Studies On The Biological Characteristics Of Human Umbilical Cord Mesenchymal Stem Cells And Acceleration Of Osteogenesis In It By Pulsed Electromagnetic Field Stimulation

Bone defection or bone fracture delaying to heal which caused by different reasons are a hard work to solution for orthopedics clinical workers. Developments of bone tissue engineering and regenerative medicine bring a new hope for the treatment to those diseases. human umbilical cord mesenchymal stem cells(hUCMSCs )are a promising source of stem cells for regenerative medicine and tissue engineering applications by virtue of their great capacity for self-renewal ,deficiency of immunogenicity and potential for differentiation into cells of various types of tissues, such as bone, cartilage, and adipose.However, the methods of induction bone tissue engineering seed cells to osteoblasts adopted more chemical regents or growth factors inducement currently. The chemical regents or growth factors can't work by stable density in the body. The growth factors in super concentration will intoxicate the cells. Exceed physiology quantity of inducement factors to osteogenesis will result in the heterotopic bone formation.Pulsed electromagnetic field used of electric and magnetic forces to treat disease has fascinated the orthopedics scientists more than 30 years. PEMFs has obtained affirmation effects in many orthopedics diseases such as fractures and osteoporosis.Making the PEMFs to be effective means that induces MSCs to osteoblasts will avoid the use of the chemical inducement that is not physiological. The mesenchymal stem cells are precursor cells of osteoblasts during restoration of bone injury. It has been proved that PEMFs can promote proliferation and differentiation of osteoblasts cell line by means of various frequencies. The biological characteristics of human umbilical cord mesenchymal stem cells are not comprehensive understanding as yet. There is no report on the influence of biological character to hUCMSCs after stimulus by PEMF at present. Whether PEMFs can accelerate differentiation to osteoblasts from hUCMSCs or not is awaiting further study.The task one is to establish a suitable cell growth and expanded of hUCMSCs in vitro conditions, expounded its basal biological characteristics, identification to their adipogenic, osteogenic and Chondrogenic differentiation potential. The task two is to confirm PEMFs accelerating Osteogenesis in hUCMSCs and repairing bone defect with the compound of hUCMSCs and Hydroxyapatite .1,Studies on the basic biological characteristics of hUCMSCsFresh human umbilical cord which were obtained after abortion and stored in balanced salt solution for 1-12hours before tissue processing to isolate cells. After removal of blood vessels, hUCMSCs were isolated off from the Wharton's jelly with collagenase-Ⅱ(0.01%)and hyaluronidase (0.05%) for 12 hours at 37℃. Finally, the cells were washed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum in 5% CO2 in a 37℃incubator.Using flow cytometric analysis, we found that mesenchymal cells isolated from the Wharton's jelly from the umbilical cord express mesenchymal stem cells markers CD29, CD44, CD73, CD105, CD166 but not hematopoietic lineage markers CD34, CD45, and not costimulatory molecules CD40, CD40L, CD80, CD86, HLA-DR is also negative expressed.DNA content analysis showed that cells were normal diploid cells, and cell cycle analysis showed that exponential phase of growth cells were over 80% in G0 to G1period.2,Studies on cell differentiation potency of hUCMSCsCurrently mesenchymal stem cells have not highly recognized specific molecular marker, there are no more separation and cultured standard methods to get mesenchymal stem cells. These experiments aim to prove that the mesenchymal stem cells have the potency to differentiate into 3 cells types included osteocyte, chondrocyte, adipocyte.(1)Adipogenic differentiationhUCMSCs with adipogenic medium resulted in expanded cell morphology and a time-dependent increase in intracellular Oil Red O staining; an established lipid dye. When total RNA was isolated and analyzed by Real-time PCR, the adipocyte marker PPARγwas not expressed in untreated cells but was expressed in adipogenic formula-treated cells.(2)Osteogenic differentiationhUCMSCs with osteogenic medium (10-7mol/L dexame thasone, 50mg/L vitamin C, 10mmol/Lβ-glycerophosphate, 10% FBS , DMEM/F12) were Induced into osteogenic cells. To confirm osteogenesis, cells were examined by Real-time PCR for the expression of osteopontin genes after induction. Using Von Kossa staining and alizarin red staining detected the formation of calcium nodus.(3)Chondrogenic differentiationChondrogenic differentiation of hUCMSCs was achieved by adding the chondrogenic medium with serum-free medium supplemented with ITS+10ng/ml of TGF-β. toluidine blue staining of an aggrecan-rich extracellular matrix was evident. A typeⅡcollagen-rich extracellular matrix was demonstrated by immunohistochemically, and collagenⅡgenes was detected by Real-time PCR.3,Acceleration of Osteogenesis in hUCMSCs by PEMF StimulationThe third generation of hUCMSCs are devided into 4 groups: No.1: The basal medium culture group; No.2 The basal medium culture combine with PEMFs group; No.3: The basal medium culture combine with chemical reagents group; No.4: The chemical reagents combine with PEMFs group. The production of alkaline Phosphatase(ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMFs treatment in both basal and chemical reagents cultures as compared to untreated controls. Furthermore, the expressions of other osteogenic genes, including Runx2 ,collagen-Ⅰand osteopontin were also modulated by PEMFs confirmed by Real-time PCR and western-blot. Our results indicate that extremely advisable frequency PEMFs stimulation may play a modulating role in hUCMSCs osteogenesis.4,PEMFs accelerate repairing bone defect with the compound of hUCMSCs and HydroxyapatiteFirst, we made the animal models of bilateral radius bone defect: we selected 40 New Zealand white rabbits. In the middle of bilateral radius, 1.5 cm bone defects were made. Randomly-selected 40 rabbits from the models of bilateral radius defects were implanted with the compound of hUCMSCs and HA or HA only. PEMFs stimulated the radius defects part 3 hours everyday. In the 2th, 4th, 8th and 12th weeks, all the rabbits had X-ray examination to observe the reparation of bone defect and growth of bone callus.Conclusively, to our knowledge, this is the first study in the literature regarding the following aspects. (a) Isolation of hUCMSCs from the Wharton's jelly which were obtained from abortion fetus in gestational age from 12 to 18 weeks with collagenase-Ⅱand hyaluronidase . (b) PEMFs accelerate Osteogenesis in hUCMSCs and repairing bone defect with the compound of hUCMSCs and Hydroxyapatite .The findings provide insights on the development of PEMFs as an effective technology for bone tissue engineering and regenerative medicine.