hydroxylysine and Disease-Models--Animal

Cartilage has rather limited capacities for self-repair and regeneration. To repair complexly shaped cartilage tissue defects, we propose the application of microtissues fabricated from bone marrow-derived mesenchymal stem cells (BMSCs) cultured in natural bionic nanofibrous microcarriers (NF-MCs). The NF-MCs were structurally and functionally designed to mimic natural extracellular matrix (ECM) by crosslinking dialdehyde bacterial cellulose (DBC) with DL-allo-hydroxylysine (DHYL) and complexing chitosan (CS) with DHYL through electrostatic interactions. The orthogonal design allows for fine tuning of fiber diameter, pore size, porosity, mechanical properties, and biodegradation rate of the NF-MC. BMSCs cultured in NF-MCs showed improved proliferation compared with those cultured in chitosan microcarriers (CS-MCs). After three-week culture under microgravity conditions, functional cartilage microtissues were generated. When implanted into a knee articular cartilage defect in mice, the microtissue showed superior in vivo cartilage repair as characterized by cell tracking, histology, micro CT image, and gait analysis. Versatile in natural biopolymer design and biomimetic in nanofibrous component embedded in macroporous microcarriers, these injectable NC-MCs demonstrate to be effective carriers for cell proliferation and differentiation. Furthermore, the functional microtissues also show their prospect in repair of cartilage tissue, and suggest their potential for other tissues in general.

Topics: Animals; Biomimetic Materials; Cartilage, Articular; Cell Proliferation; Cell Survival; Cells, Cultured; Cellulose; Disease Models, Animal; Extracellular Matrix; Femur; Gait; Green Fluorescent Proteins; Hydroxylysine; Imaging, Three-Dimensional; Mesenchymal Stem Cells; Microtechnology; Nanofibers; Rats, Sprague-Dawley; Regeneration; Tissue Engineering; X-Ray Microtomography

Topics: Amino Acid Sequence; Amino Acids; Animals; Blood Glucose; Collagen Type I; Cross-Linking Reagents; Diabetes Mellitus, Type 2; Disease Models, Animal; Glycated Hemoglobin; Glycation End Products, Advanced; Glycosylation; Hydroxylation; Hydroxylysine; Lysine; Male; Mass Spectrometry; Mice; Obesity; Protein-Lysine 6-Oxidase; Tail; Tendons

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.

Topics: Animals; Collagen; Disease Models, Animal; Ehlers-Danlos Syndrome; Gait; Hydroxylysine; Mice; Mice, Inbred Strains; Mice, Knockout; Muscle Hypotonia; Phenotype; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Skin Diseases; Tissue Distribution

Two protected derivatives of beta-D-galactopyranosyl-5-hydroxy-L-lysine, in which HO-4 of galactose has been O-methylated or replaced by fluorine, have been prepared. The building blocks were incorporated at position 264 of the peptide fragment CII259-273 from type II collagen by solid-phase synthesis. The ability of these two glycopeptides, and two CII259-273 glycopeptides in which HO-4 of galactose was either unmodified or deoxygenated, to elicit responses from T-cell hybridomas obtained in a mouse model for rheumatoid arthritis was then determined. The hybridomas were all highly sensitive towards modifications at C-4 of the beta-D-galactosyl residue of CII259-273, highlighting the role of HO-4 as an important contact point for the T-cell receptor. Most likely, this glycopeptide hydroxyl group is involved in hydrogen bonding with the T-cell receptor.

Topics: Animals; Arthritis, Rheumatoid; Collagen Type II; Disease Models, Animal; Dose-Response Relationship, Immunologic; Galactose; Glycopeptides; Hybridomas; Hydroxylysine; Magnetic Resonance Spectroscopy; Mice; Peptide Fragments; Receptors, Antigen, T-Cell; T-Lymphocytes

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.

Topics: Animals; Calcification, Physiologic; Collagen Type I; Cross-Linking Reagents; Disease Models, Animal; Female; Hydroxylysine; Male; Mice; Mice, Mutant Strains; Osteogenesis Imperfecta; Protein Denaturation; Tail; Tendons; Tensile Strength; Tibia

Five protected analogues of beta-D-galactosyl-(5R)-5-hydroxy-L-lysine were prepared, in which the galactosyl moiety was modified by monodeoxygenation or inversion of stereochemistry at C-4. The building blocks were used in the solid-phase synthesis of a set of glycopeptides related to the peptide fragment CII256-273 from type II collagen. Evaluation of the glycopeptides revealed that T-cell hybridomas obtained in collagen-induced arthritis (CIA), which is a common mouse model for rheumatoid arthritis, recognized the galactosyl moiety with high specificity for individual hydroxy groups. Moreover, T-cell hybridomas obtained in a humanized variant of CIA were also found to recognize the glycopeptides in an equally carbohydrate-specific manner. The results allowed the generation of models of the complexes formed between the appropriate class II major histocompatibility complex (MHC) molecule, glycopeptide, and the T-cell receptor, that is, of an interaction that is critical for the stimulation of T cells in the arthritis models. In the structural models, peptide side chains anchor the glycopeptide in pockets in the class II MHC molecule, whereas the galactosylated hydroxylysine residue forms the key contacts with the T-cell receptor. Importantly, the results also suggest that a T-cell response towards glycopeptide fragments from type II collagen could play an important role in the development of rheumatoid arthritis in humans.

Topics: Animals; Arthritis, Rheumatoid; Binding Sites; Collagen Type II; Disease Models, Animal; Glycopeptides; Histocompatibility Antigens Class II; Humans; Hybridomas; Hydroxylysine; Mice; Mice, Transgenic; Peptide Fragments; Structure-Activity Relationship; T-Lymphocytes, Helper-Inducer

To study the effect of streptozotocin induced diabetes on glomerular basement membrane (GBM) synthesis, an isolated rat glomerular preparation has been developed, and its metabolic properties have been defined. The chemical composition of normal rat GBM isolated from this preparation closely resembles human GBM. Incubation with [U-14C] lysine leads to prompt incorporation of label into GBM and the subsequent appearance of labeled hydroxylysine. A 1-h lag before detection of labeled hydroxylysine in GBM suggests a delay in the release of GBM precursors. Significantly lower counts appeared in the nondialyzable fraction of the medium than in insoluble GBM during pulse-chase experiments, and labeled hydroxylysine accounted for a lower portion of the total counts in the medium (0.85%) than in the GBM (1.98%). Isolated glomeruli were prepared from streptozotocin diabetic rats of 4-6 wks duration. After incubation with [ U-14C] lysine recovery of label in diabetic GBM (88.98+/-8.26 nmol/g GBM) did not differ from age matched controls (82.52 +/- 8.26 nmol/g GBM). In pulse-chase experiments recovery of label in hydroxylysine of diabetic GBM (o.473 +/- 0.082 nmol/g GBM) did not differ from age matched controls (0567+/-0.065 nmol/g GBM). These findings indicate normal rates of GBM synthesis and hydroxylation of lysine residues in animals with streptozotocin diabetes.

Topics: Amino Acids; Animals; Basement Membrane; Diabetes Mellitus; Disease Models, Animal; Hydroxylysine; In Vitro Techniques; Kidney Glomerulus; Lysine; Rats; Streptozocin