Tibia Test Research Articles

Shin Pain - Running

HISTORY A 51 year old man with a four year history of celiac disease presented with shin pain. After putting on weight with his gluten-free diet, he had commenced running and had recently completed a half marathon. Since the race he had shin pain every time he ran. He had been diagnosed with a stress fracture of the tibia and rested from running for two months. When he resumed running the pain was still present. PHYSICAL EXAMINATION He was tender to palpation over the anterior aspect of the tibia. There were no obvious biomechanical abnormalities. DIFFERENTIAL DIAGNOSIS Stress fracture tibia Stress fracture anterior cortex tibia TEST AND RESULTS X Ray lower leg cortical thickening of the antero-lateral aspect of the lower tibial shaft with a faint transverse linear lucency through the thickened cortex DEXA bone density scan Lumbar spine 1.052 gm/sq cm (T score: −1.6), femoral neck 0.835 gm/sq cm (T score: −1.8). Vit D level - lower end of normal range PTH - high Serum calcium - normal FINAL WORKING DIAGNOSIS Non-union of stress fracture of the anterior cortex of the tibia secondary to osteopenia due to the Vitamin D malabsorption associated with celiac disease TREATMENT AND OUTCOMES Vitamin D 1000 units/day Surgery to scallop out the area of non-union of the stress fracture of the tibia

Shin Pain - Running

HISTORY A 51 year old man with a four year history of celiac disease presented with shin pain. After putting on weight with his gluten-free diet, he had commenced running and had recently completed a half marathon. Since the race he had shin pain every time he ran. He had been diagnosed with a stress fracture of the tibia and rested from running for two months. When he resumed running the pain was still present. PHYSICAL EXAMINATION He was tender to palpation over the anterior aspect of the tibia. There were no obvious biomechanical abnormalities. DIFFERENTIAL DIAGNOSIS Stress fracture tibia Stress fracture anterior cortex tibia TEST AND RESULTS X Ray lower leg cortical thickening of the antero-lateral aspect of the lower tibial shaft with a faint transverse linear lucency through the thickened cortex DEXA bone density scan Lumbar spine 1.052 gm/sq cm (T score: −1.6), femoral neck 0.835 gm/sq cm (T score: −1.8). Vit D level - lower end of normal range PTH - high Serum calcium - normal FINAL WORKING DIAGNOSIS Non-union of stress fracture of the anterior cortex of the tibia secondary to osteopenia due to the Vitamin D malabsorption associated with celiac disease TREATMENT AND OUTCOMES Vitamin D 1000 units/day Surgery to scallop out the area of non-union of the stress fracture of the tibia

Fracturas de implantes de tibia. Consecuencias para los ensayos de los implantes

El objetivo de este trabajo es estudiar los modelos de fracturas de prótesis de tibia descritos en la literatura
 y, sobre la base de dichos conocimientos, desarrollar un ensayo para implantes en la cual se logren simular
 condiciones fisiológicas. El caso descrito con mayor frecuencia en la literatura es la migración de la parte
 medial de la prótesis, fenómeno que, a menudo, antecede a la fractura de la prótesis. La posición defectuosa
 resultante de dicha migración modifica el eje mecánico y provoca una sobrecarga en la parte medial de la
 prótesis. Se ha desarrollado un ensayo de implantes que simula el modelo de fallo in-vivo. Dos bases de
 material sintético de diferente dureza simulan la diferente densidad ósea medial y lateral, para permitir una
 migración del lado medial del platillo. En el ensayo dinámico el platillo tibial es sometido a una carga de un
 solo lado y el vástago de la prótesis se apuntala con un contrasoporte. Para simular el entorno fisiológico, todo
 el dispositivo se encuentra sumergido en una solución de Ringer a 37°. El implante de tibia es expuesto durante
 10 millones de ciclos a una carga que corresponde al quíntuple del peso del cuerpo (4.000 N). El punto de
 aplicación de la fuerza fue tomado de datos de análisis del caminar (marcha en superficie plana, ascendente
 y descendente, subir escaleras). Con esta carga fisiológica se efectuaron los primeros ensayos. En éstas quedó
 demostrado que productos clínicamente exitosos podían superar el ensayo. Por el contrario, los productos con
 modelos de fracturas conocidos no la superaron. Los primeros resultados con estos productos demostraron
 también que el ensayo generaba muestras de fractura semejantes a las que se producen in vivo. Debido a la
 configuración fisiológica del dispositivo se somete el sistema completo es decir el platillo, el vástago de la
 prótesis y la unión - generalmente modular - entre ambos a ensayo. El "ensayo fisiológico de tibia " descrito
 ayuda a evaluar los diseños de tibia antes de que se autorice su venta. Nuevos implantes y modificaciones de
 diseño en implantes ya existentes son puestos a la venta después de cumplir con las exigencias del ensayo.

Ultrasensitive in vivo bioassay detects bioactive human growth hormone in transduced primary human keratinocytes.

An improved in vivo body weight gain bioassay for the potency determination of human growth hormone (hGH) has been set up in "little" mice (lit/lit), a mutant derived from the C57BL/6J strain. This improved assay now has a detection limit of the order of 0.05 micrograms/mouse/day, which corresponds to a sensitivity about 20-fold higher than that of the most sensitive in vivo assay reported up to now: the tibia test in hypophysectomized rats or mice. This sensitivity was achieved mainly by introduction of a careful pre-assay selection and of a three injections per day schedule. The utilization of these conditions in a 2x2 factorial assay design allowed the potency determination of recombinant DNA-derived hGH (rec-hGH) in bacterial extracts with acceptable accuracy and precision, together with the greatest economy of material, only 0.24 mg of unknown and standard hormone preparation being sufficient for an entire 10-animal assay. This contrasts to a minimum of 2.7 mg that are necessary for an economical assay in hypophysectomized rats. The same assay procedure was also used to demonstrate the in vivo bioactivity of hGH secreted into a culture medium from transduced human primary keratinocytes. The growth curve constructed with n = 8 little mice presented a highly significant correlation (r = 0.939, p < 0.001) and a slope = 0.016 g/mouse/day. It was thus possible to prove, for the first time, the in vivo bioactivity of rec-hGH secreted by transplantable human epidermal cells, utilized as an experimental model for somatic gene therapy.

The effect of phage T7 lysozyme on the production of biologically active porcine somatotropin in Escherichia coli from a gene transcribed by T7 RNA polymerase

We have analyzed the inducible synthesis of recombinant porcine somatotropin (rPST) from the phage T7 gene 10 promoter on the vector pET3a [Rosenberg et al., Gene 56 (1987) 125–135]. Low-level synthesis of phage T7 lysozyme is crucial for high-level synthesis (40%) of rPST, which is greatly reduced if T7 lysozyme synthesis is absent or too high. The synthesis of rPST mRNA is optimized in those constructs coding for low levels of T7 lysozyme, with a reduction in mRNA levels in constructs coding for higher levels of T7 lysozyme or no lysozyme. The rPST can be readily purified following a single chromatographic step and is biologically active as determined by the tibia test following administration to hypophysectomized rats.

Comparison of the pharmacological properties of pituitary and biosynthetic human growth hormone

Biosynthetic human growth hormone was compared with pituitary human growth hormone and pituitary 22 K in the weight gain and the tibia test. The three preparations were found to be equipotent. Furthermore, the growth hormones were compared in various pharmacological test systems. All three preparations were found to have a marked antidiuretic and antinatriuretic effect in the rat and to cause a significant shortening of the hexobarbital sleeping time in mice. Biosynthetic and pituitary preparations had the same diabetogenic activity in obese mice, and the growth hormones did not differ with respect to pharmacological profiles in the test systems applied.

Structure‐function studies on human growth hormone

The relationship between the conformation of human pituitary growth hormone (hGH), biological activity, and ligand binding activity was studied by comparing conformational details previously published on in vivo and in vitro studies of identical samples of hGH and its known derivatives. In vivo assays included the rat tibia test for somatotropic activity and the pigeon crop-sac assay for lactogenic hormone activity. Relative binding affinities were compared in radioimmunoassays using 125I-hGH as tracer with 1) anti-human chorionic somatomammotropin (hCS) serum (low discriminatory hybrid assay), 2) anti-hGH sera (in conventional assays), 3) monospecific anti-hGH serum (absence of cross-reaction with hCS) and 4) human anti-hGH sera obtained from GH-deficient patients on replacement therapy. In addition, binding affinities were examined in two receptor-binding assays, one specific for somatotropic activity (rabbit liver membranes, 125I-hGH), and the other, for lactogenic hormones (rabbit mammary membranes, 125I-oPRL). The conformational properties of native hGH and various chemically and enzymatically modified derivatives of the hormone were evaluated primarily from circular dichroism spectra, while conformational stabilities were estimated from the relative rates of tryptic digestion. Unfragmented, but chemically modified derivatives, exhibited good parallelism between retention or loss of native conformation and the in vivo potencies and in vitro binding affinities. None of the fragments of hGH showed activity in any of the radioreceptor assays or radioimmunoassays. Two derivatives of hGH, which contain gaps of 6 or 12 residues in the polypeptide backbone produced by partial enzymatic digestion, had full or increased in vivo potencies, full activities in the radioimmunoassays, and were the most active derivatives in both radioreceptor assays. One of these, missing the hexapeptide corresponding to residues 135-140, was also found to retain nearly all the conformational properties of native hGH. These studies proved further evidence that 1) retention by modified forms of hGH of a high degree of in vivo biological potency or in vitro binding affinity is causally related to the retention of most of the conformation and conformational stability of the molecule, and 2) the biologically active, receptor-binding and immunoreactive sites on the hGH molecule are 3-dimensional in nature.

Further characterization of growth hormone from the chum salmon ( Oncorhynchus keta)

This report describes the isolation of growth hormone (GH) from the chum salmon ( Oncorhynchus keta) pituitary using gel, affinity, and ion exchange chromatography. Chum GH has an estimated molecular weight of 23,500 and an amino acid composition that is consistent with a vertebrate GH. The differentially charged forms of chum GH which are only apparent under alkaline conditions were separated by ion exchange and compared immunologically and biologically; Peak I, which consists of a single band ( R f = 0.35) under alkaline electrophoresis and Peak II which consists of two bands with R f's of 0.41 and 0.45. Both forms were found to be immunologically identical by immunodiffusion and to have similar growth promoting properties in intact rainbow trout ( Salmo gairdneri). Chum GH was also active in the rat tibia test at a daily dosage of 70 μg/animal. The results are discussed in relation to previous studies with chum GH and other fish GHs.

The heterogeneity of bovine growth hormone. Extraction from the pituitary of components with different biological and immunological properties

Bovine growth hormone (somatotropin) was extracted from anterior pituitaries and fractionated into four protein peaks (A-D) by chromatography on DEAE-Sephacel. Analysis by high-pressure liquid chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the homogeneity of the material increased from fraction A through to D. The properties of the fractions were examined in the following manner: immunological activity (radioimmunoassays for ruminant growth hormone and prolactin); growth-promoting activity (rat tibia test); lipolytic activity (release of glycerol from rat epididymal fat in the presence of dexamethasone); diabetogenic activity (rate of glucose transport in epididymal fat of hypophysectomized rats and intravenous insulin-tolerance tests in goats). None of the fractions contained immunoreactive prolactin and all were equally lipolytic. Although fraction A contained a small quantity of immunoreactive growth hormone it had no growth-promoting or diabetogenic activities. Both fractions B and C were diabetogenic and contained high concentrations of immunoreactive growth hormone, consistent with their growth-promoting activity. Although the growth-promoting activity of fraction D was higher than that of the other three fractions, it was not diabetogenic and was only weakly immunoreactive. These results for bovine growth hormone support the contention that growth hormone, as commonly extracted, is a mixture of different molecular forms and that these different metabolic properties of the hormone might be explained in terms of this heterogeneity.

The effect of glibenclamide on plasma insulin, plasma somatomedin bioactivity and skeletal growth in hypophysectomized rats.

Abstract. Male rats, body weight 60–75 g, were hypophysectomized. Three days after operation the animals were divided into two groups. Group B received solvent solution and group C 1 mg/kg body weight per day of glibenclamide ip for the following 9 days. Group A consisted on non-operated normal rats. Twenty-four hours after the last injections and after a 12 h overnight fast the body weights of groups B and C were not different, the increase during the 10 days being 10% in both groups. Serum insulin (IRI) was significantly higher in group C than in group B (C: 8.0 ± 0.3 μU/ml, n = 14 vs B: 4.9 ± 1.0 μU/ml, n = 14; P &lt; 0.01, mean ± sem) as was serum somatomedin bioactivity (SM)-porcine cartilage assay — (C: 1.06 ± 0.1 U/ml, n = 14 vs B:0.41 ± 0.01 U/ml, n = 14; P &lt; 0.001). Skeletal growth was determined with the tibia test and by a radiograph of each rat. The width of the proximal epiphyseal growth plate of the tibia was significantly increased in group C compared to group B (C: 204 ± 4.8 μm, n = 12 vs 181 ± 6.5 μm, n = 13; P &lt; 0.005). On the radiograph the area of the right femur was not different between the two groups of animals, while the height and the area of the first lumbar spine were significantly augmented in group C. The results show that glibenclamide stimulates IRI, SM and skeletal growth in hypophysectomized rats. Compared to the glibenclamide treated hypophysectomized animals the normal rats of group A had doubled their body weights. IRI (59 ± 5 μU/ml, n = 4) and skeletal growth (tibia test: 454 ± 5.8 μm) were greatly increased. SM did not differ between group A (1.21 ± 0.35 U/ml and group C. T4 was much lower in group B (0.64 ± 0.09 μg/100 ml, n = 5) than in group A (4.1 ± 0.3 μg/100 ml, n = 6; P &lt; 0.001). It is concluded that a normal SM concentration is not necessarily associated with appropriate growth.

Human somatotropin. Selective removal with trypsin of residues 135-145 from the hormone molecule with no loss of biological activities.

A 11-residue segment, sequence positions 135-145, has been cleaved selectively from the human somatotropin molecule by limited digestion of the hormone with trypsin at pH 6.0 in 90% (v/v) glycerol. Differences between the tertiary structures of the native and trypsin-modified hormones have been demonstrated by circular dichroism spectroscopy. Despite these conformational differences, the biological properties of intact hormone and its modified form appeared to be very similar, as tested by two receptor-binding assays, radioimmunoassay, and the rat tibia test.

Human somatotropin: covalent reconstitution of two polypeptide contiguous fragments with thrombin.

Thrombin has been shown to resynthesize the Arg134-Thr135 peptide bond between the 134- and 57-residue thrombin fragments of reduced and carbamidomethylated human somatotropin at pH 6.0 in 90% (vol/vol) glycerol. The maximal amount of synthesis was about 20% as estimated by sodium dodecyl sulfate gel electrophoresis and radioreceptor assay. The resynthesized polypeptide was isolated and shown to be indistinguishable from the reduced and carbamidomethylated hormone when tested by two receptor-binding assays, radioimmunoassay, and rat tibia test.

Isolation and partial characterization of growth hormone from fin whale pituitary glands.

A highly purified somatotropin (growth hormone) has been isolated from fin whale (Balaenoptera physalus) pituitary glands in a yield of 1,014 mg/kg of wet weight tissue. The preparation was shown to be homogenous by isoelectric focusing on polyacrylamide with growth-promoting potency comparable to that of ovine growth hormone by the rat tibia test. The molecular weight was found to be 20,000 daltons by SDS gel electrophoresis and 40,000 daltons by exclusion chromatography on Sephadex G-100 at pH 8.2. The amino acid composition and alpha-helix content of fin whale somatotropin were similar to those of human, bovine, and ovine hormones. The circular dichroism in the side chain region, however, differed from those of the mammalian hormones. The NH2-terminal amino acid sequence was nearly identical with those of the bovine and ovine hormones as determined by the fluorescein isothiocyanate method.

EVIDENCE TO SUGGEST THAT RAT GROWTH HORMONE IS MODIFIED WHEN SECRETED BY THE PITUITARY GLAND

The relationships were examined between the concentrations of GH determined by bioassay (rat tibia test) and radioimmunoassay in homogenates of rat adenohypophysial tissue and in the incubation medium in which the tissue had been maintained. Adenohypophyses from young male rats were incubated in Medium 199 with or without a putative synthetic GH-releasing factor (GRF) to achieve a dynamic state of GH secretion, but the GRF did not consistently stimulate the release of GH as measured by either bioassay or radioimmunoassay. Both assays gave closely similar values for the concentration of GH in homogenates of the adenohypophyses of undisturbed rats. When the two assays were used to measure the concentrations of GH in incubated pituitary tissue, the values showed no correlation, although the concentrations of GH in the incubation medium were again highly correlated. However, the bioassay : radioimmunoassay ratio for the secreted hormone [2.14 +/- 0.09 (S.E.M.)] was significantly higher than that for the intrapituitary form from the unincubated gland (0.93 +/- 0.06). By radioimmunoassay, secreted rat GH was found to be less stable than purified rat GH when the two forms were incubated in vitro with slices of various rat tissues. Similarly, the rate of clearance of secreted rat GH from the plasma of hypophysectomized rats, as determined by radioimmunoassay, was much faster than that of the purified GH. The biological activity of purified bovine GH was blocked by aprotinin, an inhibitor of proteolysis, but the drug had no inhibitory effect on the bioactivity of rat GH secreted into the incubation medium. Overall, these results indicate that secreted rat GH has properties intermediate between those of the intrapituitary form and the form found in the circulation.

Electrophoretic separation of forms of rat growth hormone with different bioassay and radioimmunoassay activities: comparison of intraglandular and secreted forms

Rat adenohypophysial tissue and medium in which pituitary explants had been incubated were processed by polyacrylamide disc gel electrophoresis (pH 9.5). Eluates of 6 regions of the gel columns were tested for growth hormone (GH) by a bioassay (BA; rat tibia test) and a radioimmunoassay (RIA). In the tissue, both assays detected most of the GH (80–99%) in segments with an R f range of 0.30–0.56. In the medium, both assays recorded a more cathodal distribution of most of the hormone (85–96%) in the R f region 0.00–0.42. The BA/RIA ratios of the segment containing the prominent stainable GH band (R f range 0.30–0.42) varied from 0.6–1.8 in tissue and medium. The tissue contained slow migrating (R f range 0–0.29) GH that had a high BA/RIA ratio (11.1–25.7). The medium contained high ratio GH (32.7–33.8) in a more anodal location (R f 0.57–0.68). Purified rat prolactin (RP-I) was not active in either the BA or the RIA. The results indicate that rat GH exists in electrophoretically heterogeneous forms which differ in their BA/RIA activity ratios. The proportion of these forms stored in adenohypophysial tissue and secreted by the gland may differ.

Human somatotropin: Semisynthesis of the hormone by noncovalent interaction of the NH 2-terminal fragment with synthetic analogs of the COOH-terminal fragment

Complementation of the natural NH 2-terminal fragment (consisting of 134 amino acid residues) with synthetic analogs of COOH-terminal fragments of 52 or 47 amino acids of reduced-carbamidomethylated human somatotropin gave recombinants with full growth-promoting activity as evidenced by the tibia test. Radioimmunoassay data show that the semisynthetic recombinant hormones possess nearly full immunoreactivity as compared to the native molecule.

Differential effects of elevated potassium ion concentration and of theophylline on growth hormone release by rat adenohypophyses in vitro.

The effects of 27 mM K+ and of 6.7 mM theophylline on the release of growth hormone (GH) by rat hemipituitaries in vitro were investigated using bioassay (rat tibia test) and radioimmunoassay (RIA). Both agents markedly increased the release of immunoreactive GH. High K+ also promoted the release of bioactive GH but to a much lesser degree than RIA-GH. Theophylline did not consistently affect the release of bioassay-detectable GH. The results suggest that these agents promote massive release of a form of immunoreactive GH (possibly "immature") that has little or no activity in the bioassay. Theophylline is relatively more effective in this regard than is elevated K+.

GROWTH HORMONE RELEASING FACTOR AND THE BIOASSAY-RADIOIMMUNOASSAY PARADOX REVISITED

ABSTRACT Relationships between depletion of growth hormone (GH) from the rat adenohypophysis and its release into the circulation were re-evaluated using bioassay (BA: rat tibia test) and radioimmunoassay (RIA). Anaesthetized male rats were injected with a putative growth hormone releasing factor ("GRF") and killed 15 and 30 min later. For the assays, the plasma and adenohypophyses were pooled; the latter were separated into granule and cytosol fractions prior to assay. Fifteen minutes after "GRF" injection the BA recorded a 75 % depletion of GH from the granular fraction but the RIA measured a loss of only 13 %. By 30 min both assays recorded restoration of pituitary GH. Only slight changes occurred in the levels of BA- or RIA-detectable GH in the cytosol fractions. The massive depletion of bioassayable GH from the granule fraction was accompanied by a large increase (3.2 mU/ml) in the plasma concentration of BA-detectable hormone by 15 min. The slight depletion of RIA-detectable GH that occurred during this time was associated with a meagre increase (0.05 mU/ml) in the plasma level of immunoreactive GH. The plasma concentration of BA- and RIA-detectable GH did not change significantly between 15 and 30 min. Analysis of the relationship between depletion of GH from the pituitary and its release into the extrapituitary compartment disclosed that the BA recordings of these events were in excellent quantitative agreement but RIA measurements were not. The results confirm that biologically active GH can be depleted from and released by the adenohypophysis in vivo independently of RIA-detectable GH.

A re-assessment of the effect of thyrotrophin (TSH) on the tibial plate bioassay for growth hormone (GH).

The claim has been made that thyrotrophin (TSH) can augment the action of growth hormone (GH) to stimulate growth of the epiphysial cartilage plate of the hypophysectomized rat's tibia. The TSH induces its effect via secretion of thyroid hormones which in turn enhance the stimulatory action of GH. If this is true then the employment of the tibia test, whose endpoint is the increase in thickness of the epiphysial cartilage plate in response to GH present either in crude pituitary extracts or relatively purified preparations, which also are likely to contain modest or appreciable quantities of TSH, requires further examination. The present study utilized various fractions of crude pituitary extracts from intact and thyroidectomized rats that respectively contained appreciable quantities of GH or essentially no GH. Fractional aliquots of pituitary extracts from thyroidectomized rats were administered concomitantly with graded doses of exogenous GH to hypophysectomized rats to determine the point at which TSH in the extracts was sufficiently able to stimulate significant tibial plate growth when compared to recipients given GH alone. Purified GH and TSH were also administered in various doses to hypophysectomized recipients in a further attempt to delineate the dose range at which TSH augments the action of GH to promote significant chondrogenesis of the epiphysial plate. The results indicate that the enhancement of the GH effect on the cartilage plate by TSH was evident only when quantities above 100 microng bovine GH were co-administered with 100 mU bovine TSH. As little as 40 mU TSH augmented the growth effect of 400 microng GH on the cartilage plate, demonstrating that smaller quantities of TSH could potentiate larger quantities of GH. These data, therefore, suggest that extracts equivalent to not more than one-half of a normal adult rat's anterior pituitary gland should be administered to hypophysectomized rats for bioassay of GH. Fractions of glands greater than this may contain sufficient amounts of TSH to augment the appreciable quantities of GH already present.

Human somatotropin: restoration of full biological activity by noncovalent interaction of a natural and a synthetic fragment of the hormone.

The recombinant hormone obtained by noncovalent interaction of the natural NH2-terminal fragment (consisting of 134 amino acid residues) with a synthetic COOH-terminal fragment of 52 amino acids of the reduced-carbamidomethylated human somatotropin molecule is found to exhibit full biological activity of the native hormone as evidenced by the tibia test. Radioimmunoassay data indicate that the semisynthetic recombinant hormone possesses essentially full immunoreactivity as compared to the native one. In addition, circular dichroism and fluorescence emission spectra of the semisynthetic recombinant are identical to that of the undisociated, plasmin modified, reduced-carbamidomethylated hormone.