Intracellular Na+ and Ca2+ modulation increases the tensile properties of developing engineered articular cartilage

Objective

Significant collagen content and tensile properties are difficult to achieve in tissue‐engineered articular cartilage. The aim of this study was to investigate whether treating developing tissue‐engineered cartilage constructs with modulators of intracellular Na⁺ or Ca²⁺ could increase collagen concentration and construct tensile properties.

Methods

Inhibitors of Na⁺ ion transporters and stimulators of intracellular Ca²⁺ were investigated for their ability to affect articular cartilage development in a scaffoldless, 3‐dimensional chondrocyte culture. Using a systematic approach, we applied ouabain (Na⁺/K⁺‐ATPase inhibitor), bumetanide (Na⁺/K⁺/2Cl⁻ tritransporter inhibitor), histamine (cAMP activator), and ionomycin (a Ca²⁺ ionophore) to tissue‐engineered constructs for 1 hour daily on days 10–14 of culture and examined the constructs at 2 weeks or 4 weeks. The gross morphology, biochemical content, and compressive and tensile mechanical properties of the constructs were assayed.

Results

The results of these experiments showed that 20 μM ouabain, 0.3 μM ionomycin, or their combination increased the tensile modulus by 40–95% compared with untreated controls and resulted in an increased amount of collagen normalized to construct wet weight. In constructs exposed to ouabain, the increased percentage of collagen per construct wet weight was secondary to decreased glycosaminoglycan production on a per‐cell basis. Treatment with 20 μM ouabain also increased the ultimate tensile strength of neo‐tissue by 56–86% at 4 weeks. Other construct properties, such as construct growth and type I collagen production, were affected differently by Na⁺ modulation with ouabain versus Ca²⁺ modulation with ionomycin.

Conclusion

These data are the first to show that treatments known to alter intracellular ion concentrations are a viable method for increasing the mechanical properties of engineered articular cartilage and identifying potentially important relationships to hydrostatic pressure mechanotransduction. Ouabain and ionomycin may be useful pharmacologic agents for increasing tensile integrity and directing construct maturation.

     

Type II collagen levels correlate with mineralization by articular cartilage vesicles

Objective

Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors.

Methods

ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme‐linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of ⁴⁵Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze‐thawed cartilage.

Results

As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze‐thawed cartilage. Type II collagen levels were 100‐fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase‐crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs.

Conclusion

Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro.

     

Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis

Objective

Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA).

Methods

Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low‐density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real‐time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis/N‐terminal sequencing, while its ability to activate proteinase‐activated receptor 2 (PAR‐2) was determined using a synovial perfusion assay in mice.

Results

Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP‐1 and processed proMMP‐3 to its fully active form. Exogenous matriptase significantly enhanced cytokine‐stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase‐dependent. Matriptase also induced MMP‐1, MMP‐3, and MMP‐13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR‐2, and we demonstrated that matriptase‐dependent enhancement of collagenolysis from OA cartilage is blocked by PAR‐2 inhibition.

Conclusion

Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR‐2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

     

Cartilage degradation biomarkers predict efficacy of a novel,highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: Confirmation by multivariate analysis that modulation of type ii collagen and aggrecan degradation peptides parallels pathologic changes

Objective

To demonstrate that the novel highly selective matrix metalloproteinase 13 (MMP‐13) inhibitor PF152 reduces joint lesions in adult dogs with osteoarthritis (OA) and decreases biomarkers of cartilage degradation.

Methods

The potency and selectivity of PF152 were evaluated in vitro using 16 MMPs, TACE, and ADAMTS‐4 and ADAMTS‐5, as well as ex vivo in human cartilage explants. In vivo effects were evaluated at 3 concentrations in mature beagles with partial medial meniscectomy. Gross and histologic changes in the femorotibial joints were evaluated using various measures of cartilage degeneration. Biomarkers of cartilage turnover were examined in serum, urine, or synovial fluid. Results were analyzed individually and in combination using multivariate analysis.

Results

The potent and selective MMP‐13 inhibitor PF152 decreased human cartilage degradation ex vivo in a dose‐dependent manner. PF152 treatment of dogs with OA reduced cartilage lesions and decreased biomarkers of type II collagen (type II collagen neoepitope) and aggrecan (peptides ending in ARGN or AGEG) degradation. The dose required for significant inhibition varied with the measure used, but multivariate analysis of 6 gross and histologic measures indicated that all doses differed significantly from vehicle but not from each other. Combined analysis of cartilage degradation markers showed similar results.

Conclusion

This highly selective MMP‐13 inhibitor exhibits chondroprotective effects in mature animals. Biomarkers of cartilage degradation, when evaluated in combination, parallel the joint structural changes induced by the MMP‐13 inhibitor. These data support the potential therapeutic value of selective MMP‐13 inhibitors and the use of a set of appropriate biomarkers to predict efficacy in OA clinical trials.

     

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle‐derived stem cells expressing bone morphogenetic protein 4 and soluble flt‐1

Objective

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on the chondrogenesis of skeletal muscle‐derived stem cells (MDSCs) in a rat model of osteoarthritis (OA).

Methods

We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP‐4) in combination with MDSCs expressing VEGF or sFlt‐1.

Results

In vivo, a combination of sFlt‐1– and BMP‐4–transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt‐1– and BMP‐4–transduced MDSCs compared with a combination of VEGF‐ and BMP‐4–transduced MDSCs or with BMP‐4–transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP‐4–transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes.

Conclusion

Our results demonstrate that MDSC‐based therapy involving sFlt‐1 and BMP‐4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

     

Parathyroid hormone 1–34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats

Objective

Parathyroid hormone 1–34 (PTH[1–34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone–related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1–34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA).

Methods

We studied the effect of PTH(1–34) on human articular chondrocytes with azacytidine (azaC)–induced terminal differentiation in vitro and on papain‐induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl‐2, and Bax by real‐time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees.

Results

AzaC induced terminal differentiation of human chondrocytes, including down‐regulation of aggrecan, type II collagen, and GAG and up‐regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3–10 days of treatment with 10 nM PTH(1–34). SOX9 expression was not changed by either azaC or PTH(1–34) treatment. Bcl‐2 and Bax were up‐regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1–34) treatment did not reverse this effect. Furthermore, PTH(1–34) treatment reversed papain‐induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats.

Conclusion

Our findings indicate that PTH(1–34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat 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.

     

Gremlin 1, Frizzled‐related protein,and Dkk‐1 are key regulators of human articular cartilage homeostasis

Objective

The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation.

Methods

Whole‐genome gene‐expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA.

Results

Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled‐related protein (FRP), and Dkk‐1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long‐bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single‐nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population‐based cohorts.

Conclusion

Taken together, our study identified Gremlin 1, FRP, and Dkk‐1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

     

Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA–array technology

Objective

To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)–array technology.

Methods

Nine normal, 6 early degenerated, and 6 late‐stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis.

Results

In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late‐stage specimens, reflecting the high matrix‐remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP‐3 appeared to be strongly expressed in normal and early degenerative cartilage and down‐regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP‐2 and MMP‐11, both of which were up‐regulated in late‐stage disease. MMP‐11 was up‐regulated in OA chondrocytes and, interestingly, also in the early‐stage samples. Neither MMP‐1 nor MMP‐8 was detectable, and MMP‐13 and MMP‐2 were significantly detectable only in late‐stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers.

Conclusion

This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.

     

Accelerated,aging‐dependent development of osteoarthritis in α1 integrin–deficient mice

Objective

Cell–matrix interactions regulate chondrocyte differentiation and survival. The α1β1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin α1 gene (α1‐KO mice) provide a model that can be used to address the role of cell–matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis.

Methods

Knee joints from α1‐KO and wild‐type (WT) BALB/c mice were harvested at ages 4–15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of α1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis.

Results

In WT mice, the α1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in α1‐positive chondrocytes in the superficial and upper mid‐zones in OA‐affected areas in joints from old WT mice. The α1‐KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP‐2 and MMP‐3 expression was increased in the OA‐affected areas. In cartilage from α1‐KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the α1 integrin subunit is involved in the early remodeling process in OA cartilage.

Conclusion

Deficiency in the α1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging‐dependent development of OA.

     

Stimulation of collagenase 3 expression in synovial fibroblasts of patients with rheumatoid arthritis by contact with a three‐dimensional collagen matrix or with normal cartilage when coimplanted in NOD/SCID mice

Objective

To study the expression of collagenase 3 (matrix metalloproteinase 13 [MMP‐13]) and collagenase 1 (MMP‐1) in synovial fibroblasts from patients with rheumatoid arthritis (RA) when cultured within 3‐dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice.

Methods

Messenger RNA (mRNA) and protein expression of collagenase 3 and collagenase 1 were characterized in synovial and skin fibroblasts by Northern blot and Western blot analysis. The mRNA expression of both collagenases in cell–cartilage implants in NOD/SCID mice was investigated by in situ hybridization in combination with immunohistochemistry of human fibroblasts.

Results

Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice deeply invaded adjacent cartilage tissue. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, while the expression of collagenase 1 mRNA could not be detected. Culture of synovial fibroblasts within 3‐dimensional collagen gels was a ssociated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was 1 order of magnitude higher in comparison with a 2–4‐fold increase upon treatment with interleukin‐1 β or tumor necrosis factor α. In contrast, mRNA expression and proenzyme production of collagenase 1 were increased strongly, and to a similar extent, either by contact with 3‐dimensional collagen or by proinflammatory cytokines.

Conclusion

The expression of collagenase 3, in contrast to that of collagenase 1, is preferentially stimulated in synovial fibroblasts by 3‐dimensional collagen rather than by proinflammatory cytokines. The induction of collagenase 3 by cell–matrix interactions represents a potential mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA.

     

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n‐3 fatty acids

Objective

To determine if n‐3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n‐6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage.

Methods

The metabolic profile of human OA cartilage was determined at the time of harvest and after 24‐hour exposure to n‐3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin‐1 (IL‐1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]).

Results

Supplementation with n‐3 PUFA (but not other fatty acids) reduced, in a dose‐dependent manner, the endogenous and IL‐1–induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS‐4, MMP‐13, and MMP‐3 (but not TIMP‐1, ‐2, or ‐3) was also specifically abolished with n‐3 PUFA supplementation. In addition, n‐3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5‐lipoxygenase, 5‐lipoxygenase–activating protein, tumor necrosis factor α, IL‐1α, and IL‐1β) without affecting the expression of message for several other proteins involved in normal tissue homeostasis.

Conclusion

These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n‐3 PUFA, but not to other classes of fatty acids.

     

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: Prominent role of Wnt‐induced signaling protein 1

Objective

Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA.

Methods

Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/β‐catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression.

Results

Wnt‐induced signaling protein 1 (WISP‐1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt‐16 and Wnt‐2B were also markedly up‐regulated during the course of disease. Interestingly, increased WISP‐1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP‐1 resulted in interleukin‐1–independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP‐1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage.

Conclusion

This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP‐1 expression is a feature of experimental and human OA and that WISP‐1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.