Cervical collagen and biomechanical strength in non-pregnant women with a history of cervical insufficiency.

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.

Collagen concentration and biomechanical properties of samples from the lower uterine cervix in relation to age and parity in non-pregnant women.

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.

Strontium and bone nanostructure in normal and ovariectomized rats investigated by scanning small-angle X-ray scattering.

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.

T-tube drainage stimulates the healing of choledocho-choledochostomies. An experimental study in pigs.

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.

Choledocho-choledochostomy: the natural history of healing in pigs.

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.

The influence of ultrasound on the mechanical properties of healing tendons in rabbits.

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, 1000 and 2000 mW/cm2, 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 2000 mW/cm2 than after 50 mW/cm2. 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.

The total length of myocytes and capillaries, and total number of myocyte nuclei in the rat heart are time-dependently increased by growth hormone.

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.

Subchronic toxicity of baltic herring oil and its fractions in the rat (III) bone tissue composition and dimension, and ratio of n-6/n-3 fatty acids in serum phospholipids.

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 density. 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.

Growth hormone can reverse glucocorticoid-induced low bone turnover on cortical but not on cancellous bone surfaces in adult Wistar rats.

UNLABELLED: 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 surfaces.

Biphasic effect of relaxin, inhibitable by a collagenase inhibitor, on the strength of human fetal membranes.

BACKGROUND: Relaxin has been proposed as a hormone involved in the collagen remodeling of the utero-placental unit. MATERIALS AND METHODS: Human 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. RESULTS: Biomechanical 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. CONCLUSION: H2 relaxin had an effect on human fetal membranes and this effect may be mediated through collagenases.

Simvastatin treatment partially prevents ovariectomy-induced bone loss while increasing cortical bone formation.

Statins are commonly prescribed drugs that inhibit hepatic cholesterol synthesis and thereby reduce serum cholesterol concentrations. Some of the statins are thought to possess bone anabolic properties. Effects of statin on tibia, femur, and vertebral cortical and cancellous bone were studied in ovariectomized (OVX) rats. Sixty Wistar female rats, 4 months old, were allocated into four groups: baseline control, sham + placebo group, OVX + placebo, OVX + simvastatin. Simvastatin, 20 mg/kg, or placebo was given twice daily by a gastric tube for 3 months. The rats were labeled with tetracycline at day 11 and calcein at day 4 before sacrifice. Concerning cortical bone, the tibial diaphysis bending strength was increased by 8% and the periosteal bone formation rate (BFR) at the mid-diaphysis increased by twofold in the OVX + simvastatin group compared with the OVX + placebo group, in harmony with increased serum osteocalcin concentrations. Simvastatin did not affect the endocortical bone formation. Concerning cancellous bone, the cancellous bone volumes in the proximal tibia and vertebral body were reduced in both OVX groups, but the reduction was less in the OVX + simvastatin group compared with the OVX + placebo group. This reduction in cancellous bone loss is in agreement with the 36% decreased activity of serum tartrate-resistant-acid-phosphatase 5b (TRAP-5b), indicating decreased osteoclast activity in the OVX + simvastatin group compared with the OVX + placebo group. In conclusion, simvastatin induces a moderate increase in cortical bone formation at the periosteal bone surface. The new cortical bone exhibits a normal lamellar structure, and simvastatin seems to respect the regional pattern of bone formation, bone resorption, and drift; for example, no periosteal bone formation is observed in the vertebral canal. Furthermore, simvastatin reduces the loss of cancellous bone induced by ovariectomy.

Growth hormone increases the total number of cardiac myocyte nuclei in young rats but not in old rats.

Previously we have shown that growth hormone (GH) increases the total number of myocyte nuclei of the left ventricle in adult rats (8 months old). In the present study, we investigated whether GH could increase the total number of myocyte nuclei of the left ventricle in young and old rats. Female rats, 3 months old and 20 months old, were injected with GH or vehicle for 80 days. Using immersion-fixed left ventricles, unbiased stereological methods were applied. The weight of the left ventricle was increased by 49% (P<0.001) in the GH-injected young rats and by 32% (P<0.01) in the GH-injected old rats compared with the controls. Compared with the control groups, there was a 31% increase in the total number of myocyte nuclei in the GH-injected young group (P<0.05), but no significant increase in the GH-injected old group. The total number of non-myocyte nuclei was increased by 59% in the young GH-injected group (P<0.001) and by 25% in the old GH-injected group (P<0.01). In conclusion, GH induced a substantial left ventricle growth in both young and old rats. GH increased the total number of myocyte nuclei in the left ventricle of young rats, but not in old rats. This study shows that the myocyte response to GH declines with ageing.

Growth hormone increases the total number of myocyte nuclei in the left ventricle of adult rats.

Several studies have shown that growth hormone (GH) administration can increase size and dimensions of rat hearts. The aim of the present study was to evaluate the effect of GH on the growth of myocytes, myocyte nuclei, connective tissue, connective tissue nuclei and capillaries in the adult rat heart with special reference to total number, total length and total and relative volumes. Eight-month-old female rats were injected with either GH or vehicle for 80 days. Unbiased stereological methods were applied on immersion-fixed left ventricles. GH increased the total number of myocyte nuclei by 25% and the total number of non-myocyte nuclei by 46%. The total lengths of the myocytes and capillaries were increased by 24% and 25% respectively. Furthermore, GH increased the weight of the left ventricle by 58% without changing the relative volume fraction of myocytes, connective tissue or capillaries. In conclusion, GH seems to be a potent mediator of myocardial growth in the adult rat.

Parathyroid hormone induces formation of new cancellous bone with substantial mechanical strength at a site where it had disappeared in old rats.

OBJECTIVE: The present study addresses the question--can PTH induce formation of trabeculae in areas where cancellous bone has disappeared? Two-year-old male rats were chosen, because in this aged animal model the distal femurs have almost no cancellous bone, and the marrow cavity has reached a substantial dimension. DESIGN: The rats were injected for 56 days with either PTH(1-34), 15 nmol/kg/day (62.5 microg/kg/day), or vehicle. METHODS: Transverse specimens, 2-mm high, were cut from the distal femoral metaphysis. Marrow cavity diameters and cancellous bone trabeculae were analysed by a micro-computerized tomography scanner. The cancellous bone within the cortical and endocortical rim of each specimen was submitted to a biomechanical compression test. Furthermore, the cancellous bone was studied by dynamic tetracycline labelling and histomorphometry. RESULTS: In the vehicle-injected group the trabecular bone volume was 0% (0-1.4), median (range). All PTH-injected rats had trabeculae in the distal metaphysis and the trabecular bone volume (6.7% (2.3-12.0)) was markedly increased (P<0.003). The median trabecular thickness was increased (P<0.003) in the PTH-injected rats (118 microm (104-125)) compared with the vehicle group (0 microm (0-71)). The compressive stress was increased (P<0.003) in the PTH-injected group (0.7 MPa (0.1-2.1)) compared with the vehicle-injected group (0 MPa (0-0.4)). The histomorphometry revealed that only 3 animals of the 10 in the vehicle-injected group had trabeculae in the distal femoral metaphysis. All PTH-injected animals (12 of 12) had continuous trabecular bone network in the marrow cavity. CONCLUSION: Intermittent PTH treatment induced marked formation of new cancellous bone trabeculae with substantial mechanical strength, at a site where it had disappeared in old rats.

Growth hormone influences the content and composition of collagen in the aorta from old rats.

Collagen and elastin are major components of the aortic extracellular matrix and crucial in determining the stiffness of the aorta. We recently showed that growth hormone (GH) changes the mechanical properties, content and composition of aortic collagen from young rats. In the present study, the effect of GH on aorta from old rats was investigated. Old female rats (18(1/2)-20(1/2) months) were injected with either GH (5 mg/kg per day; n=15) or vehicle (n=14) for 80 days. Mechanical and biochemical properties of the thoracic aorta were investigated. Long-term GH injections increased the body weight of female rats by 47% accompanied by a threefold increase in serum IGF-I. The diameter of the aorta was increased by 5%, resulting in a 10% increase in the cross-section of the aortic lumen. Growth hormone increased the content of collagen per sample by 6% and increased the amount of type I collagen relative to type III collagen. No changes in the mechanical properties or elastin content per sample were found. In conclusion, GH induced a substantial growth of old rats. However, although the diameter and the collagen content were increased, the mechanical properties of the aorta were preserved in the GH-injected rats.