Comparison of bone biomechanical properties after bone marrow mesenchymal stem cell or alendronate treatment in an osteoporotic animal model.

The aim of this study was to explore the effects of bone marrow mesenchymal stem cells (BMMSCs) and alendronate sodium (ALN) intervention on osteoporosis (OP). Sixty-eight 6-month-old healthy female Sprague Dawley (SD) rats were used to generate an OP model by removal of the ovaries. After 12 weeks, rats were treated with BMMSCs (BMMSC group) or ALN (ALN group) for 5 weeks. Serum type I collagen C terminal peptide (CTX_1), procollagen type I N-terminal propeptide (PINP), and bone alkaline phosphatase (BALP) were tested along with the femur bone density and other properties, including bone mineral density (BMD), BALP, percent trabecular area (BV/TV), trabecular thickness (Tb.Th), trabecular number (TbN), maximum load, maximum stress, maximum strain, and elastic modulus. BMD, BALP, BV/TV, Tb.Th, TbN, maximum load, maximum stress, maximum strain, and elastic modulus values were higher in the BMMSC group versus the ALN group relative to the control group (p < 0.05); CTX_1, PINP, trabecular separation (Tb.Sp), and osteoclast number (OC.N) were lowest in the BMMSC group versus the ALN group relative to the control group (p < 0.05). Both BMMSCs and ALN could improve the metabolic function and bone quality in osteoporotic mice while restoring the strength and toughness of bones. The intervention effects of BMMSCs are better than ALN in this model.

Chondrocytes cultured in silk-based biomaterials maintain function and cell morphology.

OBJECTIVE:: To characterize the morphology of chondrocytes and the expression and secretion of active collagen II by these cells cultured within a regenerated silk fibroin film. Silk fibroin film cytocompatibility and the effect of silk fibroin on chondrocytes in vitro were also evaluated. METHODS:: Chondrocytes were transfected with a lentivirus containing a green fluorescent protein marker and cultured within a regenerated silk fibroin film. Effects on chondrocyte adhesion, growth, and expression of functional collagen II were assessed in vitro by analysis with immunofluorescent histochemistry and laser scanning confocal microscopy. RESULTS:: The results of this study showed that the regenerated silk fibroin film had no cytotoxic effect on chondrocytes. The regenerated silk fibroin film facilitated the adhesion of chondrocytes with typical morphology. Chondrocytes cultured within silk fibroin films exhibited the expression of collagen II in vitro. CONCLUSION:: Regenerated silk fibroin film was found to be an excellent biomaterial with good cytocompatibility for chondrocytes, because these cells remained functional and maintained normal cell morphology when cultured in silk-based biomaterials. These results suggest that silk-based chondrocyte biomaterial complexes may provide a feasible and functional biomaterial for repairing clinical cartilage defects.

PINP as a biological response marker during teriparatide treatment for osteoporosis.

Postmenopausal women with severe osteoporosis may require treatment with the bone anabolic drug teriparatide. While changes in bone mineral density (BMD) are one measure of response, BMD changes often require a minimum of one year to observe measureable changes. Biochemical markers of bone turnover change within 1 to 3 months of initiating osteoporosis therapy. Monitoring with a marker such as procollagen type I N propeptide (PINP), an osteoblast-derived protein, during teriparatide treatment may provide clinically useful information for managing patients with osteoporosis. Clinical trials have shown consistent increases in PINP within 3 months of initiating teriparatide, increases that are significantly greater than placebo and significantly different from baseline. Increases in PINP concentrations during teriparatide treatment correlate well with increases in skeletal activity assessed by radioisotope bone scans and quantitative bone histomorphometry parameters. Individuals treated with teriparatide in clinical trials usually experienced an increase in PINP > 10 mcg/L from baseline, while those given placebo usually did not. In the clinical setting, patients experiencing a significant increase in PINP > 10 mcg/L after initiating teriparatide therapy may receive an earlier confirmation of anabolic effect, while those who do not may be assessed for adherence, proper injection technique, or undetected secondary conditions that might mitigate an anabolic response. PINP monitoring may provide information supplemental to BMD monitoring and be a useful aid in managing patients receiving anabolic osteoporosis treatment in the same way that biochemical markers of bone resorption are useful in monitoring antiresorptive therapy. This review examines PINP as a biological response marker during teriparatide treatment for osteoporosis.

Carotid intima-media thickness and bone turnover: the role of C-terminal telopeptide of type I collagen.

Osteoporosis and vascular disease are commonly found together in elderly people. Several common mechanisms and risk factors have been suggested to contribute to the development of osteoporosis and atherosclerosis. The present cross-sectional study was performed to determine whether the degree of bone turnover is correlated to carotid intima-media thickness (CCA-IMT), as a marker of subclinical atherosclerosis. We selected 50 outpatients (mean age 71.7 +/- 12.3), underwent to eco-Doppler evaluation of extracranial carotid tract, without history of calcium and/or vitamin D supplementation, or antireabsorptive therapy. CCA-IMT was measured by high-resolution B-mode ultrasonography. Bone turnover was evaluated by analysing serum levels of C-terminal telopeptide of type I collagen (sCTX), and bone-specific alkaline phosphatase. We also evaluated the vitamin D status by determination of the serum concentration of 25-hydroxyvitamin D [25(OH)D]. We found a prevalence of hypovitaminosis D [serum 25(OH)D levels <30 ng/mL, mean value 10.7 +/- 5.8] of 91.8%, and an increased bone resorption, with mean sCTX levels higher than reference values (mean 1.18 +/- 0.57 ng/mL). A significant positive correlation was found between CCA-IMT and age (r = 0.480, P = 0.001), erythrocyte sedimentation rate (ESR: r = 0.438, P = 0.001), high-sensitivity C-Reactive Protein (HsCRP: r = 0.482, P = 0.011), serum creatinine (r = 0.305, P = 0.031), and sCTX (r = 0.389, P = 0.006). In a multivariate linear regression, CCA-IMT was independently predicted by age (beta = 0.34, P = 0.001), ESR (beta = 0.37, P = 0.005), and sCTX (beta = 0.32, P = 0.006). The preliminary results of our study seem to indicate that after adjustment for established cardiovascular risk factors, sCTX independently predict an increased CCA-IMT in the elderly population.

Procollagen I amino-terminal propeptide as a potential marker for multiple myeloma.

OBJECTIVES: Investigating relationship between bone markers, cytokines and conventional prognostic parameters in patients with multiple myeloma (MM) and to assess the clinical application of bone turnover markers. DESIGN AND METHODS: Sixty-four patients with MM were examined before treatment and followed for survival for over 7 years. Serum concentrations of bone markers and cytokines were determined by the Roche and R&D kits, respectively. Standard deviation scores (SDS) were employed to normalize values. RESULTS: Collagen fragments (beta-CTX) were elevated in 47%, procollagen I amino-terminal propeptide (PINP)-in 28%, and osteocalcin (OC) in 11% of patients. The values of the SDS of PINP and OC, but not beta-CTX significantly decreased with MM stage. beta-CTX inversely correlated with vascular endothelial growth factor (VEGF) and albumin, and directly correlated with serum macrophage colony-stimulating factor (M-CSF). OC values correlated with albumin and beta2-microglobulin. PINP inversely correlated with LDH. The SDS values of PINP were significantly lower in MM patients with advanced bone disease. CONCLUSIONS: Circulating PINP concentration may be a useful marker for monitoring of treatment of multiple myeloma patients with bone lytic lesions, in particular, of patients treated with preoteasome inhibitors.

Type IX collagen is crucial for normal hearing.

cDNA microarray analysis indicated that COL9A1 and COL9A3 are highly expressed in the human inner ear, suggesting that type IX collagen has a crucial functional role in the inner ear. This study further confirmed, by means of real-time PCR, the presence of collagen type IX genes in the mouse inner ear. Immunocytochemical analysis also revealed that type IX collagen is distributed in the tectorial membrane, where it co-localizes with type II collagen, indicating that type IX collagen may contribute to the three-dimensional integrated structure of type II collagen molecules. Mice with targeted disruption of the col9a1 gene were shown through assessment by auditory brain stem response to have hearing loss, suggesting an important role of type IX collagen in maintaining normal hearing. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape, and electron microscopy confirmed disturbance of organization of the collagen fibrils. An antibody against type II collagen failed to detect type II collagen in the tectorial membrane of type IX collagen knock-out mice, suggesting that a lack of type IX collagen may affect the three-dimensional structure of type II collagen molecules. These findings indicate that genes encoding each chain of type IX collagen may fulfill an important function associated with the tectorial membrane in the auditory system.

Pro-collagen I COOH-terminal trimer induces directional migration and metalloproteinases in breast cancer cells.

Breast and prostatic carcinomas, melanoma, and endothelial cell lines are chemoattracted by medium conditioned by mature osteoblasts. The chemoattractant for endothelial cells was identified with C3, carboxyl-terminal trimer of pro-collagen type I. We report that C3 induces directional migration and proliferation, the expression of tissue inhibitor of metalloproteinases-2, pro-metalloproteinase-2 and -9, and their activation in MDA MB231 cells, without changing the expression of tissue inhibitor of metalloproteinases-1 and of metalloproteinase-14. Antiserum against metalloproteinase-2 or -9 or -14, tissue inhibitor of metalloproteinases-1, or GM6001 inhibits the C3-induced migration. Urokinase and its receptor are detected and unchanged upon exposure to C3. The antibody against urokinase or addition of plasminogen activator inhibitor inhibits migration. Blocking antibodies to integrins alpha(2), alpha(6), beta(1), and beta(3) inhibit chemotaxis and do not change urokinase and urokinase receptor expression. Blockage of alpha(2), beta(1), and beta(3) integrins affect differently the induction by C3 of pro-metalloproteinase-2 and -9 and of tissue inhibitor of metalloproteinases-2. Chemotaxis to C3 is also inhibited by genistein, by pertussis toxin, which also inhibits C3-induced pro-metalloproteinase -2 and -9, but not urokinase expression. Wortmannin partially inhibits C3-induced cell migration. Other, but not all, breast carcinoma lines tested responded to C3 with migration and pro-metalloproteinase-2 induction. Presently C3 is the only agent known to induce migration specifically of both endothelial and breast carcinoma cells. The mitogenic and motogenic role of C3 in vitro might prefigure a role in in vivo carcinogenesis and in the establishment of metastasis.

Type IIA procollagen in development of the human intervertebral disc: regulated expression of the NH(2)-propeptide by enzymic processing reveals a unique developmental pathway.

Type II collagen can be synthesized in two forms generated by alternative splicing of the precursor mRNA. Type IIA procollagen, which contains a cysteine-rich domain in the NH(2)-propeptide (exon 2), is produced by precartilage and noncartilage epithelial and mesenchymal cells, and type IIB procollagen, without the cysteine-rich domain, is characteristic of chondrocytes. Mice lacking type II collagen fail to develop intervertebral discs. We have previously shown that the human intervertebral disc and notochord synthesize primarily the type IIA form of procollagen. Therefore, we investigated the distribution of type IIA procollagen during early disc development in humans. By processes of radioactive in situ hybridization and fluorescence immunohistochemistry, we localized mRNA and protein of type IIA procollagen, type I collagen, and type III collagen in fetal intervertebral disc specimens ranging from day 42 (embryonic stage 17) to day 101 (week 14.5) of gestation. Antibodies to the three distinct domains of type IIA procollagen: the NH(2)-propeptide, the fibrillar domain, and the COOH-propeptide were used. The earliest stage of developing intervertebral disc (42 days, stage 17) was characterized by diffuse synthesis of types I and III collagens in the dense zone (intervertebral area) and synthesis of type IIA procollagen by the chondrocyte progenitor cells surrounding the disc. The notochord cells synthesized and deposited into the notochordal sheath all three fibrillar collagens. By 54 days (stage 22), the developing disc was clearly divided into three regions: 1.) the outer annulus, characterized by synthesis and deposition of types I and III collagens; 2.) the inner annulus, characterized by synthesis and deposition of type IIA collagen containing the NH(2)-propeptide but devoid of the COOH-propeptide (pN-procollagen); and 3.) the notochord, the cells of which synthesized and deposited of all three fibrillar collagens. In later stages of fetal development (72-101 days), a change in type IIA procollagen processing was observed in the cells of the inner annulus: even though these cells continued to synthesize type IIA procollagen, they deposited into the extracellular matrix (ECM) only the processed fibrillar domain, with the NH(2)-propeptide removed. This finding indicates that there is a developmentally regulated change in the processing of type IIA procollagen NH(2)-propeptide in the cells of the inner annulus. This mechanism is in contrast to previously shown developmental regulation of the cysteine-rich domain of the NH(2)-propeptide by alternative splicing of the precursor mRNA. Although the cells of the inner annulus have been identified as chondrocytes, based on their shape and synthesis of characteristic ECM components, they appear to represent a distinct developmental pathway characterized by their synthesis and differential processing of type IIA procollagen. This developmental pattern may prove important for disc regeneration.

Molecular mechanisms of cell-cell signaling by the Spemann-Mangold organizer.

We review how studies on the first Spemann-Mangold organizer marker, the homeobox gene goosecoid, led to the discovery of secreted factors that pattern the vertebrate embryo. Microinjection of goosecoid mRNA formed secondary axes and recruited neighboring cells. These non-cell autonomous effects are mediated in part by the expression of secreted factors such as chordin, cerberus and Frzb-1. Unexpectedly, many of the molecules secreted by the Spemann-Mangold organizer turned out to be antagonists that bind growth factors in the extracellular space and prevent them from binding to their receptors. The case of chordin is reviewed in detail, for this molecule has provided biochemical insights into how patterning by Spemann's organizer can be regulated by diffusion and proteolytic control. The study of the BMP-binding repeats of Chordin, which are present in many extracellular proteins, may provide a new paradigm for how cell-cell signaling is regulated in the extracellular space not only in embryos, but also in adult tissues.

Regulation of type I collagen genes expression.

Type I collagen is the major component of many extracellular matrices, and its accumulation characterizes most fibrotic processes. It is synthesized by a small number of discrete cell types, including fibroblasts, osteoblasts and odontoblasts. Analysis of transgenic mice harbouring different segments of the promoters of the mouse pro-alpha1 (I) and pro-alpha2 (I) genes has led to the conclusion that this tissue-specific expression is controlled by different cis-acting elements which are responsible for the expression of type I collagen genes in different type I collagen-producing cells. Transacting factors which bind to these different tissue-specific elements are still unknown, but they probably act by modifying the chromatin structure. In fibroblastic cells, various soluble molecules can modulate the transcription of type I collagen genes. Analysis of the pro-alpha1 (I) and pro-alpha2 (I) proximal promoters has led to the identification of different cis-acting elements which can modulate the expression of reporter genes, in transfection experiments. Among these cis-acting elements, a sequence located between -378 and -183 bp in the human pro-alpha2 (I) promoter appears to mediate the transcriptional effects of transforming growth factor-beta. It binds a large multimeric complex which contains Sp1, as well as AP1 and other DNA-binding proteins which have not yet been identified.

Coexpression of the lysyl oxidase-like gene (LOXL) and the gene encoding type III procollagen in induced liver fibrosis.

We have isolated a mouse lysyl oxidase-like (LOXL) cDNA from a mouse embryo cDNA library and used this cDNA to measure changes in steady state levels of LOXL mRNA during the development of carbon tetrachloride-induced liver fibrosis in adult mice. These results revealed the coincident appearance of increased steady state levels of LOXL mRNA and type III procollagen mRNA early in the development of liver fibrosis. In contrast, steady state levels of lysyl oxidase mRNA increased throughout the onset of hepatic fibrosis and appeared in parallel with the increased steady state levels of pro-alphaI (I) collagen mRNA. These findings suggest that the LOXL protein (possibly an isoform of lysyl oxidase) is involved in the development of lysine-derived cross-links in collagenous substrates. Moreover, the substrate specificity of the LOXL protein may be different to that of lysyl oxidase and this difference may be collagen-type specific.

Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice.

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (> 400 Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the alpha S1-casein mammary gland-specific promoter operatively linked to 37 Kb of the human alpha 1(I) procollagen structural gene and 3' flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(alpha 1)3] type I procollagen were detected (up to 8 mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in milk; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.

Collagen synthesis by liver stellate cells is released from its normal feedback regulation by acetaldehyde-induced modification of the carboxyl-terminal propeptide of procollagen.

Acetaldehyde stimulates collagen synthesis in stellate cells and forms adducts with procollagen in the liver of alcoholics. To assess the possibility that modification of the carboxyl-terminal propeptide by acetaldehyde affects its capacity to exert a feedback inhibition of collagen synthesis after splitting from procollagen, the propeptide was prepared by gel filtration of the bacterial collagenase digests of procollagen type I (obtained from 10(9) calvaria fibroblasts of newborn rats) and reacted with either 250 mM acetaldehyde and 100 mM CNBH3 or with 170 microM acetaldehyde without reducing agents, to mimick in vivo conditions. The unmodified propeptide produced a concentration-dependent inhibition of collagen synthesis by Ito cells. By contrast, the acetaldehyde-modified propeptide produced a lesser inhibition of procollagen synthesis in the cells, associated with a greater accumulation of collagen in the media. The incubation with 170 microM acetaldehyde and, to a lesser extent, 50 mM ethanol produced collagenase-digestible adducts in stellate cells. Thus, the formation of acetaldehyde adducts with the carboxyl-terminal propeptide of procollagen may account, at least in part, for the stimulatory effect of acetaldehyde on collagen synthesis by stellate cells and may lead to collagen accumulation through a decrease of the normal feedback regulation of collagen synthesis by the propeptide.

Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure.

Type II collagen is the most abundant collagen in articular cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock-absorbing properties and its resiliency to stress. Specific antibodies which recognize various collagen types have been very useful in the study of collagen biosynthesis, structure and metabolism in normal and pathological conditions. Antibodies which recognize epitopes of type II collagen have been described previously; however, many of these antibodies display cross-reactivity with other collagens or with type II collagen from other species, reflecting the high degree of homology of the helical domains of fibrillar collagens. In this study, we prepared antibodies to sequential determinants of human type II procollagen employing synthetic peptides with sequences deduced from the nucleotide sequence of the human alpha 1 (II) procollagen cDNA. The antibodies were highly specific for epitopes in either the C-terminal propeptide or the telopeptide of the human type II collagen and did not cross-react with other human interstitial collagens or with murine type II collagen. These antibodies were used in conjunction with biosynthetic labeling to study the secretion and processing of human type II procollagen and collagen in human chondrocytes in vitro. The results indicated that a lag period of about 90 min was required for the secretion of newly synthesized type II procollagen. Conversion of the secreted procollagen into fully processed alpha-chains and their deposition in the cell layer were first apparent 240 min following the initiation of biosynthetic labeling. The antibodies were also used to examine, by immunoelectron microscopy, the structure of the extracellular matrix produced by human chondrocytes maintained in long-term cultures under conditions which permit the preservation of the cartilage-specific phenotype. These highly specific antibodies provide valuable tools to study the metabolism and structure of human type II procollagen and collagen in normal and pathologic conditions.

The aminoterminal propeptide of type III procollagen. Studies on physiology and pathophysiology.

The aim of the present study was to examine the physiological basis for the clinical use of serum PIIINP as a marker of the deposition rate of type III collagen. The assumption was that the serum concentration of PIIINP would reflect the turnover of type III collagen and thus directly reflect the inflammatory response. For the study we assessed commercially available RIAs and optimised one of them. Porcine PIIINP was purified and compared with human PIIINP. The application of a gentle iodination procedure made it possible to perform tracer studies. An experimental model consisting of a thoracic duct-venous shunt in conscious pigs was developed. Double and triple isotope tracer techniques were used for kinetic studies in the animal model and in cultures of tubule cells. The rat model with the induction of granulation tissue was used to investigate catabolic states. The anabolic state was studied in humans receiving growth hormone therapy. We conclude: 1) That, for our purpose, the best method of determining PIIINP is the PIIINP RIA, owing to the profile of the substances determined. It was possible to improve the quality of the tracer and to increase sensitivity by changing the assay procedure. 2) That porcine PIIINP is similar to human PIIINP, therefore the human assay is suitable for studies in pigs. 3) That PIIINP most likely escapes from the extracellular space by bulk flow, similar to that of albumin. That the major part of the PIIINP synthesised is drained via the lymphatics. That intact PIIINP is not, or only to a minor extent, degraded through the lymphatics. Consequently, peak B is not a product of processes of the lymph system. 4) That in pigs intact PIIINP has a circulatory half-life of about 1 hour, and that it is degraded by at least two intermediary steps. The first step gives rises to peak B, which is found in an almost constant ratio to intact PIIINP. Peak B has a half-life of about 4 hours. Given steady state conditions peaks B and C (intact PIIINP) thus reflect the same process. In three different studies a fraction with an MW lower than that of PIIINP but higher than the col 1 domain, appeared during the degradation of intact PIIINP. This fraction (peak E) has not been described before. Furthermore, we did not observe the formation of peak D (proposed to be the col 1 domain of PIIINP) which indicates that this fraction does not originate from the metabolism of PIIINP. 5) That PIIINP rapidly distributed from the circulation to tissues, and that the liver and the kidneys are the organs mainly responsible for the degradation of PIIINP. The high hepatic clearance described previously is in part due to shunting of PIIINP directly to the lymph, but most of it is extracted by the liver for subsequent degradation or release of the intact molecule to the hepatic vein. Only a small part is irreversibly cleared and metabolised (about 5%). 6) That, given steady state conditions, the turnover of PIIINP is well reflected by changes in serum PIIINP, but also that this relation disappears when the body is in a catabolic state. Anabolic states give rise to increased serum concentrations of PIIINP as compared with normals states. The general conclusion is that serum PIIINP is a marker of type III collagen turnover under well-defined conditions. Serum PIIINP, mainly consisting of peaks B and C (intact PIIINP) may, owing to the disposal rate, reflect changes in type III collagen turnover over one day (6 half-lives). The liver and kidneys actively take part in the degradation of circulating PIIINP. Serum concentrations of PIIINP in the presence of changing body composition (weight loss) or during treatment with cytostatic drugs (cyclophosphamide) should be interpreted with caution.

Evans' syndrome, myelofibrosis and systemic lupus erythematosus: role of procollagens in myelofibrosis.

We describe an 18 yr old female with systemic lupus erythematosus presenting with Evans' syndrome (autoimmune hemolytic anemia and immune thrombocytopenia) and dense myelofibrosis. Her clinical course and response to treatment were monitored with regular blood counts, serial measurements of serum procollagen I and III and periodic bone marrow examinations. This report brings to 9 the number of well-documented cases of systemic lupus erythematosis and myelofibrosis in the literature; we discuss the pathogenesis and management of patients with this apparently rare disorder, with particular reference to the role of serum procollagen measurements as markers of myelofibrosis.

The regulation of collagen in fetal skin wounds: mRNA localization and analysis.

The deposition of collagen in fetal skin wounds has been shown in several animal models. The authors used a radiolabeled RNA antisense probe, complementary to the mRNA for the alpha-1 chain of human procollagen type I, to assess regulation of this collagen species in fetal and adult rabbit wounds. Dorsal skin wounds were placed on fetal and maternal animals at the beginning of the third trimester, and were harvested 3, 5, and 7 days later. In situ RNA/RNA hybridization was performed on suitable specimens, and morphometric analysis was carried out with a computerized LECO image analyzer. Fetal wounds exhibited an inflow of mesenchymal cells that produced collagen type I at levels higher than the surrounding tissue; this activity was highest on days 3 and 5 after wounding. Adult wounds had increased fibroblast presence by day 7, producing collagen type I at levels higher than those of adjacent unwounded tissue. Morphometric analysis of the signal produced by in situ hybridization and of the number of cells producing the signal in a given field showed that fetal wounds appear to produce collagen type I by an increase in the number of cells in the area of the wound--not by induction of the gene for procollagen type I. In contrast, adult wounds had both fibroblast migration and induction of procollagen type I mRNA synthesis. These findings imply multilevel regulation of collagen production in the adult and posttranslational regulation in the fetus.

Preferential expression of alternatively spliced mRNAs encoding type II procollagen with a cysteine-rich amino-propeptide in differentiating cartilage and nonchondrogenic tissues during early mouse development.

Type II procollagen mRNAs are alternatively spliced: type IIA mRNA contains an exon encoding a cysteine-rich domain in the amino-propeptide and type IIB mRNA lacks this exon. In mouse embryos between 9.5 and 13.5 days, type IIA mRNA was the major form of Col2a-1 transcript expressed in both prechondrogenic and nonchondrogenic tissues and type IIB mRNAs were present in small amounts. After 12.5 days, type IIB mRNA levels increased rapidly and finally exceeded type IIA mRNAs. Type IIB mRNAs became the major Col2a-1 transcript by 14.5 days, predominantly expressed in maturing chondrocytes. By 17.5 days type IIB mRNAs account for 80% of the Col2a-1 transcripts. Expression of type IIA mRNAs follows the change in the growth pattern of the cartilaginous model of the axial and appendicular skeleton and of the otic capsule and nasal septum. In nonchondrogenic tissues, type IIA mRNAs are more commonly expressed in epithelial structures of ectodermal and endodermal origin than in nonepithelial tissues. The switching of expression from type IIA to type IIB mRNA as major Col2a-1 transcript may be associated with the commitment of precursor cells to the chondrocyte lineage and sites of type IIA mRNA expression may mark regions of potential cartilage growth. The differential expression pattern of type IIA mRNAs therefore points to an association of type IIA procollagen with chondrocyte differentiation during cartilage growth and some function early in embryogenesis in the epithelial organization of nonchondrogenic tissues.