Objective As a bioactive material, the osteogenic activity of borate bioglass has been proved. To design a novel borate bioglass according to an improved formula and to investigate the effects of the borate bioglass on osteoblasts invitro for further research and potential cl inical appl ication. Methods The novel Na2O-K2O-MgO-CaO-P2O5-B2O3-SrO borate bioglass was prepared by melting process. The initial and secondary extracts were prepared according to ISO10993-12: 2007 respectively with different extract time of 0-24 hours and 24-48 hours. The osteoblasts (MC3T3-E1) of the 5th-15th passages from mouse were cocultured with the initial (initial extract group) and secondary (secondary extract group) extracts, respectively, to assess the effects of the borate bioglass on the cell prol iferation, protein synthesis, alkal ine phosphatase (ALP) activity, cell apoptosis, and cell migration; while α-MEM medium without addition of extract served as control group. Results The absorbance values at 450 nm were 0.356 0 ± 0.018 7, 0.331 0 ± 0.025 4, and 0.204 0 ± 0.013 8 in initial extract, secondary extract, and control groups, respectively, showing significant differences among 3 groups (P lt; 0.05). The total protein contents were (382.847 ± 9.521), (226.071 ± 5.847), and (220.248 ± 8.213) U in initial extract, secondary extract, and control groups, respectively; there were significant differences between initial extract group and control group, and between initial extract group and secondary group (P lt; 0.05), but there was no significant difference between secondary extract group and control group (P gt; 0.05). However, no significant difference was observed in the ALP activity [(0.013 01 ± 0.000 39), (0.012 93 ± 0.000 44), and (0.012 92 ± 0.000 35) U/ mg], apoptosis rate (7.03% ± 1.95%, 6.46% ± 2.88%, and 6.18% ± 2.21%), horizontal migration [(137.50 ± 11.43), (134.98 ± 10.50), (135.21 ± 8.66) μm], and transmembrane cell number [(10.92 ± 4.99), (10.07 ± 2.50), and (9.81 ± 2.64) cells/ field] among initial extract, secondary extract, and control groups (P gt; 0.05). Conclusion This novel borate bioglass has excellent cytocompatibil ity, which plays regulatory effects on the cell prol iferation, secretion, and migration.
Objective To review and analyze the properties, products, and appl ications of chitosan so as to explore the key molecular structure parameters which can affect the properties and appl ications significantly, and to reveal the relationship between molecular structures and properties so as to provide reference for further development of chitosan industryand scientific research. Methods Based on the collection and analysis of related l iterature, patents and medical productsderived from chitosan, as well as the author’s experiences in research and development, evaluation and standardization of chitosan, the paper was prepared to bring more attentions into the correlativity between structure and properties of chitosan. Results Potential risks in cl inical appl ication of chitosan-based preparations were seriously proposed in addition to a scientific review and analysis on relationships between chitosan structure and properties, as well as the present situations of developments and appl ications of chitosan. Conclusion The molecular structure is the crucial factor that can bring not only positive but also passive effects to the properties and appl ications of chitosan, especially for highly purified chitosan, molecular weight, and deacetylation degree are the most important parameters that should be focused more attention on.
Objective To investigate the effect of various concentration of platelet-rich plasma (PRP) on osteogenic differentiation of rabbit skeletal muscle-derived stem cells (SMSCs) cultured in vitro. Methods Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg (male and female) was used to prepare PRP (Landesberg method). Full blood count and platelet count in PRP were tested. Soleus muscle of right hindl imb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologousPRP (6.25%, 12.50%, 25.00%, 50.00%), and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, al izarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor α1 (Cbfα1) of osteogenic gene expression was tested by RT-PCR. Results The full blood PRP count and the platelet count in PRP was (3.06 ± 0.46) × 105/μL and (18.08 ± 2.10) × 105/μL, respectively. ALP staining: the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity: all the experimental groups were higher than the control group (P lt; 0.05), the experimental group at 12.50% was superior to other experimental groups at each time point (P lt; 0.05). Al izarin red staining: at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineral ization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineral ization rate (P lt; 0.05), the experimental group at 12.50% had a higher mineral ization rate than other experimental groups (P lt; 0.05). Immunofluorescence staining for osteocalcin: at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection: no obvious changes of the gene expression were noted at 4, 12, and 24 hoursafter culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. Conclusion PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.
【Abstract】 Objective To establ ish a animal model of osteonecrosis of femoral head in canine l ike human.Methods The thermal field of canine’s femoral head was three-dimensionally analyzed with fluent 6.2 software so that the best cryosurgery patent could be designed to maximize the osteonecrosis and minimize extra surgery trauma with the cryosurgery system invented by Shanghai Jiaotong University. Liquid nitrogen was pressurized to 0.5 MPa, poured into femoral head for 6.5 minutes, rewarming to 2 for 5 minutes and then repoured into it again for another 6.5 minutes. Ten three-foot canines were conducted as the animal models of osteonecrosis of femoral head according to the method above. At the end of followup,the results were reviewed by radiologic and pathologic check. Two dogs were conducted as control group. Results In the experimental group, one of the ten canines was testified to occur osteonecrosis of femoral head after one week pathologically, cell death and vessel breakage of cavitas medullaris in the femoral head was obvious under microscope; in other nine canines beingstill under follow-up, five with three-month follow-up at least progressed to the collapse of femoral head l ike human (Ficat III). In control group, no osteonecrosis was found. Conclusion Cryosurgery for osteonecrosis of the femoral head in three-foot canine model may become a method to establ ish the animal model of osteonecrosis of femoral head l ike human.
【Abstract】 Objective To measure the changes of bone mineral density and bone micro-architecture of thefemoral head that harvested from the three-foot bearing ethanol destroyed canine model for osteonecrosis of femoral head, and discuss the influences of local injection of ethanol and biomechanical loading to the structural properties of the femoral head. Methods Twenty-four Beagles were divided randomly into four-foot bearing canines and three-foot bearing canines. One fore-l imb was fixed randomly in three-foot bearing canines. Osteonecrosis was induced in all experimental animals by local injection of 5 mL pure ethanol into one side of the femoral head. The hind l imbs injected with NS were acted as control group, that of three-foot canines injected with ethanol were acted as three-foot canine group, and that of four-foot canines injected with ethanol were acted as four-foot canine group. The contralateral femoral head was injected into equal amount of NS. Animals were sacrificed at the time intervals of 1, 3, 6, and 12 weeks after the injection of ethanol. Quantitative microcomputedtomography was used to characterize changes in bone micro-architecture and bone mineral density of femoralhead. Results The clear three-dimensional model of trabecular bone of necrotic femoral head were obtained. There were no significant differences among 3 groups according to the time l ine by 1 week after ethanol injection(P gt; 0.05). At 3 weeks after injection of ethanol, in three-foot canine group and four-foot canine group, the volume of BMC, BMD, BVF, and BS/BV increased gradually as the distance to the drill ing canal increased. There were significant differences between 3 regions (P lt; 0.05). At 6 weeks, in three-foot canine group and four-foot canine group, the volume of BMC, BMD,BVF, and Tb.N of region I and II decreased significantly compared with region III (P lt; 0.05). At 12 weeks, there are no differences among 3 groups (P gt; 0.05). There were significant decreases in BMD values, BVF, BS/BV, Tb.N, Tb.Sp and Tb.Th after the injection of pure ethanol. And, the changes were more and more obvious by the time l ine. These changes were differentiable at 3 weeks after injection of ethanol, and became obvious at 6 weeks. These changes were more obvious at the part that near the injection canal. The changes in threefoot canine group were more obvious than that in four-foot canine group. Conclusion Resorption of necrotic compact bone trabecular may weaken the structural properties of the femoral head. Moreover, remodel ing and repairing process of necrotic bone trabecular may be hampered by constant biomechanical loading that presented in three-foot bearing canines, and thereby further weaken the structural properties of the femoral head. Biomechanical loading may be one of the critical reasons that lead to the collapse of femoral head.
To evaluate the cl inical results of less invasive stabil ization system (LISS) for femur supercondylar and intercondylar fractures. Methods From March 2004 to November 2005, 47 patients with 49 intercondylar and supercondylar fractures were treated. Of all the patients, there were 34 males and 13 females with an average of 39.7 years (range 19-56 years). The locations were left side in 21 cases and right side in 28 cases. Fracture was caused by traffic accident in 31 cases, fall ing in 8 cases, violence in 6 cases and others in 2 cases. Forty-nine fractures included 14 intercondylar fractures, 21supercondylar fractures and 14 intercondylar and supercondylar fractures; 32 closed fractures and 17 open fractures. According to the AO typing, there were 6 type 33-A1, 8 type 33-A2 , 10 type 33-A3, 7 type 33-C1, 3 type 33-C2 and 15 type 33-C3. The disease course was 30 minutes to 6 days. Articular surface reduction was first performed, then the LISS plate was inserted via two incisions and locking screws were used later. Results The average operation time was 126 minutes (range 48-248 minutes). The blood loss was 180 mL(range 60-1 200 mL). The average follow-up time was 18.6 months (range 12-23 months). There were 4 patients with AP angular deformity and 5 patients with lateral angular deformity (range 2-5°). External rotation deformity was presented in 2 patients. There were no plate breakage, screw loosen and fixation failure. Average bone union time was 5.6 months (range 3-8 months) without infection case. Six cases were treated with il iac bone transplantation for delayed union. Conclusion LISS is one kind of effective treatment to femoral intercondylar and supercondylar fractures.
Objective To establ ish an animal model of osteonecrosis of the femoral head (ONFH) l ike human. Methods Ten healthy adult three-leg Beagle male dogs weighing (16.0 ± 1.6) kg were conducted as the animal model of ONFH according to the schedule of cryosurgery designed in advance in which l iquid nitrogen, pressurized to 0.5 MPa, was poured into the femoral head for 16.5 minutes. After rewarmed to 0℃ for 10 minutes, the l iquid nitrogen was repoured into the femoral head for another 16.5 minutes. At the end of the follow-up, the results were reviewed by pathologic check. One dog was conducted as control group. Results The first boundary temperature of (—27.9 ± 4.3)℃ was higher than the second boundary temperature (— 31.3 ± 4.7)℃ by —3.4℃ , and there was significant difference (P lt; 0.01). The diameter of the femoral head of (17.7 ± 1.1) mm was l inearly (^ y= — 2.6 － 2.409 x) correlated to boundary temperature by Pearson analysis, and the R rate was —0.977 (Plt; 0.05). Four dogs in experimental group progressed to collapse of the femoral head l ike human in the 6th month after operation. The rate of the femoral head collapse rose to 44.4%. In the control group, osteonecrosis was never found. Conclusion Cryosurgcry for osteonecrosis of the femoral head in the three-leg canine model may become a method to establ ish an animal model of ONFH l ike human.
Objective To study an optimal ratio of small intestinal submucosa (SIS) and (hydroxyapatite-tricalcium phosphate,HA-TCP,SIS/HA-TCP) compositions according to the effect of SIS/HA-TCP compositions with different ratios on repairing rabbit femoral condyle defect. Methods Thirty-six rabbits were made into bone defect models of 6 mm in diameter and 10 mm in depth in both sides of femoral condyles. Three different ratios of SIS/HA-TCP compositions (w/w: 1, 0.5, 0.25) were implanted into rabbit femoral condyle defect. After 2, 4, 8 and 12 weeks of operation, the repair effect wasobserved grossly. The histological evaluations were performed by histological scoring system and computer imaging analysis system. Results The amount of new bone formation in SIS/HA-TCP(0.5) group was more than that in SIS/HA-TCP(1) and SIS/HA-TCP(0.25) groups. Histological observation: In SIS/HA-TCP(1) group, few new bone formation was seen and bone defect was repaired in the 12th week. In SIS/HA-TCP(0.5) group, immature woven bone was found in the defect in the 2nd week; more immature woven bone appeared and formed trabeculae in the 4th week; the regenerated bone was vigorously growing into the interspaces of the implanted materials in the 8th week; the implanted materials was basically replaced by bony structure and the lamellar bone appeared in the 12thweek. The results of SIS/HA-TCP (0.25) group were similar to that of SIS/HA-TCP(0.5) group. The histological scoring was higher in SIS/HA-TCP(0.5) and SIS／HA-TCP(0.25) groups than that in SIS/HA-TCP(1) group (Plt;0.05) in the 2nd, 4th, 8th, and 12th weeks. The scoring was higher in SIS/HA-TCP(0.5) roup than that in SIS/HA-TCP(0.25) group in the 2nd and 12th weeks(P＜0.05). In new bone formation and the degradation of HA-TCP, SIS/HA-TCP(0.5) and SIS／HA-TCPC(0.25) groups were superior to SIS/HA-TCP(1) group(Plt;0.05), SIS/HA-TCP(0.5) group was superior to SIS/HA-TCP(0.25) group (Plt;0.05). Conclusion SIS/HA-TCP(0.5) has better effects of repairing bone defect and it can be used as a reference ratio in constructing bone scaffolds.
Objective To study the effects of platelet-rich plasma(PRP) on the proliferation and osteogenetic activity of the marrow mesenchymal stem cells(MSCs) cultured in vitro to elucidate the cellular and molecularmechanism by which PRP accelerates bone repair.Methods The human MSCs were cultured in vitro and randomly divided into the experimental group(n=9) and control group(n=9). In the experimental group, the MSCs were interfused with PRP(10 μl/ml culture media). The proliferation ability of the cells was tested by flow cytometry and MTT, the osteogenetic activity by alkaline phosphatase(ALP) measuring and tetracycline fluorometry, and cbfal mRNA expression by reverse transcriptPCR.Results PRP could stimulate the MSCs proliferation. The flow cytometry assay showed that the MSCs proportion of S period of the experimental group significantly increased 14.5±0.4 in comparison with that of the control group 7.2±0.5 (P＜0.01) after 24 hours. MTT value showed that MSCs proliferatedto platform period earlier in the experimental group than in the control group. There was a significant increase in ALP activity of the experimental group 7.79±1.98,9.51±2.31and 14.03±3.02 when compared with that of the control group 2.06±0.77,2.84±0.82 and 2.58±0.84 after 3, 6 and 9 days(P＜0.05). The number of mineral nodes increased. Reverse transcript-PCR showed that the expression of cbfal mRNA were elevated gradually at 2,4 and 8 hours after interfused with PRP.Conclusion The effect of PRP on accelerating bone repair is related to its effects on stimulating the proliferation of MSCs, increasing the cbfal expression and promoting the osteogenetic activity.
Objective To investigate the possibility of constructing eukaryoticexpression vector for human angiopoietin 1（hAng-1），transfecting it to bonemarrow mesenchymal stem cells (MSCs) so as to repair bone defect. Methods The eukaryotic expression vector pcDNA3-hAng-1 was constructed by recombinant DNA technique, transfected into MSCs by liposome DOTAP, and selected with G418. The hAng-1 expression of mRNA and protein was detected by reverse transcript-PCR and Western Blot. Results After the recombinant eukaryotic expressionvector for hAng-1 was digested with Xho-I and BamH-I, electrophoresis revealed 1.4 kb fragment for hAng-1 gene and 5.4 kb fragment for pcDNA3 vector. In the transfected MSCs, the mRNA and protein expression of hAng-1 gene were detected with reverse transcriptPCR and Western Blot. Conclusion The constructed eukaryotic expression vector hAng-1 could be expressed in the transfected MSCs, thus to provide the basis for bone repair with tissue engineering.