Treatment of articular cartilage lesions remains a clinical challenge. The uses of prosthetic joint replace allograft and/or autograft transplant carry a risk of complications due to infection, loosening of its component, immunological rejection and morbidity at the donor site. There has been an increasing interest in the management of cartilage damages, owing to the introduction of new therapeutic options. Tissue engineering as a method for tissue restoration begins to provide a potential alternative therapy for autologous grafts transplantations. We aimed to evaluate how well a tissue engineered neocartilage implant, consist of human articular chondrocytes cultured with the presence of autologous serum and mixed in a fresh fibrin derived from patient, would perform in subcutaneous implantation in athymic mice.
Biomechanics ; Cartilage, Articular/injuries ; Cartilage, Articular/physiology ; Cartilage, Articular/*transplantation ; Chondrocytes/*cytology ; Culture Media ; Mice, Nude ; *Orthopedic Procedures ; Serum ; *Tissue Engineering

Culture media supplemented with animal serum e.g. fetal bovine serum; FBS is commonly used for human culture expansion. However, for clinical application, FBS is restricted as its carry a risk of viral or prion transmission. Engineering autologous cartilage with autologous human serum supplementation is seen as a better solution to reduce the risk of transmitting infectious diseases and immune rejection during cartilage transplantation. The purpose of this study is to establish and compare the effects of 10% autologous human serum (AHS) and 10% FBS on the growth of chondrocytes and the formation of tissue engineered human articular cartilage.
Cartilage, Articular/growth & development ; Cartilage, Articular/*transplantation ; Cell Count ; Cell Division/physiology ; Chondrocytes/*cytology ; Culture Media ; *Serum ; *Tissue Engineering

OBJECTIVETo obtain large amount of differentiated chondrocytes in vitro, examine and compare the biological characterization of rabbits' articular chondrocyte cultured in different density in vitvo.

METHODSFrom 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.

RESULTSWhen 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.

CONCLUSIONSCulture 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.

Animals ; Cartilage, Articular ; cytology ; Cell Culture Techniques ; methods ; Cells, Cultured ; Chondrocytes ; cytology ; Female ; Male ; Rabbits

BACKGROUNDChondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure. Microfilaments, microtubules, and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes. The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.

METHODSChondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n = 24). The isolated cells were stabilized for three days and then exposed to low, medium, and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest: colchicine for microtubules, acrylamide for intermediate filaments, and cytochalasin D for actin microfilaments. A group of control cells were treated with carrier. Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents. The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3. The mRNA levels of aggrecan (AGG) and type II collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzyme-linked immunosorbent serologic assay (ELISA) on days 3, 6, and 9.

RESULTSIn the initial drug-dose-response study, there was no significant difference in the vitality of cells treated with 0.1 µmol/L colchicine, 2.5 mmol/L acrylamide, and 10 µg/L cytochalasin D for two days when compared with the control group of cells. The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei. Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three, six, and nine days, and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells. Cellular proliferation, monolayer morphology, and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.

CONCLUSIONSThe disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis, increased matrix metalloprotease expression, and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures. Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Chondrocytes ; cytology ; Collagen ; metabolism ; Cytoskeleton ; metabolism ; Knee Joint ; cytology ; metabolism ; Microscopy, Confocal ; Rabbits

OBJECTIVETo evaluate the use of alginate three-dimensional culture system for mandibular condylar chondrocytes culture in vitro from human osteoarthritic temporomandibular joint.

METHODSThe cultured mandibular condylar chondrocytes from the operatively removed cartilage from a patient with osteoarthritic temporomandibular joint were harvested by mechanical dissection and enzyme digestion. Partial chondrocytes were suspended in the aqueous sodium alginate beads with high seeding density and cultured for 4 weeks, while the others were cultured in monolayer culture condition for 1 week. Thereafter, the alginate beads were embedded in paraffin and sectioned, then studied immunologically with type II collagen antibody and aggrecan antibody, same studies were adopted for the monolayer cultures.

RESULTSThe monolayer cultures were confirmed as chondrocytes. The chondrocytes cultured in the alginate medium showed well. These cells exhibited the excellent differentiated phenotype after 4 weeks culture in alginate gel.

CONCLUSIONSThe condylar chondrocytes from human osteoarthritic temporomandibular joint were successfully cultured in vitro. The alginate three-dimensional culture system was successfully adopted for in vitro culture of condylar chondrocytes from human osteoarthritic temporomandibular joint, in which the chondrocytes exhibited the excellent differentiated phenotype.

Cartilage, Articular ; Cell Culture Techniques ; Cells, Cultured ; Chondrocytes ; cytology ; Humans ; Mandibular Condyle ; Temporomandibular Joint

Cartilage, Articular ; cytology ; injuries ; Cells, Cultured ; Chondrocytes ; transplantation ; Humans ; Transplantation, Autologous ; Wound Healing

The transforming growth factor-beta 1 was known as having the most important influence on chondrocytes among various growth factors, being abundant in articular chondrocytes and osteocytes. We performed in vitro monolayer cultures of human articular chondrocytes from normal and osteoarthritic patients and studied the transforming growth factor-beta 1 responsiveness of those chondrocytes. The cell-growth curve indicated that the primary osteoarthritic chondrocyte culture with transforming growth factor-beta 1 showed a more rapid growth pattern than normal chondrocytes with or without TGF-beta 1 and osteoarthritic chondrocytes without TGF-beta 1. The osteoarthritic group showed a sharp decline in growth pattern with subsequent culture. The shape of osteoarthritic chondrocytes was bigger and more bizarre compared to those of normal chondrocytes. With subsequent culture, this change became prominent. The transforming growth factor-beta 1 increased the [3H]-TdR uptake in each group. The phenotypes of chondrocytes were more clearly expressed in the normal group. The chondrocytes lost their phenotype (production of collagen type II) following subculture in each group. The transforming growth factor-beta 1 could not inhibit or delay the dedifferentiation process (loss of phenotype).
Cartilage, Articular/drug effects* ; Cartilage, Articular/cytology ; Cell Division/drug effects ; Cells, Cultured ; Human ; Osteoarthritis/pathology ; Reference Values ; Transforming Growth Factor beta/pharmacology*

To investigate the estrogen receptor (ER) expression in cartilage cell in the development of osteoarthritis induced by bilateral ovariectomy in guinea pig and to find their relationship. 30 two-month-old female guinea pigs were randomly divided into two groups (n = 15 each): sham operation (control) group and ovariectomized group (OVX); Scanning electorne microscope (SEM) and transmission electron microscope (TEM) were obtained to analysis the cartilage degeneration of the hind limb knee joint after 6 and 12 weeks of ovariectomy. Dextran-Coated-Charcoal (DCC) was taken to quantitively detect the expression of ER. The serum levels of estrogen and gestone were detected by immune contest assay. The results showed that ER do exist in the cartilages of the guinea pigs, with higher expression in the control group than in OVX group at the same time point (P < 0.05). It was increased also at 12 th week after operation than that of preoperation. The blood serum levels of estrogen and gestone showed a similar tendency to the expression of ER. Joint cartilage degeneration detected by SEM and TEM could be found at 6 th week, but severe degenerative lesions at 12 th week in the OVX group compared with the control group (P < 0.01). The data suggested that bilateral ovariectomy in guinea pig lead to severe osteoarthritis which mighgt be related to the lower serum level of estrogen and the downregulation of the expression of ER in the cartilage also.
Cartilage, Articular/cytology ; Cartilage, Articular/*metabolism ; Chondrocytes/metabolism ; Estrogens/*blood ; Osteoarthritis/*etiology ; Osteoarthritis/metabolism ; Ovariectomy ; Random Allocation ; Receptors, Estrogen/*biosynthesis ; Receptors, Estrogen/genetics

OBJECTIVETo explore the method of fabricating oriental scaffolds and investigate the biocompatibility of the scaffolds as well as cells distribution within the scaffolds in vitro.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONSThe 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.

Cartilage, Articular ; cytology ; Cells, Cultured ; Chondrocytes ; cytology ; Glycolates ; Humans ; Lactic Acid ; Materials Testing ; Polyglycolic Acid ; Tissue Scaffolds

OBJECTIVETo investigate the effects in vitro of 17 beta-estradiol (E2) on the proliferation and metabolism of rabbit mandibular condylar cartilage cells.

METHODSChondrocytes were derived from neonatal rabbit mandibular condylar cartilage using a modified enzyme method. 17 beta-estradiol was added to the culture medium in a variety of concentrations. Cell growth and DNA, collagen, and proteoglycan synthesis were used as indicators of proliferation and differentiation of condylar chondrocytes. These were measured by cell number, 3H-proline and 35S-incorporation, respectively.

RESULTSE2 increased cell number and 3H-thymidine incorporation at 10(-8) to 10(-10) mol/L, and 10(-8) to 10(-11) mol/L in a dose-dependent manner, peaking at 10(-8) mol/L and 10(-9) mol/L, respectively. However, further increase in the concentration of estradiol caused inhibition of both cell number and 3H-thymidine incorporation, and this was significant at 10(-6) mol/L. The effect of E2 on proteoglycan synthesis was similar; the maximum stimulating effect was at 10(-8) mol/L, and inhibition was significant at 10(-6) mol/L. There was no obvious stimulatory effect of E2 on 3H-thymidine incorporation observed.

CONCLUSIONSEstradiol affects condylar chondrocyte cell growth, DNA, and proteoglycan synthesis in a biphasic manner depending on its concentration. This indicates that estrogen may be important in the proliferation and differentiation of mandibular condylar chondrocytes, and could be relevant to some aspects of certain temporomandibular joint diseases by modulating the function of the chondrocytes.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Cell Differentiation ; Cell Division ; drug effects ; Cells, Cultured ; Estradiol ; pharmacology ; Mandibular Condyle ; cytology ; metabolism ; Rabbits