Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

Summary The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10°). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out succesfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azocoupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix. Changes of enzyme activities in synoviocytes, chondrocytes and osteoclasts during induced arthritis are discussed.     

Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10 degrees). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out successfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azo-coupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

骨关节炎软骨细胞发生内质网应激

目的：研究骨关节炎软骨细胞是否发生内质网应激现象。方法：对关节置换术后的人类骨关节炎软骨标本和正常关节软骨标本切片进行内质网应激标志分子免疫球蛋白重链结合蛋白（BiP）的免疫组织化学检测;对小鼠膝关节进行半月板切断术诱发实验性骨关节炎,在术后1、3和6周取材,对组织切片进行番红花“O”染色、Mankin评分及BiP的免疫组织化学检测。结果：所有人类骨关节炎标本中软骨细胞BiP的表达明显升高。番红花“O”染色结果表明,在小鼠骨关节炎模型中,全部手术侧关节表面发生磨损,且随着术后时间延长关节表面磨损范围逐步扩大,手术侧Mankn分值显著高于对照侧;此外,手术侧的软骨细胞内BiP呈阳性表达,且表达量随术后时间延长而增加。结论：在人类骨关节炎标本和实验性小鼠骨关节炎模型中,关节软骨细胞均发生明显的内质网应激现象。     

Osteoarthritis in the Knee Joints of G?ttingen Minipigs after Resection of the Anterior Cruciate Ligament? Missing Correlation of MRI,Gene and Protein Expression with Histological Scoring

IntroductionThe Göttingen Minipig (GM) is used as large animal model in articular cartilage research. The aim of the study was to introduce osteoarthritis (OA) in the GM by resecting the anterior cruciate ligament (ACLR) according to Pond and Nuki, verified by histological and magnetic resonance imaging (MRI) scoring as well as analysis of gene and protein expression.

Materials and Methods

The eight included skeletally mature female GM were assessed after ACLR in the left and a sham operation in the right knee, which served as control. 26 weeks after surgery the knee joints were scanned using a 3-Tesla high-field MR tomography unit with a 3 T CP Large Flex Coil. Standard proton-density weighted fat saturated sequences in coronal and sagittal direction with a slice thickness of 3 mm were used. The MRI scans were assessed by two radiologists according to a modified WORMS-score, the X-rays of the knee joints by two evaluators. Osteochondral plugs with a diameter of 4mm were taken for histological examination from either the main loading zone or the macroscopic most degenerated parts of the tibia plateau or condyle respectively. The histological sections were blinded and scored by three experts according to Little et al. Gene expression analysis was performed from surrounding cartilage. Expression of adamts4, adamts5, acan, col1A1, col2, il-1ß, mmp1, mmp3, mmp13, vegf was determined by qRT-PCR. Immunohistochemical staining (IH) of Col I and II was performed. IH was scored using a 4 point grading (0—no staining; 3-intense staining).

Results and Discussion

Similar signs of OA were evident both in ACLR and sham operated knee joints with the histological scoring result of the ACLR joints with 6.48 ± 5.67 points and the sham joints with 6.86 ± 5.84 points (p = 0.7953) The MRI scoring yielded 0.34 ± 0.89 points for the ACLR and 0.03 ± 0.17 for the sham knee joints. There was no correlation between the histological and MRI scores (r = 0.10021). The gene expression profiles as well as the immunohistochemical findings showed no significant differences between ACLR and sham knee joints. In conclusion, both knee joints showed histological signs of OA after 26 weeks irrespective of whether the ACL was resected or not. As MRI results did not match the histological findings, MRI was obviously unsuitable to diagnose the OA in GM. The analysis of the expression patterns of the 10 genes could not shed light on the question, whether sham operation also induced cartilage erosion or if the degeneration was spontaneous. The modified Pond-Nuki model may be used with reservation in the adult minipig to induce an isolated osteoarthritis.     

Preliminary histopathological study of intra-articular injection of a novel highly cross-linked hyaluronic acid in a rabbit model of knee osteoarthritis

Osteoarthritis is a degenerative joint disease mostly occurring in the knee and commonly seen in middle-aged and elderly adults. Intra-articular injection of hyaluronic acid has been widely used for treatment of knee osteoarthritis. The aim of this study was to evaluate the efficacy of intra-articular injection of a novel highly cross-linked hyaluronic acid, alone or in combination with ropivacaine hydrochloride and triamcinolone acetonide, on knee articular cartilage in a rabbit model of collagenase-induced knee osteoarthritis. After induction of experimental osteoarthritis by intra-articular injection of collagenase, adult New Zealand white rabbits (n = 12) were divided into 3 groups. Group 1 (control group) received 0.3 ml phosphate buffered saline into the right knee joint. Group 2 received 0.3 ml cross-linked hyaluronic acid (33 mg/ml) into the right knee joint. Group 3 received a mixture of 0.15 ml cross-linked hyaluronic acid (33 mg/ml), 0.05 ml ropivacaine hydrochloride 1 % and 0.1 ml triamcinolone acetonide (10 mg/ml) into the right knee joint. Intra-articular injections were given 4 weeks after first collagenase injection and were administered once a week for 3 weeks. Gross pathology and histological evaluation of rabbits’ knee joints were performed after 16 weeks following initial collagenase injection. Histological analysis of sections of right knee joints at lesion sites showed a significant decrease in Mankin’s score in groups treated with hyaluronic acid alone or in combination with ropivacaine hydrochloride and triamcinolone acetonide versus control group (p < 0.05 and p < 0.01 respectively). This evidence was consistent with strong articular degenerative changes in control right knee joints (grade III osteoarthritis), while the treated groups revealed less severe articular degenerative changes (grade II osteoarthritis). The present results show that cross-linked hyaluronic acid, alone or in combination with ropivacaine hydrochloride and triamcinolone acetonide, produces a significant improvement in knee articular cartilage degeneration in a rabbit model of collagenase-induced osteoarthritis.     

Loss of vasomotor responsiveness to the mu-opioid receptor ligand endomorphin-1 in adjuvant monoarthritic rat knee joints

Endomorphin-1 is a short-chain neuropeptide with a high affinity fo the mu-opioid receptor and has recently been localized in acutely inflamed knee joints where it was found to reduce inflammation. The present study examined the propensity of endomorphin-1 to modulate synovial blood flow in normal and adjuvant-inflamed at knee joints. Under deep urethane anesthesia, endomorphin-1 was topically applied to exposed normal and 1 wk adjuvant monoarthritic knee joints (0.1 ml bolus; 10(-12)-10(-9) mol). Relative changes in articular blood flow were measured by laser Doppler perfusion imaging and vascular resistances in response to the opioid were calculated. In normal knees, endomorphin-1 caused a dose-dependent increase in synovial vascular resistance and this effect was significantly inhibited by the specific mu-opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-O n-Thr-Pen-Th amide (CTOP) (P < 0.0001, 2-factor ANOVA, n = 5-7). One week after adjuvant inflammation, the hypoaemic effect of endomorphin-1 was completely abolished (P < 0.0001, 2-factor ANOVA, n = 5-7). Immunohistochemical analysis of normal and adjuvant-inflamed joints showed a ninefold increase in endomorphin-1 levels in the monoarthritic knee compared with normal control. Western blotting and immunohistochemistry revealed a moderate number of mu-opioid receptors in normal knees; however, mu-opioid receptors were almost undetectable in arthritic joints. These findings demonstrate that peripheral administration of endomorphin-1 reduces knee joint blood flow and this effect is not sustainable during advanced inflammation. The loss of this hypoaemic response appears to be due to down regulation of mu-opioid receptors as a consequence of endomorphin-1 accumulation within the arthritic joint.     

重组人中期因子midkine对大鼠膝关节软骨部分损伤的修复作用

目的:通过外源注射不同剂量的重组人中期因子midkine(rhMK),研究其对大鼠膝关节软骨部分损伤的修复作用。方法:雄性SD大鼠双侧膝关节建立软骨部分损伤的动物模型,术后24小时分别向关节腔内注射生理盐水或rhMK (20μg/kg、60μg/kg、180μg/kg)。于术后8周将大鼠全部处死,取材进行组织学观察,从而确定最佳注射剂量;在药代动力学研究中,按最佳注射剂量向正常大鼠膝关节腔内注射rhMK,分别于注射后1小时、1天、3天、6天、9天、12天和15天处死大鼠,检测膝关节软骨组织中rhMK的含量。结果:不同剂量的重组蛋白对膝关节软骨部分损伤均有不同程度的修复作用,其中180μg/kg的剂量效果最佳;以180μg/kg的剂量向正常大鼠膝关节腔内注射rhMK后,经过Kinetica5.0药代动力学软件拟合后,计算得rhMK在软骨组织中的消除相半衰期为8.69天。结论:rhMK对大鼠膝关节软骨部分损伤有明显的修复作用,最佳注射剂量为180μg/kg,最佳注射时间间隔为8天。     

Alignment of the human body in standing

Alignment of the body in typical symmetrical standing was studied by photographing fifteen subjects in profile on a reaction board. Two aspects of alignment were studied: (1) the anteroposterior position of the body landmarks of knee joint, hip joint, shoulder joint, and ear, compared to the ankle joint; and (2) the positions of the partial centers of gravity above the knee and hip, as a measure of how the body is balanced above these joints. The knee, hip, shoulder, and ear were forward of the ankle in all subjects. On average, the knee was 3.8 (+/- 2.0), the hip 6.2 (+/- 1.3) the shoulder 3.8 (+/- 1.9), and the ear 5.9 (+/- 1.6) cm (+/-S.D.) anterior to the ankle. The positions of landmarks were positively correlated with one another but not highly. The position of the center of gravity could be predicted well from the positions of the landmarks within individual subjects' data, but not across subjects. The centers of gravity above the knee and hip were calculated by subtracting the mass and position of the segments below the joint from the whole-body center of gravity. The center of gravity above the knee was located on average 1.4 (+/- 1.1) cm in front of the joint, and that of the hip 1.0 (+/- 1.6) cm behind the trochanter. Thus, at both knee and hip in typical standing, there exist slight gravitational torques tending to extend the joints.     

Evaluation of cartilage damage by measuring collagen degradation products in joint extracts in a traumatic model of osteoarthritis

The objective of this work was to investigate whether collagen degradation products in protein extract from joints could provide quantitative information on cartilage damage. Osteoarthritis (OA) was surgically induced in rat knee joints. Joints were isolated 7, 14 and 28 days after surgery for protein extraction and histology. C-terminal telopeptide of type II collagen (CTX-II), CTX-I and hydroxyproline were measured in protein extracts. Matrix metalloproteinase (MMP)-2 and -9 activity was evaluated by gelatinase zymography and joint pathology was visualized by histology and immunohistochemistry. The results showed that levels of CTX-II were significantly increased in anterior cruciate ligament transection (ACLT)-operated compared with sham-operated knee joints on days 7 and 28, whereas the levels of hydroxyproline and CTX-I epitopes showed no difference. MMP activity was slightly increased in ACLT-operated joints. The CTX-II epitope was highly expressed and co-localized to damaged articular cartilage in ACLT-operated joints. We have therefore demonstrated an increased type II collagen degradation in knees after surgical induction of OA, and propose assessment of collagen degradation epitopes as a quantitative measure of cartilage damage.     

Quantification of subchondral bone changes in a murine osteoarthritis model using micro-CT

In the past few years there has been a considerable interest in the role of bone in osteoarthritis. Despite the increasing evidence of the involvement of bone in osteoarthritis, it remains very difficult to attribute the cause or effect of changes in subchondral bone to the process of osteoarthritis. Although osteoarthritis in mice provides a useful model to study changes in the subchondral bone, detailed quantification of these changes is lacking. Therefore, the goal of this study was to quantify subchondral bone changes in a murine osteoarthritis model by use of micro-computed tomography (micro-CT). We induced osteoarthritis-like characteristics in the knee joints of mice using collagenase injections, and after four weeks we calculated various 3D morphometric parameters in the epiphysis of the proximal tibia. The collagenase injections caused cartilage damage, visible in histological sections, particularly on the medial tibial plateau. Micro-CT analysis revealed that the thickness of the subchondral bone plate was decreased both at the lateral and the medial side. The trabecular compartment demonstrated a small but significant reduction in bone volume fraction compared to the contralateral control joints. Trabeculae in the collagenase-injected joints were thinner but their shape remained rod-like. Furthermore, the connectivity between trabeculae was reduced and the trabecular spacing was increased. In conclusion, four weeks after induction of osteoarthritis in the murine knee subtle but significant changes in subchondral bone architecture could be detected and quantified in 3D with micro-CT analysis.     

Evaluation of cartilage damage by measuring collagen degradation products in joint extracts in a traumatic model of osteoarthritis

The objective of this work was to investigate whether collagen degradation products in protein extract from joints could provide quantitative information on cartilage damage. Osteoarthritis (OA) was surgically induced in rat knee joints. Joints were isolated 7, 14 and 28 days after surgery for protein extraction and histology. C-terminal telopeptide of type II collagen (CTX-II), CTX-I and hydroxyproline were measured in protein extracts. Matrix metalloproteinase (MMP)-2 and -9 activity was evaluated by gelatinase zymography and joint pathology was visualized by histology and immunohistochemistry. The results showed that levels of CTX-II were significantly increased in anterior cruciate ligament transection (ACLT)-operated compared with sham-operated knee joints on days 7 and 28, whereas the levels of hydroxyproline and CTX-I epitopes showed no difference. MMP activity was slightly increased in ACLT-operated joints. The CTX-II epitope was highly expressed and co-localized to damaged articular cartilage in ACLT-operated joints. We have therefore demonstrated an increased type II collagen degradation in knees after surgical induction of OA, and propose assessment of collagen degradation epitopes as a quantitative measure of cartilage damage.     

Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.     

Matrix Metalloproteases and Tissue Inhibitors of Metalloproteinases in Medial Plica and Pannus-like Tissue Contribute to Knee Osteoarthritis Progression

Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1β, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1β, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee.     

A variable-stiffness shoe lowers the knee adduction moment in subjects with symptoms of medial compartment knee osteoarthritis

The purpose of this study was to evaluate the effectiveness of variable-stiffness shoes in lowering the peak external knee adduction moment during walking in subjects with symptomatic medial compartment knee osteoarthritis. The influence on other lower extremity joints was also investigated. The following hypotheses were tested: (1) variable-stiffness shoes will lower the knee adduction moment in the symptomatic knee compared to control shoes; (2) reductions in knee adduction moment will be greater at faster speeds; (3) subjects with higher initial knee adduction moments in control shoes will have greater reductions in knee adduction moment with the intervention shoes; and (4) variable-stiffness shoes will cause secondary changes in the hip and ankle frontal plane moments. Seventy-nine individuals were tested at self-selected slow, normal, and fast speeds with a constant-stiffness control shoe and a variable-stiffness intervention shoe. Peak moments for each condition were assessed using a motion capture system and force plate. The intervention shoes reduced the peak knee adduction moment compared to control at all walking speeds, and reductions increased with increasing walking speed. The magnitude of the knee adduction moment prior to intervention explained only 11.9% of the variance in the absolute change in maximum knee adduction moment. Secondary changes in frontal plane moments showed primarily reductions in other lower extremity joints. This study showed that the variable-stiffness shoe reduced the knee adduction moment in subjects with medial compartment knee osteoarthritis without the discomfort of a fixed wedge or overloading other joints, and thus can potentially slow the progression of knee osteoarthritis.     

Autoradiography of Water-Diffusible Substances in Sections of Whole Baby Rats

Rats, 7 days postnatal which had been injected with a radioactive nuclide, were quick frozen and sectioned in the frozen state. An adhesive cellulose tape (Sellotape) was used to support the section during cutting, through freeze-drying, and attaching to slides. Dehydration of the frozen sections consisted of 1 hr in a chilled desiccator containing silica gel, then at reduced pressure of 2-3 mm Hg until quite dry. The exposed side of the section was sprayed with celloidin dissolved in amyl acetate and allowed to dry. This side of the section was attached to a slide, previously coated with 1% gelatin containing 0.1% chrome alum, by means of an adhesive consisting of 4% gelatin and 5% formalin in 60% glycerol. In applying this adhesive it is mandatory that a border of about 3 mm of bare glass be left outside the adhesive, to allow intimate contact between the sticky side of the tape and the glass. The adhesive was allowed to set for 20 min, the slide immersed in water lor 50 sec, and the cellulose layer of the tape peeled off. The rubber base from the tape was removed with chloroform, the slide dried, and the exposed surface of the section coated with celloidin in amyl acetate, by dipping. After this treatment, the slides could be coated by dipping in autoradiographic emulsion without affecting water-soluble radioactive substances in the tissue.     

Evaluation of the cholesterol influence in type II collagen-induced arthritis in DBA/1J mice: an autoradiographic study

In order to verify the cholesterol influence in RA severity in DBA/1J mice, we quantified the cholesterol present in the knee joints of normal (N) and with collagen II induced arthritis (CIA). Forty male DBA/1J mice, were divided in normal (n=20) and CIA group (n=20). Mice in CIA group were injected with 100 μg of collagen II emulsified in Freund's complete adjuvant. Sixteen DBA/1J (8 N and 8 CIA) received an injection of 2.96 × 10⁶ Bq of 3H-cholesterol and were anesthetized and sacrificed. Semi-fine sections were covered with LM-1 emulsion, exposed for six weeks and developed. Collagen induced edema, erythema and dysfunction of knee joints in CIA group. Radioactive cholesterol was located more on the synovial membrane, where we found the greatest density of silver grains, significantly ( P <0.0001) higher in group CIA vs. controls (61±2.3 × 18±0.7). We conclude that the cholesterol deposits on the synovial membrane is related to CIA severity.     

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.     

A noninvasive technique for in vivo measurement of joint torques of biarticular muscles.

The objective of this work was to develop a noninvasive method to measure the joint torques produced by biarticular muscles at two joints simultaneously. During intramuscular stimulation of the cat medial gastrocnemius (MG) muscle, torques at the ankle and knee joints were calculated from forces measured in two dimensions at the end point of the cat paw under isometric conditions. The method was verified by the known anatomical properties of cat MG muscle and the tibialis anterior (TA) muscle. The MG muscle was shown to produce a significant flexion torque at the knee, besides an extension torque at the ankle. This was in agreement with its anatomical arrangement. The TA muscle produced primarily an ankle flexion torque. The small knee torque, due to measurement errors, yielded an estimate of measurement accuracy of 3.0 +/- 2.1% (n = 52). The coupling ratio of the MG muscle, defined as T(ankle)/T(knee), varied significantly with both knee and ankle angles. The profile of MG mechanical coupling agreed qualitatively with changes in limb configuration. The method can be used to measure recruitment properties of electrically stimulated biarticular muscles, and may potentially be used to study the biomechanics of biarticular coupling.     

Localization and cytophotometric analysis of cathepsin B activity in unfixed and undecalcified cryostat sections of whole rat knee joints

Cathepsin B activity is demonstrated histochemically with a post-coupling method using Z-Arg-Arg-4-methoxy-2-naphthylamide as substrate and Fast Blue BB as coupling reagent in unfixed and undecalcified cryostat sections of whole rat knee joints. Sections were attached to transparent tape to keep the integrity of the tissue intact, such attachment being essential for precise precipitation of the final reaction product at sites of enzyme activity. Also essential was inclusion of polyvinyl alcohol in the enzyme incubation medium. High cathepsin B activity was found in osteoclasts, chondrocytes, fibroblasts, synovial cells, and bone marrow cells in knee joints after induction of arthritis. The final reaction product was precipitated as fine cytoplasmic granules probably corresponding to lysosomes. The reaction was specific because addition to the incubation medium of selective inhibitors of cathepsin B-like activity completely blocked the activity. The amount of final reaction product in synovium and in bone marrow cells was analyzed cytophotometrically. Specific formation of final reaction product was linear with incubation time up to 60 min at 37 degrees C and with section thickness up to 12 microns. Variation of the substrate concentration in the incubation medium revealed a KM value of 1.86 +/- 0.36 mM in synovial cells and 2.48 +/- 0.51 mM in bone marrow cells and Vmax values (expressed as mean integrated absorbance) of 1.18 +/- 0.10 in synovial cells and 1.02 +/- 0.11 in bone marrow cells. Both KM and Vmax values were significantly different in synovial cells and bone marrow cells (p less than 0.01) which could be owing to the presence of different isoenzymes in these tissues. We conclude that the described post-coupling method is sufficient to yield precise localization and that the method is valid for quantitative purposes.