The Combined Analysis of Transcriptome and Antioxidant Enzymes Revealed the Mechanism of EBL and ZnO NPs Enhancing Styrax tonkinensis Seed Abiotic Stress Resistance.

As global climate change worsens, trees will have difficulties adapting to abiotic pressures, particularly in the field, where environmental characteristics are difficult to control. A prospective commercial and ornamental tree species, Styrax tonkinensis, has its seed oil output and quality reduced as a result, which lowers the economic benefits. This necessitates growers to implement efficient strategies to increase the seeds of woody biofuel species' tolerance to abiotic stress. Numerous studies have shown that ZnO nanoparticles (NPs), a new material, and BRs assist plants to increase their resilience to abiotic stress and subsequently adapt to it. However, there have not been many investigations into S. tonkinensis seed resistance. In this study, we examined the changes in antioxidant enzyme activities and transcriptomic results of S. tonkinensis seeds throughout the seed development period to investigate the effects of 24-epibrassinolide (EBL), one of the BRs, and ZnO NPs treatments alone or together on the stress resistance of S. tonkinensis seeds. On 70, 100, and 130 days after flowering (DAF), spraying EBL or ZnO NPs increased the activity of antioxidant enzymes (POD, SOD, and CAT) in S. tonkinensis seeds. Moreover, when the EBL and ZnO NPs were sprayed together, the activities of antioxidant enzymes were the strongest, which suggests that the positive effects of the two can be superimposed. On 70 and 100 DAF, the EBL and ZnO NPs treatments improved seed stress resistance, mostly through complex plant hormone crosstalk signaling, which includes IAA, JA, BR, and ABA signaling. Additionally, ABA played an essential role in hormone crosstalk, while, on 130 DAF, due to the physiological characteristics of seeds themselves in the late stage of maturity, the improvement in seed stress resistance by EBL and ZnO NPs was related to protein synthesis, especially late embryogenesis-abundant protein (LEA), and other nutrient storage in seeds. Spraying EBL and ZnO NPs during the seed growth of S. tonkinensis could significantly increase seed stress resistance. Our findings provide fresh perspectives on how cultural practices can increase abiotic stress tolerance in woody seedlings.

Molecular epidemiology of coxsackievirus A16 circulating in children in Beijing, China from 2010 to 2019.

BACKGROUND: Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot and mouth disease (HFMD). This study aimed to investigate the molecular epidemiology and evolutionary characteristics of CVA16. METHODS: Throat swabs were collected from children with HFMD and suspected HFMD during 2010-2019. Enteroviruses (EVs) were detected and typed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR. The genotype, evolutionary rate, the most recent common ancestor, population dynamics and selection pressure of CVA16 were analyzed based on viral protein gene (VP1) by bioinformatics software. RESULTS: A total of 4709 throat swabs were screened. EVs were detected in 3180 samples and 814 were CVA16 positive. More than 81% of CVA16-positive children were under 5 years old. The prevalence of CVA16 showed obvious periodic fluctuations with a high level during 2010-2012 followed by an apparent decline during 2013-2017. However, the activities of CVA16 increased gradually during 2018-2019. All the Beijing CVA16 strains belonged to sub-genotype B1, and B1b was the dominant strain. One B1c strain was detected in Beijing for the first time in 2016. The estimated mean evolutionary rate of VP1 gene was 4.49 × 10-3 substitution/site/year. Methionine gradually fixed at site-23 of VP1 since 2012. Two sites were detected under episodic positive selection, one of which (site-223) located in neutralizing linear epitope PEP71. CONCLUSIONS: The dominant strains of CVA16 belonged to clade B1b and evolved in a fast evolutionary rate during 2010-2019 in Beijing. To provide more favorable data for HFMD prevention and control, it is necessary to keep attention on molecular epidemiological and evolutionary characteristics of CVA16.

Progress in Metabolic Reprogramming of Tumor-associated Macrophages in Tumor Microenvironment / 中国实验方剂学杂志

Occupying more than half of the tumor volume in a variety of solid tumors, tumor-associated macrophages (TAMs) are an important part of the tumor microenvironment (TME) with high plasticity and heterogeneity. In the early stages of tumor development, TAMs mediate antitumor effect through phagocytosis and their antioxidant functions. However, in order to meet the needs of self-renewal and proliferation, malignant tumor cells continuously adjust their metabolic patterns, leading to the accumulation of metabolites such as lactate, reactive oxygen species, nitric oxide, arachidonic acid and prostaglandin in the TME, which results in the changes in its inflammatory profiles, thereby altering the metabolism and function of TAMs and ultimately promoting the tumor development. Therefore, further understanding of the metabolism and immune responses of TAMs in the TME during tumor progression is warranted and the investigation may lead to identification of novel potential targets for cancer immunotherapy. This review aims to clarify the close relationship between TAMs metabolism and TME immune response, to reveal the mechanism of tumor immunosuppression produced by TAMs metabolism, and to provide new treatment ideas and approaches for tumor immunotherapy.

Expression of CCR7 and Tim-3 in Childhood Patients with Acute Lymphoblastic Leukemia and Their Predictive Value for Prognosis / 中国实验血液学杂志

OBJECTIVE@#To analyze the expression of CCR7 and Tim-3 in childhood patients with acute lymphoblastic leukemia (ALL) and their predictive value for prognosis.@*METHODS@#Eighty-six newly diagnosed ALL childhood patients from January 2007 to January 2017 treated in our hospital were selected. The expression level of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients were detected by flow cytometry, all the patients were divided into the recurrence group and non-recurrence group according to the follow-up results, the differences in the expressions of CCR7, Tim-3 between the two groups were compared. The correlation between the expression of CCR7 , Tim-3 and the clinicopathologic features of ALL patients were analyzed, the predictive value of CCR7 and Tim-3 for the prognosis of newly ALL patients were evaluated by ROC curve, and the relationship between serum CCR7, Tim-3 and prognosis were analyzed.@*RESULTS@#The expression levels of CCR7 and Tim-3 in recurrence group were significantly higher than those in non-recurrence group(P0.05). The exogenous infiltration rate of patients with high expression of CCR7 and Tim-3 was significantly higher than those in low expression group (P<005). The high expression rate 76.9% of Tim-3 in patients with T-ALL was significantly higher than that of B-ALL patients with Tim-3 high expression rate 45.2% (P<0.05). The median OS of patients with CCR7 level ≥45.97% and <45.97% were 9.3 months and 13.6 months respectively(P=0.004), and the Tim-3≥53.54% and Tim-3<53.54% were 9.1 months and 13.6 months respectively(P=0.001). The results of Cox's multi-factor regression analysis showed that CCR7 level(HR=1.024, 95 CI 1.001-1.049) and Tim-3 level (HR=1.879, 95 CI 1.183- 2.985) were the independent risk factors that affect the OS in ALL patients(P<0.05).@*CONCLUSION@#The expression of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients shows good predictive value for prognosis, and the combination of CCR7 and Tim-3 can improve the sensitivity of the detection, the higher expression of CCR7 and Tim-3 can be used as potential indexes in prognosis evaluate.

MiR-92a inhibits fibroblast-like synoviocyte proliferation and migration in rheumatoid arthritis by targeting AKT2.

Growing data have indicated that the miR-17-92 cluster is implicated in inflammatory response and rheumatoid arthritis (RA). This study was aimed to investigate the effects of miR-92a on the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). Our results showed that miR-92a was significantly down-regulated in RA synovial tissue and RA-FLSs, whereas the protein level of AKT2 is increased. Restoration of miR-92a suppressed the proliferation and migration of RA-FLSs. Down-regulation of miR-92a promotes proliferation and migration of normal human FLSs. Dual luciferase reporter gene assay showed that miR-92a could specifically bind with the 30UTR of AKT2 and significantly repressed the luciferase activity. Down-regulation or up-regulation of miR-92a significantly increased or decreased the protein and phosphorylation levels of AKT2. siRNA-mediated down-regulation of AKT2 significantly prevented cell proliferation and migration of RA-FLSs, which were similar to the effects induced by overexpression of miR-92a. Moreover, AKT2 overexpression rescued miR-92a-mediated suppressive effect on proliferation and migration of RA-FLS. Thus, miR-92a could inhibit the proliferation and migration of RA-FLSs through regulation of AKT2 expression.

TNF­α increases inflammatory factor expression in synovial fibroblasts through the toll­like receptor­3­mediated ERK/AKT signaling pathway in a mouse model of rheumatoid arthritis.

Osteoarthritis is a type of joint disease that may lead to other joint diseases. Previous research has demonstrated that tumor necrosis factor (TNF)­α is associated with osteoarthritis activity and pathology. The possible mechanisms of the TNF­α­mediated signaling pathway have not been clearly elaborated in synovial fibroblasts. The present study aimed to investigate the potential mechanisms of TNF­α in a mouse model of iodoacetate­induced osteoarthritis. Reverse transcription­quantitative polymerase chain reaction, ELISA, western blotting and immunohistochemistry were performed to evaluate the role of TNF­α in the progression of osteoarthritis. The results revealed that the serum levels of TNF­α, interleukin (IL)­1ß, IL­4 and IL­6 were significantly upregulated in a mouse model of iodoacetate­induced osteoarthritis compared with healthy mice (P<0.01). TNF­α, IL­1ß, IL­4 and IL­6 mRNA and protein levels were also significantly upregulated in synovial fibroblasts in the experimental mice (P<0.01). It was demonstrated that TNF­α increased pro­inflammation factors matrix metalloproteinase (MMP)­3, MMP­9, nuclear factor (NF)­κB and receptor activator of NF­κB ligand (RANKL) in synovial fibroblasts. It was also observed that the toll­like receptor (TLR)­3 was significantly upregulated and extracellular signal­regulated kinase (ERK) and protein kinase B (AKT) were significantly downregulated in synovial fibroblasts in osteoarthritis mice (P<0.01). An in vitro assay demonstrated that TNF­α inhibitor decreased mRNA and protein levels of IL­1ß, IL­4 and IL­6 in synovial fibroblasts. The knockdown of TLR­3 abolished the TNF­α upregulated mRNA and protein levels of IL­1ß, IL­4 and IL­6 in synovial fibroblasts. In addition, the knockdown of TLR­3 also reversed TNF­α­upregulated ERK and AKT expression in synovial fibroblasts. In vivo assays demonstrated that TNF­α inhibitor significantly decreased the deposition of IL­1ß, IL­4 and IL­6 as well as bone destruction and significantly increased the body weight and osteoarthritis score for osteoarthritic mice (P<0.01). TNF­α inhibitor decreased TLR­3 and significantly increased the expression and phosphorylation of ERK and AKT in articular cartilage (P<0.01). In conclusion the results of the present study indicate that TNF­α serves an essential role in synovial fibroblasts in osteoarthritis, suggesting that inhibition of TNF­α may decrease inflammation via the TLR­3­mediated ERK/AKT signaling pathway in a mouse model of monosodium iodoacetate­induced osteoarthritis.

Overexpressed miR-145 inhibits osteoclastogenesis in RANKL-induced bone marrow-derived macrophages and ovariectomized mice by regulation of Smad3.

BACKGROUND: MicroRNAs (miRs) play an important role in osteoclastogenesis. However, no study has investigated the underlying molecular mechanisms of miR-145 in this process. The purpose of the present study was to investigate the role of miR-145 and its post-transcriptional mechanism in the progression of osteoclast differentiation. METHODS: Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) were used to induce osteoclastogenesis originated from bone marrow-derived macrophages (BMMs). Female C57BL/6J mice were divided into sham, OVX, OVX + NC-agomir and OVX + miR-145-agomir groups. Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify osteoclasts in-vitro and in-vivo. The mRNA and protein levels in osteoclast and tibia were assayed by qRT-PCR and western blotting, respectively. RESULTS: miR-145 expression was inhibited in RANKL-induced osteoclastogenesis, whereas overexpression of miR-145 attenuated it. We further found that Smad3 is a direct target gene of miR-145 by binding with its 3'-UTR. Overexpression of miR-145 significantly suppressed Smad3 mRNA and protein expression. In-vivo, miR-145 agomir treatment inhibited osteoclast activity in OVX mice by inhibiting Smad3 expression. CONCLUSION: We provide the evidence that over-expression of miR-145 could inhibit osteoclast differentiation, at least partially, by decreasing Smad3 expression.

Experimental study of super paramagnetic iron oxide labeled synovial mesenchymal stem cells.

To investigate the feasibility and changes of biological characteristics before and after synovial mesenchymal stem cells (SMSCs) labelled by super paramagnetic iron oxide (SPIO). The rabbit SMSCs were isolated, cultured, purified and identified in vitro. After adding the different concentrations of SPIO-labelled liquid, the cells were incubated 24 h in 37°C carbon dioxide incubator. The labeled-cell samples were observed by Prussian blue staining, transmission electron microscope (TEM) and the cell biology before and after the labeling was compared. The blue stained particles could be seen in the cytoplasm; the SPIO label was positive in 95% SMSC cells. With the concentration of the label liquid increasing, the blue-stained cytoplasm became darker. A large number of high electron density particles could be seen in the cytoplasm and in the pinocytosis vesicles by TEM, which suggested SPIO label positive. When the SPIO concentration was (12.5~50) µg/mL, the differences in cell proliferation and cell viability between the SMSCs after labelling and the SMSCs before labelling were not significant; when the concentration was over 100 µg/mL, the cell proliferation and cell viability were inhibited. A certain concentration range of SPIO can safely label the rabbit SMSC according to this study, which is important for solving the problem of tracing SMSCs in the joints.

[Mechanisms of autologous chondrocytes mass transplantation in the repair of cartilage defects of rabbits' knee].

OBJECTIVE: To trace the pathological changes of the cultured autologous chondrocytes mass after implanted in cartilage defects and investigate the pathophysiological mechanisms of the antologous chondrocytes mass transplantation in the repair of cartilage defects. METHODS: Twenty-four New Zealand white rabbits of 4 to 6 month-old and weighing more than 3.0 kg (female and male was unrestricted) were randomly divided into experiment group and the control group. For 12 rabbits of experiment group, the cartilage defects were repaired with the autologous chondrocytes mass and sealed with one piece of periosteum. Firstly, cartilage tissue of 10 to 30 mg was obtained from the shoulder of the rabbits after anaesthetized by 1 mg/kg 20% sumianxin. Then, chondrocytes were isolated from the cartilage tissue with 0.2% type II collagenase digestion and were cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS), 50 microg/ml ascorbic acid-2-phosphate, 0.4 mM proline, 5 microg/ml insulin and 1 mM non-essential amino acids (NEAA) in flasks in vitro. The cells were harvested until a thin film of the cells covered the bottom of the flask could be seen with naked eyes. Then the film was collected with a curled glass stick and formed a solid mass. On this time, the animal was anaesthetized again and the full-thickness cartilage square defect of 4.0 mm x 6.0 mm was fabricated in the patellar grove of distal femur, and then the cellular mass was transplanted into the defect covered by one piece of periosteum which obtained from the upper anterior of tibia and sealed with the femoral condyles. For 12 rabbits of the control group, the defects were sealed with one piece of periosteum only. The animals were sacrificed in the 1st, 3rd, 6th and 12th weeks after the operation respectively. The histologic sections were stained with safranin O-fast green, hematoxylin-eosin (H&E) and picric acid-Sirius red and immunostained for type II collagen and aggrecan. RESULTS: In the 1st week, the transplanted cells oriented to articular surface differentiated to matured hyaline chondrocytes and excrete large amount cartilage matrix. In the 3rd week, the trend was more obvious and the periosteum was union to the cell mass. In the 12th week, the defects were repaired with hyaline-like cartilage tissue, and in the 24th week, the repair tissue turned to matured hyaline cartilage. In the control group, the defects were repaired with fibrocartilage tissues. CONCLUSION: It was evidenced that the defects were repaired by the autologous chondrocytes mass transplantation. The procedure was gradual and initialed from up toward joint to down to the deep of the defect.

[Surgical treatment of previously treated thoracic and thoracolumbar spinal tuberculosis].

OBJECTIVE: To explore the surgical measurements and principles in the treatment of thoracic and thoracolumbar spinal tuberculosis. METHODS: A total of 232 cases of previously treated thoracic or thoracolumbar spinal tuberculosis in recent 7 years were retrospective analyzed. Preoperative assessments were as follows: Cobb angles of kyphosis: < 30° (n = 65), 30 - 60° (n = 147) and > 60° (n = 20); Frankel B (n = 13), C (n = 12), D (n = 41) and E (n = 166). Forty-eight cases were performed with one-stage transpedicular screw system and anterolateral debridement by single incision, 184 cases with one-stage anterior approach (debridement, fusion and plate-screw fixation) routinely. The tissues and liquor paris debrided from focus were sent for pathology, Bacillus tuberculosis detection and culture, and drug sensitivity test. The patients were given anti-tuberculosis therapy according to the results of drug sensitivity test for 1 - 1.5 years. The follow-up parameters included relapse rate, fusion of bone graft, the status of neurological restoring and kyphosis correction. RESULTS: All 232 cases recovered from perioperation and 230 cases achieved primary wound healing. Two cases undergoing single incision one-stage posterior instrumentation and anterolateral debridement were complicated with wound healing and sinus formation. There was delayed healed by changing dressings. The complications included intercostals neuralgia (n = 135) and pneumothorax or hydrothorax requiring no special measure (n = 13). The follow-up period ranged from 1.0 to 4.5 years old (mean: 2.6). There was no recurrence within the follow-up period and bone union was found in all cases. All 66 cases with neurological deficits recovered partially or totally. Kyphosis correction was achieved by 27.5° on average postoperatively and showed a mild loss of 4.2° on average during the follow-up period. All cases were confirmed pathologically as Bacillus tuberculosis infection. Bacillus tuberculosis was detected and cultured successfully in 107 cases (46.1%). Forty strains (37.4%) were drug resistant among which 8 strains (7.5%) was multi-drug resistant. CONCLUSION: For the patients with thoracic and thoracolumbar spinal tuberculosis, directional chemotherapy, one-stage anterior approach with thorough debridement, auto-rib or Ti-mesh fusion and plate-screw fixation may be the first-line therapy.

[Surgical treatment of thoracic and thoracolumbar spinal tuberculosis].

OBJECTIVE: To explore the surgical measurements and principles in the treatment of thoracic and thoracolumbar spinal tuberculosis. METHODS: From 2001 to 2008, 232 cases of thoracic or thoracolumbar spinal tuberculosis were treated by operations in the study, including 148 males and 84 females with an average age of 37.8 years ranging from 20 to 76 years. Preoperative assessment displayed as follow: Cobb angles of kyphosis < 30 degrees in 65 cases, 30 degrees to 60 degrees in 147 cases, > 60 degrees in 20 cases; Frankel B grade in 13 cases, C in 12 cases, D in 41 cases, E in 166 cases. Among them, 48 cases were performed with one-stage transpedicular screw system and anterolateral debridement by single incision, 184 cases with one-stage anterior approach (debridement, fusion, and plate-screw fixation) routinely. The tissues and liquor puris debrided from focus were sent for pathological examination, Bacillus tuberculosis detection and culture, and drug sensitivity test. The patients were given anti-tuberculosis therapy according the results of drug sensitivity test for 1 to 1.5 years. The followed-up included relapse rate, fusion of the bone graft, the status of neurological restoring, kyphosis correction etc. RESULTS: All 232 cases recovered from perioperation and 230 cases got primary wound healing, only 2 cases performed with single incision one-stage posterior instrumentation and anterolateral debridement got complications of wound healing problems and the sinus formation,which delayed healed by changing dressings. The complications included intercostals neuralgia in 135 cases and pneumothorax or hydrothorax in 13 cases, which needed not special handling. All the patients in this series got the followed-up ranging from 1.0 to 4.5 years (means 2.6 years). No recurrence within followed-up period and bone union was found in all cases. All 66 cases with the neurological deficits recovered partially or totally. Kyphosis correction were achieved by 27.5 degrees on average postoperatively and showed a mild loss of 4.2 degrees on average during followed-up period. All cases were confirmed with Bacillus tuberculosis infection by pathology. Bacillus tuberculosis was detected and culture successfully in 107 cases (46.1%), 40 strains (37.4%) were drug resistant and in which 8 strains (7.5%) were multi-drug resistant. CONCLUSION: For the treatment of thoracic and thoracolumbar spinal tuberculosis, the best treatment include directional chemotherapy, one-stage anterior approach with thorough debridement, auto-rib or Ti-mesh fusion, and plate-screw fixation.

[Surgical retreatment of thoracic and thoracolumbar spinal tuberculosis].

OBJECTIVE: To explore the surgical measures and principles in the retreatment of thoracic and thoracolumbar spinal tuberculosis. METHODS: Thirty retreatment cases of thoracic and thoracolumbar spinal tuberculosis in recent 3 years were retrospectively analyzed. The patients were hospitalized by inadequate decompression of spinal canal, tubercular toxic symptoms or sinuses. The disease course was an average of 6 months from the last operation. The patients were given anti-tuberculosis therapy according to the adjusted regimens for 2 - 8 weeks before reoperations. Ten cases were performed by anterior approach with debridement, 6 cases anterior approach (debridement, fusion & plate-screw fixation) and 4 cases dislodgment of transpedicular screw system and routine surgical treatment by anterior approach in one primary term. Debridement, Ti-mesh implantation and bone grafting, without taking out of the transpedicular screw systems was performed in 1 case of elder patient older than 70 years old with transpedicular screw system fixation. Nine cases underwent sinuses excision, debridement and dislodgment of transpedicular screw system in first attempt. After 2 - 3 weeks since incision healing, anterior approach was routinely performed. The tissues and liquor puris debrided from focus were sent for pathological examination, Bacillus tuberculosis detection and culture and drug sensitivity test. The patients were given anti-tuberculosis therapy according to the results of drug sensitivity test for 1 - 1.5 years. Follow-ups included relapse rate, fusion of bone graft and status of neurological recovery, etc. RESULTS: All 30 cases recovered from peroperation. The follow-up period ranged from 3 to 32 months (mean: 18 months). Fourteen of 21 cases with neurological deficits recovered partially or totally. All incisions had primary healing. No relapse occurred within follow-up period. All cases were confirmed with Bacillus tuberculosis infection by pathology. Bacillus tuberculosis was detected and cultured successfully in 16 cases (53.3%). Nine strains (56.3%) were drug resistant and in which 4 strains (25.0%) were multi-drug resistant. CONCLUSION: For the retreatment of thoracic and thoracolumbar spinal tuberculosis, targeted chemotherapy, thorough debridement and reasonable operative options are key to therapeutic success.

[Fabrication and characteristics of oriental poly (lactic-co-glycolic acid) scaffold: a preliminary study].

OBJECTIVE: To explore the method of fabricating oriental scaffolds and investigate the biocompatibility of the scaffolds as well as cells distribution within the scaffolds in vitro. METHODS: The oriental poly (lactic-co-glycolic acid) (PLGA) scaffolds were fabricated with modified emulsion-phase separation method. The scaffolds were treated with plasma and then anchored with collagen I. Articular chondrocytes were loaded into the scaffolds. The growth status and distributing characteristic of the cells were investigated by environmental scanning electron microscope. RESULTS: The scaffold was well compatible with the articular chondrocytes. The cells could reach to 2.5 mm depth with unilateral loading. The cells distributed evenly in the scaffold and lined along the inner pipes. CONCLUSIONS: The oriental scaffold fabricated could significantly promote the distributing characteristics of the chondrocytes. The vertical alignment of the chondrocytes within the scaffold is closely similar to that of articular cartilage.

[Biological characterization of rabbit's articular chondrocytes by confluent culture in vitro].

OBJECTIVE: To obtain large amount of differentiated chondrocytes in vitro, examine and compare the biological characterization of rabbits' articular chondrocyte cultured in different density in vitvo. METHODS: From November 2001 to June 2004, articulate tissues were obtained from the joints of the adult rabbits. Chondrocytes were isolated from the cartilage tissue with type II collagenase digestion and cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS). The chondrocytes were cultured with low density of monolayer culture and high density of confluent culture respectively. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry. RESULTS: When chondrocytes cultured in monolayer and in low density, it proliferated rapidly during the three generations, but with the same time, dedifferentiation was also rapid. After the third passage, most of the passage cells lost the phenotype, and the proliferation also stagnated. While chondrocytes cultured in high density, dedifferentiation slowed down. And even the phenotypes of the dedifferentiated chondrocyte which were cultured in low density could reduced partly by followed high density culture. CONCLUSIONS: Culture chondrocytes by high density in vitro can effectively maintain the differentiated phenotype of chondrocyte. It also keeps the proliferation character as monolayer culture. The dedifferentiated chondrocyte caused by many passages could redifferentiate partly. So it is indicated that confluent culture of original or expanded chondrocytes in high density is a better culture methods than culture in low density.

[Joint resurfacing using allograft chondrocytes embedded in alginate gel].

OBJECTIVE: To explore the feasibility of repairing the articular cartilage with allo-articular chondrocytes embedded in alginate gel. METHODS: Allo-articular chondrocytes were isolated from three adult New zealand rabbits. The cells were cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS). Chondrocytes of 2nd - 3rd passage were harvested and were diluted to 5.0 x 10(7) cells/ml with 1.2% alginate. Then alginate gel was formed by 102 mM CaCl(2). The gels were cultured subsequently for 1 week and then transferred to the full-thickness defects in the femoral condyles of adult rabbits. In control group the defects were left untreated. The animals were sacrificed in the 3rd and 6th month after operation respectively. The specimens were decalcified with 50% formic acid. The histologic sections were stained with safranin O-fast green, hematoxylin-eosin (H&E) and picric acid-Sirius red and immunohisto-stained for type II collagen and aggrecan. The repairing efficiency was evaluated according to Wakitani scoring. RESULT: In the experiment group all 8 defects acquired repair, 7/8 were repaired with mature hyaline cartilage tissue, and 1/8 was with fibrocartilage tissue for less cell-gel inputted. The thick of repaired tissues were closed to the normal and the tissue integrated smoothly with cartilage around the defects. Safranin O staining of the matrix acted in accordance with the normal and immunostaining for type II collagen and aggrecan showed positive. Picric acid-Sirius red staining showed that the chondrocytes lined in lines and the collagen aligned like Gothic architecture structure by polarization microscopy. There was no evidence of residue of alginate and inflammation in 3rd month specimens and no obvious deterioration at 6th month. But in control group, only a small amount of fibrous, fibrocartilage, or hyaline-like tissue was seen on the surface of the defects. Wakitani scoring showed 1.75 points for the experiment group and 7.65 for the control group. CONCLUSION: It is a promising way to repair the articular cartilage with homogeneous articular chondrocytes embedded in alginate gel.