Binding of calcium by proteoglycansin vitro

Summary Proteoglycans from bovine nasal septa or the Swarm rat chondrosarcoma, as potassium salts, effectively inhibit the precipitation of tricalcium phosphatein vitro at pH 7.8. The same preparations, and many other similar preparations, however, do not site bind calcium, as assessed with a calcium ion specific electrode. However, after treatment of aggregate preparations of proteoglycans with EDTA, the preparations can site bind calcium. The amount thus bound is approximately equal to one-half the sum of the equivalents of the ester sulfate and the uronic acid carboxyl groups in the preparations. This latter observation suggested the possibility that the supposed potassium salts of the proteoglycans had, in the course of preparation, acquired calcium and held onto it strongly. In checking this possibility, using neutron activation analysis, it was found that some of the preparations do contain small amounts of calcium but these amounts are insufficient to saturate the binding sites potentially available to this end. In view of the above observations, it is suggested that the proteoglycans inhibit the formation of calcium phosphate precipitatesin vitro not because the calcium is site bound but because the calcium ions are territorially bound.     

Effect of postnatal age on the ultrastructure of six anatomical areas of canine flexor digitorum profundus tendon

We report findings of a transmission electron microscopic study comparing the morphological appearance of cells and extracellular matrix of two fibrocartilaginous regions of canine flexor digitorum profundus (FDP) tendon with that for typical tendinous regions. In addition, we determined the size distribution of collagen fibrils in six anatomical areas of the tendon from animals of three different ages. Average collagen fibril diameters for each of the six anatomical sites of 11-week-old FDP tendon were consistently different from that for older tissue. As growth proceeds, fibrils in tendinous regions almost double in size and take on a broad bimodal distribution. Collagen diameters in fibrocartilaginous areas do not increase, but rather decrease in size with age. Finally, the cells and associated pericellular matrix of fibrocartilaginous areas of adolescent and mature FDP tendon are ultrastructurally distinct from those of typical tendinous regions. On the contrary, the cellular morphology of 11-week-old tendon was invariant regardless of the anatomical region examined. In summary, fibrocartilage of canine FDP tendon, although not evident at 11 weeks of age, is well established by 6-12 months after birth and is the result of cellular and extracellular matrix specialization.     

Chondroitin sulphate proteoglycan and embryonic brain enlargement in the chick

Previous studies of the early development of the neural tube have shown the existence of an intraneural fluid, which causes a positive pressure inside this primordium, and seems to play a key role in the early development of the central nervous system. In the present study we investigated the composition and synthesis of this intraneural fluid. By using a sequential method, which includes fixation with glutaraldehyde plus cetylpyridinium chloride, opening the neural cavity after critical point drying and scanning electron microscopy analysis, we found a water-soluble extracellular matrix that filled up the brain vesicles of chick embryos at the earliest stages of the neural tube. An ultrastructural study of the neural epithelium during these stages revealed the existence of a secretion process in the neural cells toward the apical side, the future neural cavity. An immunocytochemical study to asses the nature of the secreted material has shown that the intraneural matrix contains chondroitin sulphate proteoglycan, which appeared homogenously distributed throughout the neural cavity. Our findings demonstrate that the intraneural liquid is a fluid of complex composition and includes chondroitin sulphate proteoglycan as an osmotically active molecule. This suggests a morphogenetic role for the proteoglycan during early brain enlargement. The neural ectoderm is a polarized epithelium from early developmental stages and secretes the intraneural matrix.     

Elevated levels of insulin-like growth factor (IGF) binding protein-3 in rheumatoid arthritis synovial fluid inhibit stimulation by IGF-I of articular chondrocyte proteoglycan synthesis

The objective of this study was to quantify insulin-like growth factor (IGF) binding proteins (IGFBPs) in the synovial fluid (SF) and plasma of patients with rheumatic diseases and to study the role of these proteins in the regulation of cartilage proteoglycan (PG) synthesis. Immunological determination of IGFBP-2, IGFBP-3, IGF-I, IGF-II, interleukin-1β (IL-1β) and tumour necrosis fac-tor α (TNFα) was undertaken in the SF and plasma of 115 patients with rheumatoid arthritis (RA; n = 53), osteoarthritis (OA; n = 44) and other rheumatic disorders. We also determined the effects of SF on bovine cartilage PG synthesis in culture. IGFBP-2 and IGFBP-3 were elevated in the plasma (by 38% and 28%, respectively) and SF (by 56% and 59%, respectively) of patients with RA compared to age- and sex-matched OA controls (determined by RIA and confirmed by Western ligand blot). IGF-I and IGF-II did not differ significantly between the two groups. OA SF, and, to a lesser extent, RA SF stimulated cartilage PG synthesis in culture, and more than 60% of this activity was neutralised by a specific monoclonal anti-IGF-I antibody. Human IGFBP-3 dose-dependently inhibited the stimulation of cartilage PG synthesis effected by SF or human IGF-I. In RA patients, the SF concentration of IGFBP-3 was positively correlated with SF levels of IL-1β and TNFα, with the serum level of C-reactive protein and with the erythrocyte sedimentation rate. We concluded that IGF-I is, under the conditions studied, the most important anabolic factor in human SF with respect to articular cartilage PG synthesis. The bioactivity of IGF-I in joints is modulated by IGFBP-3, which is elevated in RA SF compared to OA SF. Elevated IGFBP-3 in RA SF may reduce the availability of IGF-I to articular chondrocytes, thus interfering with cartilage PG synthesis in RA. Received: 30 November 1996 / Accepted: 19 February 1997     

Oxidized lipid-mediated alterations in proteoglycan metabolism in cultured pulmonary endothelial cells

Compared to cholesterol or linoleic acid (18:2), oxidized lipids such as cholestan-3β,5,6β-triol (triol) and hydroperoxy linoleic acid (HPODE) markedly impair endothelial barrier function in culture [Hennig and Boissonneault, 1987; Hennig et al. 1986]. Because proteoglycans contribute to vascular permeability properties, the effects of cholesterol and 18:2 and their oxidation products, triol and HPODE, on endothelial proteoglycan metabolism were determined. While cholesterol was without effect, a concentration-dependent decrease in cellular proteoglycans (measured by ³⁵S incorporation) was observed after exposure to triol. Compared to control cultures, cholesterol reduced mRNA levels for the proteoglycans, perlecan and biglycan. Triol had a similar effect on biglycan but not on perlecan mRNA levels. Compared to 18:2, 1, 3 and 5 μM HPODE depressed cellular proteoglycans. Perlecan mRNA levels were reduced more by HPODE when compared to 18:2. Biglycan mRNA levels were reduced by 3 μM, but not by 5 μM HPODE. These data demonstrate that oxidized lipids such as triol and HPODE can decrease cellular proteoglycan metabolism in endothelial monolayers and alter mRNA levels of major specific proteoglycans in a concentration-dependent manner. This may have implications in lipid-mediated disruption of endothelial barrier function and atherosclerosis.     

Articular cartilage and intervertebral disc proteoglycans differ in structure: an electron microscopic study

Articular cartilage and the intervertebral disc tissues have different material and biological properties and different patterns of aging and degeneration. To determine if the proteoglycans of these tissues differ in structure, we used the electron microscopic monolayer technique to compare baboon articular cartilage proteoglycans with baboon annulus fibrosus, transition zone, and nucleus pulposus proteoglycans. Intervertebral disc and articular cartilage proteoglycans differed significantly. Articular cartilage contained large proteoglycan aggregates formed from hyaluronic acid central filaments, multiple monomers, and large nonaggregated monomers. These molecules were identical to those of nasal cartilage, growth plate cartilage, chondrosarcomas, or menisci. In contrast, the intervertebral disc tissues contained only nonaggregated proteoglycan monomers and clusters of monomers without apparent central filaments. Intervertebral disc nonaggregated monomers were shorter and more variable in length than those from articular cartilage, and nucleus pulposus nonaggregated monomers were even shorter and more variable in length than transition zone and annulus fibrosus monomers. These observations suggest that significant differences in proteoglycan metabolism exist between articular cartilage and intervertebral disc.     

24-Week study on the use of collagen hydrolysate as a dietary supplement in athletes with activity-related joint pain

ABSTRACT

Background: Collagen hydrolysate is a nutritional supplement that has been shown to exert an anabolic effect on cartilage tissue. Its administration appears beneficial in patients with osteoarthritis.

Objective: To investigate the effect of collagen hydrolysate on activity-related joint pain in athletes who are physically active and have no evidence of joint disease.

Design and setting: A prospective, randomized, placebo-controlled, double-blind study was conducted at Penn State University in University Park, Pennsylvania. Parameters including joint pain, mobility, and inflammation were evaluated with the use of a visual analogue scale during a 24-week study phase.

Study participants: Between September 2005 and June 2006, 147 subjects who competed on a varsity team or a club sport were recruited. Data from 97 of 147 subjects could be statistically evaluated.

Intervention: One hundred and forty-seven subjects (72 male, 75 female) were randomly assigned to two groups: a group (n?=?73) receiving 25?mL of a liquid formulation that contained 10?g of collagen hydrolysate (CH-Alpha)^* and a group (n?=?74) receiving a placebo, which consisted of 25?mL of liquid that contained xanthan.

Main outcome measures: The primary efficacy parameter was the change in the visual analogue scales from baseline during the study phase in relation to the parameters referring to pain, mobility, and inflammation.

Results: When data from all subjects (n?=?97) were evaluated, six parameters showed statistically significant changes with the dietary supplement collagen hydrolysate (CH) compared with placebo: joint pain at rest, assessed by the physician (CH vs. placebo (–1.37?±?1.78 vs. –0.90?±?1.74 (?p?=?0.025)) and five parameters assessed by study participants: joint pain when walking (–1.11?±?1.98 vs. –0.46?±?1.63, p?=?0.007), joint pain when standing (–0.97?±?1.92 vs. –0.43?±?1.74, p?=?0.011), joint pain at rest (–0.81?±?1.77 vs. –0.39?±?1.56, p?=?0.039), joint pain when carrying objects (–1.45?±?2.11 vs. –0.83?±?1.71, p?=?0.014) and joint pain when lifting (–1.79?±?2.11 vs. –1.26?±?2.09, p?=?0.018). When a subgroup analysis of subjects with knee arthralgia (n?=?63) was performed, the difference between the effect of collagen hydrolysate vs. placebo was more pronounced. The parameter joint pain at rest, assessed by the physician, had a statistical significance level of p?=?0.001 (–1.67?±?1.89 vs. –0.86?±?1.77), while the other five parameters based on the participants’ assessments were also statistically significant: joint pain when walking (?p?=?0.003 (– 1.38?±?2.12 vs. – 0.54?±?1.65)), joint pain when standing (?p?=?0.015 (–1.17?±?2.06 vs. –0.50?±?1.68)), joint pain at rest with (?p?=?0.021 (–1.01 ±1.92 vs. –0.47?±?1.63)), joint pain when running a straight line (?p?=?0.027 (–1.50?±?1.97 vs. –0.80?±?1.66)) and joint pain when changing direction (?p?=?0.026 (–1.87?±?2.18 vs. –1.20?±?2.10)).

Conclusion: This was the first clinical trial of 24-weeks duration to show improvement of joint pain in athletes who were treated with the dietary supplement collagen hydrolysate. The results of this study have implications for the use of collagen hydrolysate to support joint health and possibly reduce the risk of joint deterioration in a high-risk group. Despite the study's size and limitations, the results suggest that athletes consuming collagen hydrolysate can reduce parameters (such as pain) that have a negative impact on athletic performance. Future studies are needed to support these findings.     

The effects of proteoglycans from different cartilage types onin vitro hydroxyapatite proliferation

Summary Proteoglycans extracted from nasal and articular cartilages have previously been shown to inhibit hydroxyapatite proliferationin vitro. This study now demonstrates that proteoglycans isolated (dissociatively extracted and reaggregatedin vitro) from bovine fetal growth plate and bovine occipital condyle, as well as those from bovine nasal cartilage, all retard hydroxyapatite seeded-growthin vitro. On a weight basis (1 mg/ml), the growth plate proteoglycan preparation had a significantly greater inhibitory effect. The greater inhibitory effect of the growth plate proteoglycans as compared to the other cartilage proteoglycan preparations may be related to the unique properties of the proteoglycans in the growth plate, a tissue that undergoes physiological calcification.     

Human cartilage proteoglycans isolated from normally ossifying and congenitally malformed leg bones

Summary Proteoglycans were extracted with 4 M guanidine HC1 solution containing protease inhibitors from various zones of human epiphyseal cartilages of the normally ossifying fibula and cartilaginous rudiment of the tibia of a 12-month-old boy with congenital absence of the tibia, when the knee disarticulation was performed. All the proteoglycan preparations from the epiphyseal cartilages were separated with a sucrose density gradient centrifugation into two components; a heavy, major component and a light one. The molecular size and the proportion of isomeric chondroitin sulfates of polysaccharides of the heavy component differed from those of the light one. The relative amounts of isomeric chondroitin sulfates in the polysaccharide moieties of the components also varied among these zones. The glycosaminoglycan content in the rudimentary tibia was equal to that of the epiphyseal cartilage of the fibula. However, proteoglycan preparations showed neither the normal sedimentation profile with two peaks nor the zonal differences as to the proportion of isomeric chondroitin sulfates. These results suggest that the alterations in proteoglycan metabolism might be involved in the pathogenetic mechanisms producing the congenital limb defect.     

Embryonic radial glia bridge spinal cord lesions and promote functional recovery following spinal cord injury

Radial glial cells are neural stem cells (NSC) that are transiently found in the developing CNS. To study radial glia, we isolated clones following immortalization of E13.5 GFP rat neurospheres with v-myc. Clone RG3.6 exhibits polarized morphology and expresses the radial glial markers nestin and brain lipid binding protein. Both NSC and RG3.6 cells migrated extensively in the adult spinal cord. However, RG3.6 cells differentiated into astroglia slower than NSC, suggesting that immortalization can delay differentiation of radial glia. Following spinal cord contusion, implanted RG3.6 cells migrated widely in the contusion site and into spared white matter where they exhibited a highly polarized morphology. When injected immediately after injury, RG3.6 cells formed cellular bridges surrounding spinal cord lesion sites and extending into spared white matter regions in contrast to GFP fibroblasts that remained in the lesion site. Behavioral analysis indicated higher BBB scores in rats injected with RG3.6 cells than rats injected with fibroblasts or medium as early as 1 week after injury. Spinal cords transplanted with RG3.6 cells or dermal fibroblasts exhibited little accumulation of chondroitin sulfate proteoglycans (CSPG) including NG2 proteoglycans that are known to inhibit axonal growth. Reduced levels of CSPG were accompanied by little accumulation in the injury site of activated macrophages, which are a major source of CSPG. However, increased staining and organization of neurofilaments were found in injured rats transplanted with RG3.6 cells suggesting neuroprotection or regrowth. The combined results indicate that acutely transplanted radial glia can migrate to form bridges across spinal cord lesions in vivo and promote functional recovery following spinal cord injury by protecting against macrophages and secondary damage.