Hydroxyproline Analysis Research Articles

Tissue-Engineered Composites of Anulus Fibrosus and Nucleus Pulposus for Intervertebral Disc Replacement

By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Quantitation of hydroxyproline in bone by gas chromatography–mass spectrometry

A validated gas chromatography (GC)–mass spectrometric (MS) method for the analysis of hydroxyproline in rat femur is reported. Hydroxyproline in bone hydrolysates was extracted with an anion exchange resin and the N(O)- tert-butyldimethylsilyl derivatives analyzed by GC–MS. The hydroxyproline concentration was estimated relative to pipecolic acid, 3,4-dehydroproline and n-tetracosane as internal standards. The mass-to-charge ratios ( m/ z) for the ions used for quantitation by single ion monitoring were 314 m/ z for hydroxyproline, 198 m/ z for pipecolic acid, 256 m/ z for dehydroproline and 57 m/ z for n-tetracosane. A coefficient of variation of 5.8% was achieved and the limit of detection was calculated to be 0.233 μmol/l bone hydrolysate.

Intra-articular hyaluronate in experimental rabbit osteoarthritis can prevent changes in cartilage proteoglycan content

Objective: The purpose of this study was to determine the effects of intra-articular injections of high molecular weight (2000 kDa) sodium hyaluronate (HA) on the progression of articular cartilage degeneration in a rabbit partial medial meniscectomy model of osteoarthritis. Design: Six experimental groups included normal, sham operated, and operated and injected animals, the latter injected once-weekly (for two weeks or twelve weeks, beginning four weeks after surgery) with either 1% (w/v) HA or phosphate buffered saline (PBS). Following assessment of gross morphology, serial adjacent blocks of full-depth articular cartilage were prepared from the tibial condyle for analysis of total water, hydroxyproline, DNA and proteoglycan (uronic acid) content, as well as the ratio of galactosamine to glucosamine. Samples were sub-divided into inner (medial) and outer (lateral) regions. Results: No morphological differences were recognized between joints injected with PBS and those receiving HA. When analysed biochemically, there were no significant differences in hydration, hydroxyproline or DNA content between the experimental groups. In contrast, HA injection did affect changes in proteoglycan content. Expressed per tissue dry weight, uronic acid content in the operated group injected with PBS for two weeks was lower than normal ( P<0.02), a result not seen in the corresponding HA injected group. After 12 weeks of PBS injections, uronic acid content (per dry weight) was higher than normal ( P<0.01), an effect again not observed in the corresponding HA injected group. Results for the galactosamine: glucosamine ratio showed a reduction after 12 weeks of injections, but no differences between PBS and HA injected groups. Conclusions: Once-weekly, intra-articular injection of high molecular weight HA can prevent changes in proteoglycan content in tibial condylar articular cartilage, compared to PBS injected controls, in the rabbit partial meniscectomy model of osteoarthritis.

Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase.

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung and vascular tissue. Localization of EC-SOD to the matrix of the lung may protect against oxidative tissue damage that leads to pulmonary fibrosis. This study directly examines the protective role of EC-SOD in a bleomycin model of pulmonary fibrosis and the effect of this enzyme on oxidative protein fragmentation. Mice null for ec-sod display a marked increase in lung inflammation at 14 d post-bleomycin treatment as compared to their wild-type counterparts. Hydroxyproline analysis determined that both wild-type and ec-sod null mice display a marked increase in interstitial fibrosis at 14 d post-treatment, and the severity of fibrosis is significantly increased in ec-sod null mice compared to wild-type mice. To determine if the lack of EC-SOD promotes bleomycin-induced oxidative protein modification, 2-pyrrolidone content (as a measure of oxidative protein fragmentation at proline residues) was assessed in lung tissue from treated mice. 2-Pyrrolidone levels in the lung hydrolysates from ec-sod null mice were increased at both 7 and 14 d post-bleomycin treatment as compared to wild-type mice, indicating EC-SOD can inhibit oxidative fragmentation of proteins in this specific model of oxidative stress.

SPARC-Null Mice Display Abnormalities in the Dermis Characterized by Decreased Collagen Fibril Diameter and Reduced Tensile Strength

Although collagen and elastic fibers are among the major structural constituents responsible for the mechanical properties of skin, proteins that associate with these components are also important for directing formation and maintaining the stability of these fibers. We present evidence that SPARC (secreted protein acidic and rich in cysteine) contributes to collagen fibril formation in the dermis. The skin of SPARC-null adult mice had approximately half the tensile strength as that of wild-type skin. Moreover, the collagen content of SPARC-null skin, as measured by hydroxyproline analysis, was substantially reduced in adult mice. At 2 weeks of age, no differences in collagen content were observed; within 2 months, however, the dermis of SPARC-null mice displayed a reduced collagen content that persisted through adulthood until approximately 20 months, when collagen levels of SPARC-null skin approximated those of wild-type controls. The collagen fibrils present in SPARC-null skin were smaller and more uniform in diameter, in comparison with those of wild-type skin. At 5 months of age, the average fibril diameter in SPARC-null versus wild-type skin was 60.2 nm versus 87.9 nm, respectively. Extraction of soluble dermal collagen revealed a relative increase in collagen VI, accompanied by a decrease in collagen I, in SPARC-null mice. A reduction in the relative amounts of higher-molecular weight collagen complexes was also observed in extracts of dermis from SPARC-null animals. Thus the absence of SPARC compromises the mechanical properties of the dermis, an effect that we attribute, at least in part, to the changes in the structure and composition of its collagenous extracellular matrix.

Cyclosporin A Upmodulates Bleomycin-Induced Pulmonary Fibrosis in BALB/c Mice

Background: Intratracheal instillation of bleomycin (Bleo) into rodents serves as a model for human lung fibrosis. Various mouse strains respond differently to Bleo, and BALB/c mice are relatively resistant. Objective: Since T lymphocytes have been shown to play a major role in this model, the effect of the immunomodulator cyclosporin A (CyA) on lung fibrosis was studied in Bleo-‘resistant’ BALB/c mice. Methods: Pulmonary fibrosis was induced by a single intratracheal (IT) instillation of Bleo. One of the four following treatments was given to one of four groups of female BALB/c mice: (1) Bleo-CyA: IT Bleo and daily intraperitoneal (IP) injections of CyA 100 mg/day starting 1 day before IT instillation of Bleo; (2) Bleo-Sal: IT Bleo and IP injections of saline; (3) Sal-CyA: IT saline and IP CyA 100 mg/kg; (4) Sal-Sal: IT and IP saline. The animals were killed on day 14. Fibrosis was evaluated by analysis of hydroxyproline and by quantitative image analysis of the fibrosis fraction. Ex vivo IFN-γ, IL-4, IL-13 and IL-5 secretion by peribronchial lymphatic tissue lymphocytes was measured. Results: Pretreatment with CyA upmodulated Bleo-induced lung fibrosis in the Bleo-‘resistant’ BALB/c mice; hydroxyproline was higher in Bleo-CyA compared to Bleo-Sal animals and both hydroxyproline and fibrosis fraction measurements were higher in Bleo-CyA compared to Sal-Sal. Cytokine secretion by lymphocytes demonstrated increased IFN-γ/IL-4 and IFN-γ/IL-13 mean secretion ratios in the Bleo-CyA animals compared to the Bleo-Sal animals. Conclusions: These results indicate that CyA upmodulates Bleo-induced lung fibrosis in Bleo-‘resistant’ BALB/c mice by allowing the emergence of a Th1 inflammatory response.

Sheep lumbar intervertebral discs as models for human discs

Objective. To determine the water content, collagen content and collagen orientation angle in different regions of sheep lumbar discs. Design. A laboratory study of sheep discs obtained from an abattoir. Methods. A total of 21 sheep lumbar discs were obtained from three lumbar spines. Water content was determined by oven drying (60 °C) to constant mass. Collagen content was determined by hydroxyproline analysis. Fibre orientation angles were determined by X-ray diffraction. Results. Water content increased from 74% of total tissue mass in the outer annulus, to 82% in the inner annulus, to 86% in the nucleus. Collagen content decreased from 30% of total tissue mass in the outer region to 20% in the inner region of the anterior and lateral annulus; it was 16% in the posterior annulus. The orientation angle of the collagen fibres decreased from 59° in the outer region to 56° in the inner region of the anterior and lateral annulus; it was 51° in the posterior annulus. Conclusions. Sheep lumbar intervertebral discs provide a reasonable model for human lumbar intervertebral discs. Relevance Sheep lumbar discs have been used to investigate the effects of removing and replacing the nucleus. These studies indicate that removal of nucleus may lead to further disc degeneration and indicate the material properties required for an implant material. The relevance of these previous studies is increased if human and sheep lumbar discs have a similar composition and structure.

SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.

Silicon deprivation decreases collagen formation in wounds and bone, and ornithine transaminase enzyme activity in liver.

We have shown that silicon (Si) deprivation decreases the collagen concentration in bone of 9-wk-old rats. Finding that Si deprivation also affects collagen at different stages in bone development, collagen-forming enzymes, or collagen deposition in other tissues would have implications that Si is important for both wound healing and bone formation. Therefore, 42 rats in experiment 1 and 24 rats in experiment 2 were fed a basal diet containing 2 or 2.6 microg Si/g, respectively, based on ground corn and casein, and supplemented with either 0 or 10 microg Si/g as sodium metasilicate. At 3 wk, the femur was removed from 18 of the 42 rats in experiment 1 for hydroxyproline analysis. A polyvinyl sponge was implanted beneath the skin of the upper back of each of the 24 remaining rats. Sixteen hours before termination and 2 wk after the sponge had been implanted, each rat was given an oral dose of 14C-proline (1.8 microCi/100 g body wt). The total amount of hydroxyproline was significantly lower in the tibia and sponges taken from Si-deficient animals than Si-supplemented rats. The disintegrations per minute of 14C-proline were significantly higher in sponge extracts from Si- deficient rats than Si-supplemented rats. Additional evidence of aberrations in proline metabolism with Si deprivation was that liver ornithine aminotransferase was significantly decreased in Si-deprived animals in experiment 2. Findings of an increased accumulation of 14C-proline and decreased total hydroxyproline in implanted sponges and decreased activity of a key enzyme in proline synthesis (liver ornithine aminotransferase) in Si-deprived animals indicates an aberration in the formation of collagen from proline in sites other than bone that is corrected by Si. This suggests that Si is a nutrient of concern in wound healing as well as bone formation.

Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specific manner

It was hypothesized that cartilage macro-molecular characteristics are influenced by exercise intensity and by location within a joint.Objective To determine the macromolecular characteristics of carpal articular cartilage at common and uncommon sites of pathology in horses undergoing high or low intensity exercise, and to compare this composition between exercise groups.Design Twelve horses (19.3±0.9 years) were assigned to exercise groups. Each group underwent 19 weeks high-intensity treadmill training (N=6) or low-intensity exercise (N=6). Dorsal and palmar test sites were identified on radial, intermediate and third carpal articular surfaces after euthanasia. Cartilage was collected from each site, freeze-dried and assessed for water content. Hydroxyproline, glycosaminoglycan and DNA analyses were performed on cartilage from each test site. Adjacent cartilage underwent histological preparation and assessment for chondrocyte numerical density at each site and proteoglycan distribution through the depth of cartilage.Results Dorsal cartilage had a higher collagen content, DNA content, and chondrocyte numerical density, but lower glycosaminoglycan content than palmar cartilage. Cartilage from horses undergoing high-intensity training had a significantly higher glycosaminoglycan content than cartilage from horses undergoing low-intensity exercise, with maximal difference being observed in cartilage from dorsal radial and dorsal intermediate carpal articular surfaces. Overall no effect of exercise on collagen was observed, but at sites predisposed to clinical lesions cartilage from horses undergoing high-intensity training contained significantly less collagen than from horses undergoing low-intensity exercise. Distribution of proteoglycan was non-uniform in 52% of the sections examined, with superficial loss of toluidine blue staining primarily at dorsal sites and in the high-intensity exercise group.Conclusions These results indicate that topographical and exercise related differences exist in carpal cartilage composition, and that the effect of exercise on overall composition and distribution within the cartilage was maximal at sites predisposed to clinical lesions. These findings could indicate that the combined effect of exercise and local load variations within a joint may lead to a risk of exceeding the physiologic threshold at high load sites that are predisposed to clinical injury.

Development of pulmonary fibrosis in fibrinogen-deficient mice.

Bleomycin is an antineoplastic drug commonly used for the treatment of many carcinomas and lymphomas. Its toxic side effect on lung tissue is a major limitation to its use, with approximately 3-5% of patients affected. Although the number of affected patients is small, the damage incurred by bleomycin in these patients is often irreversible and, at times, fatal. A number of therapies have been shown to be effective in animal studies to minimize damage, but to date no "magic bullet" has been identified. Many proteins of the fibrinolytic system have been implicated as playing a role in the progression of the disease, one of which is fibrinogen (Fg) acting in the context of a fibroproliferative agent. Its presence correlates with an upregulation of plasminogen activator inhibitor-1 and tissue factor in alveolar cells surrounding the lesion area. It is believed that Fg participates in the activation and migration of fibroblasts and provides a scaffold, in the form of fibrin, for cell migration following induction of acute lung injury. To further understand the mechanism of injury following bleomycin treatment and the possible role of fibrinogen therein, mice have been generated with a targeted deletion of the gamma-chain of Fg, which resulted in the absence of detectable circulating Fg. The offsprings of Fg heterozygous mice (FG+/-) mice follow Mendelian distributions indicating no embryonic lethality with this deletion. Approximately one-half of the Fg-deficient (FG-/-) neonates exhibited bleeding episodes, approximately one-half of which were fatal. For the pulmonary fibrosis study, FG-/- mice and wildtype littermates were administered a bleomycin solution intratracheally and the disease was allowed to progress for two weeks. The mice were then sacrificed, the left lung was excised for hydroxyproline analysis, the right lung was processed for histologic profiling. Examination of trichrome stained sections, surprisingly, revealed no qualitative difference between wildtype and FG-/- animals. The extent and pattern of the deposition of collagen were also similar. These results were quantitatively confirmed by hydroxyproline analysis, which revealed equivalent increases in collagen content between wildtype and FG-/- animals when compared to appropriate saline controls. Analysis of the early acute inflammatory stage of the disease showed a difference in the neutrophil population between days three and five of the disease. These studies suggest that, although fibrinogen is not required for collagen deposition at the later stage of the disease, it may play a role in the early acute inflammation stage.

PG490-88, a Derivative of Triptolide, Blocks Bleomycin-Induced Lung Fibrosis

In this study we evaluate the antifibrotic properties of PG-490-88, a water-soluble derivative of triptolide. Triptolide is an oxygenated diterpene that is derived from a traditional Chinese herb that has potent immunosuppressive and antitumor activity. We used the intratracheal bleomycin mouse model and found that PG490-88 inhibits fibrosis in the bleomycin group when given the same day or 5 days after bleomycin. PG490-88 also markedly reduced the number of myofibroblasts in the bleomycin treatment group. An enzyme-linked immunosorbent assay of transforming growth factor (TGF)-beta in the bronchoalveolar lavage fluid showed a significant decrease in TGF-beta in the PG490-88-treated groups compared to the bleomycin-treated group. Additionally, triptolide blocked bleomycin-induced increase in TGF-beta mRNA in cultured normal human lung fibroblasts. The efficacy of PG490-88 when administered late after bleomycin installation suggests a potential role in the treatment of idiopathic pulmonary fibrosis.

Growth, slaughter and intra-muscular collagen characteristics in Garganica kids

Growth performance, ossification of metacarpal growth plate, and intra-muscular collagen characteristics in three muscles of 10 intact male Garganica kids were studied. Each week, starting from the 47th day of age to slaughter (75th day of age), kids were weighed and blood samples taken for plasma non-protein hydroxyproline analysis. At slaughter, metacarpal, metatarsal bones and metacarpal growth plate thickness were measured. The semitendinosus (ST), semimembranosus (SM), and rectus medialis (RM) muscle samples were analysed for collagen concentration and hydroxylysyl pyridinoline (HP) cross-links. ST muscle had a collagen amount higher than SM and RM ( P<0.01), and RM muscle had the highest ( P<0.01) HP concentration. Tenderness index was the highest ( P<0.01) in RM muscle. Animal growth rates slowed, starting from 61st day of age ( P<0.01); this trend coincided with a marked reduction in concentration of plasma non-protein hydroxyproline ( P<0.01). In addition, plasma non-protein hydroxyproline concentration was positively correlated ( r=0.61, P=0.001) with growth rate of animals, suggesting that collagen turnover decreases during a slow growth phase. Based on the metacarpal growth plate width, the rate of bone ossification appears to be slower in goats than sheep.

Changes in the weight, collagen concentration and content of the uterus and cervix of the ewe during pregnancy

The dry and wet weights of the uterus (caruncular and intercaruncular areas) and cervix were measured in non-pregnant (n = 5) and pregnant (n = 25) ewes post mortem; for the latter, five were obtained for each of the 5 months of gestation. The total collagen tissue content was measured in both areas of the uterus and cervix by hydroxyproline analysis and image analysis of Haematoxylin-Van Gieson stained tissue sections. Both wet and dry uterine weights increased significantly with gestational age (P < 0·001). The water content of uterine and cervical tissue remained constant, at between 83 per cent to 85 per cent and 76 per cent to 80 per cent, respectively. There was a close correlation between the two methods used to determine the collagen content (r = 0·96, P < 0·001), and between the increasing weight of the uterus during pregnancy and the total collagen content of tissues (r = 0·97, P < 0·001). At all stages, the total collagen content of the cervix [mean (SEM) 96·2 (5·4) mg g–1] was significantly greater (P < 0·001) than that of the caruncular mean [mean (SEM) 24·3 (1·4) mg g–1], and the intercaruncular areas [mean (SEM) 29·0 (1·0) mg g–1]. The changes in uterine and cervical weights and collagen content of the tissues were similar to those reported in other related species.

Wound healing in ovariectomized rats: effects of chemically modified tetracycline (CMT-8) and estrogen on matrix metalloproteinases -8, -13 and type I collagen expression.

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen; and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

Increased numbers of microchimeric cells of fetal origin are associated with dermal fibrosis in mice following injection of vinyl chloride.

To develop a murine model for use in examining the role of microchimeric cells and certain chemical exposures in the pathogenesis of systemic sclerosis (SSc). Female BALB/cJ retired breeder mice were bled before and after vinyl chloride injection. The DNA from their white blood cells was obtained, and the number of microchimeric cell equivalents was determined by quantitative polymerase chain reaction using DNA primers specific for the H-2Kb gene, a sequence not found in BALB/cJ mice. Skin was obtained at autopsy, embedded in paraffin, sectioned, and stained with Masson's trichrome. Hydroxyproline analyses were performed on 4-mm skin biopsy samples. Microchimeric cells were identified and quantitated before and after 20 daily intraperitoneal injections of vinyl chloride. The number of microchimeric cells in the peripheral blood increased an average of 48-fold after treatment with vinyl chloride. Histologic examination of the skin of these same mice (which had an increased number of microchimeric cells) showed inflammation, with abundant fibroblasts and a heavy mononuclear infiltration in the dermis. The collagen fibers appeared densely packed and disorganized. Histologic examination of the skin of untreated retired breeder mice and treated virgin mice appeared normal. Quantitative assays to determine the collagen content of skin biopsy samples obtained from treated microchimeric mice compared with nontreated microchimeric or with treated nonmicrochimeric mice showed a 2-3-fold increase in collagen content in the treated microchimeric mice. Extraordinary splenomegaly was present in the vinyl chloride-treated microchimeric mice, accompanied by cellular infiltration and fibrosis. The results suggest that vinyl chloride injections into BALB/cJ retired breeder mice lead to activation of microchimeric cells, which causes the cells to divide and multiply. The correlation between the 48-fold increase in microchimeric cells and the appearance of dermal inflammation and fibrosis similar to that of graft-versus-host disease suggests that activated microchimeric cells may be a necessary factor in the pathogenesis of autoimmune diseases such as SSc.

Type V collagen is increased during rabbit medial collateral ligament healing.

To understand the reparative process of medial collateral ligament (MCL), fibrillar collagen and their relative ratios in healing MCL with anterior cruciate ligament (ACL) reconstruction were analyzed. Skeletally mature New Zealand white rabbits were subjected to a mop-end tear of MCL without repair with ACL reconstruction. Rabbits were killed 6 and 52 weeks after injury. Ligamentous tissues from the injury site and sham controls were soaked in 0.5 M acetic acid for 24 h, minced, and treated with pepsin to solubilize collagen. Pepsin solubilized about 80% of the total collagen as determined by hydroxyproline analysis of the pepsin residues. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the solubilized collagen revealed presence of fibrillar collagen types I, III, and V. Densitometric scanning of the protein bands corresponding to types I, III, and V collagen indicated that in sham controls types III and V collagen represented about 8% and 12%, respectively, of the type I collagen whereas the healed MCL ligaments at 6 weeks showed significant increase in type III and V collagen to about 19% and 24%, respectively. By 52 weeks type III collagen in the healed MCL had returned to that of sham controls while type V collagen remained elevated at approximately 18%. These data suggest that presence of type V collagen in high concentration in healing ligaments may have an influence on collagen fibril diameters seen in healed ligament and should be included in the analysis when evaluating ligament healing.

Hepatic Ascorbic Acid Saturation Is the Most Stringent Response Criterion for Determining the Vitamin C Requirement of Juvenile European Sea Bass (Dicentrarchus labrax)

Our main objective was to verify whether the dietary ascorbic acid (AA) requirement of juvenile European sea bass (Dicentrarchus labrax) varies as a function of different physiological needs. Practical diets with eight (0, 5, 10, 20, 40, 80, 160, 320 mg AA/kg diet) levels of ascorbic acid polyphosphate were fed to sea bass (mean weight: 0.7 g) for 15 wk. At the beginning and at the end of the feeding trial, tissues were sampled for vitamin C and hydroxyproline (HyPro) analysis. Dose-dependent responses of skin and whole body HyPro concentrations and hepatic AA concentration to dietary vitamin C levels were observed. Skin and whole body HyPro concentrations were low in sea bass fed AA-deficient diet, 217 and 15 nmol/g tissue, respectively. HyPro levels increased with increasing dietary levels, reaching plateaus of 297 and 45 nmol/g tissue in the skin and whole body at dietary vitamin C levels of at least 5 and 31 mg AA/kg. Hepatic AA level increased with increasing dietary levels, reaching a plateau of 474 pmol/g tissue in juveniles fed at least 121 mg of AA/kg. We concluded that hepatic AA saturation is the most stringent response criterion for determination of the vitamin C requirement in juvenile European sea bass.

Left and right ventricular collagen type I/III ratios and remodeling post-myocardial infarction

Background: Types I and III collagen have different physical properties, and an increase of type I/III ratio can have a deleterious impact on myocardial compliance and left and right ventricular diastolic function. Post-myocardial infarction, these changes in collagen types may be relevant to the remodeling process and the development of heart failure. Methods and Results: In the rat coronary ligation heart failure model, we studied the time course of changes in types I and III and total collagen levels over 10 weeks postinfarction. Collagen types were separately quantified in the left (LV) and right ventricles (RV) by computerized morphometry and standard immunohistochemistry techniques, and also by hydroxyproline analysis, and these were correlated with hemodynamic changes. Compared with sham-operated rats, total collagen level increased 2.5- to 2.9-fold and 1.7- to 2.9-fold in the noninfarcted areas (NIAs) of the LV and RV, respectively, over the 10-week period and showed a good relation with changes in hydroxyproline content ( r 2 = 0.62; P < .0001). In the NIAs of both the LV and RV, type III collagen level showed a transient twofold increase at 2 weeks, which declined to normal at 4 weeks. Type I collagen level increased twofold at 4 weeks in the NIA of the LV and remained elevated at 10 weeks. In the RV, type I collagen level increased 2.7-fold to a peak at 4 weeks and declined gradually to 1.7 times baseline at 10 weeks. The patterns of change in type I collagen level in the RV correlated with the changes in LV end-diastolic pressure ( r = 0.73; P < .0001) and RV weight to body weight ratio ( r = 0.73; P < .0001). Conclusion: There is a relative greater increase of type I collagen level in the NIA and RV postinfarction, and this may lead to left and right ventricular dysfunction. Separate mechanisms might be involved in the induction of the different types of collagen deposition, with type I collagen levels apparently closely correlating with hemodynamic stress.

T cell independence of bleomycin-induced pulmonary fibrosis.

The role of T cells and cytokines in bleomycin (BLM)-induced fibrosis was evaluated in susceptible and resistant strains of normal and SCID mice. Histology and hydroxyproline analysis showed that BLM induced pulmonary fibrosis in C57BL/6 and (C57BL/6 x BALB/c)F1 mice, whereas BALB/c mice were resistant to the disease. To test whether lymphocytes were required for the induction of BLM-induced pulmonary fibrosis, SCID mice were injected intratracheally with BLM and evaluated for the development of pulmonary inflammation and fibrosis. Similar morphological changes and increases in hydroxyproline were observed in both C57BL/6 SCID and (C57BL/6 x CB.17)F1 SCID animals compared to those seen in wild-type C57BL/6 and (C57BL/6 x BALB/c)F1 mice. In contrast, CB.17 SCID mice, which are genetically similar to BALB/c mice, were resistant to disease induction. Analysis of the cellular infiltrate in BLM-treated C57Bl/6 SCID mice confirmed a lack of T cells in the lungs of SCID mice and demonstrated a pronounced accumulation of eosinophils in areas of developing pulmonary fibrosis. NK cells were significantly elevated in untreated SCID mice and did not increase further after BLM treatment. Analysis of selected cytokines 1 day after initiation of BLM-induced pulmonary fibrosis indicated that the levels of TNF-alpha and IFN-gamma appeared to segregate with fibrosis in both the SCID and wild-type mice. The data demonstrate that T cells are not required for the induction of fibrosis by BLM and suggest that responses by non-lymphoid cells may be sufficient for the induction of fibrosis.