Objective To compare the cl inical outcomes of the core decompression combined with autologous bone marrow mesenchymal stem cells (BMSCs) transplantation with the isolated core decompression for the treatment of earlyavascular necrosis of the femoral head (ANFH). Methods From May 2006 to October 2008, 8 patients (16 hips) with earlyANFH were treated. There were 7 males and 1 female with an average age of 35.7 years (range, 19-43 years). According to the system of the Association Research Circulation Osseous (ARCO): 4 hips were classified as stage II a, 2 as stage II b, 1 as stage II c, and 1 as stage III a in group A; 2 hips were classified as stage II a, 2 as stage II b, 3 as stage II c, and 1 as stage III a in group B. The average disease course was 1.1 years (range, 4 months to 2 years). The patients were randomly divided into 2 groups according to left or right side: group A, only the core decompression was used; group B, both the core decompression and autologous BMSCs transplantation were used. The Harris score and visual analogue scale (VAS) score were determined, imaging evaluation was carried out by X-rays and MRI pre- and post-operatively. The erythrocyte sedimentation rate, C-reactive protein, l iver function, renal function, and immunoglobul in were detected for safety evaluation. Results All incisions healed by first intention. Eight patients were followed up 12-42 months (23.5 months on average). The cl inical symptoms of pain and claudication were gradually improved. The Harris scores and VAS scores of all patients were increased significantly at 3, 6, and 12 months after operation (P lt; 0.05). There was no significant difference between groups A and B 3 and 6 months after operation (P gt; 0.05), but there was significant difference between groups A and B 12 months after operation (P lt; 0.05). The necrosis area of femoral head in groups A and B were 18.13% ± 2.59% and 13.25% ± 2.12%, respectively, showing significant difference (P lt; 0.05). In group A, femoral head collapsed 12 months after operation in 1 case of stage III. No compl ication of fever, local infectionoccurred. Conclusion The core decompression and the core decompression combined with BMSCs transplantation are both effective for the treatment of early ANFH. The core decompression combined with BMSCs transplantation is better than core decompression in the rel ief of pain and postponing head collapse.
ObjectiveTo investigate whether desferrioxamine (DFO) can enhance the homing of bone marrow mesenchymal stem cells (BMSCs) and improve neovascularization in random flaps of rats.MethodsBMSCs and fibroblasts (FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×106 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×106 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO [100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation. Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1 (SDF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues.ResultsThe necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation (P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries, capillaries, and hair follicles at 7 days after transplantation.ConclusionDFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.
ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
ObjectiveTo investigate the effect of microencapsulated transgenic bone marrow mesenchymal stem cells (BMSCs) transplantation on early steroid induced osteonecrosis of femoral head (SONFH) in rabbits.MethodsAlginate poly-L-lysine-sodium alginate (APA) microencapsulated transgenic BMSCs with high expression of Foxc2 were prepared by high-voltage electrostatic method. Part of the cells were cultured in osteoblasts and observed by alizarin red staining at 2 and 3 weeks. Forty New Zealand white rabbits were used to prepare SONFH models by using hormone and endotoxin. Thirty two rabbits who were successful modeling were screened out by MRI and randomly divided into 4 groups (groups A, B, C and D, n=8); another 6 normal rabbits were taken as normal control (group E). The rabbits in group A did not receive any treatment; and in groups B, C, and D were injected with normal saline, allogeneic BMSCs, and APA microencapsulated transgenic BMSCs respectively after core decompression. At 6 and 12 weeks after operation, specimens of femoral head were taken for HE staining to observe bone ingrowth; the expressions of osteocalcin (OCN), peroxisome proliferative activated receptor γ 2 (PPARγ-2), and vascular endothelial growth factor (VEGF) proteins were observed by immunohistochemistry staining. At 12 weeks after operation, the bone microstructure was observed by transmission electron microscope, and the maximum compressive strength and average elastic modulus of cancellous bone and subchondral bone were measured by biomechanics.ResultsAfter 2 and 3 weeks of induction culture, alizarin red staining showed the formation of calcium nodules, and the number of calcium nodules increased at 3 weeks when compared with 2 weeks. The rabbits in each group survived until the experiment was completed. Compared with groups A, B, and C, the trabeculae of group D were more orderly, the empty bone lacunae were less, there were abundant functional organelles, and obvious osteogenesis was observed, and the necrotic area was completely repaired at 12 weeks. Immunohistochemical staining showed that, at 6 and 12 weeks after operation, the expressions of OCN and VEGF in groups A, B, and C were significantly lower than those in groups D and E, while those in groups B and C were significantly higher than those in group A, and in group E than in group D (P<0.05). The expression of PPARγ-2 was significantly higher in groups A, B, and C than in groups D and E, and in group A than in groups B and C, and in group D than in group E (P<0.05). At 12 weeks after operation, biomechanical test showed that the average elastic modulus and maximum compressive strength of cancellous bone and subchondral bone in groups D and E were significantly higher than those in groups A, B, and C (P<0.05); there was no significant difference between groups A, B, and C and between groups D and E (P>0.05).ConclusionIn vivo transplantation of microencapsulated transgenic BMSCs can repair early SONFH in rabbits.
This study aimed to comprehensively evaluate the biological activity in different passage populations of mesenchymal stem cells (BMSCs) derived from bone marrow in ovariectomy osteoporotic rats (named OVX-rBMSCs), providing experimental basis for new osteoporotic drug development and research. OVX-rBMSCs were isolated and cultured in vitro by the whole bone marrow adherent screening method. The morphological observation, cell surface markers (CD29, CD45, CD90) detection, cell proliferation, induced differentiation experimental detection were performed to evaluate the biological activity of Passage 1, 2, 3, 4 populations (P1, P2, P3, P4) OVX-rBMSCs. The results showed that whole bone marrow adherent culture method isolated and differentially subcultured OVX-The morphology of P4 OVX-rBMSCs was identical fibroblast-like and had the characteristics of ultrastructure of stem cells. The CD29 positive cells rate, CD90 positive cells rate, cell proliferation index, and the osteogenic, adipogenic, chondrogenic differentiation capacities of P4 OVX-rBMSCs were significantly better than those of other populations (P < 0.05). OVX-rBMSCs purity and biological activity were gradually optimized with the passaged, and among them P4 cells were superior to all the other populations. Based on these results, we report that the P4 OVX-rBMSCs model developed in this study can be used to develop a new and effective medical method for osteoporotic drug screening.
Objective To investigate the ability of gene-loaded lipopolysaccharide-amine nanopolymersomes (LNPs) in inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by in vitro gene transfection, where LNPs were used as a non-viral cationic carrier, and their properties were optimized during synthesis. Methods LNPs were synthesized by a graft-copolymerization method, and the effects of different pH environments during synthesis on physicochemical properties of LNPs and LNPs/plasmid of bone morphogenetic protein 2-green fluorescent protein (pBMP-2-GFP) complexes were explored. Then, optimized LNPs with maximum transfection efficiency and safe cytotoxicity in rat BMSCs were identified by cytotoxicity and transfection experiments in vitro. Thereafter, the optimized LNPs were used to mediate pBMP-2-GFP to transfect rat BMSCs, and the influences of LNPs/pBMP-2-GFP on osteogenic differentiation of BMSCs were evaluated by monitoring the cell morphology, concentration of BMP-2 protein, activity of alkaline phosphatase (ALP), and the formation of calcium nodules. Results The nitrogen content, particle size, and zeta potential of LNPs synthesized at pH 8.5 were lower than those of the other pH groups, with the lowest cytotoxicity (96.5%±1.4%) and the highest transfection efficiency (98.8%±0.1%). After transfection treatment, within the first 4 days, BMSCs treated by LNPs/pBMP-2-GFP expressed BMP-2 protein significantly higher than that treated by Lipofectamine2000 (Lipo)/pBMP-2-GFP, polyethylenimine 25K/pBMP-2-GFP, and the blank (non-treated). At 14 days after transfection, ALP activity in BMSCs treated by LNPs/pBMP-2-GFP was higher than that treated by Lipo/pBMP-2-GFP and the blank, comparable to that induced by osteogenic medium; with alizarin red staining, visible calcium nodules were found in BMSCs treated by LNPs/pBMP-2-GFP or osteogenic medium, but absent in BMSCs treated by Lipo/pBMP-2-GFP or the blank with apoptosis. At 21 days after transfection, transparent massive nodules were discovered in BMSCs treated by LNPs/pBMP-2-GFP, and BMSCs exhibited the morphologic features of osteoblasts. Conclusion LNPs synthesized at pH 8.5 has optimal transfection efficiency and cytotoxicity, they can efficiently mediate pBMP-2-GFP to transfect BMSCs, and successfully induce their directional osteogenic differentiation, whose inducing effect is comparable to the osteogenic medium. The results suggest that gene transfection mediated by LNPs may be a convenient and effective strategy in inducing directional differentiation of stem cells.
ObjectiveTo prepare dopamine modified and cartilage derived morphogenetic protein 1 (CDMP1) laden polycaprolactone-hydroxyapatite (PCL-HA) composite scaffolds by three-dimensional (3D) printing and evaluate the effect of 3D scaffolds on in vitro chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsA dimensional porous PCL-HA scaffold was fabricated by 3D printing. Dopamine was used to modify the surface of PCL-HA and then CDMP-1 was loaded into scaffolds. The surface microstructure was observed by scanning electron microscope (SEM) and porosity and water static contact angle were also detected. The cytological experiment in vitro were randomly divided into 3 groups: group A (PCL-HA scaffolds), group B (dopamine modified PCL-HA scaffolds), and group C (dopamine modified and CDMP-1 laden PCL-HA scaffolds). The hBMSCs were seeded into three scaffolds, in chondrogenic culture conditions, the cell adhesive rate, the cell proliferation (MTT assay), and cell activity (Live-Dead staining) were analyzed; and the gene expressions of collagen type Ⅱ and Aggrecan were detected by real-time fluorescent quantitative PCR.ResultsThe scaffolds in 3 groups were all showed a cross-linked and pore interconnected with pore size of 400–500 μm, porosity of 56%, and fiber orientation of 0°/90°. For dopamine modification, the scaffolds in groups B and C were dark brown while in group A was white. Similarly, water static contact angle was from 76° of group A to 0° of groups B and C. After cultured for 24 hours, the cell adhesion rate of groups A, B, and C was 34.3%±3.5%, 48.3%±1.5%, and 57.4%±2.5% respectively, showing significant differences between groups (P<0.05). Live/Dead staining showed good cell activity of cells in 3 groups. MTT test showed that hBMSCs proliferated well in 3 groups and the absorbance (A) value was increased with time. The A value in group C was significantly higher than that in groups B and A, and in group B than in group A after cultured for 4, 7, 14, and 21 days, all showing significant differences (P<0.05). The mRNA relative expression of collagen type Ⅱ and Aggrecan increased gradually with time in 3 groups. The mRNA relative expression of collagen type Ⅱafter cultured for 7, 14, and 21 days, and the mRNA relative expression of Aggrecan after cultured for 14 and 21 days in group C were significantly higher than those in groups A and B, and in group B than in group A, all showing significant differences (P<0.05).ConclusionCo-culture of dopamine modified and CDMP1 laden PCL-HA scaffolds and hBMSCs in vitro can promote hBMSCs’ adhesion, proliferation, and chondrogenic differentiation.
Objective To review the research progress of exosomes (EXOs) derived from different cells in the treatment of osteoporosis (OP). Methods Recent relevant literature about EXOs for OP therapy was extensively reviewed. And the related mechanism and clinical application prospect of EXOs derived from different cells in OP therapy were summarized and analyzed. Results EXOs derived from various cells, including bone marrow mesenchymal stem cells, osteoblasts, osteoclasts, osteocytes, and endothelial cells, et al, can participate in many links in the process of bone remodeling, and their mechanisms involve the regulation of proliferation and differentiation of bone-related cells, the promotion of vascular regeneration and immune regulation, and the suppression of inflammatory reactions. A variety of bioactive substances contained in EXOs are the basis of regulating the process of bone remodeling, and the combination of genetic engineering technology and EXOs-based drug delivery can further improve the therapeutic effect of OP. Conclusion EXOs derived from different cells have great therapeutic effects on OP, and have the advantages of low immunogenicity, high stability, strong targeting ability, and easy storage. EXOs has broad clinical application prospects and is expected to become a new strategy for OP treatment.
ObjectiveTo explore the effects of concentrated growth factor (CGF) combined with mineralized collagen (MC) materials on the adhesion, proliferation, and differentiation of bone marrow mesenchymal stem cells (BMSCs) and their osteogenic effects in vivo, and to provide a theoretical basis for the combined application of CGF and MC materials in bone defect regeneration and repair.MethodsCGF was prepared from venous blood of healthy volunteers, and then CGF extracts (CGFe) were prepared. In vitro experiment: human BMSCs (hBMSCs) were divided into 4 groups. Groups A, B, and C were cultured with α-MEM medium [containing 10% fetal bovine serum (FBS) and 1% double antibody] containing 2%, 5%, and 10%CGFe, respectively; group D was cultured with α-MEM medium (containing 10%FBS and 1% double antibody) without CGFe. Scanning electron microscopy was used to observe the effect of CGFe on cell adhesion. Cell counting kit 8 (CCK-8) was used to detect the effect of CGFe on cell proliferation. After osteogenic induction, alkaline phosphatase (ALP) activity was detected and Western blot was performed to detect osteopontin (OPN) expression. In vivo experiment: Eighteen New Zealand big-eared rabbits were used to prepare circular bone defect models on the left and right mandibles, and implant CGF gel (prepared from autologous venous blood)+MC material (volume ratio 1∶1, experimental group) and simple MC material (control group), respectively. At 4, 8, and 12 weeks after operation, 6 rabbits were sacrificed respectively to obtain materials, and Micro-CT scanning was performed to observe the formation of new bone and material degradation in vivo.ResultsIn vitro experiments: Scanning electron microscopy showed that the cells of groups A, B, and C spread better on MC materials than group D, with more pseudopodia. CCK-8 method showed that different concentrations of CGFe could promote cell proliferation, and the absorbance (A) value of cells cultured for 2, 3, 5, and 7 days was in the order of group C>group B>group A>group D, the differences were significant (P<0.05). ALP activity test showed that its activity was proportional to the osteogenic induction time and CGFe concentration (P<0.05). Western blot analysis of osteogenic induction culture for 14 days showed that the relative expression of OPN protein in groups A, B, and C was significantly higher than that in group D, and the higher the CGFe concentration, the higher the relative expression of OPN protein (P<0.05). In vivo experiment: Micro-CT observation showed that the new bone formation and material degradation of the experimental group were better than those of the control group at 4, 8, and 12 weeks after operation. Quantitative detection showed that the volume of new bone volume, new bone volume fraction, trabeculae number, and trabecular thickness of the experimental group were significantly higher than those of the control group at each time point, the residual material volume, residual material volume fraction, and trabecular separation were significantly lower than those of the control group, all showing significant differences (P<0.05).ConclusionCGF can effectively promote the adhesion, proliferation, and osteogenic differentiation of BMSCs on MC materials, and 10%CGFe has the most significant effect. The combined application of CGF and MC material can significantly promote bone formation in vivo.
Objective To investigate the effect of solid lipid nanoparticles (SLNs) on enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro by resveratrol (Res), and provide a method for the treatment of bone homeostasis disorders. MethodsRes-SLNs were prepared by high-temperature emulsification and low-temperature solidification method, and then the 2nd-3rd generation BMSCs from Sprague Dawley rat were co-cultured with different concentrations (0, 0.1, 1, 5, 10, 20 μmol/L) of Res and Res-SLNs. The effects of Res and Res-SLNs on the cell viability of BMSCs were detected by cell counting kit 8 (CCK-8) and live/dead cell staining; the effects of Res and Res-SLNs on the osteogenic differentiation of BMSCs were detected by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining after osteogenic differentiation induction, and the optimal concentration of Res-SLNs for gene detection was determined. Anti-osteocalcin (OCN) immunofluorescence staining and real-time fluorescent quantitative PCR (RT-qPCR) were used to detect the effect of Res and Res-SLNs on osteoblast-related genes (ALP and OCN) of BMSCs. ResultsLive/dead cell staining showed that there was no significant difference in the number of dead cells between Res and Res-SLNs groups; CCK-8 detection showed that the activity of BMSCs in Res group was significantly reduced at the concentration of 20 μmol/L (P<0.05), while Res-SLNs activity was not affected by Res concentration (P>0.05). After osteogenic differentiation, the staining intensity of ALP and ARS in both groups was dose-dependent. The percentage of ALP positive staining area and the percentage of mineralized nodule area in Res group and Res-SLNs group reached the maximum at the concentrations of 10 μmol/L and 1 μmol/L, respectively (P<0.05), and then decreased gradually; the most effective concentration of Res-SLNs was 1 μmol/L. The expression of OCN and the relative expression of ALP and OCN mRNA in Res-SLNs group were significantly higher than those in Res group (P<0.05). ConclusionEncapsulation of SLNs can improve the effect of Res on promoting osteogenesis, and achieve the best effect of osteogenic differentiation of BMSCs at a lower concentration, which is expected to be used in the treatment of bone homeostasis imbalance diseases.