Systemic inhibition or global deletion of CaMKK2 protects against post-traumatic osteoarthritis.

OBJECTIVE: To investigate the role of Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2) in post-traumatic osteoarthritis (PTOA). METHODS: Destabilization of the medial meniscus (DMM) or sham surgeries were performed on 10-week-old male wild-type (WT) and Camkk2-/- mice. Half of the DMM-WT mice and all other cohorts (n = 6/group) received tri-weekly intraperitoneal (i.p.) injections of saline whereas the remaining DMM-WT mice (n = 6/group) received i.p. injections of the CaMKK2 inhibitor STO-609 (0.033 mg/kg body weight) thrice a week. Study was terminated at 8- or 12-weeks post-surgery, and knee joints processed for microcomputed tomography imaging followed by histology and immunohistochemistry. Primary articular chondrocytes were isolated from knee joints of 4-6-day-old WT and Camkk2-/- mice, and treated with 10 ng/ml interleukin-1ß (IL)-1ß for 24 or 48 h to investigate gene and protein expression. RESULTS: CaMKK2 levels and activity became elevated in articular chondrocytes following IL-1ß treatment or DMM surgery. Inhibition or absence of CaMKK2 protected against DMM-associated destruction of the cartilage, subchondral bone alterations and synovial inflammation. When challenged with IL-1ß, chondrocytes lacking CaMKK2 displayed attenuated inflammation, cartilage catabolism, and resistance to suppression of matrix synthesis. IL-1ß-treated CaMKK2-null chondrocytes displayed decreased IL-6 production, activation of signal transducer and activator of transcription 3 (Stat3) and matrix metalloproteinase 13 (MMP13), indicating a potential mechanism for the regulation of inflammatory responses in chondrocytes by CaMKK2. CONCLUSIONS: Our findings reveal a novel function for CaMKK2 in chondrocytes and highlight the potential for its inhibition as an innovative therapeutic strategy in the prevention of PTOA.

Noninvasive dualMRI-based strains vary by depth and region in human osteoarthritic articular cartilage.

OBJECTIVE: To noninvasively assay the mechanical and structural characteristics of articular cartilage from patients with osteoarthritis (OA) by magnetic resonance imaging (MRI), and to further relate spatial patterns of MRI-based mechanical strain to joint (depth-wise, regional) locations and disease severity. METHODS: Cylindrical osteochondral explants harvested from human tissue obtained during total knee replacement surgery were loaded in unconfined compression and 2D deformation data was acquired at 14.1 T using a displacements under applied loading by MRI (dualMRI) approach. After imaging, samples were histologically assessed for OA severity. Strains were determined by depth, and statistically analyzed for dependence on region in the joint and OA severity. RESULTS: Von Mises, axial, and transverse strains were highly depth-dependent. After accounting for other factors, Von Mises, axial, and shear strains varied significantly by region, with largest strain magnitudes observed in explants harvested from the tibial plateau and anterior condyle near exposed bone. Additionally, in all cases, strains in late-stage OA were significantly greater than either early- or mid-stage OA. Transverse strain in mid-stage OA explants, measured near the articular surface, was significantly higher than early-stage OA explants. CONCLUSION: dualMRI was demonstrated in human OA tissue to quantify the effects of depth, joint region, and OA severity, on strains resulting from mechanical compression. These data suggest dualMRI may possess a wide range of utility, such as validating computational models of soft tissue deformation, assaying changes in cartilage function over time, and perhaps, once implemented for cartilage imaging in vivo, as a new paradigm for diagnosis of early- to mid-stage OA.

Enhanced repair of articular cartilage defects in vivo by transplanted chondrocytes overexpressing insulin-like growth factor I (IGF-I).

Traumatic articular cartilage lesions have a limited capacity to heal. We tested the hypothesis that overexpression of a human insulin-like growth factor I (IGF-I) cDNA by transplanted articular chondrocytes enhances the repair of full-thickness (osteochondral) cartilage defects in vivo. Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or the human IGF-I gene and were encapsulated in alginate. The expression patterns of the transgenes in these implants were monitored in vitro for 36 days. Transfected allogeneic chondrocytes in alginate were transplanted into osteochondral defects in the trochlear groove of rabbits. At three and 14 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively. In vitro, IGF-I secretion by implants constructed from IGF-I-transfected chondrocytes and alginate was 123.2+/-22.3 ng/10(7) cells/24 h at day 4 post transfection and remained elevated at day 36, the longest time point evaluated. In vivo, transplantation of IGF-I implants improved articular cartilage repair and accelerated the formation of the subchondral bone at both time points compared to lacZ implants. The data indicate that allogeneic chondrocytes, transfected by a nonviral method and cultured in alginate, are able to secrete biologically relevant amounts of IGF-I over a prolonged period of time in vitro. The data further demonstrate that implantation of these composites into deep articular cartilage defects is sufficient to augment cartilage defect repair in vivo. These results suggest that therapeutic growth factor gene delivery using encapsulated and transplanted genetically modified chondrocytes may be applicable to sites of focal articular cartilage damage.

Gene-based approaches for the repair of articular cartilage.

Gene transfer technology has opened novel treatment avenues toward the treatment of damaged musculoskeletal tissues, and may be particularly beneficial to articular cartilage. There is no natural repair mechanism to heal damaged or diseased cartilage. Existing pharmacologic, surgical and cell based treatments may offer temporary relief but are incapable of restoring damaged cartilage to its normal phenotype. Gene transfer provides the capability to achieve sustained, localized presentation of bioactive proteins or gene products to sites of tissue damage. A variety of cDNAs have been cloned which may be used to stimulate biological processes that could improve cartilage healing by (1) inducing mitosis and the synthesis and deposition of cartilage extracellular matrix components by chondrocytes, (2) induction of chondrogenesis by mesenchymal progenitor cells, or (3) inhibiting cellular responses to inflammatory stimuli. The challenge is to adapt this technology into a useful clinical treatment modality. Using different marker genes, the principle of gene delivery to synovium, chondrocytes and mesenchymal progenitor cells has been convincingly demonstrated. Following this, research efforts have begun to move to functional studies. This involves the identification of appropriate gene or gene combinations, incorporation of these cDNAs into appropriate vectors and delivery to specific target cells within the proper biological context to achieve a meaningful therapeutic response. Methods currently being explored range from those as simple as direct delivery of a vector to a cartilage defect, to synthesis of cartilaginous implants through gene-enhanced tissue engineering. Data from recent efficacy studies provide optimism that gene delivery can be harnessed to guide biological processes toward both accelerated and improved articular cartilage repair.

Overexpression of human insulin-like growth factor-I promotes new tissue formation in an ex vivo model of articular chondrocyte transplantation.

Articular cartilage, the tissue that forms the gliding surface of joints, has a poor regenerative capacity. Insulin-like growth factor-I (IGF-I) is a polypeptide that is anabolic and mitogenic for cartilage. Transfection of articular chondrocytes with an expression plasmid vector containing the cDNA for human IGF-I under the control of the cytomegalovirus promoter/enhancer led to expression of the transgene and synthesis of biologically relevant amounts of IGF-I protein. Transplantation of transfected articular chondrocytes on to the surface of articular cartilage explants led to the formation of a new tissue layer on the cartilage explant surface. The new tissue was characterized by the presence of type II collagen and proteoglycan and by the absence of type I collagen, consistent with hyaline-like cartilage. The tissue formed by the chondrocytes expressing IGF-I was thicker and contained more cells than controls transfected with an expression plasmid vector containing the Escherichia coli (E. coli) beta-galactosidase (lacZ) gene. Transplantation of articular chondrocytes that overexpress human IGF-I also increased DNA synthesis and the synthesis of glycosaminoglycans by the underlying explant cartilage chondrocytes. These results identify a mechanism by which IGF-I may simultaneously promote chondrogenesis and shift cartilage homeostasis in an anabolic direction. The data further suggest that therapeutic growth factor gene transfer may be applicable to articular cartilage.

The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I.

Articular cartilage is routinely subjected to mechanical forces and to cell-regulatory molecules. Previous studies have shown that mechanical stimuli can influence articular chondrocyte metabolic activity, and biochemical studies have shown that growth factors and cytokines control many of the same cell functions. Little is known, however, of the relationships or interplay, if any, between these two key components of the articular environment. This study investigated the comparative and interactive effects of low amplitude, sinusoidal, dynamic compression and insulin-like growth factor-I (IGF-I), a polypeptide in synovial fluid that is anabolic for cartilage. In bovine patellofemoral cartilage explants, IGF-I increased protein and proteoglycan synthesis 90% and 120%, respectively while dynamic compression increased protein and proteoglycan synthesis 40% and 90%, respectively. Stimulation by IGF-I was significantly greater than by dynamic compression for both protein and proteoglycan synthesis. When applied together, the two stimuli enhanced protein and proteoglycan synthesis by 180% and 290%, respectively, a degree greater than that achieved by either stimulus alone. IGF-I augmented protein synthesis with a time constant of 12.2 h. Dynamic compression increased protein synthesis with a time constant of 2.9 h, a rate significantly faster than that of IGF-I, suggesting that these signals act via distinct cell activation pathways. When used together, dynamic compression and IGF-I acted with a time constant of 5.6 h. Thus, dynamic compression accelerated the biosynthetic response to IGF-I and increased transport of IGF-I into the articular cartilage matrix, suggesting that, in addition to independently stimulating articular chondrocytes, cyclic compression may improve the access of soluble growth factors to these relatively isolated cells.

Regulation by IGF-I and TGF-beta1 of Swarm-rat chondrosarcoma chondrocytes.

The growth factors transforming growth factor-beta 1 and insulin-like growth factor-I influence a wide range of cellular actions, including the growth of several neoplastic cell types. Their role in the regulation of neoplastic chondrocytes remains unclear. We tested the hypotheses that transforming growth factor-beta 1 and insulin-like growth factor-I differentially regulate neoplastic chondrocytes and interact to modulate the mitotic and matrix synthetic activities of neoplastic chondrocytes. We used Swarm-rat chondrosarcoma chondrocytes to investigate the effect of each factor individually and of both factors in combination on [(3)H]thymidine incorporation into DNA and on [(35)S]sulfate incorporation into glycosaminoglycans. Each factor increased [(3)H]thymidine incorporation 2.7-fold: transforming growth factor-beta 1 achieved this effect at a 20-fold lower concentration than insulin-like growth factor-I. In contrast, insulin-like growth factor-I stimulated [(35)S]sulfate incorporation 3.5-fold; this was twice the maximal effect of transforming growth factor-beta 1. Transforming growth factor-beta 1 and insulin-like growth factor-I each decreased the proportion of newly synthesized glycosaminoglycans that were retained in the cells and pericellular matrix, indicating that the anabolic effect of these factors is only partly directed toward cell-associated matrix production. The mitogenic and matrix synthetic actions of insulin-like growth factor-I and transforming growth factor-beta 1 were synergistic. In concert, they increased [(3)H]thymidine incorporation approximately 12-fold, an effect three times greater than the sum of the maximal stimulation achieved by each factor individually. Similarly, transforming growth factor-beta 1 and insulin-like growth factor-I together increased glycosaminoglycan synthesis approximately two times more than the sum of their maximal individual effects. Taken together, these data indicate that these chondrosarcoma chondrocytes are positively regulated by insulin-like growth factor-I and transforming growth factor-beta 1 and that these growth factors interact to augment the mitotic and matrix synthetic actions of the chondrocytes. If supported in human models, the sensitivity to growth factors of these cells suggests that interventions directed toward growth factor inhibition may be of therapeutic value.

Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage.

The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0-50% static compression, and the transient and steady-state incorporation of [(3)H]proline and [(35)S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0-300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [(3)H]proline and [(35)S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of (125)I-labeled IGF-I into cartilage explants. Static compression decreased both [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (tau) for IGF-I stimulation of cartilage explants was approximately 24 h, while tau for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter tau for (125)I-labeled IGF-I transport into cartilage but decreased the concentration of (125)I-labeled IGF-I in the tissue at equilibrium.

Efficient lipid-mediated gene transfer to articular chondrocytes.

We examined nonviral, lipid-mediated gene transfer methods as potential tools for efficient transfection of articular chondrocytes. Transfection conditions were determined for primary cultures of normal human articular, osteoarthritic human articular and normal bovine articular chondrocytes using a lacZ reporter gene construct with the commercially available cationic liposomes Cellfectin, DMRIE-C, LipofectAmine, Lipofectin, LipoTaxi, TransFast and the lipid-based reagent FuGENE 6. Optimized conditions were then evaluated in an ex vivo model of chondrocyte transplantation. FuGENE 6 transfection produced the maximum levels of transgene expression. Transfection efficiency was cell type specific and affected by DNA concentration, lipid/DNA ratio and the presence of hyaluronidase, a matrix-degrading enzyme. Analysis of X-gal staining demonstrated an efficiency of 41.0% in normal bovine articular chondrocytes, 20.7% in normal human articular chondrocytes and 7.8% in osteoarthritic human chondrocytes. Transfected chondrocytes were found to successfully populate the articular cartilage surface in explant cultures. Transplanted genetically modified chondrocytes adhered to the articular cartilage and continued to produce beta-galactosidase for 2 weeks. This evaluation and optimization of lipid-based gene transfer into articular chondrocytes may serve as a useful tool in studies of genes involved in articular cartilage damage and repair and as a potential delivery method for therapeutic genes. Gene Therapy (2000) 7, 286-291.

The unmet anti-inflammatory needs in orthopedics.

Approximately half of Americans 70 years of age or older suffer from arthritis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most effective nonsurgical therapies for arthritis, but usage often causes harmful side effects, especially in the gastrointestinal tract. Such effects require supplemental therapy that adds an economic burden and may even cause death. The benefits derived from NSAIDs are believed to be due to suppression of cyclooxygenase-2 (COX-2), while the harmful side effects are believed to be due to suppression of cyclooxygenase-1 (COX-1). COX-2-specific inhibitors that do not inhibit COX-1 may meet arthritis sufferers' needs for therapies that are safe, convenient, and as effective as conventional NSAIDs.

Cartilage degeneration in relation to repetitive pressure: case study of a unilateral hip hemiarthroplasty patient.

In vivo acetabular contact pressures were measured over 32 months in an elderly man with a pressure instrumented hemiarthroplasty. After death, left (hemiarthroplasty) and right (control) acetabula were explanted. Cartilage thickness and degeneration were quantified from magnetic resonance imaging and histological analysis. Highest repetitive in vivo contact pressures during gait (4.5 to 6.5 MPa) were measured in the superior dome of the acetabulum and decreased at a rate of approximately 1 MPa per year after implant (R2 = 0.48, P < .001). Contact pressure magnitudes measured during gait correlated positively with regional histology score (R2 = 0.34, P < .0001) and negatively with cartilage thickness (R2 = 0.35, P < .0001). Although histology scores were typical of early osteoarthritis (histological grade of 4-6), there were no significant differences in overall histology score for the left and right acetabula (P = .23). We conclude that acetabular cartilage degeneration was explained, in part, by repetitive stress, but the degeneration did not appear to be mediated solely by articulation with the metallic endoprosthesis.

Transport of tissue inhibitor of metalloproteinases-1 through cartilage: contributions of fluid flow and electrical migration.

The preservation of the structure of articular cartilage depends on the availability of inhibitors of matrix-degrading enzymes. Tissue inhibitor of metalloproteinases-1 is thought to be an important contributor to the integrity of the matrix of articular cartilage, but the mechanisms that regulate its availability within the tissue are poorly understood. These studies elucidate the contributions of diffusion, fluid flow, and electrical migration to the transport of iodinated recombinant human tissue inhibitor of metalloproteinases-1 through explants of adult bovine articular cartilage under conditions relevant to the loading of cartilage. With use of measured partition coefficients of the cartilage explants, the diffusivity of the inhibitor was 0.5-1.6 x 10(-7) cm2/sec. Fluid velocities that were induced by applying an electrical current across the cartilage disks increased the flux of the inhibitor by approximately 20 to more than 150-fold compared with the effect of diffusion alone for the range of current densities that were applied. We examined the contribution of electrophoretic migration by titrating the charge on the inhibitor during measurements of flux and found that flux in the presence of the applied current decreased as the inhibitor became more negatively charged. Enhancements in the flux of the inhibitor were observed relative to the flux during diffusion alone even under conditions in which electrophoretic migration opposed the flux due to fluid flow, suggesting that of the transport mechanisms tested, fluid flow was dominant. These results suggest that the physical phenomena present during physiologic loading conditions (e.g., fluid flows and streaming currents) can affect the transport of tissue inhibitor of metalloproteinases-1 through the matrix of cartilage.

Longitudinal analysis of the relationship between serum insulin-like growth factor-I and radiographic knee osteoarthritis.

OBJECTIVE: To examine the relation between serum insulin-like growth factor I (IGF-I) levels and both incident and progressive radiographic knee osteoarthritis (OA) in the Framingham Osteoarthritis Study. DESIGN: Subjects had bilateral weight-bearing, anterior-posterior knee radiographs performed in 1983-1985 and again in 1992-1993. IGF-I levels were measured from blood specimens obtained in 1988-1989 by a competitive binding radio-immunoassay (RIA) after separation with octadecasilyl-silica cartridges of serum IGF-I from binding proteins. Participants without baseline radiographic OA [Kellgren and Lawrence grades (K&L) = 0-1] were classified as having incident disease if they had K&L > or = 2 grades at follow-up. Progressive OA was defined as an increase in K&L score of > or = 1 in knees with baseline OA (K&L > or = 2). All analyses were knee-based and sex-specific. We examined IGF-I tertiles in relation to the risk of incident and progressive radiographic OA separately, adjusting for age, body mass index (BMI), and baseline K&L score, and used generalized estimating equations to adjust for the correlation between fellow knees. RESULTS: Four hundred and forty-one participants had knee radiographs and serum IGF-I levels measured. No associations were found for serum IGF-I levels and incident [women: OR = 0.9 (0.6-1.7), men OR = 1.2 (0.6-2.6)] or progressive [women OR = 0.9 (0.6-1.6), men OR = 0.9 (0.3-3.0)] radiographic knee OA in either sex. Neither did we observe any association between IGF-I and worsening of individual radiographic features of OA (i.e., osteophyte growth and joint space loss). CONCLUSION: In summary, this longitudinal study did not demonstrate any association of serum IGF-I and incident or progressive radiographic knee OA. Further studies are needed to clarify the role of IGF-I in OA.

Potential role of insulinlike growth factors in fracture healing.

Several lines of evidence suggest that the insulinlike growth factors play a role in fracture healing. They promote cell proliferation and matrix synthesis by chondrocytes and osteoblasts, the two cell types largely responsible for the formation of fracture callus. Circulating levels of insulinlike growth factor I and bone mineral density decrease with increasing age, and administration of insulinlike growth factor I increases bone turnover in patients with low bone mineral density. Insulinlike growth factor I may accelerate the normal healing of intramembranous bone defects, inducing the healing of defects that otherwise would not heal. An important role of insulinlike growth factor I is to mediate many of the actions of growth hormone on the skeleton. Considerable effort has been devoted to testing the effect of growth hormone and, thereby, indirectly that of insulinlike growth factor I on fracture healing. These studies have yielded such disparate results that no general conclusions regarding the effect of growth hormone (or of growth hormone dependent insulinlike growth factor I) on fracture healing currently can be drawn. Additional studies are needed to clarify the role of the insulinlike growth factors in the fracture healing process and to determine how their anabolic actions can be enlisted in the clinical enhancement of fracture healing.

Interaction of epidermal growth factor and insulin-like growth factor-I in the regulation of growth plate chondrocytes.

The action of growth factors on the cells of the epiphyseal growth plate is an important mechanism in the regulation of skeletal growth. Insulin-like growth factor-I (IGF-I) is known to play a central role in the regulation of bone growth. In contrast, the role, if any, of epidermal growth factor (EGF) is not yet clear. In these studies, we tested the hypothesis that EGF interacts with IGF-I in the regulation of growth plate chondrocyte mitotic and metabolic activities. Chondrocytes isolated from bovine radioulnar growth plates and incubated in suspension culture were analyzed for their responsiveness to EGF with respect to synthesis of DNA, proteins, and proteoglycans, responsiveness to IGF-I, and ability to specifically bind [125I]IGF-I. Treatment of growth plate chondrocytes with maximally effective concentrations (10-100 ng/ml) of EGF produced a 16-27% increase in specific binding of [125I]IGF-I. Scatchard analysis indicated that this increase in specific binding was due to an increase in the number of receptors/cell with no change in receptor affinity. EGF stimulated protein synthesis by 30-35%. Pretreatment with EGF increased the responsiveness of chondrocytes to IGF-I, resulting in 90 and 60% augmentation of IGF-I-stimulated mitotic activity and proteoglycan synthesis, respectively. Given the prominent role of IGF-I in skeletal development and the presence of EGF in the growth plate, this study suggests an important role for interactions between these growth factors in the regulation of skeletal growth.