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Due to good mechanical properties and biocompatibility, tissue engineering scaffolds have become the vital method for repairing and regenerating articular cartilage defects. With the continuous development of tissue engineering technology, many scaffolds preparation and formation methods have been developed and tested in the past decade, however, the preparation of ideal regenerative scaffolds remain controversial. As load-bearing tissue inside the body joints, the matrix structure and cell composition of articular cartilage are hierarchical, and there are several smooth natural gradients from the cartilage surface to the subchondral bone layer, including cell phenotype and number, specific growth factors, matrix composition, fiber arrangement, mechanical properties, nutrient and oxygen consumption. Therefore, in the design of regenerative scaffolds, it is necessary to achieve these gradients to regenerate articular cartilage in situ. In recent studies, many new biomimetic gradient scaffolds have been used to simulate the natural gradient of articular cartilage. These scaffolds show different mechanical, physicochemical or biological gradients in the structure, and have achieved good repair effects. The related articles on tissue engineering for the treatment of articular cartilage defects were retrieved by searching databases with key wordsarticular cartilage injury, cartilage repair and gradient scaffolds. In this work,the structural, biochemical, biomechanical and nutrient metabolism gradients of natural articular cartilage were studied and summarized firstly. Then, the latest design and construction of articular cartilage gradient scaffolds were classified. Besides that, the material composition (such as hydrogels, nanomaterials, etc.) and the preparation process (such as electrospinning, 3D printing, etc.) of grandient scaffolds were further enhanced. Finally, the prospect and challenge of biomimetic gradient scaffolds in cartilage engineering are discussed, which provides a theoretical basis for the successful application of gradient scaffolds in clinical transformation.
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The treatment of articular cartilage (AC) injury caused by various reasons is still a major clinical problem. The emergence of cartilage tissue engineering brings new hope for the treatment of AC injury. In general, AC tissue engineering can be divided into two categories, including cell-based tissue engineering and cell-free tissue engineering. Although cell-based tissue engineering can repair cartilage damage to a certain extent, existing therapeutic strategies still suffer from limited cell sources, high costs, risk of disease transmission, and complex procedures. However, the cell-free tissue engineering avoids these shortcomings and brings hope for in-situ AC regeneration. Non-cellular tissue engineering is mainly used to recruit endogenous stem cells/progenitor cells (SCPCs) to reach the site of cartilage injury, and provide a suitable regenerative microenvironment to promote cell proliferation and chondrogenic differentiation, then the maturation of new cartilage tissue was promoted. Therefore, it is also called as cell-homing in situ tissue engineering. Successful recruitment of endogenous SCPCs is the first step in in-situ cartilage tissue engineering. This review aims to introduce chemokine response of cartilage injury, systematically summarize traditional chemoattractant (chemokines and growth factors etc.) and emerging chemoattractant (functional peptides, exosomes and nucleic acid adapters etc.), evaluate the combination mode between chemoattractant and delivery devices, discuss the prospects and challenges of chemoattractant-mediated in situ tissue engineering and provide theoretical basis for the design of endogenous SCPCs homing-based in situ tissue engineering.
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Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide (mHSP/P) vaccine in combination with a programmed death ligand 1 (PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and en-zyme-linked immunosorbent assay (Elisa) was used to quantitatively identify the expression of heat shock proteins (HSP70, HSP90, Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide (Grp94/P) sarcoma vac-cines were isolated using chromatography and were identified by Western blot (WB). Flow cytometry was used to determine their cy-totoxic effects. The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes (HSP70, HSP90, Grp94). We successfully isolated sarco-ma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cy-totoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation (P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor (P<0.05). Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune eva-sion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.
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BACKGROUND:Soft tissue engineering mainly includes seed cells,scaffolds,cytokines and bioreactors,among which,the scaffolds are the key link in the construction of tissue-engineered cartilage.OBJECTIVE:To prepare an articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold,and to evaluate its physicochemical properties and biocompatibility.METHODS:The articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold was prepared by freeze thawing drying method using porcine articular cartilage extracellular matrix and human umbilical cord Wharton glue as raw materials.The porosity,water absorption,tissue composition and longitudinal compressive elastic modulus of the scaffold were measured and histologically stained.Rabbit chondrocytes were co-cultured with the articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold for 7 days.Then,scanning electron microscopy,live-dead cell staining and hematoxylin-eosin staining were performed.In addition,rabbit chondrocytes were cultured in the extract of the articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffold and cell culture medium for 6 days,respectively;and MTT assay was used to detect cell proliferation.RESULTS AND CONCLUSION:The articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffolds had a cross-section of uniform porous network structure and a vertical cross-section of the vertical tubular structure,and the pore wall was densely covered with cartilage fibers.The composite porous scaffold was positive for hematoxylin-eosin staining,safranin O staining and toluidine blue staining,and contained collagen and glycosaminoglycan ingredients.The water absorption,porosity and longitudinal compressive elastic modulus of the scaffolds were (17.418 8±0.909 0)％,(81.495 1±6.621 0)％ and (2.833 3±0.456 4) kPa,respectively.After 7 days of co-culture,rabbit chondrocytes adhered to the scaffold and proliferated,and further grew into the pores of the scaffold.Moreover,the scaffold was non-toxic to the rabbit chondrocytes.To conclude,the physiochemical properties and biochemical components of articular cartilage extracellular matrix/human umbilical cord Wharton gel porous scaffolds are similar to those of natural cartilage,and the scaffold has good biocompatibility.
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BACKGROUND:Accumulative evidence supports that co-culture technology can be applied to construct the tissue-engineered cartilage with excellent biological characters. OBJECTIVE:To elaborate the co-culture concept and conclude and analyze seed cell sources, cel mixed ratio, spatial y-defined co-culture models and biomaterials in co-culture systems to conclude and analyze the biological characters of tissue-engineered cartilage, and to prospect progression of co-culture systems in cartilage tissue engineering. METHODS:The first author retrieved the databases of PubMed, Web of Science, and CNKI for relative papers published from January 1976 to May 2016 using the keywords ofco-culture, co-culture systems;articular cartilage, chondrocytes, mesenchymal stem cells;tissue engineering, articular cartilage tissue engineeringin English and Chinese, respectively. Finally 60 literatures were included in result analysis, including 1 Chinese and 59 English articles. RESULTS AND CONCLUSION:Co-culture technology emphasizes the role of microenvironment in terms of various physical, chemical and biological factors in the cell processing. In cartilage tissue engineering, co-culture systems contribute to maintain the viability and natural cell phenotype of chondrocytes and induce cartilage differentiation of mesenchymal stem cells. In addition, co-culture technology provides a novel way for cartilage tissue engineering to overcome the shortage of chondrocytes and repair injury to the cartilage-subchondral bone. However, the mechanisms of cell-cell interaction in co-culture systems still need to be explored in depth, so as to optimize the co-culturing conditions and construct perfect tissue-engineered cartilage.
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BACKGROUND:Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE:To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS: Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracelular matrix in the concentration of 1×106/L to prepare cel-scaffold composites. Cel-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz, 3 hours per day, 10% compression) as experimental group for 7, 14, 24, 28 days or cultured staticaly for 1 day as control group. RESULTS AND CONCLUSION:Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P < 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradualy with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that mRNA expressions of colagen type I and colagen type II were significantly higher in the experimental group than the control group (P < 0.05). The experimental group showed the highest mRNA expression of colagen type I and colagen type II at 21 and 28 days of mechanical stimulation, respectively (P < 0.05). With the mechanical stimulation of bioreactor, the cel-scaffold composite can produce more extracelular matrix, such as colagen and proteoglycan, strengthen the mechanical properties to be more coincident with thein vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage wil be achieved.
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BACKGROUND:Magnesium can be degraded voluntarily in vivo, so a second surgery is avoided. However, its aloys have not been widely used in the clinical orthopedics because there is a lack of accurate and reliable methods to assess its degradationin vivo. OBJECTIVE:To explore the degradation of micro-arc-oxidized AZ31 magnesium aloy in the femoral condyle of rabbits based on micro-CT images and relative data. METHODS:Forty micro-arc-oxidized AZ31 magnesium aloys were implanted into the right femoral condyle of 40 New Zealand rabbits. Then 10 right femoral condyles were removed at 5, 10, 15 and 20 weeks after surgery, respectively, to quantitatively analyze and evaluate the degradation of AZ31 magnesium aloys by micro-CT images and relative data. RESULTS AND CONCLUSION:The surface of AZ31 aloys was corroded progressively with dark color and distorted appearance at 5-20 weeks post implantation. Micro-CT images showed that in the first 5 weeks, the degradation was inactive, and at the 10th week, it turned active; at the 15th week, the corrosion pits were obviously increased in number, and the corrosion area and corrosion speed were enlarged and fastened, respectively. Up to the 20th week, the aloy surfaces were ful of corrosion pits besides roughness and discontinuity. Relevant data analysis showed that the volume fraction of magnesium aloy was 98.6%, 97.1% and 86.4% at the 5th, 10th and 20th weeks after implantation, respectively, and it had a significant decrease from the 10th to 15th week and from the 15th to 20th week (P < 0.05). Within 15-20 weeks, the volume fraction of magnesium aloy was decreased by 6.5% that was the maximum volume reduction per unit cycle. With the progress of corrosion, the surface continuously became rough and vague, and its surface area was enlarged; the ratio of surface area to volume continuously increased, and there was a significant difference at 15 and 20 weeks (P < 0.05). Because of the increasing number of corrosion pits, the cross-sectional radius decreased, which was reflected by the trabecular thickness decreasing from 1.00 to 0.87 mm. From the view of the slope of curve, the trabecular thickness decreased most rapidly at 10-15 weeks. The mineral density of magnesium aloy continuously decreased from 649.302 to 356.445 mg/cm3 during the whole experiment period (P< 0.05). In addition, the micro-CT image density decreased from 679.710 to 644.947 mg/cm3, but there was no significant difference. To conclude, the degradation speed is peaked at 10-20 weeks after implantation, and the content of magnesium aloys decrease with degradation, but the magnesium density has no significant change.
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BACKGROUND:At present, a variety of extracel ular matrix-derived scaffolds have been successful y applied for cartilage tissue engineering in experiment and clinical practice. OBJECTIVE:To summarize the application and research status of extracel ular matrix-derived scaffolds in cartilage tissue engineering. METHODS:A computer-based online search in PubMed, CNKI, CqVip and WanFang databases was performed using the keywords of“tissue engineering, cartilage, extracel ular matrix, scaffolds”in English and Chinese, respectively. A total of 1 140 literatures were retrieved, and final y 65 eligible literatures were included. RESULTS AND CONCLUSION:In terms of the components, extracel ular matrix-derived scaffolds are divided into monomeric natural polymers, mixed natural polymers, natural polymers compositing with synthetic polymers as wel as acel ular extracel ular matrix-derived materials. Extracel ular matrix-derived scaffolds hold good biocompatibility and degradability, and can promote proliferation and differentiation of choncrodytes;therefore, they as good bionic scaffolds have been applied for cartilage tissue engineering in clinical practice, However, poor mechanical properties and difficulty to molding should never be ignored. Further research should focus on improving the preparation technology by combining synthetic materials with extracel ular matrix-derived scaffolds for cartilage tissue engineering.
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BACKGROUND:Cartilage extracelular matrix with a large number of signaling molecule proteins and factors is likely to be an ideal material for tissue engineering cartilage. OBJECTIVE: To investigate the possibility of calcium alginate and cartilage extracelular matrix combined with microencapsulated stem cels derived from human umbilical cord Wharton’s jely to construct ectopic tissue-engineered cartilage in nude mice. METHODS: Microfilament suspension of the cartilage extracelular matrix was prepared. Human stem cels derived from Wharton’s jely of the umbilical cord were inoculated in to calcium alginate and cartilage extracelular matrix gel microspheres as experimental group. Stem cels derived from human umbilical cord Wharton’s jely were incubated in simple alginate gel microspheres as control group. After in vitro culture, the microspheres wereimplanted into the dorsal subcutaneous tissue of nude mice. Samples were taken after 4 weeks, respectively, for gross and histological observation. RESULTS AND CONCLUSION:The stem cels exhibited paralel-chondrocyte morphology in microspheres, which grew and proliferated quite wel during in vitro culture. A new paralel-cartilaginous tissue was found in the subcutaneous tissue 4 weeks after surgery in the experimental group, and the tissue was positive for hematoxylin-eosin, safranine O, toluidine blue and colagen II. A large number of paralel-chondrocytes and cartilage lacuna-like structures were observed under a microscope with no obvious inflammatory reaction around the microspheres. The control group showed the partial degradation of microspheres, surrounded by only a smal number of inflammatory cels and lymphocytes. Calcium alginate and cartilage extracelular matrix microspheres have a rather good histocompatibility which can be used to construct paralel-cartilaginous tissues by implanting stem cel-microspheric compound into the subcutaneous tissue of nude mice.
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Objective To investigate the effects of the novel scaffold on repairing large,high-loadbearing osteochondral defects of femoral head in a canine model.Methods The biphasic scaffolds were fabricated using cartilage extracellular matrix (ECM)-derived scaffold (cartilage layer) and acellular bone matrix (bone layer) by phase separation technique.Articular high-load-bearing osteochondral defects with a diameter of 11-mm and the depth of 10-mm were created in femoral heads.The defects were treated with constructs of a biphasic scaffold seeded with chondrogenically induced bone marrow-derived mesenehymal stem cells (BMSCs).The outcomes were evaluated for gross morphology,histological,biomechanical and micro-CT analysis at the third and sixth month after implantation.Results The gross and X-ray results showed femoral head slightly collapsed at the third month and severely collapse at the sixth month.Histological analysis showed cartilage defects were repaired with fibrous tissue or fibrocartilage with severe osteoarthritis and the varied degrees of the collapse of femoral heads were presented.Micro-CT showed that the values of bone volume fraction in defect area were always lower than those of the normal area in the femoral heads.Biomechanical analysis showed rigidity of the subchondral bone in defect area was significantly lower than that in normal area in the femoral heads at the sixth month.Conclusion The ECM-derived,integrated biphasic scaffold seeded with chondrogenically induced BMSCs could not successfully repair the large high-load-bearing osteochondral defects of the femoral head.
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Objective To probe the immunological traits of mesenchymal stem cells derived from umbilical cord Wharton's jelly (WJMSCs). Methods The diced Wharton's jelly which was from healthy fullterm birth human umbilical cord was cultured. The mesenchymal stem cells were identified with mesenchymal stem cells markers expression by flow cytometry and multiple differentiation ability. The expression of MHC- Ⅰ / Ⅱ, costimulatory molecules (CD40, CD80 and CD86) was detected with flow cytomctry, immunocytochemistry, and RT-PCR. The expression of immune inhibitors like HLA-G, IDO, and PGE2 was detected by immunocytochemistry and RT-PCR. The expression of immune-related molecules as IL-10, TGF-β, FGF and VEGF was detected with antibody microarray and western blot. Further more, to clarify the in vivo immune reaction of hWJMSCs, we fabricated the hWJMSC-scaffold constructs and implanted them into the rabbit backs. The lymphocyte infiltration and implanted cell survival observed with immunofluorescence. Results After culturinge of diced Wharton's jelly tissue, we obtained spindle-shaped cells. With differentiation medium, the cells can differentiate into osteoblasts, chongdrocytes, adipose cells and schwann cells. Expression of MHC, costimulatory molecules, and a series of immune suppressive-related molecules was found. Immune inhibitors as HLA-G, 1DO, PGE2, and immune suppressive related molecules as HGF, VEGF, TGFand IL-10 were positively expressed. But the cells did not express MHC-Ⅱ. No immune rejection was observed in vivo after implantation of hWJMSC-scaffold constructs. Conclusion It can be concluded that hWJMSCs have very low immunogenicity, which means the cells have potential to induce immune tolerance.The hWJMSCs do not provoke immune rejection in vivo.
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Objective To explore the feasibility of fabricating a novel cartilage acellular matrix/chitosan hybrid scaffold for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and made into 1% suspension after decellularization. The suspension was mixed with 2% chitosan acetic acid solution, and then hybrid scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-linked and were investigated by histological staining,SEM observation, porosity measurement, water absorption rate, biomechanical properties, and biocompatibility analysis. MTT test was also done to assess the cytotoxicity of scaffold leaching liquor. Canine chondrocytes were isolated and seeded into the scaffold. Cell proliferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed no chondrocyte fragments remained in the scaffolds, and anti-col Ⅱ immunohistochemistry staining were positive. SEM observation show the scaffold has good pore interconnectivity with pore diameter (136.2±34.9) μm, 81.4%±3.5% porosity and 1525.7%±129.3% water absorption rate. The longitudinal elastic modulus of the scaffold was (1.940±0.335) MPa. MTT test showed that the scaffold leaching liquor did not exert any cytotoxic effect on BMSCs. Inverted microscope and SEM micrographs indicatod that cells covered the scaffolds uniformly, and majority of the cells showed the round or elliptic morphology with much matrix secretion. Conclusion Novel cartilage acellular matrix/chitosan hybrid scaffold had similar extracellular matrix as cartilage, good pore diameter and porosity,appropriate biomechanical character, non-toxicity and good biocompatibility, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.
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Objective To fabricate cartilage extracellular matrix (ECM) oriented scaffolds and investigate the attachment, proliferation, distribution and orientation of bone marrow mesenchymal stem cells (BMSCs) cultured within the scaffolds in vitro. Methods Cartilage slices were shattered in sterile phosphate-buffered saline (PBS) and the suspension were differentially centrifugated untill the micro- fiber of the cartilage extracellular matrix was disassociated from the residue cartilage fragments. At last the supernatant were centrifugated, the precipitation were collected and were made into 2%-3% suspension. Using unidirectional solidification as a freezing process and freeze-dried method, the cartilage extracellular matrix derived oriented scaffolds was fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution. By light microscope and scan electron microscope (SEM) observation, histological staining, and biomechanical test, the traits of scaffolds were studied. After being labelled with PKH26 fluorescent dye, rabbit BMSCs were seeded onto the scaffolds. The attachment, proliferation and differentiation of the cells were analyzed using inverted fluorescent microscope. Results The histological staining showed that toluidine blue, safranin O, alcian blue and anti-collagen Ⅱ immunohistochemistry staining of the scaffolds were positive. A perpendicular pore-channel structures which has a diameter of 100 μm were verified by light microscope and SEM analysis. The cell-free scaffolds showed the compression moduli were (2.02±0.02) MPa in the mechanical testing. Inverted fluorescent microscope showed that most of the cells attached to the scaffold. Cells were found to be widely distributed within the scaffold, which acted as a columnar arrangement. The formation of a surface cells layer was found on the surface of the scaffolds which resembled natural cartilage. Coclusion The cartilage extracellular matrix derived oriented scaffolds have promising biological, structural, and mechanical properties.
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BACKGROUND:Choose an ideal vector for amplified chondrocytes arouses more and more attention during the construction of epiphyseal cartilage tissue engineering. OBJECTIVE:To explore the feasibility and performance of epiphyseal cartilage tissue engineering scaffold constructed by chitosan and extracellular matrix of cartilage. DESIGN,TIME AND SETTING:An in vitro study was performed at Department of Orthopedics,General Hospital of Chinese PLA from December 2007 to March 2003. MATERIALS:Chitosan(deacetylation:90%;Mr10?105) was provided by Haihui Bioengineering Company,Qingdao;articular cartilage of swine was collected from market. METHODS:Fresh porcine articular cartilages were obtained and shattered in the iso-osmia liquid. After pulverization and gradient centrifugation,3% artilage microfilament suspension was equally mixed with 2% chitosan. Three-dimensional porous scaffolds were fabricated using a simple freeze-drying method. MAIN OUTCOME MEASURES:After the second gradient ethanol treatment,the scaffolds were investigated by histological staining and scanning electron microscopy to measure aperture,porosity,and water absorption rate. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by transforming growth factor-?1(TGF-?1) ,bone marrow mesenchymal stem cells(BMSCs) of rabbits were incubated onto the scaffolds. Cell proliferation and differentiation were analyzed using inverted microscopy and scanning electron microscopy. RESULTS:The three-dimensional porous scaffold had good pore interconnectivity with pore diameter(161?31) ?m,(90.1?1.6) % porosity and(2 361?132) % water absorption rate. The histological staining showed that toluidine blue,safranin O and anti-collagen II immunohistochemistry staining were positive. The intrinsic cytotoxicity assessment of the scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Most of the BMSCs attached and covered the surface of the scaffolds with matrix secretion. CONCLUSION:The three-dimensional porous scaffold constructed by extracellular matrix of cartilage and chitosan has good pore diameter and porosity,non-toxicity and good biocompatibility,so it is a suitable scaffold for epiphyseal cartilage tissue engineering.
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0.05).[Conclusion]Porcine articular cartilage extracellular matrix derived scaffold used in heterogenic transplantation or allograft have no immunologic rejection.This research provides a feasibility for application of xenogenic acellular cartilage to clinic.
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[Objective]To analyze factors related with surviving rate and to evaluate effectiveness of the adjuvant chemotherapy in the treatment of osteosareoma.[Method]Eighty-four patients aging from 9 to 47 years(averaged,21 years)were analysed respectively:52 of them were male and 32 were female.The tumors were located at the femur in 42,the tibia in 29,the humerus in 13.There were 22 patients classified as stage ⅡA and 62 patients as ⅡB.The pathological study,of subtype of osteosarcoma revealed that 47 were osteoblastic,11 chondroblastic,19 fibroblastic and 7 other subtypes.There were 46 patients who received the chemotherapy;38 patients without chemotherapy,49 of the 84 patients treated surgically had limb salvage procedures,35 had amputations.Multivariate analsis was done by using the proportional hazards model of Cox,categoric data were analyzed by using the chi-square statistic.[Result]All cases were followed up from 6 to 74 months(with an average of 25.5 months).Cox model analysis showed that age,sex,site,and subtype were not significant prognostic variables in this group of patients;the significant affecting prognosis in patients was Enncking staging and chemotherapy.Chi-square showed significant difference in the higher metastasis rates of lung in the group without chemotherapy than in those with chemotherapy group(P
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[Objective]To establish a method for obtaining well-differentiated goat chondrocytes quickly in large scale by RCSS using microcarriers technique. [Method]Articular chondrocytes were harvested from goat by sequential digestion with trypsin and collagenase , and then grown in RCSS bioreactor culture system which contained the Cytodex-3 microcarriers in the culture medium (DMEM) . Growth of chondrocytes on Cytodex-3 microcarriers was observed dynamically under phase contrast microscope . Immunocytochemical analysis was performed for type Ⅰcollagen and type Ⅱcollagen. [Result]The articular chondrocytes attached rapidly to the surface of Cytodex-3 microcarriers. Quick growth of these cells was observed after they fully spread onto the microcarriers. The density of chondrocytes increased by 15～17 times in later stage of culture as compared with the initial density. The harvested chondrocytes had no detectable staining for collagen type Ⅰ, but stained intensively for collagen type Ⅱ. [Conclusion]Microcarrier culture of chondrocytes can yield a large quantity of goat cells within a short time ,which will be of benefit for banking cartilage cells for reconstruction of impaired cartilage by way of tissue engineering.