OBJECTIVE This paper aims to investigate the suitable cell density and the best formation time of tissue engineered autologous cartilage and to provide theoretical basis and parameters for clinical application. METHODS The chondrocytes isolated from mini swines’ ears were mixed with injectable biocompatible matrix (Pluronic), and the density of cell suspensions were 10, 20, 30, 40, 50, 60, 70 x 10(4)/ml. The chondrocyte-polymer constructs were subcutaneously injected into the abdomen of autologous swine. The specimens were observed grossly and histologically after 6 weeks, and investigated the suitable cell density. Then the chondrocyte-polymer constructs with suitable cell density were transplanted into the abdomen of autologous swine and evaluated grossly and histologically in 1, 3, 6, 9, 15 weeks after transplantation to investigate the best formation time of tissue engineered cartilage. RESULTS The experiments demonstrated that the tissue engineered autologous cartilage was similar to the natural cartilage on animals with normal immune system in histological characteristics. The optimal chondrocyte density is 50 x 10(6)/ml, and the proper harvest time is the sixth week. CONCLUSION With tissue engineering skills, we have identified the optimal chondrocyte density and the proper harvest time.
OBJECTIVE: To observe the effects of silks on attachment, shape and function of chondrocytes cultured in vitro. METHODS: The silks from silk worm cocoons were digested by trypsin and coated with polylactic acid to from three dimensional scaffolds for rabbit rib chondrocyte culture. The growth and shape of chondrocytes were observed with phase contrast microscopy, scanning electron microscopy. RESULTS: The chondrocytes were adhered to silks slowly after chondrocytes were seeded into silk scaffolds and cells fixed on silks well 1 or 2 days later. Cells began to proliferate after 3 days and multiplicative growth was observed on the 6th day. Microholes of silk scaffolds were filled with chondrocytes 2 weeks later. Scanning electron microscopy showed that there was a lot of extracellular matrix surrounding cells. CONCLUSION: Silks are ideal for attachment, growth and function maintenance of chondrocytes, and silks can be used as scaffolds for chondrocytes in three dimensional culture.
Objective To study the effects of the periosteum，synovium andcartilage tissues on the gene expressions of proteoglycan, collagen Ⅱ, andnuclear factor kappa B (NF-κB) and to investigate the different effects of these tissues on cartilage regeneration. Methods In 20 New Zealand white rabbits, 20 cartilage explants were taken from the knee joints in each rabbit, the sizeof which was 4 mm×4 mm×4 mm. All the cartilages were divided into the following 4 groups and cultured for 7 days: Group A, with 5 pieces (2 mm×2 mm) of the synovium of theknee joints in each dish; Group B, with 5 pieces (2 mm×2 mm) of the periosteum ineach dish; Group C, with 5 pieces (2 mm×2 mm×2 mm) of the cartilage in each dish; and Group D, with no addition of other tissues (control group). RNA was extracted from the cells of the cartilage explants (4 mm×4 mm×4 mm) in all the dishes. Thegene expressions of proteoglycan, collagen Ⅱ and NF-κB were defected by a reversetranscription-polymerase chain reaction (RT-PCR).Results In group A, the gene expression of proteoglycan was significantly decreased. The relative density of this gene expression had a significant difference when compared with that in group D (1.09±0.21 vs. 1.25±0.25, Plt;0.05); the gene expressions of collagen Ⅱ and NF-κB were also decreased, but they had no significant differences when compared with those in group D (Pgt;0.05). In groupB, the gene expressions of proteoglycan, collagen Ⅱ, and NF-κB were significantly increased. The relative densities of these gene expressions were 1.60±0.26, 1.57±0.24, and 4.20±2.22, respectively, which had significant differences when compared with those in group D (Plt;0.05). In group C, the relative density of the gene expression of collagen Ⅱ was 1.43±0.28, which had a significant difference when compared with that in group D (Plt;0.05), but therelative densities of the gene expressions of proteoglycan and NF-κB had no significant differences when compared with those in group D (Pgt;0.05). Conclusion The results indicate that the periosteum can up-regulate the gene expressions of proteoglycan, collagen Ⅱ and NF-κB. The NF-κB is likely to be an important nuclear transcription factor related to cartilage regeneration. The results also suggest that the periosteum maybe better in facilitating the cartilage repair and regeneration in clinical practice.
Objective To explore the ability of insulin-like growth factor-Ⅰ (IGF-Ⅰ) and hyaluracan acid in prompting chondrogenesis of engineering cartilage tissue.Methods Human articular chondrocytes were isolatedand cultured in DMEM plus 10% fetal bovine serum. They were divided into three groups：hyaluracan acid+chondrocytes + IGF-Ⅰ group(IGF-Ⅰ group), hyaluracan acid+chondrocytes group(cell group), hyaluracan acid group(control group). The ability of chondrogenesis was investigated by HE and toluidine blue staining, human collagen Ⅱ immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).Results Both cell group and IGF-Ⅰ group could develop into cartilage tissue in the sixth week while control group could not. The number of cartilage lacuna in IGF-Ⅰ group were more than that in cell group. Human collagen Ⅱ immunohistochemistry showed that there were ber positive cell in IGF-Ⅰ group than in cell group, collagen Ⅱ mRNA expression was more higher and collagen Ⅰ mRNA expression was lower in IGF-Ⅰ group than in cell group. Conclusion Insulin growth factorⅠ can prompt chondrogenesis of engineering cartilage tissue and ameliorate the quality of engineering cartilage tissue in vitro.
OBJECTIVE: To investigate apoptosis of chondrocytes cultured in vitro and related expression of caspase-3. METHODS: Apoptosis of chondrocytes were detected by flow cytometry analysis and TUNEL staining. The expression of caspase-3 was determined by RT-PCR and Western blot, and caspase-3 protein activity was determined by ELISA. RESULTS: Apoptosis was observed in chondrocytes cultured in vitro from passage 1 to passage 4 at various degrees. The percentage of apoptosis of chondrocytes on day 7 was much higher than that on day 3 (15.7% +/- 0.3% vs 8.9% +/- 0.6%, P lt; 0.01). caspase-3 mRNA and protein expressed in chondrocytes during whole culture process. Along with the culture time extension in vitro, caspase-3 expression and protein activity up-regulated, coincident with apoptosis of chondrocyte. caspase-3 was activated and a fragment of 20 kDa was detected after 7 days of culture. CONCLUSION: caspase-3 is involved in apoptosis of chondrocytes cultured in vitro.
OBJECTIVE: To study the feasibility of the formation of allogeneic tissue-engineered cartilage of certain shape in immunocompetent animal using the injectable biomaterial. METHODS: Fresh newborn rabbits’ articular cartilages were obtained under sterile condition (lt; 6 hours after death) and incubated in the sterile 0.3% type II collagenase solution. After digestion of 8 to 12 hours, the solution was filtered through a 150 micron nylon mesh and centrifuged, then the chondrocytes were washed twice with phosphate buffered saline (PBS) and mixed with the biomaterial to create a final cell density of 5 x 107/ml. The cell-biomaterial admixture was injected into rabbits subcutaneously 0.3 ml each point while we drew the needle back in order to form the neocartilage in the shape of cudgel, and the control groups were injected with only the biomaterial or the suspension of chondrocytes with the density of 5 x 10(7)/ml. After 4, 6, 8 and 12 weeks, the neocartilages were harvested to analyze. RESULTS: The new nodes could be touched subcutaneously after 2 weeks. In the sections of the samples harvested after 4 weeks, it was found that the matrix secreted and the collagen formed. After 6 weeks and later than that, the neocartilages were mature and the biomaterial was almost completely degraded. The cudgel-shaped samples of neocartilage could be formed by injection. In the experiment group, there was no obvious immune rejection response. On the contrary, there were no neocartilage formed in the control group. CONCLUSION: The injectable biomaterial is a relatively ideal biomaterial for tissue engineering, and it is feasible to form allogeneic tissue engineered cartilage of certain shape by injection in an immunocompetent animal.
Objective To isolate，culture and expand bone marrow mesenchymal stem cells （MSCs） in vitro，induce MSCs to differentiate directionally towards chondrocytes，and provide experimental basis for clinical application of MSCs and construction of tissue engineering tracheal cartilage. Methods Cultured MSCs were isolated from bone marrow of Sprague-Dawley rats，purified using adherence separation，and identified by flow cytometry analysis. Transforming growth factor β1 （TGF-β1）and insulin-like growth factor 1 （IGF-1） were used as main induction factors to induce MSCs to differentiate directionally towards chondrocytes. The expression of collagen typeⅡwas evaluated by immunocytochemical staining 21 days after induction. Light microscope and electron microscope were used to observe tiny and ultrastructural changes of the cells before and after induction. Results The expression of collagen typeⅡwas positive by immunocytochemical staining 21 days after induction. MSCs were fusiform before induction under light microscope and electron microscope. After induction，the cells became larger，polygon，star-shaped or triangular. Transmission electron microscope showed that the cells had abundant organelles，larger nuclei and more nucleoli after induction. Conclusion Abundant organelles，larger nuclei and more nucleoli are the ultrastructure changes of chondrocytes differentiated from MSCs，indicating that the cells are active in differentiation and metabolism.
Objective
To explore the effect of rolling compression loading bioreactor on chondrogenesis of rabbit bone marrow mesenchymal stem cells (BMSCs) with different loading parameters.
Methods
BMSCs were isolated from New Zealand rabbits, aged 2.5 months. BMSCs at passage 3 were used to prepare BMSCs-agarose gels (4 mm in diameter and height, respectively). Samples were divided into 8 groups: 10% (group A1), 20% (group A2), and 30% (group A3) compression groups (0.4 Hz, 3 h/ d) and 20 minutes (group B1), 3 hours (group B2), and 12 hours (group B3) rolling time groups and static culture (control groups). The living cell rate, the collagen type II and Aggrecan gene expressions, and glycosaminoglycan (GAG) content were determined, and histological staining was done at 24 hours, 7 days, 14 days, and 21 days after culture.
Results
At 14 and 21 days, the living cell rates of groups A1 and A2 were significantly higher than that of group A3 (P lt; 0.05), groups B1 and B2 were significantly higher than group B3 (P lt; 0.05). Collagen type II and Aggrecan gene expressions of the experimental groups at each time point were significantly higher than those of the control groups (P lt; 0.05); at 14 and 21 days, collagen type II and Aggrecan gene expressions of groups A1 and A2 were significantly higher than those of group A3, and groups B1 and B2 were also significantly higher than group B3 (P lt; 0.05). At 14 and 21 days, the GAG contents of groups A1 and A2 were significantly higher than those of group A3 (P lt; 0.05); groups B1 and B2 were also significantly higher than group B3 (P lt; 0.05). At 21 days, toluidine blue staining showed that obvious blue-staining and even cartilage lacunae were seen in groups A2 and B2, but light and quite rare blue-staining in groups A1, A3, B1, and B3.
Conclusion
The rolling compression loading bioreactor has great promotion effect on chondrogenesis of rabbit BMSCs with rolling parameters of 0.4 Hz, 3 hours, and 20% compression.
ObjectiveTo review the chondrocyte survival microenvironment and the research progress of the application of microfluidic chips in constructing the chondrocyte microenvironment.
MethodsRecent literature about the role of microenvironment in the regulation of chondrocytes and the application of microfluidic chips in constructing the chondrocyte microenvironment was reviewed and analyzed.
ResultsRegulating the microenvironment of chondrocyte mainly involves extracellular matrix microenvironment, mechanical microenvironment, electric microenvironment, and hypoxic microenvironment. Currently, the related research of chondrocyte microenvironment based on microfluidic system mainly involves biochemical stimuli, mechanical stimuli, production of biomimetic scaffold materials, and so on.
ConclusionIt will be helpful for constructing cartilage tissue being closer to the physiological function in the future to deeply understand chondrocyte survival environment and to mimic the microenvironment in vivo required by chondrocyte development as possible by using microfluidic chips.
【Abstract】 Objective To investigate the possibil ity of BMSCs seeded into collagen Ⅰ -glycosaminoglycan (CG)matrices to form the tissue engineered cartilage through chondrocyte inducing culture. Methods Bone marrow aspirate of dogs was cultured and expanded to the 3rd passage. BMSCs were harvested and seeded into the dehydrothemal treatment (DHT)cross-l inked CG matrices at 1×106 cells per 9 mm diameter sample. The samples were divided into experimental group and control group. In the experimental group, chondrogenic differentiation was achieved by the induction media for 2 weeks. Medium was changed every other day in both experimental group and control group. The formation of cartilage was assessed by HE staining and collagen Ⅱ immunohistochemical staining. Results The examinations under the inverted phase contrast microscopeindicated the 2nd and 3nd passage BMSCs had the similar morphology. HE staining showed the BMSCs in the experimental group appeared polygon or irregular morphology in the CG matrices, while BMSCs in the control group appeared fibroblast-l ike spindle or round morphology in the CG matrices. Extracellular matrix could be found around cells in the experimental group. Two weeks after seeded, the cells grew in the CG matrices, and positive collagen Ⅱ staining appeared around the cells in the experimentalgroup. There was no positive collagen Ⅱ staining appeared in the control group. Conclusion It is demonstrated that BMSCs seeded CG matrices can be induced toward cartilage by induction media.
