ObjectiveTo study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD). MethodsPHBV and PHBHHx films were fabricated by solvent evaporation.Scanning electronic microscope (SEM) was used to study the morphology of the films.PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively.The hydrophilicity of the surface were detected by the contact angle measurements.Septal cartilage cells obtained from human septal cartilage were cultured in vitro.The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1,PHBV modified with PhaP-RGD in group A2,PHBHHx unmodified with PhaP-RGD in group B1,PHBHHx modified with PhaP-RGD in group B2,and on the cell culture plates in group C.After cultured for 3 days,the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days,the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. ResultsSEM observation showed that PHBV and PHBHHx films had porous structures.The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P<0.05).Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups.After 3 days of cultivation in vitro,the cell proliferation and viability of group B2 were the strongest among all groups (P<0.05); the cell proliferation after cultured for 7 days was significantly stronger than that after cultured for 3 days in groups A1,A2,B1,and B2 (P<0.05); and the cell proliferation was significantly stronger in groups B1 and B2 than groups A1,A2 and C,in group B2 than group B1,and in group A1 than group A2 (P<0.05).The results of toluidine blue staining showed that blue metachromasia matrixes were observed in groups A1,A2,B1,and B2; group A1 and group A2 had similar staining degree,and the staining of group B2 was deeper than that of group B1.The adhesion of cells in all groups was good through SEM observation; and the connection of cells formed and stretched into the pores of the materials. ConclusionThe biomaterial films of PHBHHx modified with PhaP-RGD fusion protein can promote its biocompatibility with chondrocytes.
ObjectiveTo review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. MethodsThe literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. ResultsArticular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. ConclusionDue to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.
ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
Objective To review the research progress of the current methods of inducing bone marrow mesenchymal stem cells (BMSCs) to chondrogenic differentiation in vitro so as to provide references for researches in cartilage tissue engineering. Methods Various methods of inducing BMSCs differentiation into the chondrogenic l ineage in vitro inrecent years were extensively reviewed and analyzed. Results Adding exogenous growth factors is still the mainly methodof inducing BMSCs differentiation into the chondrogenic l ineage; among the members, transforming growth factor β (TGF-β) family is recognized as the most important chondrogenic induction factor. Other important inducing factors include various chemical factors, physical factors, transgenic methods, and the microenvironmental induction. But the problems of low inducing efficiency and unstable inducing effects still exist. Conclusion The progress of chondrogenic induction of BMSCs promotes its util ization in cartilage tissue engineering. Further researches are needed for establ ishing more efficient, simpler, and safer inducing methods.
Cartilage with limited self-repairing ability is a kind of tissue with relatively hypocellular structure, low nerve distribution and vascular nutrient. Cartilage tissue engineering provides a new therapeutic idea for cartilage injured cartilage repairing in clinical practice. Electrospinning fibrous scaffold with three-dimensional structure like extracellular matrix is suitable for cell growth and bioactive factor loading for cartilage tissue engineering. This paper introduces studies of the application of electrospinning technology in repairing damaged cartilage by simulating highly hierarchical structures and mechanical features from the aspects of composition optimization, structure optimization and multi-technology combination.
ObjectiveTo construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2(BMP-2) and transforming growth factor β3(TGF-β3) genes,to observe the expressions of BMP-2 and TGF-β3 after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs. MethodsBMP-2 cDNA and TGF-β3 cDNA were amplified by PCR,and were subcloned into the pEC3.1(+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β3 plasmid respectively.They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β3.The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured.The 3rd passage BMSCs were transfered with Ad-BMP-2(group A),Ad-TGF-β3(group B),Ad-BMP-2+Ad-TGF-β3(group C),and untransfected cells served as a control (group D).The expressions of BMP-2 and TGF-β3 genes and proteins were detected by PCR,immunofluorescence,and Western blot.The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type Ⅱ. ResultsThe Ad-BMP-2 and Ad-TGF-β3 were constructed successfully and confirmed by PCR and sequencing.The expression clones of Ad-BMP-2 and Ad-TGF-β3 were packaged into maturated adenovirus successfully,the titer was 5.6×108 and 1.6×108 pfu/mL respectively.The PCR results showed a light band at 310 bp in group A and at 114 bp in group B,and both 310 bp and 114 bp bands in group C,but no band in group D.The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B,respectively;in group C,both red and green fluorescence expressions were detected,and no red or green fluorescence was detected in group D.The results of Western blot showed that there was a light band at 18×103 in group A and at 50×103 in group B;both 18×103 and 50×103 bands were detected in group C;but no band was detected in group D.The cells were positive for collagen type Ⅱ in groups A,B,and C;group C acquired strong collagen type Ⅱ staining when compared with group A and group B;in group D,the cells were negative for collagen type Ⅱ staining. ConclusionThe recombinant adenovirus vector expressing BMP-2 and TGF-β3 are constructed successfully.The BMP-2 and TGF-β3 genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection,which could afford modified seeding cells for cartilage tissue engineering.
【Abstract】 Objective To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-β1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering. Methods The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-β1 were ampl ified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-β1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-β1 and IGF-1 (Group E). After being transfected, the cells were selected, then the prol iferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot. Results The result of electrophoresis showedthat sequence of two bands of the target genes, IGF-1 and TGF-β1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hoursof transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were0.432 ± 0.038 in group A, 0.428 ± 0.041 in group B, 0.664 ± 0.086 in group C, 0.655 ± 0.045 in group D and 0.833 ± 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P lt; 0.01). The differences between groups A and B or between C, D and E were not significant (P gt; 0.05)。RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-β1 was the highest in group C, 0.925 0 ± 0.022 0, 124.341 7 ± 2.982 0, followed by group E, 0.771 7 ± 0.012 0, 101.766 7 ± 1.241 0（P lt; 0.01）; The expression of IGF-1 was the highest in group E, 1.020 0 ± 0.026 0, 128.171 7 ± 9.152 0, followed by group D, 0.465 0 ± 0.042 0, 111.045 0 ± 6.248 0 （P lt; 0.01）. And the expression of collagen II was the hignest in group E, 0.980 0 ± 0.034 0, 120.355 0 ± 12.550 0, followed by group C, 0.720 0 ± 0.026 0, 72.246 7 ± 7.364 0（P lt; 0.01）. Conclusion The repairment of cartilage defects by BMSCs transfected with TGF-β1 and IGF-1 gene together hasa good prospect and important significance of cl inic appl ication in cartilage tissue engineering.
ObjectiveTo construct a transgenic cell sheet of cartilage-derived morphogenetic protein 1 (CDMP-1) by adenovirus vector in vitro and to identify its biological activity. MethodsThe bone mesenchymal stem cells (BMSCs) were isolated from bone marrow of 1-month-old rabbit, and cultured in vitro. The 3rd-6th generation of BMSCs were used for experiment. The experiment was divided into 3 groups:BMSCs transfected by adenovirus (Ad)-cytomegalovirus (CMV)-human CDMP1 (hCDMP1)-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) in group A, BMSCs transfected by Ad-CMV-EGFP in group B, and untransfected BMSCs in group C. The expression of green fluorescence was observed in 3 groups under fluorescent inverted microscope. MTT assay was used to detect the proliferation of the cells. The cell sheet was obtained by means of temperature-responsive culture dish for 14 days. The morphological and HE staining observations of the cell sheet were carried out. RT-PCR and Western blot were used to detect the expressions of hCDMP1 and collagen type II at gene and protein levels, while alcian blue staining was used to detect the expression of glycosaminoglycans (GAG). ResultsBright green fluorescence was observed in transfected cells at 72 hours under fluorescent inverted microscope, and the transfection efficiency was up to 90%. MTT assay showed approximate S-shaped growth curves in 3 groups, showing no significant difference in the absorbance (A) value among 3 groups within 9 days (P>0.05). The three-dimensional cell sheets were successfully harvested in vitro. The RT-PCR and Western blot showed that there were positive expressions of hCDMP1 and collagen type II in group A and negative expression in other 2 groups. HE staining and alcian blue staining showed that there were rich fibrous tissues, mass extracellular matrix, and dark blue metachromatic granules in group A, but there was less fibrous tissues and no specific blue metachromatic granules in other 2 groups; and the positive expression area was significantly lower and gray scale of GAG was significantly higher in group A than that in groups B and C (P<0.05). ConclusionA transgenic cell sheet of exogenous recombinant hCDMP1 by adenovirus vector can express collagen type II and GAG, so it has chondrogenic capacity. This technology that overcomes limitations in traditional tissue engineering, such as low cell-attachment efficiency and inflammatory reaction, may be a new tissue engineering approach for hard tissue reconstruction and is hopeful to build a large density of tissue engineered cartilage.
Objective To review the research progress of articular cartilage scaffold materials and look into the future development prospects. Methods Recent literature about articular cartilage scaffold for tissue engineering was reviewed, and the results from experiments and clinical application about natural and synthetic scaffold materials were analyzed. Results The design of articular cartilage scaffold for tissue engineering is vital to articular cartilage defects repair. The ideal scaffold can promote the progress of the cartilage repair, but the scaffold materials still have their limitations. Conclusion It is necessary to pay more attention to the research of the articular cartilage scaffold, which is significant to the repair of cartilage defects in the future.
Objective To prepare the silk fibroin (SF)-chitosan (CS) scaffolds by adjusting the mass ratio between CS and SF, and test and compare the properties of the scaffolds at different mass ratios. Methods According to the mass ratios of 6 ∶ 4 (group A), 6 ∶ 8 (group B), and 6 ∶ 16 (group C) between SF and CS, CS-SF scaffolds were prepared by freeze-drying method, respectively. The material properties, porosity, the dissolubility in hot water, the modulus elasticity, and the water absorption expansion rate were measured; the aperture size and shape of scaffolds were observed by scanning electron microscope (SEM). Density gradient centrifugation method was used to isolate the bone marrow mesenchymal stell cells (BMSCs) of 4-week-old male Sprague Dawley rats. The BMSCs at passage 3 were seeded onto 3 scaffolds respectively, and then the proliferation of cells on the scaffolds was detected by MTS method. Results The results of fourier transform infrared spectroscopy proved that with the increased content of CS, the absorption peak of random coil/α helix structure (1 654 cm-1 and 1 540 cm-1) constantly decreased, but the absorption peak of corresponding to β-fold structure (1 628 cm-1 and 1 516 cm- 1) increased. The porosity was 87.36% ± 2.15% in group A, 77.82% ± 1.37% in group B, and 72.22% ± 1.37% in group C; the porosity of group A was significantly higher than that of groups B and C (P lt; 0.05), and the porosity of group B was significantly higher than that of group C (P lt; 0.05). The dissolubility in hot water was 0 in groups A and B, and was 3.12% ± 1.26% in group C. The scaffolds had good viscoelasticity in 3 groups; the modulus elasticity of 3 groups were consistent with the range of normal articular cartilage (4-15 kPa); no significant difference was found among 3 groups (F=5.523, P=0.054). The water absorption expansion rate was 1 528.52% ± 194.63% in group A, 1 078.22% ± 100.52% in group B, and 1 320.05% ± 179.97% in group C; the rate of group A was significantly higher than that of group B (P=0.05), but there was no significant difference between groups A and C and between groups B and C (P gt; 0.05). SEM results showed the aperture size of group A was between 50-250 μm, with good connectivity of pores; however, groups B and C had structure disturbance, with non-uniform aperture size and poor connectivity of pores. The growth curve results showed the number of living cells of group A was significantly higher than that of groups B and C at 1, 3, 5, and 7 days (P lt; 0.05); and there were significant differences between groups B and C at 3, 5, and 7 days (P lt; 0.05). Conclusion The CS-SF scaffold at a mass ratio of 6 ∶ 4 is applicable for cartilage tissue engineering.