Abnormal procollagen synthesis in fibroblasts from three patients of the same family with a severe form of osteogenesis imperfecta (type III).

Dermal fibroblast cultures from three siblings with a severe form of osteogenesis imperfecta were established in order to analyze their procollagen and collagen synthesis. Cell strains from clinically normal consanguineous parents (first cousins), were also obtained for comparison. Total collagen production in culture media was diminished by 55% in the patients fibroblasts and to a lesser extent in the parents. This decrease was specific for collagenous proteins. From polyacrylamide gel electrophoresis, it appeared that the three children had not only the same defective secretion of pro alpha 1(I) molecules but that their pro alpha 1(I) migrated slightly faster than the parental and control counterparts. Analysis of secretion confirmed a reduced rate in procollagen synthesis and the absence of intracellular storage. Upon pepsin treatment, extracellular alpha 1(I) and alpha 2(I) chains were found in the expected ratio of 2:1 and migrated normally, suggesting that the altered mobility of pro alpha 1(I) chains was related to COOH or NH2 terminal propeptides. In agreement with the reduced type I collagen production, an increase in the alpha 1(III)/alpha 1(I) ratio was also detected. Furthermore, after a 2.5-h labelling followed by alkylation with iodoacetamide, free intracellular pro alpha 2(I) and alpha 1(I) chains were detected in the absence of reduction, consistent with an abnormal intracellular ratio of pro alpha 1(I)/pro alpha 2(I) that was measured after dithiothreitol reduction. Analysis of intracellular collagen chains from parental strains following a 4-h incubation demonstrated that pro alpha 1(I) appeared as a doublet, one band with normal mobility and a less intense band migrating faster and corresponding to the defective chain found in the patients. Absence of the abnormal molecules in culture media was related to the demonstration of a defective collagen secretion by parental fibroblasts. Correlation between these biochemical findings and clinical data strongly support a recessive inheritance of the disease that could be classified as a type III form of osteogenesis imperfecta. Patients would be homozygous for the same defective allele and the asymptomatic parents would most likely be heterozygous carriers of the mutation. Although the exact location of the alteration is not yet elucidated, a splicing mutation is suggested.

Influence of collagen denaturation on the chemorheological properties of skin, assessed by differential scanning calorimetry and hydrothermal isometric tension measurement.

The curves obtained for skin samples of different ages and species by hydrothermal isometric tension ("HIT") measurement are compared to those obtained by differential scanning calorimetry (DSC) under the same thermal conditions (for a rise in temperature at a rate of 1.0 degrees C/min). Collagen denaturation, observed by DSC, directly affects the kinetics of the tension variations in the first part of the "HIT" curves, including the early peak due to the presence and destruction of the heat-labile cross-links in the collagen network. The presence of cross-links is in term shown to delay collagen denaturation to an extent which depends in part on their heat-stability. The final part of the "HIT" curves reflecting the effects of heat in the stable polymeric collagen network is no longer affected by collagen denaturation. Thus, both "HIT" and DSC are useful methods to evaluate collagen reticulation in connective tissues.

Type II collagen defect in two sibs with the Goldblatt syndrome, a chondrodysplasia with dentinogenesis imperfecta, and joint laxity.

We report on a syndrome of spondylo-epimetaphyseal dysplasia, dentinogenesis imperfecta, and ligamentous hyperextensibility in two sibs born to nonconsanguineous parents. This chondrodysplasia was characterized by severe shortness of stature and an osteoporosis without fractures. Electron microscopic examination of the cartilage documented large vacuoles of dilated rough endoplasmic reticulum within the cytoplasm of chondrocytes. Gel electrophoresis of pepsin-soluble collagen extracted from cartilage demonstrated the presence of type II collagen chains with an abnormal mobility. Prolyl and lysyl hydroxylations were slightly increased. The abnormal molecules melted at a higher temperature than the normal ones. CNBr peptide mapping of type II collagen showed an altered electrophoretic migration of peptides CB 11, CB 8, and CB 10,5 whereas CB 9,7 looked normal. In addition, two small non-collagenous proteins isolated from cartilage were not found in an age-matched control individual but were detected in a normal newborn infant. The quantitation of proline-labelled collagen synthesized by dermal fibroblasts demonstrated a 50% reduction of total collagen. This decrease essentially affected the amount of extracellular type I collagen, which was secreted less efficiently than in control cells. Nevertheless, type I collagen chains behaved normally on 5% polyacrylamide gels. The reduced mRNA levels of alpha 1I and alpha 2I chains might reflect either a transcriptional defect or a decreased stability of mRNA transcripts. We suggest that the association of both pathological chondrocytes producing altered collagen type II and decreased synthesis of type I could be responsible for this peculiar phenotype. The overmodification of alpha 1II CNBr peptides is consistent with the presence of a single-base substitution in the COL2A1 gene. Whether there is a direct causal relationship between the type II collagen defect and the underexpression of type I collagen will require clarification.

A new lethal brittle bone syndrome with increased amount of type V collagen in a patient.

A new lethal brittle bone disease is described in three patients with slender long bones, thin ribs, hypomineralized calvaria, and normal facial appearance. In spite of several limb fractures this syndrome can be differentiated from the lethal forms of osteogenesis imperfecta and is better related to the thin-bone group of lethal dysplasias. Biochemical investigation of collagen from one of the patients by the use of gel electrophoresis and high-pressure liquid chromatography analyses failed to demonstrate any evident defect in the structure of type I collagen chains. Nevertheless collagen extractability from the dermis was altered owing to an increase in the proportion of acid-soluble material. Tritium-proline labeling of cultured fibroblasts confirmed the reduction in total collagen synthesis. This was attributed to a lower type I and type III amount whereas type V collagen level was markedly increased in the cell layer. RNA analysis of the three collagen types with the appropriate cDNA probes confirmed the protein data. Electron microscopic examination of bone and skin showed morphologically abnormal fibroblasts and osteoblasts with an abundant distended rough endoplasmic reticulum and an altered plasma membrane. Unexpected thin fibrils with a banding pattern and surrounding the type I fibrils were observed. They might represent type V collagen. We suggest that, in this patient, the moderate decrease in type I collagen amount is insufficient to account for the radiological findings and that type V collagen overproduction could play a role in the bone brittleness by interfering with the process of mineralization.

Observations on the staining of ceruloplasmin following disc-electrophoresis utilizing polyacrylamide gels.

In our hands the dianisidine technique for the staining of ceruloplasmin in polyacrylamide gels following disc electrophoresis has proven unsatisfactory for three reasons. First, staining of ceruloplasmin could not be achieved with physiological amounts of the glycoglobulin present in serum. Second, even when relatively massive quantities of pure ceruloplasmin permitted effective staining there was loss of stain intensity with increasing electrophoresis time. Third, the necessity of utilizing dianisidine in alcoholic solution produces undesirable shrinkage and distortion of the gels. However, a modification of the p-phenylenediamine technique for the staining of ceruloplasmin in agar gels has been found to stain ceruloplasmin very effectively in polyacrylamide gels following disc electrophoresis.

[Tissular collagen polymerization (author's transl)]. / Polymérisation du collagène tissulaire.

The type of collagen reticulation exerts an influence on biological, physiological and mechanical properties of tissues, thus the utility of its study. Chemical techniques allow both localization and evaluation of levels and origin (Schiff base or aldol) of reducible bonds in collagen. Physical techniques allow evaluation of the thermolability or thermostability, as well as the degree of collagen reticulation in a given tissue for all collagen bridges. Results by these methods have shown that the mode of tissular collagen reticulation evolves from the embryonic phase to senescence, the rate varying as a function of the tissue and species of the donor. Thus, during tissular reparation, the newly formed collagen network itself undergoes transformations. Cicatricial tissue thus gives rise to an anisotropic zone in particular in terms of mechanical properties, in the organ. This can have grave pathological consequences. In addition to these wide areas of study, a certain number of specific diseases, frequently genetic, show pathological changes in collagen polymerization, the molecular mechanisms for which are now becoming better known.

[Action of carbon tetrachloride on the quality of hepatic collagen]. / Effet exercé par du tétrachlourure de carbone sur la qualilé du collagène hépatique

The collagen, abundantly accumulated in the liver of carbon tetrachloride treated rats, is highly resistant to dissociative agents, indicating a stron cross-linkage. However, even after a very long treatment, such collagen can be resorbed.

Age related evolution of stable collagen reticulation in human skin.

The relaxation time of hydrothermal isometric tensions (HIT) was measured in different tissues during collagen hydrolysis. This hydrolysis was shown to have the same activation energy in all skin samples studied, whatever the species and age. Therefore, variations in the HIT relaxation half-time at the boiling point, exclusively reflected variations in the reticulation degree of the stable infinite network of denatured collagen. Stable collagen reticulation was studied in human skin from birth to 70 yrs of age in 113 biopsies, by measuring the relaxation half-time of hydrothermal isometric tension (HIT) at boiling point. This time was observed to vary in three different ways as a function of age: 1) from birth to eight or 10 yrs and from 14-15 yrs until adulthood, it increased constantly, and at the same rate; 2) during early puberty, the striking observation was the dramatic decrease of relaxation time value which remained low for 4-5 yrs thereafter, and 3) from adulthood to 70 yrs of age, relaxation time values were scattered along several plateaux, apparently separated by similar incremental steps. A parallel study on rat and pig skin showed a similar age-related evolution for collagen stable reticulation i.e., a sudden drop of reticulation at puberty, and low values for several months thereafter, followed by a linear increase, at the same rate as that observed for human skin. The study of collagen reticulation in various pathological situations may help to clarify and increase our understanding of the different mechanisms involved in collagen modifications during in vivo growth and aging.

Consequences of nonpenetrating projectile impact on a protected head: study of rear effects of protections.

BACKGROUND: Police and armed forces have helmets that can now stop handgun bullets and even a certain category of rifle bullets. The trend is to increase the ballistic limits of helmets, but injuries caused by nonpenetrating impacts are not well understood. The helmet defeats the projectile and creates a local cone of deformation that impacts the head a second time. The term "rear effects" describes the behind-armor blunt trauma caused by the nonpenetrating impact. METHODS: To analyze rear effects on the skull, an experimental study was associated with parametric simulations on a three-dimensional finite element model. Transfer of energy throughout the head was tested on 30 human skulls filled with a silicone gel. The magnitude of contact forces on the skull surface and the pressure levels in the skull were recorded during a reference impact. RESULTS: A biomedical approach by pathologic findings and radiographs showed very localized fractures. The protection brought by the diploe in the multilayered bone was confirmed and characterized by numerical simulations. CONCLUSION: This first step toward a better understanding of the rear effect phenomenon in relation to its consequences on brain tissue will lead to the design of more efficient protections.

[Variations in the hydrothermic contraction of the rat skin as a function of the age of the animals]. / Variations de la contraction hydrothermique de la peau du Rat en fonction d l'âge des animaux.

The tension developed in Rat skin by hydrothermal shrinkage is modified with ageing of the animals: the temperature of maximum tension decreases from birth to 1 month, then very slowly increases with age. At higher temperatures than that of maximum tension a relaxation occurs very rapidly in young Rats and in not yet senescent adult skins. These modifications appeared to be related to those of the nature of the intermolecular collagen cross-links with ageing.

Hydrothermal isometric tension curves from different connective tissues. Role of collagen genetic types and noncollagenous components.

Variations in hydrothermal isometric tension (HIT) were recorded in tendons, ligaments, skin, blood vessels, nerves, palatal mucosa, lungs, muscles, cartilages, demineralized bones and dentine from donors of different ages and species. The curves obtained during a linear rise in temperature from 37 degrees C to 100 degrees C at a rate of 1.15 degrees C/min were classified into three major families, A, B and C, depending on whether these curves displayed an early maximum, two shoulders or a late maximum. The ratio of heat-labile to heat-stable cross-links in the tissue's collagen network was shown to determine the type of curve obtained, but the genetic types of collagen in the tissue and the amount and quality of its noncollagenous components were not important in this respect. These results are discussed in the light of the accepted view that HIT variations are due to the rubber-elastic properties of gelatin.

Isometric tensions developed during the hydrothermal swelling of rat skin.

Isometric tensions developed in connective tissue under the influence of temperature have previously been measured by their effect on a "shrinkage" phenomenon observed along the main axis of the collagen fibers. A new device, built and tested in our laboratory enabled us to obtain isometric tension curves by measuring a "swelling" phenomenon observed in other directions. Curves recorded in the two systems have been compared. Shapes were unchanged and the parameters chosen to define these morphologies showed the same values in both types of experiments. The parameters, which are independent of maximal tension, were also found to be independent of the total collagen content of the sample. Interpretation of the results suggests that these parameters depend not only on the nature but also on the density of the collagen crosslinks. The new device is applicable to minute tissue samples, whatever their fragility and fiber orientation.

The rate of collagen maturation in rat and human skin.

Hydrothermal Isometric Tension (HIT) relaxation was observed at the boiling point in rat, cat and human skin samples of different ages, after raising the temperature from 37 degrees to 100 degrees C at a rate of 1.15 degrees C/min. A Maxwellian relaxation to nearly zero was observed at the boiling point. A given velocity constant characterized a given tissue whether experiments were performed under pressure or under tensile stress. However, the velocity constant of the relaxation decreased as a function of age for all three species during the period studied. Moreover, the velocity constant decreased at the same constant rate for skin samples of cats aged 1 to nearly 5 years, of rats aged from 9 to 28 months and of children from birth to 10 years. We propose a model, based on the well known rubber-elastic properties of the denatured collagen network. In this model (1) HIT decreases as an exponential function of time during bond scission along the polymeric chains; (2) the velocity constant of the relaxation process is proportional to the rate of bond rupture and inversely proportional to the number of stable polymeric chains originally present per unit volume; (3) the evolution of the velocity constant as a function of aging finds an explanation. The HIT test should find useful applications in pathology and pharmacology, since it provides rapid, precise information on the stable state of collagen reticulation in small biopsies.

Tendon adaptation to different long term stresses and collagen reticulation in soleus muscle.

Leg immobilization with or without soleus muscle denervation was studied in young rabbits. Muscle and tendon were maintained in extension, i.e., in the most lengthened position. Length measurements performed on sarcomeres, muscle fibers and tendon-plus-muscle complexes suggest the following progression in tendon growth rates: normal less than denervated-extended less than innervated-extended. Collagen reticulation was studied as a function of fiber location along these tendons by measuring hydrothermal isometric tension (HIT). Large variations were observed depending on fiber location, and significant modifications were induced by immobilization in extension. The results are discussed in relation to stress as a possible factor controlling collagen maturation in connective tissues.