In Vitro Anti-Inflammation and Chondrogenic Differentiation Effects of Inclusion Nanocomplexes of Hyaluronic Acid-Beta Cyclodextrin and Simvastatin

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.

Effect of eletroacupuncture with close-to-bone needling treatment on expression of Sox9, VEGF and ColX in impaired cartilage of rabbits with knee osteoarthritis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of eletroacupuncture with close-to-bone needling treatment on expression of Sox9, vascular endothelial growth factor (VEGF) and type X collagen (ColX) in impaired cartilage of rabbits with knee osteoarthritis (KOA) and explore its possible mechanisms.</p><p><b>METHODS</b>Forty New Zealand rabbits were randomized equally into normal control group, KOA model group, eletroacupuncture with close-to-bone needling group (CN group), and normal thrust needing group (NTN group). In the latter 3 groups, KOA was induced by Hulth-Telhag treatment and evaluated with X-ray examination, and 6 weeks after the modeling, eletroacupuncture for 20 min was administered in CN and NTN groups at the acupoints "Zusanli", "Waixiyan", "Neixiyan", "Liangqiu" and "Yinlingquan" in the left knee joints once daily for 5 days as a treatment cycle. After 5 treatment cycles, the rabbits were examined for behavioral changes, cartilage morphology, and Mankin scores; The protein and mRNA expressions of S0x9, VEGF, and ColX were examined using Westen blotting, immunohistochemistry, and RT-PCR as appropriate.</p><p><b>RESULTS</b>The rabbits in the model, CN and NTN groups showed significant changes in behaviors and cartilage histomorphology after the modeling and after the treatments. HE staining showed that cartilage injury was repaired and tended to recovery in CN and NTN groups. The cartilage pathologies was severer in the model group than in the normal control, CN and NTN groups (P<0.01); Sox9 protein increased and VEGF mRNA level decreased in CN and NTN groups after treatment as compared with those in the model group (P<0.01).</p><p><b>CONCLUSION</b>Eletroacupuncture with close-to-bone needling can effectively improve KOA in rabbits probably by enhancing Sox9 and reducing VEGF and ColX expressions in the cartilage to inhibit hypertrophic differentiation of the chondrocytes, maintain chondrogenic phenotype and repair cartilage cells.</p>

5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture / 대한해부학회지

This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product.

Condrodisplasia metafisaria tipo Schmid: un caso de presentación familiar / Schmid type metaphyseal chondrodysplasia: a case of familial presentation

Resumen: dentro de las displasias óseas hay cuadros clínicos que hacen parte de las denominadas condrodisplasias metafisarias, conocidas también como disostosis metafisarias, las cuales presentan mínimas diferencias entre sí, lo que las hace susceptibles de ser confundidas con otros cuadros clínicos como la acondroplasia y el raquitismo. En este artículo se presenta un caso clínico de condrodisplasiametafisaria tipo Schmid de un paciente de Popayán, Colombia, al igual que algunas consideracionessobre las principales características clínicas, radiológicas, de diagnóstico y tipo de herencia de esta enfermedad. El caso clínico corresponde a un paciente de género masculino de 23 meses de edad, en quien se inician estudios por la presencia de talla baja desproporcionada. Los resultados mostraron coxa vara, genu varo y extremidades cortas, con un fenotipo similar en la madre y el abuelo materno. Las radiografías evidencian la presencia de irregularidad con “deshilachamiento” de las metáfisis de huesos largos; además, ensanchamiento y esclerosis en las metáfisis proximales de ambos fémur. La meta final es ser confirmado por medio de pruebas genéticas. En conclusión, las condrodisplasias metafisarias, especialmente la tipo Schmid, son enfermedades caracterizadas por talla baja y hallazgos radiológicos especiales, dados principalmente por el compromiso metafisario a nivel de los huesos largos, que en conjunto con las características fenotípicas pueden conducir a la sospecha e identificación de este tipo de patología.

Abstract: between the dysplastic bone pathologies there are some medical conditions that belong to so-called metaphyseal chondrodysplasias, also known as metaphyseal dysostosis. These differ slightly from each other, making them capable of being confused with other medical conditions such as achondroplasia and rickets. This article presents a case of Schmid type metaphyseal chondrodysplasiafrom Popayan, Colombia, as well as some considerations about the main clinical characteristics, radiological, diagnosis, and type of inheritance of this disease. The clinical case corresponds to a male patient, 23 months old, who was studied by the presence of disproportionate short stature. Findings showed coxa vara, genu varus, and short limbs, with similar phenotype to the mother and maternal grandfather. The radiological images showed the presence of irregularity with ®fraying¼ of the metaphysis of long bones, in addition to widening and sclerosis in the proximal metaphysis of both femurs. The ultimate goal is to be confirmed by genetic testing. In conclusion, the metaphyseal chondrodysplasias, especially Schmid type, are diseases characterized by short stature and by special radiological findings, mainly given by the metaphyseal affectation of long bones, which together with the phenotypic characteristics may lead to the suspicion and identification of this disease.Keywords: Schmid type metaphyseal chondrodysplasia, osteochondrodysplasias, collagen type.

Construction of self-assembled cartilage tissue from bone marrow mesenchymal stem cells induced by hypoxia combined with GDF-5 / 华中科技大学学报（医学）（英德文版）

It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.

Characterization of human primary chondrocytes of osteoarthritic cartilage at varying severity / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>There 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.</p><p><b>METHODS</b>Cartilage 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.</p><p><b>RESULTS</b>Both 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.</p><p><b>CONCLUSIONS</b>Expression 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.</p>

Developmental changes of acetabular cartilage complex: an experimental study of a straight-leg swaddle model of newborn rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>By establishing a model of straight-leg swaddle of newborn rats and observing the experimental animals'hips morphologically and pathologically, this study explored the changes of gross appearance of the acetabulum and the maturity of cartilage cells in the different regions of acetabular cartilage complex.</p><p><b>METHODS</b>The legs and hips were fixed by adhesive tape for 10 days in the position of hip extension and adduction in 31 newborn Wistar rats (experimental group). The other 31 newborn rats without legs and hips treatment were used as the control group. After 10 days raising in the same condition, all the rats were sacrificed. The gross appearance, histological observations and VEGF and type X collagen immunohistochemistry were used for examining the acetabulum changes.</p><p><b>RESULTS</b>A straight leg swaddle model of newborn rats was established successfully. In the experimental group the acetabulum became shallow and small and surrounded by more soft tissues. There were 49 dislocated hips (49/54) in the experimental group and 2 hips dislocated (2/60) in the control group (p<0.01). Fake acetabulum appeared in the experimental group. In the control group, the shape of the acetabulum was normol, and no fake acetabulum was found. The safranin O-fast green staining showed that the orange-red cartilage in the experimental group was wider than the control group. Immunohistochemistry observations showed VEGF and type X collagen immunoreactivities in the hypertrophic layer of the acetabular cartilage complex in the experimental group were lower than those in the control group. The percentages of VEGF positive and type X collagen positive cells in the iliac hypertrophic layer of the acetabular articular cartilage were significantly higher than those in the ischiadic ramus and the pubic branch in the experimental group.</p><p><b>CONCLUSIONS</b>VEGF and type X collagen immunoreactivities in acetabular cartilage cells decrease in a straight-leg swaddle model of newborn rats. This suggests that this position might lead to dysmaturity of the acetabular cartilage cells and affect the development of the acetabulum.</p>

Indexes of intervertebral disc degeneration in rats during the aging process / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the indexes for evaluating intervertebral disc degeneration in rats during the aging process.</p><p><b>METHODS</b>Nine 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The 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.</p>

Gene expression of collagen types IX and X in the lumbar disc / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Fetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSIONS</b>Obvious 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.</p>

Expression of Fibroblast Growth Factor Receptors at Different Stages of Differentiation in Chick Embryo Chondrocytes / 소아과

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.

Gene expression of type X collagen in the intervertebral disc of idiopathic scoliosis patients / 中国医学科学院学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHOD</b>Investigating 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The 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.</p>

The Role of Growth Factors to Rabbit Chondrocytes and Subtypes of Collagen in Three Dimensional High Density Culture

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.

The Effect of IGF-I on Collagen Synthesis in Cultured Chondrocytes

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.