Objective To investigate the effect and mechanism of leptin (LEP) on the osteoblastic differentiation of hBMSCs in vitro. Methods Whole bone marrow culture method was appl ied to culture hMBSCs and hBMSCs at passage 3 were divided into groups A, B, C, D, E and F, and when cell attachment was evident, 400, 200, 100 and 50 ng/mL LEP, 100 ng/mLBMP and common nutrient medium were added into each group, respectively. ALP staining and mineral ized nodules staining were conducted at 7 and 21 days after culture, respectively. And inverted phase contrast microscope observation was performed. ALP activity and osteocalcin (OCN) level of hBMSCs in each group was detected at 7, 14 and 21 days after culture to select the best induced concentration of LEP on osteoblastic differentiation. For groups of B, E and F at 7 days after culture, RT-PCR was adopted to detect the expression of such osteogenesis-related genes as core-binding factor α 1 (Cbfα1), ALP, Col I and OCN mRNA. Results At 7 days after induced culture, the ALP staining result showed that the endochylema in groups A, B, C, D and E were stained blue and the endochylema in the group F was sl ightly positive. At 21 days after induced culture, the mineralized nodules staining showed that cells in groups A, B, C, D and E were stained positively and cells in group F were negative. At 7, 14 and 21 days after culture, ALP and OCN activities in group B were less than that of group E (P lt; 0.05), but significant higher than that of groups A, C, D and F (P lt; 0.05), the optimal concentration of LEP was 200 ng/mL. At 7 days after culture, group F witnessed no expression of Cbfα1, ALP, Col I and OCN mRNA, while groups B and E witnessed expressions of all those indexes, but the expressions in group B were less than those of group E. Conclusion LEP can stimulate osteoblastic differentiation of hBMSCs in vitro, and the possible mechanism is its role of promoting the expression of osteoblastic related genes.
ObjectiveTo investigate the feasibility and mechanical properties of polymethyl methacrylate (PMMA) bone cement and allogeneic bone mixture to strengthen sheep vertebrae with osteoporotic compression fracture.MethodsA total of 75 lumbar vertebrae (L1-L5) of adult goats was harvested to prepare the osteoporotic vertebral body model by decalcification. The volume of vertebral body and the weight and bone density before and after decalcification were measured. And the failure strength, failure displacement, and stiffness were tested by using a mechanical tester. Then the vertebral compression fracture models were prepared and divided into 3 groups (n=25). The vertebral bodies were injected with allogeneic bone in group A, PMMA bone cement in group B, and mixture of allogeneic bone and PMMA bone cement in a ratio of 1∶1 in group C. After CT observation of the implant distribution in the vertebral body, the failure strength, failure displacement, and stiffness of the vertebral body were measured again.ResultsThere was no significant difference in weight, bone density, and volume of vertebral bodies before decalcification between groups (P>0.05). After decalcification, there was no significant difference in bone density, decreasing rate, and weight between groups (P>0.05). There were significant differences in vertebral body weight and bone mineral density between pre- and post-decalcification in 3 groups (P<0.05). CT showed that the implants in each group were evenly distributed in the vertebral body with no leakage. Before fracture, the differences in vertebral body failure strength, failure displacement, and stiffness between groups were not significant (P>0.05). After augmentation, the failure displacement of group A was significantly greater than that of groups B and C, and the failure strength and stiffness were less than those of groups B and C, the failure displacement of group C was greater than that of group B, and the failure strength and stiffness were less than those of group B, the differences between groups were significant (P<0.05). Except for the failure strength of group A (P>0.05), the differences in the failure strength, failure displacement, and stiffness before fracture and after augmentation in the other groups were significant (P<0.05).ConclusionThe mixture of allogeneic bone and PMMA bone cement in a ratio of 1∶1 can improve the strength of the vertebral body of sheep osteoporotic compression fractures and restore the initial stiffness of the vertebral body. It has good mechanical properties and can be used as one of the filling materials in percutaneous vertebroplasty.
Objective To study the osteogenic effects of a new type of peptides anchored aminated-poly-D, L-lactide acid (PA/PDLLA) scaffold in repairing femoral defect in rats. Methods The PDLLA scaffolds were treated by ammonia plasma and subsequent anchor of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide linkage formation. Thus PA/PDLLA scaffolds were prepared. The bone marrow was harvested from the femur and tibia of 4 4-week-old Sprague Dawley (SD) rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured by whole bone marrow adherence method. BMSCs-scaffold composites were prepared by seeding osteogenic-induced BMSCs at passages 3-6 on the PA/PDLLA and PDLLA scaffolds. The right femoral defects of 8 mm in length were prepared in 45 adult male SD rats (weighing, 350-500 g) and the rats were divided into 3 groups (n=15) randomly. BMSCs-PA/PDLLA (PA/PDLLA group) or BMSCs-PDLLA (PDLLA group) composites were used to repair defects respectively, while defects were not treated as blank control (blank control group). General state of the rats after operation was observed. At 4, 8, and 12 weeks after operation, general, radiological, histological, micro-CT observations and real-time fluorescent quantitative PCR were performed. Results Two rats died after operation, which was added; the other rats survived to the end of the experiment. At each time point after operation, general and radiological observations showed more quick and obvious restoration in PA/PDLLA group than in PDLLA group; no bone repair was observed in blank control group. The X-ray scores were the highest in PA/PDLLA group, higher in PDLLA group, and the lowest in blank control group; showing significant difference in multiple comparison at the other time (P lt; 0.05) except between blank control group and PDLLA group at 4 weeks (P gt; 0.05). The X-ray scores showed an increasing trend in PDLLA group and PA/PDLLA group with time (P lt; 0.05). Histological and micro-CT observations showed the best osteogenesis in PA/PDLLA group, better in PDLLA group, and worst in blank control group. Comparison between groups had significant differences (P lt; 0.05) in bone mineral density, bone volume/total volume of range of interest, trabecular number, and structure model index. Significant differences (P lt; 0.05) were found in the expression levels of osteogenesis-related genes, such as osteocalcin, alkaline phosphatase, collagen type I, bone morphogenetic protein 2, and osteopontin when compared PA/PDLLA group with the other groups by real-time fluorescent quantitative PCR analysis. Conclusion The PA/PDLLA scaffolds can accelerate the repair of femoral defects in rats.
Objective To study the feasibil ity of preparation of the poly-D, L-lactide acid (PDLLA) scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide l inkage formation. Methods PDLLA scaffolds (8 mm diameter, 1 mm thickness) were prepared by solvent casting/particulate leaching procedure and then treated by ammonia plasma. The consequent scaffolds were labeled as aminated PDLLA (A/ PDLLA). The pore size, porosity, and surface water contact angle of groups 0 (un-treated control), 5, 10, and 20 minutes A/ PDLLA were measured. A/PDLLA scaffolds in groups above were immersed into the FITC labelled GRGDS aqueous solutionwhich contain 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide(NHS), the molar ratio of peptides/EDC.HCL /NHS was 1.5 ∶ 1.5 ∶ 1.0, then brachytely sloshed for 24 hours in roomtemperature. The consequent scaffolds were labelled as peptides conjugated A/PDLLA (PA/PDLLA). The scaffolds in groups 0, 5, 10, and 20 minutes A/PDLLA and groups correspondingly conjugation of peptides were detected using X-ray photoelectron spectroscopy (XPS). The scaffolds in groups of conjugation of peptides were measured by confocal laser scanning microscope and high performance l iquid chromatography (HPLC), un-treated and un-conjugated scaffolds employed as control. Bone marrow mesenchymal stem cells (BMSCs) from SD rats were isolated and cultured by whole bone marrow adherent culture method. BMSCs at the 3rd–6th passages were seeded to the scaffolds as follows: 20 minutes ammonia plasma treatment (group A/PDLLA), 20 minutes ammonia plasma treatment and conjugation of GRGDS (group PA/PDLLA), and untreated PDLLA control (group PDLLA). After 16 hours of culture, the adhesive cells on scaffolds and the adhesive rate were calculated. After 4 and 8 days of culture, the BMSCs/scaffold composites was observed by scanning electron micorscope (SEM). Results No significant difference in pore size and porosity of PDLLA were observed between before and after ammonia plasma treatments (P gt; 0.05). With increased time of ammonia plasma treatment, the water contact angle of A/PDLLA scaffolds surface was decreased, and the hydrophil icity in the treated scaffolds was improved gradually, showing significant differences when these groups were compared with each other (P lt; 0.001). XPS results indicated that element nitrogen appeared on the surface of PDLLA treated by ammonia plasma. With time passing, the peak N1s became more visible, and the ratio of N/C increased more obviously. AfterPDLLA scaffolds treated for 0, 5, 10, and 20 minutes with ammonia plasma and subsequent conjugation of peptides, the ratio of N/C increased and the peak of S2p appeared on the surface. The confocal laser scanning microscope observation showed that the fluorescence intensity of PA/PDLLA scaffolds increased obviously with treatment time. The amount of peptides conjugated for 10 minutes and 20 minutes PA/PDLLA was detected by HPLC successfully, showing significant differences between 10 minutes and 20 minutes groups (P lt; 0.001). However, the amount of peptides conjugated in un-treated control and 0, 5 minutes PA/PDLLA scaffolds was too small to detect. After 16 hours co-culture of BMSCs/scaffolds, the adhesive cells and the adhesive rates of A/PDLLA and PA/PDLLA scaffolds were higher than those of PDLLA scaffolds, showing significant difference between every 2 groups (P lt; 0.01). Also, SEM observation confirmed that BMSCs proliferation in A/PDLLA and PA/PDLLA groups was more detectable than that in PDLLA group, especially in PA/PDLLA group. Conclusion Ammonia plasma treatment will significantly increase the amount of FITC-GRGDS peptides conjugated to surface of PDLLA via amide l inkage formation. This new type of biomimetic bone has stablized bioactivities and has proved to promote the adhesion and proliferation of BMSCs in PDLLA.