Mouse Skeletal Tissue Research Articles

Spatial transcriptomics implicates impaired BMP signaling in NF1 fracture pseudarthrosis in murine and patient tissues.

The Neurofibromatosis Type 1 (NF1) RASopathy is associated with persistent fibrotic nonunions (pseudarthrosis) in human and mouse skeletal tissue. Here, we first performed spatial transcriptomics to define the molecular signatures across normal endochondral healing following fracture in mice. Within the control fracture callus, we observed spatially restricted activation of morphogenetic pathways, such as TGF-β, WNT, and BMP. To investigate the molecular mechanisms contributing to Nf1-deficient delayed fracture healing, we performed spatial transcriptomic analysis on a Postn-cre;Nf1flox/- (Nf1Postn) fracture callus. Transcriptional analyses, subsequently confirmed through p-SMAD1/5/8 immunohistochemistry, demonstrated a lack of BMP pathway induction in Nf1Postn mice. To further inform the human disease, we performed spatial transcriptomic analysis of fracture pseudarthrosis tissue from a NF1 patient. Analyses detected increased MAPK signaling at the fibrocartilaginous-osseus junction. Similar to the Nf1Postn fracture, BMP pathway activation was absent within the pseudarthrosis tissue. Our results demonstrate the feasibility to delineate the molecular and tissue-specific heterogeneity inherent in complex regenerative processes, such as fracture healing, and to reconstruct phase transitions representing endochondral bone formation in vivo. Furthermore, our results provide in situ molecular evidence of impaired BMP signaling underlying NF1 pseudarthrosis, potentially informing the clinical relevance of off-label BMP2 as a therapeutic intervention.

Identification of conserved skeletal enhancers associated with craniosynostosis risk genes.

Craniosynostosis, defined by premature fusion of one or multiple cranial sutures, is a common congenital defect affecting more than 1/2000 infants and results in restricted brain expansion. Single gene mutations account for 15%-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. We hypothesized that the two noncoding genomic regions identified by a GWAS for craniosynostosis contain distal regulatory elements for the risk genes BMPER and BMP2. To identify such regulatory elements, we surveyed conserved noncoding sequences from both risk loci for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous expression of bmper and bmp2. For each locus, we also found a skeletal enhancer that also contains a sequence variant associated with craniosynostosis risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. The same enhancer is active in mouse skeletal tissues, demonstrating evolutionarily conserved activity. Using enhanced yeast one-hybrid assays, we identified transcription factors that bind each enhancer and observed differential binding between alleles, implicating multiple signaling pathways. Our findings help unveil the genetic mechanism of the two craniosynostosis risk loci. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

Preparation of Adult Mouse Skeletal Tissue Sections for RNA In Situ Hybridization.

The RNA in situ hybridization assay is essential in many studies to evaluate gene expression in vivo. It consists ofgenerating tissue sections and subsequently hybridizing these sections with RNA probes. Keeping RNA intact is a challenge while harvesting tissue samples, processing through embedding, sectioning them, and conditioning the sections for hybridization. These challenges are particularly strong for adult skeletal tissues due to their copious, dense, and mineralized extracellular matrices. Here, we describe a method optimized to successfully hybridize RNA species, even of low abundance, in adult mouse bone and cartilage samples. This method involves tissue fixation with paraformaldehyde, demineralization with Morse's solution and paraffin embedding, all of which can be completed in 4days. Sections are then generated and hybridized using a 1-day standard protocol. Sections prepared using this method are compatible with immunostaining and standard staining procedures for skeletal tissues.

EdU-Based Assay of Cell Proliferation and Stem Cell Quiescence in Skeletal Tissue Sections.

Identifying and tracking proliferating and quiescent cells in situ is an important phenotyping component of skeletal tissues in development, physiology and disease. Among all the methods that exist, which include immunostaining for cell cycle-specific proteins, the gold standards use thymidine analogs. These compounds label proliferating cells by being incorporated into de novo-synthesized genomic DNA. 5-bromo-2'-deoxyuridine (BrdU) has traditionally been used for this purpose, but its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to DNA. An alternative, more recently developed, uses 5-ethynyl-2'-deoxyuridine (EdU). This thymidine analog is detected by click chemistry, that is, covalent cross-linking of its ethynyl group with a fluorescent azide that is small enough to easily penetrate native tissues and reach DNA. In addition to being simple and quick, this EdU-based assay is compatible with other protocols, such as immunostaining, on the same tissue sections. We here describe an EdU-based protocol optimized to label and functionally assess actively proliferating cells as well as slowly dividing cells, including stem cells, in mouse skeletal tissues.

Immunofluorescent Staining of Adult Murine Paraffin-Embedded Skeletal Tissue.

Immunohistochemistry, or immunolabeling, is a key method for the identification of protein expression and localization. Successful detection relies on a low signal-to-noise ratio, which is affected greatly by antibody specificity as well as the staining protocol. Immunohistochemistry in the mouse is challenging, particularly in adult skeletal tissue, due to the need for long decalcification, high autofluorescence and high levels of endogenous peroxidase. Here, we describe a highly sensitive protocol for protein detection in decalcified paraffin-embedded sections from adult mouse skeletal tissue. By using four levels of amplification, this method allows for the identification of even low-abundance proteins.

Trps1 transcription factor represses phosphate-induced expression of SerpinB2 in osteogenic cells.

Serine protease inhibitor SerpinB2 is one of the most upregulated proteins following cellular stress. This multifunctional serpin has been attributed a number of pleiotropic activities, including roles in cell survival, proliferation, differentiation, immunity and extracellular matrix (ECM) remodeling. Studies of cancer cells demonstrated that expression of SerpinB2 is directly repressed by the Trps1 transcription factor, which is a regulator of skeletal and dental tissues mineralization. In our previous studies, we identified SerpinB2 as one of the novel genes highly upregulated by phosphate (Pi) at the initiation of the mineralization process, however SerpinB2 has never been implicated in formation nor homeostasis of mineralized tissues. The aim of this study was to establish, if SerpinB2 is involved in function of cells producing mineralized ECM and to determine the interplay between Pi signaling and Trps1 in the regulation of SerpinB2 expression specifically in cells producing mineralized ECM. Analyses of the SerpinB2 expression pattern in mouse skeletal and dental tissues detected high SerpinB2 protein levels specifically in cells producing mineralized ECM. qRT-PCR and Western blot analyses demonstrated that SerpinB2 expression is activated by elevated Pi specifically in osteogenic cells. However, the Pi-induced SerpinB2 expression was diminished by overexpression of Trps1. Decreased SerpinB2 levels were also detected in osteoblasts and odontoblasts of 2.3Col1a1-Trps1 transgenic mice. Chromatin immunoprecipitation assay (ChIP) revealed that the occupancy of Trps1 on regulatory elements in the SerpinB2 gene changes in response to Pi. In vitro functional assessment of the consequences of SerpinB2 deficiency in cells producing mineralized ECM detected impaired mineralization in SerpinB2-deficient cells in comparison with controls. In conclusion, high and specific expression of SerpinB2 in cells producing mineralized ECM, the impaired mineralization of SerpinB2-deficient cells and regulation of SerpinB2 expression by two molecules regulating formation of mineralized tissues suggest involvement of SerpinB2 in physiological mineralization.

Extending the Scope of 1H NMR Spectroscopy for the Analysis of Cellular Coenzyme A and Acetyl Coenzyme A.

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) are ubiquitous cellular molecules, which mediate hundreds of anabolic and catabolic reactions including energy metabolism. Highly sensitive methods including absorption spectroscopy and mass spectrometry enable their analysis, albeit with many limitations. To date, however, NMR spectroscopy has not been used to analyze these important molecules. Building on our recent efforts, which enabled simultaneous analysis of a large number of metabolites in tissue and blood including many coenzymes and antioxidants ( Anal. Chem. 2016, 88, 4817-24; ibid 2017, 89, 4620-4627), we describe here a new method for identification and quantitation of CoA and acetyl-CoA ex vivo in tissue. Using mouse heart, kidney, liver, brain, and skeletal tissue, we show that a simple 1H NMR experiment can simultaneously measure these molecules. Identification of the two species involved a comprehensive analysis of the different tissue types using 1D and 2D NMR, in combination with spectral databases for standards, as well as spiking with authentic compounds. Time dependent studies showed that while the acetyl-CoA levels remain unaltered, CoA levels diminish by more than 50% within 24 h, which indicates that CoA is labile in solution; however, degassing the sample with helium gas halted its oxidation. Further, interestingly, we also identified endogenous coenzyme A glutathione disulfide (CoA-S-S-G) in tissue for the first time by NMR and show that CoA, when oxidized in tissue extract, also forms the same disulfide metabolite. The ability to simultaneously visualize absolute concentrations of CoA, acetyl-CoA, and endogenous CoA-S-S-G along with redox coenzymes (NAD+, NADH, NADP+, NADPH), energy coenzymes (ATP, ADP, AMP), antioxidants (GSH, GSSG), and a vast pool of other metabolites using a single 1D NMR spectrum offers a new avenue in the metabolomics field for investigation of cellular function in health and disease.

LC-MS Analyses of Lipid Species in Skeletal Muscle Cells and Tissue.

Liquid chromatography-mass spectrometry (LC-MS) is a widely used methodology for measuring lipids at a global level. Combined with an optimal extraction method LC-MS enables the detection and characterization of a wide range of lipid species even of low abundance. Here, we describe two extraction- and LC-MS-based quantitative analytical methods for lipid, acyl-CoA, and acyl-carnitine analyses from either mouse C2C12 myotubes or mouse skeletal tissue. We also describe the use of 13C16-palmitate and its incorporation into acyl-carnitines to show how stable isotope tracers are metabolized within cells and therefore can be implemented for lipidomic flux analysis.

P 177 - αB-crystallin activation in cardiac muscle by acute exercise mirrors the sHSP kinetic in oxidative skeletal muscle fibers: animal and cellular study

Alpha-B-Crystallin (CRYAB), a Small Heat Shock Protein sensitive to oxidative stress, is implicated in various biological processes in many tissues. In cardiac muscle, CRYAB exerts a cardio protective role in ischemia-induced damage preventing apoptosis and necrosis. We aimed to study αB-crystallin’ response in mouse cardiac tissue (H), at different time of recovery from an acute aerobic exercise (1 hour), correlating its modulation with oxidative stress level. We found that a single bout exercise lead to a specific short-term increase of phospho-αB-crystallin level (pCRYAB), without changes of its total expression. Further, the level of 4-hydroxynonenal, a marker of lipidic peroxidation, has shown a similar trend of pCRYAB enhancement. This may indicate that CRYAB in cardiac muscle is activated and it has a putative role in oxidative stress during exercise. These results are supported by our previous data obtained in mouse skeletal tissues (i.e. gastrocnemius, soleus) and in H₂O₂-treated C2C12 myotubes. In particular, we observed not only a fiber-dependent response of pCRYAB, but also its translocation into myofibrillar compartment. Experiments are in progress to further investigate on CRYAB role during exercise and its interactions with cytoskeletal structures.

An animal and cellular study on αB-crystallin activation in cardiac muscle by acute exercise

Alpha-B-Crystallin (CRYAB), a Small Heat Shock Protein sensitive to oxidative stress, is expressed in many tissues and implicated in various biological processes. In cardiac muscle, CRYAB exerts a cardio protective role in ischemia-induced damage preventing apoptosis and necrosis. In the present study we used forty young (7-weeks old) healthy male mice (BALB/c AnNHsd), which after 1 week of acclimatization to the new housing environment, runned 2 days per 10 minutes. The TR mice ran for 60 min at a speed of 5.5 m/min. Mice were sacrificed immediately after, 15 and 120 minutes after the end of the acute bout of endurance exercise (TR-0’, TR-15’ and TR-120’, respectively). We prepared samples from the heart and from the group of posterior muscles study αB-crystallin’ response at different time of recovery from an acute aerobic exercise (1 hour), correlating its modulation with oxidative stress level. We found that a single bout exercise lead to a specific short-term increase of phospho-αB-crystallin level (pCRYAB), without changes of its total expression. Further, the level of 4-hydroxynonenal, a marker of lipidic peroxidation, has shown a similar trend of pCRYAB enhancement. This may indicate that CRYAB in cardiac muscle is activated and it has a putative role in oxidative stress during exercise. These results are supported by our previous data obtained in mouse skeletal tissues (i.e. gastrocnemius, soleus) and in H₂O₂-treated C2C12 myotubes. In particular we observed not only a fiber-dependent response of pCRYAB, but also its translocation into myofibrillar compartment. Experiments are in progress to further investigate on CRYAB role during exercise and its interactions with cytoskeletal structures.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Skeletal Tissues (Bones, Joints, and Teeth).

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is an initiative of the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the skeletal tissues and teeth of laboratory rats and mice, with color photomicrographs illustrating examples of many common lesions. The standardized nomenclature presented in this document is also available on the internet (http://www.goreni.org/). Sources of material were databases from government, academic and industrial laboratories throughout the world.

Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue.

High-resolution confocal imaging is a vital tool for analyzing the 3D architecture and detailed spatial distribution of cells in situ. However, imaging of skeletal tissue has remained technically challenging because of its calcified nature. Here we describe a protocol that allows high-resolution imaging of skeletal tissue with preservation of cellular morphology and tissue architecture. The procedure involves tissue fixation, decalcification and cryosectioning of the mouse skeletal tissue to generate thick sections. The thick sections generated by this procedure are not only compatible with the analysis of genetically expressed fluorescent proteins but they also preserve antigenicity, thus enabling diverse combinations of antibody labeling. Further, this procedure also permits other fluorescence techniques such as TUNEL and ethynyl deoxyuridine (EdU) incorporation assays. Images resulting from the confocal imaging can be assessed qualitatively and quantitatively to analyze various parameters such as distribution and interrelationships of cell types. The technique is straightforward and robust, highly reproducible and can be completed in ∼11 d.

The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity

Studies in sarcolipin knockout mice have led to the suggestion that skeletal muscle sarcolipin plays a role in thermogenesis. The mechanism proposed is uncoupling of the sarcoplasmic reticulum calcium pump. However, in other work sarcolipin was not detected in mouse skeletal tissue. We have therefore measured sarcolipin levels in mouse skeletal muscle using semi-quantitative western blotting and synthetic mouse sarcolipin. Sarcolipin levels were so low that it is unlikely that knocking out sarcolipin would have a measurable effect on thermogenesis by SERCA. In addition, overexpression of neither wild type nor FLAG-tagged variants of mouse sarcolipin in transgenic mice had any major significant effects on body mass, energy expenditure, even when mice were fed on a high fat diet.

Characterization of a murine type IIB procollagen-specific antibody

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1+ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1+ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.

In vivo association of CReMM/CHD9 with promoters in osteogenic cells

Molecular mechanisms that control cell differentiation involve with chromatin remodeling activities. We recently identified Chromatin Related Mesenchymal Modulator (CReMM), a CHD protein expressed by mesenchymal cells. In this study, we analyzed CReMM expression on RNA and protein levels during embryonic development in mouse skeletal tissues. CReMM appears transiently during mesenchymal cell differentiation, being detected first in osteoprogenitors and declining in mature cells. A novel aspect of the study elaborates on in vivo association of CReMM with promoters in cells obtained by laser capture micro-dissection (LCM) technique from periosteum and endochondreal ossification regions. Using chromatin immunoprecipitation (ChIP), we proved that CReMM binds to skeletal tissue-specific promoters: CBFA1, biglycan, osteocalcin (OC), collagen-II, and myosin in a differential manner. The results imply that CReMM selectively interacts with analyzed promoters activated in the tissue at the appropriate time of development. The identification of CReMM and its tissue distribution and function provides an attractive clue for the study of transcriptional regulation of osteogenic cells' maturation.

Differentiation of cultured mice bone marrow into osteoblast-like cells results in acquisition of sex-specific responsiveness to gonadal steroids

We have previously demonstrated that mouse skeletal tissue, rat bone as well as rat or human derived bone cells in culture, show a sex-specific response to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK). This response could be modified by manipulation of the endocrine environment during early postnatal development. Moreover, pretreatment with vitamin D up-regulated the sex-specific responsiveness and sensitivity to gonadal steroids. In the present study we examine the differentiation pattern into osteoblast-like cells using dexamethasone (DEX) and 1,25 dihydroxy vitamin D3 (1,25D) and their effect on the acquisition of responsiveness to gonadal steroids by the differentiated cells. Cultured femoral bone marrow in the presence of DEX or 1,25D or both, were examined for their response to gonadal steroids by measuring the specific activities of alkaline phosphatase (AP) and CK BB. The constitutive level of CK in both male- and female-derived bone cells was decreased by DEX, by 1,25D or by both, whereas the constitutive level of AP was increased by DEX while decreased by 1,25D or by both. Following incubation of the bone marrow cultures with DEX, treatment with estradiol 17beta (E2, 30 nM, 24 h) stimulated CK activity in female derived bone cells, with no effect of treatment with dihydrotestosterone (DHT, 300 nM). In contrast, in male derived bone cells, DHT but not E2 increased CK activity. This sex-specific response was also achieved upon culturing with 1,25D and was significantly augmented by culturing with both. No response to gonadal steroids was seen with undifferentiated bone marrow cells. All cultures responded to IGF-I when cultured with or without DEX and/or 1,25D but with no augmentation by 1,25D. Gonadal steroids increased AP to a much lesser extent; but enzyme activity decreased in the presence of 1,25D. IGF-I stimulated AP slightly with no effect of 1,25D. These findings suggest that manipulation of the hormonal milieu in early stages of differentiation sequence of osteoblast-like cells, determines the subsequent selective responsiveness of the developing bone tissue to gonadal steroids.

Expression of matrilins during maturation of mouse skeletal tissues.

The matrilins are a recently discovered family of non-collagenous extracellular matrix proteins. During embryogenesis, all matrilins are expressed in skeletal tissues. Additionally, matrilin-2 and -4 are expressed in the dermis and in connective tissues of internal organs, e.g. of the lung and kidney. After birth, the expression of matrilin-1 and -3 remains specific for cartilage and bone whereas matrilin-2 and -4 display a broader tissue distribution and could be detected in epithelial, muscle, and nervous tissue as well as in loose and dense connective tissue. In epiphyseal cartilage of growing long bones, matrilin-1 and -3 are present in all cartilage regions, in contrast to matrilin-2, which is expressed in the proliferative and the upper hypertrophic zones. Similarly matrilin-4 was detected all over the epiphyseal cartilage, with the weakest expression in the hypertrophic zone. Although it was shown that matrilin-1 and -3 can form hetero-oligomers and are often co-localized in tissue, clear differences in their spatial distribution could be demonstrated by double-immunolabelling. During joint development matrilin-2 and matrilin-4 are present at the developing joint surface, while in articular cartilage of 6-week-old mice all matrilins are only weakly expressed.

Analysis of aggrecan and tenascin gene expression in mouse skeletal tissues by Northern and in situ hybridization using species specific cDNA probes

Cartilage matrix is an interacting multicomponent system of collagen fibrils, fibril-associated small proteoglycans, and large proteoglycans and glycoproteins entrapped within the fibrillar network. In order to better understand the relationships between these different components we have constructed short cDNA clones for detection of mRNAs for two major noncollagenous macromolecules of cartilage matrix, aggrecan and tenascin. We subsequently determined their corresponding mRNA levels by Northern analysis in a panel of total RNAs isolated from several newborn mouse tissues. The expression of aggrecan was strictly restricted to cartilages while tenascin mRNA was present at variable levels in most of the tissues studied. The cDNA clones were also used to identify the cells responsible for aggrecan and tenascin production in newborn mouse tissues by in situ hybridization. With this technique aggrecan mRNA was detected in chondrocytes throughout the developing skeleton in a pattern very similar but not identical to those of type II and IX collagen mRNAs. In the newborn mouse skeleton tenascin and aggrecan mRNAs were expressed essentially in a mutually exclusive manner, tenascin transcripts being present in osteoblasts, periosteal and perichondrial cells, and in cells at articular surfaces. None of these cells expressed the cartilage specific collagen or aggrecan genes. The results further suggest different patterns of gene expression in chondrocytes based on their location in the different cartilages.

Fulvic acid supplementation and selenium deficiency disturb the structural integrity of mouse skeletal tissue. An animal model to study the molecular defects of Kashin-Beck disease

High concentrations of fulvic acid and selenium deficiency are the main causative factors of Kashin-Beck disease, an endemic, chronic and degenerative osteoarticular disorder found in China. In the search for an animal model of this disease, mice were exposed to these pathogenetic conditions for two generations and the collagen types from skin, bone and cartilage were analysed. The growth of the treated mice was slightly retarded, and the rate of reproduction was lower in animals maintained on a fulvic acid-supplemented and/or selenium-deficient diet. Irregular bone formation was seen by radiography and morphometry. Biochemical analysis indicated that lysine residues in collagen I from bone and in collagen II from cartilage were overmodified. The values of Hyl/(Hyl+Lys) in bone collagen alpha 1(I) chains from treated mice were about 0.434-0.484, i.e. substantially higher than that of the control (0.277). The values of this parameter for collagen II were 0.482 for control and 0.546-0.566 for treated mice. The melting temperature of collagen I from bones of treated mice was 1 degrees C lower than that of control collagen, indicating decreased thermal stability. The breakage point of the tibiae of treated mice occurred at a lower preload force than for controls, suggesting that the overmodified and thermally less stable collagen molecules are causally related to a lower mechanical strength of bones.