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Objective:To prepare a progressive gradient-aperture scaffold composed of silk fibroin(SF)-chitosan(CS)-nano-hydroxyapatite(nHAp)for osteochondral repair.Method:The SF solution, CS solution and nHA suspension were mixed in vitro at equal proportions.The progressive gradient osteochondral(OC)scaffold-1(2%), scaffold-2(3%)and scaffold-3(4%)was respectively prepared by using centrifugation, vacuum freeze-drying, chemical cross-linking and three shaping steps.General conditions, porosity, hot water dissolution rate, water swelling rate, compression water swelling rate, water swelling rate after dissolution, mechanical properties, internal structure observation and pore size were measured.Rat bone marrow mesenchymal stem cells(BMSCs)were cultured and the scaffold extract was prepared.The effect of scaffold extract on the proliferation of BMSCs was detected by the cell counting kit-8(CCK-8)method.BMSCs were co-cultured with the scaffold, and the distribution and morphology of the cells around the scaffold were observed.Results:The structure of scaffold was regular in each group and the porosity was more than 80%.Along with the increase of the material concentration, the water swelling rate of the scaffold was decreased gradually( P<0.05). Compared with before compression, the water swelling rate of scaffold-1 was decreased after compression( P<0.05). There was no significant difference in the hot water dissolution rate among all groups( all P>0.05), and the complete dissolution of the scaffold-1, scaffold-2 and scaffold-3 in vitro required 65.9, 60.9, and 73.9weeks, respectively.The elastic modulus of scaffolds in above three groups were 0.0955, 0.1762 and 0.3468 MPa, respectively.The examination results of scanning electron microscope(SEM)showed that the internal structure of scaffold was honeycomb in each group, the pore shape was regular, which showed an inter-connected pore network.The pore distribution was gradually dense and the pore diameter gradually decreased from the cartilage side to the osteogenic side( P<0.05), and the nHAp content increased gradually.The scaffold extract had no obvious toxicity to the growth and proliferation of BMSCs in each group.After BMSCs were seeded on scaffolds and co-cultured for 5 days, the cells grew well without obvious cell death or morphological abnormalities. Conclusions:In this study, a progressive gradient pore size OC scaffold is successfully prepared with good physical properties and biocompatibility, which is expected to be a new bio-mimetic composite scaffold material for repairing OC defects.
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Objective To explore the feasibility and methods of preparing silk fibroin(SF)/ chitosan(CS)/nano hydroxyapatite(nHA)composite osteochondral scaffolds with a gradient pore size structure.Methods We prepared an SF solution,a CS solution and an nHA suspension,all with a 2％ concentration,and mixed them with equal proportions.The mixture was used to prepare SF/CS/nHA composite osteochondral scaffolds with a pore size gradient through a centrifugal freeze drying and chemical cross linking method.The porosity,hot water loss rate,water swelling rate and mechanical properties of the scaffold were measured,and the dissolution curve and stress strain curve were drawn.The internal structure and morphology of the scaffold were observed by scanning electron microscopy(SEM)and the sizes of pores in the scaffold were measured.Results The porosity of the scaffold was(91.30± 3.35)％;The five week hot water loss rate was(16.57± 3.18)％;And the water swelling rate was (3218.53 ± 84.37)％.Mechanical test results demonstrated a good compression performance of the scaffold.SEM showed that the internal pores of the scaffolds were honeycomb structured with communicating passages;The density of pores gradually increased with decreasing pore sizes from the top to the bottom(pore sizes at four different levels:(141.11± 11.85)μm,(119.94± 9.05)μm,(93.10 ± 14.98) μm,and (79.95 ± 8.65)μm,respectively,F =22.973,P =0.000).Scaffold cytotoxicity test results indicated no significant difference between A values of the extract group and of the negative control group at any time point(t24 h =0.520,P =0.610;t48 h =0.665,P =0.515;t72 h =0.439,P =0.666),and all RGR values were greater than 100％.Conclusions SF/CS/nHA composite osteochondral scaffolds with a gradient pore size structure can be prepared with a centrifugal-freeze drying and chemical cross-linking method.Scaffolds prepared this way have a three-dimensional structure,a gradient pore size structure,high porosity,strong water absorption,suitable degradation rates and good compressive resistance.Besides,the good cell compatibility and low cytotoxicity of the scaffolds satisfy the requirements for osteochondral tissue engineering materials.
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BACKGROUND:Bone morphogenetic protein-2 (BMP-2) is a key to bone formation and repair.However,it has some disadvantages such as easy to lose and degrade and difficult to sustain continuous effect.OBJECTIVE:To study the preparation and properties of silk fibroin/chitosan/nano-hydroxyapatite (SF/CS/nHA) scaffold loading BMP-2.METHODS:After silk degumming,dissolution and purification,2％ SF solution was obtained.BMP-2 was dissolved in 2％ CS solution,and then fully mixed with equal volume of SF solution and proper amount of nHA.At last,the SF/CS/nHA scaffold loading BMP-2 was prepared using freeze-drying method as experimental group.The SF/CS/nHA scaffold was soaked in the BMP-2 solution as control group.The scaffold porosity was measured by Archimedes method,the surface morphology of the scaffold was observed by scanning electron microscope,the compressive strength was measured by universal testing machine.Scaffolds in the two groups were soaked in PBS,and the release of BMP-2 was measured by ELISA method at different time points.RESULTS AND CONCLUSION:(1) The scaffolds in the two groups had irregular porous structure,interconnected pores and uneven pore wall.There was no significant difference between the two groups in mean pore diameter,porosity and maximum compressive strength.(2) On the 1st day,the release rate of BMP-2 was 4.63％ in the experimental group,and the release curve increased slowly.After 28 days,the release curve of BMP-2 was transferred to the plateau stage.But in the control group,the release rate of BMP-2 on the 1stday was 58.84％,and it was a significant initial burst release.The release curve increased rapidly,and was transferred to the platform stage on the 10th day.The release rate of BMP-2 release was significantly different between the two groups at days 1,2,4,10 (P ＜ 0.05).These results show that the SF/CS/nHA scaffold loading BMP-2 could sustainably and slowly release BMP-2.
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Objective To investigate the influence of recombinant human bone morphogenetic protein‐2(rhBMP‐2)of poly lactic acid(PLA) release microspheres for compatibility of rabbit bone marrow mesenchymal stem cells(BMSCs) .Methods The rh‐BMP‐2‐PLA release microspheres were prepared by w/o/w multiple emulsion volatilizing method and then cocultured BMSCs .The effects of rhBM P‐2‐PLA release microspheres on the cytotoxicity and relative proliferation rate by MTTassay .Evaluation of mate‐rials biocompatibility by inverted microscope and scanning electron microscope(SEM) .Results The rhBMP‐2‐PLA release micro‐spheres in various concentration of leaching solution and BMSCs training of uninfected cells .Experimental group and control group in 4 different time cell proliferation OD values by analysis of repeated measurement variance between time OD values were statisti‐cally significant(P=0 .000) ,the experimental group and control group OD values are statistically significant(P=0 .025) ,the exper‐imental group higher than the control group ,experimental group OD value time there was a significant interaction effect and the group number ,the change trend are obviously different(P=0 .006) .Inverted microscope to observe materials normal cell prolifera‐tion ,SEM found that vaccination cells surrounding rhBMP‐2‐PLA release microspheres of 7 days later ,the cells grew well and split proliferation activity .Conclusion rhBMP‐2‐PLA release microspheres of BMSCs has non‐toxic and has compatibility .
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BACKGROUND:The low oxygen environment after femoral fracture and cerebral trauma wil induce series of related cytokines expression, including hypoxia-inducible factor 1αand core binding factorα1, which play key roles in regulating bone healing. However, whether the accelerated bone healing is correlated with the expression of hypoxia-inducible factor 1αand core binding factorα1 is stil unknown. OBJECTIVE:To construct rat models of brain injury, to compare the expression level of hypoxia-inducible factor 1αand core binding factorα1 in femoral fracture combined with cerebral trauma rats and simple femoral fracture rats, and to assess the influence of cerebral trauma on bone healing. METHODS:Rats were randomly divided into blank group, simple femoral fracture group and femoral fracture combined with cerebral trauma group. At 1, 2, 3 and 5 weeks after modeling, rats were executed. Bone healing was evaluated using femoral fracture end X-ray score and hematoxylin and eosin staining at cal us tissues. Besides, the expression levels of hypoxia-inducible factor 1αand core binding factorα1 of three groups were determined with immunohistochemistry. RESULTS AND CONCLUSION:Bone healing in the femoral fracture combined with cerebral trauma group was better than that of simple femoral fracture group. There was significant difference in the expression level of hypoxia-inducible factor 1αand core binding factorα1 between the simple femoral fracture group and femoral fracture combined with cerebral trauma group (P<0.05). At the same time, the level of simple femoral fracture group and femoral fracture combined with cerebral trauma group was significantly higher than that of blank group, and that in femoral fracture combined with cerebral trauma group was significantly higher than that of simple femoral fracture group (P<0.05). Results verified that the expression levels of hypoxia-inducible factor 1αand core binding factorα1 of rats with femoral fracture combined with cerebral trauma were significantly high, which may be the major reason why the bone healing was accelerated after fracture combined with brain injury.
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BACKGROUND:Poly lactic acid as an excellent delivery has good biocompatibility. OBJECTIVE:To prepare recombinant human bone morphogenetic protein-2 (rhBMP-2)/poly lactic acid (PLA) sustained release microspheres, and to study its physical and chemical properties. METHODS:The rhBMP-2/PLA sustained release microspheres were prepared using w/o/w solvent evaporation method. Scanning electron microscopy, laser particle size, zeta potential, and swel ing properties were detected. ELISA kit was utilized for measurement of encapsulation efficiency, drug-loading rate and in vitro drug release rate. RESULTS AND CONCLUSION:Under the scanning electron microscope, rhBMP-2/PLA sustained release microspheres were approximately circle with excellent dispersion. The uniform spheres were visible with a mean particle size of 839.6 nm. The zeta potential were (-32.93±3.74) mV. The swel ing coefficient was 1.157±0.059. The drug-loading rate and encapsulation efficiency of rhBMP-2/PLA sustained release microspheres were (88.943±2.878)%and (0.026±0.001)%respectively. The drug release rate at 1 day was about 10.199%, then the drug release was relatively constant, and til 19 days, the cumulative drug release rate was 54.643%. These findings indicate that the constructed rhBMP-2/PLA sustained release microspheres meet the requirement of the Chinese Pharmacopoeia (10th edition) that the encapsulation efficiency is not less than 80%and the microspheres have a good slow-release function in vitro.
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BACKGROUND:Silk fibroin/chitosan/nano hydroxyapatite (SF/CS/nHA) composite scaffold constructed in preliminary experiments has good physical and chemical properties. OBJECTIVE:To study the capacity and mechanism of SF/CS/nHA composite scaffold for repair of rabbit radial bone defects. METHODS:Thirty-six New Zealand white rabbits were selected to make animal models of right radial bone defects, and then randomly divided into SF/CS/nHA group, SF/CS group and blank control group. Blank control group had no treatment after modeling. X-ray radiography, gross observation and histopathological observation were performed at 4, 8, 12, 16 weeks postoperatively. RESULTS AND CONCLUSION:Sixteen weeks after surgery, bone defects in the SF/CS/nHA group were completed replaced by normal bone tissue on X-ray images, and the bone marrow cavity showed complete recanalization with new bone formation;hematoxylin-eosin staining showed bone trabecula and many fusiform bone cells. In the SF/CS group, the bone mineral density in the defect area was slightly lower than that of the normal bone tissues, the bone marrow cavity was partly rehabilitated, and many chondrocytes were seen around bone cells that arranged irregularly with no bone trabecula or bone lamel a. In the blank control group, the images of bone calcification were consistent with normal bone tissues, and a closed bone ununion was formed at each end;hematoxylin-eosin staining showed that the blank control group was fil ed by fibrous connective tissue and a smal amount of bone-like tissues. SF/CS/nHA composite scaffold is better for repair of rabbit radial bone defects.
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BACKGROUND:Silk fibroin, chitosan, and nano hydroxyapatite are natural materials, and they al have good biological activity and physical or chemical properties. As tissue engineering materials, they have been already widely used in clinic or research work, but there are some defects in the application of these three kinds of materials. OBJECTIVE:To discuss the preparation and characteristics of silk fibroin/chitosan/nano hydroxyapatite complicated scaffolds which could be used in bone tissue engineering. METHODS:Silk fibroin, chitosan, and nano hydroxyapatite were separately prepared into 2%solution, and then mixed at the ratio of 1:1:0.5, 1:1:1, 1:1:1.5 respectively. The three-dimensional complicated scaffolds were prepared by those mixed liquids through repeated freeze drying and chemical crosslinking technology. Scanning electron microscope was used to detect the pore size of the scaffolds. Porosity, water absorption rate, and hot-water loss rate were determined. Mechanical tester was used to measure the tensile and compressive modulus of dried three-dimensional scaffolds. RESULTS AND CONCLUSION:The silk fibroin/chitosan/nano hydroxyapatite complicated scaffold in the dry state had no special smel , appeared to be a stabilized solid cylinder, and exhibited clear resiliency and flexibility with a touch. With the increased content of nano hydroxyapatite, the porosity, water absorption rate and average pore size of the scaffolds appeared to be decreased, while the hot-water loss rate and compressive strength were increased. The scaffold prepared at 1:1:1 was better for bone tissue engineering, and the average pore size, water absorption rate and hot-water loss rate were 85.67 μm, (135.65±4.56)%and (22.84±1.06)%, respectively, closer to the needs of the bone tissue engineering. Uniform pores were found within the scaffold at 1:1:1, showing the network structure, developed transport among pores, and the network structure was approximately 10μm.
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Objective To investigate the feasibility of restoration of cartilage defect with silk fibrin/chitosan(SF-CS)biological scaffold compound by induced bone marrow mesenchymal stem cells (BMSCs)in the elderly rabbits.Methods BMSCs were extracted,cultured and induced to differentiate,then inoculated into SF-CS three-dimensional scaffold restoration.54 rabbits(aged 16-18months)were divided into scaffold restoration,single scaffold and control groups(n=18 per group).The right knee joint was used for building cartilage defect model and implanted by scaffolds.General observation,tissue staining and modified Wakitani histological scoring were performed at 4,8 and 12weeks after operation.Results SF-CS scaffold was structured by multiple interlinked pores.The average pore size was 151.72 μm.The porosity was(92.72±4.78)％.The imbibition rate was (141.10± 6.87)％.BMSCs was grown well and proliferated dynamically in SF-CS scaffold after induction.At 12 weeks,the cartilage defect was basically repaired,type Ⅱ collagen was positively expressed and the scaffold was almost assimilated in scaffold restoration group.In single scaffold group,the cartilage defect was repaired mainly by fiber tissue,type Ⅱ collagen was less expressed and the scaffold almost degraded while the cartilage defect was repaired badly in control group.The scaffold restoration group was superior to single scaffold and control groups(P＜0.05)in improving the Wakitani score.Conclusions The SF-CS scaffold as BMSCs carrier may restore cartilage defect in knee joint of the elderly rabbits.