Collagen Protein Abnormalities Produced by Site-Directed Mutagenesis of the Proal(I) Gene

Site-directed mutagenesis of collagen genes offers a powerful new approach for studying structure-function relationships. The construction of engineered mutant collagen genes coding for glycine substitutions and their expression giving rise to the osteogenesis imperfecta type II phenotype in cells and transgenic mice has recently been achieved^I. This paper further defines the molecular abnormalities of collagen and bone pathology resulting from the expression of the mutant genes.     

Osteoporosis and Familial Idiopathic Scoliosis: Association with an Abnormal Alpha 2(I) Collagen

A positive family history is considered a risk factor for osteoporosis (OP) although the genetic or biochemical basis for this relationship remains undefined. Various mutations affecting normal synthesis of type I collagen have been reported in osteogenesis imperfecta (OI), a heritable disorder of connective tissue. Family A, in which the proband and a daughter are afflicted with OP and idiopathic scoliosis was examined for defects in collagen metabolism. Dermal fibroblast cultures were established to investigate de novo collagen synthesis. SDS-PAGE revealed an abnormally migrating alpha 2(I) chain and procollagen in two generations. Examination of the kinetics of type I collagen pC & N-propeptide processing demonstrated a rate 2x control in the proband. The phenotype family A is not OI. It shares features with families B & C, having familial clustering of OP. However, collagen synthesis was not abnormal in family B & C. These data suggest that in family A the alpha 2(I) structural defect may be related to defective skeletal matrix formation.     

Osteoblastic Behavior of Human Bone Marrow Cells Cultured Over Adsorbed Collagen Layer,Over Surface of Collagen Gels,and Inside Collagen Gels

While collagen type I is often used as a substrate for cell culturing and as a coating in biomedical implants, as far as we know a simple systematic study comparing the effects of the different presentations of collagen type I on the osteoblastic behavior of cells is missing. In this work, human bone marrow cells (hBMCs) were cultured under osteoblastic-inducing conditions, for 21 days, over a layer of adsorbed collagen (monomeric) and on the surface and inside collagen gels (fibrillar). Comparison was made based on three classical parameters; cell proliferation/viability, alkaline phosphatase (ALP) activity, and production of mineral deposits. The three types of collagen type I substrates allowed the adhesion, proliferation, and the osteoblastic differentiation of cells. However, hBMCs behavior was influenced by the monomeric/fibrillar and 2-/3-dimensional nature of the collagen substrates, namely: monomeric collagen favored cell attachment; cells on 2D substrates presented higher proliferation rates during the exponential phase of growth with formation of spiral-like multilayered structures; cells seeded inside 3D collagen gels formed a regular dense cellular mesh and had a low proliferating rate; cells cultured over or inside fibrillar collagen differentiated faster, with the 3D cultures presenting higher levels of ALP activity; and the extension of mineralization was greater for the cultures done over or inside fibrillar collagen. Thus, cells cultured over collagen gels showed both the ability for cell proliferation and for earlier differentiation, a fact that can be exploited in the biomaterials field.     

Short-term efficacy of monthly pamidronate infusion in patients with osteogenesis imperfecta

We evaluated the efficacy of a monthly infusion of pamidronate on the frequency of fractures, biochemical effects, and bone mineral density in children with osteogenesis imperfecta. Eleven patients from 0.9 to 13.8 yr of age were included in this study. The patients were administered pamidronate intravenously (30 mg/m(2)) over a 4-hr period monthly for a period ranging from 6 to 37 months. Height and weight Z-scores did not change significantly. The frequency of fractures was decreased from 2.3+/-1.01 times per year before treatment to 0.6+/-0.69 times per year during treatment. There were no long-term changes in biochemical markers during pamidronate therapy. The mean bone mineral density of the spine and femur increased significantly. Monthly intravenous pamidronate therapy decreased frequencies of fracture and increased bone mineral density without significant adverse events in Korean patients with osteogenesis imperfecta.     

Odontoblast Dysfunction in Osteogenesis Imperfecta: An LM,SEM, and Ultrastructural Study

The inherited dentin defect dentinogenesis imperfecta (DI), while clinically obvious in osteogenesis imperfecta (OI) Types IB and IC, II, III, and IVB, is now thought to be present in all children with OI, in a continuum from minimal to severe dentin pathology. This collaborative study further clarifies the structural and ultrastructural dentin changes in the teeth of OI children with clinically obvious DI, and attempts to explain these in terms of odontoblast dysfunction. Collaborative studies were carried out in Melbourne, Australia, and Strasbourg, France, using light and polarized-light microscopy, scanning and transmission electron microscopy (SEM, TEM), selected-area diffraction (SAD), and x-ray spectroscopy (EDX). These showed structurally normal enamel (but containing long and broad lamellae) and a normally scalloped dentino-enamel junction (DEJ), but severe pathologic changes in the dentin. An initial narrow band of normal-appearing dentin tubules (including the mantle layer) ceased abruptly and was replaced by a wavelike laminar zone parallel to the DEJ with occluded tubules. Multiple parallel channels of 5-10 &#119 m diameter were present at right angles to the DEJ indenting this zone, some terminating in retro-curved "processes." The abnormal dentin containing these channels almost completely occluded the pulp chamber. The structural and ultrastructural changes seen can be explained on the basis of the collagen defect in OI resulting in odontoblast dysfunction, which produces a distinct phenotype and one that is different from that in bone.     

Increase of Interstitial Collagen in the Mouse Endometrium During Decidualization

Decidualization in the mouse consists of an extensive remodeling of the endometrial extracellular matrix, resulting in a reduction of the extracellular spaces, an increase in the diameter of collagen fibrils, and changes in the relative ratio of different types of glycosaminoglycans. To assess the dynamic changes of the endometrial extracellular matrix during decidualization, collagen was analyzed biochemically and immunochemically in the endometrium of nulliparous and day 5 to day 8 pregnant mice. The amount of collagen per gram dry weight was higher in the endometrium of implantation sites than in interimplantation sites. Collagen types I, III, and V were the main components of the endometrium of nulliparous and pregnant animals. The amount of collagen type V was higher in the endometrium of pregnant animals than in nulliparous ones. A relative unusual homotrimeric form of collagen type V, probably formed by [ &#102 1(V)] 3 , was detected in pregnant endometrium by gel eletrophoresis and immunoblotting.     

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation.     

Age-Related Changes of Noncalcified Collagen in Human Cortical Bone

It is well known that osteoid, which contains a noncalcified collagenous matrix, is formed during the initial stage of bone formation during the bone remodeling process. Thus, synthesis of defective collagen molecules in osteoid may cause abnormal bone formation, thereby leading to changes in bone quality. The objective of this study was to investigate age-related changes in noncalcified collagen molecules in osteoid and its likely effects on the mechanical integrity of human cortical bone. Thirty human cadaveric femurs were divided into three age groups: young adults, middle age, and the elderly, respectively. A novel high performance liquid chromatography approach was employed to quantify the denaturation of noncalcified collagenous matrix in addition to mechanical tests of bone. Bulk concentrations of both enzymatic and nonenzymatic collagen cross links in bone also were measured. Moreover, the number of newly formed osteons per unit area and the bony area fraction of these osteons were estimated. The results of this study indicate that denaturation of the noncalcified collagenous matrix in bone increases with increasing age. In addition, such collagen denaturation in osteoid exhibited a correlation with nonenzymatic collagen cross links as well as the strength and toughness of bone. These results suggest that age-related changes in the noncalcified collagenous matrix induced by bone remodeling may have likely effects on bone quality. © 2003 Biomedical Engineering Society. PAC2003: 8719Rr, 8718La, 8280Bg     

胶原海绵及其止血性能的研究

利用酸碱溶解法，从牛腱中提取了可溶性胶原材料。冻干法制得了胶原海绵止血材料。对胶原材料的氨基酸分析和紫外吸收光谱分析，证实了可溶性胶原的结构和氨基酸组成。物理性能测试和止血效果实验结果表明，胶原海绵亲水性强，而且具有优良的止血和粘附创而的能力。     

胶原对TTCP/MCPM骨水泥性能的影响

以混合后钙磷比为1.67的磷酸四钙(TTCP)和一水磷酸二氢钙(MCPM)粉体为原料,按固液比3∶1分别用水和5.24 mg/ml自制Ⅰ型胶原溶胶作固化液制备骨水泥试样,测定其凝结时间和抗压强度。结果显示,用胶原作固化液可使TTCP/MCPM骨水泥的抗压强度由17.8±1.9 MPa增至22.7±1.6 MPa,对凝结时间则没有明显影响;两种试样经模拟体液(SBF)浸泡抗压强度均有增加,尤以胶原溶胶固化的试样增幅较大,SBF浸泡4 d和14 d,抗压强度分别增至31.8±3.9 MPa(胶原溶胶)/19.5±1.3 MPa(水)和38.1±3.1 MPa/21.9±2.2 MPa。对比胶原矿化前后的红外谱图发现,矿化后胶原的酰胺Ⅰ带特征峰红移,酰胺Ⅱ带、Ⅲ带几乎消失,提示在胶原与羟基磷灰石(HA)之间发生了明显的化学作用,这应是胶原对TTCP/MCPM骨水泥增强作用的基础;而SBF浸泡前后试样表面的SEM和XRD图谱则显示,SBF浸泡在使透钙磷石(DCPD)转化为HA的同时,又沉积生成了大量新的HA,使试样表面更加致密、光滑,这既是SBF浸泡增强的机理,也揭示TTCP/MCPM骨水泥的凝结硬化是先生成DCPD...     

Quantification of the Temporal Evolution of Collagen Orientation in Mechanically Conditioned Engineered Cardiovascular Tissues

Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network. The work was performed at the Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.     

Development of Ligament-Like Structural Organization and Properties in Cell-Seeded Collagen Scaffolds in vitro

Acute anterior cruciate ligament (ACL) injuries lead to poor joint function, instability, and eventually osteoarthritis if left untreated. Current surgical treatment options are not ideal; however, tissue engineering may provide mechanically sound, biocompatible reconstructions. Collagen fiber scaffolds were combined with fibroblast-seeded collagen gels and maintained in culture for up to 20 days. The tensile and viscoelastic behavior of the constructs closely mimicked that of natural ligament. Constructs’ mechanical and viscoelastic properties did not degrade over time in culture, and peak stress was significantly higher for constructs with embedded fibroblasts. Immunocytochemical and histological analyses demonstrated cell proliferation and ligament-like organization. We have created an engineered tissue that closely approaches key mechanical and viscoelastic properties of the ACL, does not degrade after 20 days in culture, and is histologically similar to the native tissue. This study should aid in developing effective treatments for ACL injury.     

Properties of collagen in OIM mouse tissues

The deletion of the alpha2 chain from type I collagen in the oim mouse model of osteogenesis imperfecta has been shown to result in a significant reduction in the mechanical strength of the tail tendon and bone tissue. However, the exact role of the alpha2 chain in reducing the mechanical properties is not clear. We now report that the stabilizing intermolecular cross-links in bone are significantly reduced by 27%, thereby contributing to the loss of tensile strength and the change in stress-strain profile. We also report that, in contrast to previous studies, the denaturation temperature of the triple helical molecule and the intact fibers are 2.6 degrees and 1.9 degrees C higher than the corresponding tail tendon collagen from wild-type mice. The increase in hydroxyproline content accounts, at least in part, for the increase in denaturation temperature. The alpha2 chain clearly plays an important part in stabilizing the type I collagen triple helix and fiber packing, but further studies are required to determine the precise mechanism.     

胶原止血海绵的结构与理化性能分析

对从牛腱中制备的胶原止因海绵进行了结构分析,结果表明所制备的止血海绵具有胶原蛋白的特征结构与组成,是纯度较高的胶原蛋白;理化性能测试表明原止血海绵质量稳定,从而证明了胶原止血海绵的制备工艺合理可靠。     

Distribution of Collagen Types I,III, and V in Pregnant Mouse Endometrium

Decidualization in mice comprises a deep remodeling of extracellular matrix (ECM) components of the endometrium. In a previous biochemical study we showed that collagen types I and III are present in both pregnant and nonpregnant mouse endometrium, whereas collagen type V is expressed exclusively after the onset of decidualization. The distribution of collagen types in the pregnant mouse endometrium and possible changes of these molecular types in the different regions of the decidua is, however, not known. Using immunofluorescence and confocal microscopy we showed the presence of collagen types I, III, and V in the endometrial stroma of implantation and interimplantation sites from days 5 to 8 of pregnancy in the mouse. Collagen type III was chiefly expressed in the implantation sites and was the only collagen type to be present in the materno-fetal interface on the day of the embryo implantation. However, collagen type I was the predominant collagen in the interimplantation sites. Collagen type V was weakly expressed in the nondecidualized stroma during all periods but was expressed in larger amounts in the decidualized areas on day 7 of pregnancy, simultaneously with the accumulation of thick collagen fibrils in the same region. The highest immunofluorescence labeling for the three types of collagen was observed on day 7 when the antimesometrial decidual tissue achieved its greatest development. These data support previous studies that showed an intense ECM remodeling of the mouse endometrial stroma during the beginning of pregnancy. This outstanding remodeling may be important to stabilize placental anchorage.     

Synthesis and Evaluation of Long-Acting D-Penicillamine Derivatives

This study extends the use of two lathyrogens, β-aminopropionitrile (BAPN) and D-penicillamine (DPA) from daily systemic or local-topical administration to long-time acting agents. This was achieved by converting the hydrophilic drugs into lipophilic derivatives. The synthesis of functional derivatives of DPA consisted in esterification with methyl-, hexyl-, or benzyl alcohols in the presence of thionylchloride. The esters formed were hydrochlorides, acidic and soluble in water. During neutralization in vitro or in vivo by tissue fluid, an oily substance is formed that elutes from a hydrogel polymer at a much slower rate than hydroplilic DPA itself. The degree of lipophilicity, measured as a partition coefficient between octanol/water, was highest for hexyl ester and lowest for methyl ester DPA. A single injection of either DPA hexyl ester HCl or 3-hexyl(amino) propionitrile into the full thickness skin incision wound in rats significantly lowered the breaking strength of the wound 12 days after injection, indicating the interference with collagen cross-linking. Both agents injected into the breast adenocarcinoma in Fisher rats significantly inhibited tumor growth without any signs of local or systemic toxicity. We conclude that these lipophilic lathyrogens with prolonged effectiveness are suitable in the treatment of pathologies, consisting of excessively cross-linked or deposited collagen (fibrotic adhesions, strictures, stenosis, and scar contractures) and in the treatment of single, solitary tumors, malignant and benign.     

Characterization of skin abnormalities in a mouse model of osteogenesis imperfecta using high resolution magnetic resonance imaging and Fourier transform infrared imaging spectroscopy

Evaluation of the skin phenotype in osteogenesis imperfecta (OI) typically involves biochemical measurements, such as histologic or biochemical assessment of the collagen produced from biopsy-derived dermal fibroblasts. As an alternative, the current study utilized non-invasive magnetic resonance imaging (MRI) microscopy and optical spectroscopy to define biophysical characteristics of skin in an animal model of OI. MRI of skin harvested from control, homozygous oim/oim and heterozygous oim/+ mice demonstrated several differences in anatomic and biophysical properties. Fourier transform infrared imaging spectroscopy (FT-IRIS) was used to interpret observed MRI signal characteristics in terms of chemical composition. Differences between wild-type and OI mouse skin included the appearance of a collagen-depleted lower dermal layer containing prominent hair follicles in the oim/oim mice, accounting for 55% of skin thickness in these. The MRI magnetization transfer rate was lower by 50% in this layer as compared to the upper dermis, consistent with lower collagen content. The MRI transverse relaxation time, T2, was greater by 30% in the dermis of the oim/oim mice compared to controls, consistent with a more highly hydrated collagen network. Similarly, an FT-IRIS-defined measure of collagen integrity was 30% lower in the oim/oim mice. We conclude that characterization of phenotypic differences between the skin of OI and wild-type mice by MRI and FT-IRIS is feasible, and that these techniques provide powerful complementary approaches for the analysis of the skin phenotype in animal models of disease.     

Obesity Affects Collagen Fibril Diameter and Mechanical Properties of Tendons in Zucker Rats

Obesity is currently considered to be a world epidemic and one of the major public health problems in many countries, whose incidence is increasing at alarming rates. Genetically obese Zucker rats are used as a model of obesity and were employed in the present study. Tendons transmit contractile force from muscles to bone, thus permitting articular movement. The objective of our study was to analyze the ultrastructural, biochemical, and biomechanical alterations that occur in the deep digital flexor tendon of obese Zucker rats compared to lean animals. Ultrastructural analysis showed differences in collagen fibril diameter distribution and mass-average diameter between obese and lean animals. Regarding mechanical parameters, there was a significant difference in maximum displacement and strain. Hydroxyproline content was higher in obese animals. In view of the excess weight and peculiar conditions to which the tendon of obese animals is submitted, we concluded that obesity provokes alterations in the composition and organization of tendon extracellular matrix components. These alterations might be related to organizational and structural modifications in the collagen bundles, influencing the mechanical properties of the tendon and the progression to a pathological state.     

Living Donor Liver Transplantation for an Infant with Osteogenesis Imperfecta and Intrahepatic Cholestasis: Report of a Case

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by bone fragility and connective tissue manifestations. We report a successful liver transplantation (LT) in an 8-month-old boy with OI and cholestatic biliary cirrhosis. After 4 cycles of intravenous pamidronate, LT was performed under intravenous anesthesia using a left lateral section from his mother without mechanical retractors. The operation time was 420 min and estimated blood loss was 520 mL requiring one unit of RBC transfusion. He was discharged without surgical complications. Therefore, LT should be considered for patients with end stage liver disease and OI under organic multidisciplinary cooperation.

Graphical Abstract

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Paternal mosaicism for a COL1A1 dominant mutation (α1 Ser-415) causes recurrent osteogenesis imperfecta

We describe a dominant point mutation in the COL1A1 gene causing extremely severe osteogenesis imperfecta (OI type II/III) which was detected in the dermal fibroblasts of a proband, diagnosed by ultrasonography at 24 weeks of gestation. Type I collagen secretion was reduced and proαl(I) chains were overmodified. The mutation was localised in one COL1A1 allele by chemical cleavage of mismatched bases in normal cDNA/proband's mRNA heteroduplexes, and identified by cloning and sequencing. A G-to-A transition which causes the Substitution of Gly-415 with serine in the a α1(I) triple helical domain was found. The same mutation was detected in the father's spermatozoa and lymphocytes. Mosaicism in the father's germline explains the occurrence in the family of two additional OI pregnancies, which were documented by X-ray and ultrasound investigations. © 1993 Wiley-Liss, Inc.