Identification of a nucleotide pyrophosphohydrolase from articular tissues in human serum

Objective. To characterize the nucleotide pyro-phosphohydrolase (NTPPHase) in human serum. Methods. NTPPHase activity and kinetic analysis were performed using thymidine monophosphate paranitrophenyl ester (TMPNP) or ³²Pγ-labeled ATP as substrate. Sera were chromatographed (dye column), and peak fractions were analyzed kinetically and by immunoblot using antibodies to 127-kd articular cartilage vesicle (ACV) NTPPHase as well as to PC-1 and to 58 kd, two plasma membrane ecto-NTPPHases. Enzyme activity was measured before and after sample ultracentrifugation. Results. NTPPHase activity was found in all sera tested (2 normal subjects, 9 arthritis patients). Specific activity was increased 9–32-fold after chromatography; 60–80% of total activity was recovered in a single peak containing an ∼100-kd soluble peptide related to the 127-kd ACV enzyme. The apparent K_m of this peptide (TMPNP) was virtually identical to that of the porcine ACV 127-kd enzyme. No immunoreactivity against PC-1 or 58-kd NTPPHase was found. Conclusion. Human serum NTPPHase is derived from 127-kd ACV-related enzyme.     

A comparison of the effect of transforming growth factor β1 on pyrophosphate elaboration from various articular tissues

Objective. The purpose of this study was to determine the effect of transforming growth factor β1 (TGFβ1) on inorganic pyrophosphate (PPi) elaboration from articular tissues to better understand the pathophysiology of calcium pyrophosphate dihydrate (CPPD) crystal deposition in the joint. Methods. PPi was measured in the media of adult porcine articular tissue in organ culture and monolayer cultures. Results. TGF/β1 strongly stimulated PPi elaboration by porcine fibrocartilage and hyaline cartilage. It modestly increased PPi elaboration by ligament, and had no effect on PPi elaborated by synovium. Of all cell types tested in cell culture, only chondrocytes responded to TGF/β1 by significantly increasing PPi elaboration. Conclusion. TGF/β1 stimulates PPi elaboration from hyaline cartilage, fibrocartilage, and ligament, indicating that there is in situ CPPD crystal formation in these tissues. The ability of tissues to respond to TGFβ1 by increasing PPi elaboration correlates with the prevalence of CPPD crystal deposition found clinically. The unique response of chondrocyte monolayers to TGF/β1 reinforces the key role of the chondrocyte in PPi elaboration in the joint. These findings support an etiologic role for responsiveness to TGFβ1 in CPPD disease.     

Articular Cartilage Vesicles Generate Calcium Pyrophosphate Dihydrate-Like Crystals in Vitro

Objective. To identify the morphology of a mineral-forming fraction of adult porcine hyaline articular cartilage digest and characterize the mineral it forms. Methods. Electron microscopy, Fourier transform infrared (FTIR) spectroscopy, x-ray microanalysis, compensated polarized light microscopy, and biochemical studies including ¹⁴C-labeled UDPG pyrophosphohydrolase radiometric assay. Results. This fraction of articular cartilage digest contained membrane-limited vesicles resembling growth plate cartilage matrix vesicles and formed mineral after only 24 hours in physiologic salt solution containing 1 mM ATP. The mineral contained inorganic pyrophosphate, 95% of which derived from ATP, and phosphate, 93% of which derived from inorganic phosphate in the medium. The FTIR spectrum of this mineral closely resembled the spectrum of standard calcium pyrophosphate dihydrate (CPPD) crystals. Compensated polarized light microscopy showed positively birefringent, rod-shaped crystals morphologically identical to CPPD. Ca:P ratios, defined by energy-dispersive microanalysis, were also consistent with CPPD. Conclusion. The articular cartilage vesicle fraction of porcine hyaline cartilage is capable of generating mineral that strongly resembles CPPD.     

Novel fluorescent compound (DDP) in calf, rabbit, and human articular cartilage and synovial fluid

OBJECTIVE: To investigate the presence of and quantify 2,6-dimethyldifuro-8-pyrone (DDP), a novel fluorescent compound identified as in various calf, rabbit, and human tissue/fluid samples, to determine the DDP level in articular cartilage (AC) laminae, and to investigate the changes in cartilage DDP content with cartilage maturation. METHODS: Samples were obtained from calf (< 2 years), rabbit (< 2 weeks to 2 years) or human AC and synovial fluid (SF) as well as other non-cartilaginous tissues. SF and tissue samples were hydrolyzed with 6 M HCl (24 hours at 110 degrees C), lyophilized, and dissolved in HPLC mobile phase. DDP and collagen crosslink peaks were measured using a fluorescence detector at excitation and emission wavelengths of 295 and 395 nm, respectively. RESULTS: DDP was detected from calf metacarpophalangeal joint AC (362 +/- 48 pmol/mg dry weight), SF (4.5 +/- 0.3 pmol/microl SF), and intervertebral disc (24 +/- 4 pmol/mg). DDP was not detected in calf ligament, tendon, bone, ocular lens, cornea, or elastic cartilage. The DDP amount was greater in mid-deep cartilage lamina (448 +/- 63 pmol/mg) than superficial-mid lamina (129 +/- 52 pmol/mg) (p = 0.008). DDP level decreased with maturation in rabbit knee joint AC from 185 +/- 40 (< 2 weeks) to 27 +/- 3 (2 years) pmol/mg dry weight. DDP was not detected in adult rabbit ligament, tendon, meniscus, or bone. DDP was detected in human knee joint AC and SF. The DDP level in osteoarthritic lesions was present in lower concentrations (range: 0 to 96 pmol/mg dried AC) compared to intact AC (range: 63 to 236 pmol/mg) of the same knee. CONCLUSION: DDP is a hyaline cartilage specific compound present in all articular cartilage samples from various articulating joints/animal species. DDP level increases with AC depth and decreases with cartilage maturation. DDP is a potential indicator of cartilage metabolism during normal growth, ageing, and cartilage disease.     

The role of cartilage canals in the pathogenesis of experimentally induced polyarthritis

Summary Porcine articular cartilage from cases of experimentally induced Erysipelas polyarthritis, a comparative model of rheumatoid arthritis (RA) in man, was examined with different histological and immunohistochemical techniques. The preexisting canals in articular cartilage played a crucial role during the flooding and deposition of arthritogenic microorganisms deep into the cartilage matrix. Subsequently this vascularized tissue mediated the same inflammatory reactions in hyaline cartilage of young animals as seen in other connective tissues. However, these stereotypical responses to injury were modulated by the unique composition and structure of articular cartilage.     

Surgical approaches to the repair of cartilage defects

Traumatic defects in articular cartilage are common in the young athletic population. They may occur in association with other injuries to the knee, particularly anterior cruciate ligament disruption. We understand that both theoretically and clinically, these defects may lead to an increased incidence of ongoing painful symptoms in the short term and degenerative joint disease in the long term. The orthopedic surgeon has several options to deal with theses injuries. The choice is dependent on the size and position of the lesion in the knee and any associated pathology. The most commonly practised methods aim to evoke a ‘repair’ response of fibrocartilage formation. This often helps in the short term, but the fibrocartilage degenerates over time, resulting in the return of symptoms. These ‘repair’ methods can be classified as: 1. Marrow stimulation techniques– these include abrasion chondroplasty, subchondral drilling and microfracture techniques. 2. Scaffold induced repair techniques. More recently, attempts to ‘re‐generate’ normal articular cartilage have been introduced to clinical practice. These are: 1. Mosaicplasty– autogenous osteochondral plug transplantation. 2. Autologous chondrocyte implantation (ACI)– this involves the harvest and culturing of autologous chondrocytes and then re‐implantation via injection under a periosteal or porcine collagen patch. The newest technology allow implantation of the collagen patch which has had chondrocytes cultured within its structure. The results of these autologous chondrocyte implantation show encouraging short to medium term results clinically. 3. Some orthopaedic centres have recently been advocating ‘articular cartilage paste grafting’– this is a simple technique based upon the theory that a paste of autogenous articular cartilage and subchondral bone should contain chondrocytes, stem cells and growth factors in its own scaffold. Orthopedic surgeons await with interest the outcome of pre‐clinical work in examining the use of growth factors, scaffold technology and gene therapy in helping with the clinical problem of articular cartilage defects.     

Characteristics of anti-type ii collagen antibody binding to articular cartilage

Objective. Previous studies suggested that it was possible to characterize the intact surface of articular cartilage by probing it with antibodies against matrix macromolecules. The present studies were undertaken to investigate type II collagen (CII) epitope availability on the intact surface of articular cartilage. Methods. Normal bovine, rabbit, and human cartilage specimens were used to measure binding of anti-CII antibodies to the articular and cut surfaces of cartilage. Antisera were raised against the material obtained after brief extraction of the cartilage surface with 4M guanidine solution. Results. Anti-CII did not bind to the intact surface of rabbit articular cartilage when compared with control rat sera, but did bind significantly to the cut surface. With normal human articular cartilage, the cut surfaces bound approximately 4 times as much anti-CII antibody as the intact articular surfaces. These findings suggested that the CII epitopes are normally protected by the superficial cartilage layer. In experiments carried out to characterize this layer, binding of anti-CII antibodies was unchanged after exhaustive washing of bovine or rabbit cartilage with phosphate buffered saline or 1M NaCl solution, whereas it was significantly increased after brief exposure to 4M guanidine solution or after incubation with neutrophil elastase. No restoration of the protection of CII epitopes in guanidine-treated cartilage could be achieved by incubation with undiluted normal bovine synovial fluid; however, 8-day culture of elastase-treated cartilage explants resulted in partial restoration of protection of the CII epitopes. Rat and rabbit antisera raised against the cartilage surface material extracted with 4M guanidine contained antibodies that bound to the cartilage surface. By Western blotting, rat antibodies identified 50–65-kd protein bands present in the guanidine extract, but not present either in the material obtained from brief digestion of cartilage with neutrophil elastase or in synovial fluid. The rabbit antisera identified a broad 70–95-kd band. Exposure of elastase-treated cartilage to the guanidine-extracted material resulted in a partial decrease of anti-CII antibody binding. Conclusion. These results suggest that CII on intact cartilage is protected from antibody binding, and that the protective material is at least partly proteinaceous in nature, is unlikely to be derived from synovial fluid, is noncovalently bound to the underlying intercellular matrix, and is synthesized by resident chondrocytes. Further characterization of the protective material may provide important information on the mechanisms of early cartilage damage in inflammatory arthritis.     

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.     

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.

     

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.     

Electron microscopic morphology of immunoglobulin aggregates and their interactions in rheumatoid articular collagenous tissues

Immunoelectron microscopy for IgG and IgA was combined with plain electron microscopy and light microscopy to study a variety of articular collagenous tissues from 14 cases of classic rheumatoid arthritis. Numerous extensive aggregates of IgG and IgA positive material were found widely spread in locations that coincided with immunofluorescent staining for these immunoglobulins and B1c. This Ig positive material is thought to represent deposits of immune complexes. As great and greater amounts were seen in fibrocartilage menisci as in hyaline cartilage. A single specimen of disease tendon was also positive. Severe pathologic changes of the matrix characterized by loss of normal morphology, alterations in collagen, and overall reduced density were observed in regions of aggregate deposition with a consistent replacement and disappearance of collagen in the locale of the aggregates themselves. Polymorphonuclear cells in regions of degraded hyaline and tendon matrix were seen to have phagocytosed aggregates. These data give some credence to a direct role of immune aggregates in rheumatoid arthritis articular collagenous tissues in disease pathogenesis.     

Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles

Objective. Transglutaminases (TGases) (E.G. 2.3.2.13) catalyze a posttranslational modification of proteins and are associated with biomineralization in growth plate cartilage. Type II TGase participates in the activation of latent transforming growth factor β (TGFß), a crucial factor for both normal cartilage mineralization and the pathologic mineralization that results in calcium pyrophosphate dihydrate (CPPD) crystal formation in aging articular cartilage. To explore a possible association between TGase levels and CPPD crystal formation in mature articular cartilage, TGase activity in articular chondrocytes from old and young pigs and in the articular cartilage vesicle (ACV) fraction of porcine articular cartilage was examined. In addition, the effects of TGase inhibitors on the production of inorganic pyrophosphate (PPi), a process necessary for CPPD crystallogenesis, were determined. Methods. TGase activity was measured with a radiometric assay in cultured articular chondrocytes from the knee joints of old (3–5 years old) and young (2–6 weeks old) pigs and in the ACVs. PPi levels were measured in chondrocyte-conditioned media in the presence of TGase inhibitors or control compounds. Results. Levels of TGase activity in the cytosolic fraction of old chondrocytes were 7-fold higher than those in identically cultured young chondrocytes. The mean ± SD activity level in the membrane fraction of lysed chondrocytes was 6.0 ± 0.6 units/mg protein in old articular chondrocytes and was undetectable in young chondrocytes. In ACVs, the mean ± SD TGase activity level was 1.23 ± 0.1 units/mg protein. Type II TGase protein was present in chondrocyte cytosol and in ACVs. TGase activity was increased by TGFß to 120% of control values (P < 0.01), and decreased by insulin-like growth factor 1 to 80% of control values (P < 0.01). TGase inhibitors blocked media accumulation of PPi, an essential precursor of CPPD crystal formation, and a sensitive marker of TGFß effect. Conclusion. These data suggest a potential link between TGase activity and processes of pathologic biomineralization that result in CPPD crystal formation in aging articular cartilage.     

ATP-induced chondrocalcinosis

Objective. To determine whether adult articular cartilage mineralizes in the presence of ATP. Methods. Intact adult porcine articular cartilage and monolayers of chondrocytes were cultured in physiologic media containing ATP, and mineralization was measured as retention of ⁴⁵Ca. Cartilage was analyzed by electron microscopy. Results. Articular cartilage sequestered ⁴⁵Ca when incubated with 100 γM ATP. Use of the ATP analog α,β-methylene ATP did not promote mineralization and addition of pyrophosphatase inhibited mineralization, indicating that hydrolysis of ATP to AMP and inorganic pyrophosphate is necessary for the process to occur. Mineral was concentrated in articular cartilage vesicles in the perichondral area. Conclusion. Adult articular cartilage mineralizes in the presence of ATP, in a manner similar to that found with isolated matrix or articular cartilage vesicles. This supports the notion that these structures have a role in chondrocalcinosis.     

Articular cartilage vesicles generate calcium pyrophosphate dihydrate-like crystals in vitro.

OBJECTIVE. To identify the morphology of a mineral-forming of adult porcine hyaline articular cartilage digest and characterize the mineral it forms. METHODS. Electron microscopy, Fourier transform infrared (FTIR) spectroscopy, x-ray microanalysis, compensated polarized light microscopy, and biochemical studies including 14C-labeled UDPG pyrophosphohydrolase radiometric assay. RESULTS. This fraction of articular cartilage digest contained membrane-limited vesicles resembling growth plate cartilage matrix vesicles and formed mineral after only 24 hours in physiologic salt solution containing 1 mM ATP. The mineral contained inorganic pyrophosphate, 95% of which derived from ATP, and phosphate, 93% of which derived from inorganic phosphate in the medium. The FTIR spectrum of this mineral closely resembled the spectrum of standard calcium pyrophosphate dihydrate (CPPD) crystals. Compensated polarized light microscopy showed positively birefringent, rod-shaped crystals morphologically identical to CPPD. Ca:P ratios, defined by energy-dispersive microanalysis, were also consistent with CPPD. CONCLUSION. The articular cartilage vesicle fraction of porcine hyaline cartilage is capable of generating mineral that strongly resembles CPPD.     

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.

     

Repair of articular cartilage defects in rabbits using CDMP1 gene-transfected autologous mesenchymal cells derived from bone marrow

OBJECTIVE: Cartilage-derived morphogenetic protein 1 (CDMP1), which is a member of the transforming growth factor-beta superfamily, is an essential molecule for the aggregation of mesenchymal cells and acceleration of chondrocyte differentiation. In this study, we investigated whether CDMP1-transfected autologous bone marrow-derived mesenchymal cells (BMMCs) enhance in vivo cartilage repair in a rabbit model. METHODS: BMMCs, which had a fibroblastic morphology and pluripotency for differentiation, were isolated from bone marrow of the tibia of rabbits, grown in monolayer culture, and transfected with the CDMP1 gene or a control gene (GFP) by the lipofection method. The autologous cells were then implanted into full-thickness articular cartilage defects in the knee joints of each rabbit. RESULTS: During in vivo repair of full-thickness articular cartilage defects, cartilage regeneration was enhanced by the implantation of CDMP1-transfected autologous BMMCs. The defects were filled by hyaline cartilage and the deeper zone showed remodelling to subchondral bone over time. The repair and reconstitution of zones of hyaline articular cartilage was superior to simple BMMC implantation. The histological score of the CDMP1-transfected BMMC group was significantly better than those of the control BMMC group and the empty control group. CONCLUSION: Modulation of BMMCs by factors such as CDMP1 allows enhanced repair and remodelling compatible with hyaline articular cartilage.     

Immobilization of the knee prevents osteoarthritis after anterior cruciate ligament transection

The effect of immobilization of the knee in flexion on the development of osteoarthritis was studied in dogs that had undergone transection of the anterior cruciate ligament. Knees of dogs that were permitted ad libitum ambulation in a pen for 12 weeks after transection of the ligament showed osteophytes and fibrillation and a decrease in proteoglycan (uronic acid) content, although thickness of the articular cartilage was normal. Proteoglycan synthesis was 80% greater (P < 0.01) than that in cartilage from the contralateral knee, and more than twice the normal proportion of the total proteoglycans was present in the medium of cultures of cartilage from the unstable knee (P < 0.01). Similarly, the proportion of total tissue proteoglycans extracted with 0.4M guanidinium chloride was 3 times greater than normal (P < 0.01). In contrast, osteophytes were not seen when the leg was immobilized in flexion immediately after transection of the ligament, and the articular surface remained intact. The cartilage became atrophic, however, and its proteoglycan content (uronic acid) was diminished; proteoglycan synthesis was decreased to 61% of the level in cartilage from the contralateral knee, although proteoglycan extractability was normal. Knee cartilage from these legs that were immobilized after transection, therefore, resembled that from dogs whose leg had been immobilized without cruciate ligament transection. In cartilage from all operated and/or immobilized joints the proportion of proteoglycans that interacted with hyaluronic acid in vitro was diminished, because of a defect in the hyaluronate-binding region of the proteoglycan.     

Histochemical investigation of adjuvant-induced arthritis

Rats with adjuvant-induced arthritis were observed to have increased alkaline phosphatase, acid phosphatase (two isozymes), and ATPase activity in the radial zone of articular cartilage, at the osteochondral junction, and in the bone marrow elements. A qualitative and quantitative reduction of azure A, PAS, colloidal iron, alcian blue critical electrolyte concentration staining (0.4 and 0.9 M (mg Cl₂) was also observed in corresponding areas. These findings suggest the degradation of the articular cartilage matrix with possible simultaneous or resultant calcification.     

Proliferating cell nuclear antigen (PCNA) expression in regenerating rat liver after partial hepatectomy

Proliferating cell nuclear antigen (PCNA) is a nuclear protein maximally elevated in the S phase of proliferating and transformed cells and is recognized by the monoclonal antibody PC-10 in paraffin tissue sections. The liver regenerative process after partial hepatectomy in rats was estimated with thein vivo incorporation of [³H]thymidine into liver DNA and the liver thymidine kinase activity. The expression of PCNA in rat liver after partial hepatectomy was performed by immunohistochemical staining with PC-10 in paraffin embedded tissues, at different time intervals up to 240 hr. Proliferating cell nuclear antigen expression, [³H]thymidine incorporation into DNA, and liver thymidine kinase activity exhibited marked oscillations during the liver regenerative process. A close relationship was demonstrated among DNA synthesis, thymidine kinase activity, and PC-10 score. Our results suggest that PC-10 monoclonal antibody may be used as a worthwhile proliferation index in the evaluation of the rate of liver regeneration in rats.