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Biomedical subjects

R S Carvalho

Publications and source records attributed to R S Carvalho.

14 recordsLinked to original sources
PubMed2004

The role of angiogenesis in a murine tibial model of distraction osteogenesis.

Distraction osteogenesis (DO) is one of the most dramatic in vivo applications of mechanical stimulation as a means of inducing bone regeneration. A simple and reproducible murine model of tibia distraction osteogenesis was developed using a monolateral fixator. Bone formation was assessed histologically over a 35-day time course. The steady state expression of a broad family of angiogenesis-associated genes was assessed by microarray hybridization analyses over the same time course, while the immediate gene response that was induced during each cycle of distraction was assessed at 30 min and 8 h after the first and last rounds of activation of the fixator. Distraction osteogenesis promoted new bone formation primarily through an intramembranous process with maximal osteogenesis during the active distraction period. Histological analysis also showed that dense cortical bone continued to be formed, during the consolidation phase, for 2 weeks after distraction ended. The analysis of steady state mRNA expression levels over the time course of DO showed that VEGF-A and neuropilin, an alternate receptor for VEGF-A, both angiopoietin (Ang) 1 and 2 factors, and the Ang receptor Tie2 were the critical angiogenic factors during DO. A key transcriptional regulator of many of the angiogenic factors, hypoxia-induced factor1alpha (Hif-1a), the FGF binding protein pleiotropin/OSF1, and multiple MMP(s), were also induced during the active distraction period. Examination of the expression of angiogenic factors that were induced after each cycle of activation, demonstrated that Hif-1a, Nrp1, and VEGF-A were all cyclically induced after each increment of distraction. These results suggest that these factors are early mediators that are produced by distraction and contribute toward the processes that promote bone formation. These experiments represent the first step in defining the molecular mechanisms that regulate skeletal regeneration and the functional relationship between angiogenesis and osteogenesis during distraction osteogenesis.

Animals↗
PubMed2003

Selective adhesion of osteoblastic cells to different integrin ligands induces osteopontin gene expression.

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.

Animals↗
PubMed2002

Predominant integrin ligands expressed by osteoblasts show preferential regulation in response to both cell adhesion and mechanical perturbation.

Previous studies have demonstrated that both mechanical perturbation and cell adhesion induced the expression of osteopontin (opn) by osteoblasts (Carvalho et al. [1998] J. Cell. Biochem. 70:376-390). The present study examined if these same stimuli on osteoblasts would induce the expression of other integrin binding proteins, specifically fibronectin (fn) and bone sialoprotein (bsp). All three genes showed three- to four-fold maximal induction in response to both cell adhesion and a single 2-h period of an applied spatially uniform, dynamic biaxial strain of 1.3% at 0.25 Hz. Each gene, however, responded with a different time course of induction to mechanical strain, with bsp, fn, and opn showing their maximal response at 1, 3, and 9 h, respectively, after the perturbation period. In contrast, peak induction to cell adhesion was observed at 24 h for bsp and opn, while fn levels peaked at 8 h. Interestingly, while both opn and fn mRNA expression returned to base line after cell adhesion, bsp mRNA levels remained elevated. Examination of collagen type I and osteocalcin mRNAs showed unaltered levels of expression in response to either type of perturbation. A common feature of the signal transduction pathways, which mediate the gene expression in response to both cell adhesion and mechanical perturbation, was the activation of specific tyrosine kinases based on the ablation of the induction of these genes by the tyrosine kinase inhibitor genistein. While cycloheximide blocked the induction of all three mRNAs in response cell adhesion, it failed to block the induction of any of these genes in response to mechanical perturbation. Such results suggest that the induction of these genes after mechanical perturbation was mediated by an immediate response to signal transduction, while cell adhesion mediated effects secondary to signal transduction. Depolymerization of microfilaments with cytochalasin D had no effect on the overall expression of any of these genes in response to cell adhesion and only blocked the induction of opn expression in response to mechanical perturbation. These results suggest that cytoskeletal integrity is only selectively important in the signal transduction of certain types of stimuli and for the regulation of certain genes. In summary, both mechanical perturbation and cell adhesion stimulated the expression of integrin binding proteins. Furthermore, while there are common features in the signal transduction processes that mediate the induction of these genes in response to both stimuli, specific genes are separately regulated by precise mechanisms that are unique to both forms of stimuli.

Actin Cytoskeleton↗
PubMed1998

Osteoblasts induce osteopontin expression in response to attachment on fibronectin: demonstration of a common role for integrin receptors in the signal transduction processes of cell attachment and mechanical stimulation.

Osteopontin is a predominant integrin binding protein of bone and its expression has been shown to be induced by mechanical stimuli within osteoblasts (Toma et al. [1997] J. Bone Miner. Res. 12:1626-1636). The present studies examined if the cell adhesion would mimic the mechano-transduction that stimulated opn mRNA expression and whether integrin receptors were involved in these processes. Osteopontin mRNA expression was induced three- to four-fold, 24 hours after embryonic chicken calvaria osteoblast attachment to fibronectin (FN), however no induction was observed if the cells were plated on tissue culture plastic alone. Osteopontin mRNA induction in response to cell attachment on FN was dependent on new protein synthesis and the activation of a tyrosine protein kinase(s) but unlike mechano-induction was independent of the maintenance of the cell's microfilament structure. Integrin receptor(s) were shown to be involved in mediating the signal transduction processes of both cell attachment and mechanical stimulation since incubation of osteoblasts with the integrin binding peptide RGDS partially blocked the induction of opn expression in response to both stimuli. Interestingly, incubation of the osteoblasts that were adherent on tissue culture plastic alone with the RGDS peptide lead to an induction in opn expression suggesting that integrin occupancy by itself was sufficient to initiate the signal transduction process that induced opn expression. In order to assess the role of integrin occupancy vs. focal adhesion complex formation that accompanies cell attachment, in the signal transduction process that induces opn expression, receptor clustering was stimulated pharmacologically with bombesin or lysophosphatidic acid in osteoblasts attached to tissue culture plastic. Neither compound in the absence of occupancy of the integrin receptors was capable of stimulating opn expression in attached cells, however if the cells were placed in suspension pharmacological mediation of receptor clustering and integrin occupancy were additive in their effect of inducing opn expression. These data demonstrate that induction of opn expression by mechanical stimuli and cell attachment are commonly mediated through integrin receptor(s). However, when cells are attached receptor clustering alone which accompanies focal adhesion formation was incapable of mediating signal transduction suggesting that receptor occupancy was a prerequisite to the signal transduction process that leads to the induction of opn mRNA expression.

Animals↗
PubMed1997

Collagen synthesis from human PDL cells following orthodontic tooth movement.

The dynamic remodelling processes in the periodontal ligament (PDL) account for the reaction of PDL cells to different orthodontic force simulations. These occur mostly by degradation and synthesis of collagen types I, III, V, VI, XII and XIV. The purpose of this study was to quantify specific collagen types in the PDL from zones of tension and compression of experimental teeth. Such changes could then be correlated with the processes of orthodontic-stimulated tissue breakdown. Maxillary and mandibular premolars of three females and one male patient were orthodontically moved with a box loop for a total of 14 days, prior to tooth extraction. Teeth from the contralateral side of either the maxilla or the mandible served as the untreated controls. A total of seven experimental and seven control teeth were used in this investigation. PDL fibroblasts from the cervical third of the roots corresponding to the compression and tension zones of the experimental and control teeth, respectively, were scraped and cultured in vitro at 37 degrees C in a humidified incubator with 5 per cent CO2/95 per cent air. Collagen synthesis of types I, III, V and VI was quantified by using an ELISA. Application of orthodontic forces in the experimental teeth showed a significant increase (P < 0.05) of the synthesis of all collagen types in the compression as opposed to the tension zones. Collagen synthesis on the compression zone of experimental teeth was not significantly different in the mandible when compared with those of the maxilla. In addition, the proportional distribution of different types of collagen was also not significantly different in the PDL fibroblasts from either zone of experimental teeth of either the maxilla or the mandible. Collagen metabolism in response to orthodontic stimulation appears to be higher in the compression zones and lower in the tension zones. Contrary to what is traditionally assumed in the literature, such findings indicate that in addition to bone resorption, tissue remodelling is very active in zones of compression following the disappearance of the hyalinized areas. These findings constitute a model for future studies on collagen metabolism during orthodontic-stimulated tooth movement.

Adolescent↗
PubMed1995

Conformational changes upon binding of a receptor loop to lipid structures: possible role in signal transduction.

The mas oncogene codes for a seven transmembrane helix protein. The amino acid sequence 253-266, from the third extracellular loop and beginning of helix 7, was synthesized either blocked or carrying an amino acid spin label at the N-terminus. Peptide binding to bilayers and micelles was monitored by ESR, fluorescence and circular dichroism. Binding induced tighter lipid packing, and caused an increase of peptide secondary structure. While binding to bilayers occurred only when peptide and phospholipid bore opposite charges, in micelles the interaction took place irrespective of charge. The results suggest that changes in lipid packing could modulate conformational changes in receptor loops related to the triggering of signal transduction.

Amino Acid Sequence↗
PubMed1995

The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cells.

Mechanical stimulation of the skeleton alters the metabolism of bone cells, but the effects of mechanical strain on the cytoskeleton of osteoblasts are poorly understood. While changes in the distribution of the cytoskeleton in mechanically strained cells have been reported, little is known about the pathways by which these changes are transduced into cell functions. Human osteosarcoma (HOS) TE-85 cells were cultured in Dubelcco's modified Eagle's medium/F-12 and grown to confluency in Flexercell type I dishes in a humidified incubator with 5% CO2 and 95% air. Intermittent strain (3 cycles/min) was applied to the cells for periods of 15 and 30 min, 2, 4 and 24 h, and 3, 5, 7, 10, 14, 20 and 28 days. Unstrained cells were used as controls. The distribution of beta 1 integrin was studied immunocytochemically. Total RNA was isolated at every period of time and Northern blots were used to study the effects of strain on the levels of beta 1-integrin expression. The results indicated that mechanical strain increased the synthesis of beta 1 integrin. Northern blots showed that beta 1 mRNA expression was increased significantly (p < 0.005) at 30 min and 3 days of strain application. Strain also affected beta 1 distribution markedly in 24-h cultures. The response of HOS cells to mechanical strain demonstrates that the cytoskeleton of the osteoblast adapts to strain through the stimulation of specific cytoskeletal and receptor proteins. These results suggest a pathway through which mechanical strain is transmitted to the osteoblastic-like cells.

Analysis of Variance↗
PubMed1995

Glycosaminoglycan synthesis in the rat articular disk in response to mechanical stress.

The mechanism by which compressive mechanical stress affects glycosaminoglycan synthesis in the articular disk was investigated with a modified organ culture technique. Forty-eight male Sprague-Dawley rats were divided into three experimental groups and one control group of 12 animals each, aged 7 and 9 weeks. The experimental groups followed different regimens of stress applied for 25%, 75%, or 100% of the time during the total test period of 24 hours. Articular disks were stressed with flexible bottomed dishes (Flex I dishes, Flexcell Corp., McKeesport, Pa.) using the Flexercell Strain Unit (Flexcell Corp., McKeesport, Pa.) and incubated with [3H]-glucosamine for 24 hours. Samples were then collected, digested with Pronase-E, and after precipitation with cetylpyridinium chloride (CPC) and ethanol, the different glycosaminoglycans (GAGs) were separated by using cellulose acetate electrophoresis. The significant GAG types with stress were chondroitin6sulfate (C6S), hyaluronic acid (HA), and dermatan sulfate (DS). There was no significant relationship in the experimental groups between age and regimen of stress applied in either age. Higher stress regimens showed significantly higher proportions of C6S when compared with the controls, whereas HA appeared to decrease slightly and DS was not affected. Since C6S is the major component of hyaline cartilage, the results of this study suggest that compressive forces in the articular disk may stimulate the development of more cartilagenous-like properties with respect to GAG content.

Age Factors↗
PubMed1994

Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone.

Second-messenger systems have been implicated to transmit mechanical stimulation into cellular signals; however, there is no information on how mechanical stimulation is affected by such systemic factors as parathyroid hormone (PTH). Regulation of adenylyl cyclase and phosphatidylinositol pathways in rat dentoalveolar bone cells by mechanical strain and PTH was investigated. Two different cell populations were isolated after sequential enzyme digestions from dentoalveolar bone (group I and group II) to study potential differences in response. Mechanical strain was applied with 20 kPa of vacuum intermittently at 0.05 Hz for periods of 0.5, 1, 5, 10, and 30 minutes and 1, 3, and 7 days using the Flexercell system. Levels of cAMP, measured by RIA, and levels of inositol 1,4,5-triphosphate (IP3) and protein kinase C activity (PKC), measured by assay systems, increased with mechanical strain. When PTH was added to the cells, there was a significant increase in levels of all the intracellular signals, which appeared to potentiate the response to mechanical strain. IP3 levels (0.5 minute) peaked before those of PKC activity (5 minutes), which in turn peaked before those of cAMP (10 minutes). Group II cells showed higher levels of cAMP and IP3 than the group I cells. This suggests that the former may ultimately play the predominant roles in skeletal remodeling in response to strain. Immunolocalization of the cytoskeleton proteins vimentin and alpha-actinin, focal contact protein vinculin, and PKC showed a marked difference between strained and nonstrained cells. However, the addition of PTH did not cause any significant effect in cytoskeleton reorganization. Staining of PKC and vimentin, alpha-actinin, and vinculin suggests that PKC participates actively in the transduction of mechanical signals to the cell through focal adhesions and the cytoskeleton, although only PKC seemed to change with short time periods of strain. In conclusion, dentoalveolar osteoblasts responded to mechanical strain initially through increases in levels of IP3, PKC activity, and later cAMP, and this response was potentiated when PTH was applied together with mechanical strain.

Adenylyl Cyclases↗
PubMed1994

Binding of peptide fragments from a seven helix membrane receptor to lipid bilayers and to micelles.

Membrane proteins influence the organizational and motional properties of lipids, while the conformation and function of these proteins (receptors, channels, enzymes, pumps) are affected by the lipid environment. Model systems consisting of peptides and lipids can provide information at a molecular level about the interactions between proteins and lipids in biological membranes. We have synthesized peptides (residues 253-266 (EYWSTFGNLHHISL) from the seven-helix receptor expressed by the mas oncogene), having free or blocked N- and C-terminals. An analog was obtained by linking a spin-labeled amino acid to the N-terminal via a peptide bond. Several spectroscopic techniques were employed to study the interaction between the peptides and lipophilic systems (zwitterionic and negatively charged phospholipid bilayers, and negatively charged, positively charged, zwitterionic and nonionic micelles). Peptide conformational changes were monitored by circular dichroism (CD). The peptides acquired an increased secondary structure upon binding to the lipid systems. Additional evidence for peptide incorporation into micelles came from fluorescence measurements which indicated a blue shift of the tryptophan's emission wavelength, and from ESR spectra of the spin-labeled analog. While narrow lines were obtained in the aqueous phase, line broadening indicative of slower motion was observed in the presence of the lipophilic aggregates. The slow exchange between the two media allowed the evaluation of partition coefficients. The spectra in aqueous solution were also sensitive to conformational changes as a function of pH, allowing the determination of the N-terminal pK. ESR spectra of lipid spin probes incorporated into phospholipid bilayers indicated that the lipids became more immobilized upon binding of the peptides.

Amino Acid Sequence↗
PubMed1993

The effect of growth on collagen and glycosaminoglycans in the articular disc of the rat temporomandibular joint.

Newly synthesized collagen and glycosaminoglycans (GAG) were studied in the temporomandibular discs of male Sprague-Dawley rats of 3-13 weeks of age. Each age group had eight animals and [14C]glycine or [3H]glucosamine were used to determine the proportion of newly synthesized type III to type I collagens or the proportion of different types of newly synthesized GAGs during 4 h of labelling in organ culture. Separation of newly synthesized collagen bands from rat disc by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a peak in type III at the ages of 7 and 8 weeks. Type III collagen synthesis and the rate of mandibular growth were strongly related through all ages studied. GAG chains were separated by cellulose-acetate electrophoresis. Calculation of disintegrations/min per mg of wet disc tissue for each GAG peak showed that hyaluronic acid (HA), chondroitin-6-sulphate and keratan sulphate/chondroitin-4-sulphate (KS/C4S) were the predominant molecules synthesized in the disc. There was also a steady increase in newly synthesized HA and C6S synthesis up to 6 and 7 weeks respectively. Proportions of newly synthesized C4S/KS, HA and C6S were significantly higher than those of other GAGs with respect to ageing. From these observations it appears that the articular disc shows more of the characteristics of cartilage, as evidenced by the increased amounts of C6S and KS/C4S during the mandibular growth spurt at the ages of 6 and 7 weeks, similar to that of type III collagen. There were also increased amounts of HA, suggesting that during 5-7 weeks of age the rat disc is undergoing more active remodelling. This study provides baseline data for further analysis of the effects of mechanical loading and trauma on articular disc responses.

Animals↗
PubMed1989

An evaluation of the changes in soft-tissue profile form induced by orthodontic therapy.

In view of the growing concensus that traditional cephalometric appraisals yield data of dubious scientific value, the changes in soft-tissue profile forms were evaluated by an alternative technique. This involved dividing the soft-tissue profile form into a series of triangular finite elements spanning homologous datum points. With the use of techniques derived from continuum mechanics, the changes in soft-tissue profile form were evaluated by computing the degree of distortion in each triangle after treatment (target element) compared with the triangle before treatment (reference element) in terms of specific size and shape parameters. In a comparison of samples of patients with Class I and II malocclusion, varying patterns of facial profile change were noted, depending on the parameters analyzed. The results, therefore, showed that although such techniques offer rigorous cephalometric potential, further investigation is required to identify more appropriate datum points.

Cephalometry↗