Articular cartilage deformation following experimental synovitis in the rabbit hip

The development of transient synovitis in the hips of young children occurs quite frequently. This experiment examined the effects of a model synovitis on the deformability of articular cartilage of the immature rabbit hip. At 1, 2, 3, and 4 weeks following synovitis, there was an increase of cartilage deformability on both the acetabular and femoral sides of the joint. This increased deformability may alter force transmission to the underlying bone and its contained vascular structures.     

同种透明软骨移植后蛋白多糖动态变化的实验观察

通过动物实验,利用组织学、组织化学和生物化学等手段,对同种透明软骨移植后不同时期基质中的蛋白多糖含量进行了动态变化观察。结果显示,蛋白多糖含量的高低对移植的异体软骨能否存活极为重要。     

同种透明软骨移植后蛋白多糖动态变化的实验观察

通过动物实验，利用组织学、组织化学和生物化学等手段，对同种透明软骨移植后不同时期基质中的蛋白多糖含量进行了动态变化观察。结果显示，蛋白多糖含量的高低对移植的异体软骨能否存活极为重要。     

Identification of rectal ganglion cells using monoclonal antibodies

A monoclonal antibody has been developed that reacts specifically with ganglion cells of the myenteric and submucous plexuses of the gastrointestinal tract. This antibody also recognizes an axonal antigen that is distributed throughout the circular muscle of normal colon and rectum. Aganglionic segments of colon and rectum from patients with Hirschsprung's disease showed no specific antibody binding using a fluorescent labelled assay. The use of monoclonal antibodies should provide further insights into the pathophysiology of Hirschsprung's disease and allied disorders.     

Immunological analysis of proteoglycan structural changes in the early stage of experimental osteoarthritic canine cartilage lesions.

Specific modifications of the proteoglycan (PG) structure of osteoarthritic (OA) dog cartilage in relation to endogenous metalloprotease activity were examined using murine anti-proteoglycan monoclonal antibodies (MoAbs). OA lesions were induced over a period of 8 weeks in crossbred dogs (Pond-Nuki model). The articular cartilage was removed and homogenized in a Tris buffer, pH 7.5, and then divided into four groups: direct PG extraction, no addition, presence of 1 mM p-aminophenyl mercuric acetate (APMA), and presence of 1 mM APMA and 10 mM o-phenanthroline, incubated for 42 h at 37 degrees C followed by PG extraction. MoAbs reactive with PG protein and carbohydrate epitopes included 1C6, 3B3, 5D4, D1B2, and m4D6. The results showed marked alterations induced by APMA activation of the endogenous metalloproteases. PG changes were apparent at at least three sites: one was either in the hyaluronic acid-binding region or between the hyaluronic-binding region and the G2 globular domain, another was between the keratan-sulfate-rich domain and the chondroitin sulfate-attachment domain, and a third was in the chondroitin sulfate-attachment domain. Constitutive metalloprotease activity resulted in less marked PG alterations with preservation of functional PG aggregation to hyaluronan.     

Production and characterization of new monoclonal antibodies to human osteoclasts

Summary Two monoclonal antibodies raised against human osteoclastoma were found to show antiosteoclastic activity on frozen sections of tumor. Immunoreactivity was localized on the membrane surface. These antibodies exhibited no activity against tissue macrophages and human visceral tissue except kidney, where they stained tubules but not glomeruli. In addition, no activity was observed against rabbit or rat osteoclasts, suggesting that they might react with unique epitopes on human osteoclasts.     

Immunomodulation of dog islets using a cocktail of monoclonal antibodies

Islet allografts are particularly vulnerable to rejection, and current immunosuppressive agents are deleterious to their function. They are, however, highly suitable for ‘immunomodulation’, i.e., the removal or inactivation of passenger leukocytes to reduce their immunogenicity. For this purpose we have used 3 rat anti-dog monoclonal antibodies (Mabs) which are synergistic for leukocytolysis in the presence of autologous dog serum. Spleen cells or purified islets treated with these Mabs together with autologous serum were tested in mixed leukocyte and islet co-culture assays. The stimulatory properties of the Mab-pretreated splenocytes or islets were markedly reduced; moreover, the Mab cytolytic activity was shown to be confined to the leukocyte target cells and did not affect islet secretory function upon glucose stimulation. We conclude that this method of modifying the immunogenicity of dog islets could lead to successful islet grafting in vivo, allowing the reduction of conventional immunosuppression. Successful in vivo studies in this model, which are currently in progress, could have implications for clinical islet transplantation.     

Keratan sulfate content and articular cartilage maturation during postnatal rabbit growth

This article describes the macromolecular changes in keratan sulfate and proteoglycan that occur in rabbit articular cartilage during postnatal development. Articular cartilage glycosaminoglycans from femoral condyles and the tibial plateaus of rabbits at 8, 12, 18, and 26 weeks and 2 years of age were extracted, fractionated, and quantified. The predominant glycosaminoglycan present in articular cartilage at 8 weeks was chondroitin sulfate. During subsequent maturation the relative proportions of keratan sulfate and chondroitin sulfate varied inversely. The greatest increase in the amount of keratan sulfate present in cartilage was observed between 12 and 26 weeks of age. Hyaluronic acid content was measurable at 12 weeks; afterward the amount remained relatively constant with age. Proteoglycans, extracted from 6-, 12-, and 22-week-old rabbit femoral and tibial cartilage in the presence of protease inhibitors, were analyzed on columns of Sepharose CL-2B. Cartilage proteoglycans decreased in hydrodynamic size between 12 and 22 weeks, corresponding to the period of maximal change in content of keratan and chondroitin sulfate.     

The effect of molecular weight on hyaluronan's cartilage boundary lubricating ability--alone and in combination with proteoglycan 4

Objectives

(1) assess the molecular weight dependence of hyaluronan’s (HA) cartilage boundary lubricating ability, alone and in combination with proteoglycan 4 (PRG4), at physiological concentrations; (2) determine if HA and PRG4 interact in solution via electrophoretic mobility shift assay (EMSA).

Methods

The cartilage boundary lubricating ability of a broad range of MW HA (20 kDa, 132 kDa, 780 kDa, 1.5 MDa, and 5 MDa) at 3.33 mg/ml, both alone and in combination with PRG4 at 450 μg/ml, was assessed using a previously described cartilage-on-cartilage friction test. Static, μ_{static, Neq}, and kinetic, <μ_{kinetic, Neq}>, were calculated. An EMSA was conducted with PRG4 and monodisperse 150 kDa and 1,000 kDa HA.

Results

Friction coefficients were reduced by HA, in a MW-dependent manner. Values of <μ_{kinetic, Neq}> in 20 kDa HA, 0.098 (0.089, 0.108), were significantly greater compared to both 780 kDa, 0.080 (0.072, 0.088), and 5 MDa, 0.079 (0.070, 0.089). Linear regression showed a significant correlation between both μ_{static, Neq} and <μ_{kinetic, Neq}>, and log HA MW. Friction coefficients were also reduced by PRG4, and with subsequent addition of HA; however the synergistic effect was not dependent on HA MW. Values of <μ_{kinetic, Neq}> in PRG4, 0.080 (0.047, 0.113), were significantly greater than values of PRG4 + various MW HA (similar in value, averaging 0.040 (0.033, 0.047)). EMSA indicated that migration of 150 kDa and 1,000 kDa HA was retarded when combined with PRG4 at high PRG4:HA ratios.

Conclusions

These results suggest alterations in HA MW could significantly affect synovial fluid’s cartilage boundary lubricating ability, yet this diminishment in function could be circumvented by physiological levels of PRG4 forming a complex, potentially in solution, with HA.     

Immunohistochemical localization of osteonectin in developing human and calf bone using monoclonal antibodies

Summary Osteonectin was immunolocalized in human fetal and calf neonatal developing bone using newly developed monoclonal antibodies. The protein was localized to the cytoplasm of osteoblasts and young osteocytes. In bone matrix, strong reactivity was found in newly laid down osteoid. Bone matrix immunoreactivity was enhanced by pretreatment of sections with proteases, possibly because of an unmasking of epitopes engaged in protein-protein interactions. Osteonectin immuno-reactivity was also found in preosteoblasts in all types of human fetal osteogenesis (membranous, endochondral, subperiosteal, and mantellar (Meckel's cartilage) ossification), and in some chondrocytes of metaphyseal growth plate, posibly modulating towards an osteoblastic phenotype.     

Identification of osteoblast-specific monoclonal antibodies

Summary A series of four antibodies against rat osteoblasts have been produced using the hybridoma technique. After bone cells isolated from newborn rat calvariae by a sequential digestion procedure were cultured for 3 days, the cells were trypsinized and further maintained in rotation cultures overnight. Out of the cultured bone cells alkaline phosphatase-positive cells were sorted by flow cytometry and used as immunogens. The clones secreting the antibodies were selected on the basis of the abilities of these antibodies to bind to the bone cells but not to fibroblasts from neonatal rat head skins, in an enzyme-linked immunosorbent assay. Clones of two hybridomas, designated AOB-1 and AOB-2, were used to characterize the antigenic determinant(s) in osteogenic cells. The antibody showed the reactivity with isolated alkaline phosphatase-positive cells, osteogenic tissue cells in newborn rat calvaria, and mandibula, but not with the cells in head skin, lung, kidney, liver, or stomach as determined by immunofluorescence study. Western blot analysis has identified the antigenic determinants possessing apparent molecular weights of 210,000, 110,000, 65,000, 58,000, 40,000, 36,000, 32,000, 28,000, 25,000, 17,000, and 15,000 of osteoblast-rich monolayer cultured cells. According to the cell surface detection with biotin-avidin protein blotting technique, these fractions appear to be present as components of the cell surface of the osteoblast.     

Demonstration of increased proteoglycan turnover in cartilage explants from dogs with experimental osteoarthritis

The turnover of proteoglycans (assessed by the release into the medium of newly synthesised [35S]-proteoglycan) in explant cultures of articular cartilage from various anatomical sites of the knee joints (stifle) of mature beagles with experimental osteoarthritis has been studied with the following findings: (a) The proportion of newly synthesised proteoglycans released from cartilage explants maintained in vitro was generally increased for cartilage from operated compared with nonoperated control joints. (b) At 3 weeks after surgery there was a significant increase in the release of [35S]-proteoglycans from explants of the lateral and medial tibial plateaux of operated joints compared with sham-operated joints but not from other sites. On the other hand, when this comparison was made at 3 to 6 months after surgery, significant increases in the release of [35S]-proteoglycans were observed from cartilage of all anatomical areas except the patellar groove. (c) The release of [35S]-proteoglycan from cartilage explant cultures was dependent on live chondrocytes, since freeze-thawing the tissue immediately after labelling markedly reduced the release from both normal and osteoarthritic cartilage.     

颈椎间盘压缩力学性能改变与终板蛋白多糖变化关系的实验研究

目的:探讨颈椎间盘退变时基质各成分的变化及其对椎间盘力学性能的影响。方法:将24只家兔随机分为对照组和模型组(n=12),模型组保持45°低屈曲位5h/次/d,最长持续3个月。分别于造模后1、2、3个月时各处死4只动物,取C5/6椎间盘测定终板糖胺多糖(glycosaminoglycan,GAG)总量、硫酸软骨素(chongdroitinsulphate,CS)与硫酸角质素(keratansulphate,KS)比值和透明质酸(hyaluronicacid,HA)含量;同时作力学测定。结果:模型组终板抗压强度、断裂时的最大形变、终板基质GAG总含量、CS/KS比值、HA含量都低于对照组(P<0.05),并随造模时间的延长两组差异越显著。结论:长时间的应力不均可造成椎间盘终板材料力学特性改变;终板蛋白多糖含量和成分改变可能是颈椎间盘生物力学性能减退的主要原因之一。     

Correlation of lysozyme activity with proteoglycan biosynthesis in epiphyseal cartilage

Summary Pig epiphyseal cartilage (proximal ulna epiphysis) previously incubated in vitro in the presence of sodium [³⁵S]sulfate or [³H]thymidine was either analyzed by autoradiography or separated into 9 morphologically defined consecutive layers and investigated for³⁵S-incorporation into the guanidinium chloride-extractable proteoglycans and for lysozyme activity.The lowest³⁵S incorporation and lysozyme activity were determined in the zone of resting cells, but there is a consecutive increase in the rate of proteoglycan synthesis and lysozyme activity toward the diaphyseal cartilage-bone junction, with the maximum at the lower columnar cell zone and a sharp reduction of both parameters at the hypertrophic zone. The maxima of³⁵S incorporation and [³H]thymidine incorporation do not coincide.The guanidinium chloride-soluble proteoglycans exhibit macromolecular polydispersity. Fractions excluded from as well as retarded by Sepharose 2B gel could be separated and were detected in all zones.The results indicate a correlation of proteoglycan biosynthesis and lysozyme activity in epiphyseal cartilage.     

Production and characterization of monoclonal antibodies against rabbit growth cartilage

Summary Growth cartilage (GC) cells of young rabbits were cultured in vitro and their homogenates were injected into mice. Hybridomas were prepared by the cell fusion technique between the myeloma cells and the spleen cells of the immunized mice. Monoclonal antibodies (MoAbs) were produced by the hybridomas in the peritoneal cavities of the mice, and some of these, temporarily named MoAbs A, B, D, N, P, and S, were studied. The localization of the antigens of each of the MoAbs in the GC or adjacent resting cartilage (RC) was examined by indirect fluorescent antibody staining. The molecular weight of the antigens was examined by immunoblot staining after SDS-polyacrylamide gel electrophoresis: MoAb A and MoAb N stained RC cells and GC cells, except calcified GC. MoAb B stained the hypertrophic and calcified GC, and matrices in the RC and proliferating GC. MoAb D stained the calcified GC. MoAb P and MoAb S stained the RC cells and the matrices in the GC, intensively in the hypertrophic GC and perichondrium. The molecular weights of the antigens of MoAbs A, P, and S were 40–70 KD, 35–40 KD and 30 KD, respectively.

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Résumé Des cellules de cartilage de croissance de jeunes lapins ont été cultivées in vitro et leurs homogénats ont été injectés à la souris. Des hybridomes ont été préparés par la technique de fusion des cellules de myélome et des cellules de la rate de souris immunisée. Des anticorps monoclonaux (MoAbs) ont été produits par les hybridomes dans la cavité péritonéale de la souris et certains d'entre eux, provisoirement appelés MoAbs A, B, D, N, P et S, ont été étudiés. La localisation des antigènes de chaque MoAbs dans le cartilage de croissance ou dans le cartilage au repos avoisinant a été recherchée par coloration fluorescente indirecte des anticorps. Le poids moléculaire des antigènes a été évalué par coloration immunogène après électrophorèse au gel polycrylamide SDS. Le MoAb A et le MoAb N ont coloré les cellules du cartilage au repos et les cellules du cartilage de croissance à l'exception du cartilage de croissance calcifié. Le MoAb B a coloré le cartilage de croissance hypertrophique et calcifié ainsi que les matrices du cartilage au repos et le cartilage de croissance proliférant. Le MoAb D a coloré le cartilage de croissance calcifié. Le MoAb P et le MoAb S ont coloré les cellules du cartilage au repos et les matrices dans le cartilage de croissance, de manière particulièrement intensive dans le cartilage de croissance hypertrophique et le périchondre. Le poids moléculaire de l'antigène des MoAbs A, P et S s'est trouvé être respectivement 40–70 KD, 35–40 KD et 30 KD.

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Supported by Japan Orthopaedics and Traumatology Foundation, Inc. (JOTF), Grant No. 0021     

Thermochondroplasty of rabbit ear cartilage using the carbon dioxide laser

Remodelling of deformed cartilaginous tissue in the head and neck presents a difficult surgical problem since cartilage tends to return to its original shape because of its natural internal stresses. This is also true of cartilage autografts. Cartilage can be remodelled using heat and this paper describes the use of light from a carbon dioxide laser (λ=10.6 μm) to remodel cartilaginous tissue, in a procedure which we have called laser thermochondroplasty. Straight cartilage samples were removed from the ears of 12 rabbits, moulded with the CO₂ laser at an output power of 3 W with a spot diameter of 2 mm and exposure times of 0.5 s. After remoulding, the cartilage, together with a control, unmoulded sample, were implanted into the rabbits' back and retrieved 6–8 months later. Histological and morphological analysis showed that the treated cartilages retained both their shape and viability. This may well be a useful clinical technique for the in situ remoulding of difficult cartilage, such as that found in the nasal septum, with minimal damage, and the transplanting of autologous cartilage which has been remoulded in vitro.

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Перιλхψη H αпокαтάστααση τoν σχήμс τоς τωv παραμoρϕωμέvωv μoρϕoλoγικώv χóvδρωv, óπως επίσης και η μεταβoλή τoν σχήματoς τωv χóvδριvωv μoσχενμάτωv, εξακoλoνϑoύv vα πρoβάλoνv σημαvτικά πρoβλήματα. Aιτία είvαι η ύπαρξη εvóς σνστήματoς εσωτερικώv στoνς χóvδρoνς, πoν αvϑίσταται στη μεταβoλή τoν σχήματóς τoνς και τείvει vα τoνς επαvαϕέρει στηv αρχική τoνς μoρϕή. H περμoχovδρoπλαστική με laser ειvαι μια ελπιδoϕoρα vεα τεχvικη για τη μεταβoλη τℴν σχηματoς τωv χovδρωv. Eνϑεα χovδριvα τμηματα πoν παρεληϕϑησαv απo αντια κoνvελιωv, αλλαξαv τℴ σχημα τoνς μετα τηv ακτιvoβoληση τoνς με εvα CO₂ laser, σε επιλεγμέvες πνκvóτητες ισχύoς. Tα δείγματα αντά εμϕντεύϕηκαv στo νπoδóρειo της πλάτης τωv κoνvελιώv και παρελήϕϑησαv μετά απó 6–12 μήvες. Oλα τα δείγματα διατηρoύσαv τo vέo τoνς σχήμα, εvώ η ιστoλoγική μελέτη τωv δειγμάτωv με oπτικó και ηλεκτρovικó μικρoσκóπιo έδειξε έvα ζωvταvó και καλά αvεκτó απó τoνς περιϕάλλovτες ιστoύς χóvδρo. Πιστεύvνμε óτι με τηv τεχvική μας, είvαι εϕικτή η απoκατάσταση τωv παραμoρϕωμέωv μoρϕoλoγικώv χóvδρωv (π.χ. σκóλιωv ριvικ<v διαϕραγμάτωv) καϑώς και η τρoπoπoίηση τoν σχήματoς χóvδριvωv μoσχεvνμάτωv με ελάχιστη δνvατή καταστρoϕή τoν χóvδρoν.

     

Electron microscopic analysis of articular cartilage proteoglycan degradation by growth plate enzymes

To assess the effect of intracellular growth plate chondrocyte enzymes on proteoglycan structure, we examined enzyme-treated articular cartilage proteoglycans and untreated articular cartilage proteoglycans with the electron microscopic monolayer technique. The untreated proteoglycan monomers ranged in length from less than 20 nm to more than 700 nm, with a mean length of 224.5 +/- 101.6 nm in one experiment and 224.6 +/- 95.7 nm in a second experiment. Incubation with growth plate enzymes reduced proteoglycan monomers to fragments with lengths that varied from less than 5 nm to 143 nm, increased the variability in monomer length, and destroyed proteoglycan aggregates. The enzyme treated monomers had an average length of 29.5 +/- 17.9 nm in one experiment and 35.2 +/- 17.0 nm in a second experiment. The smallest common fragments were 15 nm long and would be expected to contain about 15 glycosaminoglycan chains. This experiment demonstrates that enzymes extracted from growth plate chondrocytes can degrade the chondroitin sulfate-rich region of proteoglycan monomer core proteins, produce a range of monomer fragment sizes with less than 2% of the fragments shorter than 5 nm or longer than 100 nm, increase the variability in monomer length, and degrade proteoglycan aggregates.     

Flow cytometric determination of spermatozoa binding monoclonal antibodies does not improve the prediction of fertility potential

Summary. In a previous publication the determination of binding of several monoclonal antibodies to human spermatozoa by flow cytometry was described in 223 patients. In normozoo-spermic samples the percentage of spermatozoa binding antibodies was higher than in samples with oligozoospermia. However, only weak correlations were found between the percentage of antibody binding spermatozoa and sperm count, motility and morphology. Thus the antibody binding may represent a property different from the classical parameters and might improve the prediction of fertility in the patients. A questionnaire was sent to all the patients inquiring about a conception in their female partners. One hundred and twenty one out of the 223 patients replied, 103 of whom we were able to evaluate. Forty five of them had induced a pregnancy in their partners. The mean values of sperm count, motility and morphological normal cells, as well as the percentages of cells that bound antibodies, were significantly higher in the group of men whose partners became pregnant, than in those who had not achieved conception. The differences, however, were only marginal. We conclude from our results that the determination of the percentage of spermatozoa binding the monoclonal antibodies used in our study is not likely to improve the prediction of conception probability.