Cartilage Of Guinea Pigs Research Articles

The study of targeted blocking SDF-1/CXCR4 signaling pathway with three antagonists on MMPs, type II collagen, and aggrecan levels in articular cartilage of guinea pigs

ObjectiveTo explore the possibility and mechanism of targeted blocking SDF-1/CXCR4 signaling pathway using three antagonists TN14003, T140, and AMD3100 in vivo, and to investigate the function of three antagonists in delay degeneration process of articular cartilage.MethodsNinety-six male Duncan-Hartley guinea pigs (6 months old) were divided into groups A, B, C, and D randomly. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group A, and TN14003 with concentration of 180 μg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group B, and T140 with concentration of 180 μg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group C, and AMD3100 with concentration of 180 μg/ml was pumped every day. Hartley guinea pigs in group D remained untreated as the blank control group. At 2, 4, 6, 8, 10, and 12 weeks of treatment, 5 to 8 animals in each group were randomly chosen for blood collection via cardiac puncture. SDF-1 content using enzyme-linked immunosorbent assay (ELISA). At 12 weeks, all guinea pigs were sacrificed by injecting pentobarbital sodium (30 mg/kg) into the peritoneal cavity. Cartilages from the tibial plateau in each group were harvested for PCR testing and western blot analysis. SPSS19.0 was used for data analysis.ResultsResult of ELISA: the serum levels of SDF-1 of groups A, B, and C decreased gradually with time. Significant drop of SDF-1 level was seen in group A while increased SDF-1 was shown in group D. At the same time, the serum levels of SDF-1 of the group A were significantly lower than that of group B; those of group B were significantly lower than that of group C, which was significantly lower than that of group D, and their difference is statistically significant (P < 0.05). Real time quantitative PCR result: The mRNA levels of MMPs in group A were significantly lower than group B, and those of group B were significantly lower than group C, which was significantly lower than group D, and there was statistically significant (P < 0.05). The mRNA levels of type II collagen, aggrecan in group A were significantly more than group B; those of group B were significantly more than group C, which was significantly more than group D, and the difference was statistically significant (P < 0.05). H&E staining result: cartilage of group C was more significantly degenerative than other groups.ConclusionsThe three antagonists can target SDF-1/CXCR4 signaling pathway in vivo, reduce the expression and secretion of MMP-3, MMP-9, and MMP-13 in cartilage tissue, and reduce the degradation of collagen II and aggregating proteoglycan, thus delaying the degeneration of articular cartilage, of which TN14003 has the strongest regulatory effect. Targeted blockade of SDF-1/CXCR4 signaling pathway by TN14003 in vivo delays articular cartilage degeneration more effectively than T140 and AMD3100.

Effectiveness of Radiofrequency Hyperthermia for Treating Cartilage in Guinea Pigs with Primary Osteoarthritis.

Objective Autophagy was reported to be essential for maintaining chondrocyte function, and reduced autophagy leads to osteoarthritis (OA). Previous studies showed involvement of heat shock stress in the control of autophagy in cells. This study sought to investigate the effect of hyperthermia on the expression of autophagy-related proteins in articular cartilage and the progression of naturally occurring OA in Hartley guinea pigs. Design Radiofrequency pulses of 13.56 MHz were applied to the animals' knees for 20 minutes to induce hyperthermia. The knee joints were resected at 8 hours, 24 hours, 72 hours, 7 days, and 6 months after hyperthermia. Serial sections of knees were examined for histopathological changes. The expression levels of Unc-51-like kinase 1 (ULK1) and Beclin1 were analyzed by immunohistochemistry. Results Analysis of the distribution of positive cells showed that, in cases of moderate OA, ULK1 and Beclin1 expression levels were significantly decreased in the superficial zone (SZ) and middle zone (MZ) ( P < 0.01) compared with normal cartilage. Seven days after exposure to radiofrequency waves, expression levels of ULK1 and Beclin1 were augmented in the SZ in animals with mild OA. The severity of cartilage degradation was significantly reduced ( P < 0.01) in the radiofrequency-treated knees versus the untreated knees. Conclusions This study showed that heat stimulation enhanced autophagy in healthy knee chondrocytes and chondrocytes in knees with mild OA. The study also showed that long-term periodic application of hyperthermia suppresses aging-related progression of OA. The activation of autophagy by radiofrequency hyperthermia may be an effective therapeutic approach for osteoarthritis.

Evaluation of the ability of two transfection reagents to deliver small interfering RNA molecules to equine and guinea pig cartilage in vitro

To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses. Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses. Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested amount of a proprietary siRNA molecule. Full-thickness cartilage explants were treated with 1× (1μM), 2× (2μM), and 4× (4μM) or 1× (0.13μM), 4× (0.52μM), and 8× (1.04μM) the recommended concentrations of the proprietary siRNA and the cationic liposome siRNA, respectively, in equivalent media volumes. Use of fluorescent siRNA duplexes allowed quantification of transfected cells via flow cytometry and direct visualization of the depth and distribution of in situ transfection via fluorescent microscopy. With both transfection reagents, > 90% of monolayer chondrocytes were transfected. In explants, only use of the proprietary molecule achieved > 50% transfection efficiency, whereas use of the cationic liposome achieved < 20%. Only the proprietary molecule-treated cartilage consistently contained fluorescent cells throughout all zones; the cationic liposome-transfected chondrocytes were restricted to explant surfaces. Robust transfection of chondrocytes in monolayer was achieved with both reagents, but only use of the proprietary molecule attained effective full-thickness transfection of explants that may allow relevant transcript reduction via RNAi.

Detectable reporter gene expression following transduction of adenovirus and adeno‐associated virus serotype 2 vectors within full‐thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo.

Osmotic loading to determine the intrinsic material properties of guinea pig knee cartilage

Few methods exist to study cartilage mechanics in small animal joints due to the difficulties associated with handling small tissue samples. In this study, we apply an osmotic loading method to quantify the intrinsic material properties of articular cartilage in small animal joints. Cartilage samples were studied from the femoral condyle and tibial plateau of two-month old guinea pigs. Swelling strains were measured using confocal fluorescence scanning microscopy in samples subjected to osmotic loading. A histochemical staining method was developed and calibrated for quantification of negative fixed charge density in guinea pig cartilage. Site-matched swelling strain data and fixed charge density values were then used with a triphasic theoretical model for cartilage swelling to determine the uniaxial modulus of the cartilage solid matrix. Moduli obtained in this study (7.2 MPa femoral condyle; 10.8 MPa, tibial plateau) compare well with previously reported values for the tensile moduli of human and other animal cartilages determined from uniaxial tension experiments. This study provides the first available data for material properties and fixed charge density in cartilage from the guinea pig knee and suggests a promising method for tracking changes in cartilage mechanics in small animal models of degeneration.

The effects of ascorbic acid on cartilage metabolism in guinea pig articular cartilage explants.

Ascorbic acid has been associated with the slowing of osteoarthritis progression in guinea pig and man. The goal of this study was to evaluate transcriptional and translational regulation of cartilage matrix components by ascorbic acid. Guinea pig articular cartilage explants were grown in the presence of L-ascorbic acid (L-Asc), D-isoascorbic acid (D-Asc), sodium L-ascorbate (Na L-Asc), sodium D-isoascorbate (Na D-Asc), or ascorbyl-2-phosphate (A2P) to isolate and analyze the acidic and nutrient effects of ascorbic acid. Transcription of type II collagen, prolyl 4-hydroxylase (alpha subunit), and aggrecan increased in response to the antiscorbutic forms of ascorbic acid (L-Asc, Na L-Asc, and A2P) and was stereospecific to the L-forms. Collagen and aggrecan synthesis also increased in response to the antiscorbutic forms but only in the absence of acidity. All ascorbic acid forms tended to increase oxidative damage over control. This was especially true for the non-nutrient D-forms and the high dose L-Asc. Finally, we investigated the ability of chondrocytes to express the newly described sodium-dependent vitamin C transporters (SVCTs). We identified transcripts for SVCT2 but not SVCT1 in guinea pig cartilage explants. This represents the first characterization of SVCTs in chondrocytes. This study confirms that ascorbic acid stimulates collagen synthesis and in addition modestly stimulates aggrecan synthesis. These effects are exerted at both transcriptional and post-transcriptional levels. The stereospecificity of these effects is consistent with chondrocyte expression of SVCT2, shown previously to transport L-Asc more efficiently than D-Asc. Therefore, this transporter may be the primary mechanism by which the L-forms of ascorbic acid enter the chondrocyte to control matrix gene activity.

ONO-4817, an orally active matrix metalloproteinase inhibitor, prevents lipopolysaccharide-induced proteoglycan release from the joint cartilage in guinea pigs.

To evaluate the effect of a newly developed inhibitor of matrix metalloproteinases (MMPs), ONO-4817, on the degradation of cartilage in the guinea pig arthritis model. 42 guinea pigs were used in the arthritis model. Lipopolysaccharide (LPS) was injected into guinea pig knee joints. The content of proteoglycan released in synovial cavity was measured as a marker of cartilage degradation. ONO-4817, dexamethasone or indomethacin were administered orally to the animals. ONO-4817 showed a broad inhibitory activity against MMPs except MMP-1 and MMP-7. The oral administration of ONO-4817 dose-dependently suppressed the release of proteoglycan from the cartilage of the knee joints. This study suggests the possibility that a novel MMP inhibitor, ONO-4817 may have a therapeutic utility for MMP-related diseases.

Correlation of morphologic and biochemical changes in the natural history of spontaneous osteoarthrosis in guinea pigs.

To study how the concentrations of proteoglycans (PGs) and collagen change in various parts of tibial articular cartilage during aging, and to evaluate the development of spontaneous osteoarthrosis (OA) in guinea pigs. PGs were extracted from guinea pig cartilage samples using 4M guanidine hydrochloride, and the amount of hydroxyproline was determined in the extraction remainder. The molecular size and aggregation of PGs were analyzed by electrophoresis, and the glycosaminoglycan composition was assessed by high-performance liquid chromatography. The PG concentration was proportional to the load distribution. However, when OA became histologically manifest, the PG concentration decreased by 50% (from a mean of 44 microg to 22 microg per mg fresh tissue) and the collagen level decreased by 40% (from a mean of 17 microg to 10 microg per mg fresh tissue), while the proportion of water increased by 13% (from a mean of 710 mg to 800 mg per mg fresh tissue). Unmineralized cartilage can, within physiologic load limits, respond to increased mechanical demands by increasing the PG and collagen concentrations. Beyond a certain limit, however, the cartilage can no longer compensate for further increases in stress, which results in cartilage degeneration and losses of matrix constituents. These losses seemed to appear earlier in the disease process than has been described in previous animal models of secondary OA.

Anin vivo investigation of the effect of anthraquinones on the turnover of aggrecans in spontaneous osteoarthritis in the guinea pig

We present details of a method which allows for the determination of chondroitin sulphate turnover in vivo using the guinea pig. Such methods have been utilised to examine the effects of diacetyl rhein, a compound with purported anti-osteoarthritic activity, and several related anthraquinone analogues on the turnover of chondroitin. Since the guinea pig develops spontaneous osteoarthritis, this may give useful information on the potential for such compounds to inhibit the progression of osteoarthritis. The results show that several of the anthraquinones are capable of reducing the turnover of chondroitin 4- but not 6-sulphate. This may indicate potential mechanisms for the breakdown of guinea pig cartilage aggrecans. We propose that these techniques could be useful for the screening of chemical agents with useful activity against osteoarthritis.

Proteoglycan structure and metabolism in normal and osteoarthritic cartilage of guinea pigs.

Sulfated proteoglycan structure and metabolism in osteoarthritic articular cartilage from the hind-limb joints of guinea pigs in which the disease had been surgically induced were evaluated and compared with those from nonoperated or sham-operated controls. In these studies, conducted on animals receiving high and low dietary levels of ascorbic acid, the effect of vitamin C on these variables was also examined. Sulfated proteoglycan biosynthesis per unit of DNA as reflected by in vivo uptake of 35S-sulfate was stimulated both by arthritic development and high dietary vitamin C. Under these conditions, the specific activities, 35S-cpm/micrograms uronic acid, of the proteoglycans were elevated. Neither arthritic development nor variation in dietary levels of vitamin C altered the structure of the proteoglycans. Two species of proteoglycans that exhibited the same electrophoretic mobilities in agarose/acrylamide gels, the same elution profiles on high performance liquid chromatography,, and the same galactosamine/glucosamine ratios were found in all normal and osteoarthritic cartilage specimens tested. The specific activities of these two specimens tested. The specific activities of these two species were dissimilar. In all samples, the larger of the two species had a higher specific activity.

Aging changes in ultrastructure and enzymatic activity of articular cartilage of guinea pigs.

Aging changes in ultrastructure and enzymatic activity of articular cartilage of guinea pigs.

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage.

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.

Effect of Manganese Deficiency on the Acid Mucopolysaccharides in Cartilage of Guinea Pigs

Skeletal abnormalities observed in the manganese-deficient guinea pig at birth were studied from the standpoint of defects in biosynthesis of the organic matrix of cartilage during fetal development. When the adult female was provided with less than 3 ppm of Mn throughout gestation there was a significant reduction in the concentration of all acid mucopolysaccharides (AMPS) tested in rib and epiphyseal cartilage. The mixture of chondroitin sulfates which makes up the major constituent of the total AMPS present was significantly reduced, with chondroitin 4-sulfate and chondroitin 6-sulfate being affected about equally in epiphyseal cartilage. Chondroitin 6-sulfate was significantly reduced in rib cartilage. Hyaluronic acid and heparin were likewise significantly lowerd in the case of manganese-deficient animals at birth. Skeletal defects associated with manganese deficiency are believed to be brought about by defective metabolism of cartilage matrix constituents.

Uptake of S35 in the costal cartilage of the guinea pig under the influence of different glucocorticoids

The uptake of35S, in the chondroitin sulfate of the costal cartilage of guinea pigs, is inhibited by cortisone, methyl-prednisolon, triamcinolon, and dexamethason. No inhibition could be seen under the influence of the natural adrenal hormone hydrocortisone and its nearest relation, prednisolon.

An experimental test of the protective action of glycerol on frozen cartilage

Summary Guinea-pig cartilage, with or without perichondrium, pre-treated with 15 and 30 per cent. glycerol for half, one, and two hours is non-viable after one month's storage at − 79° C.

Influence of vitamin C on the utilization of sulphate labelled with sulphur-35 in the synthesis of chondroitin sulphate of the costal cartilage of the guinea pig.

IT has been reported that sulphate when administered to the rat is retained by cartilage, bone and bone marrow1, and it is now further established that the sulphate is mainly incorporated into the chondroitin sulphate of the cartilage2–4. Since the utilization of the sulphate administered in the formation of chondroitin sulphate lends itself to quantitative determinations, this was used by us for studying the influence of vitamin C on the formation of this sulphuric ester in the costal cartilage of the guinea pig. The evidence so far obtained by employing the histological techniques is not conclusive. Thus, Hojer5, who was the first to observe the atrophy of collagen in the cartilage in scurvy, was not able to find any difference from the normal in regard to chondroitin sulphate by staining the sections with methylene blue. Contrary to the above observation, Meyer6, using basic metachromatic dyes, did not find any chondroitin sulphate in the cartilage of scorbutic guinea pigs.

Ossification of Auricles of External Ears Associated with Acromegaly

In 1931, J. Erdheim (4) first showed that the cartilage in acromegaly undergoes changes of both a proliferative and degenerative nature. The examples cited in his study are the cartilages of the ribs and vertebrae. The pathological process consisted of an increase in cartilaginous cells and intercellular substance, leading to a distention of the transitional zone between bone and cartilage. Proliferation was followed by degeneration with liquefaction and formation of vacuoles which were filled with deficient cartilaginous tissue. Later, ossification took place with or without previous calcification. According to Erdheim, this process of calcification and ossification may take place in the ribs and intervertebral disks in acromegaly. Another localization of these proliferative and degenerative changes in the cartilage of acromegalics was observed by H. v. Meyenburg (5), namely, the trachea and larynx. To his knowledge, his was the first such report. Experimental evidence of the action of anterior pituitary extract of cattle upon the articular cartilage of guinea-pigs was published by Silberberg in 1936 (6). He produced what he called an “acromegalic arthropathy.” Hypertrophy and hyperplasia started in the transitional zone of the cartilage, and areas of liquefaction subsequently developed. This was at times followed by calcification. When calcification did not occur, the areas of liquefaction broke down and ulcerated. To the above known localizations of calcification and ossification occurring in acromegaly, we wish to add a report of similar involvement of the auricles of the external ear in a long-standing case. Case Report B. R., a 63-year-old textile dealer, first exhibited symptoms of acromegaly thirty years ago, when he noticed that his hands were becoming gradually larger and his facial features distorted. He consulted Dr. Harvey Cushing, and a definite diagnosis of acromegaly was made. Surgery was suggested but refused. The patient subsequently received a course of radiation therapy to the pituitary gland at the Post Graduate Hospital in New York. He was then able to continue with his work for the following fifteen years, although symptoms became more pronounced. At the present time physical examination reveals a typical acromegalic facies, kyphosis, and spade-like hands and feet. General motor weakness is a prominent symptom; the skin is pale, and hair is absent from the chest, abdomen, and axilla. The voice is slow, indistinct, and high-pitched. The supra-orbital ridges are prominent; the eyebrows are almost absent and the palpebral fissures are narrow and slit-like. Examination of the eyes reveals bilateral cataracts. The lips are thick and bulging, and the mouth and tongue are enormously enlarged. The ears are likewise enlarged, displaced upward and backward, and are rigid. The chest is barrel-shaped, and there is a marked kyphosis. The angle of Ludwig measures about 90 degrees. The heart, lungs, and abdomen appear normal.