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.
ObjectiveTo study the preparation method of acellular dermal matrix (ADM) for cartilage tissue engineering and analyze its biocompatibility. MethodsThe dermal tissues of the calf back were harvested, and decelluarized with 0.5% SDS, and the ADM was reconstructed with 0.5% trypsin, cross-linked with formaldehyde, and modified with 0.5% chondroitin sulfate which can promote the proliferation of chondrocytes. And the porosity, cytotoxicity, and biocompatibility were determined. Co-cultured 2nd passage chondrocytes and bone marrow stromal cells in a proportion of 3 to 7 were used as seed cells. The cells were seeded on ADM (experimental group) for 48 hours to observe the cell adhesion. The expressions of mRNA and protein of collagen type Ⅱ were tested by RT-PCR and Western blot methods, respectively. And the expressions were compared between the cells seeded on the scaffold and cultured in monolayer (control group). ResultsAfter modification of 0.5% trypsin, the surface of ADM was smooth and had uniform pores; the porosity (85.4%±2.8%) was significantly higher than that without modification (72.8%±5.8%) (t=-4.384, P=0.005). The cell toxicity was grade 1, which accords to the requirements for cartilage tissue engineering scaffolds. With time passing, the number of inflammatory cells decreased after implanted in the back of the rats (P<0.05). The scanning electron microscope observation showed that lots of seed cells adhered to the scaffold, the cells were well stacked, displaying surface microvilli and secretion. The expressions of mRNA and protein of collagen type Ⅱ were not significantly different between experimental and control groups (t=1.265, P=0.235;t=0.935, P=0.372). ConclusionThe ADM prepared by acellular treatment, reconstruction, cross-linking, and modification shows perfect characters. And the seed cells maintain chondrogenic phenotype on the scaffold. So it is a proper choice for cartilage tissue engineering.
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.
Objective To investigate the feasibility of fabricating an oriented scaffold combined with chondrogenic-induced bone marrow mesenchymal stem cells (BMSCs) for enhancement of the biomechanical property of tissue engineered cartilage in vivo. Methods Temperature gradient-guided thermal-induced phase separation was used to fabricate an oriented cartilage extracellular matrix-derived scaffold composed of microtubules arranged in parallel in vertical section. No-oriented scaffold was fabricated by simple freeze-drying. Mechanical property of oriented and non-oriented scaffold was determined by measurement of compressive modulus. Oriented and non-oriented scaffolds were seeded with chondrogenic-induced BMSCs, which were obtained from the New Zealand white rabbits. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay and scanning electron microscope. Then cell-scaffold composites were implanted subcutaneously in the dorsa of nude mice. At 2 and 4 weeks after implantation, the samples were harvested for evaluating biochemical, histological, and biomechanical properties. Results The compressive modulus of oriented scaffold was significantly higher than that of non-oriented scaffold (t=201.099, P=0.000). The cell proliferation on the oriented scaffold was significantly higher than that on the non-oriented scaffold from 3 to 9 days (P lt; 0.05). At 4 weeks, collagen type II immunohistochemical staining, safranin O staining, and toluidine blue staining showed positive results in all samples, but negative for collagen type I. There were numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells on the oriented-structure constructs. Total DNA, glycosaminoglycan (GAG), and collagen contents increased with time, and no significant difference was found between 2 groups (P gt; 0.05). The compressive modulus of the oriented tissue engineered cartilage was significantly higher than that of the non-oriented tissue engineered cartilage at 2 and 4 weeks after implantation (P lt; 0.05). Total DNA, GAG, collagen contents, and compressive modulus in the 2 tissue engineered cartilages were significantly lower than those in normal cartilage (P lt; 0.05). Conclusion Oriented extracellular matrix-derived scaffold can enhance the biomechanical property of tissue engineered cartilage and thus it represents a promising approach to cartilage tissue engineering.
Objective To review the recent research progress of the bioreactor biophysical factors in cartilage tissue engineering. Methods The related literature concerning the biophysical factors of bioreactor in cartilage tissue engineering was reviewed, analyzed, and summarized. Results Oxygen concentration, hydrostatic pressure, compressive force, and shear load in the bioreactor system have no unified standard parameters. Hydrostatic pressure and shear load have been in controversy, which restricts the application of bioreactors. Conclusion The biophysical factors of broreactor in cartilage tissue engineering have to be studied deeply.
Objective To explore the impact of basic fibroblast growth factor (bFGF) and parathyroid hormone-related protein (PTHrP) on early and late chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) induced by transforming growth factor β1 (TGF-β1). Methods BMSCs were isolated from 3 healthy Japanese rabbits (2-month-old, weighing 1.6-2.1 kg, male or female), and were clutured to passage 3. The cells were put into pellet culture system and were divided into 5 groups according to different induce conditions: TGF-β1 group (group A), TGF-β1/bFGF group (group B), TGF-β1/21 days bFGF group (group C), TGF-β1/PTHrP group (group D), and TGF-β1/21 days PTHrP group (group E). At the beginning, TGF-β1 (10 ng/mL) was added to all groups, then bFGF and PTHrP (10 ng/mL) were added to groups B and D respectively; bFGF and PTHrP (10 ng/mL) were added to groups C and E at 21 days respectively. The gene expressions of collagen type I (Col I), Col II, Col X, matrix metalloproteinases (MMP)-13, and alkaline phosphatase (ALP) activity were detected once every week for 6 weeks. The 1, 9-dimethylmethylene blue (DMMB) staining was used to observe the extracellular matrix secretion at 6 weeks. Results The expression of Col I in groups C and E showed a significant downward trend after 3 weeks; the expression in group A was significantly higher than that in groups C and E at 4 and 5 weeks (P lt; 0.05), and than that in groups B and D at 3-6 weeks (P lt; 0.05); and significant differences were found between groups B and C at 3 and 4 weeks, and between groups D and E at 3 weeks (P lt; 0.05). After 3 weeks, the expressions of Col II and Col X in groups C and E gradually decreased, and were significantly lower than those in group A at 4-6 weeks (P lt; 0.05). Groups B and D showed no significant difference in the expressions of Col II and Col X at all time points, but there was significant difference when compared with group A (P lt; 0.05). MMP-13 had no obvious expression at all time points in group A; significant differences were found between group B and groups A, C at 3 weeks (P lt; 0.05); and the expression was significantly higher in group D than in groups A and E (P lt; 0.05). ALP activity gradually increased with time in group A; after 4 weeks, ALP activity in groups C and E obviously decreased, and was significantly lower than that in group A (P lt; 0.05); there were significant differences between groups B and C, and between groups D and E at 2 and 3 weeks (P lt; 0.05). DMMB staining showed more cartilage lacuna in group A than in the other groups at 6 weeks. Conclusion bFGF and PTHrP can inhibit early and late chondrogenic differentiation of BMSCs by changing synthesis and decomposition of the cartilage extracellular matrix. The inhibition is not only by suppressing Col X expression, but also possibly by suppressing other chondrogenic protein.
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 recent progress of the researches in the field of cartilage tissue engineering, and to discuss the challenges in construction of tissue engineered cartilage. Methods Literature related with cartilage tissue engineering was reviewed and analyzed. Results Some techniques have been appl ied in cl inical. As far as the seeding cells, induced pluripotent stem cells have attracted much more attention. Current strategies of scaffold designing are trying to imitate both component and structure of natural extracellular matrix. Cartilage regeneration through the autologous cell homing technique el iminate the transplantation of exotic cells and has become the hot topic. Conclusion Successful treatment of the damaged cartilage using tissue engineering method will depend on the advances of stem cell technology development, biomimetic scaffolds fabrication and proper appl ication of growth factors.
Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
Objective To review the appl ication of and the research progress on acellular matrix (ACM) in cartilage tissue engineering. Methods Related l iteratures both at home and abroad were retrospected and analyzed. Results Manyresearchers improved the properties of cartilage ACM scaffold through co-appl ication of solution diosmosis method, freezedrying method and physical and chemical cross-l inking method etc., and the experimental results of applying cartilage ACM scaffold for the construction of tissue engineered cartilage were closely related to the properties of ACM. Conclusion ACM has a wide appl ication prospect for the construction of tissue engineered cartilage, and further in-depth studies are needed to improve its property.