OBJECTIVES:To investigate whether treatment with a bisphosphonate would influence the subchondral bone plate stiffness and the development of cartilage damage in Dunkin Hartley guinea pigs, which develop osteoarthritis (OA) spontaneously.METHOD:Fifty-six 3-month-old male Dunkin Hartley guinea pigs were randomized into a baseline group and six groups receiving either the bisphosphonate risedronate (30 µg/kg) or vehicle five times a week for 6, 12, or 24 weeks. The medial condyle of the right stifle joint was investigated by histology, using the Osteoarthritis Research Society International (OARSI) score, along with static and dynamic histomorphometry. The subchondral bone plate of the left tibia was tested mechanically with indentation testing. Degradation products of C-terminal telopeptides of type II collagen (CTX-II) were measured in serum.RESULTS:The OARSI score did not differ between risedronate-treated and control animals at any time point. The fraction of bone surfaces covered with osteoclasts (Oc.S/BS) was significantly suppressed in risedronate-treated animals at all time points, as were the fractions of mineralizing surfaces (MS/BS) and osteoid-covered surfaces (OS/BS), and also serum CTX-II. This was accompanied by a significant increase in the epiphyseal content of calcified tissue and in the thickness of the subchondral bone plate. However, this did not result in a stiffer subchondral bone at any time point.DISCUSSION:The risedronate treatment inhibited osteoclastic resorption of calcified cartilage in the primary spongiosa under the epiphyseal growth plate, explaining the risedronate-mediated decrease in CTX-II. Moreover, the serum CTX-II level was not related to the OA-induced articular cartilage degradation seen in this model.CONCLUSIONS:Risedronate did not influence the OARSI score and subchondral plate stiffness, but decreased serum CTX-II in Dunkin Hartley guinea pigs.

Objectives: To describe the age-related changes of articular cartilage, subchondral bone morphology, and stiffness. Furthermore, to investigate whether subchondral bone histological and mechanical properties and meniscal histological properties are related to articular cartilage damage in the Dunkin Hartley guinea pig model of osteoarthritis (OA).Methods: Forty male Dunkin Hartley guinea pigs aged 2, 6, 9, and 12 months were studied. The right stifle joints and the left menisci were embedded undecalcified and the tibial articular cartilage and subchondral bone and the menisci were examined using histology. The stiffness of the left tibial subchondral bone was determined with indentation testing.Results: The Osteoarthritis Research Society International (OARSI) grade of the osteoarthritic cartilage lesions of the medial (p < 0.001) and lateral (p < 0.001) condyle and the ossification of the medial (p < 0.001) and lateral (p < 0.001) meniscus increased significantly with age and was significantly more pronounced at the medial condyle than at the lateral condyle. The grade of the osteoarthritic cartilage lesions was significantly correlated (r == 0.78, p < 0.001) with the meniscal ossification, weakly correlated (r == 0.34, p < 0.007) with the subchondral bone plate thickness, and not correlated with the subchondral bone density (r == -0.010, p == 0.94) and the subchondral bone stiffness (r == -0.13, p == 0.30).Conclusion: The meniscal ossification observed in Dunkin Hartley guinea pigs may play an important role in the pathogenesis of OA in these animals.

The effect of SrCl(2) treatment on bone nanostructure in a rat ovariectomy model was studied using scanning small-angle X-ray scattering (sSAXS). Twelve 6-month-old female Wistar rats were used. Six animals were ovariectomized (+ovx) and six were left intact after sham surgery (-ovx). Six animals, three +ovx and three -ovx, were treated with 4 mmol SrCl(2) (aq)/kg/day (+Sr), whereas the remaining six received placebo (-Sr) for 140 days. Rats were labeled with flourochromes at days 7, 126, and 136. Femoral cross sections were studied using fluorescence microscopy, scanning electron microscopy including energy-dispersive X-ray analysis, and sSAXS. The SAXS data comprised about 5,500 measurements and provided information about mineral crystal thickness and orientation in new and old bone. The newly formed bone contained higher levels of Sr(2+) in +Sr than in -Sr animals, indicating that the Sr(2+) was incorporated into the new bone. Mineral plates were significantly thicker in old bone, 2.62 nm (95% CI 2.58-2.66), than in new bone, 2.41 nm (95% CI 2.36-2.46). Surprisingly, mineral plates in new bone were significantly thicker (2.52 [95% CI 2.47-2.57] nm vs. 2.41 [95% CI 2.36-2.46] nm, P = 0.017) in +ovx rats than in -ovx rats. However, no significant effect of SrCl(2) on mineral plate thicknesses in new bone was observed. The statistical model yielded estimates of the difference in bone mineral plate thickness induced by Sr. The estimated effect of Sr was -0.09 (95% CI -0.21 to 0.03) and 0.02 (95% CI -0.10 to 0.14) nm for new bone in -ovx and +ovx rats, respectively.

Background: During normal pregnancy the cervix has a load bearing function. The cervical tissue consists mainly of an extracellular matrix (ECM) rich in collagen; important for the biomechanical properties. The aim of the present study was to evaluate how the biomechanical strength of samples from the distal cervix is associated with collagen content in relation to age and parity. This study demonstrates a method to investigate cervical tissue from women who still have their uterus in situ.Methods: Cervical punch biopsies (2 x 15 mm) were obtained from 57 healthy women (median age: 39 years, range: 29-49 years). Biomechanical tensile testing was performed, and collagen concentration (as % of dry defatted weight (DDW)) and content (mg of collagen per mm of specimen length) was determined. Histomorphometry was used to determine the volume densities of extracellular matrix and smooth muscle cells. Smooth muscle cells were identified by immunohistochemistry. Finally, orientation of collagen fibers was estimated. Data are given as mean +/- SD.Results: The mean collagen concentration (62.2 +/- 6.6%) increased with age (0.5% per year, r = 0.45, p = 0.003) and decreased with parity (1.7% per birth, r = -0.45, p = 0.033). Maximum load was positively correlated with collagen content (mg of collagen per mm of specimen length) (r = 0.76, p < 0.001). Normalized maximum stiffness was increased with age (r = 0.32, p = 0.017), whereas no correlation was found with regard to parity. In tissue samples with a length of approximately one cm, volume density of smooth muscle cells increased gradually from 8.9% in the distal part near the epithelium, to 15.5% in the proximal part (p < 0.001).Conclusions: The present study shows that cervical collagen concentration increases with age and decreases with parity in non-pregnant women. In addition, collagen stiffness increased with age, whereas no change in collagen tensile strength with respect to age and parity was found. These results show that collagen contributes to cervical tissue tensile strength and age and parity should be considered confounding factors.

BACKGROUND:It has been suggested that cervical insufficiency (CI) is characterized by a "muscular cervix" with low collagen and high smooth muscle concentrations also in the non-pregnant state. Therefore, the aim of this study was to investigate the biomechanical properties, collagen concentration, smooth muscle cell density, and collagen fiber orientation in cervical biopsies from non-pregnant women with a history of CI.METHODS:Cervical punch biopsies (2 x 15 mm) were obtained from 57 normal non-pregnant women and 22 women with a history of CI. Biomechanical tensile testing was performed, and collagen content was determined by hydroxyproline quantification. Histomorphometry was used to determine the volume densities of extracellular matrix and smooth muscle cells from the distal to the proximal part of each sample. Smooth muscle cells were identified using immunohistochemistry. Finally, collagen fiber orientation was investigated. Data are given as mean +/- SD.RESULTS:Collagen concentration was lower in the CI group (58.6 +/- 8.8%) compared with the control group (62.2 +/- 6.6%) (p = 0.033). However, when data were adjusted for age and parity, no difference in collagen concentration was found between the two groups. Maximum load of the specimens did not differ between the groups (p = 0.78). The tensile strength of cervical collagen, i.e. maximum load normalized per unit collagen (mg of collagen per mm of specimen length), was increased in the CI group compared with controls (p = 0.033). No differences in the volume density of extracellular matrix or smooth muscle cells were found between the two groups. Fibers not oriented in the plane of sectioning were increased in CI patients compared with controls.CONCLUSIONS:Cervical insufficiency does not appear to be associated with a constitutionally low collagen concentration or collagen of inferior mechanical quality. Furthermore, the hypothesis that a "muscular cervix" with an abundance of smooth muscle cells contributes to the development of cervical insufficiency is not supported by the present study.

BACKGROUND/PURPOSE:We investigated the effect of T-tube drainage on the healing of choledocho-choledochostomies in pigs.METHODS:Twenty pigs with a median weight of 56 kg were used for the experiments. The pigs were randomized to two groups of ten. In all pigs the gallbladder was removed and the common bile duct was transected. In both groups continuity was re-established by standardized single-line, interrupted, and inverted sutures. In one group a T-tube for decompression was inserted. On postoperative day 6, a laparotomy was performed. Pigs were investigated for signs of cholascos, and an intraoperative cholangiography was performed. The excised anastomosis was examined for breaking strength and collagen content. Blood samples were drawn prior to the first and the final operations.RESULTS:In both groups standard liver parameters were unaffected by surgery, and cholangiography showed no signs of extrahepatic stenosis or intrahepatic dilatation. The T-tube-drained choledocho-choledochostomies showed a significantly higher breaking strength (P = 0.035) compared to the group which had no drainage. Collagen content per volume was unaffected by T-tube drainage.CONCLUSIONS:T-tube drainage had a significant stimulatory effect on the breaking strength of choledocho-choledochostomies in pigs on postoperative day 6, but was without effect on collagen content.

BACKGROUND/PURPOSE:Aiming to investigate the natural history of the healing of choledocho-choledochostomies.METHODS:Fifty-five female pigs of 57 kg median weight were used for the experiments. The gallbladder was removed and the common bile duct transected. Continuity was re-established by standardized single-line, interrupted, and inverted sutures. The pigs had a planned postoperative survival of up to 14 days with a subsequent laparotomy for evaluation. Blood samples were drawn prior to the first and the final operations. During laparotomy the animals were investigated for signs of cholascos, and an intraoperative cholangiography was performed. The excised anastomosis was examined for breaking strength and collagen content.RESULTS:Standard liver parameters were not significantly affected by the surgery, and cholangiography showed no signs of extrahepatic stenosis or intrahepatic dilatation. Breaking strength showed a decrease for the initial 3 postoperative days (PODs), then an increase to a stable level on PODs 6 to 14. Collagen content per volume showed a rise on PODs 0 to 1, then no change until POD 4, followed by a gradual rise until day 6. Subsequently a stable level was reached until POD 14. Two pigs were excluded due to minor cholascos.CONCLUSIONS:The present study on pigs shows that choledocho-choledochostomies, judged by breaking strength and collagen content, regain a stable level of strength 6 days after operation.

Glucocorticoids (GC) are used for the treatment of a wide spectrum of diseases because of their potent anti-inflammatory and immunosuppressive effects, and they are serious and common causes of secondary osteoporosis. Administration of intermittent parathyroid hormone (PTH) may induce formation of new bone and may counteract the bone loss induced by GC treatment. Effects of simultaneous PTH and GC treatment were investigated on bone biomechanics, static and dynamic histomorphometry, and bone metabolism. Twenty-seven-month-old female rats were divided randomly into the following groups: baseline, vehicle, PTH, GC, and PTH + GC. PTH (1-34) 25 mu g/kg and GC (methylprednisolone) 2.5 mg/kg were injected subcutaneously each day for a treatment period of 8 weeks. The rats were labeled with fluorochromes 3 times during the experiment. Bone sections were studied by fluorescence microscopy. The PTH injections resulted in a 5-fold increase in cancellous bone volume. At the proximal tibia, PTH induced a pronounced formation of new cancellous bone which originated from the endocortical bone surfaces and from thin trabeculae. Formation and modeling of connections between trabeculae were observed. Similar but less pronounced structural changes were seen in the PTH + GC group. The compressive strength of the cancellous bone was increased by 6-fold in the PTH group compared with the vehicle group. GC partially inhibited the increase in compressive strength induced by PTH. Concerning cortical bone, PTH induced a pronounced increase in the endocortical bone formation rate (BFR) and a smaller increase in periosteal BFR. The combination of PTH + GC resulted in a partial inhibition of the PTH-induced increase in bone formation. Serum-osteocalcin was increased by 65% in the PTH group and reduced by 39% in the GC group. The pronounced anabolic effect of PTH injections on the endocortical and trabecular bone surfaces and less pronounced anabolic effect on periosteal surfaces were partially inhibited, but not prevented, by simultaneous GC treatment in old rats. Both cortical and cancellous bone possessed full mechanical competence after treatment with PTH + GC. (c) 2006 Elsevier Inc. All rights reserved.

Growth hormone (GH) can increase size and dimensions of rat hearts. The aim was to study how GH administration influences the growth of cardiac myocytes and capillaries in relation to time. Three-month-old female rats were divided into 10 groups (n=3), and injected with either GH (5mg/kg/day) or vehicle for 5, 10, 20, 40, or 80 days. From the left ventricle (LV) histological sections were made and stereological methods applied. Linear regression showed that GH time-dependently increased: LV volume (r=0.96, P<0.001), total volume of myocytes (r=0.96, P<0.001) and capillaries (r=0.64, P<0.05), total length of myocytes (r=0.90, P<0.001) and capillaries (r=0.78, P<0.001), and total number of myocyte nuclei (r=0.85, P<0.001). In conclusion, during 80 days of GH treatment the total volume and length of myocytes and capillaries, and total number of myocyte nuclei increased in a linear way. The results indicate that GH is a potent mediator of myocardial growth.

We evaluated the effect of glucocorticoids (GC) and growth hormone (GH) on cortical and cancellous bone turnover in adult rats using random vertical sections giving valid measurements of bone surfaces and bone formation parameters. GH administration Could reverse GC-induced osteopenia and low bone turnover of cortical bone. However, GH could not reverse the GC-induced low bone turnover of cancellous bone. Methods: Seventy female Wistar rats, 7 months of age, were divided into five groups: (1) start control, (2) saline, (3) GC 9 mg/kg/day (Solu Medrol), (4) GH 5 mg/kg/day, and (5) GC 9 mg/kg/day + GH 5 mg/kg/day, and injected for 3 months. The vertebral body was examined Using dynamic histomorphometry and biomechanical tests. Nonparametric methods were used. Results: Glucocorticoid administration induced a low bone turnover state of both the cortical and cancellous bone of the vertebral body, without altering the absolute amount of bone or the biomechanical competence of the vertebral body. GH administration induced a small increase in longitudinal bone growth and ventral modeling drift. This growth increased the total amount of cortical, endocortical, and cancellous bone in the vertebra. The biomechanical competence of a 3.5-mm-high cylinder of the central vertebral body was also increased due to an increase in the amount of cortical bone, whereas the total amount of cancellous bone in the cylinder was unaltered. The cancellous bone density (CBV) was, however, increased due to thicker trabeculae probably induced by an accelerated mineral appositional rate (MAR) induced by GH. GH also increased longitudinal and ventral modeling drifts in the GC-injected animals. GH increased the amount of cortical bone and also the amount of cancellous bone close to the epiphyseal growth plate, whereas the cancellous bone volume of the central vertebral cylinder was unaffected by GH administration in GC-injected animals. GH Could also increase parameters of bone formation (bone mineralizing surface (MS) and MAR) on cortical bone surfaces in GC-injected animals, whereas parameters of bone formation [MS and bone formation rates (BFR)] on cancellous bone surfaces were even lower than those of animals injected with GC alone. Conclusion: GH can reverse GC-induced low bone turnover on cortical but not On cancellous bone SUI-faces. (C) 2004 Elsevier Inc. All rights reserved.

Background Therapeutic ultrasound is commonly used for treatment of injuries to tendons, ligaments and joint capsules. Opinions still differ, however, as to the beneficial effects of ultrasound treatment of the above- mentioned conditions.Methods We studied the effect of various ultrasound intensities on the healing of tenotomized, sutured and immobilized Achilles tendons of adult rabbits. Different intensities of pulsating ultrasound ( 0, 50, 100, 200, 500, 750, 1 000 and 2 000 mW/cm(2), frequency 3 MHz) were given over the healing tendons for 5 min daily, using a gel as the coupling agent between the ultrasound probe and the skin. Eleven days after the tenotomy, the healing tendons were analyzed in a materials testing machine.Results The extensibility of the healing tendons was greater after sonication at an intensity of 2 000 mW/ cm(2) than after 50 mW/cm(2). We found no significant effect on the load at rupture and normalized load at rupture of the ultrasound- treated healing tendons compared with mock- sonicated healing tendons. A gradual decline was observed, however, in the stiffness and collagen content of the healing tendons with increasing intensity of the ultrasound treatment.Interpretation The pulsating ultrasound treatment applied did not improve the mechanical properties of healing Achilles tendons at day 11 after the operation. On the other hand, a slight decline in stiffness was observed with increasing intensity of ultrasound treatment.

Abstract: Changes in total bone mineral density determined by the bone-ash method were recently demonstrated in rats, exposed to Herring oil from the contaminated southern part of the Baltic Sea. In the present study more detailed analysis of bone structure and biomechanics was performed and obtained results were evaluated in the context of dietary factors, such as polyunsaturated fatty acids, vitamin D and vitamin A. Baltic Sea herring oil was fractionated into one relatively pollutant-free fraction (F1), and two fractions with pronounced enrichment of pollutants (F2 and F3). Female Sprague-Dawley rats were fed diets supplemented with Baltic Herring oil, its fractions, Nordic Sea capelin oil or soy oil. Femur was scanned with peripheral quantitative computed tomography (pQCT) and also tested by a mechanical compression analysis. Polyunsaturated fatty acids, vitamin A and D were analysed in serum. Rats fed the high dose of herring oil exhibited shorter femur length with decreased diaphyseal cortical bone mineral density, as well as lowered metaphyseal cross-sectional area compared to the soy oil group. Rats fed the high dose of F1 diet had increased cortical and decreased trabecular area, and higher total and trabecular bone mineral dencity. Rats fed the low dose of F2 diet showed similar changes associated with increased maximum load and energy absorption in compression test of the femoral metaphysis. In summary, our findings in changes of bone geometry and density could not be linked to any isolated exposure parameter, suggesting synergistic or antagonistic effects of several components of the test diets.

BACKGROUNDRelaxin has been proposed as a hormone involved in the collagen remodeling of the utero-placental unit.MATERIALS AND METHODSHuman fetal membrane explants were incubated with H1 or H2 relaxin for 48 hours and stretched until rupture in a materials testing machine. Co-incubation with a synthetic collagenase inhibitor was performed in order to examine whether the effects of relaxin could be inhibited. The effects on hydroxyproline and histology were evaluated.RESULTSBiomechanical testing showed that H2 relaxin induced a biphasic weakening of human fetal membranes, an effect that was abolished after co-incubation with a collagenase inhibitor. H1 relaxin produced no significant effects on the biomechanical properties. The effects of H2 relaxin on the biomechanical properties were, however, not followed by changes in the hydroxyproline concentration or the histology.CONCLUSIONH2 relaxin had an effect on human fetal membranes and this effect may be mediated through collagenases.

The studies behind this thesis were carried out at the Laboratory of Biochemical Pathology, Arhus Sygehus. The thesis is based on an original paper investigating the effects of diabetic-like concentrations of components of the metabolic disorders of diabetes mellitus. In 1971 the term diabetic macroangiopathy was introduced to emphasize the presence of non-atherosclerotic large vessel disease in diabetic patients. Diabetic macroangiopathy does not indicate a set of abnormalities restricted to certain areas of the large vessels as in atherosclerosis, but a number of specific vessel wall changes seen in the entire large vessel system. These changes are regarded to be a consequence of the diabetic metabolism. In areas of aorta without atherosclerotic plaque an increased proportion of HA is observed in tunica media in diabetic individuals. Furthermore the HA receptor, CD44, is induced on vascular SMCs after arterial wall injury and may mediate proliferation and migration of SMCs into the tunica intima. Thereby HA and its receptor CD44 may be an important factor in the pathogenesis of cardiovascular disease in diabetic patients. The aim of this thesis was to evaluate the influence of diabetic-like concentrations of glucose, insulin, IGF-1 and hGH on the expression of CD44std and the variant isoforms CD44v3 and CD44v6 on the arterial SMCs in vitro. Furthermore the effect of diabetic – like concentrations of the four metabolic components on SMC migration is examined to determine whether CD44 receptor is important for SMC migration. In conclusion, these in vitro studies confirm that the CD44 receptor plays a role in SMC migration at diabetic-like concentrations of glucose, insulin and IGF-1. Therefore CD44 receptor might contribute to SMC accumulation in the intima in patients with diabetes. Furthermore the results support the notion that insulin might have a significant influence on HA binding and thereby be a factor modulating vascular SMC function during the development of large vessel disease in diabetic subjects. Finally the expression of CD44 and CD44v3 showed significant but small changes after metabolic manipulation with some of the components.