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

The growth plate is responsible for longitudinal bone growth. The problem of repair of damaged growth plate in children has never been adequately solved. The purpose of this study is to investigate the ability of the cultured chondrocyte for the prevention of bony bridge and repairment of damaged growth plate. Chondrocytes were obtained from the new born canine epiphyseal plate and was cultured in high density. Fourteen days later they formed micromass easily removable from the culture flask. Twenty dogs were divided into two groups; in group I, the medial proximal tibial growth plate was destroyed and then cultured chondrocytes were transplanted into the defect, and in group II, the medial proximal tibial growth plate was left in destroyed state. Each left leg was remained as a control. The growth pattern was observed radiographically and histologically until 16 weeks after graft. 4 weeks after the operation, the angular deformity had been observed, and 31 degrees of angulation was noted at the 16th week in group II, while there was less than 8 degrees of angulation and nearly normal growth in most of dogs of group I (8 of 10 dogs). The other 2 dogs had shown 20 degrees angulation. In group II, there was definite bony bridge on the medical proximal growth plate. In group I, initially, the cultured chondrocyte remained as a amorphous cartilagenous mass, but as time progressed, amorphous cartilagenous mass had formed cartilagenous matrix which was proved by Safranin-O staining. Although this study showed the role of cultured chondrocyte as a method of preventation of bony bridge formation and possibility to repair of growth plate, further studies should be done to prove the reconstruction of the growth plate.
Animal ; Cartilage/*cytology ; *Cell Transplantation ; Cells, Cultured ; Dogs ; Growth Plate/injuries/*surgery ; Transplantation, Homologous

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*

OBJECTIVETo study the effect of growth hormone (GH) on the proliferation and type II collagen secretion of chondrocytes of mandibular condyle in rabbit in vitro.

METHODSFlow cytometry (FCM) and immunohistochemical technique were employed to observe the possible changes.

RESULTS(1) The exogenic GH can enhance the proliferation and synthesis of DNA of the chondrocytes of mandibular condyle in rabbit in vitro. The suitable concentration of GH is 10 microg/ml. The synthesis of DNA reaches the highest level after 12 hours, while the proliferation index (PI) hits the highest after 24 hours. (2) GH (10 microg/ml) can stimulate the secretion of type II collagen of the chondrocytes.

CONCLUSIONThe exogenic GH can enhance the proliferation, the synthesis of DNA and the secretion of type II collagen of the chondrocytes of mandibular condyle in rabbit in vitro.

Animals ; Cartilage, Articular ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chondrocytes ; drug effects ; secretion ; Collagen Type II ; drug effects ; Growth Hormone ; pharmacology ; Mandibular Condyle ; cytology ; Rabbits

OBJECTIVETo evaluate the use of cancellous bone matrix gelatin (BMG) combined with chondrocytes in constructing tissue-engineered cartilage by observing the growth, proliferation and differentiation of chondrocytes on allogeneic cancellous BMG.

METHODSThe articular chondrocytes isolated from a 1-month-old rabbit were multiplied to a monolayer and seeded onto cancellous BMG to construct tissue-engineered cartilage in vitro during a period of 6 weeks. Samples were taken from the construct after 1, 2, 4, and 6 weeks of culture and evaluated by histology, immunohistochemistry and transmission electron microscopy (TEM).

RESULTSThe chondrocytes excreted matrix proteoglycan and collagen on cancellous BMG. With the prolongation of the culture time, the cells proliferated in the construct and the cells in the lacunae increased. Numerous chondrocytes were present the central region of the cancellous BMG and surrounded by extracellular matrix. By 6 weeks of culture, the BMG was covered with 15-20 layers of chondrocytes and cartilaginous tissue occurred in the pores throughout the cancellous BMG. Immunohistochemical staining showed rich and evenly distributed type II collagen around the chondrocytes, and TEM revealed an ultrastructure of the chondrocyte similar to that of native chondroctyes, with abundant extracellular matrix produced around the cells.

CONCLUSIONTissue-engineered cartilage can be constructed in vitro using allogeneic cancellous BMG combined with chondrocytes. Allogeneic cancellous BMG serves as a good scaffold material for tissue-engineered cartilage to promote the growth and proliferation of the seeded chondrocytes and allows maintenance of the differentiation phenotype of the cells.

Absorbable Implants ; Animals ; Bone Matrix ; chemistry ; Cartilage ; cytology ; growth & development ; Cells, Cultured ; Chondrocytes ; cytology ; physiology ; Gelatin ; chemistry ; Rabbits ; Tissue Engineering ; methods ; Tissue Scaffolds

Osteogenesis and angiogenesis are two closely correlated processes during bone growth, development, remodelling and repair.Vascular endothelial growth factor (VEGF) is an essential mediator during the process of angiogenesis. Based on an extensive literature search, which was carried out using the PubMed database and the keywords of osteogenesis, VEGF, endochondral ossification and intramembranous ossification, this manuscript reviews the role of VEGF in ossification, with emphasis on its effect in endochondral and intramembranous ossification. Osteogenesis and angiogenesis are closely correlated processes. VEGF acts as an essential mediator during these processes. It not only functions in bone angiogenesis but also in various aspects of bone development.
Animals ; Bone Remodeling ; physiology ; Bone and Bones ; cytology ; physiology ; Calcification, Physiologic ; physiology ; Cartilage ; cytology ; physiology ; Humans ; Neovascularization, Physiologic ; physiology ; Osteoclasts ; physiology ; Osteogenesis ; physiology ; Vascular Endothelial Growth Factor A ; physiology

OBJECTIVEThe purpose of this study was to investigate effects of static tension-stress and TGF-beta 1 on proliferation of mandibular condylar chondrocytes in vitro.

METHODSThe fourth-passage chondrocytes were harvested from the mandibular condyles of 2-week-old SD rats for this study, and a cellular static tension-stress device was used to apply stress on cells. The effects of continuous static tension-stress and/or transforming growth factor-beta 1 (TGF-beta 1) on the proliferation of mandibular condylar chondrocytes were examined using flow cytometry. The experiment was divided into two parts. The first part consisted of 100 specimens which were divided into 20 groups with different TGF-beta 1 dosage (0 ng/ml, 0.1 ng/ml, 1 ng/ml and 10 ng/ml) for 0, 6, 12, 18 and 24 hours respectively. The second part consisted of 30 specimens which were divided into six groups under continuous static tension-stress (5 kPa) and different TGF-beta 1 dosage (0.1 ng/ml, 1 ng/ml, 10 ng/ml) for 0, 6 and 12 hours. Multivariable analyses were conducted to test for associations between proliferation of mandibular condylar chondrocytes and TGF-beta 1 and/or different stresses.

RESULTSThe results showed that TGF-beta 1 had a mitogenic effect on rat mandibular condyle at the concentrations of 0.1, 1 and 10 ng/ml, and the mitogenic effects of TGF-beta 1 on condylar chondrocytes were demonstrated in 12 to 18 hours after application of stresses, and the peak of mitogenic effects appeared at 18 hour (P < 0.05). The most active mitogenesis happened in the group with continuous static tension-stress (5 kPa) combined with TGF-beta 1.

CONCLUSIONThese results prove mechanical stimulates and TGF-beta 1 in vitro could influence and regulate the growth of condylar chondrocytes.

Animals ; Cartilage, Articular ; cytology ; Cell Division ; Cells, Cultured ; Chondrocytes ; cytology ; Mandibular Condyle ; cytology ; Orthodontic Appliances, Functional ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factor beta1

The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-beta 1 genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The full-length rat TGF-beta 1 cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418, a synthetic neomycin analog. The transient and stable expression of TGF-beta 1 by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently transfected MSCs in vitro with the TGF-beta 1 gene causing a marked up-regulation in TGF-beta 1 expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were susceptible to in vitro lipofectamine mediated TGF-beta 1 gene transfer and that transgene expression persisted for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, i.e. molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineering, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness articular cartilage defects that occur as a result of injury and osteoarthritis.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cartilage, Articular ; cytology ; Cells, Cultured ; Chondrocytes ; cytology ; Gene Transfer Techniques ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; Stem Cells ; cytology ; metabolism ; Tissue Engineering ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics

OBJECTIVESeed cell study is an essential area in the research of tissue engineering. To evaluate the potentiality of marrow stromal cell(MSCs) as seed cell in the regeneration of tissue engineered cartilage, formation of cartilage nodules by culture expanded MSCs pellets under the induction of TGF-beta was investigated.

METHODSMSCs were cultured and expanded in vitro. Cell pellets containing 1 x 10(6) MSCs were obtained by centrifuging MSCs solution at 1,000 r/min in 5 ml centrifugation tube. Pellets were exposed to cell culture media containing 20 ng/ml TGF-beta for 7 days and then cultured for another 7 and 21 days. The nodules were moved out of the tube and cartilage formation was observed by stereomicroscope, light microscope and electronic microscope.

RESULTS10 days after exposure to TGF-beta, pellets contracted and formed small and round nodules on the bottom of the tubes. The nodules grew bigger slowly and reached maximal diameter of 1.8 mm in 28 days. The surface of the nodules was smooth and bright white. Histological examination showed that extra cellular matrix formed in 14 days and in some areas cells situated in lacuna. In 28 days' specimens, a lot of cells situated in lacuna could be observed and the histological appearance looked much similar to cartilage. Electronic microscope observation demonstrated that in 28 days' specimens a large amount of collagen fiber existed.

CONCLUSIONUnder the induction of TGF-beta, MSCs could differentiate into chondrogenesis cell and form cartilaginous nodules in vitro. This indicated that MSCs could be the potential seed cells in the regeneration of cartilage employing method of tissue engineering.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cartilage ; cytology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Chondrogenesis ; drug effects ; Rabbits ; Stromal Cells ; cytology ; metabolism ; Tissue Engineering ; Transforming Growth Factor beta ; pharmacology

BACKGROUNDNatural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-beta1 (TGF-beta1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-beta1 delivery and the effect of released TGF-beta1 on the chondrogenic potential of chondrocytes.

METHODSChitosan scaffolds and chitosan microspheres loaded with TGF-beta1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-beta1 microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-beta1 on cell adhesivity, proliferation and synthesis of the extracellular matrix.

RESULTSTGF-beta1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-beta1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-beta1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-beta1 was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7% +/- 10.4%) was significantly lower than that in the controlled TGF-beta1 release group (92.4% +/- 4.8%, P < 0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-beta1 microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-beta1 was retained during microsphere fabrication and after growth factor release.

CONCLUSIONChitosan microspheres can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-beta1 possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.

Animals ; Cartilage ; metabolism ; Cell Proliferation ; Chitosan ; administration & dosage ; Chondrocytes ; cytology ; Drug Carriers ; Microspheres ; Rabbits ; Tissue Engineering ; methods ; Transforming Growth Factor beta1 ; administration & dosage ; chemistry