RESUMO
Small intestinal starch digestion (SISD) in cattle is often limited; however, greater postruminal flow of high-quality protein (e.g., casein) can increase SISD, and Glu can mimic responses to casein for SISD. We evaluated effects of increasing Glu flows to the duodenum on SISD and N retention in cattle. Cattle received (DM basis) continuous duodenal infusion of raw cornstarch (1.5 ± 0.08 kg/d) and 0, 30.9 ± 0.6, 62.4 ± 1.2, or 120.4 ± 3.4 g/d Glu or 387.9 ± 17.5 g/d casein. As expected, the positive control (i.e., casein) increased ( = 0.05) SISD. Interestingly, SISD linearly increased ( = 0.02) with increasing amounts of Glu. Starch flow to the ileum linearly decreased ( = 0.04) in response to greater postruminal Glu and tended to decrease ( = 0.07) with duodenal casein infusion. Ileal flow of ethanol-soluble starch was not affected by duodenal Glu ( = 0.16) or casein ( = 0.42). There was a tendency ( = 0.08) for a quadratic response to Glu for ileal glucose flow with greater flows for intermediate levels of Glu, but casein had no effect ( = 0.81) on glucose flows to the ileum. Greater postruminal supplies of Glu (linear, = 0.05) and casein ( = 0.02) decreased fecal starch flow. Postruminal starch digestion was increased by both casein ( = 0.03) and Glu (linear, = 0.05). Nitrogen intake from feed was not different among treatments ( ≥ 0.23). By design, infusate N increased from 0 to 13 ± 1.5 g/d with greater amounts of Glu, and casein provided 61 ± 1.3 g N/d. Urinary N excretion was not affected ( ≥ 0.30) by postruminal Glu flow, but urine N was increased by casein ( < 0.01). Glutamic acid did not affect N retention ( ≥ 0.34), but casein increased N retention ( < 0.01). However, N retained as a percent of N intake (26.7 ± 1.7%) was not different when cattle were provided Glu ( ≥ 0.16) or casein ( = 0.38).
Assuntos
Bovinos/fisiologia , Digestão/fisiologia , Ácido Glutâmico/farmacologia , Intestino Delgado/fisiologia , Nitrogênio/metabolismo , Animais , Caseínas/metabolismo , Glucose/metabolismo , Rúmen/metabolismo , Amido/metabolismoRESUMO
The osteoconductive properties of calcium phosphate cements (CPCs) may be improved by the addition of growth factors, such as recombinant human transforming growth factor-beta1 (rhTGF-beta1). Previously we have shown that rhTGF-beta1 was released from cement enriched with rhTGF-beta1 and subsequently stimulated the differentiation of pre-osteoblastic cells from adult rat long bones. It is unknown whether the addition of rhTGF-beta1 changes the material properties of this alpha-tricalcium-phosphate (alpha-TCP)/tetracalcium-phosphate-monoxide (TeCP)/dicalcium-phosphate-dihydrate (DCPD) cement, and what the characteristics of the release of rhTGF-beta1 from this CPC are. Therefore, in the present study we determined the release of rhTGF-beta1 from cement pellets in vitro. The possible intervening effects of the CPC modification for intermixing rhTGF-beta1 on physicochemical properties were studied by assessing the compressive strength and setting time, as well as crystallinity, calcium to phosphorus ratio, porosity and microscopic structure. Most of the previously incorporated rhTGF-beta1 in the cement pellets was released within the first 48 h. For all concentrations of rhTGF-beta1 intermixed (100 ng-2.5 mg/g CPC), approximately 0.5% of the amount of rhTGF-beta1 incorporated initially was released in the first 2 h, increasing to 1.0% after 48 h. The release of rhTGF-beta1 continued hereafter at a rate of about 0.1% up to 1 week, after which no additional release was found. The initial setting time, nor the final setting time was changed in control cement without rhTGF-beta1 (standard CPC) or in cement modified for rhTGF-beta1 (modified CPC) at 20 degrees C and 37 degrees C. Setting times were more than six times decreased at 37 degrees C compared to 20 degrees C. The compressive strength was initially low for both standard CPC and modified CPC, after which it increased between 24 h and 8 weeks. The compressive strength for the modified CPC was significantly higher compared with standard at 1, 2, and 8 weeks after mixing. X-ray diffraction revealed that both standard and modified CPC changed similarly from the original components into crystalline apatite. The calcium to phosphorus ratio as determined by an electron microprobe did not differ at all time points measured for standard CPC and modified CPC. In both standard CPC and modified CPC the separated particles became connected by crystals, forming a structure in which the particles could hardly be recognised in a densifying matrix with some small pores. The present study shows that the calcium phosphate cement is not severely changed by modification for the addition of rhTGF-beta1. The addition of rhTGF-beta1 in CPC enhances the biologic response as shown in our previous study and did not interfere with the aimed physical and chemical properties as shown in this study. We conclude that the addition of rhTGF-beta1 enlarges the potential of the CPC in bone replacement therapy.
Assuntos
Cimentos Ósseos , Fosfatos de Cálcio , Fator de Crescimento Transformador beta/administração & dosagem , Animais , Fenômenos Químicos , Físico-Química , Força Compressiva , Preparações de Ação Retardada , Humanos , Técnicas In Vitro , Teste de Materiais , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Ratos , Proteínas Recombinantes/administração & dosagem , Fator de Crescimento Transformador beta1 , Difração de Raios XRESUMO
The bone regenerative properties of calcium phosphate cements (CPCs) may be improved by the addition of growth factors, such as recombinant human transforming growth factor-beta1 (rhTGF-beta1). Previously, we showed that rhTGF-beta1 in CPC stimulated the differentiation of preosteoblastic cells from adult rat long bones. The intermixing of rhTGF-beta1 in CPC, which was subsequently applied to rat calvarial defects, enhanced bone growth around the cement and increased the degradation of the cement. However, it is unknown whether the addition of rhTGF-beta1 changes the material properties of CPC and what the characteristics of the release of rhTGF-beta1 from CPC are. Therefore, we determined in this study the release of rhTGF-beta1, in vitro, from the cement pellets as implanted in the rat calvariae. The possible intervening effects of rhTGF-beta1 intermixing on the clinical compliance of CPC were studied through an assessment of its compressive strength and setting time, as well as its crystallinity, calcium-to-phosphorus ratio, porosity, and microscopic structure. We prepared CPC by mixing calcium phosphate powder (58% alpha-tricalcium phosphate, 25% anhydrous dicalcium phosphate, 8.5% calcium carbonate, and 8.5% hydroxyapatite) with a liquid (3 g/mL). The liquid for standard CPC consisted of water with 4% disodium hydrogen phosphate, whereas the liquid for modified CPC was mixed with an equal amount of 4 mM hydrochloride with 0.2% bovine serum albumin. The hydrochloride liquid contained rhTGF-beta1 in different concentrations for the release experiments. Most of the rhTGF-beta1 incorporated in the cement pellets was released within the first 48 h. For all concentrations of intermixed rhTGF-beta1 (100 ng to 2.5 mg/g of CPC), approximately 0.5% was released in the first 4 h, increasing to 1.0% after 48 h. Further release was only about 0.1% from 2 days to 8 weeks. CPC modification slightly increased the initial setting time at 20 degrees C from 2.6 to 5 min but had no effect on the final setting time of CPC at 20 degrees C or the initial and final setting times at 37 degrees C. The compressive strength was increased from 18 MPa in the standard CPC to 28 MPa in the modified CPC only 4 h after mixing. The compressive strength diminished in the modified CPC between 24 h and 8 weeks from 55 to 25 MPa. No other significant change was found with the CPC modification for rhTGF-beta1. X-ray diffraction revealed that standard and modified CPCs changed similarly from the original components, alpha-tricalcium phosphate and anhydrous dicalcium phosphate, into an apatite cement. The calcium-to-phosphorus ratio, as determined with an electron microprobe, did not differ for standard CPC and modified CPC. Standard and modified CPCs became dense and homogeneous structures after 24 h, but the modified CPC contained more crystal plaques than the standard CPC, as observed with scanning electron microscopy (SEM). SEM and back- scattered electron images revealed that after 8 weeks the cements showed equally and uniformly dense structures with microscopic pores (<1 microm). Both CPCs showed fewer crystal plaques at 8 weeks than at 24 h. This study shows that CPC is not severely changed by its modification for rhTGF-beta1. The prolonged setting time of modified cement may affect the clinical handling but is still within acceptable limits. The compressive strength for both standard and modified cements was within the range of thin trabecular bone; therefore, both CPCs can withstand equal mechanical loading. The faster diminishing compressive strength of modified cement from 24 h to 8 weeks likely results in early breakdown and so might be favorable for bone regeneration. Together with the beneficial effects on bone regeneration from the addition of rhTGF-beta1 to CPC, as shown in our previous studies, we conclude that the envisaged applications for CPC in bone defects are upgraded by the intermixing of rhTGF-beta1. Therefore, the combination of CPC and rhTGF-beta1 forms a promising synthetic bone graft.
Assuntos
Cimentos Ósseos , Fosfatos de Cálcio , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/farmacocinética , Substitutos Ósseos , Força Compressiva , Preparações de Ação Retardada , Humanos , Técnicas In Vitro , Teste de Materiais , Microscopia Eletrônica de Varredura , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacocinética , Difração de Raios XRESUMO
Bone regeneration of the alveolar crest around dental implants is an important factor in the success of implant use. Calcium phosphate cement can be used as a bone substitute and applied clinically as a paste to fill micro- and macroscopic bone defects. We have shown earlier that the intermixing of the recombinant human transforming growth factor-beta1 (rhTGF-beta1) in hardening calcium phosphate cement stimulated osteoblastic differentiation of rat primary bone cells in vitro. The aim of the present study was to examine whether the similar enrichment with rhTGF-beta1 affects the replacement of calcium phosphate cement by bone (osteotransduction) in calvarial critical size defects (csd) of adult rats. Two bone defects of 5 mm diameter were created bilaterally in each skull of 10 adult male rats. Both defects were filled with 53 mg of calcium phosphate cement without rhTGF-beta1 (control) at one side, and with 10 or 20 ng rhTGF-beta1 at the other side. After 8 weeks, defects with surrounding skull were analysed histologically and histomorphometrically. The addition of rhTGF-beta1 in the cement increased the amount of bone in rat skull defects. This finding coincidences with our in vitro observations, that intermixing of rhTGF-beta1 in calcium phosphate cement stimulates bone cell differentiation. Addition of rhTGF-beta1 stimulated bone formation as indicated by an increased bone volume of 50% and an increased bone/cement contact of 65%, in comparison to control defects with cement without rhTGF-beta1. In addition, rhTGF-beta1 reduced the remaining volume of cement, by 11% at 10 ng rhTGF-beta1, and by 20% at 20 ng rhTGF-beta1 in the cement. Defect closure was not affected. We conclude that the intermixing of rhTGF-beta1 in a fast-setting calcium phosphate cement stimulates bone growth and the osteotransduction of the cement. For bone regeneration procedures around endosseous implants, calcium phosphate cement with rhTGF-beta1 might be an appropriate combination for early osseointegration and implant use.
Assuntos
Cimentos Ósseos/química , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio , Fator de Crescimento Transformador beta/farmacologia , Animais , Substitutos Ósseos , Humanos , Masculino , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Crânio/cirurgiaRESUMO
Growth stimulation of periimplant tissues by growth factors like transforming growth factor-beta1 (TGF-beta1) may increase the indication for and success of implant use. Calcium phosphate as a material for implants or for coating of implants is known for its good biologic interaction with bone. Therefore, calcium phosphate implants combined with TGF-beta1 might improve osseointegration. In this study we hypothesise that the addition of recombinant human TGF-beta1 (rhTGF-beta1) to calcium phosphate cement (CPC) affects the differentiation of bone cells growing on the cement layer. rhTGF-beta1 incorporated during setting in a CPC layer at 20 ng rhTGF-beta1/60 mg cement was found to be gradually released into tissue culturing medium leading to a 20% release after 24 h. Two cell populations were obtained from collagenase-treated fragments of adult rat long bones: preosteoblastic cells, which were released by the collagenase treatment, and osteoblastic cells, which grew from the collagenase-stripped bone fragments. Both cell populations were tested for their osteoblastic characteristic phenotype by measuring their alkaline phosphatase (ALP) activity after vitamin D treatment and cyclic AMP after parathyroid hormone stimulation. After preculture the cells were plated on a layer of CPC containing 0 (control), 10, or 20 ng rhTGF-beta1/60 mg CPC. Bone cell differentiation was analyzed after 10 days by measuring the ALP activity, as well as the protein content of the cell layer. Incorporation of rhTGF-beta1 in the CPC did not change the ALP activity in osteoblastic cells, but a significant (analyzed by multivariate analysis of variance) increase was observed in preosteoblastic cells. Incorporation of 10 ng of rhTGF-beta1 in 60 mg of CPC increased the ALP activity in preosteoblastic cells by threefold and 20 ng rhTGF-beta1/60 mg CPC increased it by fivefold. The total protein content was not affected by rhTGF-beta1 in either of the cell populations. We conclude that rhTGF-beta1 incorporated during setting in CPC stimulates the differentiation of preosteoblastic cells in vitro. These results provide a basis for further studies on the use of this combination as an implant material in vivo.
Assuntos
Cimentos Ósseos , Osso e Ossos/citologia , Fosfatos de Cálcio , Osteoblastos/citologia , Fator de Crescimento Transformador beta/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Fosfatase Alcalina/análise , Animais , Biomarcadores , Regeneração Óssea , Calcitriol/farmacologia , Diferenciação Celular , Células Cultivadas , Humanos , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Ratos , Proteínas Recombinantes/farmacologiaRESUMO
Dental implants are successfully used for tissue-integrated protheses, but the long-term survival in the maxilla is shorter than in the mandible [Cune MS, Thesis, University of Utrecht, 1993; Jaffin RA, Berman CL. J Periodontol 1991;62:2-4]. However, by adding growth hormone at implantation, increased bone apposition may be expected, since it is known that growth hormone has a stimulating effect on the differentiation and proliferation of osteoblastic cells [Ernst M, Froesch ER. Biochem Biophys Res Commun 1988;151:142-47; Scheven BAA et al. Growth Regul 1991;1:160-67; Stracke H et al. Acta Endocrinol 1984;107:16-24]. We studied bone ingrowth and bone contact of grooved implants impregnated with growth hormone in the cortex of femurs of female goats. We compared the effect of growth hormone on grooved implants with or without calcium phosphate coatings at the bottom of the grooves. The coatings used were hydroxyapatite, fluorapatite and heat-treated hydroxyapatite. The implants had both small and large grooves. The implants were positioned in the cortex of one femur and were treated with recombinant human growth hormone, while the implants on the opposite femur served as controls. After 6 weeks, the implants and surrounding tissues were dissected and evaluated histomorphologically and morphometrically by light microscopy. The bone ingrowth and the bone contact in the grooves were quantified by digital image analysis. Calcium phosphate coating at the bottom of the grooves resulted in a significant increase of bone ingrowth and bone contact. Small grooves had significantly more bone ingrowth and bone contact than the larger grooves. However, all implants impregnated with growth hormone showed inhibition of bone contact and bone ingrowth. We conclude that recombinant human growth hormone inhibits bone formation in the grooves coated with calcium phosphate. Without the addition of growth hormone, the calcium phosphate coatings improved bone ingrowth and bone contact in the grooves. Further studies are required to determine whether growth hormone could also possibly act as a bone growth promoting factor in these implants.
Assuntos
Fosfatos de Cálcio , Fêmur/crescimento & desenvolvimento , Hormônio do Crescimento Humano/administração & dosagem , Implantes Experimentais , Osseointegração/efeitos dos fármacos , Ligas , Animais , Desenvolvimento Ósseo/efeitos dos fármacos , Feminino , Fêmur/efeitos dos fármacos , Cabras , Hormônio do Crescimento Humano/farmacologia , Humanos , TitânioRESUMO
Urine from untreated patients with various tumours and controls has been examined for the excretion of beta-aminoisobutyric acid and uric acid. The patients were classified into four groups: I, beta-aminoisobutyric acid and uric acid both normal; II, beta-aminoisobutyric acid normal, uric acid elevated; III, beta-aminoisobutyric acid elevated, uric acid normal; IV, beta-aminoisobutyric acid and uric acid both elevated. Uric acid was used as an indicator for tissue-breakdown. Pseudouridine being a specific parameter for t-RNA degradation was estimated for comparison. Increased urinary concentrations of beta-aminoisobutyric acid were frequently found in tumour patients, especially in patients with leukaemia and non-Hodgkin lymphoma. Tissue breakdown being the cause of the beta-aminoisobutyric aciduria could only be considered in part of the patients. Moreover, strongly elevated ratios of beta-aminoisobutyric acid to uric acid were found. Urinary patterns of pyrimidines and purines were determined in order to exclude other abnormalities. The results are discussed in relation to thymine metabolism and renal function.
Assuntos
Aminobutiratos/urina , Neoplasias/urina , Timina/urina , Adolescente , Criança , Pré-Escolar , Humanos , Lactente , Contagem de Leucócitos , Purinas/urina , Pirimidinas/urina , Ácido Úrico/urinaRESUMO
Excretion patterns of pyrimidines and purines in patients with various types of hyperammonemia have been investigated by 2-dimensional thin-layer chromatography and high pressure liquid chromatography (HPLC). For the quantitative analysis of pseudouridine, uracil and uridine a new procedure has been developed, consisting of pre-fractionation with Dowex 1 X 8, followed by dual column HPLC on a strong anion-exchanger and a reverse phase column. Thymine has also been analyzed in the pre-fractionated urine by a new HPLC method using the reverse phase column in combination with a strong cation-exchange column. Quantitative data for urinary pyrimidines and uric acid in hyperammonemia are given. In patients with a defect in one of the urea cycle enzymes, the level of pyrimidine excretion was found to depend on plasma ammonia concentrations. In other hyperammonemic patients, an increased excretion of orotic acid, uracil and uridine has only been found in one of the two patients with lysinuric protein intolerance, all other patients showing normal excretion patterns. Elevated uric acid excretions have been found frequently in our patients with hyperammonemia, but they did not always coincide with high plasma ammonia levels. A possible explanation for the difference in the excretion levels of the various pyrimidines is discussed.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/urina , Amônia/sangue , Purinas/urina , Pirimidinas/urina , Argininossuccinato Sintase/deficiência , Acidúria Argininossuccínica , Carbamoil-Fosfato Sintase (Amônia)/deficiência , Criança , Pré-Escolar , Feminino , Humanos , Recém-Nascido , Lisina/urina , Masculino , Doença da Deficiência de Ornitina Carbomoiltransferase , Síndrome de Reye/enzimologiaRESUMO
Chromatographic characteristics of urinary metabolites of theophylline were studied by two-dimensional thin-layer chromatography, high-performance liquid chromatography and gas chromatography--mass spectrometry. Quantitative date for the urinary metabolites of theophylline in asthmatic children are given. It was shown that 1,3-dimethyluric acid is the predominant excretory product. In addition, smaller amounts of 1-methyluric acid, 3-methylxanthine and unchanged theophylline were found. Excretory patterns after theophylline ingestion before and during the administration of allopurinol in asthma patients and in rats suggest the existence of three metabolic pathways of theophylline. The administration of this drug to a patient with xanthine oxidase of theophylline. The administration of this drug to a patient with xanthine oxidase deficiency resulted in the excretion of 1-methyluric acid in addition to 1,3-dimethyluric acid, 3-methylxanthine, 1-methylxanthine and unchanged theophylline. It was concluded that in man the oxidation of theophylline is not catalysed by xanthine oxidase.