Ratio Of Collagen Type Research Articles

Lamina Propria Cellularity and Collagen Composition: An Integrated Assessment of Structure in Humans

In this study, we quantitatively examined cell density, collagen types I and III, and regional variations in collagen fiber thickness and orientation in the human midmembranous vocal fold lamina propria (LP). Lamina propria samples were solubilized with proteinase K or with cyanogen bromide. Cell density was assessed in proteinase K digests by measuring DNA and normalizing it to tissue total protein. Collagen types I and III were quantified by enzyme-linked immunosorbent assay-based detection of collagen type-specific peptides generated by cyanogen bromide digestion. In addition, LP total collagen was determined by measuring sample hydroxyproline levels. Variations in collagen fiber thickness and orientation with LP region were evaluated by examining picrosirius red-stained LP sections with circularly polarized light. The mean (+/-SEM) cell density in the LP and associated epithelium was approximately 0.57 +/- 0.09 million cells per milligram of tissue total protein. Collagen type III composed an average of 34% to 40% of LP total collagen. Quantitative histology indicated that the superficial LP contained an average of 70% thin, 26% intermediate, and 4% thick collagen fibers. This is in contrast to the intermediate and deep LPs, each of which contained less than 25% thin and more than 50% thick collagen fibers. The angular deviations in collagen fiber orientation were relatively large and were similar in magnitude across all LP layers. The total cell density of the LP and associated epithelium was intermediate between that of hyaline cartilage and dermis. The ratio of collagen type III to total collagen in the LP was similar to that of highly elastic lung parenchyma and roughly twice that of the comparatively less-elastic dermis. The average thickness of collagen fibers increased markedly with increasing LP depth, and the relatively large angular deviations in fiber orientation appeared to correspond in part to the crimped nature of LP collagen fibers.

Muscle senescence in short‐lived wild mammals, the soricine shrews Blarina brevicauda and Sorex palustris

Red-toothed (soricine) shrews are consummate predators exhibiting the highest energy turnovers and shortest life spans (ca. 18 months) of any mammal, yet virtually nothing is known regarding their physiological aging. We assessed the emerging pattern of skeletal muscle senescence (contractile/connective tissue components) in sympatric species, the semi-aquatic water shrew (WS), Sorex palustris, and the terrestrial short-tailed shrew (STS), Blarina brevicauda, to determine if muscle aging occurs in wild, short-lived mammals (H(0): shrews do not survive to an age where senescence occurs), and if so, whether these alterations are species-specific. Gracilis muscles were collected from first-year (n=17) and second-year (n=17) field-caught shrews. Consistent with typical mammalian aging, collagen content (% area) increased with age in both species (S. palustris: approximately 50%; B. brevicauda: approximately 60%). Muscle was dominated by stiffer Type I collagen, and the ratio of collagen Type I:Type III more than doubled with age. The area ratio of muscle:collagen decreased with age in both species, but was considerably lower in adult STS, suggesting species-specificity of senescence. Extracellular space was age-elevated in B. brevicauda, but was preserved in S. palustris ( approximately 50 vs. 10% elevation). Though juvenile interspecific comparisons revealed no significance, adult WS myocytes had 68% larger cross-sectional area and occurred at 28% lower fibers/area than those of adult STS. We demonstrate that age-related muscle senescence does occur in wild-caught, short-lived mammals, and we therefore reject this classic aging theory tenet. Our findings moreover illustrate that differential age adjustments in contractile/connective tissue components of muscle occur in the two species of wild-caught shrews.

Absence of circulating aldosterone attenuates foreign body reaction around surgical sutures

Adrenal hormones influence inflammatory and fibrotic activity and thereby are involved in wound-healing process. Any excess as well as any shortage of glucocorticoids leads to a delayed wound healing. Mineralocorticoids like aldosterone have a pro-fibrotic and pro-inflammatory impact; thus, reduction of circulating aldosterone should result in an attenuated inflammatory response to implanted foreign bodies. Eighteen rats were bilaterally adrenalectomized and substituted with dexamethasone (12 microg/kg per day) and 1% salt in their drinking water; 22 rats were sham-operated. The surgical suture material was removed after 3 weeks and analyzed for size of granuloma, ratio of collagen type I/III, apoptotic cells (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling), expression of matrix metalloproteinase (MMP)-2, cyclooxygenase 2, tumor necrosis factor receptor 2 (TNF-R2), cluster of differentiation 68 (CD68), Ki67, and cold shock protein Y box binding protein 1 (YB-1). Cell expression was scored according to Remmele. All animals developed foreign body granulomas around the sutures. Absence of circulating aldosterone after adrenalectomy (ADX) was associated with smaller granuloma size and a reduced ratio of collagen type I/III. Ki67 and MMP-2 showed the strongest expression in cells of the infiltrate around suture. In adrenalectomized rats, we observed significantly less CD68-positive macrophages and less Ki67-positive cells but no significant differences in the expression of YB-1, TNF-R2, or MMP-2. Looking for correlations and co-expressions of proteins, the number of significant Spearman correlations was reduced in the ADX group compared to controls (one and four, respectively). The absence of circulating aldosterone attenuates inflammatory intensity around suture material. Foreign body granuloma seems to be an appropriate model to study chronic inflammatory process.

Effects of Methotrexate on Plasma Cytokines and Cardiac Remodeling and Function in Postmyocarditis Rats

Excessive immune activation and inflammatory mediators may play a critical role in the pathogenesis of chronic heart failure. Methotrexate is a commonly used anti-inflammatory and immunosuppressive drug. In this study, we used a rat model of cardiac myosin-induced experimental autoimmune myocarditis to investigate the effects of low-dose methotrexate (0.1 mg/kg/d for 30 d) on the plasma level of cytokines and cardiac remodeling and function. Our study showed that levels of tumor necrosis factor-(TNF-)alpha and interleukin-6 (IL-6) are significantly increased in postmyocarditis rats, compared with the control rats. Methotrexate treatment reduced the plasma levels of TNF-alpha and IL-6 and increased IL-10 level, compared to saline treatment. In addition, postmyocarditis rats showed significant cardiac fibrosis characterized by increased myocardial collagen volume fraction, perivascular collagen area, and the ratio of collagen type I to type III, compared with the control rats. However, MTX treatment not only markedly attenuated cardiac fibrosis, diminished the left ventricular end-diastolic dimension, but also increased the left ventricular ejection fraction and fractional shortening. Collectively, these results suggest that low-dose methotrexate has ability to regulate inflammatory responses and improves cardiac function and hence contributes to prevent the development of postmyocarditis dilated cardiomyopathy.

Intradiscal injection of simvastatin retards progression of intervertebral disc degeneration induced by stab injury

IntroductionEarlier work indicates that the cholesterol-lowering drug, simvastatin, is anabolic to chondrogenic expression of rat intervertebral disc (IVD) cells, which suggests a potential role for simvastatin in IVD regeneration. In this study, we expand on our earlier work to test the effectiveness of simvastatin on disc degeneration utilizing a rat tail disc degeneration model.Methods30 rats that underwent 21 G needle-puncture at rat tail discs were injected with simvastatin-loaded poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PEG-PLGA-PEG) gel (5 mg/ml) or vehicle control at 4 weeks after needle injury. All animals were sacrificed 2 weeks after simvastatin injection. Bone morphogenetic protein-2 (BMP-2), aggrecan, collagen type II, and collagen type I messenger ribonucleic acid (mRNA) expression in the rat nucleus pulposus (NP) were measured by real-time polymerase chain reaction (PCR). In vivo magnetic resonance imaging (MRI) was performed to monitor changes in disc degeneration. Rat discs were also assessed by histology using hematoxylin and eosin (H&E) and safranin O staining. In addition, the NP weight, glycosaminoglycan (sGAG) and DNA content were also measured.ResultsA single dose of simvastatin loaded in thermo-sensitive PEG-PLGA-PEG gel injected into the NP had the trend to increase aggrecan expression and sGAG content, and significantly increased mRNA levels of BMP-2, collagen type II, and the differentiation index (the ratio of collagen type II to collagen type I). The decreased NP weight, T2 intensity, as well as MRI index in the rat tail discs induced by needle puncture were significantly reversed after 2 weeks of simvastatin treatment. In addition, simvastatin treatment also improved histological changes induced by needle puncture.ConclusionsA single injection of simvastatin loaded in PEG-PLGA-PEG gel into rat tail discs had the potential to retard or regenerate the degenerative disc.

Collagen content, but not the ratios of collagen type III/I mRNAs, differs among hypertensive, alcoholic, and idiopathic dilated cardiomyopathy

Cardiac interstitial fibrosis may contribute to ventricular dysfunction and the prognosis of patients with dilated cardiomyopathy. The objective of the present study was to determine if total myocardial collagen content and collagen type III/I (III/I ratio) mRNAs differ in hypertensive, alcoholic, and idiopathic dilated cardiomyopathy subjects. Echocardiography and exercise cardiopulmonary testing were performed in patients with idiopathic (N = 22), hypertensive (N = 12), and alcoholic (N = 11) dilated cardiomyopathy. Morphometric analysis of collagen was performed in fragments obtained by endomyocardial biopsy with picrosirius red staining. The collagen III/I ratio was determined by reverse transcription polymerase chain reaction. Samples of controls (N = 10) were obtained from autopsy. Echocardiographic variables and maximal oxygen uptake were not different among dilated cardiomyopathy groups. Collagen was higher in all dilated cardiomyopathy groups (idiopathic, hypertensive and alcoholic, 7.36 +/- 1.09%) versus controls (1.12 +/- 0.18%), P < 0.05. Collagen was lower in idiopathic dilated cardiomyopathy (4.97 +/- 0.83%) than hypertensive (8.50 +/- 1.11%) and alcoholic (10.77 +/- 2.09%) samples (P < 0.005 for both). The collagen III/I ratio in all samples from dilated cardiomyopathy patients was higher compared to that in controls (0.29 +/- 0.04, P < 0.05) but was the same in the samples from idiopathic (0.77 +/- 0.07), hypertensive (0.75 +/- 0.07), and alcoholic (0.81 +/- 0.16) dilated cardiomyopathy groups. Because of the different physical properties of the types of collagen, the higher III/I ratio may contribute to progressive ventricular dilation and dysfunction in dilated cardiomyopathy patients.

Cell–Matrix Interactions Improve β-Cell Survival and Insulin Secretion in Three-Dimensional Culture

Controlled matrix interactions were presented to pancreatic beta-cells in three-dimensional culture within poly(ethylene glycol) hydrogels. Dispersed MIN6 beta-cells were encapsulated in gel environments containing the following entrapped extracellular matrix (ECM) proteins: collagen type I, collagen type IV, fibrinogen, fibronectin, laminin, and vitronectin. In ECM-containing gels, beta-cell survival was significantly better than in gels without ECM over 10 days. Correspondingly, apoptosis in encapsulated beta-cells was less in the presence of each matrix protein, suggesting the ability of individual matrix interactions to prevent matrix signaling-related apoptosis (anoikis). MIN6 beta-cells cultured in gels containing collagen type IV or laminin secreted more insulin in response to glucose stimulation than beta-cells in all other experimental conditions. Variations in collagen type IV or laminin concentration between 10 microg/mL and 250 microg/mL did not affect insulin secretion. Finally, beta-cell function in hydrogels presenting both collagen type IV and laminin revealed synergistic interactions. With a total protein concentration of 100 microg/mL, three gel compositions of varying ratios of collagen type IV to laminin (25:75, 50:50, and 75:25) were tested. In the presence of 25 microg/mL of collagen type IV and 75 microg/mL of laminin, beta-cell insulin secretion was greater than with laminin or collagen type IV individually. These results demonstrate that specific, rationally designed extracellular environments promote isolated beta-cell survival and function.

Histological Characterization of the Urethral Edges in Patients Who Underwent Bulbar Anastomotic Urethroplasty

Meticulous excision of the stricture and any associated spongiofibrosis is critical to the success of urethral reconstruction. However, normal urethral limits are determined during surgical reconstruction based on macroscopic appearance only. We evaluated structural changes of the presumed healthy urethral ends. Samples were obtained from 29 patients with a mean age of 40 years who underwent end-to-end anastomotic urethroplasty. Suprapubic catheter drainage was performed for at least 30 days in 15 patients. After the urethral stricture was excised biopsy was performed of the proximal and distal healthy ends. Structural characterization was evaluated by staining histological sections with Masson's trichrome, Sirius red and Weigert's resorcin-fuchsin method. The control group consisted of 10 cadaver bulbar urethras. All samples of presumed normal urethral ends showed histological changes. Chronic and acute inflammatory reactions were the most common findings. In the proximal urethral end intense cellularity was found in 18 patients (62.1%). Fibroblast and inflammatory cells were most common and related to the lack of suprapubic cystostomy (p = 0.001). Between proximal urethral edges with and without a suprapubic urinary catheter the elastic fiber distribution and the ratio of collagen types III and I showed a significant change (p = 0.045 and <0.001, respectively). The supposedly healthy urethral ends of end-to-end anastomotic urethroplasty show structural changes. Urethral obstruction and its relief by suprapubic urinary drainage can affect extracellular matrix turnover regulation. Therefore, these changes can lead to urethral remodeling in the proximal bulbar urethra.

Local Delivery of 17-Beta-Estradiol Modulates Collagen Content in Coronary Porcine Arteries after PTCA and Stent Implantation

Background: Percutaneous transluminal coronary angioplasty (PTCA) and stent implantation are associated with intimal hyperplasia and extracellular matrix (ECM) accumulation, resulting in restenosis. We showed that local delivery of 17-beta-estradiol (17βE) reduced restenosis following PTCA and stent implantation by 47 and 23%, respectively. Because estrogens decreased type I and type III collagen synthesis in vitro, we hypothesized that local delivery of 17βE may influence intimal hyperplasia formation by modulating ECM expression. Methods: Porcine coronary arteries underwent PTCA or stenting and were randomly assigned to 17βE or placebo. After 28 days, animals were sacrificed for histology and collagen type I and III content analysis. Results: Both collagen subtypes increased in the media by 1.7 to 2.6-fold after PTCA and by 15.7 to 16.1-fold after stenting, as compared to PTCA segments. In the neointima, the ratio of collagen type III to type I was 2.7 in stented arteries and only 0.3 in PTCA arteries. In the neointima of 17βE-treated animals, collagen type I (but not type III) content upregulation was limited by 53% after PTCA and by 74% after stenting. Conclusion: Local delivery of 17βE reduces restenosis, in part by decreasing the density of collagen type I in the neointima in PTCA and stented arteries.

Therapeutic ultrasound effects on interleukin-1β stimulated cartilage construct in vitro

A low-intensity ultrasound (LIUS) was examined for its possible therapeutic effects on degenerative osteoarthritic cartilage. Along with the daily treatment of 5 ng interleukin-1β (IL−1β) for 5 d, an engineered 3D neocartilage construct was used as an in vitro OA model. Followed by 24 h preincubation with the first dose of IL−1β, the constructs were then given ultrasonic stimulation (frequency 1.5 MHz and SATA 30 mW/cm 2) once a day up to 5 d for the predetermined time. Fresh IL−1β was added before the stimulation. The difference in the cell number and viability was insignificant between control (US−/IL+) and LIUS-stimulated groups. As the daily stimulation time was extended, the GAG contents in the constructs themselves significantly increased with 50 min stimulation but those released into the culture medium remained unaffected by LIUS. While the gene expression level of aggrecan was similar between control and LIUS (50 min) group, the ratio of collagen type II to type I was found to be higher in the control. The mRNA level of matrix metalloproteinase (MMP)-1 was substantially downregulated in the stimulated construct and that of MMP-13 was indifferent between control and stimulated one. The endogenous expression of transforming growth factor (TGF)-β1 and β3 was barely responsive to the LIUS stimulation. From histologic analysis, more intense GAG deposition was clearly identified with the LIUS-stimulated constructs. This study indicates that LIUS may have a significant potential to be a chondroprotective stimulant for osteoarthritic cartilage. (E-mail: kpark@kist.re.kr)

Quantitative Analysis of Temporal and Spatial Variations of Chondrocyte Behavior in Engineered Cartilage during Long-Term Culture

In this work, we present the fact that chondrocyte activity differs in relation to their position in an engineered cartilage construct. Chondrocytes from porcine articular cartilage were cultured in a monolayer. Then the cell/extracellular matrix (ECM) membrane was peeled off and centrifuged into a three-dimensional (3D) pellet-type construct. Cultivated in a static condition, the constructs were harvested at specific time intervals (1, 2, 3, and 5 weeks) and manually cored using a biopsy punch to separate the core from the remaining construct. The resultant parts, core and peripheral remnant were thus obtained and subjected to analysis individually. Cell density (10(6 )cells/cm(3)) of the core was significantly higher at 1 week than that of the periphery but this trend was reversed at later time points. Cell viability was remarkably better in the peripheral tissue. Alcian blue staining of glycosaminoglycan (GAG) revealed an intense blue staining from the periphery, exhibiting a steep gradient in distribution of GAG concentration. The gene expression ratio of collagen type II to I appeared to be more altered in the periphery, possibly suggesting cell dedifferentiation, especially at later time points (>2 weeks). The mRNA levels of matrix metalloproteinase-1 (MMP-1) and MMP-13 remained in the normal range, whereas collagen type X expression was more significantly upregulated at the periphery. This study showed that chondrocyte behavior could be highly variable in the extent of their proliferation, differentiation and dedifferentiation, depending on their physical location within 3D engineered cartilage construct.

Impact of collagen subtype proportions in peritoneal tissues on inguinal hernia formation in adults and infants

Impaired collagen subtype proportions in the matrices results in the alteration of tissue tensile strength, and is an underlying cause of adult inguinal hernia formation; however, it remains unclear if this alteration is also responsible for infantile inguinal hernia formation. We correlated the ratios of collagen type I to type III between adults and infants with and those without an inguinal hernia, in an attempt to clarify the pathogenesis of this disorder. We extracted collagen from the hernial sacs of patients with an inguinal hernia, and from the normal peritoneum of patients without an inguinal hernia. After separation by electrophoresis, the collagen bands were quantified and we compared the ratios of collagen type I to type III between the patients with and those without an inguinal hernia. The ratio of collagen type I to type III was significantly lower in the adults with an inguinal hernia (n=8, 6.17+/-2.69) than in those without an inguinal hernia (n=8, 9.38+/-2.99, P=0.041), whereas the ratios were similar in infants with (n=10, 5.72+/-2.66) and those without an inguinal hernia (n=6, 5.60+/-1.88, P=0.925). Our results showed that tissue weakness was not involved in the pathogenesis of infantile inguinal hernia formation, confirming that simple herniotomy is adequate treatment.

Phenotypic Drift in Human Tenocyte Culture

Introduction: Tendon ruptures are increasingly common, repair can be difficult and healing poorly understood. Tissue engineering approaches often require expansion of cell numbers to populate a construct, and maintenance of cell phenotoype is essential for tissue regeneration. Methods: In this study we characterise the phenotype of human Achilles tenocytes and assess how this is affected by passaging. Tenocytes, isolated from tendon samples from 6 patients receiving surgery for rupture of the Achilles tendon, were passaged 8 times. Proliferation rates and cell morphology were recorded at passages 1, 4 and 8. Total collagen, the ratio of collagen types I and III and decorin were used as indicators of matrix formation and expression of the integrin ‘alpha’1 subunit as a marker of cell-matrix interactions. Results: With increasing passage number, cells became more rounded, were more widely spaced at confluence and confluent cell density declined from 18700 /cm2 to 16100 /cm2 (P=0.009). No change to total cell layer collagen was observed but the ratio of type III to type I collagen increased from 0.60 at passage 1 to 0.89 at passage 8 (P Conclusion: We conclude that the phenotype of tenocytes in culture rapidly drifts with progressive passage.

Phenotypic Drift in Human Tenocyte Culture

Tendon ruptures are increasingly common, repair can be difficult, and healing is poorly understood. Tissue engineering approaches often require expansion of cell numbers to populate a construct, and maintenance of cell phenotype is essential for tissue regeneration. Here, we characterize the phenotype of human Achilles tenocytes and assess how this is affected by passaging. Tenocytes, isolated from tendon samples from 6 patients receiving surgery for rupture of the Achilles tendon, were passaged 8 times. Proliferation rates and cell morphology were recorded at passages 1, 4, and 8. Total collagen, the ratio of collagen types I and III, and decorin were used as indicators of matrix formation, and expression of the integrin beta1 subunit as a marker of cell-matrix interactions. With increasing passage number, cells became more rounded, were more widely spaced at confluence, and confluent cell density declined from 18,700/cm2 to 16,100/cm2 ( p = 0.009). No change to total cell layer collagen was observed but the ratio of type III to type I collagen increased from 0.60 at passage 1 to 0.89 at passage 8 ( p < 0.001). Decorin expression significantly decreased with passage number, from 22.9 +/- 3.1 ng/ng of DNA at passage 1, to 9.1 +/- 1.8 ng/ng of DNA at passage 8 ( p < 0.001). Integrin expression did not change. We conclude that the phenotype of tenocytes in culture rapidly drifts with progressive passage.

Collagen I:III ratio in canine heterologous bladder acellular matrix grafts

The objective of this study was to investigate the limitations of a heterologous bladder acellular matrix graft (BAMG) and the influence of the collagen ratio on functional regeneration in a large animal model. Ten female dogs underwent partial cystectomy; eight received BAMG (two homologous; six heterologous) and two partial cystectomy only. A cystometry was performed prior to surgery and 7 months postoperatively when all animals underwent sacral root stimulation. Tissue specimens were studied by histologic and immunohistochemical techniques and for collagen types. At month 7, all animals survived and bladder capacity in the grafted animals was increased. All grafts demonstrated all components of a normal bladder wall. Nerves were seen with the density decreasing with distance from the anastomosis. The BAMG processing and follow-up demonstrated no changes in the homologous tissue, whereas in the heterologous tissue, the amount of collagen changed with the processing during implantation. None of these heterologous specimens demonstrated a similar collagen ratio to the hosts'. The homologous BAMG undergoes more complete regeneration. In the heterologous BAMG, collagen seems not to be replaced. The amounts and ratio of collagen types I and III seem to influence smooth muscle regeneration.

Treatment with Bioscaffold Enhances the the Fibril Morphology and the Collagen Composition of Healing Medial Collateral Ligament in Rabbits

Porcine small intestinal submucosa (SIS) was shown to be an effective bioscaffold in enhancing the mechanical properties of healing medial collateral ligaments (MCL). The purpose of this study was to investigate whether there are corresponding improvements in morphology and tissue compositions. Fourteen rabbits were equally divided into two groups. In the SIS-treated group, a 6 mm gap was surgically created in the right MCL and a layer of SIS was sutured covering the gap. For the nontreated group, the gap-injured MCLs remained untreated. All the left MCLs were sham operated and used as controls. At 12 weeks, the status of collagen types I and V was evaluated with immunofluorescent staining. The collagen type V/I ratios were obtained using SDS-PAGE. Collagen fibril diameters were calculated from the transmission electron micrographs. The results revealed that in the SIS-treated group, the collagen fibers were more regularly aligned as were the cell nuclei. The collagen fibril diameters were 22.2% larger and the ratio of collagen type V/I was 28.4% lower than those for the nontreated group (p < 0.05). These improvements in the morphological characteristics and biochemical constituents of healing MCLs following SIS treatment are the likely reasons for improved mechanical properties.

Long-term effects of porcine small intestine submucosa on the healing of medial collateral ligament: A functional tissue engineering study

Porcine small intestinal submucosa (SIS) was previously shown to enhance the mechanical properties of healing medial collateral ligaments (MCL), and the histomorphological appearance and collagen type V/I ratio were found to be close to those of normal MCL. We hypothesized that at a longer term, 26 weeks, SIS could guide a better organized neo-ligament formation, increasing mechanical properties and increasing collagen fibril diameters mediated by a reduction in collagen type V. A 6 mm gap injury in the right MCL was surgically created in 38 rabbits, while the contralateral intact MCL served as a sham-operated control. In half the animals, a strip of SIS was sutured onto the severed ends. In the other half, no SIS was applied. The cross-sectional area (CSA) was determined with a laser micrometer system. The femur-MCL-tibia complex was mechanically tested in uniaxial tension. Histomorphology was determined through H&E and immunofluorescent staining and transmission electron microscopy (TEM). Sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine collagen type V/I ratio. SIS-treated MCLs displayed a 28% reduction in CSA, a 33% increase in tangent modulus, and a 50% increase in tensile strength compared with the nontreated group (p < 0.05). TEM showed groups of collagen fibrils with larger diameters in the SIS-treated ligaments in comparison with uniformly small fibrils for the nontreated group. H&E staining showed more densely stained collagen fibers in the SIS-treated group aligned along the longitudinal axis with more interspersed spindle-shaped cells. Immunofluorescent staining showed less collagen type V signals, confirmed by a 5% lower ratio of collagen type V/I compared with the nontreated controls (p < 0.05). The findings extend the shorter term 12-week results, and support the potential of porcine SIS as a bioscaffold to enhance ligament healing.

Mechanical Unloading During Left Ventricular Assist Device Support Increases Left Ventricular Collagen Cross-Linking and Myocardial Stiffness

Left ventricular assist devices (LVADs) induce reverse remodeling of the failing heart except for the extracellular matrix, which exhibits additional pathophysiological changes, although their mechanisms and functional consequences are unknown. Hearts were obtained at transplant from patients with idiopathic dilated cardiomyopathy (DCM) not requiring LVAD support (n=30), patients requiring LVAD support (n=16; LVAD duration, 145+/-33 days), and 5 nonfailing hearts. Left (LV) and right ventricular (RV) ex vivo pressure-volume relationships were measured, and chamber and myocardial stiffness constants were determined. Myocardial tissue content of total and cross-linked collagen, collagen types I and III, MMP-1, MMP-9, TIMP-1, and angiotensin (Ang) I and II were measured. LV size, mass, and myocyte diameter decreased after LVAD compared with DCM without LVAD (P<0.05). Total and cross-linked collagen and ratio of type I to III collagen increased in DCM compared with nonfailing hearts and increased further after LVAD (P<0.05 versus DCM and nonfailing). Concomitantly, chamber and myocardial stiffness increased with LVAD. The ratio of MMP-1 to TIMP-1 increased in DCM and almost normalized after LVAD, favoring decreased collagen degradation. Tissue Ang I and II also increased during LVAD. There was no significant change in the RV of LVAD-supported heart compared with DCM. LVAD support increases LV collagen cross-linking and the ratio of collagen type I to III, which is associated with increased myocardial stiffness. Decreased tissue MMP-1-to-TIMP-1 ratio (decreased degradation) and increased Ang levels (stimulants of synthesis) are likely mechanisms for these changes. Lack of significant effects on the RV suggest that hemodynamic unloading of the LV (not provided to the RV) might be the primary factor that regulates these extracellular matrix changes.

Fibroblast growth factor-2 in serum-free medium is a potent mitogen and reduces dedifferentiation of human ear chondrocytes in monolayer culture

The loss of the differentiated phenotype (dedifferentiation) during the expansion culture of donor chondrocytes remains a large problem in cartilage tissue engineering. Dedifferentiated chondrocytes produce other matrix components and therefore the tissue produced will be of less suitable quality. Previously, the addition of fibroblast growth factor-2 (FGF2) to a serum-containing medium (SCM) during expansion culture was shown to have positive effects on the phenotype of articular chondrocytes. In the present study, we focused on a more defined, serum-free medium (SFM), to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Adult human ear chondrocytes were expanded in serum-free medium supplemented with 100 ng/ml FGF2. Expansion culture in a conventional serum-containing medium (10% FCS) served as control. The cell yield during expansion culture in serum-free medium with FGF2 was significantly higher compared to serum-containing medium. In addition, chondrocytes expanded in the serum-free medium with FGF2 expressed a more differentiated phenotype at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I and aggrecan to versican. Also, a higher gene expression of Sox9 was found. Next, suspension in alginate and subsequent culture in vitro or subcutaneous implantation in nude mice was used to evaluate the capacity of the chondrocytes, expanded in either medium, to re-express the differentiated phenotype (redifferentiation) and to form cartilage. The observed beneficial effects of the serum-free medium with FGF2 on the chondrocyte phenotype at the end of monolayer culture were sustained on both transcriptional and extracellular level throughout both redifferentiation methods.

Changes in tendon extracellular matrix composition with age

Introduction A major aspect of the normal ageing process is the loss of suppleness of tissues; skin becomes wrinkled and there is a decline in joint flexibility. These gradual changes predominantly result due to long‐term alterations in the extracellular matrices (ECMs) of structures including skin, tendons/ligaments, bones, cartilage and blood vessels. ECMs are generally remodelled during an individual's life, but for tendons/ligaments this occurs at a slow rate. Parameters such as slow turnover, long‐term post‐translational modifications and extensive cell–matrix interactions are three aspects of ECM biology, which influence the mechanisms of ageing. Ageing of tendon/ligament is a major problem affecting the mobility of an increasingly ageing population, and age‐related changes lead to numerous musculoskeletal pathologies in old age. Therefore, the initial objective of these studies was to compare the biochemical composition of young vs. old tendon, with an aim of elucidating the mechanisms controlling the ageing process in tendon ECM.Materials and methods Tail tendons were dissected from 2‐, 9‐, 12‐ or 22‐month‐old C57/Blacks, and both protein and RNA were analysed for differences in ECM tendon composition with age (n = 4). Proteoglycans were extracted in 4 m guanidine hydrochloride and sulphated glycosaminoglycans measured using the DMMB assay. Collagens were extracted by acid hydrolysis and total collagen measured using the hydroxyproline assay; additionally, the ratio of collagen types in the ageing tail tendons was analysed by Western blotting with antibodies raised against collagen types I, III and V. The degree of tissue hydration was determined by measuring the water content of the tendons. Matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 expression and activation were detected by gelatin substrate zymography, and the presence of their inhibitors was assessed by reverse zymography. To assess age changes at the transcriptional level, RNA was extracted from tendons, labelled with biotin and cDNA arrays (Affymetrix) performed.Results The results indicate that there is a change in tendon matrix composition with increasing age (Table 1). There is a significant loss in tissue hydration and a reduction in the amount of sulphated GAGs present in the 22‐month tendons compared with young tissue, whilst the amount of collagen present does not significantly alter. 2 months 9 months 12 months 22 months sGAG (µg/mg)* 49.6 ± 8.7 — 40.1 ± 4.6 28.9 ± 0.9 Collagen (µg/mg)† 48.4 ± 5.3 39.9 ± 3.4 40.2 ± 4.7 47.2 ± 4.3 Water content (%) 80.4 ± 3.9 74.6 ± 2.4 74.5 ± 3.8 69.3 ± 2.8 µg/mg wet weight tissue. µg/mg dry weight tissue. Analysis of MMP expression by gelatin zymography demonstrated that there was a significant loss of MMP‐9 expression in the tendons of 9‐ and 12‐month‐old animals. MMP‐9 was only evident in the 2‐ and 22‐month‐old tendons indicative of developmental turnover and a remodelling response, respectively. MMP‐2 expression and activation was evident in all tendon ages analysed, as were the MMP inhibitors – TIMP‐1 and TIMP‐2.Discussion The data demonstrate that during the ageing process, the composition of tendon ECM is modified. This may compromise the response of the tissue to application of mechanical loads hence the increased incidences of musculoskeletal injuries, e.g. tendon/ligament sprains in elderly people. The reduction in both water and sGAGs, and the increased expression of MMP‐9 may be responsible for changes observed in biomechanical properties where the tendons become more brittle and less able to withstand load. Understanding changes in the biochemical composition may allow us to manipulate cell activity and ultimately ECM structure and function to combat these age‐related effects and the musculoskeletal pathologies incurred.