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C Ohlsson

Publications and source records attributed to C Ohlsson.

108 records · Page 6Linked to original sources
PubMed1995

Expression of functional growth hormone receptors in cultured human osteoblast-like cells.

Recent clinical studies indicate that GH is important for bone remodeling. Patients with GH deficiency exhibit decreased bone density, and GH substitution increases bone density in these patients. The aim of the present study was to investigate the presence of GH receptors and the effects of GH on cultured human osteoblast-like cells. Primary cultures of human osteoblast-like cells were established from trabecular bone. Northern blot analysis, using a probe recognizing exon 10 of the human GH receptor, revealed a 4.7-kilobase transcript corresponding to the human GH receptor. Cultured osteoblast-like cells expressed, as determined by RNase protection assay, approximately one fourth of the GH receptor messenger RNA levels found in liver. Binding studies using 125I-labeled GH revealed a single class of receptors with approximately 2000 binding sites per cell and an association constant (Ka) of 2.6 x 10(9) M-1. GH stimulation of the cultured cells resulted in increased [3H]thymidine incorporation, suggesting that the GH receptors are functional. In summary, the present study shows that cultured human osteoblast-like cells express functional GH receptors.

Base Sequence↗
PubMed1995

Expression of exon 3-retaining and exon 3-excluding isoforms of the human growth hormone-receptor is regulated in an interindividual, rather than a tissue-specific, manner.

GH has multiple effects on growth and metabolism, and these functions are mediated through binding to specific cell surface receptors. The human GH receptor (GHR) exists in two known isoforms; in one form exon 3 is present (GHR3+), and in the other, exon 3 is absent (GHR3-). Recent reports have suggested that the expression of the two isoforms is tissue specific and/or developmentally regulated. We used a reverse transcription-polymerase chain reaction assay to study the expression pattern of the two isoforms in a variety of tissues from normal subjects and patients with acromegaly. In skeletal muscle from both normal subjects and patients with acromegaly, the GHR3+ transcript was expressed, either alone or together with the shorter (GHR3-) transcript. When multiple tissues from six subjects were tested, the expression of the two isoforms varied among subjects, whereas different tissues from the same subject showed the same expression pattern. These results indicate that the expression of the GHR isoforms is not tissue specific. Instead, the expression of the GHR isoforms appears to be specific for each individual, suggesting that it is under the control of factors that affect all tissues in the body.

Adipose Tissue↗
PubMed1995

[Autologous cartilage cell transplantation. The goal is pain relief and restored joint function].

Chondral and osteochondral damage is a common result of trauma to the joints. The capacity of cartilage to heal such damage is poor, and repetitive wear on joint surfaces that do not heal results in impaired joint function, which can culminate in full blown arthrosis. Thus, it is important to improve our knowledge of cartilage regenerative potential, and develop methods to forestall progression to arthrosis by promoting the early healing of cartilage damage. Autologous cartilage cell transplantation may be a mean of healing cartilage damage. A method of cultivating autologous chondrocytes for transplantation in the treatment of isolated damage to articular cartilage of the knee is presented in the article.

Cartilage↗
PubMed1994

Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.

BACKGROUND: Full-thickness defects of articular cartilage in the knee have a poor capacity for repair. They may progress to osteoarthritis and require total knee replacement. We performed autologous chondrocyte transplantation in 23 people with deep cartilage defects in the knee. METHODS: The patients ranged in age from 14 to 48 years and had full-thickness cartilage defects that ranged in size from 1.6 to 6.5 cm2. Healthy chondrocytes obtained from an uninvolved area of the injured knee during arthroscopy were isolated and cultured in the laboratory for 14 to 21 days. The cultured chondrocytes were then injected into the area of the defect. The defect was covered with a sutured periosteal flap taken from the proximal medial tibia. Evaluation included clinical examination according to explicit criteria and arthroscopic examination with a biopsy of the transplantation site. RESULTS: Patients were followed for 16 to 66 months (mean, 39). Initially, the transplants eliminated knee locking and reduced pain and swelling in all patients. After three months, arthroscopy showed that the transplants were level with the surrounding tissue and spongy when probed, with visible borders. A second arthroscopic examination showed that in many instances the transplants had the same macroscopic appearance as they had earlier but were firmer when probed and similar in appearance to the surrounding cartilage. Two years after transplantation, 14 of the 16 patients with femoral condylar transplants had good-to-excellent results. Two patients required a second operation because of severe central wear in the transplants, with locking and pain. A mean of 36 months after transplantation, the results were excellent or good in two of the seven patients with patellar transplants, fair in three, and poor in two; two patients required a second operation because of severe chondromalacia. Biopsies showed that 11 of the 15 femoral transplants and 1 of the 7 patellar transplants had the appearance of hyaline cartilage. CONCLUSION: Cultured autologous chondrocytes can be used to repair deep cartilage defects in the femorotibial articular surface of the knee joint.

Adolescent↗
PubMed1994

Hormonal regulation of longitudinal bone growth.

The regulation of postnatal somatic growth is complex. Genetic, nutritional factors and hormones exert regulatory functions. Hormones that have an established role in the regulation include growth hormone (GH), thyroid hormone and sex steroids. GH promotes mainly the growth of the long bones in terms of final height, while the action of the sex steroids and thyroid hormone is less well known. Longitudinal bone growth is the result of chondrocyte proliferation and subsequent endochondral ossification in the epiphyseal growth-plates. The growth-plate is a cartilaginous template that is located between the epiphysis and the metaphysis of the long bones. GH and insulin-like growth factor-I (IGF-I) have different target cells in the epiphyseal growth-plate. GH stimulates the slowly dividing prechondrocytes in the germinative cell layer while IGF-I promotes the clonal expansion in the proliferative cell layer of a GH primed cell. Thyroid hormone blocks the clonal expansion and stimulates chondrocyte maturation. IGF-I mRNA is primarily regulated by GH, and IGF-I is produced in several tissues such as the liver, muscle, fat and epiphyseal growth plates. However, IGF-I mRNA is also increased during compensatory growth of heart and kidneys and by estrogen in the Fallopian tube in the rat. Nutrition, i.e. energy from fat and carbohydrates and proteins, also influences the final height, but the cellular mechanism of action is not known. The aim of this article is to review hormonal action on longitudinal bone growth.

Animals↗
PubMed1994

Disproportional bone growth and reduced weight gain in gonadectomized male bovine growth hormone transgenic and normal mice.

Sex steroids have been shown to influence longitudinal bone growth during sexual maturation, partially by increased GH secretion. Mice transgenic for metallothionein promoter bovine GH were developed by pronuclear injection as a model with sex steroid-independent GH secretion. Prepubertal normal and transgenic, male and female mice were either gonadectomized or sham operated. The growth was divided into two segments: peripubertal growth from 30-60 days of age and adult growth from 60-90 days of age. Orchidectomy resulted in a decreased growth rate of the lumbar spine and a decreased weight gain during the peripubertal growth, whereas tibia growth was unaffected. The alteration in proportions between the lumbar spine and the tibia was apparent for both normal and bovine GH transgenic mice, suggesting that the effect was not mediated via decreased GH secretion. Orchidectomy resulted in increased adult tibial growth, whereas weight gain and lumbar growth were unaffected. In female mice, gonadectomy did not influence these parameters during either time period studied. In summary, we present data indicating that the male gonads in a GH secretion-independent manner stimulate pubertal growth of the spine and inhibit the tibial growth of adult animals.

Animals↗
PubMed1994

Clonal analysis of rat tibia growth plate chondrocytes in suspension culture--differential effects of growth hormone and insulin-like growth factor I.

The number of growth hormone (GH) receptors in cultured rat epiphyseal chondrocytes are increased with numbers of cell divisions in monolayer. We wanted to study if increased number of cell divisions in monolayer influence GH or insulin-like growth factor I (IGF-I) response in a subsequent suspension culture. Primary isolated chondrocytes from rat tibia growth plates were cultured in monolayer at different seeding densities (4000, 8000 and 24,000 cells/cm2). After a culture period of 7 days, cells were trypsinized, counted and subcultured at 50,000 cells per dish in suspension stabilized with 0.5% agarose. 14 days later the agarose cultures were dried, stained and the number of clones with a diameter exceeding 50 microns was counted. Individual clones were classified as undifferentiated or differentiated according to the following criteria: cell clusters with a diameter of 50 microns and without matrix staining were classified as undifferentiated; cell clusters with a diameter over 50 microns consisting of 4 cells or more and with matrix stained by Alcian Blue were classified as differentiated clones. Human growth hormone (hGH) added to the suspension culture medium increased the number of undifferentiated clones if cells had been precultured at 4000 and 8000 cells/cm2 but hGH had no stimulatory effect on either clone type at 24,000 cells/cm2. IGF-I significantly increased the number of differentiated clones at all seeding densities while no effect was demonstrated on the number of undifferentiated clones. The results from the present study suggest that an increased number of cell divisions during primary monolayer culture increases GH responsiveness in a subsequent suspension culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Cellular aspects on treatment of cartilage injuries.

Cellular aspects on articular cartilage growth and development are discussed. Cells with chondrogenic potential are described and current treatment models for cartilage injuries are considered. A rabbit model for treatment of articular cartilage defects with autologous cultured and transplanted chondrocytes for treatment of knee cartilage defects in humans are discussed.

Animals↗
PubMed1993

Establishment of a growth hormone responsive chondrogenic cell line from fetal rat tibia.

Reproducible effects of growth hormone (GH) on primary isolated cells in monolayer are highly dependent on the culture conditions and/or the fraction of GH responsive cells. To study the effect of GH at the cellular level, a homogenous cell line with both GH responsiveness and chondrogenic properties was established. Primary isolated cells from 18-day-old fetal rat tibia were subcultured using a strict protocol for passages (every third day and a seeding density of 15,000/cm2). Of six established cell lines, one fetal tibia cell line No. 5 (FTC 5) expressed adipogenic and chondrogenic properties at a low frequency. Cells from FTC 5 were subcultured in soft agar suspension with the addition of bovine GH (100 ng/ml). After 14 days in culture eight monoclonal cell lines were established from individual large colonies. Two subclones, FTC 5:3 and FTC 5:6, expressed a chondrogenic phenotype as demonstrated by chondrocyte foci, alcian blue staining and production of type II collagen. Further characterization of FTC 5:3 revealed specific binding of bovine GH with an affinity of 1.7 x 10(9) M-1, and approximately 7300 receptors/cell. Northern blot analysis of FTC 5:3 with a 32P-labeled RNA probe complementary to an extracellular part of the rat GH receptor, revealed two major labeled bands (4.0 and 1.2 kilobases). Both GH and insulin-like growth factor-I (IGF-I) stimulated 3H-thymidine uptake in FTC 5:3 (194 +/- 28% and 405 +/- 127% over control, respectively), while proteoglycan synthesis, as measured by [35S]sulphate uptake, was stimulated by IGF-I only (101 +/- 18% over control).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Embryonic stem cells express growth hormone receptors: regulation by retinoic acid.

Embryonic and fetal growth are generally considered to be independent of pituitary GH. However, it has been demonstrated recently that 18-day-old rat embryos and rat fetuses express GH receptors, suggesting that GH could play a role in early development. The aim of the present investigation was to determine whether preimplantation embryos also express GH receptors. Germ line competent mouse embryonic stem (ES) cells and cultured mouse preimplantation embryos were examined with Northern blot analysis, RNAse-protection solution-hybridization assays, reverse transcription/polymerase chain reaction assays and immunohistochemistry for the detection of GH receptors. Northern blot analysis of ES cells using a probe corresponding to the extracellular domain of the GH receptor demonstrated the presence of two transcripts (1.2 and 4.5 kilobases). The RNAse-protection solution-hybridization assay revealed that ES cells express approximately one sixth of the GH-receptor messenger RNA (mRNA) levels expressed in liver from pregnant mice. Treatment of cultured ES cells with retinoic acid (100 nM) for 6 days increased GH-receptor mRNA levels (P < 0.01). GH-receptor mRNA was further identified in ES cells, preimplantation embryos, muscle, liver, and placenta by a reverse transcription/polymerase chain reaction assay. In humans it has previously been shown that exon 3 of the GH-receptor is deleted in the placenta. However, none of the studied mouse tissues had a deletion of the GH-receptor mRNA corresponding to exon 3 of the human GH receptor. GH-receptor immunoreactivity was identified on the cultured ES-cells by immunohistochemistry. In conclusion, we have in the present study shown that germline competent ES cells and preimplantation mouse embryos express the GH receptor transcript and that this transcription is increased by retinoic acid in ES cells. Furthermore, the presence of GH-receptor immunoreactivity on the ES cells indicates that the GH-receptor transcript is translated.

Animals↗
PubMed1993

A double-staining technique for detection of growth hormone and insulin-like growth factor-I binding to rat tibial epiphyseal chondrocytes.

In the present study a double-staining technique was developed to investigate simultaneous GH and insulin-like growth factor-I (IGF-I) binding to chondrocytes in a monolayer cell culture. Rat tibial epiphyseal chondrocytes were isolated by enzymatic digestion and cultured in monolayer. GH and IGF-I were labelled with biotin. The affinity of the biotin-labelled ligands was compared with unlabelled ligands in a radioreceptor assay. To study the distribution of GH and IGF-I binding in the monolayer, chondrocytes were incubated with biotinylated ligands with or without an excess of unlabelled ligands, followed by incubation with Vectastain ABC complex, which was then reacted with diaminobenzidine (DAB). Double staining was accomplished by carrying out the first reaction with DAB in the presence of nickel ammonium sulphate to give a black precipitate, followed by incubation with the second ligand, then ABC complex and finally DAB in the absence of nickel ammonium sulphate to give a brown stain. The presence of type-II collagen was demonstrated by immunohistochemistry and used as a marker for differentiated chondrocytes. Biotin-labelled GH and biotin-labelled IGF-I exhibited dose-dependent displacements of 125I-labelled GH and 125I-labelled IGF-I respectively from the chondrocytes in a radioreceptor assay. The displacement curves were identical to those of unlabelled ligands indicating that the affinity was unaltered. Binding of biotinylated GH to cells was seen throughout the culture in regions where there was little or no type-II collagen staining. IGF-I binding was predominantly localized to cells at high density; areas which also showed a high degree of staining for type-II collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Growth hormone induces multiplication of the slowly cycling germinal cells of the rat tibial growth plate.

To study the effect of locally infused growth hormone (GH) or insulin-like growth factor I(IGF-I) on slowly cycling cells in the germinal cell layer of the tibial growth plate, osmotic minipumps delivering 14.3 microCi of [3H]thymidine per day were implanted s.c. into hypophysectomized rats, and GH (1 microgram) or IGF-I (10 micrograms) was injected daily through a cannula implanted in the proximal tibia. The opposite leg served as a control. After 12 days of treatment, the osmotic minipumps were removed, and three rats in each group were given GH (20 micrograms/day, s.c.) for an additional 14 days to chase the labeled cells out of the proliferative layers. Labeled cells remained in the germinal layer, in the perichondrial ring, and on the surface of the articular cartilage close to the epiphyseal plate. GH administered together with labeled thymidine significantly increased the number of labeled cells in the germinal cell layer compared to that in the control leg (ratio = 1.95 +/- 0.13), whereas IGF-I showed no stimulatory effect (ratio = 0.96 +/- 0.04). Therefore GH but not IGF-I stimulates the multiplication of the slowly cycling (label-retaining) cells in the germinal layer of the epiphyseal plate. IGF-I acts only on the proliferation of the resulting chondrocytes.

Animals↗
PubMed1992

Effect of growth hormone and insulin-like growth factor-I on DNA synthesis and matrix production in rat epiphyseal chondrocytes in monolayer culture.

The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([3H]thymidine incorporation) and matrix production ([35S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture. Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 micrograms/l (60 +/- 11% stimulation over control) and for IGF-I at 10 micrograms/l (162 +/- 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [35S]sulphate uptake were 135 +/- 25 and 320 +/- 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2.5 days showed that GH but not IGF-I produced a sustained effect on [3H]thymidine uptake. In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5-15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150,000-300,000 cells/cm2. GH had no significant effect at a low (less than 100,000 cells/cm2) or a high (greater than 400,000 cells/cm2) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10,000 and 250,000 cells/cm2, but was reduced at higher cell densities (over 250,000 cells/cm2). Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Effects of tri-iodothyronine and insulin-like growth factor-I (IGF-I) on alkaline phosphatase activity, [3H]thymidine incorporation and IGF-I receptor mRNA in cultured rat epiphyseal chondrocytes.

The effects of tri-iodothyronine (T3) and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation, alkaline phosphatase (ALP) activity and IGF-I receptor mRNA levels were studied in rat epiphyseal chondrocytes cultured in monolayer. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in monolayer culture and precultured for 7-14 days in Ham's F-12 medium supplemented with 10% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture the medium was changed to Ham's F-12 medium containing 1% (v/v) serum from hypophysectomized rats, and the effects of T3 and/or IGF-I on DNA synthesis ([3H]thymidine incorporation), ALP activity (a late marker of differentiated epiphyseal chondrocytes) and IGF-I receptor mRNA levels were studied. ALP activity was increased by T3 in a dose-dependent manner with a maximal response at 10 micrograms T3/l (678 +/- 86% compared with control culture). The increase in ALP activity was accompanied by a concomitant decrease in [3H]thymidine incorporation (52 +/- 14% compared with control culture). Human GH (hGH; 50 micrograms/l) and IGF-I (25 micrograms/l) had no stimulatory effect on ALP activity. However IGF-I (10 micrograms/l) exerted an inhibition on the T3 (10 micrograms/l)-induced increase in ALP activity (64 +/- 9% compared with T3-treated culture). T3 (3 micrograms/l) inhibited the increase in [3H]thymidine incorporation caused by 25 micrograms IGF-I/l (51 +/- 13% compared with IGF-I-treated culture). Furthermore, IGF-I receptor mRNA levels were increased by 10 micrograms T3/l (137 +/- 4.2% compared with control culture) while no effect of hGH (50 micrograms/l) or IGF-I (25 micrograms/l) was demonstrated. Both T3 and IGF-I were shown to interact with epiphyseal chondrocytes and both substances seemed to affect cell proliferation and maturation and therefore longitudinal bone growth. Furthermore, the results indicated that IGF-I is important for proliferation of the cells while T3 initiates the terminal differentiation of epiphyseal chondrocytes.

Alkaline Phosphatase↗
PubMed1991

Regulation of cartilage growth by growth hormone and insulin-like growth factor I.

A number of studies have shown that growth hormone (GH) and insulin-like growth factor-I (IGF-I) have important regulatory roles for skeletal growth. However, it has been a matter of controversy whether GH acts directly on cells in the growth plate or if the growth-promoting effects of GH are mediated by liver-derived (endocrine-acting) IGF-I. With the recognition that GH regulates the production of IGF-I in multiple extra-hepatic tissues, autocrine and paracrine functions of IGF-I have been suggested as important components of GH action. This review focuses on recent developments in our understanding of the cellular mechanisms by which GH promotes longitudinal bone growth and the inter-relationship between GH and IGF-I in the growth plate.

Animals↗
PubMed1991

Expression and physiological significance of growth hormone receptors and growth hormone binding proteins in rat and man.

The molecular structure of the GH receptor has recently been characterized and the receptor identified as a member of a new receptor superfamily that includes the prolactin receptor and several cytokine receptors. No obvious signal transducing domain has been identified on any of these related receptors. One possible signalling mechanism involves receptor interaction with other membrane-associated proteins that function as mediators of signal transduction. Whether such a mechanism is involved in signal transduction of the GH receptor is not known. Another common feature of these receptors is the presence of soluble forms such as the GHBP. The functions of these proteins in the circulation and at the level of the target cell remain to be resolved.

Animals↗