Kenneth P.H. Pritzker

Subchondral bone in osteoarthritis.

SummaryTo determine whether subchondral bone in osteoarthritis differs from that seen in normal human aging, osteoarthritic femoral heads removed for total hip arthroplasty were compared with normal age-matched and young autopsy controls. Standardized, 1-cm deep, weight-bearing and nonweight-bearing subchondral bone blocks, as well as cancellous core bone, 2–4 cm deep to the articular surface, were examined in each femoral head. Mineralization was assessed using density fractionation and chemical analysis, and compared to histomorphometry. In osteoarthritis, both weight-bearing and nonweight-bearing surface subchondral bone showed a lower degree of mineralization than age-matched and young controls. Histomorphometric analysis showed that subchondral bone thickness, as well as all osteoid parameters and eroded surfaces, were increased in osteoarthritic samples versus controls. Mineralization in the deep cancellous core bone increased with normal aging but underwent less change with osteoarthritis. Histomorphometry of the cancellous core showed that osteoid parameters, but not bone volume, were increased in osteoarthritis versus controls. In conclusion, osteoarthritis is associated with a thickening of the subchondral bone with an abnormally low mineralization pattern.

TLR2 Signaling in Chondrocytes Drives Calcium Pyrophosphate Dihydrate and Monosodium Urate Crystal-Induced Nitric Oxide Generation

Microcrystals of calcium pyrophosphate dihydrate (CPPD) and monosodium urate (MSU) deposited in synovium and articular cartilage initiate joint inflammation and cartilage degradation in large part by binding and directly activating resident cells. TLRs trigger innate host defense responses to infectious pathogens, and the expression of certain TLRs by synovial fibroblasts has revealed the potential for innate immune responses to be triggered by mesenchymally derived resident cells in the joint. In this study we tested the hypothesis that chondrocytes also express TLRs and that one or more TLRs centrally mediate chondrocyte responsiveness to CPPD and MSU crystals in vitro. We detected TLR2 expression in normal articular chondrocytes and up-regulation of TLR2 in osteoarthritic cartilage chondrocytes in situ. We demonstrated that transient transfection of TLR2 signaling-negative regulator Toll-interacting protein or treatment with TLR2-blocking Ab suppressed CPPD and MSU crystal-induced chondrocyte release of NO, an inflammatory mediator that promotes cartilage degeneration. Conversely, gain-of-function of TLR2 in normal chondrocytes via transfection was associated with increased CPPD and MSU crystal-induced NO release. Canonical TLR signaling by parallel pathways involving MyD88, IL-1R-associated kinase 1, TNF receptor-associated factor 6, and IκB kinase and Rac1, PI3K, and Akt critically mediated NO release in chondrocytes stimulated by both CPPD and MSU crystals. We conclude that CPPD and MSU crystals critically use TLR2-mediated signaling in chondrocytes to trigger NO generation. Our results indicate the potential for innate immunity at the level of the articular chondrocyte to directly contribute to inflammatory and degenerative tissue reactions associated with both gout and pseudogout.

The immunogenicity of fresh and frozen allogeneic bone

Both fresh and frozen allogeneic bone elicit both acellular and humoral immune response. This response includes the development of enhancing factors which block detectable immunity and probably protect the graft from rejection. There seems to be no evidence of an alteration in immunogenicity by freezing of the graft. The importance of these observations lies in the potential technique of employing fresh viable allografts; prior freezing and tissue matching for HL-A transplantation antigens should not be necessary.

Articular cartilage transplantation

This report describes the biopsy findings in four of 30 patients treated with cadaver osteochondral shell allografts for osteoarthritis in the knee. This study demonstrates that graft cartilage cells can survive in excess of 25 months, and that host bone can completely replace graft bone by creeping substitution. An inflammatory reaction in synovium and bone marrow was found in only one of four cases. Graft failure was related to prolonged down time of donor cartilage in one case and mechanical factors related to osteoarthritis in the apposing femoral surface in other cases. The clinical success of these grafts is attributed to the prolonged viability of cartilage cells, the capacity of host bone to join graft cartilage without histologic reaction, and the hosts immunologic tolerance, which obviates the need for immunosuppressive therapy.

Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification

OBJECTIVE Elevated cartilage inorganic pyrophosphate (PPi) production and PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity are strongly linked with aging-related cartilage calcification in meniscal and articular cartilages. We hypothesized that there were divergent relationships of 3 NTPPPH isozymes with cartilage matrix calcification and sought to identify them. METHODS We studied knee medial meniscal expression in situ of 3 NTPPPH isozymes of the phosphodiesterase nucleotide pyrophosphatase (PDNP) family: plasma cell membrane glycoprotein 1 (PC-1, or PDNP1), autotaxin (ATX, or PDNP2), and B10/PDNP3. We also used complementary DNA transfection to assess differential functions in matrix calcification of each NTPPPH isozyme in vitro in meniscal cells. RESULTS We observed diffuse cell-associated ATX and B10/PDNP3 expression in central (chondrocytic) and, to a lesser degree, peripheral (fibroblastic) regions of normal, degenerative uncalcified, and degenerative calcified menisci. In contrast, PC-1 expression was only robust at sites of apoptotic cells and calcification in central regions of degenerative menisci. Only PC-1 was abundant at the perimeter of meniscal cells and in association with meniscal cell-derived matrix vesicles (MVs). Because each PDNP-family isozyme was expressed by cells near calcifications, we transfected the isozymes in nonadherent knee meniscal cells cultured with ascorbic acid, beta-glycerophosphate, and dexamethasone supplementation to stimulate them to calcify the matrix. PC-1, but not ATX or B10/PDNP3, consistently promoted increased MV NTPPPH, MV-associated PPi, and extracellular PPi. PC-1 also increased matrix calcification (with hydroxyapatite crystals) by meniscal cells. ATX uniquely induced alkaline phosphatase activity, but promoted only moderately increased matrix calcification. CONCLUSION We identified divergent effects of 3 PDNP-family NTPPPH isozymes on meniscal cell matrix calcification. Increased expression of PC-1 is both a marker and a potential pathogenic factor for knee meniscal cartilage matrix calcification.

Matrix Vesicle Plasma Cell Membrane Glycoprotein-1 Regulates Mineralization by Murine Osteoblastic MC3T3 Cells†

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)‐generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC‐1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in “tiptoe walking” (ttw) mice. Thus, we tested the hypothesis that PC‐1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC‐1 mRNA and produced hydroxyapatite deposits at 12–14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor‐β and 1,25‐dihydroxyvitamin D3 increased PC‐1 and NTPPPH in matrix vesicles (MVs). Because PC‐1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2‐ to 6‐fold more PC‐1. PC‐1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC‐1. Furthermore, MC3T3 cells transfected with PC‐1 deposited ∼80–90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC‐1. ATP‐dependent45Ca precipitation by MVs from cells overexpressing active PC‐1 was comparably diminished. Thus, regulation of PC‐1 controls the PPi content and function of osteoblast‐derived MVs and matrix hydroxyapatite deposition. PC‐1 may provide a novel therapeutic target in certain disorders of bone mineralization.

Interleukin-1 Induces Pro-Mineralizing Activity of Cartilage Tissue Transglutaminase and Factor XIIIa

Two transglutaminases (TGases), Factor XIIIa and tissue TGase (tTGase), are expressed in temporal-spatial association with matrix calcification in growth plates. Meniscal and articular cartilage matrix calcification are prevalent in osteoarthritis (OA) and aging. Here, we demonstrated up-regulation of tTGase and Factor XIIIa in superficial and deep zones of knee OA articular cartilage and the central (chondrocytic) zone of OA menisci. Transforming growth factor-beta and interleukin (IL)-1beta induced Factor XIIIa and tTGase expression in cartilage and meniscal organ cultures. Thus, we studied TGase activity. Donor age-dependent, OA severity-related, and IL-1-induced increases in TGase activity were demonstrated in both knee menisci and cultured meniscal cells. Meniscal cell TGase activity was stimulated by nitric oxide donors and tumor necrosis factor-alpha, but transforming growth factor-beta did not stimulate TGase activity. The iNOS inhibitor N-monomethylarginine (NMMA) and an inhibitor of tumor necrosis factor receptor-associated factor (TRAF)2 and TRAF6 signaling (the zinc finger protein A20) suppressed IL-1 induction of TGase activity. Increased Factor XIIIa and tTGase activities, achieved via direct transfection of chondrocytic TC28 and meniscal cells, both induced matrix apatite deposition. Thus, Factor XIIIa and tTGase activities were increased in aging, degenerative cartilages and induced by IL-1. Because TGase activity promoted apatite deposition, our findings potentially implicate inflammation in the pathogenesis of cartilage matrix calcification.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rabbit.

AIM The primary goal of this body of work is to suggest a standardized system for histopathological assessment of experimental surgical instability models of osteoarthritis (OA) in rabbits, building on past experience, to achieve comparability of studies from different centres. An additional objective is to review methodologies that have been employed in the past for assessing OA in rabbits with particular reference to the surgical anterior cruciate ligament transection (ACLT) model. METHODS A panel of scientists and clinician-scientists with recognized expertise in assessing rabbit models of OA reviewed the literature to provide a critical appraisal of the methods that have been employed to assess both macroscopic and microscopic changes occurring in rabbit joint tissues in experimental OA. In addition, a validation of the proposed histologic histochemical grading system was performed. RESULTS The ACLT variant of the surgical instability model in skeletally mature rabbits is the variation most capable of reproducing the entire range of cartilage, synovial and bone lesions recognized to be associated with OA. These lesions can be semiquantitatively graded using macroscopic and microscopic techniques. Further, as well as cartilage lesions, this ACLT model can produce synovial and bone lesions similar to that of human OA. CONCLUSIONS The ACLT variant of the surgical instability model in rabbits is a reproducible and effective model of OA. The cartilage lesions in this model and their response to therapy can be graded according to an adapted histological and histochemical grading system, though also this system is to some extent subjective and, thus, neither objective nor entirely reproducible.

Effects of the steroidal aromatase inhibitor Exemestane and the nonsteroidal aromatase inhibitor letrozole on bone and lipid metabolism in ovariectomized rats

Purpose: Exemestane (EXE) and letrozole (LET) are third-generation aromatase inhibitors currently prescribed for postmenopausal hormone-dependent breast cancer. The impact on end organs of estrogen depletion in menopausal women is of significant clinical importance. We studied the effects of EXE, its principal metabolite, 17-hydroexemestane (17-H-EXE), and LET on bone and lipid metabolism in ovariectomized (OVX) rats. Experimental Design: OVX rats were treated by weekly intramuscular injection for 16 weeks with 20, 50, and 100 mg/kg EXE, 20 mg/kg 17-H-EXE, and daily oral gavage of 1 mg/kg LET. At the end of the treatment period, bone mineral density (BMD), the bone resorption marker serum pyridinoline, the bone formation marker serum osteocalcin, bone mechanical properties, histomorphometry, and serum lipid concentrations were determined. Results: Lumbar vertebral and femoral BMD, bending strength of the femur, compressive strength of the fifth lumbar vertebra, and trabecular bone volume were significantly higher in OVX animals given EXE and 17-H-EXE than in OVX controls. EXE and 17-H-EXE significantly reduced an ovariectomy-induced increase in serum pyridinoline and serum osteocalcin. EXE and 17-H-EXE given to OVX rats caused significant reductions of serum cholesterol and low-density lipoprotein cholesterol. In contrast, OVX rats treated with LET had BMD, bone biomarkers, mechanical failure properties, and lipid levels similar to those of OVX controls. Conclusions: EXE and 17-H-EXE significantly prevent bone loss, enhance bone mechanical strength, and lower serum cholesterol and low-density lipoprotein levels in OVX rats. These protective effects on end-organ function are not seen with the nonsteroidal inhibitor LET.

Recommendations for the use of preclinical models in the study and treatment of osteoarthritis

yCanadian Arthritis Network, Canada zDepartment of Surgery, McGill University, Montreal, Quebec, Canada xCentocor Pharmaceuticals, Malvern, PA, USA k Ecole Polytechnique, Montreal, Canada {Harvard University, Boston, MA, USA #Universite de Montreal, Montreal, Canada yyConsumer Advisory Council, Canadian Arthritis Network, Canada zzUniversity of Calgary, Calgary, Canada xxMount Sinai Hospital, Toronto, Canada kk Sanofi-Aventis, Frankfurt, FRG, Germany {{University Hospital Nijmegen, Nijmegen, The Netherlands ##University of California, San Diego, USA