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.
Mechanical signal transduction are crucial for chondrocyte in response to mechanical cues during the growth, development and osteoarthritis (OA) of articular cartilage. Extracellular matrix (ECM) turnover regulates the matrix mechanical microenvironment of chondrocytes. Thus, understanding the mechanotransduction mechanisms during chondrocyte sensing the matrix mechanical microenvironment can develop effective targeted therapy for OA. In recent decades, growing evidences are rapidly advancing our understanding of the mechanical force-dependent cartilage remodeling and injury responses mediated by TRPV4 and PIEZOs. In this review, we highlighted the mechanosensing mechanism mediated by TRPV4 and PIEZOs during chondrocytes sensing mechanical microenvironment of the ECM. Additionally, the latest progress in the regulation of OA by inflammatory signals mediated by TRPV4 and PIEZOs was also introduced. These recent insights provide the potential mechanotheraputic strategies to target these channels and prevent cartilage degeneration associated with OA. This review will shed light on the pathogenesis of articular cartilage, searching clinical targeted therapies, and designing cell-induced biomaterials.
Objective To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro andto investigate the possibility of using cultured cartilage as a substitute for meniscus.Methods Chondrocytes isolated from articular cartilage and meniscus of rabbits aged 3 weeks were respectively passaged in monolayer and cultured in centrifuge tube. Cartilages cultured in centrifuge tube and meniscus of rabbit aged 6 weeks were detected by histological examination and transmission electron microscopy. Growth curves of articular chondrocytes and meniscalfibrochondrocytes were compared; meanwhile, cell cycles of articular chondrocytes and meniscal fibrochondrocytes in passage 2and 4 were separately measured by flow cytometry.Results Articular chondrocytes in passage 4 were dedifferentiated. Articular chondrocytes formed cartilage 2 weeks after cultivation in centrifuge tube, but meniscal fibrochondrocytes could not generate cartilage. The differences in ultrastructure and histology obviously existed between cultured cartilage and meniscus; moreover, apoptosis of chondrocytes appeared in cultured cartilage. Proportion of subdiploid cells in articular chondrocytes passage 2 and 4 was markedly higher than that in passage 2 and 4 fibrochondrocytes(Plt;0.05). Conclusion Meniscal fibrochondrocytes can not form cartilage after cultivationin centrifuge tube, while cartilage cultured in centrifuge tube from articular chondrocytes can not be used as graft material for meniscus. Articular cartilage ismarkedly different from meniscus.
Objective To investigate the role of transforming growth factor β(TGF-β)in the regulation of the gene expression of matrix metalloproteinase 13(MMP-13)in the human hyaline chondrocytes. Methods The human hyaline chondrocytes harvested enzymatically and cultured in DMEM supplemented with 20% fetus calf serum were divided into 7 groups. Group 1 was used as a contol, and 1 ng/ml TGF-β(group 2), 10 ng/ml TGF-β(group 3), 100 ng/ml TGF-β(group 4), 1 ng/ml TGF-β＋10 ng/ml IL-1β(group 5), 10 ng/ml TGF-β＋10 ng/ml IL-1β(group 6),and 100 ng/ml TGF-β＋10 ng/ml IL1β(group 7) were given for 12-hour coculture. The MMP-13 mRNA levels of passaged human hyaline chondrocytes were assessed by reverse transcriptionpolymerase chain reaction(RT-PCR) and real-time fluorescent quantitative PCR. Results TGF-β can increase the MMP-13 mRNA level respectively in the passagedhyaline chondrocytes. In the multifactor treated groups, TGF-β can decrease the MMP-13 mRNA level respectively and there was significant difference between groups (Plt;0.05).The level of MMP-13 mRNA expression had significant coherence withthe dosage of TGF-β. Conclusion The above results show that human chondrocytes express MMP-13 mRNA. TGF-β could cause a dosedependent stimulation on MMP-13 gene expression in human chondrocytes and have a potent effect of antagonizing IL-1β in osteoarthritis. TGF-β may play a crucial role in the occurrence anddevelopment of osteoarthritis through regulating MMP-13.
ObjectiveTo assess the role and effect of Wharton's jelly of human umbilical cord oriented scaffold on chondrocytes co-cultured in vitro.
MethodsChondrocytes from shoulder cartilage of adult New Zealand rabbits were isolated,cultured,amplified,and labelled using fluorescent dye PKH26.Cells were extracted from human umbilical cord tissue using wet-grinding chemical technology to prepare the Wharton's jelly of human umbilical cord oriented scaffold by freeze-drying and cross-linking technology.Second generation of chondrocytes were cultured with Wharton's jelly of human umbilical cord oriented scaffold.Inverted microscope and scanning electron microscope (SEM) were used to observe the cell distribution and adhesion on the scaffold; extracellular matrix secretion of the chondrocytes were observed by toluidine blue and safranin O staining.Cells distribution and proliferation on the scaffold were assessed by fluorescein diacetate-propidium iodide (FDA-PI) and Hoechst33258 staining.The viability of the in vitro cultured and PKH26 fluorescence labelled chondrocytes on the scaffold were assessed via fluorescence microscope.
ResultsInverted microscope showed that the cells cultured on the scaffold for 3 days were round or oval shaped and evenly distributed into space of the scaffold.SEM observation showed that large number of cultured cells adhered to the pores between the scaffolds and were round or oval shape,which aggregated,proliferated,and arranged vertically on longitudinally oriented scaffold at 7 days after culture.Histological observation showed that cells distributed and proliferated on the scaffold,and secreted large amount of extracellular matrix at 7 days.Scaffold could guide cell migration and proliferation,and could effectively preserve and promote the secretion of extracellular matrix.Cell viability assessments at 3 days after culture showed most of the adhered cells were living and the viability was more than 90%.PKH26 labelled chondrocytes were seen,which distributed uniformly along the pore of oriented scaffold,and exuberantly proliferated.
ConclusionWharton's jelly of human umbilical cord oriented scaffold favors adhesion,proliferation,and survival of chondrocytes.It possesses a favorable affinity and cell compatibility.Thus,it is an ideal scaffold for cartilage tissue engineering.
Objective To observe the replicative senescence of rat articular chondrocyte cultured in vitro so as to provide reference for the succeeding experiment of using medicine interfere and reverse the cataplasia of tissue engineering cartilage or probing cataplasia mechanism．Methods Different generations(P1, P2, P3 and P4) of the chondrocytes were detected with the methods of histochemistry for β-galactosidase (β-gal)， electronmicroscope for ultromicrostructure， immunocytochemistry for proliferating cell nuclear antigen (PCNA)，alcian blue stain for content and structure of sulfatglycosaminoglycan (GAG) of extracellular matrix (ECM)，reverse transcriptionpolymerase chain reaction (RTPCR) for content of collagen Ⅱ，flow cytometry for cell life cycle and proliferative index(PI) to observe senescence of chondrocytes．Results In the 4th passage，the chondrocytes emerging quantitively positive express of β-gal，cyto-architecture cataplasia such as caryoplasm ratio increasing and karyopycnosis emerging under electronmicroscope ，cell life cycle being detented on G1 phase(83．8％)，while in P1, P2, P3 the content of G1 phase was 79.1%, 79.2%, 80.8% respectively. In the 4th passage, PI decreased(16．2％)，while in P1, P2, P3, it was 20.9%, 20.8%, 19.2%. The positive percentage of PCNA，the content of GAG(long chain molecule) and the positive expression of collagen Ⅱ diminished,all detections above were significantly different (Plt;0.01) when compared the 4th passage with the preceding passages．Conclusion Chondrocytes show the onset of senescence in the 4th passage．
ObjectiveTo investigate the mechanism of Semaphorin 3A (Sema3A) in fracture healing after nerve injury by observing the expression of Sema3A in the tibia fracture healing after traumatic brain injury (TBI).
MethodsA total of 192 Wistar female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into tibia fracture group (group A, n=48), TBI group (group B, n=48), TBI with tibia fracture group (group C, n=48), and control group (group D, n=48). The tibia fracture model was established at the right side of group A; TBI model was made in group B by the improved Feeney method; the TBI and tibia fracture model was made in group C; no treatment was given in group D. The tissue samples were respectively collected at 3, 5, 7, 14, 21, and 28 days after operation; HE staining, immunohistochemistry staining, and Western blot method were used for the location and quantitative detection of Sema3A in callus tissue.
ResultsHE staining showed that no obvious changes were observed at each time point in groups B and D. At 3 and 5 days, there was no obvious callus growth at fracture site with inflammatory cells and fibrous tissue filling in groups A and C. At 7 and 14 days, fibrous tissue grew from periosteum to fracture site in groups A and C; the proliferation of chondrocytes in exterior periosteum gradually formed osteoid callus at fracture site in groups A and C. The chondrocyte had bigger size, looser arrangement, and more osteoid in group C than group A. Group B had disorder periosteum, slight subperiosteal bone hyperplasia, and no obvious change of bone trabecula in group B when compared with group D. At 21 and 28 days, cartilage callus was gradually replaced by new bone trabecula in groups A and C. Group C had loose arrange, disorder structure, and low density of bone trabecula, big callus area and few chondrocyte and osteoid when compared with group A; group B was similar to Group D. Immunohistochemistry staining showed that Sema3A expression in chondrocytes in group C was higher than that in group A, particularly at 7, 14, and 21 day. Sema3A was significantly higher in osteoblasts of new bone trabecula in group A than group C, especially at 14 and 21 days (P<0.05). Western blot results showed that the Sema3A had the same expression trend during fracture healing in groups A and C. However, the expression of Sema3A protein was significantly higher in group C than group A (P<0.05) and in group B than group D (P<0.05) at 7, 14, 21, and 28 days.
ConclusionAbnormal expression of Sema3A may play a role in fracture healing after nerve injury by promoting the chondrocytes proliferation and reducing the distribution of sensory nerve fibers and osteoblast differentiation.
ObjectiveTo summarize the research progress of pathological manifestations and mechanism of endochondral ossification in osteoarthritis (OA).
MethodsThe literature about endochondral ossification, bone-cartilage remodeling in OA, and joints development was reviewed, analyzed, and summarized.
ResultsChondrocyte hypertrophy and apoptosis, vascular invasion, replication of the tidemark, thickening calcified cartilage, and thinning superficial cartilage are the characteristics of cartilage degeneration in OA. Articular cartilage and growth plate are similar in structure, and cartilage degeneration in OA is similar to a process of endochondral ossification of the growth plate.
ConclusionLoss of stability characterization from resting metabolic balance to a high conversion state of temporary cartilage in stimulation of abnormal mechanical stresses and cytokines would subsequently contributed to continual calcification and remodeling of articular cartilage, which may be the key link of the initiation and development of OA.
Objective To explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects. Methods SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articularcartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multipl ication method were diluted by sodium alginate to (5-7) × 107 cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n=20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was establ ished in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation. Results Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the inferface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in theimplant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly andactive cell metabol ism and prol iferation were evident 9 months after operation. While in the control group, no repair of thedefect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P lt; 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P lt; 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P lt; 0.05). Conclusion Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in l ine with physiological repair process of articular cartilage.
Objective To establish a kind of gene therapy method of rheumatoid arthritis, to construct the interleukin-18-PE38 fusion gene expression vectorand to explore the expression of the fusion gene in the chondrocytes and 3T3 cells. Methods Interleukin-18-PE38 fusion gene was cleaved from plasmid PRKL459k-IL-18-PE38 by restriction enzyme digestion,then linked with vectors PsecTag2B and transformed into competence bacteria, positive clones were selected and confimed by restrictive enzyme(EcoRI) digestion assay. The rearrangement plasmid PsecTag2B-IL-18-PE38 was transfected into 3T3 cells and mouse chondrocytes by liposome protocol(experimental group),null vector was used as negative control, and the transient expression was identified by fluorescence immunocytochemical assay. Results Restrictive enzymes digestion analysis revealed thatthe length of theinterleukin-18-PE38 fusion gene was 6 000 bp. Fluorescence immunocytochemical method showed that fluorescence intensity of the experimental group is b,whilefluorescence intensity of the control group is weak. Conclusion the eukaryoticexpression vector PsecTag2B-IL-18-PE38 is established successfully which canbeexpressed in the 3T3 cells and mouse chodrocytes. Our results lay a foundationfor the further investigation for rheumatoid arthritis therapy.
