltration of subcutaneous tissues was severe and there were a large number of inflammatory cells in the wound but few formations of collagen fibers. There was less dried blood on the skin surface in the normal BMMSCs group, and the subcutaneous inflammation environment was improved significantly which presented as fewer inflammatory cells and more formation of collagen fibers compared with the control group and the PBS group. But the improvement of subcutaneous inflammation environment and the formation of collagen fibers in the diabetic BMMSCs group were not as obviously as in the normal BMMSCs group. On the fourteenth day, dried blood on the skin surface could also be observed in the control group and the PBS group, the subcutaneous inflammation environment was improved slightly, and there was a small amount of collagen fibers found in the wound. The wounds in the normal BMMSCs group were almost recovered, and the formation of collagen fibers was obvious and thick. In the diabetic BMMSCs group, there still were small amount of inflammatory cells in the wound, and the accumulation of collagen was less than the normal BMMSCs group.6. The rate of wound healingDigital cameras were used to record overall wound healing condition in each group at Od,7d,14d. We utilized Image J software to measure the wound area, and then the7d and14d wound healing rates were calculated in each group. Statistical analysis showed that the rate of wound healing on the seventh day in the control group, the PBS group, the diabetic BMMSCs group and the normal BMMSCs group were54.46±6.27%,52.71±5.51%,67.06±6.94%,78.13±7.38%respectively; and the rate of wound healing on the fourteenth day in four groups were61.21+6.16%,63.61±7.82%,90.31±2.37%,97.46±2.00%respectively. One-Way ANOVA was adopted to compare the rate of wound healing among the four groups. The analysis results suggested that there were no differences between the control group and the PBS group. The groups which had been transplanted BMMSC were significantly better than the control group and PBS group. In addition, the normal BMMSCs group was better than the diabetes BMMSCs group, and there were significant differences between the groups.Conclusions1. Compared with the control group and the PBS group, the effect of BMMSCs on promoting wound healing is obvious.2. The detection of VEGF expression by immunofluorescence assay showed that the expression level of VEGF was increased in BMMSCs transplantation groups. It indicated that the effect of BMMSCs on promoting wound healing may be closely associated with the increasing of angiogenesis in the wounds.3. Compared with the BMMSCs derived from normal mice, the cytoactive of diabetic BMMSCs was declined in vitro, and the ability of differentiation into osteogenic and adipose cell was also deteriorated. Moreover, the effect of BMMSCs derived from diabetic mice on promoting wound healing was declined. |
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