OBJECTIVE: To explore the molecular basis for a pedigree affected with spondyloepiphyseal dysplasia congenita (SEDC). METHODS: The proband was subjected to whole exome sequencing. Suspected variant was verified by Sanger sequencing. RESULTS: All patients from the pedigree were found to carry a novel missense variant c.1394G>C (p.Gly465Ala) of the COL2A1 gene. The variant was not reported previously. Provean, Polyphen-2 and Mutation Taster software predicted that the variant is highly likely to be pathogenic. CONCLUSION The c.1394G>C (p.Gly465Ala) variant of the COL2A1 gene probably underlies the SEDC in this pedigree.
Asian Continental Ancestry Group ; Collagen Type II ; genetics ; Humans ; Osteochondrodysplasias ; congenital ; genetics ; Pedigree

OBJECTIVEThe aim of this study was to investigate the expression of collagen in the process of masseter muscle reattachment to the cortical and cancellous bones of mandible.

METHODSA total of nine adult goats were used in the study. One was the control. The other eight were treated with bilateral detachment of the masseter muscles. The biopsies of bone and muscle were taken at 2, 4, 8 and 12 weeks after the operation. The characteristics of the healing muscle-bone interfaces were examined using immunohistochemical techniques.

RESULTSImmunohistochemical analysis illustrated that the locations of collagen type I, II and III were different during the healing process, but similar in the cortical and cancellous bones.

CONCLUSIONThis study demonstrates that the distribution of the three types of collagens at the muscle-bone interfaces is associated with time, but not related with their locations.

Animals ; Collagen ; biosynthesis ; genetics ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type II ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Female ; Goats ; Male ; Mandible ; metabolism ; pathology ; surgery ; Masseter Muscle ; metabolism ; pathology ; surgery ; Wound Healing ; physiology

Cartilage is regularly needed for reconstructive surgery. Basic research in tissue engineering is necessary to develop its full potential. We presented here the expression profile of type II collagen gene and type I collagen gene in human auricular monolayer culture expansion. Cultured chondrocytes documented a reduction in the expression level of collagen type II gene whilst collagen type I gene was gradually expressed through all the passages. This study demonstrated that human auricular chondrocytes lose its phenotypic expression during monolayer culture expansion. Further studies are required to enhance cartilage specific gene expression, collagen type II throughout the in vitro culture.
Cells, Cultured ; Chondrocytes/*cytology ; Collagen Type I/*genetics ; Collagen Type II/*genetics ; Ear, External ; Fibroblasts/cytology ; *Phenotype ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Engineering/*methods

BACKGROUNDThere is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.

METHODSCartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.

RESULTSBoth the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable.

CONCLUSIONSExpression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.

ADAM Proteins ; ADAMTS5 Protein ; Cartilage ; pathology ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Chondrocytes ; metabolism ; Collagen Type II ; genetics ; Collagen Type X ; genetics ; Glycosaminoglycans ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; genetics ; Osteoarthritis ; genetics ; pathology

Adult ; Asian Continental Ancestry Group ; Collagen Type II ; genetics ; Exome ; genetics ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Male ; Osteochondrodysplasias ; diagnosis ; genetics ; Pedigree ; Sequence Analysis, DNA

OBJECTIVETo investigate effect of connective tissue growth factors (CTGF) on secretion of extracellular matrix synthesis of meniscal fibrochondrocytes, expression of vascular endothelial growth factors (VEGF), and angiogenesis during the repair of meniscal tearing damage.

METHODSMeniscal fibrochondrocytes were isolated from the inner--1/2 of rabbits' meniscus by collagenase enzymatic digestion, centrifugal separation, and treated with 100 ng/ml CTGF in vitro. Characterization of fibrochondrocytes was identified by flow cytometry analysising CD31, CD44, CD45 and CD105, and was further tested by type II collagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. In vitro, the sections of the 3 mm of the longitudinal teared in the middle of the rabbit's meniscus, and then the defects were dealed with simple suture, suture and implanting with PBS-fibrin glue, sutured and implanting with 1.5 microg CTGF respectively. Expression and distribution of type I and II collagen and VEGF, the tearing healing were observed by fluorescence-immunohistochemisty analysis on the 1st week, the 4th week and the 10th week.

RESULTSQuantitative RT-PCR assays showed that type I and type II collagen,and VEGF mRNA expression in the 100 ng/ml CTGF group had been remarkably enhanced than in the PBS group on the 14th day. Consistent with these effects in vitro, fluorescence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue, type I collagen, type I collagen and capillaries completely filled the defect on the 10th week postoperatively. In contrast, only soft tissue repair occurred after the PBS-fibrin glue was implanted.

CONCLUSIONCTGF can significantly promote extracellular matrix (I collagen, II collagen) of the meniscal avascular zone synthesis, and CTGF can greatly heighten the expression of VEGF activity at the same time in vitro, so that it can further enhance the repair of meniscal tearing damage in the avascular zone.

Animals ; Collagen Type I ; genetics ; Collagen Type II ; genetics ; Connective Tissue Growth Factor ; therapeutic use ; Gene Expression Regulation ; drug effects ; Male ; Menisci, Tibial ; surgery ; Rabbits ; Tibial Meniscus Injuries ; Vascular Endothelial Growth Factor A ; genetics ; Wound Healing ; drug effects

This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.
Actins/genetics ; Cartilage/transplantation ; Cell Aging/physiology ; Cells, Cultured ; Chondrocytes/*cytology ; Collagen Type I/*genetics ; Collagen Type II/*genetics ; Ear, External ; Fibroblasts/cytology ; Gene Expression/physiology ; Mice, Nude ; *Phenotype ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Engineering/*methods

OBJECTIVETo investigate the effects of excessive intake of fluoride on the expression of type II collagen gene and types and morphological change of collagen fiber in the bone tissues of rats.

METHODSA rat model with fluorosis was established by adding 221 mg/L of sodium fluoride (NaF) to drinking water for the rats for 15 days, 30 days and two months, respectively. Type II collagen alpha1 (II) cDNA probe was prepared, and cDNA-mRNA in-situ hybridization was employed to detect change in expression of type II collagen mRNA in the bone tissues of rats with excessive intake of fluoride (221 mg/L NaF). Picrosirius-polarization method was used to observe types of collagen and morphology of collagen fiber in the bone tissues.

RESULTSChondroblasts were found in the femur and other bone tissues of the rats after exposure to fluoride. cDNA-mRNA in-situ hybridization showed that expression of type II collagen gene could be observed in the cytoplasm of chondrocytic lacuna and chondrified bone tissues. mRNA in collagen of chondrocytes of the rib cartilage reached the peak level 15 days after exposure to fluoride, and decreased gradually one month and two months after exposure. Polychromatic type II collagen, breakage of collagen fiber, disorder array and reduced content of type II collagen could be found in the bone tissues with picrosirius-polarization method.

CONCLUSIONSExcessive intake of fluoride could lead to changes in types and structure of collagen (cross-linkage) of bone tissues, which caused expression of type II collagen gene in the chondrified bone tissues and enhanced its expression in the rib cartilage tissues.

Animals ; Bone Diseases ; metabolism ; pathology ; Chondrocytes ; metabolism ; Collagen Type II ; biosynthesis ; genetics ; Fluoride Poisoning ; genetics ; metabolism ; pathology ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo detect potential mutation of COL2A1 gene in two children suspected for Kniest dysplasia.

METHODSThe 54 exons and splicing regions of the COL2A1 gene were amplified with PCR and the product was subjected to direct sequencing.

RESULTSA missense mutation (c.905C>T, p.Ala302Val) was found in the coding region of the COL2A1 gene, which has been previously reported in abroad. The patients appeared to have short trunk dwarfism, enlarged joints and midface hypoplasia.

CONCLUSIONThe probands are the first cases of Kniest dysplasia described in China, and so was the p.Ala302Val mutation.

Base Sequence ; Child, Preschool ; China ; Cleft Palate ; genetics ; Collagen Diseases ; genetics ; Collagen Type II ; genetics ; Dwarfism ; genetics ; Exons ; Face ; abnormalities ; Humans ; Hyaline Membrane Disease ; genetics ; Male ; Molecular Sequence Data ; Mutation, Missense ; Open Reading Frames ; Osteochondrodysplasias ; genetics ; RNA Splicing

BACKGROUND: The mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes. METHODS: The Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the (35)S-sulfate incorporation. RESULTS: No difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. (35)S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells. CONCLUSIONS: Mutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.
Aggrecans/analysis/*biosynthesis ; Animals ; Cells, Cultured ; Chondrocytes/*metabolism ; Chondrosarcoma/metabolism ; Collagen Type II/*biosynthesis ; Extracellular Matrix/*metabolism ; Extracellular Matrix Proteins/*genetics ; Glycoproteins/*genetics ; Humans ; Mutation ; Rats ; Transfection