| Background and objective Demineralized bone matrix (DBM) widely used in clinical tissue-engineering strategies has the ability to support human mesenchymal stem cells (hMSCs) proliferation, osteogenic differentiation in vitro and bone-forming in vivo. hMSCs , which is considered as one of the best seed cells in bone tissue engineering, cultured with demineralized bone matrix can be exposed to the soluble bone growth factors to speed up their own proliferation and osteogenic differentiation。Pulsed electromagnetic fields (PEMF) with special biological bone-electrical effect has been used for the treatment of nonunion, delayed union, pseudoarthrosis successfully in orthopedics. PEMF of specific frequencies and field intensity can stimulate osteoblast proliferation, differentiation and may stimulate the production of local bone growth factors to accelerate bone matrix mineralization, which has been confirmed by domestic and foreign scholars. However, the study of PEMF and DBM co-stimulating hMSCs proliferation and osteogenic differentiation has not been reported.This study investigated the effects of PEMF on proliferation and osteogenic differentiation in DBM-induced hMSCs and discussed the underlying mechanism through the observation of alkaline phosphatase (ALP) activity and osteocalcin (OC) level, so as to provide new theoretical basis for the clinical application of PEMF and DBM.Materials and methods1. Femoral heads of patients with femoral neck fracture were collected after hip replacement.2. The femoral head was cut into 3mm×5mm×5mm bone blocks, and made into DBM. The appearance was observed and the pore diameter was measured.3. Human bone marrow was obtained from iliac crest of healthy volunteer with consent. hMSCs were isolated with lymphocyte separation medium (LSM). The passage 3 hMSCs were cultured on glass slide and reacted with rabbit anti-human CD105, CD44 and DAB coloration.4. 1×104 hMSCs per well were plated in 24-well tissue culture plates (8wells per group), and 1×105 hMSCs per well in 6-well plates (2wells per group) after they were expanded until passage 3. Those 24-well plates and 6-well plates were divided into 4 groups, cell control group (C group), cell-material group (CD group), cell-PEMF group (CP group) and cell-material-PEMF group (CDP group). CD group and CDP group were added with one DBM (size of 5 mm×5 mm×3 mm), CP and CDP groups were exposed to the PEMF (frequency of 15 Hz, intensity of 5 Gs).5. The supernatant was collected after culture medium every well was centrifugalizated at 2000 r/min. hMSCs proliferation was detected by MTT colorimetry at 1, 3, 7, 10 and 14d. ALP activity and OC level were tested with the method of ELISA and radioimmunoassay at 1, 7, 14 and 21d.6. Cover glasses in 6-well tissue culture plates for 21days were stained with alizarin red-Tris-Hcl. Histological observation was performed under Olympus microscope, and calcium nodules were counted in microscopic vision randomly.Results1. DBM was yellowish and soft. The pore surface of DBM was smooth and the pore diameter was 331.87μm-596.45μm, average pore diameter was 479.80±91.93μm. The calcium content of DBM was 11.49±0.84%.2. hMSCs isolated from bone marrow were spheric shape. Spindle adherent cells could be seen after 24-hour cultivation. hMSCs arranged like whirlpool when they reached 80% confluence. The membranes of hMSCs were brown when surface antigen CD44, CD105 reacted with the rabbit-anti-human CD44, CD105 and then with DAB.3. According to MTT results, the growth curve's of CD group, CP group and the CDP were antedisplaced compared with C group and the higher proliferation peak of CDP group compared with C group, CD group, and CP group (p <0.01).4. At 1d after planting, ALP activity of all groups had no significant difference. ALP activity of CD group, CP Group and CDP group at 7d after planting was significantly higher (p <0.01), then CP group's began to decline, while at 14d ALP activity of CD group and CDP group reached the highest value. In addition, ALP activity of CDP group at 7d and 14d was significantly higher than the CD group's and CP group's (p <0.01), at 14d the ALP activity of CD group was significantly higher than the CP group's (p <0.01), at 21d interclass ALP activity of CD group, CP Group and CDP group had no significant difference (p <0.01).5. At 7d after planting, OC values in each group were significantly higher than C (p <0.01). There was no significant difference between the CD group and CP group's OC value during the first 14d, while at 21d the OC value of CD group was significantly higher than CP group's (p <0.01). However, OC value of CDP group at 7,14d and 21d was significantly higher than the CD group and CP group (p <0.01).6. At 21d, calcium nodules of alizarin red staining shown in the 40×microscope. The number of calcium nodules of the average field of vision was 0 of C group, 13.50±1.93 of CD group, 9.88±1.55 of CP group, 19.13±3.27 of CDP group , the calcium nodules number of CDP group was significantly more than other 3 groups (p <0.01), CD group significantly more than CP group (p <0.01).Conclusion1. In vitro, the congenerous or separate stimulation of DBM and PEMF all can speed up hMSCs proliferation. In promoting hMSCs to proliferate, DBM is stronger than PEMF the co-stimulation of DBM and PEMF is stronger than their separate stimulation.2. The processes of of hMSCs osteogenic differentiation between CD group and CP group are almost consistent, but the differentiation-promoting role of DBM is stronger; Compared to CD group and CP group, it is not only earlier, but more stronger that DBM and PEMF co-stimulate hMSCs to differentiate.3. The role of DBM is stronger than PEMF in promoting calcium deposition latterly. The intensity is stronger and the duration is longer when DBM and PEMF co-promote hMSCs to differentiate and calcium to deposit.4. PEMF's stimulation on DBM inducing osteogenic differentiation of hMSCs has significant synergistic effect, and exogenous PEMF can be used in bone tissue engineering.
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