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.
The repair of defects of articular cartilage has continued to be a difficult problem. This article provided a collective review from literature pertaining to the advances gained in the repair of cartilaginous defects. In the spontaneous repair, if the defect of the cartilage was less than 3 mm, might result in complete or partial repair, but in those the diameter was more than 3 mm, the defect could not be repaired by normal cartilage. Although the cartilaginous autograft could give good result, but it could not be widely applied because short of supply of the autogenous cartilage. Cartilagious allograft could not be taken to repair cartilaginous defect because of reaction from tissue rejection. The transplantation of periosteal or perichondral graft had been tried but was eventually abandoned because of poor long-term result. The transplantation of free chondrocytes might be a method of hope. In general, transplantation of free chondrocytes into the cartilaginous defect will be lost. The supply of autogenous chondrocytes was very limited, and the heterogenous chondrocytes would inflict immunoreaction after being transplanted. In late of 1980, a new concept of tissue engineering was proposed. The problem that a scaffold of appropriate material which could hold the free chondrocytes in place from being lost might undergo proliferation and differentiation into new cartilage was far from being solved. Although tissue engineering still had various problems needed further investigation, but it will probably be the main direction of development in this field.
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 observe the biological characters of chondrocytes in articular loose body and to find out seeding cells for cartilage tissue engineering. Methods Samples from 5 loose body cartilages, 2 normal articular cartilages and 6 osteoarthritis articular cartilages were collected. Part of each sample’s cartilage was histologically studied to observe the chondrocytes distribution the morphologic changes by toluidine-blue staining, chondrocytes’ apoptosis by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL). The rest of each cartilage was digested and isolated by 0.25% trypsin and 0.2% collagenase Ⅱ, and then were cultivated in 10%DMEM. Their morphologic changes were observed 24h later.Comparison was made btween three cartilages. Results Compared with normal cartilage and osteoarthritis articular cartilage, the cells density was higher, their lacunars were larger, cells distribution was irregular, and apoptosis was more apparent in loose body cartilage. Conclusion The characters of chondrocytes from loose body is more like fibroblasts so they can not serve as seeding cells directly for cartilage tissue engineering.
Objective To investigate the possibility of differentiation of theisolated and cultured adipose-derived adult stem cells into chondrocytes, which is induced by the recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods The rabbit adipose tissue was minced and digested by collagenase Type Ⅰ. The adposederived adult stem cells were obtained and then they were cultured inthe micropellet condition respectively in the rhBMP-2 group, the rhTGF-β1 group, the combination group, and the control group for 14 days. The differentiation of the adiposederived stem cells into chondrocytes was identifiedby the histological methods including HE, Alcian blue, Von kossa, and immunohistochemical stainings. Results After the continuous induction by rhBMP-2 and continuous culture for 14 days, the HE staining revealed a formation of the cartilage lacuna; Alcian blue indicated that proteoglycan existed in the extracellular matrix; the immunohistochemical staining indicated that collagen Ⅱ was in the cellular matrix; and Von kossa indicated that the adipose-derived stem cells couldnot differentiate into the osteoblasts by an induction of rhBMP-2. Conclusion In the micropellet condition, the adipose-derived adult stemcells can differentiate into the chondrocytes, which is initially induced by rhBMP-2. This differentiation can provide a foundation for the repair of the cartilage injury.
Objective To investigate the feasibil ity and effect of inducing adi pose-derived stem cells (ADSCs) treated with growth differentiation factor 5 (GDF-5) to undergo chondrogenic differentiation in vitro. Methods Six healthy Japanese rabbits aged 3 months (2-3 kg) of clean grade were chosen, irrespective of sex. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by vimentin immunohistochemistry and CD44, CD49d, CD106immunofluorescence staining. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 106/mL, then the ADSCs were treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium, respectively. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTT. The mRNA quantities of Col II and proteoglycan expressed by the induced ADSCs were detected with RT-PCR. The Col II proteoglycan synthesized by the induced ADSCs were detected with alcian blue staining, toluidine blue staining, immunohistochemistry staining, and Western blot method. Results ADSCs mostly presented small sphere, fusiform and polygon shape with positive expression of CD44 and CD49d and negative expression of CD106 and vimentin. The ADSCs treated with 100 ng/mL GDF-5 presented sphere or sphere-l ike change and vigorous prol iferation. The mRNA quantities of Col II and proteoglycan synthesized by the induced ADSCs treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium increased in a dose-dependent manner at 7 days. There were significant differences among all the groups (P lt; 0.05), except that no significant difference was evident between the 0 ng/mL group and the 10 ng/mL group (P gt; 0.05). When ADSCs were treated with 100 ng/mL GDF-5 for 14 days, the Col II and the mRNA and protein quantities of ptoteoglycan reached the peak, and the results of alcian blue, toluidine blue and Col IIimmunohistochemistry staining were positive. Conclusion ADSCs treated with certain concentration of GDF-5 have higher expression of Col II and proteoglycan and possess partial biological function of chondrocyte.
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 gap junction and phenotype of cultured chondrocyte of rabbit, and the gap junction between the chondrocytes in the same cartilage cavities in human femoral head articular cartilage. METHODS: CFDA-AM was added into the medium of the fifth passage of chondrocyte of rabbit in the 96-well plate. The fluorescent in spherical and fibroblast-like chondrocytes was detected separately. The recurrence of the fluorescent in accordant with time in 16 minutes was recorded after blanching the fluorescent with laser. And the fluorescent after blanching of chondrocyte in the cartilage cavities in the proliferative zone of articular cartilage of adult human femoral head was recorded, too. RESULTS: The average fluorescent of the single layer of the fibroblast-like chondrocyte was 83(ranged from 1 to 274), the highest was found in the spherical shaped cell (averaged 2,057, ranged from 340 to 3,538). The recurrence of the fluorescent after the blanching appeared only in the spherical chondrocyte, the gap junctions reappeared only in the spherical chondrocytes, as well as in the cells in the cartilage cavities in the articular cartilage of the human femoral head. CONCLUSION: The appearance of the gap junction is corresponded with the spherical shape, secretion of the cartilage matrix of the chondrocyte. There are gap junctions in the cells in the same cartilage cavities in the articular cartilage of the human femoral head, while no gap junctions in the isolated chondrocytes in the cartilage.