Thermal modification of collagen.

Shoulder capsular shrinkage has recently been proposed as a therapeutic modality in a select group of patients with instability. Basic science research studying the mechanism of collagen shrinkage and the effect of shrinkage on the tissue's mechanical properties is essential to define the ideal process by which to achieve optimal tissue shrinkage. Tissue shrinkage is a function of both time and temperature. This relationship was studied, and a model was derived to describe the relationship mathematically. Tissue shrinkage rate was extremely sensitive to temperature changes. The purpose of this study, was to shrink collagenous tissue thermally and then to measure the mechanical property changes as a function of tissue shrinkage. Uniaxial tensile testing of normal and heat-shrunken bovine tendon was carried out, and a model was developed to express the relationship between shrinkage and mechanical properties. We found that the mechanical properties decreased with increasing shrinkage, and that the maximal allowable shrinkage before significant material property changes occurred was between 15% to 20%. Ultrastructural analysis with transmission electron microscopy showed denaturation of the collagen fibrillar structure and provided direct support for the observed material changes.     

Pattern of collagen types and molecular structure of collagen in acute post-traumatic pulmonary fibrosis.

Analyses of collagen types and their amino acid structures have been made with lungs obtained from eight patients who died from the adult respiratory distress syndrome after non-pulmonary trauma. Collagen in lungs from patients with the adult respiratory distress syndrome was twice as soluble as that in control lungs (p less than 0.01). The proportion of type III collagen in the whole organ as well as in the pepsin solubilised fraction was slightly but significantly raised (net increase of about 5-10% type III collagen (p less than 0.05]. Both type I and type III collagen from the patients contained less hydroxylysine than collagen from control lungs. The alterations in tissue composition described here and observed in normal wound healing support the notion that acute post-traumatic pulmonary fibrosis resembles a wound healing process in the lungs.     

Microfibrillar collagen in the oval window.

Compressed microfibrillar collagen was used to seal openings in cat oval windows. Histologic examination showed the material was well tolerated and produced a good oval window seal. Because of its hemostatic properties, this material should prove to be useful when bleeding is encountered during a stapedectomy.     

Spectroscopic characterization of collagen cross-links in bone.

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.     

Type VII collagen in Alport syndrome.

BACKGROUND: Absence or segmental distribution of the alpha5(IV) collagen chain along the epidermal basement membrane (EBM) is diagnostic of X-linked Alport syndrome (X-AS), but the typical morphologic alterations usually observed along the glomerular basement membrane (GBM) are lacking. However, several differences in protein composition exist between GBM and EBM, and such differences could account for a different phenotype with the same genetic defect. Type VII collagen is one of the major collagenous components of the EBM; the purpose of this study was to investigate the modifications of protein synthesis and expression of type VII collagen in the skin of patients with X-AS. METHODS: The distribution of type VII collagen has been studied in 15 skin biopsies (10 from X-AS patients and 5 controls) by means of electron microscopy, immunofluorescence and confocal microscopy; type VII collagen mRNA expression was also measured by RT-PCR on the same skin fragments. RESULTS: Protein and mRNA amounts for type VII collagen were significantly higher in skin samples from X-AS patients than in controls (P < 0.001); highest values were in cases in which alpha5(IV) was completely absent. CONCLUSIONS: Our results indicate that lack of alpha5(IV) molecule significantly alters the assembly of extracellular matrix molecules other than alphax(IV) chains also at the EBM level. We suggest that the increased synthesis and deposition of type VII collagen is likely to balance the absence of stabilizing activity normally exerted by alpha5(IV).     

Porcine dermal collagen graft in abdominal-wall reconstruction.

We describe the use of a porcine dermal collagen graft in the reconstruction of a large abdominal-wall defect in a woman. The graft was not rejected and, after 1 year, was not associated with incisional hernia. This graft may become an alternative to synthetic-mesh and flap reconstructions because, despite being of a similar tensile strength, it promotes less adhesion, is incorporated into the host tissue and is less prone to infection.     

Bone fragility and collagen cross-links.

Infrared imaging analysis of iliac crest biopsy specimens from patients with osteoporotic and multiple spontaneous fractures shows significant differences in the spatial variation of the nonreducible:reducible collagen cross-links at bone-forming trabecular surfaces compared with normal bone. INTRODUCTION: Although the role of BMC and bone mineral quality in determining fracture risk has been extensively studied, considerably less attention has been paid to the quality of collagen in fragile bone. MATERIALS AND METHODS: In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections, from human iliac crest biopsy specimens (N = 27) at bone-forming trabecular surfaces. The biopsy specimens were obtained from patients that had been diagnosed as high- or low-turnover osteoporosis, as well as premenopausal women <40 years of age, with normal BMD and biochemistry, who suffered multiple spontaneous fractures. The obtained values were compared with previously published analyses of trabecular bone from normal non-osteoporotic subjects (N = 14, 6 males and 8 females; age range, 51-70 years). RESULTS AND CONCLUSIONS: Collagen cross-links distribution within the first 50 microm at forming trabecular surfaces in patients with fragile bone was markedly different compared with normal bone.     

Alteration of collagen IV in acutely deteriorated renal allografts.

BACKGROUNDS: The changes in the basement membrane occurring in acutely deteriorated renal allografts (ADR) have not been extensively investigated. Our purpose is to elucidate the alteration of collagen IV, a main constituent of the basement membrane in ADR. METHODS: Fifty biopsy specimens of ADR and 10 of chronic transplant nephropathy (CTN) were examined with two monoclonal antibodies specific for collagen IV. JK199 and JK132 are monoclonal antibodies that recognize triple helical collagen IV containing the alpha1 chain. JK199 recognizes all the basement membrane containing [alpha1 (IV)]2alpha2(IV), although JK132 reacts only with a limited portion of it. In the normal kidney, JK199 reacts with the mesangial matrix, the basement membrane of Bowman's capsule (BBM), and the tubular basement membrane, as well as with the glomelular basement membrane (GBM). JK132 reacts with the mesangial matrix, BBM, and the tubular basement membrane. RESULTS: In ADR, increased intensity of JK199 was observed in GBM, the mesangial matrix, BBM, the tubular basement membrane, and the interstitium. Increased intensity of JK132 was observed in the mesangial matrix, BBM, and the tubular basement membrane, but was not remarkable in GBM or the interstitium. In contrast, biopsy specimens of CTN showed increased intensity of JK132 in GBM, the mesangial matrix, BBM, the tubular basement membrane and the interstitium. CONCLUSION: These results suggest that collagen IV is up-regulated in ADR. Differential staining of collagen IV with JK199 and JK132 in GBM and the interstitium may contribute to diagnose CTN.     

Apparent accelerated aging of human collagen in diabetes mellitus.

The chronologic ages of human subjects were determined experimentally by enzymatic digestion of tendon collagen samples. Determined age closely matched actual age for individuals dying with a variety of major diseases. Juvenile diabetics did not fit this pattern; their experimentally determined ages were significantly greater than their actual ages. This raises the possibility of relationships between diabetes mellitus, changes in connective tissue, and accelerated aging.     

Segregation of structural collagen genes in adolescent idiopathic scoliosis.

The etiology of idiopathic scoliosis remains unknown. The condition results in a characteristic deformity of the spine and surrounding tissues. Both Types I and II collagen are important constituents of the affected tissues, and thus defective collagens are reasonable candidates for the primary abnormality in adolescent idiopathic scoliosis (AIS). Direct analyses of the amount and solubility of collagen have revealed differences between normal individuals and those with AIS. However, these changes may be secondary to the mechanical effects of the spinal deformity. Segregation analysis was done of genetic markers linked to the structural genes encoding Types I and II collagen to test these candidate loci in four pedigrees with dominantly inherited AIS. In one pedigree, markers linked to both of the Type I collagen loci (COL1A1 and COL1A2) were found to be inherited independently of the abnormal phenotype. Two pedigrees were discordant at one of the Type I loci. The condition also segregated independently of the locus for Type II collagen (COL2A1) in three pedigrees. This is evidence against idiopathic scoliosis generally being caused by mutations in the Types I and II collagen genes.     

Distribution of collagen cross-links in normal human trabecular bone.

Infrared imaging analysis of normal human iliac crest biopsy specimens shows a characteristic spatial variation in the nonreducible:reducible collagen cross-links at trabecular surfaces, depending on the surfaces' metabolic status. INTRODUCTION: Bone is a composite material consisting of mineral, collagen, non-collagenous proteins, and lipids. Bone collagen, mainly type I, provides the scaffold on which mineral is deposited and imparts specific mechanical properties, determined in part by the amount of collagen present, its orientation and fibril diameter, and the distribution of its cross-links. MATERIALS AND METHODS: In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections from human iliac crest biopsy specimens (N = 14) at trabecular surfaces as a function of surface activity (forming versus resorbing), with an approximately 6.3-mm spatial resolution. The biopsy specimens were obtained from patients devoid of any metabolic bone disease based on histomorphometric and bone densitometric parameters. RESULTS AND CONCLUSIONS: Distributions of collagen cross-links within the first 50 mm at forming trabecular surfaces demonstrated a progressive increase in the nonreducible:reducible collagen cross-link ratio, unlike in the case of resorbing surfaces, in which the collagen cross-links ratio (as defined for the purposes of the present report) was relatively constant.     

Changes in type I collagen following laser welding.

Selection of ideal laser parameters for tissue welding is inhibited by poor understanding of the mechanism. We investigated structural changes in collagen molecules extracted from rat tail tendon (> 90% type I collagen) after tissue welding using an 808 nm diode laser and indocyanine green dye applied to the weld site. Mobility patterns on SDS-PAGE were identical in the lasered and untreated tendon extracts with urea or acetic acid. Pepsin incubation after acetic acid extraction revealed a reduction of collagen alpha and beta bands in lasered compared with untreated specimens. Circular dichroism studies of rat tail tendon showed absence of helical structure in collagen from lasered tendon. No evidence for covalent bonding was present in laser-treated tissues. Collagen molecules are denatured by the laser wavelength and parameters used in this study. No significant amount of helical structure is regenerated on cooling. We conclude that non-covalent interactions between denatured collagen molecules may be responsible for the creation of tissue welding.     

Causes of mechanically induced collagen damage in articular cartilage.

Osteoarthritis (OA) is a multifactorial disease, associated with articular cartilage degeneration and eventually joint destruction. The phases of the disease have been described in detail, and mechanical factors play an important role in the initiation of OA, but many questions remain about its etiology. Swelling of cartilage, one of the earliest signs of damage, is proportional to the amount of collagen damage. This strongly suggests that damage to the collagen network is an early event in cartilage degeneration. The goal of this study was to determine the mechanical cause of early collagen damage in articular cartilage after mechanical overloading. Both the shear strain along the fibrils and the maximum fibril strains were evaluated as possible candidates for causing collagen damage. This evaluation was done by comparing the locations of maximum shear and tensile strains with the locations of initial collagen damage after mechanical overloading in bovine explants as found using antibodies directed against denatured type II collagen (Col2-3/4M). Collagen damage could be initiated by excessive shear strains along the collagen fibrils, and by excessive fibrils strains. The locations of collagen damage after mechanical overloading were highly dependent on the cartilage thickness, with thinner cartilage being more susceptible to damage than thicker samples.     

Synthesis of type I collagen in healing wounds in humans.

To quantify wound healing in surgical patients, samples of wound fluid were collected through a silicone rubber tube for 7 postoperative days and their concentrations of the carboxyterminal propeptide of type I procollagen (PICP) and the aminoterminal propeptide of type III procollagen (PIIINP) were measured with specific radioimmunoassays. The mean concentration of PICP in would fluid on day 1 was 207 +/- 92 (SD) micrograms/L, and on day 2 908 +/- 469 micrograms/L (p less than 0.001, signed rank test). On day 7, the mean concentration reached was 380 times higher than that of day 1 (79,330 +/- 54,151 micrograms/L). Only one peak of PICP antigenicity, corresponding to the intact propeptide as set free during synthesis of type I procollagen, was detected on Sephacryl S-300 gel filtration analysis of wound fluid samples. The mean concentration of PIIINP was 70 +/- 61 micrograms/L on day 1, 86 +/- 88 micrograms/L on day 2, and 180 +/- 129 micrograms/L on day 3 (p less than 0.001 when compared with day 1). Finally on day 7, a 250-fold concentration (17,812 +/- 9839 micrograms/L), compared with day 1, was reached. Methods described in the present paper allow separate and repetitive quantification of the synthesis of both type I and type III procollagen during human wound healing.     

The role of collagen in determining bone mechanical properties.

The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37-200 degrees C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three-point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone, thereby supporting the hypothesis of this study.     

衰老真皮成纤维细胞胶原基因调控研究

目的针对衰老真皮成纤维细胞胶原基因表达减少的特点，应用胎儿皮肤源性活性蛋白（ＳＣＦ）进行调控，以增加胶原基因的表达。方法应用真皮成纤维细胞培养、胶原基因ｍＲＮＡ裂隙杂交和图像分析技术，观察胎儿皮肤源性活性蛋白对真皮胶原基因的调控作用。结果以Ａｃｔｉｎ作为对照，ＳＣＦ对老人成纤维细胞胶原基因有明显的调控作用，可以使Ⅰ、Ⅲ型胶原基因表达增加，处理后，Ｉ型胶原基因表达增加１５０％，Ⅲ型胶原基因增加５０％。结论ＳＣＦ可以促进衰老真皮成纤维细胞胶原基因的表达。