Inactivation of one allele of the type II collagen gene alters the collagen network in murine articular cartilage and makes cartilage softer

OBJECTIVE: To evaluate the influence of inactivation of one allele ("heterozygous knockout" or "heterozygous inactivation") of the type II procollagen gene (Col2a1) on the biomechanical properties and structure of the articular cartilage and subchondral bone in 15 month old mice. METHODS: Indentation stiffness of the humerus head articular cartilage was measured by a microindentation method. Cartilage and subchondral bone were prepared for digital densitometry of proteoglycans (PGs), polarised light microscopy (PLM) of collagen, and osteoarthrosis (OA) grading. RESULTS: Heterozygous inactivation of the Col2a1 gene softened articular cartilage (p=0.002) as measured by indentation stiffness ((mean (SEM) 0.50 (0.07) MPa v 0.94 (0.13) MPa in controls). Fibrillar collagen network exhibited lower birefringence in the intermediate (p=0.04) and deep zones (p=0.01) of cartilage by PLM, indicating either decreased collagen content or a lower degree of fibril parallelism in the knockout mice. The total and zonal thicknesses of articular cartilage were unchanged. Zonal PG contents did not differ significantly. In knockout mice, the prevalence of superficial fibrillation-that is, a sign of OA, was higher than in controls (73% v 21%, p=0.002). The collagen induced birefringence of the superficial zone was not reduced. The subchondral bone volume fraction was lower in knockout mice than in controls, 31% v 43% (p=0.01), and optical retardation values in PLM of bone collagen were slightly but significantly lower (p=0.01). CONCLUSION: Heterozygous inactivation of the Col2a1 gene made articular cartilage softer, altered the collagenous network, reduced subchondral bone volume, and altered its microstructure. Changes in the cartilage collagen network probably contributed to increased susceptibility to OA.     

Lifelong voluntary joint loading increases osteoarthritis in mice housing a deletion mutation in type II procollagen gene,and slightly also in non-transgenic mice

Objectives: To investigate the effects of voluntary running on the incidence and severity of osteoarthritis (OA) and associated changes in cartilage matrix and subchondral bone in a transgenic Del1 mouse model for OA.

Methods: Del1 mice and their non-transgenic littermate controls were housed from the age of 5–6 weeks to 15 months in individual cages with running wheels. The running activity of each mouse was monitored for the entire 12 month period. Additional Del1 and control mice were housed in individual cages without running wheels. At the end of the experiment the severity of OA was evaluated by light microscopy, and the articular cartilage matrix changes by digital densitometry and quantitative polarised light microscopy.

Results: Lifelong voluntary running increased the incidence and severity of OA significantly in Del1 mice (transgenic runners), and slightly also in non-transgenic runners. Severe OA changes increased from 39% in transgenic non-runners to 90% in transgenic runners (p=0.006) in lateral tibial condyles, and from 24% to 80% (p=0.013) in lateral femoral condyles, respectively. The proteoglycan content of articular cartilage was reduced in transgenic runners in comparison with transgenic non-runners (p=0.0167), but a similar effect was not seen in non-transgenic runners compared with non-transgenic non-runners. No attributable differences were seen in the collagen network of articular cartilage or in the subchondral bone between any of the groups.

Conclusion: The Del1 mutation has earlier been shown to disturb the assembly of the cartilage collagen network and thereby increase the incidence and severity of OA with age. In this study, voluntary running was shown to increase further cartilage damage in the lateral compartments of the knee. This suggests that articular cartilage in Del1 mice is less resistant to physical loading than in control mice. Despite severe OA lesions in the knee joint at the age of 15 months, Del1 mice continued to run voluntarily 2–3 km every night.

     

软骨下骨与骨性关节炎病变的发生发展：从基础研究到临床治疗

骨性关节炎（OA）是软骨与骨的退行性变过程。软骨下骨是构成关节结构和功能的基本单位之一,维持软骨正常结构和功能。在复杂应力和生物学作用下,OA软骨下骨的重塑和结构改变使软骨承受更高的应力。软骨下骨内血管生成和结构病变扩大了骨软骨异常交流途径,软骨下骨产生的代谢调节因子通过异常生物学交流直接促进软骨退变。研究软骨下骨内细胞及其变化,血管生成与骨软骨间生物学交流,揭示软骨下骨重塑和结构变化特点,探寻改善骨重塑治疗方法,将有利于延缓OA病变进展,有效防治OA。     

The role of subchondral bone remodeling in osteoarthritis: reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model

OBJECTIVE: It has been suggested that subchondral bone remodeling plays a role in the progression of osteoarthritis (OA). To test this hypothesis, we characterized the changes in the rat anterior cruciate ligament transection (ACLT) model of OA and evaluated the effects of alendronate (ALN), a potent inhibitor of bone resorption, on cartilage degradation and on osteophyte formation. METHODS: Male Sprague-Dawley rats underwent ACLT or sham operation of the right knee. Animals were then treated with ALN (0.03 and 0.24 microg/kg/week subcutaneously) and necropsied at 2 or 10 weeks postsurgery. OA changes were evaluated. Subchondral bone volume and osteophyte area were measured by histomorphometric analysis. Coimmunostaining for transforming growth factor beta (TGF beta), matrix metalloproteinase 9 (MMP-9), and MMP-13 was performed to investigate the effect of ALN on local activation of TGF beta. RESULTS: ALN was chondroprotective at both dosages, as determined by histologic criteria and collagen degradation markers. ALN suppressed subchondral bone resorption, which was markedly increased 2 weeks postsurgery, and prevented the subsequent increase in bone formation 10 weeks postsurgery, in the untreated tibial plateau of ACLT joints. Furthermore, ALN reduced the incidence and area of osteophytes in a dose-dependent manner. ALN also inhibited vascular invasion into the calcified cartilage in rats with OA and blocked osteoclast recruitment to subchondral bone and osteophytes. ALN treatment reduced the local release of active TGF beta, possibly via inhibition of MMP-13 expression in articular cartilage and MMP-9 expression in subchondral bone. CONCLUSION: Subchondral bone remodeling plays an important role in the pathogenesis of OA. ALN or other inhibitors of bone resorption could potentially be used as disease-modifying agents in the treatment of OA.     

Apoptotic chondrocytes from osteoarthrotic human articular cartilage and abnormal calcification of subchondral bone

OBJECTIVE: Osteoarthrosis (OA) is accompanied by altered subchondral bone remodeling. We investigated the role of chondrocytes in the mechanism of abnormal cartilage calcification. METHODS: Knee articular cartilage samples from OA and normal tissue were studied. Macroscopic and microscopic observations, alkaline phosphatase staining for light and electron microscopy (bright and dark fields). TUNEL technique, electron diffraction, and x-ray microanalyses were performed. RESULTS: Chondrocytes from patients displayed a morphology of apoptosis and showed abundant alkaline phosphatase (ALP)-rich matrix vesicles (MV) budding from the plasma membrane with hydroxyapatite microcrystals on their surface. Farther from the cells, hydroxyapatite crystals were detected on the MV surface and increased as they approached the subchondral bone. The concentration of Ca and P and their ratio increased inside the ALP-rich MV in relation to the proximity to subchondral bone. In the OA subchondral bone the ratio Ca/P varied from 3.936 to 0.974. In normal tissue the ratio was very homogeneous (maximum 1.973, minimum 1.781). CONCLUSION: In situ, apoptotic chondrocytes correlate with factors known to be involved in the calcification of the extracellular matrix. This suggests that apoptosis is involved in the abnormal calcification of OA cartilage, and consequently in the altered remodeling of the subchondral bone.     

Differential expression patterns of matrix metalloproteinases and their inhibitors during development of osteoarthritis in a transgenic mouse model

Objective: To characterise the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during degeneration of articular cartilage in a transgenic Del1 mouse model for osteoarthritis.

Methods: Northern analysis was used to measure mRNA levels of MMP-2, -3, -8, -9, -13, and -14, and TIMP-1, -2, and -3 in total RNA extracted from knee joints of transgenic Del1 mice, harbouring a 15 amino acid deletion in the triple helical domain of the α1(II) collagen chain, using their non-transgenic littermates as controls. Immunohistochemistry was used to study the presence of cleavage products (neoepitopes) of type II collagen, and the distribution of MMP-13 and TIMP-1 in degenerating cartilage.

Results: Each of the MMP and TIMP mRNAs analysed exhibited distinct expression patterns during development and osteoarthritic degeneration of the knee joint. The most striking change was up regulation of MMP-13 mRNA expression in the knee joints of Del1 mice at the onset of cartilage degeneration. However, the strongest immunostaining for MMP-13 and its inhibitor TIMP-1 was not seen in the degenerating articular cartilage but in synovial tissue, deep calcified cartilage, and subchondral bone. The localisation of type II collagen neoepitopes in chondrocytes and their pericellular matrix followed a similar pattern; they were not seen in cartilage fibrillations, but in adjacent unaffected cartilage.

Conclusion: The primary localisation of MMP-13 and TIMP-1 in hyperplastic synovial tissue, subchondral bone, and calcified cartilage suggests that up regulation of MMP-13 expression during early degeneration of articular cartilage is a secondary response to cartilage erosion. This interpretation is supported by the distribution of type II collagen neoepitopes. Synovial production of MMP-13 may be related to removal of tissue debris released from articular cartilage. In the deep calcified cartilage and adjacent subchondral bone, MMP-13 probably participates in tissue remodelling.

     

A longitudinal study of subchondral plate and trabecular bone in cruciate-deficient dogs with osteoarthritis followed up for 54 months

Objective. To evaluate the sequence of changes in articular cartilage, trabecular bone, and subchondral plate in dogs with osteoarthritis (OA), 3 months, 18 months, and 54 months after anterior cruciate ligament transection (ACLT). Methods. Specimens of the medial tibial plateau were analyzed with microscopic computed tomography (micro-CT) at a resolution of 60 μm, and biochemical and morphologic changes in the femoral articular cartilage were assessed. Results. At 3 months and 18 months after ACLT, the articular cartilage in the unstable knee showed histologic changes typical of early OA and increased water content and uronic acid concentration; by 54 months, full-thickness ulceration had developed. Micro-CT analysis showed a loss of trabecular bone in the unstable knee, compared with the contralateral knee, at all time points. At both 18 and 54 months, the differences in trabecular thickness and surface-to-volume ratio were greater than at 3 months. Although the mean subchondral plate thickness, especially in the medial aspect of the medial tibial plateau, was greater in the OA knee than in the contralateral knee 18 months and 54 months after ACLT, these differences were not statistically significant; however, the difference was significantly greater at 54 months than at 3 months. Conclusion. Thickening of the subchondral bone is not required for the development of cartilage changes of OA in this model. The bony changes that develop after ACLT, however, could result in abnormal transmission of stress to the overlying cartilage and thereby contribute to the progression of cartilage degeneration.     

The relative importance of cysteine peptidases in osteoarthritis

OBJECTIVE: To assess the activity of cysteine peptidases in cultured human articular chondrocytes as well as in osteoarthritic (OA) cartilage and subchondral bone, and to interpret their relative importance in cartilage destruction and remodeling of the subchondral region. METHODS: Intracellular and secreted cysteine peptidase activity was measured in chondrocytes using fluorimetric assays, and enzymes were immunolocalized using monospecific antibodies. Enzyme histochemistry in normal and OA femoral heads was used to characterize enzymatic activity in full thickness samples containing cartilage and subchondral bone. The zonal distribution of cathepsin activity was measured in tissue slices of normal and OA femoral heads cut parallel to the joint surface, using fluorogenic substrates. RESULTS: Cathepsins B and L were localized by immunohistochemistry with lysosome-like structures in dedifferentiated chondrocytes. Free cysteine peptidase activity (i.e., not requiring prior activation), secreted and intracellularly stored by chondrocytes, was due to cathepsin B, while cathepsin L contributed a minor fraction of the total activity, and was seen only after activation at acidic pH. Histochemistry and activity measurements confirmed cathepsin B as the major, active cysteine peptidase in OA cartilage, particularly at sites where matrix neosynthesis took place. However, free cathepsin L and/or cathepsin K activity was found subchondrally in association with cathepsin B in osteophytes, in zones undergoing bone remodeling, and at sites of inflammation. CONCLUSION: Cathepsin B, not cathepsin L or cathepsin K, is a candidate for articular cartilage catabolism in OA. While cathepsin K is the major osteoclastic cysteine peptidase, cathepsin L and cathepsin B may also participate in the remodeling processes of bone as well as in bone erosion by inflammatory cells.     

Microfractures and microcracks in subchondral bone: are they relevant to osteoarthrosis?

The existing data are consistent with the view that reactivation of the secondary center of ossification and not the stiffening of the metaphyseal trabecular bone is a mechanism of cartilage loss in idiopathic OA. The stiffening of the subchondral calcified structures would appear to be etiologically incidental and, as the arthrotic process progresses, sometimes locally transient. It is also now clear that although the apparent density of the subchondral cortical plate increases because of thickening of the plate as the OA process progresses, the elastic modulus of the bone might be reduced locally because of increases in vascularization and in the rate of bony remodeling subjacent to the cartilage. Microcracks in the subchondral mineralized tissues might contribute to degeneration of the hyaline cartilage by initiating vascular invasion of the calcified cartilage, leading to reactivation of the tidemark and enchondral ossification with subsequent thinning of the overlying articular cartilage. The thinning would tend to increase shear stresses at the base of the articular cartilage [38], overwhelming the ability of the cartilage to repair itself, resulting in cartilage degeneration. The pathogenesis of cartilage breakdown in OA is a biological and a mechanical process. OA can be understood only if the relationship between the mechanics and the biology is fully appreciated. Failure to properly absorb impact leads to microdamage in the subchondral plate and calcified cartilage. The authors believe that this action causes the secondary center of ossification at the tidemark to advance by enchondral ossification, leading to thickening of the mineralized tissues and thinning of the overlying hyaline articular cartilage. Microcracks will cause the initiation of targeted remodeling, accounting for the increased turnover and reduced material density of the subchondral plate. The resultant thinning of the articular cartilage might lead to initiation of further microdamage in bone and cartilage through a positive feedback mechanism, which can ultimately lead to complete loss of the articular cartilage. In this view, the mechanical overload that initiates microdamage of the subchondral bone provokes a biological response that potentiates the progression of articular cartilage damage in OA.     

Nitrite and nitrotyrosine concentrations in articular cartilage, subchondral bone, and trabecular bone of normal juvenile, normal adult, and osteoarthritic adult equine metacarpophalangeal joints

OBJECTIVE: Osteoarthritis (OA) is a chronic debilitating joint disorder in which the importance of inflammation is increasingly recognized. In advanced cases, both the articular cartilage and the underlying bony layers are affected, but the exact sequence of events and their localization in the initial phase of pathogenesis remain uncertain. We measured nitric oxide (NO) end products in tissue layers that constitute the bearing surface of the joint, as possible indicators of physiological and pathological processes. METHODS: Nitrite as a measure for NO and nitrotyrosine was measured in articular cartilage, subchondral bone, and the underlying trabecular bone of the proximal articular surface of the first phalanx of healthy mature horses (n = 15; age range 5-18 yrs), mature horses affected by OA (n = 15; age range 8-22 yrs), and unaffected juvenile horses (n = 13; age range 6 months-4 yrs). Data were correlated with cartilage damage, as quantified by the Cartilage Degeneration Index. RESULTS: In all 3 layers the nitrite concentration was higher in OA joints (cartilage, p < 0.001; subchondral and trabecular bone, p < 0.05). The concentration of nitrite was significantly higher in cartilage and subchondral bone of juvenile horses compared with mature horses (p < 0.001). Nitrotyrosine concentrations were significantly higher in subchondral bone of OA horses compared with healthy controls (p < 0.001), but significantly lower in trabecular bone of juvenile horses (p < 0.01). CONCLUSION: The similarities observed over the 3 tissue layers support the concept of the bearing surface of the joint as a functional entity. Nitrite concentration seems to be a good indicator of tissue metabolic activity, but cannot discriminate between physiological (juvenile animals) and pathological (OA cases) processes. The increased nitrotyrosine levels in subchondral bone of OA-affected animals suggest that this layer is important in early or moderate OA, and implies a role of oxidative stress in the development of the disease.     

Vascular pathology and osteoarthritis

There is mounting evidence that vascular pathology plays a role in the initiation and/or progression of the major disease of joints: osteoarthritis (OA). Potential mechanisms are: episodically reduced blood flow through the small vessels in the subchondral bone at the ends of long bones, and related to this, reduced interstitial fluid flow in subchondral bone. Blood flow may be reduced by venous occlusion and stasis or by the development of microemboli in the subchondral vessels. There are several likely effects of subchondral ischaemia: the first of these is compromised nutrient and gas exchange into the articular cartilage, a potential initiator of degradative changes in the cartilage. The second is apoptosis of osteocytes in regions of the subchondral bone, which would initiate osteoclastic resorption of that bone and at least temporarily reduce the bony support for the overlying cartilage. It may be important to recognize these potential aetiological factors in order to develop more effective treatments to inhibit the progression of OA.     

Biochemical evidence for altered subchondral bone collagen metabolism in osteoarthritis of the hip

Osteoarthritis (OA) of the hip is invariably viewed as a disease primarily affecting the articular cartilage. Data presented in this report, however, demonstrate changes in the metabolic activity of the underlying trabecular bone tissue, the processes of which may represent a significant factor in the pathogenesis of hip OA. Trabecular bone tissue from OA subjects expressed significantly more matrix metalloproteinase (MMP)-2 (gelatinase A, 72 kDa type IV collagenase) when compared to age-matched osteoporotic (OP) and normal bone tissue. Alkaline phosphatase was also significantly elevated in OA bone tissue. The combination of increased MMP-2 and alkaline phosphatase indicates heightened collagen turnover in the subchondral bone compartment of osteoarthritic hips. The data obtained from this study warrant a closer investigation into the significance of these changes in OA and emphasize the multifactorial elements of the whole joint in the whole joint in the overall disease process.      

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

OBJECTIVE: Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. METHODS: We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. RESULTS: Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. CONCLUSION: These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.     

Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis

OBJECTIVE: Chondromodulin-I (ChM-I), a cartilage derived anti-angiogenic factor, has been shown to regulate the vascular invasion during endochondral bone formation. We evaluated the expression and localization of ChM-I in articular cartilage during the progression of osteoarthritis (OA) in the rat, and correlated ChM-I expression with the increase in vascular invasion into OA articular cartilage. METHODS: Expression of ChM-I, type II collagen, basic fibroblast growth factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases MMP-9 and MMP-13 were examined in articular cartilage of intact growing and adult rats and in the surgically induced OA model using in situ hybridization, Western blot analysis, and immunohistochemistry. Co-immunostaining for ChM-I and CD-31 was performed to localize ChM-I and neovascularization in articular cartilage at advanced stage of OA. RESULTS: Abundant expression of ChM-I protein was detected in avascular regions of the developing and adult healthy articular cartilage. In early OA, ChM-I expression decreased in the superficial zone of articular cartilage, while levels of proteoglycan and type II collagen were comparable to control. In advanced OA, ChM-I expression was reduced in all zones of articular cartilage, and the number of VEGF-expressing cells was increased. Immunohistochemical studies showed that vascular invasion occurred in proximity to chondrocytes with high expression of pro-angiogenic markers, and decreased expression of ChM-I. CONCLUSION: High expression of ChM-I was detected in articular cartilage of growing and normal adult joints, implicating its role in the maintenance of avascularity of intact articular cartilage. Expression of ChM-I decreased, while expression of VEGF and other pro-angiogenic factors increased, in OA cartilage. These findings suggest the loss of ChM-I from articular cartilage might be responsible in part for promoting blood vessel invasion into the cartilage during progression of OA.     

In vivo bone‐specific EphB4 overexpression in mice protects both subchondral bone and cartilage during osteoarthritis

Objective

In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone‐specific EphB4 overexpression on OA pathophysiology.

Methods

Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10‐week‐old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10‐week‐old male EphB4 homozygous transgenic (EphB4‐Tg) and wild‐type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro–computed tomography.

Results

There was no obvious phenotype difference in skeletal development between EphB4‐Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post‐DMM, the EphB4‐Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4‐Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate‐resistant acid phosphatase–positive osteoclasts at both times assessed post‐DMM in the EphB4‐Tg mice than in the WT mice.

Conclusion

This is the first in vivo evidence that bone‐specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity.

     

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1)

OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.     

Osteoarthritis-like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho)

OBJECTIVE: To investigate whether heterozygosity for a loss-of-function mutation in the gene encoding the alpha1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints. METHODS: Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild-type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP-3) and MMP-13 in knee joints. In 6-month-old animals, fixed-charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique. RESULTS: The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA-like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP-3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP-13 was higher in knee joints from cho/+ mice than in joints from their wild-type littermates, and negative fixed-charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration. CONCLUSION: Heterozygosity for a loss-of-function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.     

Subchondral bone in osteoarthritis: a biologic link with articular cartilage leading to abnormal remodeling

PURPOSE OF REVIEW: This review deals with new findings highlighting the concept of cross-talk between subchondral bone tissue and articular cartilage that may be crucial for the initiation and/or progression of osteoarthritis. In this review, new factors either produced by subchondral bone tissue or modifying osteoblast metabolism, yet implicated in osteoarthritis, are discussed. RECENT FINDINGS: The development of cartilage degeneration is concomitant with subchondral bone thickness in osteoarthritis, whereas it is related to higher subchondral bone activity and dysregulation in the synthesis of bone proteins. As an immediate consequence, homotrimers of type 1 collagen are formed that could lead to undermineralization of this tissue. This dysregulation also leads to abnormal production of different factors by osteoblasts such as prostaglandins, leukotrienes, and growth factors. Because microcracks or neovascularization provide a link between the subchondral bone tissue and articular cartilage, these factors could contribute to the abnormal remodeling of osteoarthritic cartilage. SUMMARY: These findings have an immediate implication for research because new tools need to be developed to study the subchondral bone-cartilage functional unit. Moreover, it could lead to a possible cure for osteoarthritis because this pathology should be considered both a bone and cartilage disease.     

Effects of a bisphosphonate on bone histomorphometry and dynamics in the canine cruciate deficiency model of osteoarthritis

OBJECTIVE: To examine the effect of the bisphosphonate NE- 10035 on bone histomorphometry and bone dynamics in dogs after transection of the anterior cruciate ligament (ACL), and to determine, in a placebo controlled trial, whether treatment modified the severity of pathologic changes of osteoarthritis (OA) in the unstable joint. METHODS: Ten adult male mongrel dogs underwent ipsilateral ACL transection. Five dogs then received daily subcutaneous injections of NE-10035 on 5 days per week for 12 weeks beginning the day after surgery. The other 5 dogs served as concurrent OA controls and received subcutaneous injections of saline on the same schedule. At sacrifice, 12 weeks after ACL transection, the articular cartilage and synovium of both knees of each dog were evaluated grossly and histologically and the water content and uronic acid concentration of the articular cartilage was determined. Fifteen days before sacrifice, each dog was injected with the fluorochrome label calcein. The injection regimen was repeated 10 days after the initial date. At sacrifice, static and dynamic variables of bone formation were assessed and bone resorption was quantified. RESULTS: In the OA knee of the control group, bone formation and resorption were markedly increased. NE-10035 markedly reduced both formation and resorption of cancellous subchondral bone, but had no effect on osteophyte formation or pathologic changes of OA in the articular cartilage, which were mild in both treatment groups. Water content of the OA cartilage was increased by about 8% in both treatment groups. However, among the controls, the mean uronic acid concentration of the OA cartilage was increased by about 30% in comparison with values for the contralateral knee, while in the NE-10035 treatment group the mean uronic acid concentration of OA knee cartilage was about 15% lower in the active treatment group than in cartilage from the contralateral knee (p = 0.003 for the difference in OA knee uronic acid concentration between the 2 treatment groups, relative to that in the contralateral knee). CONCLUSION: The antiresorptive agent employed in this study effectively reduced turnover of subchondral bone in the OA joint, consistent with the coupling of bone formation to bone resorption at that site. Nonetheless, over the 12 week period of the study it had no effect on osteophyte formation, in which bone formation occurs via enchondral ossification and is not linked to bone resorption, and, despite the clear inhibition of bone turnover in the OA knee of the active treatment group, did not affect the severity of cartilage changes of OA. It should be noted, however, that although treatment with this antiresorptive agent did not affect the level of chondropathy, the cartilage changes in both treatment groups were relatively mild and the sample size relatively small. Additional studies with a larger number of animals and a longer period of observation (to increase the severity of pathology) are warranted to determine whether the inhibition of bone turnover and the decrease in proteoglycan concentration that resulted from therapy will affect articular cartilage degeneration in the OA joint.