OBJECTIVETo study the indexes for evaluating intervertebral disc degeneration in rats during the aging process.

METHODSNine SD rats were fed for 6 months and 12 for 22 months as the young and aged groups, respectively. The Miyamoto's grade of the rats was calculated, and the quantity and relative area of the vascular buds as well as the thickness of the calcified and non-calcified layers of the cartilage endplate were measured using the stereoscopic method. Immunohistochemistry with monoclonal antibodies was used to determine the expressions of collagens II and X in the endplate.

RESULTSThe quantity and relative area of the vascular buds, non-calcified layer/calcified layer ratio, type II collagen expression in the calcified layer and nucleus pulposus of the cartilage endplate were all significantly decreased in the aged rats as compared with those of the youth rats (P<0.05), but the collagen X expression in the non-calcified layer was significantly higher in the aged rats (P=0.003). No significant difference was found in the Miyamoto's grade between the aged and young rats (P=1.130).

CONCLUSIONThe relative area of the vascular buds, non-calcified layer/calcified layer ratio, phenotypic expressions of collagens II and X in the cartilage endplate, but not the Miyamoto's grade, are sensitive indexes for evaluating intervertebral disc degeneration in rats during the aging process.

Aging ; Animals ; Cartilage ; pathology ; Collagen Type II ; analysis ; Collagen Type X ; analysis ; Intervertebral Disc Degeneration ; pathology ; Rats ; Rats, Sprague-Dawley

PURPOSE: Proliferative chondrocytes and prehypertrophic chondrocytes secrete significant amounts of type II collagen in an extracellular matrix. In contrast, hypertrophic chondrocytes secrete type X collagen. In addition, fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) also appear to play an important role during differentiation. Accordingly, the current study identified and characterized the chondrocytes and FGFR mRNA expressed at different stages of differentiation. METHODS: Chondrocytes were isolated from the caudal one-third portion (LS) of the sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and the lower portion of the proximal tibial growth plate (Ti). Chondrocytes from the LS, USP, USC, and Ti of 17-day-old chick embryo sterna and tibia were cultured and type II and type X collagen mRNA and FGFR1, FGFR2, and FGFR3 mRNA were isolated and analyzed by Northern blotting. RESULTS: Generally, the cells were larger in size after two days of culture than after seven days of culture and the cells from the USC and Ti were larger and more mature than those from the LS and USP. Type II collagen genes were found to be expressed in all the chondrocyte types, while type X collagen was strongly expressed in the USC and Ti. Therefore, the LS was identified as a resting or proliferative zone, the USP as a postproliferative or prehypertrophic zone, and the USC or Ti as a hypertrophic zone. FGFR1 was expressed only in hypertrophic chondrocytes in proportion to the culture time, FGFR2 was not expressed in any of the chondrocyte types, and FGFR3 was expressed in all the chondrocyte types. CONCLUSION: As such, it is possible that the different receptors play distinct roles during chondrocyte differentiation.
Animals ; Blotting, Northern ; Chick Embryo* ; Chondrocytes* ; Collagen ; Collagen Type II ; Collagen Type X ; Extracellular Matrix ; Fibroblast Growth Factors* ; Fibroblasts* ; Growth Plate ; Receptors, Fibroblast Growth Factor* ; RNA, Messenger ; Tibia

Cartilage is commonly used autogenous material for aesthetic and reconstructive surgery and major donor sites of cartilage are ear, nasal septum, and rib. As the cartilage correlates with ossification and can be used for joint reconstruction. Many growth factors influencing growth and differentiation of chondrocytes have been reported, and matrix composition produced by chondrocytes may vary in types and quantity according to culture duration. Initially the chondrocytes in culture aggregate, then secrete type I collagen. Type II collagen is produced during differentiation process, and synthesis of type X collagen is the last step. In this study, chondrocytes were isolated from ear cartilage of the New Zealand white rabbit weighing 400 gm. We performed high density culture using penicylinder and pellet method. The cells were polygonal in morphology and viable under the inverted microscope. This experiment was designed to evaluate the effect of IGF-I, TGF- p, and b- FGF on the synthesis of collagen in chondrocyte culture. Optimal concentration of growth factors was determined using H-thymidine incorporation into DNA. After the addition of optimal concentration of each growth factors in experimental groups, the uptake of H-proline was measured. Only IGF-I showed a statistically significant increase of collagen synthesis. We observed how subtypes of collagen were influenced by growth factors in two culture methods and by differing the addition timing of growth factors. SDS-PAGE was adopted for subtyping of collagen. All subtypes of collagen were found in both culture methods and all growth factors facilitated the production of type II and type X collagen and may be devoted to the differentiation of chondrocytes. Immunohistochemical staining for type I, and type II collagen was examined to confirm the above result. In pellet culture, type II collagen was stained densely in response to the addition of three kinds of growth factors. The results of penicylinder culture showed similar outcome to those from pellet cultured group. From the above results, we concluded as follows; First, IGF-I generally influence the synthesis of type I and II collagen. Second, TGF beta increased the synthesis of collagen. Third, b-FGF increased the synthesis of type II and type X collagen. We concluded that IFG-I is the only growth factor which is effective regardless of culture duration and method. TGF- beta and b-FGF, which are potent mitogen, facilitate the secretion of collagen.
Cartilage ; Chondrocytes* ; Collagen Type I ; Collagen Type II ; Collagen Type X ; Collagen* ; DNA ; Ear ; Ear Cartilage ; Electrophoresis, Polyacrylamide Gel ; Humans ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins* ; Joints ; Nasal Septum ; New Zealand ; Ribs ; Tissue Donors

Cartilage is one of the most commonly manipulated tissue in esthetic and reconstructive surgery. Cartilage has an important role in longitudinal bone growth. Anabolic hormones and locally produced peptide growth factors are known to influence this process Matrix composition changes through proliferation, maturation, and differentiation of chondrocytes, and endochondral ossification thereafter. Defined cartilage matrix is synthesized during the maturation of chondrocytes where the major change is the increment of type II collagen. Variable sulfated mucololysaccharides and hyaluronic acid are also synthesized during this maturation. IGF-I(insulin like growth factor-I), so called somatomedin C, is a prominent growth factor in serum. IGF-I is known to be involved in long growth. IGF-I is affected by pituitary growth hormone. There are few studies done on IGF-I effect in cartilage matrix formation and possible changes of collagen subtypes. This experiment was designed to see the IGF-I effect on the colagen synthesis of cultured chondrocytes. Optimal concentration of IGF-I for the experiment was determined using H3-thymidine incorporation into DNA. The IGF-I effect on collagen synthesis was studied using H3-proline. The IGF-I effect on the synthesis of subtypes of collagen was studied using SDS-PAGE and immunocytochemical staining. Chondrocytes were isolated from the ears of New Zealand white rabbit and cultured in 2 X 10(5) cells/300 microgram density. IGF-I increased DNA synthesis, and optimal concentration of IGF-I was determined by dose-relationship curve as 10ng/ml. Collagen synthesis was increased by IGF-I. Type II collagen was increased on SDS-PAGE with IGF-I and this gel electrophoresis showed type X collagen, also. The increase in type II collagen was confirmed with immunocytochemical staining, the reaction becoming stronger with the addition of IGF-I. Type I collagen was not changed with IGF-I on immunocytochemistry. We conclude that IGE-I is an important modulator influencing not only proliferation and maturation but also terminal different-iation of chondrocytes.
Bone Development ; Cartilage ; Chondrocytes* ; Collagen Type I ; Collagen Type II ; Collagen Type X ; Collagen* ; DNA ; Ear ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Growth Hormone ; Hyaluronic Acid ; Immunohistochemistry ; Insulin-Like Growth Factor I* ; Intercellular Signaling Peptides and Proteins ; New Zealand

OBJECTIVETo study gene expression of collagen types IX and X in human lumbar intervertebral discs during aging and degeneration and to explore the role of collagen types IX and X in disc degeneration.

METHODSFetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene.

RESULTSIn fetal intervertebral discs, positive mRNA hybridization signals of type IX collagen were concentrated in the nucleus pulposus and the inner layer of anulus fibrosus. Interstitial matrix of the nucleus pulposus also showed positive type X collagen staining. Positive mRNA hybridization signals of types IX and X were not detected in the middle and outer layers of anulus fibrosus. In adult specimens, expression of type IX collagen mRNA was markedly decreased. No hybridization signals of type X collagen was observed. As for pathological specimens, there was no gene expression of type IX collagen. In severe degenerated discs from adults, there were focal positive expressions of type X collagen.

CONCLUSIONSObvious changes of collagen gene expression occur with aging. Expression of type IX collagen decreases in adult and pathological discs. Results of type X collagen expression suggest that type X collagen is expressed only in older adult and senile discs (i.e., when disc degeneration has already reached a terminal stage), indicating the terminal stage of degeneration.

Adolescent ; Adult ; Collagen Type IX ; metabolism ; Collagen Type X ; metabolism ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Intervertebral Disc ; embryology ; metabolism ; Lumbar Vertebrae ; Male

The aim of this study was to prepare inclusion nanocomplexes of hyaluronic acid-β-cyclodextrin and simvastatin (HA-β-CD/SIM) and evaluate in vitro anti-inflammation effects on lipopolysaccharide (LPS)-activated synoviocytes and chondrogenic differentiation effects on rat adipose-derived stem cells (rADSCs). The β-CD moieties in HA-β-CD could incorporate SIM to form HA-β-CD/SIM nanocomplexes with diameters of 297–350 nm. HA-β-CD/SIM resulted in long-term release of SIM from the nanocomplexes for up to 63 days in a sustained manner. In vitro studies revealed that HA-β-CD/SIM nanocomplexes were able to effectively and dose-dependently suppress the mRNA expression levels of proinflammatory markers such as matrix metallopeptidase-3 (MMP-3), MMP-13, cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in LPS-stimulated synoviocytes. HA-β-CD/SIM-treated rADSCs significantly and dose-dependently enhanced mRNA expressions of aggrecan, collagen type II (COL2A1), and collagen type X (COL10A1), implying that HA-β-CD/SIM greatly induced the chondrogenic differentiation of rADSCs. Conclusively, HA-β-CD/SIM nanocomplexes will be a promising therapeutic material to alleviate inflammation as well as promote chondrogenesis.
Aggrecans ; Animals ; Chondrogenesis ; Collagen Type II ; Collagen Type X ; Cyclooxygenase 2 ; In Vitro Techniques* ; Inflammation ; Interleukin-6 ; Rats ; RNA, Messenger ; Simvastatin* ; Stem Cells ; Thrombospondins ; Tumor Necrosis Factor-alpha

OBJECTIVETo observe the characteristics of gene expression of type X collagen in the cartilage of end-plate and the fibrous annulus in the intervertebral disc of idiopathic scoliosis (IS) patients.

METHODInvestigating the expression of type X collagen in the peak disc and the lower end disc of 21 IS patients, the peak disc of 16 congenital scoliosis (CS) and the lumbar disc of 3 normal people (according with the principle of medical ethnics) by reverse transcript polymerase chain reaction.

RESULTSThe expression of type X collagen in the concave side of IS peak disc was higher than the convex side (P < 0.05). There was no significant difference of gene expression of type X collagen between the convex side and the concave side of the lower end disc (P > 0.05). The gene expression of type X collagen in the IS peak disc was higher than those of lower end disc (P < 0.05). For the CS peak discs, the expression of type X collagen of the concave side was higher than the convex side (P < 0.05).

CONCLUSIONThe expression of type X collagen of the IS peak disc increases, and the expression of type X collagen of the concave side is higher than the convex side. These changes may be secondary.

Adolescent ; Child ; Collagen Type X ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Intervertebral Disc ; metabolism ; Male ; Scoliosis ; genetics ; metabolism

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

OBJECTIVE: The purpose of this study was to verify the appropriateness of ovariectomized rats as the osteoporosis animal model. METHODS: Twelve female Sprague-Dawley rats underwent a sham operation (the sham group) or bilateral ovariectomy [the ovariectomy (OVX) group]. Eight weeks after operations, serum biochemical markers of bone turnover were analyzed; osteocalcin and alkaline phosphatase, which are sensitive biochemical markers of bone formation, and C-terminal telopeptide fragment of type I collagen C-terminus (CTX), which is a sensitive biochemical marker of bone resorption. Bone histomorphometric parameters and microarchitectural properties of 4th lumbar vertebrae were determined by micro-computed tomographic (CT) scan. RESULTS: The OVX group showed on average 75.4% higher osteocalcin and 72.5% higher CTX levels than the sham group, indicating increased bone turnover. Micro-CT analysis showed significantly lower bone mineral density (BMD) (p=0.005) and cortical BMD (p=0.021) in the OVX group. Furthermore, the OVX group was found to have a significantly lower trabecular bone volume fraction (p=0.002). CONCLUSION: Our results showed that bone turnover was significantly increased and bone mass was significantly decreased 8 weeks after ovariectomy in rats. Thus, we propose that the ovariectomized rat model be considered a reproducible and reliable model of osteoporosis.
Alkaline Phosphatase ; Animals ; Biomarkers ; Bone Density ; Bone Resorption ; Collagen Type I ; Female ; Humans ; Lumbar Vertebrae ; Osteocalcin ; Osteogenesis ; Osteoporosis ; Ovariectomy ; Rats ; Rats, Sprague-Dawley ; Salicylamides ; X-Ray Microtomography

Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo.
Amino Acids ; Animals ; Bone Marrow ; Bone Regeneration ; Calcium ; Ceramics* ; Collagen Type I ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Mice ; Miners ; Osteocalcin ; Osteogenesis ; Photoelectron Spectroscopy ; Regeneration* ; X-Ray Microtomography