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Objective::To explore the effects of the biomimetic network membrane prepared by chitosan/gelatin/pectin on the proliferation and mineralization of mesenchymal stem cells (MSCs) , and to evaluate its feasibility of constructing tissue engineering bone.Methods:Chitosan, gelatin and pectin were made into a new biomimetic network membrane in a certain ratio by biomimetics.The experiment was divided into control group (MSCs+conventional medium) , material group (MSCs+network membrane+conventional medium) and material+OS group (MSCs+network membrane+OS medium) .The cell morphology was observed by inverted phase contrast microscope;the growth and secretion of extracellular matrix of the MSCs were observed under scanning electron microscope (SEM) .The proliferation of cells was determined by MTT assay (The MSCs were divided into negative control group and material group, and they were cultivated with blank medium and medium including materials) .The expression of calcium in MSCs was detected by Alizarin Red staining.Real-time polymerase chain reaction (RT-PCR) was used to determine the expression levels of osteocalcin (OC) mRNA and osteopontin (OPN) mRNA in the MSCs.Results:The network membrane was semitransparent thin film.The MSCs were short shuttle and clustered under inverted phase contrast microscope.After cultured for 7d, the MSCs were shuttle;after cultured for 14d, the number of MSCs was increased, with pseudo feet on the membrane;after cultured for21d, the MSCs clustered with a lot of neo-formed extracellular matrix.The MTT results showed that there was no significant difference in the proliferation level of MSCs between material group and negative control group (P＞0.05) .The Alizarin Red staining results showed that the MSCs in the network membrane were dyed orange red.The RT-PCR results showed that the expression levels of OC mRNA in the MSCs in material group and material+OS group were lower on the 7th and 14th days, but on the 21th day, the expression levels were significantly increased and reached the peak;the expression level of OC mRNA in the MSCs in material group was significantly increased on the 7th day, and the expression level reached the peak on the 14th day, then fell slightly on the 21th day;compared with control group, the expression levels of OC mRNA and OPN mRNA in the cells in material group and material+OS group at different time points were significantly increased (P＜0.01) , but there were no significant differences between material group and material+OS group (P＞0.05) .Conclusion:Chitosan/gelatin/pectin biomimetic network membrane has good biocompatibility, and MSCs can grow and proliferate well on the membrane.The membrane can induce the MSCs to express mineralization-related genes and proteins without inducers.
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Objective:To evaluate the anti-adhesion properties of xyloglucan(mXG)hydrogel in the L929 mouse fibroblasts,to establish the animal model of recurrent adhesion in the rats after adhesionlysis,and to investigate the prevention effect of mXG hydrogel in recurrent adhesion and its influence in the expressions of transforming growth factor-β1(TGF-β1)and connective tissue growth factor(CTGF).Methods:After seeding on the surface of mXG gel,the adhesion property of L929 mouse fibroblasts was detected with fluorescence staining.The rat models of recurrent adhesion were established after adhesionlysis.Fourty-eight SD rats were randomly divided into 3 groups respectively(n=16).In adhesion group,1 mL saline was injected into the abdominal wall and cecum of the rats;in commercial membrane group,the 2 cm×3 cm chitosan anti-adhesion membrane was used to cover the wound of abdominal wall of the rats;in mXG hydrogel group,1 mL 4% mXG hydrogel was injected into the abdominal wall and cecal wound of the rats,and abdominal surgery was ended after the complete formation of the hydrogel(3 min).Eight rats were killed in each group 7 and 14 d after the second operation,and the degrees of adhesion were evaluated and the histological changes were observed. Immunohistochemical staining was used to observe the expression levels of CTGF and TGF-β1.Results:A large number of L929 mouse fibroblasts proliferated well and exhibited a spherical morphology in control group;but in mXG hydrogel group,only very little L929 mouse fibroblasts were globular,showing the mXG hydrogel had good resistance to the adhesion of L929 mouse fibroblasts.Blunt separate adhesion surface formed in all of the rats after operation.7 d after the second operation,4-5 score adhesion with fibrous connective tissue hyperplasia formed in adhesion group;moderate adhesion formed in commercial membrane group with more connective tissue;most cecum and abdominal wall began to heal in mXG hydrogel group with less connective tissue.14 d after the second operation,more severe peritoneal adhesions presented in adhesion group with proliferated fiber connetive tissue and collegan;severe adhesions also presented in commercial membrane group;mild adhesion was found in two rats mXG group,the other rats had no adhesion,and the defects were almost completely recovered.On days 7 and 14 after the second operation,the mean adhesion score of the rats in mXG group was significantly lower than those in adhesion group and commercial membrane group(P<0.05).The expression levels of TGF-β1 and CTGF were increased with the increase of peritoneal adhesion scores and collagen deposition;the expression levels of TGF-β1 and CTGF were the highest in adgesion group and the lowest in mXG group.Conclusion:mXG hydrogels have good resistance to fibroblast adhesion,and mXG hydrogel is effective in reducing the formation of recurrent adhesion in the rats after adhesionlysis and can decrease the expression levels of adhesion-related factors TGF-β1 and CTGF.
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Objective: To investigate the effect of cartilage tissue engineering scaffold PVA/ι-CA on the biological behavior of the ATDC-5 cells,and to evaluate its feasibility on constructing tissue engineering cartilage. Methods:The polyvinyl alcohol (PVA)and carrageenan were used to make the composite scaffold material PVA/ι-CA according to a certain proportion by physical blending technology and repeated freezing thawing method,and the porosity and pore size of PVA/ι-CA were detected.The ATDC-5 cells were seeded into the composite scaffold and its growth was observed; the expressions of collagen type Ⅱ in the ATDC-5 cells were tested by immunohistochemical staining and immunofluorescence staining; the morphology of the ATDC-5 cells was confirmed by Toluidine blue staining.The growth and secretion of extracellular matrix of the ATDC-5 cells were observed under scanning electron microscope (SEM);the proliferative rates of ATDC-5 cells in composite scaffold materials in negative control group (added with DMEM culture media)and experimental group (added with DMEM contain scaffold)were determined by MTT assay.The composite scaffolds were implanted subcutaneously in the SD rats.The histocompatibility and vascularization in vivo of the composite scaffolds were evaluated.Results:The average porosity of cartilage tissue engineering scaffold PVA/ι-CA was (86.88±3.88)%,and the average pore size was 20-40 μm.The HE staining results showed that the ATDC-5 cells grew well with the polygon and plumpness morphology. All the samples were stained positive for collagen type Ⅱ by immunohistochemistry and immunofluorescence staining,which verified the normal phenotype of chondrocytes on the scaffolds. All the sample were stained positive for toluidine blue staining,which verified ECM deposition of the ATDC-5 cells on the scaffolds.The number of the positive cells was significantly increased with the prolongation of time.After cultured for 7 d,few of the ATDC-5 cells presented polygonal;after cultured for 14 d,the ATDC-5 cells distributed more densely,and contacted with each other on the scaffold;after cultured for 21 - 28 d,the ATDC-5 cells filled the interconnected pores of the scaffolds,synthesizing a significant amount of neo-formed ECM.The proliferation of ATDC-5 cells in PVA/ι-CA grew fast during 7-14 d,and it became slow during 21-28 d;the difference was not statistically significant compared with control group (P >0.05).The subcutaneous implantation results showed the inflammatory reactions were slight at the early stage and eviated gradually,there was an increasing angiogenesis at the late stage,and the degradation and absorption of the meterial were slight.Conclusion:PVA/ι-CA composite material will be an ideal material for the cartilage tissue engineering.
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BACKGROUND:Polyvinyl alcohol (PVA) hydrogel with similar porous structure and mechanical properties to the natural cartilage is very suitable for the repair of articular cartilage. However, the pure PVA hydrogel after lyophilization wil be accompanied by the shrinkage of the polymer network and the col apse of the pores, leading to the inhomogeneous performance of the material even in the state of re-swel ing. Addition of the active polymer wil increase the cel adhesion ability of PVA hydrogel. OBJECTIVE:To construct PVA/lota-carrageenan (l-CA) composite materials with different mass fractions of l-CA and evaluate the biocompatibility with vascular endothelial cel s. METHODS:PVA/l-CA composite films with different contents of l-CA were fabricated and then co-cultured with vascular endothelial cel s. Attachment, proliferation and morphological changes of vascular endothelial cel s on the composite were observed by scanning electron microscope and MTT assay to evaluate its biocompatibility. PVA/l-CA three-dimensional scaffold with different contents of l-CA were constructed, and hemolysis experiment was conducted according to the biological evaluation standards of medical devices, and the porosity and pore size were observed using scanning electron microscope. RESULTS AND CONCLUSION:In vitro experimental results showed that the addition of l-CA could significantly increase the biological activity of PVA hydrogel, and promote the cel attachment and proliferation on the scaffold. The hemolysis rate of each experimental group was less than 5%(the accepted safety standard), suggesting that the composite materials were in accordance with the standard of medical devices for hemolysis experiment. These findings indicate that the composite scaffolds with 20%-30%l-CA possess the pore size suitable for cel growth and proliferation and the porosity beneficial for transportation of nutrients and metabolites, which can serve as an excel ent scaffold for tissue engineering.
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Objective To evaluate the feasibilty of modified osteogenic culturing of goat adipose derived stromal cells(ADSCs).Methods From March,2013 to September,2014,platelet-rich plasma(PRP) was made from goat autogenous vein blood,and abodimoneal fat was aspirated,following aspetic procedure,primary and series passage of ADSCs was established.Osteoinduced ADSCs was carried out according to following group:combinative osteoinduction group(PRP+rhBMP-2 +ADSCs),growth factor group(rhBMP-2+ADSCs),conventional inductive group(dexamathasone+ascorbic acid + ADSCs) and noninductive group(blank group).Converted microscope was used to observe cellular pattern,cell activity,osteocalcin and collagen type Ⅰ level were detected at 1,3,5,7,9,13,17,21 days.Red Alazarin and Von Kossa staining were also assayed at different interval.Results Under observation of converted microscopy,remarkable cell proliferation with abundant ECM was noticed in combinative osteoinduction group,compared with other groups,level of cell activity,osteocalcin,collagen type Ⅰ [(0.82 ± 2.19)AU,(79.82 ± 1.36)U/L at,(78.51 ± 4.32)μ g/ml at]were significantly higher than other groups (P ＜0.05),and remarkable ALP expression and calcifed nodules were also seen.Conclusion PRP can enhance the inductive effect of rhBMP-2,and combinative osteoinduction procedure acts as a satisfactory culturing method.
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Objective To construct novel 3-D composite bionic network and evaluate the histocompatibility . Meth?ods The novel 3-D composite bionic network was prepared from chitosan, hydroxyapatite, gelatin and pectin in certain ra?tio by biomimtic approach, which was co-cultured with MC3T3-E1. The cell compatibility was studied by using inverted phase contrast microscope, routine paraffin section staining, scanning electron microscopy and F-DA staining. The resultant scaffold material was implanted into the dorsal subcutaneous space of SD rats. The histocompatibility, blood vessel capabili?ties and the degradation of the material were observed 2, 4, 8 and 12 weeks after surgery. Results The structure of novel 3-D composite bionic network was three-dimensional and porous. The cells attached on scaffolds attached and grew well with polygonal or fusiform form. It was found that inflammatory reactions were alleviated gradually in the early stage . There was an increasing angiogenesis at late stage. Materials degraded and absorbed more slowly. Conclusion The present study sug?gests that the novel 3-D composite bionic network has good histocompatibility with easy vascularization, and will be a candi?date scaffold for bone tissue engineering.
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Objective To evaluate the biological potential of surface topography of biomimetic matrix combined PRP gels with RGD modification. Methods Surface topography of nβ-TCP/Cs/PCL matrix was made by Nd: YAG laser, tissue-engineered bone was constructed in the following ways: ADSCs were loaded to nβ-TCP/Cs/PCL matrix with PRP gels plus RGD modification (group A), ADSCs were cultured to nβ-TCP/Cs/PCL matrix with RGD modification (group B), ADSCs implantation with topography-surfaced treatment of matrix (group C), ADSCs cultivation with smooth-surfaced treatment of matrix (group D). SEM and CLM were used to observe cellular pattern, survival rate, cell activity, ALP and collagen type Ⅰ level were detected at 1, 4, 8, 12, 16, 20, 24,28 days. Runx2 and OPG expressions were assayed at different interval. Results Under observation of SEM and CLM, new tissue showed more remarkable cell proliferation with abundant ECM in group A. Compared with other groups, the survival rate in group A was significantly higher (88.16 ± 1.29, P ＜ 0.05),and the level of cell activity, ALP, collagen type Ⅰ were significantly higher (0.92± 0.13, 87.27 ± 3.08, 93.27 ± 3.91, P＜ 0.05), and remarkable expression of Runx2 and OPG was also seen. Conclusion Topography-surfaced treatment of matrix combined PRP gels with RGD modification enhances the cell proliferation and acts as a feasible osteopromotive method.
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The construction of biologic functional surfaces of materials, from the visual angle of material science, is aimed to make the biomaterials adapted by tissues, and to endow them with dynamic conformity; moreover, from the view-point of clinical applications, it is the functional surface to join the environmental tissues with the implanted material, playing the role of artificial extracellular matrix (ECM). The architecture of biologic functional surface is very important in tissue engineering science. Here the primary concepts of biological surface science and the construction and application of biofunctional surfaces in tissue engineering are reviewed.