Minna Sandberg

Gene expression during bone repair.

Detailed understanding of the basic events in fracture healing constitutes a foundation for the development of new approaches to stimulate bone healing. Since the fracture healing process repeats, in an adult organism, several stages of skeletal growth in the same temporal order, it offers an interesting model for developmental regulation of cellular phenotypes and tissue-specific genes. Molecular biology has introduced new methods to study the regulatory phenomena during the process of fracture repair. Gene technology has also produced purified growth factors for research, which will help to understand their roles in fracture healing. This review summarizes data on the regulation of genes coding for extracellular matrix components and growth regulatory molecules during fracture healing. The information available focuses on the sequential expression of genes coding for collagens, proteoglycans, and some other matrix proteins during secondary (callus) healing. The temporal and spatial appearance of the different connective tissue components, mesenchyme, cartilage, and bone, are closely linked to the expression of genes coding for their characteristic constituents. Members of the transforming growth factor-beta superfamily, such as the bone morphogenetic proteins (BMP), are currently the most interesting ones among the factors that regulate chondrogenesis and osteogenesis. In the coming years, the availability of new cloned probes combined with sensitive analytical methods, as reviewed here, will add greatly to our understanding of the various aspects of gene expression during bone repair. This information should provide answers to some of the unresolved questions in fracture callus development.

In situ localization of collagen production by chondrocytes and osteoblasts in fracture callus.

An experimental model of fracture-healing was used to study the production of types-I and II collagen by in situ hybridization. The distribution of cartilage matrix in callus was determined by histochemical staining. Messenger RNA (mRNA) for cartilage-specific type-II collagen was detectable as early as the fifth day in a small number of cells that had acquired a chondrocyte phenotype but that also contained type-I collagen mRNA, suggesting an ongoing change in the expression of collagen genes. The location of the first chondrocytes, which were adjacent to cortical bone, suggested that they originated from cells that had derived from the periosteum by differentiation. On the seventh day of callus formation, the presence of both type-I and type-II collagen mRNA in chondrocytes of expanding cartilage suggested that most growth occurred by differentiation of mesenchymal cells and less by proliferation of differentiated chondrocytes. Expansion continued until the tenth to fourteenth day, after which the cartilage was replaced by woven bone. This was characterized by the presence of osteoblasts that were active in the synthesis of type-I collagen.

Localization of osteonectin expression in human fetal skeletal tissues by in situ hybridization

SummaryThe expression of osteonectin gene was studied in developing human fetuses by Northern analysis andin situ hybridization. The highest levels of osteonectin mRNA were detected in RNA extracted from calvarial bones, growth plates, and skin. Low mRNA levels were present in several parenchymal tissues.In situ hybridization of developing long bones revealed three cell types with high osteonectin mRNA levels: osteoblasts, cells of the periosteum, and hypertrophic chondrocytes. Weaker signals were detected in osteocytes, fibroblasts of tendons, ligaments and skin, and in cells of the epidermis. Apart from the hypertrophic chondrocytes, only low osteonectin mRNA levels were seen in cartilage. The localization of osteonectin mRNA in fetal growth plates is consistent with the hypothesis that the protein plays a role in the mineralization of bone and cartilage matrices.

Construction of a Human Proα1(III) Collagen cDNA Clone and Localization of Type III Collagen Expression in Human Fetal Tissues

A cDNA clone for human pro alpha 1(III) collagen mRNA was isolated from a cDNA library constructed for human fetal skin RNA. The clone, pHFS3, was identified by restriction mapping and sequencing. Comparison with previously published human type III collagen sequences revealed some differences which may reflect individual variation. The clone was used to study the expression of type III collagen mRNA in various fetal tissues in comparison to the expression of type I collagen mRNAs. In 15-18-week fetal skin the ratio of alpha 1(I) to alpha 1(III) collagen mRNAs was 0.8. Diaphyseal and calvarial bone contained high amounts of type I collagen mRNA and low levels of type III collagen mRNA, resulting in high type I/type III ratios. In situ hybridization of sections of skeletal tissues was employed to identify the cells containing the mRNAs for types I, II and III procollagens. The results revealed differential expression patterns for these three collagen types in various human fetal tissues. Lack of coordinate expression suggests that production of type I and type III collagens is under different regulatory mechanisms in developing skeletal tissues.

Matrix in Cartilage and Bone Development: Current Views on the Function and Regulation of Major Organic Components

Study of the growth and development of cartilage and bone has been difficult because the structure of the tissues makes biological experiments hard to conduct. Recent advances in molecular biology have offered new possibilities for studying these processes. Many cartilage and bone specific cDNAs have been cloned and characterized and consequently used to localize the corresponding mRNAs in tissue sections. Developing cartilage and bone serve as a model for the study of extracellular matrix gene regulation during the proliferation, growth and differentiation of connective tissue cells. Normal skeletal growth and development are regulated by both systemic and local factors. The effects of many systemic hormones on bone metabolism have been studied extensively, but the pathways triggered by these hormones in the target cells are less well known. Recent evidence suggests that some growth factors, such as TGF-beta, IGFs and PDGF, act as local regulators of cartilage and bone metabolism. The different extracellular matrix components, e.g. collagens, are expressed differently in distinct cell types and developmental stages during cartilage and bone development. This model, therefore, facilitates the study of relations between the production of the various extracellular matrix components and the growth factors and the proto-oncogenes which may regulate them. Existing knowledge of the expression of major cartilage and bone components and their regulation during growth, differentiation and development is reviewed. An understanding of the normal growth and development of cartilage and bone is fundamental for elucidating the mechanisms underlying the various diseases -- both hereditary and acquired -- affecting the human skeleton.

Transient Expression of Type III Collagen by Odontoblasts: Developmental Changes in the Distribution of Pro-α1 (III) and Pro-α1 (I) Collagen mRNAs in Dental Tissues

Abstract The expression of pro- α1 (III) and pro- α1 (I) collagen mRNAs in mouse and human dental tissues during tooth development and after its completion was analyzed by in situ hybridization, with use of [ 35 S]-labeled RNA probes. The expression of pro- α1 (III) mRNA was also compared to that of the protein product, as localized by immunostaining with polyclonal antibodies to type III collagen and the N-terminal propeptide of type III procollagen. Contrary to many previous reports, our results suggest that odontoblasts express type III collagen. While proal (III) transcripts were less intensely expressed in odontoblasts than pro- α1 (I) transcripts, the amounts of both mRNAs increased in odontoblasts with progressing dentin formation, and decreased toward its completion. In contrast to pro- α1 (III) mRNA, pro- α1 (I) mRNA was still detectable in odontoblasts of fully developed teeth. Type III collagen immunoreactivity was observed in the early predentin, and again in predentin toward the completion of dentinogenesis, when mRNA was no longer detected. Also in the pulp, the protein product, unlike pro- α1 (III) mRNA, was relatively strongly expressed. Hence, these immunostaining patterns were inversely related to the expression of pro- α1 (III) mRNA, suggesting accumulation of the protein. The mesenchymal cells, when condensed in the region of the future mandibular bone, expressed proal (III) mRNA intensely, whereas osteoblasts expressed pro- α1 (I) but not pro α1 (III) transcripts strongly. Cell type- and developmental stage-related differences in the expression of the two mRNAs suggest that type I1type III collagen ratio influences the structure of dental tissues.

Expression of Nucleoprotein mRNAs During Rat Spermiogenesis

DNA-associated proteins of spermatozoa are different from somatic cell histones. In mammals, arginine-rich protamines replace histones and transition proteins in spermatids during compaction of the chromatin (Poccia, 1986). The mouse protamine mRNAs are expressed in early spermatids by a haploid genome (Kleene et al., 1983). To find the exact stages of differentiation where the expression of spermatidal nucleoprotein mRNAs occur, we used three different cDNA-hybridization techniques to measure protamine 1, protamine 2 and transition protein (TP1) mRNA levels.