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Biomedical subjects

E Vuorio

Publications and source records attributed to E Vuorio.

At least 55 records · Page 3Linked to original sources
PubMed1998

Production of cartilage collagens during metaphyseal bone healing in the mouse.

Small defects of unfractured bone are believed to heal without a cartilaginous intermediate. We have determined the extent of cartilage production in an experimental model of metaphyseal bone repair involving defects in both cortical and cancellous bone, but no fracture. Northern analyses revealed the presence of mRNAs for type X and II collagens in the repair tissue. Immunohistology confirmed subperiosteal deposition of both collagen types adjacent to the defect. While the mRNAs for the two collagen types peaked by one week of defect healing, immunodetectable type X collagen was not observed until the second week. The data suggest that reactivity of periosteum and activation of chondrogenesis and subsequent endochondral ossification programs are involved in murine bone repair regardless of defect type.

Animals↗
PubMed1998

Incorporation of cortical bone allografts and autografts in rats: expression patterns of mRNAs for the TGF-betas.

Healing of bone grafts is dependent on the rate of new bone formation. To understand better the regulation of new bone formation in the graft we have studied local production of TGF-beta1, 2 and 3, and of the small proteoglycans by determining their mRNA levels in a rat bone graft model. These mRNA levels were compared to the healing rates of autografts and allografts, as determined by histology, UV-microscopic evaluation of tetracycline-labeled new bone formation, microradiography and mechanical testing at 1, 2, 4 and 8 weeks of healing. Analyses showed that, analogous to slower bone formation in allografts, the induction of TGF-beta1 gene expression was slower than in allografts, when compared with autografts. A similar delay was seen in decorin gene expression. The results agree with the suggested role of TGF-beta1 in induction of type I collagen and osteonectin production. Our findings thus support the view that locally produced TGF-beta1 plays a role in normal graft incorporation, while local production of TGF-beta3, and particularly TGF-beta2, may be less important in this respect.

Animals↗
PubMed1997

Developmental regulation of mRNA species for types II, IX and XI collagens during mouse embryogenesis.

Several techniques were used to study the co-ordination of mRNA levels for five constituent chains of cartilage collagen fibrils during mouse development. Short cDNA clones were first constructed for mouse and human alpha3(IX) and for mouse proalpha1(XI) collagen mRNA species. Northern analysis of developing mouse embryos revealed that the mRNA species for alpha1, alpha2 and alpha3 chains of type IX collagen peaked earlier than those for proalpha1(II) and proalpha1(XI) collagen chains. Quantification of these mRNA species by slot-blot hybridization confirmed this developmental regulation: the mRNA ratios for type II/type IX/type XI collagens changed from 5.7:1:0.6 (at embryonic day 12.5) to 10.6:1:0.9 (in newborn mice). However, the genes coding for the three chains of type IX collagen seemed to be under more co-ordinated regulation during mouse development. In addition to high mRNA levels in cartilages and the eye, low levels of type IX collagen transcripts were identified in brain and skin of newborn mouse using RNase protection and reverse transcriptase-PCR assays. Finally, hybridization in situ revealed identical tissue distributions of the three type IX collagen mRNA species during early chondrogenesis but somewhat more widespread expression of the alpha1(IX) and alpha3(IX) mRNA species during endochondral ossification at day 16.5 of embryonic development. These results suggest a relatively tight co-ordination of the alpha1(IX), alpha2(IX), and alpha3(IX) collagen mRNA species in chondrocytes, but a lack of co-ordination in several non-cartilaginous tissues.

Amino Acid Sequence↗
PubMed1997

Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development.

Collagenase-3 (MMP-13) is a novel matrix metalloproteinase, the expression of which has so far only been documented in human breast carcinomas and osteoarthritic cartilage. In this study we have examined the expression of MMP-13 during human fetal development. Northern blot hybridizations revealed abundant expression of MMP-13 mRNAs in total RNA from fetal cartilage and calvaria at gestational age of 15 weeks. By in situ hybridization MMP-13 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column and in the dorsal end of ribs undergoing ossification, as well as in osteoblasts and periosteal cells below the inner periosteal region of ossified ribs. In contrast, no expression of MMP-13 could be detected in osteoclasts. Furthermore, expression of MMP-13 mRNA was detected in osteoblasts and fibroblasts primarily on the inner side of calvarial bone of the skull at 16 weeks of gestation. Expression of MMP-13 mRNA by primary human fetal chondrocytes in culture was enhanced by transforming growth factor-beta (TGF-beta) and inhibited by bone morphogenetic protein-2 (BMP-2). No expression of MMP-13 mRNA could be noted in other fetal tissues, including the skin, lungs, neural tissue, muscle, and liver. These results suggest that MMP-13 plays an important role in the extracellular matrix remodeling during fetal bone development both via endochondral and intramembranous ossification.

Blotting, Northern↗
PubMed1997

Histomorphometric and molecular biologic comparison of bioactive glass granules and autogenous bone grafts in augmentation of bone defect healing.

The applicability of bioactive glass (BG) granules as a substitute for bone grafts was tested by comparing the histologic, histomorphometric, and molecular biologic healing patterns to those of bone autografts and ungrafted bone defects in a rat model. The cellular response in defects filled with BG granules was characterized by continuous overexpression of type III collagen. Osteogenic mesenchymal cells, prior to their differentiation to osteoblasts, organized as a dense periosteumlike layer on the surface of the BG granules. By day 14 new bone formation was more extensive in autografted defects than in BG filled defects (p = 0.039). No cartilage-specific type II collagen mRNA was detectable, confirming the uniformity of intramembranous bone formation. The difference in the initiation of new bone formation was further confirmed by the mRNA analyses of the de novo production of TGF-beta 1 and type I collagen. Autografted defects demonstrated the highest levels of TGF-beta 1 and type I collagen mRNAs during the first 2 weeks of healing, whereas BG-filled defects showed biphasic expression patterns of the same genes. Spontaneous new bone formation in ungrafted bone defects was also characterized by biphasic expression of type I collagen gene. Osteonectin mRNA declined gradually over time in autografted and BG filled defects, whereas unfilled defects showed a gradual increase of osteonectin mRNA during healing. By 8 weeks, about 70% of the BG surface showed evidence of direct new bone contact. Energy-dispersing X-ray analyses confirmed the presence of silica-rich and CaP-rich zones at the bonding interface. In conclusion, the osteoconductive surface of bioactive glass granules efficiently bonds to ongrowing new bone but the material does not reach the capacity of autogenous bone graft in promotion of osteogenesis.

Animals↗
PubMed1997

Different healing rates of bone autografts, syngeneic grafts, and allografts in an experimental rat model.

Matching of donors and recipients for tissue antigens is vitally important for successful transplantation of essentially all organs and tissues, the major exception being bone. The importance of tissue-typing for the healing of bone allografts remains, however, a controversial issue as development of both humoral and cell-mediated immunity against the grafted bone has been observed in some experimental systems. In the present study, we compared the healing patterns of frozen antigen-mismatched allografts, frozen antigen-matched allografts (syngeneic grafts), and fresh cortical bone autografts in an experimental rat model. Histomorphometry of the graft-host interface revealed that new bone formation started significantly earlier in autografts than in allografts or syngeneic grafts. By 2 weeks, the level of new bone formation in the syngeneic grafts had reached that in autografts. Antigen-mismatched allografts, however, continued to exhibit a retarded formation of new bone throughout the union process. These histomorphometric observations were confirmed by molecular biologic analyses for the mRNA levels of type I collagen, which increased earlier and reached a higher level in autografts than in allografts. Use of syngeneic grafts resulted in a longer persistence of type I collagen mRNA expression in the healing tissue than in antigen-mismatched allografts. No apparent differences were seen between allografts and autografts in the expression of type III collagen. No cartilage-specific type II collagen mRNA was observed, indicating that antigen-mismatching or preservation by freezing did not alter the basic mechanism of the interface healing process, although it did slow down the beginning of the process. The experiments suggest that a major antigen mismatch between donor and recipient affects the temporal gene expression of extracellular bone matrix and delays new bone formation at the graft-host interface of cortical bone allografts.

Animals↗
PubMed1997

Abnormal craniofacial growth and early mandibular osteoarthritis in mice harbouring a mutant type II collagen transgene.

Skull morphology and histology in the heterozygous offspring of a transgenic founder mouse Del1, harbouring 6 copies of deletion mutation in Col2a1 gene, were compared with those in normal siblings. On visual observation and roentgenocephalometric examination the heads of heterozygous Del1 mice were smaller than normal. Histologically the sizes of cartilaginous structures of the cranial base were reduced. Severe defects were seen in the temporomandibular joint as progressive osteoarthritic lesions. These observations elucidate the relationship between the genotype and phenotype and demonstrate that heterozygous Del1 mice are a useful model for studies on a genetic disturbance where 'clinical' manifestations are not evident until adult age.

Animals↗
PubMed1997

Gene therapy of single-gene disorders: preface to the special section.

Gene therapy was introduced into clinical practice with great excitement, much publicity and considerable optimism in the early 1990s. Scientific evaluation of the early clinical trials has, however, greatly reduced the initial optimism. Follow-up studies have revealed that many early gene therapy trials mainly represented gene transfer into patients, possibly with short-term effects, but not true gene therapy where the course of the disease is permanently affected. This has lead to critical re-evaluation of the approaches taken. Clearly, more basic understanding is needed of the molecular mechanisms of the diseases treated. For this purpose, better animal models for human diseases are necessary. One of the biggest obstacles for gene therapy has been the lack of adequate vector systems. Development of new vectors for efficient and targeted delivery and uptake of therapeutic genes is a crucial area where progress needs to be made. The rationale for gene therapy depends largely on the type of disease to be treated. Recessively inherited single-gene disorders represent diseases where the concept of gene therapy--addition of a therapeutic gene to restore the lost function of two mutant alleles--is easily understood and rarely questioned. However, most gene therapy protocols are focused on multifactorial diseases such as malignancies where the therapeutic approach is quite different. While gene transfer technologies are being developed into truly effective gene therapy, the fight against inherited single-gene disorders also continues at population level by carrier screening and prenatal diagnostics where rapid methodological developments are taking place.

Alleles↗
PubMed1997

Localization of type II collagen mRNA isoforms in the developing eyes of normal and transgenic mice with a mutation in type II collagen gene.

PURPOSE: To elucidate the function of type II collagen in the development and diseases of the eye by analyzing the temporospatial expression of the long (IIA) and short (IIB) isoforms of type II collagen in the normal and transgenic Dell mice. METHODS: Normal and Dell transgenic embryos harboring a deletion mutation in the pro alpha 1 (II) collagen chain were studied from day 10.5 of embryonic development up to day 10 postpartum. Northern and in situ hybridizations and RNase protection assays were used to study the developmental and temporospatial expression of type II collagen isoforms. RESULTS: Expression of type II collagen mRNAs was observed at all developmental stages with maximum expression at 16.5 days of embryonic development. RNase protection analyses confirmed that both wild type and transgene-derived mRNAs underwent similar alternative splicing of exon 2 in the eye. By in situ hybridization, both isoforms were observed in the cornea, sclera, vitreous, ganglion cell layer of retina, developing ciliary body-iris, and in the retinal pigment epithelium-Bruch's membrane as well as in the lens and conjunctiva. Differences were observed between eyes of Dell mice and of control subjects in the levels and temporal expression patterns of type II collagen mRNA, which resulted in structural abnormalities in histologic analysis. CONCLUSIONS: Widespread expression of type II collagen mRNAs in ocular structures suggests an important role for type II collagen in structural development of the eye. As the expression patterns observed correspond to structural abnormalities in the eyes of Dell mice, the current results offer a promising basis for further development of mouse models for arthroophthalmopathies.

Abnormalities, Multiple↗
PubMed1997

Mutation in type II collagen gene disturbs spinal development and gene expression patterns in transgenic Del1 mice.

Transgenic Del1 mice harboring a deletion mutation in the cartilage-specific type II collagen gene were used for a systematic study on the dose-dependent effects of this dominant mutation on the embryonic development and growth of the vertebral column. Skeletal staining of homozygous and heterozygous Del1 mice and their nontransgenic littermates with Alcian blue/Alizarin red revealed not only a dose-dependent retardation in the appearance of ossification centers in transgene-positive offspring but also abnormal shapes and proportions of their vertebral columns. Histologic analysis confirmed these findings and demonstrated also retarded removal of the notochord, abnormal shapes and sizes of vertebral bodies and intervertebral discs, and the presence of an occult spina bifida in homozygous Del1 mice. In situ hybridization revealed abnormalities in the expression patterns of type I, II, IX, and X collagens and aggrecan, corresponding to the disorganization of the columnar chondrocyte architecture of the growth zones, increased appositional growth activity along the periphery of the vertebrae, increased numbers of hypertrophic chondrocytes, and development of necrotic areas in the central cartilaginous areas of vertebral bodies of homozygous Del1 embryos. Many of these findings parallel those seen in human chondrodysplasias and help us to understand the pathogenetic mechanisms involved in these developmental abnormalities.

Animals↗
PubMed1996

Expression and distribution of two alternatively spliced transcripts from the chicken alpha 2 (VI) collagen gene.

Two types of mRNA molecules with different 3' ends are transcribed from the chicken alpha 2(VI) collagen gene. The major splice variant encodes a polypeptide with a von Willebrand factor A domain at its carboxyl terminus. In the minor splice variant, this A domain is replaced by a novel motif which reveals some similarity to a fibronectin type III repeat. In situ hybridization experiments demonstrate that the major transcript is ubiquitously expressed. Substantial amounts are found in skeletal and cardiac muscle, gizzard, skin, tendon, liver, the wall of blood vessels, and the connective tissue of peripheral nerves. In contrast, the minor transcript is expressed at a very low level and can hardly be detected in any tissue by in situ hybridization. Only the aortic wall contains a considerable amount of this splice variant. However, no difference is observed by Northern blotting and the polymerase chain reaction in the ratio of the two transcripts when aorta and the other tissues are compared. Thus, the minor splice variant is not expressed in a tissue specific manner and, consequently, it is unlikely that it plays a tissue specific role. It might rather serve a general function in the structure and assembly of type VI collagen microfibrils.

Alternative Splicing↗
PubMed1996

Mouse cathepsin K: cDNA cloning and predominant expression of the gene in osteoclasts, and in some hypertrophying chondrocytes during mouse development.

We have constructed cDNA clones covering the entire coding region of mouse, human and rabbit preprocathepsin K mRNA for studies on bone turnover. The clone pMCatK-1 for mouse cathepsin K shares 87% nucleotide homology with the corresponding human and rabbit sequences. Analysis of a panel of mouse tissues for tissue distribution of cathepsin K mRNA revealed the highest levels in musculoskeletal tissues: bone, cartilage and skeletal muscle. In situ hybridization of developing mouse embryos was performed to identify the cellular source of cathepsin K mRNA. The strongest mRNA signal was detected in osteoclasts of bone, identified in serial sections by positive TRAP staining. Cathepsin K mRNA was also observed in some hypertrophic chondrocytes of growth cartilages. Association of cathepsin K production with degradation of bone and cartilage matrix suggests that this enzyme and its mRNA levels could serve as markers for matrix degradation in diseases affecting these tissues.

Aging↗
PubMed1996

Systemic versus cartilage-specific expression of a type II collagen-specific T-cell epitope determines the level of tolerance and susceptibility to arthritis.

Immunization of mice with rat type II collagen (CII), a cartilage-specific protein, leads to development of collagen-induced arthritis (CIA), a model for rheumatoid arthritis. To define the interaction between the immune system and cartilage, we produced two sets of transgenic mice. In the first we point mutated the mouse CII gene to express an earlier defined T-cell epitope, CII-(256-270), present in rat CII. In the second we mutated the mouse type I collagen gene to express the same T-cell epitope. The mice with mutated type I collagen showed no T-cell reactivity to rat CII and were resistant to CIA. Thus, the CII-(256-270) epitope is immunodominant and critical for development of CIA. In contrast, the mice with mutated CII had an intact B-cell response and had T cells which could produce gamma interferon, but not proliferate, in response to CII. They developed CIA, albeit with a reduced incidence. Thus, we conclude that T cells recognize CII derived from endogenous cartilage and are partially tolerized but may still be capable of mediating CIA.

Amino Acid Sequence↗
PubMed1996

Evidence for insufficient chondrocytic differentiation during repair of full-thickness defects of articular cartilage.

The main objective of this study was to characterize the cellular phenotypes in the repair tissue of full-thickness defects of articular cartilage by histologic and molecular biologic techniques. Healing of the defects in the articular cartilage of the knee joints of 12 rabbits was analyzed at days 3, 7, 14, 28 and 50 using histology and Northern analysis of mRNA levels for type I, II and III collagens and osteonectin. The cellular source of each mRNA was determined by in situ hybridization. Two novel cDNA clones for rabbit type II and III collagen mRNAs were constructed to obtain species-specific hybridization probes. The repair tissue of full-thickness defects consisted of two types of tissue. At the bottom of the defect, bone-derived cells with high levels of type I collagen and osteonectin mRNA were actively producing new osteoid, while superficially a slow transition from a fibrin clot into undifferentiated mesenchyme with cells containing type III collagen mRNA was observed. This tissue subsequently became fibrocartilaginous, with small groups of cells turning on the transcription of the type II collagen gene and acquiring a phenotype typical for hyaline cartilage. The data suggest that small clusters of cells in the repair tissue of full-thickness articular cartilage defects are capable of turning on an apparently correct chondrocytic phenotype. The low transcription level of the type II collagen gene suggests, however, that insufficient amounts of fundamentally important regulatory factors or progenitor cells are present in the repair tissue. In the future, such factors should be administrable into the joint by novel therapeutic means.

Amino Acid Sequence↗
PubMed1996

Variability in the upstream promoter and intron sequences of the human, mouse and chick type X collagen genes.

The type X collagen gene is specifically expressed in hypertrophic chondrocytes during endochondral ossification. Transcription of the type X collagen gene by these differentiated cells is turned on at the same time as transcription of several other cartilage specific genes is switched off and before mineralization of the matrix begins. Analysis of type X collagen promoters for regulatory regions in different cell culture systems and in transgenic mice has given contradictory results suggesting major differences among species. To approach this problem, we have determined the nucleotide sequences of the two introns and upstream promoter sequences of the human and mouse type X collagen genes and compared them with those of bovine and chick. Within the promoter regions, we found three boxes of homology which are nearly continuous in the human gene but have interruptions in the murine gene. One of these interruptions was identified as a complex 1.9 kb repetitive element with homology to LINE, B1, B2 and long terminal repeat sequences. Regulatory elements of the human type X collagen gene are located upstream of the region where the repetitive element is inserted in the mouse gene, making it likely that the repetitive element is inserted between the coding region and regulatory sequences of the murine gene without interfering with its expression pattern. We also compared the sequences of the introns of both genes and found strong conservation. Comparisons of the mammalian sequences with promoter and first intron sequences of the chicken type X collagen gene revealed that only the proximal 120 nucleotides of the promoter were conserved, whereas all other sequences displayed no obvious homology to the murine and human sequences.

Animals↗
PubMed1996

Towards genomic drug therapy with antisense oligonucleotides.

Antisense oligonucleotides represent a novel class of potential drugs for highly selective blocking of genes. The basic concept of antisense strategy is simple: an antisense molecule recognizes a complementary mRNA (or DNA) by sequence-specific base pairing, and hence prevents translation (or transcription), resulting in a selective inhibition of protein synthesis. Because of these properties, antisense oligonucleotides have great potential as therapeutic agents in several human diseases, such as viral diseases, malignancies and dominant hereditary diseases. However, technical difficulties have slowed down their use as drugs: structural modifications are needed to increase the stability and potency of synthetic oligonucleotides, specific delivery systems are required to facilitate their entry into target cells, and more information is needed to their mechanism of action. Much of the current research on antisense oligonucleotides takes place at the interface of chemistry and biomedical sciences, a multidisciplinary field where finding a common language is sometimes difficult. The aim of this review is to present an overview of the antisense strategy in terms which should be understandable for chemists, biologists and physicians.

Animals↗