| Background:Diabetes mellitus (DM), as a systemic disease, causes endocrine and metabolic disorders due to high blood glucose, including the injury of bone, nerve, kidney and other organs.Diabetic patients showed the relative or absolute lack of insulin, the disorder of mineral tissue metabolism. Diabetes mellitus, has been shown to alter the biomechanical properties of bone and to impair fracture healing in both clinical and experimental settings. Several clinical series have noted that the healing time for diabetic patients is approximately twice as long as that of nondiabetic patients. Although millions of fractures occur annually and the majority heals satisfactorily, 5-10% would go on to delayed union or nonunion.In addition, impaired fracture healing might result in pseudoarthrosis or skeletal deformity that may cause functional disability all throughout the patient's life. How to treat diabetes fracture healing has been paid more and more attention.Human umbilical cord mesenchymal stem cells (hUCMSCs)are sorts of stem cells with the ability of self-renewal and multi-differentiation, such as bone marrow. They can differentiate into, and contribute to the regeneration of, mesenchymal tissues, such as bone, cartilage, ligament, tendon, muscle and adipose tissue. However, autogenous hBMSCs require an invasive procedure to harvest and are limited in cell numbers. Furthermore, hBMSCs have lower self-renewal and proliferative ability due to patient aging and diseases such as osteoporosis and arthritis.Therefore, other sources of stem cells are needed for tissue engineering.Recently, hUCMSCs were derived and shown to differentiate into adipocytes, osteoblasts,chondrocytes,neurons, and endothelial cells. Umbilical cords can provide an inexhaustible and low cost source of stem cells,without the invasive procedure of hBMSCs. In addition, in preliminary studies the hUCMSCs had minimal immunorejection in vivo and were not tumorigenic.These advantages make hUCMSCs a highly desirable stem cell source for tissue regeneration. Therefore, it has broad application prospects in tissue engineering, stem cell transplantation, gene therapy and other fields, and it has become a hot spot in stem cell research. It also provides a new way for the treatment of fracture healing of diabetes.There are many factors that affect fracture healing, including cell proliferation, collagen synthesis, bone growth factor reduction, and so on.Transforming growth factor beta 1 (TGF-?1) and bone morphogenetic proteins-2 (BMP-2) are important regulators of bone repair and regeneration. TGF-?1 enhances the proliferation of mesenchymal cells and osteoblasts in fractures. BMP-2 plays a key role influencing chondrogenesis and osteogenesis. TGF-?1 and BMP-2 are very important factors in the process of bone healing. However, little is known about the expression of TGF-betal and BMP2 in the bone healing processes of the bones in the setting of diabetes. Less is known about Application of human umbilical cord mesenchymal stem cells in the treatment of diabetes fracture healing research.Tibia fracture was created in diabetic rats and control rats. The expression of TGF-?1 and BMP2 in the fractured tibia were measured by immunohistochemistry and qRT-PCR weekly in the first 5 weeks after fracture. Human umbilical cord mesenchymal stem cells were used to induce the differentiation into osteoblasts, and then transplanted to the site of fracture, and the changes of BMP-2 and TGF-?1 were observed in the healing process. We hope the research could provide a theoretical basis for the clinical treatment of diabetic fractures.Objective:1.Culture and osteogenic differentiation of human umbilical cord mesenchymal stem cells.2.To observe the expression of TGF-?1 and BMP-2 in the fractured tibia by immunohistochemistry and qRT-PCR weekly in the first 5 weeks after fracture.3.To observe the effect of human umbilical cord mesenchymal stem cells on fracture healing of diabetic fracture.Methods:1 The Isolation, identification and differentiation of Human umbilical cord mesenchymal stem cells.1.11. Isolation, culture and detection of hUCMSCs Fresh human umbilical cords were obtained after full-term natural births, Umbilical arteries and vein were removed, and the remaining tissue was diced into small fragments. The hUCMSCs were isolated from Wharton's jelly by collagenase digestion. Then hUCMSCs were cultured, proliferated and differentiated in vitro. Cell morphology was observed under inverted microscope.1.2 Immunophenotyping of hUCMSCs by flow cytometryThe third passage of hUC-MSCs BMSCs were harvested and incubated with the following mouse anti-human antibodies:anti-CD34,-CD44,-CD45,-CD105,-CD73,-CD90,-CD14, and major histocompatibility complex class II (MHC II). These antibodies were conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE). FITC-or PE-conjugated IgG1 was used as isotype control. The immune phenotype of hUC-MSCs was identified by flow cytometry within 24 hours.1.3 Evaluation of the osteogenic differentiation and adipogenic differentiation1.3.1 The third hUC-MSCs were incubated and exposed to osteogenic induction medium. The medium was changed every 3 days.1 weeks later, mineralization was detected by alkaline phosphatase calcium-cobalt staining, and Alizarin red staining was conducted After 3 weeks.1.3.2 The third hUC-MSCs were incubated and replaced with adipogenic induction medium. Two weeks later, intracellular lipid accumulation was visualised using Oil-Red-O staining..2. Model of diabetic animal Tibia fracture2.1 Animal modelA total of 75 adult male Wistar rats were used in this study. There were three groups with 25 diabetes rats each of control group, diabetes group and hUCMSCs group.The animals were housed in groups and were fed with standard purina laboratory feed, and were weighed daily for seven days. Diabetes rats was induced by the intraperitoneal injection of alloxan (160 mg/kg). Diabetes was defined as blood glucose levels of more than 16.7 mmol/L determined two days after the alloxan injection. If the desired glucose level was not achieved an additional amount of injection was given to achieve the appropriate level. Control group rats received intraperitoneal injection of placebo and served as controls. Before the surgery, all animals, control group, diabetes group and and hUCMSCs group, were weighed and anesthetized. The tibia fracture was created using a wire saw in the left tibia in all rats. A one-off dose of 10,000 units penicillin was injected three days after the fracture for infection prevention. Fractures were allowed to heal for periods of 5 weeks.2.3 BMD (bone mineral density) assessment5 rats of each group were killed randomly after 1 weeks,2 weeks,3 weeks,4 weeks,5 weeks.Then the tibias were harvested in order to measure the bone mineral density (BMD) with dual energy X-ray absorptiometry (DEXA). The bone density instrument was set as the experimental small animal model. The callus was the center and was scanned from the near to the far about 1cm range. Each sample was measured three times and resetted each time before the measurement.The average value was calculated.2.4 Histomorphometry studiesAt 1,2,3,4,5 weeks of the fracture the rats were sacrificed in each group. Tibia samples(n=5) were extracted each group. Half of each sample was put in liquid nitrogen(storage fo RT-PCR detection) immediately, the other half sample was immediately placed in 10%formalin fixed for 24 hours, decalcification with 10%EDTA, embedded. Samples were cut into 5?m thick bone tissue slices by paraffin section machine. Routine pathological sections were made, and the immunohistochemistry test was carried out according to the specification. Gray scale of TGF-?1 and BMP-2 was recorded by Leica-QwinV3 image analysis software (Germany) automatically.2.5 Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)5 tibia samples each group were homogenized in a liquid nitrogen-cooled mortar. Tissue powders were then processed for RNA extraction using the TriZol method. Using the SuperScriptTM first-strand synthesis system, Total RNA was reverse-transcribed and the products of reverse-transcription were treated with RNase H. Real-time PCR was performed on a real-time PCR detection system. Using Sybr(?) green PCR Master Mix reagents. No-template and no-reverse-transcription reactions were included in each PCR plate as negative controls. GAPDH was used as an internal standard in each PCR plate. The Real-Time PCR System was used to detect real-time PCR. Expression level was normalized against endogenous GAPDHs for related gene expression.Results:1.The isolation of hUCMSCs by enzyme digestion with collagenase was efficient. After seeded for 24 hours, the adherent cells showed spindle shape and fibroblast-like morphology and the size of hUCMSCs was homogeneous. After 9 days, they formed a squamous eddy-like tructure. Flow cytometry analysis revealed that CD44?CD73? CD90 and CD 105 were highly expressed on the surface of passages3 cells, but the expression was negative for D14?CD34?CD45?and MHCII.2.After injected alloxan solution, diabetic rsts appeared polydipsia, polydipsia, hyperdiuresis, hyperglycemia, apositia, sluggishness, loose fur and weight loss. The levels of blood glucose of remaining diabetic rats lasted greater than 16.7mmol/L, The rats whose blood glucose did not reach the standard experiment and died was added in time throughout the experimental process.3.BMD assessment results showed that the measured values were no significantly statistical difference between local bone callus of three groups after first weeks; but in 2,3,4,5 weeks after surgery,Local bone callus density increased with time in diabetes group, but the density value was significantly lower than the control group at the same time, the difference was statistically significant; Local bone callus density of HUCMSCs group was significantly higher (P<0.05) than that of the diabetic group at the 2,3,4,5week, but the difference was not statistically significant (P>0.05) compared with the control group.4.Histology showed that the mature of chondrocyte and osteoblast was delayed in diabetic rats, and the formation of callus was slow.Immunohistochemical staining showed that BMP-2 TGF-?1 was widely distributed in normal rats 1 and 3 weeks after fracture, especially in the callus, whereas much less BMP-2?TGF-?1 was detected in diabetic rats. There was no significant difference between hUCMSCs group and the diabetic group.5.BMP-2 mRNA expression achieved its highest level at 3 week for the diabetes group but reached its highest level at 2 weeks for the control group. TGF-?1 mRNA expression achieved its highest level at 4 week for the diabetes group but reached its highest level at 3 weeks for the control group.Peak TGF-?1 and BMP-2 expression was delayed 1 week in the diabetes group than the control group. There was no significant difference between hUCMSCs group and the diabetic group.Conclusions:1. HUCMSCs were isolated from human umbilical cord by digested with collagenase,and could be cultured and proliferated in vitro.Human umbilical cord mesenchymal stem cells have multiple differentiation capacity, which can be differentiated into osteoblasts and adipocytes.2. The fracture healing rate of diabetic rats was slow, and the healing time was delayed.3. Diabetes reduce bone mineral density of local bone callus, and the callus formation decreaced.4. Diabetes significantly reduced the expression of TGF-beta 1 and BMP-2, and affected the formation of callus. It is one of the reasons for the delayed healing of diabetic fracture.5. After application of human umbilical cord mesenchymal stem cells, The expression of BMP-2 and TGF-beta 1 in diabetic callus enhanced;Besides, bone mineral density increased. which may be one of the molecular mechanisms of stem cell therapy to improve diabetic fracture healing. These may provide theoretical support for clinical treatment of diabetes fracture with HUCMSCs. |
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