Proteoglycans synthesized during the cartilage to bone transition in developing chick embryos

Osteogenesis of the long bone involves a series of cellular and molecular events culminating in the fabrication of a mineralized matrix of the long bone. These studies focus upon the chemical identity and morphology of newly synthesized proteoglycans formed during the cartilage to bone transition in embryonic chick tibia development. As early as the onset of major ossification events (day 13; Hamburger-Hamilton Stage 39), synthesis of a small, highly 4-sulfated chondroitin sulfate proteoglycan was detected in the shaft of the tibia. The characterization of the chondroitin sulfate-rich proteoglycans from cartilage, hypertrophic cartilage, bone marrow, and new bone lead to the suggestion that these molecules can be used as distinctive markers for the cellular phenotypes involved in the cartilage to bone transition.     

Transforming growth factor alpha controls the transition from hypertrophic cartilage to bone during endochondral bone growth

We have recently identified transforming growth factor alpha (TGFα) as a novel growth factor involved in the joint disease osteoarthritis. The role of TGFα in normal cartilage and bone physiology however, has not been well defined.PurposeThe objective of this study was to determine the role of TGFα in bone development through investigation of the Tgfa knockout mouse.MethodsThe gross skeletons as well as the cartilage growth plates of Tgfa knockout mice and their control littermates were examined during several developmental stages ranging from newborn to ten weeks old.ResultsKnockout mice experienced skeletal growth retardation and expansion of the hypertrophic zone of the growth plate. These phenotypes were transient and spontaneously resolved by ten weeks of age. Tgfa knockout growth plates also had fewer osteoclasts along the cartilage/bone interface. Furthermore, knockout mice expressed less RUNX2, RANKL, and MMP13 mRNA in their cartilage growth plates than controls did.ConclusionsTgfa knockout mice experience a delay in bone development, specifically the conversion of hypertrophic cartilage to true bone. The persistence of the hypertrophic zone of the growth plate appears to be mediated by a decrease in MMP13 and RANKL expression in hypertrophic chondrocytes and a resulting reduction in osteoclast recruitment. Overall, TGFα appears to be an important growth factor regulating the conversion of cartilage to bone during the process of endochondral ossification.     

Alterations in the collagen framework of osteoarthritic cartilage and subchondral bone

Summary Specimens obtained from human osteoarthritic knee joints and dog knees with experimentally induced osteoarthritis were used to study the collagenous framework of articular cartilage and subchondral bone in relation to osteoarthritic changes using scanning electron microscopy and light microscopy. Degenerative articular cartilage in osteoarthritic joints showed radial orientation of the collagen fibrils, which were usually discernible as fibrillar bundle formations. Cartilage with extensive lesions often showed cleavages or fissures down to the calcified layer. The cartilagenous collagen fibrils in osteoarthritic specimens merged into the subchondral bone plate making the tidemark and the osteochondral junction irregular or obscure. The trabecular orientation of subchondral bone changed with alteration in the articular cartilage and with reactive changes in the subchondral bone, showing the effect of cartilaginous degeneration on its ultrastructure.

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Résumé Des prélèvements obtenus à partir de genoux humains arthrosiques et de genoux de chien porteurs de lésions arthrosiques expérimentales ont permis l'étude de la charpente de collagène du cartilage articulaire et de l'os sous-chondral, par microscopie conventionnelle et par microscopie électronique.Dans les lésions dégénératives, les fibrilles de collagène du cartilage ont une orientation radiale, alors qu'elles sont habituellement groupées en faisceaux. En cas de lésions étendues, le cartilage présente souvent des clivages ou des fissures menant jusqu'aux couches calcifiées. Les fibrilles de collagène du cartilage arthrosique se fondent dans la plaque osseuse souschondrale, rendant la jonction ostéo-cartilagineuse irrégulière ou mal définie.L'orientation trabéculaire de l'os sous-chondral se modifie en fonction de l'altération du cartilage articulaire. Ces modifications réactionnelles montrent que, même au niveau ultrastructural, l'architecture de l'os sous-chondral reflète la dégénérescence cartilagineuse.

     

Antiangiogenic treatment delays chondrocyte maturation and bone formation during limb skeletogenesis.

Hypertrophic chondrocytes have important roles in promoting invasion of cartilage by blood vessels and its replacement with bone. However, it is unclear whether blood vessels exert reciprocal positive influences on chondrocyte maturation and function. Therefore, we implanted beads containing the antiangiogenic molecule squalamine around humeral anlagen in chick embryo wing buds and monitored the effects over time. Fluorescence microscopy showed that the drug diffused from the beads and accumulated in humeral perichondrial tissues, indicating that these tissues were the predominant targets of drug action. Diaphyseal chondrocyte maturation was indeed delayed in squalamine-treated humeri, as indicated by reduced cell hypertrophy and expression of type X collagen, transferrin, and Indian hedgehog (Ihh). Although reduced in amount, Ihh maintained a striking distribution in treated and control humeri, being associated with diaphyseal chondrocytes as well as inner perichondrial layer. These decreases were accompanied by lack of cartilage invasion and tartrate-resistant acid phosphatase-positive (TRAP+) cells and a significant longitudinal growth retardation. Recovery occurred at later developmental times, when in fact expression in treated humeri of markers such as matrix metalloproteinase 9 (MMP-9) and connective tissue growth factor (CTGF) appeared to exceed that in controls. Treating primary cultures of hypertrophic chondrocytes and osteoblasts with squalamine revealed no obvious changes in cell phenotype. These data provide evidence that perichondrial tissues and blood vessels in particular influence chondrocyte maturation in a positive manner and may cooperate with hypertrophic chondrocytes in dictating the normal pace and location of the transition from cartilage to bone.     

Bone morphogenetic protein signals are required for cartilage formation and differently regulate joint development during skeletogenesis.

The bone morphogenetic protein (BMP) family consists of a large number of members and has diverse biological activities during development. Various tissues express pleural BMP family members, which seem to cooperatively regulate developmental events. Here, multiple BMP signals were inactivated in chondrocytes to clarify the function of BMPs during skeletogenesis. To obtain tissue-specific inactivation, Noggin gene (Nog) was overexpressed in cartilage under the control of a2(XI) collagen gene (Collla2) promoter/enhancer sequences. The resultant transgenic mice lacked most of their cartilaginous components, suggesting that cartilage does not develop without BMP signals. These effects seem to be mediated through down-regulation of Sox9 expression. Conversely, specific BMP signals were activated in the skeleton by targeted expression of Bmp4 in cartilage and the resultant phenotype was compared with that of transgenic mice expressing growth and differentiation factor-5 (GDF-5), another BMP family member. Overactivity of Bmp4 in the skeleton caused an increase of cartilage production and enhanced chondrocyte differentiation, as GDF5 expression did, but it did not disturb joint formation as GDF5 did. During skeletogenesis, unique roles of each BMP may reside in the regulation of joint development. Together with the common effect on the cartilage overproduction by Bmp4 and GDF5 overactivation, loss of cartilage by inactivation of multiple BMPs in Noggin transgenic mice indicates that signals for cartilage production are reinforced by multiple BMPs exclusively. These conclusions may account for the reason why multiple BMPs are coexpressed in cartilage.     

Type II collagen synthesis in the articular cartilage of a rabbit model of osteoarthritis: expression of type II collagen C-propeptide and mRNA especially during early-stage osteoarthritis

Background The aim of this study was to observe time course changes in type II collagen synthesis in various regions of articular cartilage affected with osteoarthritis (OA) by examining the expression of type II collagen C-propeptide (pCOL II-C) and mRNA in a rabbit OA model. Methods Osteoarthritis was experimentally induced by partial lateral meniscectomy in the knees of Japanese white rabbits. The cartilage of the animals was then examined histologically over time. The degenerative area of articular cartilage was divided into three areas, according to the degree of degeneration. The ability to synthesize type II collagen was estimated by the immunohistological staining of pCOL II-C and the in situ hybridization of mRNA in type II collagen. Results The positive rate of pCOL II-C immunostaining in chondrocytes was highest in the central-degenerative region 1 week after surgery, and the highest rate in the para-degenerative region was observed 2 and 4 weeks after surgery. The percentage of pCOL II-C positive cells increased as the histological degeneration score increased to moderate degeneration and then decreased with further progression of the severity of cartilage degeneration. Examination by in situ hybridization revealed that the regions marked by strong pCOL II-C mRNA expression were similar to those indicated by the immunohistology results. Conclusions These results suggest that the type II collagen-synthesizing potential of chondrocytes is highest in moderately degenerated areas of OA articular cartilage. Cartilage repair continues to be seen even as OA advances, although the reaction varies depending on the stage of OA.     

Viscoelastic properties of bovine articular cartilage attached to subchondral bone at high frequencies

Background  

Articular cartilage is a viscoelastic material, but its exact behaviour under the full range of physiological loading frequencies is unknown. The objective of this study was to measure the viscoelastic properties of bovine articular cartilage at loading frequencies of up to 92 Hz.     

Type III collagen modulates fracture callus bone formation and early remodeling

Type III collagen (Col3) has been proposed to play a key role in tissue repair based upon its temporospatial expression during the healing process of many tissues, including bone. Given our previous finding that Col3 regulates the quality of cutaneous repair, as well as our recent data supporting its role in regulating osteoblast differentiation and trabecular bone quantity, we hypothesized that mice with diminished Col3 expression would exhibit altered long‐bone fracture healing. To determine the role of Col3 in bone repair, young adult wild‐type (Col3+/+) and haploinsufficent (Col3+/?) mice underwent bilateral tibial fractures. Healing was assessed 7, 14, 21, and 28 days following fracture utilizing microcomputed tomography (microCT), immunohistochemistry, and histomorphometry. MicroCT analysis revealed a small but significant increase in bone volume fraction in Col3+/? mice at day 21. However, histological analysis revealed that Col3+/? mice have less bone within the callus at days 21 and 28, which is consistent with the established role for Col3 in osteogenesis. Finally, a reduction in fracture callus osteoclastic activity in Col3+/? mice suggests Col3 also modulates callus remodeling. Although Col3 haploinsufficiency affected biological aspects of bone repair, it did not affect the regain of mechanical function in the young mice that were evaluated in this study. These findings provide evidence for a modulatory role for Col3 in fracture repair and support further investigations into its role in impaired bone healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:675–684, 2015.

     

Contribution of collagen network features to functional properties of engineered cartilage

BACKGROUND: Damage to articular cartilage is one of the features of osteoarthritis (OA). Cartilage damage is characterised by a net loss of collagen and proteoglycans. The collagen network is considered highly important for cartilage function but little is known about processes that control composition and function of the cartilage collagen network in cartilage tissue engineering. Therefore, our aim was to study the contribution of collagen amount and number of crosslinks on the functionality of newly formed matrix during cartilage repair. METHODS: Bovine articular chondrocytes were cultured in alginate beads. Collagen network formation was modulated using the crosslink inhibitor beta-aminopropionitrile (BAPN; 0.25mM). Constructs were cultured for 10 weeks with/without BAPN or for 5 weeks with BAPN followed by 5 weeks without. Collagen deposition, number of crosslinks and susceptibility to degradation by matrix metalloproteinase-1 (MMP-1) were examined. Mechanical properties of the constructs were determined by unconfined compression. RESULTS: BAPN for 5 weeks increased collagen deposition accompanied by increased construct stiffness, despite the absence of crosslinks. BAPN for 10 weeks further increased collagen amounts. Absence of collagen crosslinks did not affect stiffness but ability to hold water was lower and susceptibility to MMP-mediated degradation was increased. Removal of BAPN after 5 weeks increased collagen amounts, allowed crosslink formation and increased stiffness. DISCUSSION: This study demonstrates that both collagen amounts and its proper crosslinking are important for a functional cartilage matrix. Even in conditions with elevated collagen deposition, crosslinks are needed to provide matrix stiffness. Crosslinks also contribute to the ability to hold water and to the resistance against degradation by MMP-1.     

Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth

The longitudinal growth of bone depends on the activities of individual chondrocytes of the growth plate. Each chondrocyte remains in a fixed location throughout its life, and there accomplishes all of its functions. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during a particular phase of its life. The two most prominent stages are those of cellular proliferation and hypertrophy (including the mineralization of matrix) before the resorption of tissue during vascular invasion. By applying recently developed stereological procedures and improved methods for the fixation of cartilage, we compared cellular shape modulation, various ultrastructural parameters (surface areas or volumes of endoplasmic reticulum, Golgi membranes, and mitochondria), the production of matrix, and cellular turnover for proliferating and hypertrophic chondrocytes within the proximal tibial growth plate of the rat. By the late hypertrophic stage, fourfold and tenfold increases in the mean cellular height and volume, respectively, and a threefold increase in the mean volume of the matrix per cell were achieved. The high metabolic activity of hypertrophic cells was reflected by a twofold to fivefold increase in the mean cellular surface area of rough endoplasmic reticulum, the Golgi membranes, and the mean cellular mitochondrial volume. Rates of longitudinal growth were determined by fluorochrome labeling and incident-light fluorescence microscopy. Using these values and the stereological estimators describing cellular height, the rates of cellular turnover were calculated. The rapid progression of the vascular invasion front was found to eliminate, for each column of cells, one chondrocyte every three hours; that is, eight cells a day. The maintenance of a steady-state structure for growth-plate cartilage in rats in a steady state of growth thus necessitates efficient compensation for these losses, which is achieved by a high rate of cellular proliferation and rapid hypertrophy.     

Intra-operative contamination of bone and cartilage during an Austin bunionectomy.

During an Austin bunionectomy the potential exists for the head of the metatarsal to leave the sterile field and become contaminated. The authors present a case in which this occurred, with a 20-month follow-up. Possible options for decontamination are reviewed.     

A type XI collagen mutation leads to increased degradation of type II collagen in articular cartilage

OBJECTIVE: To determine in articular cartilage whether degraded type II collagen is more abundant in Col11a1 mutant cho/+ than in age-matched +/+ mice and whether collagen degradation occurs in a generalized or localized fashion. DESIGN: Knee joints from cho/+ and +/+ mice at 6, 9, 12 and 15 months of age were dissected, fixed, cryosectioned, and stained with antibody COL2-3/4m against denatured type II collagen using a FITC-conjugated secondary antibody. Sections were viewed and photographed under a fluorescence microscope and areas of staining were quantified. RESULTS: Before 12 months of age, little degraded collagen staining was detectable in +/+ or cho/+ mice. By 15 months, however, cho/+ mice showed significantly more degraded type II collagen than age-matched controls. Degraded collagen staining was localized at the articular surface, not distributed generally throughout the articular cartilage. CONCLUSIONS: The results suggest a model in which cumulative biomechanical stresses trigger increased collagen synthesis and degradation in both +/+ and cho/+ mice at around 12 months of age. Cho/+ mice, however, are less able to synthesize and assemble normal replacement collagen fibrils because of the Col11a1 mutation. Degradation is further activated, resulting in the accumulation of degraded type II collagen in the articular cartilage extracellular matrix. Similar mutations that do not overtly affect skeletal development may likewise predispose humans to increased collagen degradation and resultant osteoarthritis.     

胶原在软骨组织工程中的应用

软骨组织工程的出现,为解决软骨修复这个临床难题提供了新的方法.然而寻找一种合适的生物载体材料是目前软骨组织工程的热点.胶原是一种天然支架材料,具有良好的生物相容性、可降解性和生物活性,可作为细胞三维生长支架,并且有维持种子细胞增殖、黏附和分化的能力.本文就胶原及其复合材料在软骨组织工程方面的应用状况与前景作一综述.     

Malondialdehyde oxidation of cartilage collagen by chondrocytes

OBJECTIVE: The damage to cartilage collagen is a central event in the pathogenesis of cartilage aging and osteoarthritis (OA). We have previously developed an in vitro model of cartilage degradation which shows that chondrocyte-dependent lipid peroxidation mediates cartilage collagen degradation. The goal of our study was to investigate the role of vitamin C in this degradation model and to investigate effect of chondrocyte-dependent lipid peroxidation in the oxidation of cartilage collagen. METHODS: We studied primary articular chondrocytes. Effect of vitamin C was investigated in the previously described model. Serum-free stimulated and unstimulated chondrocyte-matrix extracts were subjected to SDS-PAGE and immunoblot analysis. Malondialdehyde (MDA)-protein oxidation of cartilage proteins was demonstrated by the reactivity of chondrocyte extracts to a monoclonal antibody, MDA2, which detects MDA-lysine adducts. RESULTS: Vitamin C treatment of chondrocyte cultures resulted in significant enhanced incorporation of 3H-proline label in cell-matrix. Cells treated with vitamin C, as compared to control untreated cells showed decreased spontaneous release of labeled matrix. Vitamin C treated or not treated chondrocytes responded comparably to stimulation with the agonist calcium ionophore A23187. The serum-free in vitro culture of chondrocytes resulted in MDA-protein oxidation. The treatment of chondrocytes with A23187 resulted in the enhancement of MDA-protein oxidation. The immunoblot reactivity pattern of extracts to MDA2 antibody and to polyclonal anti-type II collagen antibody was somewhat similar, which suggests that these two different types of antisera exhibit a crossreaction to chondrocyte proteins. Chondrocyte extracts were pretreated both with and without pure collagenase, and then subjected to immunoblot analysis. Only collagenase treated extracts showed a disappearance, or significant reduction, of larger than 60 kDa size MDA2 immunoreactive proteins. This suggests that the proteins that disappeared after the enzyme treatment were collagen proteins and which had also been modified by MDA oxidation. CONCLUSIONS: These observations suggest that collagen hydroxylation of matrix by vitamin C does not play a role in this model of chondrocyte-dependent collagen degradation. Also, this study demonstrates that chondrocyte-derived lipid peroxidation product MDA mediates oxidation of cartilage collagens. Oxidative modification of cartilage collagen in vivo could result in alteration of biochemical and biophysical properties of cartilage collagen fibrils, making them prone to degradation, thus initiating the changes observed in aging and OA.     

不同年龄肋软骨的组织形态学及Ⅱ型胶原蛋白含量变化的研究

目的 探讨肋软骨的组织形态学及II型胶原蛋白在不同年龄阶段的变化规律.方法 将2005年至2006年行耳廓再造的患者按年龄不同分为3组.5～10岁为儿奄组,11～17岁为青少年组,18～29岁为成人组,每组30例.观察不同年龄组肋软骨的组织形态学变化及Ⅱ型胶原免疫组织化学染色的定量分析,采用Motie Med6.0 A数码医学图像分析系统进行免疫组织化学图像定星测定,测量指标为平均积分光密度,对分析结果 进行统计学处理.结果 儿童组肋软骨膜血管最丰富,软骨基质染色均匀,软骨细胞数目最多,Ⅱ型胶原蛋白表达最活跃,平均积分光密度值最高;青少年组软骨膜内血管减少,软骨基质染色出现明显的不均质状,软骨陷窝体积变大,并呈分隔状,陷窝内软骨细胞数目减少,II型胶原蛋白表达较儿童组减弱;成人组软骨膜血管、细胞成分明显减少,软骨膜内的纤维成分明显玻璃样变,钙盐沉积较青少年组时明显增多,Ⅱ型胶原蛋白表达较青少年组减弱.经统计学分析,3组间差异有统计学意义(P＜0.01).结论 肋软骨的组织形态学随年龄增长发生变化,Ⅱ型胶原蛋白含量随年龄增长呈递减趋势.     

Type II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats

OBJECTIVE: To investigate the kinetics of early cartilage changes in mechanically induced osteoarthritis (OA) and the association of these changes with damage to the type II collagen network. METHODS: Experimental OA was induced by anterior cruciate ligament transsection in the rat knee joint (ACLT-OA). Animals were sacrificed after 2, 7, 14, 28 and 70 days. Knee joints were evaluated using routine histology and immunohistochemistry for denatured (unwound) type II collagen to detect collagen damage. An antibody recognizing the collagenase cleavage site in type II collagen was used to study the role of collagenase in this process. RESULTS: The first changes of the articular cartilage after anterior cruciate ligament transection occurred in the superficial zone. These changes included loss of superficial chondrocytes, swelling of the remaining chondrocytes and superficial fibrillation. The swelling of the chondrocytes did not result from a change towards the hypertrophic phenotype, since these cells did not stain for type X collagen. A marked increase in denatured type II collagen staining was present in the fibrillated areas. Staining of the collagenase cleavage site showed the same distribution as denatured collagen but was clearly less intense. Collagen damage could never be detected before fibrillation occurred and was not present in non-fibrillated areas. CONCLUSIONS: These results indicate that in this model cartilage degeneration starts at the articular surface and that this degeneration is associated with a localized expression of type II collagen degradation products.     

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification

The role of Wnt signaling is extensively studied in skeletal development and postnatal bone remodeling, mostly based on the genetic approaches of β-catenin manipulation. However, given their independent function, a requirement for β-catenin is not the same as that for Wnt. Here, we investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Taken together, our studies reveal that Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate.