Fourier-transform infrared study on effects of ageing, oestrogen level and altered dietary loading on rat mandibular condylar cartilage.

OBJECTIVE: Mandibular condylar cartilage (MCC) of the rat was examined with the Fourier-transform infrared (FITR) spectroscopic imaging to study the effects of ageing, oestrogen level and altered dietary loading on the structure of MCC. MATERIALS AND METHODS: The Sprague-Dawley rats (n = 96) aged 5 and 14 months were divided into 12 subgroups according to age, oestrogen status (ovariectomized [OVX], non-ovariectomized [non-OVX)]) and diet (hard, normal, soft). Specimens of the MCC were examined with FTIR spectroscopic imaging to quantify the distribution of collagens and proteoglycans. MCC was divided sagittally into three segments: anterior, most superior and posterior. From each segment, the collagen and proteoglycan contents at different depths of cartilage were statistically compared between the groups using an N-way analysis of variance (ANOVA). RESULTS: The amount of collagen content was significantly associated with old age in the deep layer of the anterior segment and in the middle layer of the posterior segment of MCC. In the deep layer of the most superior segment, the collagen content also increased with ageing. The amount of proteoglycan content increased significantly when dietary loading increased, and the oestrogen level decreased in the deep layer of the most superior segment of MCC. CONCLUSION: Ageing, oestrogen level and altered dietary loading have a significant effect on the location and content of collagens and proteoglycans of rat MCC. Ageing significantly increased the amount of collagen content in the superior and posterior segments, being highest in the older soft-diet rats. Decreased oestrogen levels and increased dietary loading increased the amount of proteoglycan content.

No adverse effects on periarticular tissue by intra-articular vitamin D analogue calcipotriol in a reduced-dose zymosan-induced arthritis model in rats.

Calcipotriol, a vitamin D analogue, is an antiproliferative and anti-inflammatory drug currently used in psoriasis. Here, our aim was to analyse the safety of calcipotriol for cartilage and bone in alleviated-dose (0.1 mg instead of usual ≥1mg dose) zymosan-induced arthritis in rats. Theoretically, high doses of vitamin D or analogues could have detrimental effects on bone or cartilage. The rats were divided into four groups: vehicle (n = 9), dexamethasone 0.1 mg/kg (n = 9), calcipotriol 0.1 mg/kg (n = 8) and negative control (n = 10) with no injections. Arthritic rats were given phosphate-buffered saline (PBS) injections to left knees as a control. After euthanasia on day 8, all knees were imaged with micro-computed tomography for surface lesions and decalcified for histological analyses. Contrary to our expectations, no significant changes could be observed in the tomography data and histological scores among the three treatment groups or between the vehicle-treated and non-arthritic group. Calcipotriol did not cause adverse effects on cartilage or subchondral bone within a week, suggesting that it could be safely used in local treatment of arthritis. The alleviated model caused synovitis with local and systemic inflammatory response without cartilage erosions, which might be useful in studying self-limiting synovitis where cartilage or bone effects are not of primary interest.

Central nervous system distribution of buprenorphine in pregnant sheep, fetuses and newborn lambs after continuous transdermal and single subcutaneous extended-release dosing.

Buprenorphine is used during pregnancy for the treatment of opioid use disorder. Limited data exist on the central nervous system (CNS) permeation and distribution, and on the fetal exposure to buprenorphine. The aim of our study was to determine the extent of buprenorphine distribution to CNS in the pregnant sheep, and their fetus at steady-state, and their newborn lambs postdelivery, using three different dosing regimens. Twenty-eight pregnant ewes in late gestation received buprenorphine via 7-day transdermal patch releasing buprenorphine 20 µg/h (n=9) or 40 µg/h (n=11), or an extended-release 8 mg/week subcutaneous injection (n=8). Plasma, cerebrospinal fluid, and CNS tissue samples were collected at steady-state from ewes and fetuses, and from lambs 0.33 - 45 hours after delivery. High accumulation of buprenorphine was observed in all CNS tissues. The median CNS/plasma concentration -ratios of buprenorphine in different CNS areas ranged between 13 and 50 in the ewes, and between 26 and 198 in the fetuses. In the ewes the CNS/plasma -ratios were similar after the three dosing regimens, but higher in the fetuses in the 40 µg/h dosing group, medians 65 - 122, than in the 20 µg/h group, medians 26 - 54. The subcutaneous injection (theoretical release rate 47.6 µg/h) produced higher concentrations than observed after 40 µg/h transdermal patch dosing. The median fetal/maternal concentration -ratios in different dosing groups ranged between 0.21 and 0.54 in plasma, and between 0.38 and 1.3 in CNS tissues, respectively, with the highest ratios observed in the spinal cord. Buprenorphine concentrations in the cerebrospinal fluid were 8 - 13 % of the concurrent plasma concentration in the ewes and 28 % in the fetuses. Buprenorphine was quantifiable in the newborn lambs' plasma and CNS tissues two days postdelivery. Norbuprenorphine was analyzed from all plasma, cerebrospinal fluid, and CNS tissue samples but was nondetectable or below the LLOQ in most. The current study demonstrates that buprenorphine accumulates into CNS tissues at much higher concentrations than in plasma in pregnant sheep, fetuses, and their newborn lambs even 45 hours after delivery.

A single intra-articular dose of vitamin D analog calcipotriol alleviates synovitis without adverse effects in rats.

1,25-dihydroxyvitamin-D3 and its derivatives have shown anti-arthritic and chondroprotective effects in experimental animal models with prophylactic dosing. The purpose of this preliminary study was to test the efficacy and safety of calcipotriol, vitamin D analog, as a treatment for a fully-developed knee arthritis in Zymosan-induced arthritis (ZIA) model. Forty 5-month-old male Sprague-Dawley rats were randomized into three arthritis groups and a non-arthritic control group with no injections (10 rats/group). A day after Zymosan (0.1 mg) had been administrated into the right knee joints, the same knees were injected with calcipotriol (0.1 mg/kg), dexamethasone (0.1 mg/kg) or vehicle in a 100 µl volume. The left control knees were injected with saline (PBS) on two consecutive days. All injections, blood sampling and measurements were performed under general anesthesia on days 0, 1, 3 and 8. Internal organs and knees were harvested on day 8 and the histology of the whole knees was assessed blinded. Joints treated with calcipotriol showed a milder histological synovitis than those treated with vehicle (p = 0.041), but there was no statistically significant difference between the dexamethasone and vehicle groups. The clinical severity of arthritis did not differ between the arthritis groups measured by body temperature, swelling of the knee, thermal imaging, clinical scoring or cytokine levels on days 1, 3 and 8. Weight loss was bigger in rats treated with dexamethasone, propably due to loss of appetite,compared to other arthritis groups on days 2-3 (p<0.05). Study drugs did not influence serum calcium ion and glucose levels. Taken together, this preliminary study shows that a single intra-articular injection of calcipotriol reduces histological grade of synovitis a week after the local injection, but dexamethasone did not differ from the vehicle. Calcipotriol may have an early disease-modifying effect in the rat ZIA model without obvious side effects.

Effects of Estrogen Level, Dietary Loading, and Aging on Types I, II, and X Collagen Expression and Structure of Rat Mandibular Condylar Cartilage.

AIMS: To investigate how estrogen level, dietary loading, and aging affect cartilage structure and the expression of major collagens (types I, II, and X) in rat mandibular condylar cartilage (MCC). METHODS: A total of 96 outbred Sprague Dawley female rats were randomly divided into two groups by ovariectomy (OVX) at 7 weeks old. One week later, the rats in each group were further divided into three subgroups on the basis of food hardness: hard food (diet board), normal food (pellet), and soft food (powder). The rats were sacrificed at the age of 5 or 14 months. The thickness of the fibrous, proliferative, and chondroblastic layers of the mandibular condylar cartilage were measured after toluidine blue staining. Immunohistochemical analysis was performed to evaluate the expression levels of types I, II, and X collagen. A linear regression model was used to investigate the main factors affecting changes in thickness and collagen expression. RESULTS: The expression levels of types II and X collagen were decreased by ovarian estrogen deficiency and increased by dietary loading. Increased dietary loading was the main factor affecting an increase in thickness of the cartilage layers, while aging was the main factor affecting a decrease in thickness of the fibrous layer. A significant age-related increase was found in the expression of type I collagen. There was some degree of interaction between aging and dietary loading that affected the thickness of the chondroblastic layer and the expression of type X collagen. CONCLUSION: The physiologic level of estrogen plays a role in MCC development by promoting the expression of types II and X collagen. Dietary loading is essential to increase the expression of types II and X collagen, as well as the thickness of cellular layers, to maintain the integrity of the MCC. Aging seems to reduce the ability of the MCC to withstand occlusal loading.

MMP-3 and MMP-8 in rat mandibular condylar cartilage associated with dietary loading, estrogen level, and aging.

OBJECTIVES: The structure of the mandibular condylar cartilage (MCC) is regulated by dynamic and multifactorial processes. The aim of this study was to examine the effects of altered dietary loading, estrogen level, and aging on the structure of the condylar cartilage and the expressions of matrix metalloproteinase (MMP) -3 and MMP-8 of rat MCC. METHODS: In this study, Crl:CD (SD) female rats were randomly divided into 3 groups according to dietary hardness: hard diet (diet board), normal diet (pellet), and soft diet (powder). In each group, the rats were further divided into 2 subgroups by ovariectomy at the age of 7 weeks. The rats were sacrificed at 5- and 14-month-old. Histomorphometric analysis of the MCC thickness was performed after toluidine blue staining. Immunochemical staining was done for MMP-3 and MMP-8. A linear mixed model was used to assess the effects of dietary loading, estrogen level, and aging. RESULTS: Increased dietary loading was the main factor to increase the MMP-3 expression and the anterior and central thickness of the MCC. Lack of estrogen was the main factor associated with decreased MMP-8. Aging was associated with the thickness changes of the whole condylar cartilage and the reduced expression of MMP-8. CONCLUSION: The condylar cartilage structure and metabolism of the female rats are sensitive to dietary loading changes, estrogen level as well as aging. The proper balance of these factors seems to be essential for the maintenance of the condylar cartilage.

Pharmacokinetics of intra-articular vitamin D analogue calcipotriol in sheep and metabolism in human synovial and mesenchymal stromal cells.

Calcipotriol (MC903) is a side chain analogue of the biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Due to its anti-inflammatory and anti-proliferative effects on stromal cells, calcipotriol is a promising candidate for the local treatment of arthritis. In this preliminary work, we studied the pharmacokinetics and safety of calcipotriol after an IV (0.1 mg/kg given to one sheep) and intra-articular dose (0.054 mg/kg, 0.216 mg/kg and 0.560 mg/kg given to three sheep). The terminal half-life of calcipotriol was approximately 1 h after an IV dose. After intra-articular dosing, the systemic absorption was between 1 and 13% during the observed 24 h. Hypercalcemia or other clinical adverse effects did not occur in any animal during the study, and no macroscopic or microscopic alterations were seen in the synovium of the calcipotriol-injected knees compared to the vehicle knees. The in vitro metabolism of calcipotriol was analyzed with LC-MS from human synovial and mesenchymal stromal cell cultures. Both cell types were able to metabolize calcipotriol with MC1080 and MC1046 as the main metabolites. CYP24A1 transcripts were strongly induced by a 48-hour calcipotriol exposure in mesenchymal stromal cells, but not consistently in synovial stromal cells, as determined by RT-qPCR. Calcipotriol proved to be safe after a single intra-articular dose with applied concentrations, and it is metabolized by the cells of the joint. Slow dissolution of calcipotriol crystals in the joint can extend the pharmaceutical impact on the synovium, cartilage and subcortical bone.

Changes in Type I and Type II Collagen Expression in Rat Mandibular Condylar Cartilage Associated with Aging and Dietary Loading.

AIMS: To evaluate the usefulness of diet board feeding as a model for temporomandibular joint (TMJ) research, characterize dietary loading-related morphometric changes in the mandibular condylar cartilage of aging rats, and investigate changes in type I and type II collagen expression in different age, sex, and diet groups. METHODS: Material was collected from a study that examined the effects of 1-year and 2-year diet board feeding on rats. In diet board feeding, rats must gnaw wood to reach their food, leading to a higher masticatory workload. The material analyzed was comprised of 150 TMJ samples from 75 Hsd:Sprague Dawley rats grouped according to feeding method (diet board [experimental group] or ad libitum [control group]), sex, and experiment length (1 or 2 years). The rats were sacrificed at the age of 15 or 26 months (15-M rats or 26-M rats). From the TMJ samples, 5-µm-thick sections were cut parallel to the sagittal plane of the mandibular condyle. Histomorphometric analysis of the thickness of the condylar cartilage and the number of cartilage cells was performed after toluidine blue staining. Immunohistochemical staining included type I and type II collagen antigens. Differences in the thickness of the cellular layer and the number of cells in the condylar cartilage were analyzed by means of a repeated-measures analysis of variance (ANOVA) model, and differences in the type of collagen with a one-way random-effects ANOVA model. RESULTS: Condylar cartilage was significantly thicker in the 15-M diet board-fed rats than in the 15-M control rats and in the 26-M rats than in the 15-M rats. The number of cells was larger in the 26-M female rats than in the 26-M male rats. Type I collagen expression was significantly higher in the 15-M diet board-fed female rats than in the 15-M controls. Type II collagen showed increased expression in older rats compared to younger rats. CONCLUSION: Condylar cartilage is sensitive to the interplay between loading, aging, and sex of middle-aged and older rats. High loading of condylar cartilage increased the thickness of cartilage in younger rats.

Diurnal rhythms of blood glucose, serum ghrelin, faecal IgA and faecal corticosterone in rats subjected to restricted feeding using the diet board.

Laboratory rats are generally fed ad libitum, although this method is associated with obesity and an increased frequency of spontaneous tumours. It has been challenging looking for ways to limit feed consumption in group-housed rats without any setbacks to animal welfare and scientific results. The diet board, as a method of dietary restriction, was used in the present study. Diet board feeding allows group housing and should result in enhanced welfare compared with traditional methods of dietary restriction. With respect to animal model robustness and translatability of results it is important that the feeding regime does not affect diurnal rhythmicity of biological parameters. In the present study the effects of diet board feeding on diurnal rhythms of blood glucose, serum ghrelin, faecal immunoglobulin A (IgA) and faecal corticosterone were assessed. The diet board did not alter diurnal rhythms, and adds weight to the use of this method for dietary restriction which should benefit animal health and the validity of scientific results generated from the animals.

Fentanyl Pharmacokinetics in Pregnant Sheep after Intravenous and Transdermal Administration to the Ewe.

Fentanyl is used for pain treatment during pregnancy in human beings and animals. However, fentanyl pharmacokinetics during pregnancy has not been fully established. The aim of this study was to characterize fentanyl pharmacokinetics in pregnant sheep after intravenous and transdermal dosing during surgical procedure performed to ewe and foetus. Pharmacokinetic parameters reported for non-pregnant sheep and nominal transdermal dose rate were utilized for a priori calculation to achieve analgesic fentanyl concentration (0.5-2 ng/ml) in maternal plasma. A total of 20 Aland landrace ewes at 118-127 gestational days were used. In the first protocol, 1 week before surgery, 10 animals received 2 µg/kg fentanyl intravenous bolus, and on the operation day, transdermal fentanyl patches at nominal dose rate of 2 µg/kg/hr were applied to antebrachium, and ewes were then given a 2 µg/kg intravenous bolus followed by an intra-operative 2.5 µg/kg/hr infusion. In the second protocol, 10 animals received fentanyl only as transdermal patches on the operation day and oxycodone for rescue analgesia. The data were analysed with population pharmacokinetic modelling. Intra- and post-operative fentanyl concentrations were similar and slightly lower than the a priori predictions, and elimination and distribution clearances appeared slower during than before or after the surgery. Transdermal patches provided sustained fentanyl absorption for up to 5 days, but the absorption rate was slower than the nominal dose rate and showed a high interindividual variability. Further research is warranted to evaluate the clinical relevance of the observations made in sheep.