In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.

Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro

Animals have evolved limb proportions adapted to different environments, but it is not yet clear to what extent these proportions are directly influenced by the environment during prenatal development. The developing skeleton experiences mechanical loading resulting from embryo movement. We tested the hypothesis that environmentally-induced changes in prenatal movement influence embryonic limb growth to alter proportions. We show that incubation temperature influences motility and limb bone growth in West African Dwarf crocodiles, producing altered limb proportions which may, influence post-hatching performance. Pharmacological immobilisation of embryonic chickens revealed that altered motility, independent of temperature, may underpin this growth regulation. Use of the chick also allowed us to merge histological, immunochemical and cell proliferation labelling studies to evaluate changes in growth plate organisation, and unbiased array profiling to identify specific cellular and transcriptional targets of embryo movement. This disclosed that movement alters limb proportions and regulates chondrocyte proliferation in only specific growth plates. This selective targeting is related to intrinsic mTOR (mechanistic target of rapamycin) pathway activity in individual growth plates. Our findings provide new insights into how environmental factors can be integrated to influence cellular activity in growing bones and ultimately gross limb morphology, to generate phenotypic variation during prenatal development.

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Limb proportions show developmental plasticity in response to embryo movement.

Subclinical thyroid disease (SCTD) is defined as serum free T4 and free T3 levels within their respective reference ranges in the presence of abnormal serum TSH levels. SCTD is being diagnosed more frequently in clinical practice in young and middle-aged people as well as in the elderly. However, the clinical significance of subclinical thyroid dysfunction is much debated. Subclinical hyper- and hypothyroidism can have repercussions on the cardiovascular system and bone, as well as on other organs and systems. However, the treatment and management of SCTD and population screening are controversial despite the potential risk of progression to overt disease, and there is no consensus on the thyroid hormone and thyrotropin cutoff values at which treatment should be contemplated. Opinions differ regarding tissue effects, symptoms, signs, and cardiovascular risk. Here, we critically review the data on the prevalence and progression of SCTD, its tissue effects, and its prognostic implications. We also examine t...

The clinical significance of subclinical thyroid dysfunction.

Statistics--with confidence?

SUMMARY
A technique for the bioassay of adrenocorticotrophic hormone (ACTH) in human plasma is described, and its experimental validation discussed.

Segments of guinea-pig adrenal are maintained individually for 5 hr in nonproliferative organ culture in a medium reinforced with 10-3m sodium ascorbate. At the end of this time each segment is exposed to either standard or unknown ACTH for 3 min. The segments are then rapidly chilled to - 70oC and sections cut in a cryostat. The sections are stained by the ferric ferrocyanide technique for reducing groups, and the degree of staining in the zona reticularis measured by scanning and integrating micro-densitometry. There is an inverse linear correlation between the intensity of the stain and the logarithmic concentration of ACTH over the concentration 0.0025-2.5 pg/ml of culture medium.

The assay is precise (λ= 0.09) and extremely sensitive. Concentrations of ACTH down to 1 pg/ml may be determined on as little as 0.1 ml of plasma without extraction. Results obtained are in a range similar to that given by the Lipscomb-Nelson bioassay and by radioimmunoassay.

A sensitive bioassay for adrenocorticotrophic hormone in human plasma.

Cell proliferation and enzyme activities associated with the development of avian tibial dyschondroplasia: an in situ biochemical study.

The expression of transforming growth factor-beta and the c-myc proto-oncogene was studied in situ in the chondrocytes of the tibial growth plate of normal chicks and those with avian tibial dyschondroplasia in which the chondrocytes are developmentally arrested in the transitional phase between proliferation and differentiation This results in an accumulation of unmineralised and avascular cartilage Dyschondroplastic chicks showed reduced c-myc expression in the transitional chondrocytes but unaltered levels in the proliferating chondrocytes Transforming growth factor-beta expression was reduced in the transitional chondrocytes of dyschondroplastic chicks In areas where the lesion was being repaired there was evidence of increased expression of both c-myc protein and transforming growth factor-beta Addition of 1,25-dihydroxyvitamin D to the diet, which is known to reduce the incidence of dyschondroplasia, resulted in an increase in c-myc production These results suggest that both transforming growth factor-beta and the proto-oncogene c-myc may be important elements of the cascade of events that lead to chondrocyte differentiation, hypertrophy and mineralisation

The control of chondrocyte differentiation during endochondral bone growth in vivo: changes in TGF-beta and the proto-oncogene c-myc.

Neonatal pig bone marrow stromal cells (PBMSC) were tested in vivo and in vitro to establish their use as a large-animal model for the study of skeletogenesis. When implanted in diffusion chambers in athymic mice for 6-8 weeks, both freshly isolated pig bone marrow and passage 2 PBMSC formed partially mineralized cartilage, bone-like material, and fibrous tissue. The cartilage showed metachromatic, perilacunar staining with toluidine blue and safronin O, alcian blue staining for chondroitin and keratan sulfate, and intense immunostaining for type II collagen. Osteocalcin was immunolocalized to the mineralized regions, consistent with the formation of bone. Alkaline phosphatase was primarily observed in cell layers at boundaries between tissue types. Unstimulated monolayer cultures of PBMSC produced type I but not type II collagen, responded to dexamethasone (10(-8) M) with a 1.7-fold increase in alkaline phosphatase activity, and were stimulated to divide by basic fibroblast growth factor (1.5-fold; EC50 1 ng/ml). Transforming growth factor beta (TGF-beta) blocked both dexamethasone-induced alkaline phosphatase expression (EC50, 1 ng/ml of TGF-beta) and the mitogenic effects of bFGF (EC50 0.06 ng/ml of TGF-beta). When incubated for 10-14 days in medium containing dexamethasone, beta-glycerophosphate and ascorbate PBMSC formed mineralized nodules. Calcification occurred in the middle of the aggregates and was associated with intensely alkaline phosphatase positive cells and a dense type I collagen-rich matrix. PBMSC also displayed colony-forming unit-fibroblastic activity, with approximately 1 in 80 of the plated cells formed colonies > 128 cells over 14-21 days. PBMSC therefore mimic the known activities of stromal cells from other species, including the human, suggesting that they are a valid model for skeletal research.

Preliminary characterization of porcine bone marrow stromal cells: Skeletogenic potential, colony‐forming activity, and response to dexamethasone, transforming growth factor β, and basic fibroblast growth factor

Parietal cell antibodies (PCA) are found in up to 90% of sera from pernicious anaemia patients but it is often stated that they could represent an epiphenomenon without being directly responsible for the achlorhydria. In the present studies a direct effect of these antibodies on the secretory function of gastric acid-secreting cells has been demonstrated in two different experimental systems. In one set of experiments IgGs containing PCA activity were shown to inhibit acid secretion specifically in the living gastric mucosa of the bull frog suspended as a diaphragm between two chambers. The other system demonstrated their inhibition of carbonic anhydrase activity in a cytochemical bioassay for human G17-gastrin, suggesting a blocking effect on the gastrin receptors in the canalicular microvilli or the cell membrane. These experiments suggest a direct pathogenic role for PCA in autoimmune fundal gastritis and in pernicious anaemia.

Nigel Loveridge

Papers

A sensitive bioassay for adrenocorticotrophic hormone in human plasma.

Cell proliferation and enzyme activities associated with the development of avian tibial dyschondroplasia: an in situ biochemical study.

The control of chondrocyte differentiation during endochondral bone growth in vivo: changes in TGF-beta and the proto-oncogene c-myc.

Preliminary characterization of porcine bone marrow stromal cells: Skeletogenic potential, colony‐forming activity, and response to dexamethasone, transforming growth factor β, and basic fibroblast growth factor

Inhibition of parietal cell function by human gammaglobulin containing gastric parietal cell antibodies.