The mechanical and morphological properties of systemic and pulmonary arteries differ in the Madagascar ground boa, a snake without ventricular pressure separation.

The walls of the mammalian aorta and pulmonary artery are characterized by diverging morphologies and mechanical properties, which have been correlated with high systemic and low pulmonary blood pressure, as a result of intraventricular pressure separation. However, the relationship between intraventricular pressure separation and diverging aortic and pulmonary artery wall morphologies and mechanical characteristics is not understood. The snake cardiovascular system poses a unique model for the study of this relationship, as representatives both with and without intraventricular pressure separation exist. In this study, we performed uniaxial tensile testing on vessel samples taken from the aortas and pulmonary arteries of the Madagascar ground boa, Acrantophis madagascariensis, a species without intraventricular pressure separation. We then compared these morphological and mechanical characteristics with samples from the ball python, Python regius, and the yellow anaconda, Eunectes notaeus - species with and without intraventricular pressure separation, respectively. Our data suggest that although the aortas and pulmonary arteries of A. madagascariensis respond similarly to the same intramural blood pressure, they diverge in morphology, and that this attribute extends to E. notaeus. In contrast, P. regius aortas and pulmonary arteries diverge both morphologically and in terms of their mechanical properties. Our data indicate that intraventricular pressure separation cannot fully explain diverging aortic and pulmonary artery morphologies. Following the law of Laplace, we propose that pulmonary arteries of small luminal diameter represent a mechanism to protect the fragile pulmonary vasculature by reducing the blood volume that passes through, to which genetic factors may contribute more strongly than physiological parameters.

Endothelial Dysfunction and Passive Changes in the Aorta and Coronary Arteries of Diabetic db/db Mice.

Endothelial cell dysfunction and vessel stiffening are associated with a worsened prognosis in diabetic patients with cardiovascular diseases. The present study hypothesized that sex impacts endothelial dysfunction and structural changes in arteries from diabetic mice. In diabetic (db/db) and normoglycaemic (db/db+) mice, the mechanical properties were investigated in pressurized isolated left anterior descending coronary arteries and aorta segments that were subjected to tensile testing. Functional studies were performed on wire-mounted vascular segments. The male and female db/db mice were hyperglycaemic and had markedly increased body weight. In isolated aorta segments without the contribution of smooth muscle cells, load to rupture, viscoelasticity, and collagen content were decreased suggesting larger distensibility of the arterial wall in both male and female db/db mice. In male db/db aorta segments with smooth muscle cell contribution, lumen diameter was smaller and the passive stretch-tension curve was leftward-shifted, while they were unaltered in female db/db aorta segments versus control db/db+ mice. In contrast to female db/db mice, coronary arteries from male db/db mice had altered stress-strain relationships and increased distensibility. Transthoracic echocardiography revealed a dilated left ventricle with unaltered cardiac output, while aortic flow velocity was decreased in male db/db mice. Impairment of acetylcholine relaxation was aggravated in aorta from female db/db compared to control and male db/db mice, while impairment of sodium nitroprusside relaxations was only observed in aorta from male db/db mice. The remodeling in the coronary arteries and aorta suggests an adaptation of the arterial wall to the reduced flow velocity with sex-specific differences in the passive properties of aorta and coronary arteries. The findings of less distensible arteries and more pronounced endothelial dysfunction in female compared to male diabetic mice may have implications for the observed higher incidence of macrovascular complications in diabetic women.

Analysis of vascular mechanical properties of the yellow anaconda reveals increased elasticity and distensibility of the pulmonary artery during digestion.

In animals with functional division of blood systemic and pulmonary pressures, such as mammals, birds, crocodilians and a few non-crocodilian reptiles, the vessel walls of systemic and pulmonary arteries are exquisitely adapted to endure different pressures during the cardiac cycle, systemic arteries being stronger and stiffer than pulmonary arteries. However, the typical non-crocodilian reptile heart possesses an undivided ventricle that provides similar systolic blood pressure to both circuits. This raises the question whether in these species the systemic and pulmonary mechanical vascular properties are similar. Snakes also display large organ plasticity and increased cardiac output in response to digestion, and we speculate how the vascular circuit would respond to this further stress. We addressed these questions by testing the mechanical vascular properties of the dorsal aorta and the right pulmonary artery of fasted and fed yellow anacondas, Eunectes notaeus, a snake without functional ventricular separation that also exhibits large metabolic and cardiovascular responses to digestion. Similar to previous studies, the dorsal aorta was thicker, stronger, stiffer and more elastic than the pulmonary artery. However, unlike any other species studied so far, the vascular distensibility (i.e. the relative volume change given a pressure change) was similar for the two circuits. Most striking, the pulmonary artery elasticity (i.e. its capacity to resume its original form after being stretched) and distensibility increased during digestion, which suggests that this circuit is remodeled to accommodate the larger stroke volume and enhance the Windkessel effect, thus providing a more constant blood perfusion during digestion.

Impact of chronic hypoxia on proximal pulmonary artery wave propagation and mechanical properties in rats.

Arterial stiffness and wave reflection are important components of the ventricular afterload. Therefore, we aimed to assess the arterial wave characteristics and mechanical properties of the proximal pulmonary arteries (PAs) in the hypoxic pulmonary hypertensive rat model. After 21 days in normoxic or hypoxic chambers (24 animals/group), animals underwent transthoracic echocardiography and PA catheterization with a dual-tipped pressure and Doppler flow sensor wire. Wave intensity analysis was performed. Artery rings obtained from the pulmonary trunk, right and left PAs, and aorta were subjected to a tensile test to rupture. Collagen and elastin content were determined. In hypoxic rats, proximal PA wall thickness, collagen content, tensile strength per unit collagen, maximal elastic modulus, and wall viscosity increased, whereas the elastin-to-collagen ratio and arterial distensibility decreased. Arterial pulse wave velocity was also increased, and the increase was more prominent in vivo than ex vivo. Wave intensity was similar in hypoxic and normoxic animals with negligible wave reflection. In contrast, the aortic maximal elastic modulus remained unchanged, whereas wall viscosity decreased. In conclusion, there was no evidence of altered arterial wave propagation in proximal PAs of hypoxic rats while the extracellular matrix protein composition was altered and collagen tensile strength increased. This was accompanied by altered mechanical properties in vivo and ex vivo. NEW & NOTEWORTHY In rats exposed to chronic hypoxia, we have shown that pulse wave velocity in the proximal pulmonary arteries increased and pressure dependence of the pulse wave velocity was steeper in vivo than ex vivo leading to a more prominent increase in vivo.

Electrospun biodegradable microfibers induce new collagen formation in a rat abdominal wall defect model: A possible treatment for pelvic floor repair?

Half of the female population over age 50 years will experience pelvic organ prolapse. We suggest a new approach based on tissue engineering principles to functionally reconstruct the anatomical structures of the pelvic floor. The aim of this study is to investigate the mechanical performance and effect on collagen and elastin production of a degradable mesh releasing basic fibroblast growth factor (bFGF). Implantation of biodegradable mesh with or without bFGF in their core has been conducted in 40 rats in an abdominal wall defect model. Samples were explanted after 4, 8, and 24 weeks, and tested for mechanical properties and the composition of connective tissue. The study showed an increase in mRNA expression for collagen-I (p = 0.0060) and collagen-III (p = 0.0086) in the 4 weeks group with bFGF. The difference was equalized at 8 and 24 weeks. No difference was found at any time for protein amount for collagen-I, collagen-III, and fibronectin. The amount of collagen decreased from 4 to 24 weeks but the fraction of collagen increased. The maximal load of the newly formed tissue showed no effect of bFGF at any time. Exclusively, histology showed a limited ingrowth of collagen fibers after 4 weeks with bFGF but signs of elastin fibers were seen at 24 weeks. The investigation showed that a biodegradable mesh promotes tissue formation with a promising strength. The mesh with bFGF did not represent any advantage on either long or short term in comparison to the mesh without bFGF. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 680-688, 2018.

Mechanisms involved in extracellular matrix remodeling and arterial stiffness induced by hyaluronan accumulation.

BACKGROUND AND AIMS: Hyperglycemia induces hyaluronan (HA) accumulation in the vasculature. Excessive accumulation of HA around the vascular smooth muscle cells (VSMC) results in increased aortic stiffness and strength and accelerated atherosclerosis in ApoE(-)/(-) mice. We hypothesized that HA accumulation primes the vasculature for atherosclerosis by crosslinking and reorganizing the extracellular matrix (ECM) and by pushing VSMC differentiation towards a less mature phenotype. METHODS: Aortas from HAS-2 transgenic (Tg) mice and wild type mice were used for all experiments. Biomechanics and cross-sectional area measurements were performed before and after HA digestion. The vessel and ECM composition was examined by immunoblotting and electron microscopy. Primary VSMC cultures were examined by qPCR and thymidine incorporation. RESULTS: Tg mice aorta cross-sectional area was increased before (14%, p = 0.0148), but not after HA digestion (p = 0.3437). The increase in vessel stiffness (32%, p = 0.0217) and strength (31%, p = 0.0043) in the Tg aorta persisted after HA digestion. Crosslinking of HA by heavy chains from Inter-α-Inhibitor was increased (175%, p = 0.0006). The Tg VSMCs have the appearance of a synthetic phenotype supported by a 40% decrease in α-smooth muscle actin isoform X1 (p = 0.0296) and an increase in proliferation (63%, p = 0.0048) and osteoprotegerin production (133%, p = 0.0010) in cultured Tg VSMCs. CONCLUSIONS: Our results show that induced HA accumulation is followed by increased HA crosslinking and create a shift in VSMC phenotype and proliferation. These findings may provide a mechanism for how hyperglycemia through HA accumulation prime the vascular wall for cholesterol and leucocyte accumulation and development of atherosclerosis.

Topography-Guided Proliferation: Distinct Surface Microtopography Increases Proliferation of Chondrocytes In Vitro.

Chondrocyte-based cartilage repair techniques require control of articular chondrocyte expansion ex vivo. Articular chondrocytes have limited availability, and prolonged culturing to obtain a cell number sufficient for clinical use often results in phenotypic alterations and increased costs. In this study, we applied a screening library consisting of micrometer-sized topographical features, termed biosurface structure array (BSSA), to identify specific topographical microstructures affecting the proliferation of human chondrocytes in passage 1 (P1) or 2 (P2). The BSSA library comprised 10 patterns and 16 combinations of pillar size (X) and interpillar gap size (Y). Specific microstructures significantly increased the chondrocytes' proliferative responsiveness in term of patterns, X and Y for P2 compared with P1. The P1 and P2 chondrocytes responded independently to similar patterns after 4 days of culturing, whereas only chondrocytes at P2 responded to specific microstructures with Y = 1 µm and X = 2, 4 µm by a 2.3- and 4.4-fold increased proliferation, respectively. In conclusion, these findings indicate that specific surface topographies promote chondrocyte proliferation and may, indeed, be a tool to control the behavior of chondrocytes in vitro.

The mechanical properties of the systemic and pulmonary arteries of Python regius correlate with blood pressures.

Pythons are unique amongst snakes in having different pressures in the aortas and pulmonary arteries because of intraventricular pressure separation. In this study, we investigate whether this correlates with different blood vessel strength in the ball python Python regius. We excised segments from the left, right, and dorsal aortas, and from the two pulmonary arteries. These were subjected to tensile testing. We show that the aortic vessel wall is significantly stronger than the pulmonary artery wall in P. regius. Gross morphological characteristics (vessel wall thickness and correlated absolute amount of collagen content) are likely the most influential factors. Collagen fiber thickness and orientation are likely to have an effect, though the effect of collagen fiber type and cross-links between fibers will need further study.

Differential effects of Smad3 targeting in a murine model of chronic kidney disease.

Transforming growth factor (TGF)-ß1 has a pivotal role in the pathogenesis of progressive kidney diseases that are characterized by fibrosis. The main intracellular signaling pathway of TGF-ß1 is the Smad system, where Smad2 and Smad3 play a central role in transcriptional regulation of target genes involved in extracellular matrix (ECM) metabolism. This study analyzes the hypothesis that blockade of Smad3 attenuates the development of TGF-ß1-driven renal fibrosis. This was examined in vivo in a transgenic model of TGF-ß1-induced chronic kidney disease with Smad3 or without Smad3 expression and in vitro in mesangial cells and glomerular endothelial cells with Smad2/3 inhibitors or Smad3-knockdown. Electron microscopy was used for evaluation of morphological changes, real-time polymerase chain reaction for detection of RNA expression, and immunohistochemistry for localization of ECM components. Matrix metalloproteinase (MMP) level was assessed by gelatin zymography electrophoresis and located by in situ zymography. The results show TGF-ß1-induced mesangial matrix expansion, tubulointerstitial fibrosis, and tubular basement membrane thickening that are attenuated by Smad3 deletion, whereas TGF-ß1-induced glomerular basement membrane thickening is not shown. The amount and distribution profile of MMP-2 may suggest a role of the enzyme herein. We conclude that Smad3 targeting is not exclusively beneficial as Smad3 has diverse transcriptional regulatory effects in different cell types in the kidney.

Three-dimensional microarchitecture of adolescent cancellous bone.

This study investigated microarchitectural, mechanical, collagen and mineral properties of normal adolescent cancellous bone, and compared them with adult and aging cancellous bone, to obtain more insight into the subchondral bone adaptations during development and growth. Twenty-three human proximal tibiae were harvested and divided into 3 groups according to their ages: adolescence (9 to 17 years, n=6), young adult (18 to 24 years, n=9), and adult (25 to 30 years, n=8). Twelve cubic cancellous bone samples with dimensions of 8×8×8 mm(3) were produced from each tibia, 6 from each medial and lateral condyle. These samples were micro-CT scanned (vivaCT 40, Scanco Medical AG, Switzerland) resulting in cubic voxel sizes of 10.5*10.5*10.5 µm(3). Microarchitectural properties were calculated. The samples were then tested in compression followed by collagen and mineral determination. Interestingly, the adolescent cancellous bone had similar bone volume fraction (BV/TV), structure type (plate, rod or mixtures), and connectivity (3-D trabecular networks) as the adult cancellous bone. The adolescent cancellous bone had significantly lower bone surface density (bone surface per total volume of specimen) but higher collagen concentration (collagen weight per dry weight of specimen) than the adult cancellous bone; and significant greater trabecular separation (mean distance between trabeculae), significant lower trabecular number (number of trabeculae per volume), tissue density (dry weight per volume of bone matrix excluding marrow space) and mineral concentration (ash weight per dry weight of specimen) than the young adult and adult cancellous bones. Despite these differences, ultimate stress and failure energy were not significantly different among the three groups, only the Young's modulus in anterior-posterior direction was significantly lower in adolescence. Apparent density appears to be the single best predictor of mechanical properties. In conclusion, adolescent cancellous bone has similar bone volume fraction, structure type, and connectivity as the young adult and adult cancellous bones, and significant lower tissue density, bone surface density and mineral concentration but higher collagen concentration than in the young adult and adult bone. Despite these differences, the mechanical properties did not show significant difference among the three groups except less stiffness in anterior-posterior direction in the adolescents.

The effects of glucocorticoid on microarchitecture, collagen, mineral and mechanical properties of sheep femur cortical bone.

In this study, 18 female skeletally mature sheep were randomly allocated into three groups of six each. Group 1 (glucocorticoid-1) received prednisolone treatment (0.60 mg/kg/day, five times weekly) for 7 months. Group 2 (glucocorticoid-2) received the same treatment regime followed by observation of 3 months without treatment. Group 3 was left untreated and served as controls. All sheep received a restricted diet with low calcium and phosphorus. At sacrifice, cortical bone samples from the femur midshaft of each sheep were harvested, micro-CT scanned and subjected to three-point bending and tensile strength testing. Bone collagen and mineral were determined. Cortical porosity was significantly increased in the glucocorticoid-2 compared with the glucocorticoid-1 and control groups. Apparent density was significantly decreased in the glucocorticoid-2 compared with the glucocorticoid-1 group. Collagen content was significantly increased in the glucocorticoid-2 compared with the glucocorticoid-1 and control groups. Bone mineral content did not differ between the groups. Neither the three-point bending mechanical properties nor the tensile mechanical properties differed significantly between the groups, while there was a trend towards decreasing bending mechanical properties in the glucocorticoid-2 group. In conclusion, 7 months of glucocorticoid treatment with malnutrition had a significant impact on the cortical microarchitecture of the sheep femur midshaft. These observed changes occurred 3 months after glucocorticoid cessation, suggesting a delayed effect of glucocorticoid on cortical bone. Thus, changes in cortical bone beyond cancellous bone might further increase fracture risk in patients treated with glucocorticoids. This model might be used as a glucocorticoid-induced osteoporotic model for orthopaedic biomaterial, joint prosthesis and medical device researches.

A biomechanical, histological and biochemical study in an experimental rabbit hypospadias repair model using scanning acoustic microscopy.

OBJECTIVE: To investigate the biomechanical, histological and biochemical properties of rabbit urethra at long-term follow up after hypospadias simulation and acute repair. MATERIALS AND METHODS: Thirty-eight white New Zealand male rabbits underwent experimental creation of a hypospadias-like defect and acute repair (mobilization and advancement, tubularized incised posterior urethral plate (TIP), modified TIP) and sham operation. After 23 weeks all groups + controls underwent biomechanical, histological and biochemical assessments. RESULTS: The mobilization and advancement group showed a higher stiffness compared to the TIP groups (P < 0.05) in the posterior urethra, whereas the TIP group was stiffer compared to the other two operative groups (P < 0.001) in the ventral urethra. In the dorsal urethra, the mobilization and advancement group and the modified TIP group had a higher collagen content compared to shams (P < 0.05). No differences in collagen content were found between groups in the ventral urethra. A correlation between acoustic and histological layers was found, partially related to collagen content. CONCLUSION: The urethras had different microelastic properties in different layers of the dorsal and ventral urethra, with higher stiffness in the connective tissue layers surrounding and within the urethra. The repaired urethras had partially recovered their elasticity at micrometer resolution at long-term follow up. Scanning acoustic microscopy elucidated structure-function relationships at microscopic level in normal and operated urethra.

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.

Balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the cervical mucus plug estimated by determination of free non-complexed TIMP.

BACKGROUND: The cervical mucus plug (CMP) is a semi-solid structure with antibacterial properties positioned in the cervical canal during pregnancy. The CMP contains high concentrations of matrix metalloproteinase 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). This indicates a potential to degrade extracellular matrix components depending on the balance between free non-complexed inhibitors and active enzymes. METHODS: Thirty-two CMPs collected during active labor at term were analyzed. Twelve CMPs were separated into a cellular and an extracellular/fluid phase and analyzed by gelatin and reverse zymography to reveal MMP and TIMP location. Twenty samples were homogenized, extracted and studied by the TIMP activity assay based on gelatin zymography. Enzyme-linked immunosorbent assay (ELISA) was used to determine TIMP-1, MMP-8 and MMP-9 protein concentrations, and gelatin and reverse zymography used to identify gelatinases and TIMPs, respectively. The Western blotting technique was applied for semi-quantification of alpha2-macroglobulin. An ELISA activity assay was used to detect MMP-8 and MMP-9 activity. RESULTS: ProMMP-2, proMMP-9, TIMP-1 and TIMP-2 were almost exclusively located in the fluid phase compared to the cellular phase of the CMP. All the extracted samples contained MMP-8, MMP-9, TIMP-1, TIMP-2 and alpha2-macroglobulin. Free non-complexed TIMP was detected in all the samples analyzed by the TIMP activity assay and was associated with TIMP-1 protein (R = 0.71, p < 0.001) and with the TIMP/MMP molar ratio (1.7 (1.1-2.5) (mean (95% confidence interval)) (R = 0.65, p = 0.002). The ELISA activity assay showed no activity from MMP-8 or MMP-9. CONCLUSION: Due to their extracellular location, potential proteolytic activity from neutrophil-derived MMPs in the CMP could exert a biological impact on cervical dilatation and fetal membrane rupture at term. The functional TIMP activity assay, revealing excess non-complexed TIMP, and a molar inhibitor/enzyme ratio above unity, indicate that refined MMP control prevents CMP-originated proteolytic activity in the surrounding tissue.

The effects of bone remodeling inhibition by alendronate on three-dimensional microarchitecture of subchondral bone tissues in guinea pig primary osteoarthrosis.

We assessed whether increase of subchondral bone density enhances cartilage stress during impact loading, leading to progressive cartilage degeneration and accelerated osteoarthrosis (OA) progression. Sixty-six male guinea pigs were randomly divided into six groups. During a 9-week treatment period, four groups received twice-weekly subcutaneous injections of alendronate (ALN) in two doses: two groups received 10 microg/kg and two groups received 50 microg/kg. The two control groups received vehicle. After 9 weeks, one 10 microg/kg ALN group, one 50 microg/kg ALN group, and one control group were killed. The remaining three groups (17-week groups) were left for an additional 8 weeks, receiving the same treatment regimen before death. The left proximal tibiae were scanned by micro-computed tomography to quantify the microarchitecture of subchondral bone, followed by mechanical testing and determination of collagen and mineral. The control groups had typical OA-related cartilage degeneration at 9 and 17 weeks, whereas the 50 microg/kg ALN group had even worse degeneration in the medial condyle. It is unclear whether there is a direct or a secondary effect of ALN on the cartilage. The 9-week ALN group had significantly greater subchondral plate thickness. The 9- and 17-week groups had similar changes of cancellous bone microarchitecture, with greater volume fraction and connectivity and an extremely plate-like structure. The 9-week ALN group had greater bone mineral concentration, and the 17-week ALN group had reduced collagen concentration and greater mineral concentration. Treatment with ALN did not significantly change the mechanical properties of the cancellous bone.

Biomechanical and biochemical assessment of properties of the anterior urethra after hypospadias repair in a rabbit model.

PURPOSE: We created a rabbit model to test hypospadias operations and investigate the biomechanical properties of the urethra at long-term followup using biomechanical and biochemical assessments. MATERIALS AND METHODS: A total of 38 New Zealand White rabbits were randomized into 4 groups, including controls, sham operation and 2 operation groups (experimental creation of a hypospadias-like defect and acute repair, respectively). In operation group 1 the ventral urethral wall and dorsal plate were longitudinally incised, half of the ventral urethral wall was excised (hypospadias-like defect) and the incised urethra was tubularized (tubularized incised posterior plate urethroplasty group). In operation group 2 the urethra was mobilized from the corpora cavernosa, excised in its distal end (hypospadias-like defect) and advanced to the glanular tip (mobilization and advancement group). At 23 weeks postoperatively biochemical and biomechanical assessments were performed. RESULTS: Maximum urethral strength and stiffness, strain at maximum load and the collagen weight fraction were not significantly different among the groups. Urethral diameter was larger and the total amount of collagen was higher in the mobilization and advancement group only (p <0.05). The mechanical quality of urethral collagen was decreased in the 2 operation groups (p <0.05). CONCLUSIONS: This animal model proved to be useful for testing hypospadias operations and urethral mechanical properties. At long-term followup after experimental hypospadias repair biochemical and biomechanical assessments showed no differences among the groups in mechanical strength, strain and stiffness, and no indication of fibrosis. Consequently testing new hypospadias repair techniques and evaluating their biomechanical long-term results could be performed using hypospadiac animal models before clinical use.

Biglycan deficiency causes spontaneous aortic dissection and rupture in mice.

BACKGROUND: For the majority of cases, the cause of spontaneous aortic dissection and rupture is unknown. An inherited risk is associated with Marfan syndrome, Ehlers-Danlos syndrome type IV, and loci mapped to diverse autosomal chromosomes. Analysis of pedigrees however has indicated that it may be also inherited as an X-linked trait. The biglycan gene, found on chromosome X in humans and mice, encodes a small leucine-rich proteoglycan involved in the integrity of the extracellular matrix. A vascular phenotype has never been described in mice deficient in the gene for small leucine-rich proteoglycans. In the breeding of BALB/cA mice homozygous for a null mutation of the biglycan gene, we observed that 50% of biglycan-deficient male mice died suddenly within the first 3 months of life. METHODS AND RESULTS: Necropsies revealed a major hemorrhage in the thoracic or abdominal cavity, and histology showed aortic rupture that involved an intimal and medial tear as well as dissection between the media and adventitia. By transmission electron microscopy and biomechanical testing, the aortas of biglycan-deficient mice showed structural abnormalities of collagen fibrils and reduced tensile strength. Similar collagen fibril changes were observed in male as well as in female biglycan-deficient mice, which implies a role of additional determinants such as gender-related response to stress in the development of this vascular catastrophe only in male mice. CONCLUSIONS: The spontaneous death of biglycan-deficient male mice from aortic rupture implicates biglycan as essential for the structural and functional integrity of the aortic wall and suggests a potential role of biglycan gene defects in the pathogenesis of aortic dissection and rupture in humans.

MMP-2 in the left rat ventricle is increased by growth hormone.

In cardiac hypertrophy induced by GH, the turn-over of collagen seems to be increased. Matrix metalloproteinases (MMP) are enzymes suggested to contribute to the remodelling of the extracellular matrix in the myocardium. The aim of the present experiment was to investigate how GH influenced MMP concentration, collagen concentration, and structure of the connective tissue of the LV in young rats in relation to time. Three-month-old female rats were injected with GH (5mg/kg/day) or vehicle for 5, 10, 20, 40, or 80 days. MMPs and structural changes of the connective tissue were analysed using zymography and stereology, respectively. Wet weight of the LV was increased time-dependently by GH (r = 0.89, P < 0.001). Furthermore, GH increased the MMP-2 concentration (P < 0.01, two-way ANOVA), whereas no collagenases (MMP-1, MMP-8, or MMP-13) could be demonstrated. The increase in MMP-2 was accompanied by a time-dependent decrease in the collagen concentration (r = -0.46, P < 0.05), whereas the total collagen content (r = 0.85, P < 0.01) and total number of non-myocyte nuclei (GH: r = 0.89, P < 0.001) were time-dependently increased. These results indicate that MMP-2 may be involved in the remodelling process of the extracellular matrix in GH-induced cardiac hypertrophy.

Plasminogen activator inhibitor-1 gene deficiency attenuates TGF-beta1-induced kidney disease.

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) stimulates the deposition of extracellular matrix (ECM), which is a hallmark in end-stage renal disease. The importance of TGF-beta1-induced changes in protease activity in this process is not fully elucidated. TGF-beta1 up-regulates plasminogen activator inhibitor type 1 (PAI-1), which lowers matrix degradation. Our aim was to investigate the importance of PAI-1 in TGF-beta1-induced kidney disease. METHODS: TGF-beta1 transgenic mice were bred with PAI-1 gene deficient mice. The effect of PAI-1 gene knockout on TGF-beta1-induced glomerular disease was investigated by measuring morphologic changes in the glomeruli. Interstitial changes were assessed by measurement of total collagen content and expression and localization of ECM components. Finally, protease activity was evaluated by plasmin activity measurement and by gel and in situ gelatin zymography. RESULTS: TGF-beta1 elevated PAI-1 expression fourfold. PAI-1 gene deficiency attenuated the TGF-beta1-induced mesangial expansion and basement membrane thickening. Furthermore, PAI-1 knockout diminished collagen accumulation in TGF-beta1-positive mice. The expression of both collagen type I and III were reduced. Interestingly, no difference in protease activity could be ascertained as cause of the decreased ECM accumulation. CONCLUSION: We show that PAI-1 gene deficiency attenuates TGF-beta1-induced kidney disease, decreasing both glomerular and interstitial ECM deposition. Thus, PAI-1 mediates some of the biological effects of TGF-beta1 in vivo. However, we could not find evidence supporting the notion that the effect was mediated through increased protease activity.