M. H. V. Fernandes

Rodent models in bone-related research: the relevance of calvarial defects in the assessment of bone regeneration strategies

In vivo research with animal models has been a preferred experimental system in bone-related biomedical research since, by approximation, it allows relevant data gathering regarding physiological and pathological conditions that could be of use to establish more effective clinical interventions. Animal models, and more specifically rodent models, have been extensively used and have contributed greatly to the development and establishment of a wide range of translational approaches aiming to regenerate the bone tissue. In this regard, the calvarial defect model has found great application in basic and applied research, nonetheless the controversial rationalization for the use of critical size defects – defects that are unable to report spontaneous healing – or subcritical size defects in the proposed applications. Accordingly, this work aims to review the advantages and limitations of the use of rodent models in biomedical bone-related research, emphasizing the problematic issues of the use of calvarial critical and subcritical size defects. Additionally, surgical protocols for the establishment of both defects in rat calvarial bone, as well as the description and exemplification of the most frequently used techniques to access the bone tissue repair, are portrayed.

Assessment of renal dopaminergic system activity in the nitric oxide‐deprived hypertensive rat model

1 The present paper reports changes in the urinary excretion of dopamine, 5‐hydroxytryptamine and amine metabolites in nitric oxide deprived hypertensive rats during long‐term administration of NGnitro‐L‐arginine methyl ester (l‐NAME). Aromatic L‐amino acid decarboxylase (AAAD) activity in renal tissues and the ability of newly‐formed dopamine to leave the cellular compartment where the synthesis of the amine has occurred were also determined.

Effect of Sterilization Methods on Electrospun Poly(lactic acid) (PLA) Fiber Alignment for Biomedical Applications

Medically approved sterility methods should be a major concern when developing a polymeric scaffold, mainly when commercialization is envisaged. In the present work, poly(lactic acid) (PLA) fiber membranes were processed by electrospinning with random and aligned fiber alignment and sterilized under UV, ethylene oxide (EO), and γ-radiation, the most common ones for clinical applications. It was observed that UV light and γ-radiation do not influence fiber morphology or alignment, while electrospun samples treated with EO lead to fiber orientation loss and morphology changing from cylindrical fibers to ribbon-like structures, accompanied to an increase of polymer crystallinity up to 28%. UV light and γ-radiation sterilization methods showed to be less harmful to polymer morphology, without significant changes in polymer thermal and mechanical properties, but a slight increase of polymer wettability was detected, especially for the samples treated with UV radiation. In vitro results indicate that both UV and γ-radiation treatments of PLA membranes allow the adhesion and proliferation of MG 63 osteoblastic cells in a close interaction with the fiber meshes and with a growth pattern highly sensitive to the underlying random or aligned fiber orientation. These results are suggestive of the potential of both γ-radiation sterilized PLA membranes for clinical applications in regenerative medicine, especially those where customized membrane morphology and fiber alignment is an important issue.

Assessment of renal dopaminergic system activity during cyclosporine A administration in the rat.

1 Administration of cyclosporine A (CsA; 50 mg kg−1 day−1, s.c.) for 14 days produced an increase in both systolic (SBP) and diastolic (DBP) blood pressure by 60 and 25 mmHg, respectively. The urinary excretion of dopamine, DOPAC and HVA was reduced from day 5–6 of CsA administration onwards (dopamine from 19 to 46%, DOPAC from 16 to 48%; HVA from 18 to 42%). In vehicle‐treated rats, the urinary excretion of dopamine and DOPAC increased (from 7 to 60%) from day 5 onwards; by contrast, the urinary excretion of HVA was reduced (from 27 to 60%) during the second week. 2 No significant difference was observed between the Vmax and Km values of renal aromatic L‐amino acid decarboxylase (AAAD) in rats treated with CsA for 7 and 14 days or with vehicle. 3 Km and Vmax of monoamine oxidase types A and B did not differ significantly between rats treated with CsA for 7 and 14 days or with vehicle. 4 Maximal catechol‐O‐methyltransferase activity (Vmax) in homogenates of renal tissues obtained from rats treated with CsA for 7 or 14 days was significantly higher than that in vehicle‐treated rats; Km(22.3 ± 1.5 μm) values for COMT did not differ between the three groups of rats. 5 The accumulation of newly‐formed dopamine and DOPAC in cortical tissues of rats treated with CsA for 14 days was three to four times higher than in controls. The outflow of both dopamine and DOPAC declined progressively with time and reflected the amine and amine metabolite tissue contents. No significant difference was observed between the DOPAC/dopamine ratios in the perifusate of renal tissues obtained from CsA‐ and vehicle‐treated rats. In addition, no significant differences were observed in k values or in the slope of decline of both DA and DOPAC between experiments performed with CsA and vehicle‐treated animals. 6 The Vmax for the saturable component of L‐3, 4‐dihydroxyphenylalanine (L‐DOPA) uptake in renal tubules from rats treated with CsA was twice that of vehicle‐treated animals. Km in CsA‐ and vehicle‐treated rats did not differ. 7 The decrease in the urinary excretion of sodium and an increase in blood pressure during CsA treatment was accompanied by a reduction in daily urinary excretion of dopamine. This appears to result from a reduction in the amount of L‐DOPA made available to the kidney and does not involve changes in tubular AAAD, the availability of dopamine to leave the renal cells and dopamine metabolism. The enhanced ability of the renal tissues of CsA‐treated animals to synthesize dopamine, when exogenous L‐DOPA is provided, results from an enhanced activity of the uptake process of L‐DOPA in renal tubular cells.

Tailoring the morphology of high molecular weight PLLA scaffolds through bioglass addition

Thermally induced phase separation (TIPS) has proven to be a suitable method for the preparation of porous structures for tissue engineering applications, and particular attention has been paid to increasing the pore size without the use of possible toxic surfactants. Within this context, an alternative method to control the porosity of polymeric scaffolds via the combination with a bioglass is proposed in this work. The addition of a bioactive glass from the 3CaO x P2O5-MgO-SiO2 system enables the porous structure of high molecular weight poly(l-lactic) acid (PLLA) scaffolds prepared by TIPS to be tailored. Bioglass acts as a nucleating catalyst agent of the PLLA matrix, promoting its crystallization, and the glass solubility controls the pore size. A significant increase in the pore size is observed as the bioglass content increases and scaffolds with large pore size (approximately 150 microm) can be prepared. In addition, the bioactive character of the scaffolds is proved by in vitro tests in synthetic plasma. The importance of this approach resides on the combination of the ability to tailor the porosity of polymeric scaffolds via the tunable solubility of bioglasses, without the use of toxic surfactants, leading to a composite structure with suitable properties for bone tissue engineering applications.

Sodium-dependence and ouabain-sensitivity of the synthesis of dopamine in renal tissues of the rat.

1 The present study has examined the influence of sodium chloride (0–160 mm) and ouabain (100 and 500 μm), an inhibitor of the enzyme Na+–K+ ATPase, on the synthesis of dopamine in slices of rat renal cortex loaded with exogenous l‐dihydroxyphenylalanine (l‐DOPA). The deamination of newly‐formed dopamine into 3,4‐dihydroxyphenylacetic acid (DOPAC) was also examined. The assay of l‐DOPA, dopamine and DOPAC in kidney slices was performed by high performance liquid chromatography (h.p.l.c.). with electrochemical detection. 2 The accumulation of newly‐formed dopamine and DOPAC in kidney slices loaded with l‐DOPA (50 and 100 μm) was found to be dependent on the concentration of NaCl in the medium. A similar picture could be observed for DOPAC. The fractional rate of accumulation (k; mm NaCl−1) was at 50 and 100 μm l‐DOPA, respectively, 0.00305 ± 0.00036 and 0.00328 ± 0.00029 for dopamine and 0.00672 ± 0.00072 and 0.00641 ±0.00069 for DOPAC. The sodium‐dependent formation of dopamine was completely abolished when the experiments were performed in the absence of oxygen. 3 In experiments performed in the presence of 120 mm NaCl, but not in conditions of low sodium (20 mm NaCl in the medium), ouabain (100 and 500 μm) was found to inhibit the accumulation of newly‐formed dopamine and DOPAC (14–57% reduction; P < 0.05); this effect was more marked at 50 and 100 μm l‐DOPA. When the experiments were performed in the absence of oxygen the renal production of dopamine and DOPAC became markedly decreased (respectively, 40% and 77% reduction; P < 0.05) and ouabain (100 and 500 μm) was no longer able to reduce the accumulation of both newly‐formed dopamine and DOPAC. 4 The formation of dopamine and its deamination to DOPAC in kidney homogenates closely depended on the concentration of l‐DOPA added to the medium; ouabain (100, 500 and 1000 μm) was found to affect neither the formation of dopamine nor its deamination to DOPAC when homogenates were used instead of kidney slices. 5 In conclusion, the results presented here show that the formation of dopamine in kidney slices loaded with l‐DOPA is sodium‐dependent and ouabain‐sensitive suggesting the involvement of a co‐transport system of sodium and DOPA into the tubular epithelial cell.

Mineralization in serially passaged human alveolar bone cells

Well-characterized human bone cell cultures have been regarded as auseful tool to study bone control mechanisms and also to analysebone/biomaterials interactions. In the present study, human alveolar bone cellswere cultured in α-minimal essential medium (α-MEM) containing 10%foetal bovine serum (FBS), 50 μg/ml ascorbic acid, 10 mM sodiumβ-glycerophosphate and either in the presence or in the absence of 10 nMdexamethasone (Dexa). Cultures were characterized concerning cellviability/proliferation, alkaline phosphatase (ALP), acid phosphatase (ACP) andtartraric acid resistant phosphatase (TRAP) activities, and formation ofmineralized areas. Cell proliferation increased gradually for approximately 20days. In the presence of Dexa, cells formed isolated or interconnectedmultilayered clusters that increased with culture time. Histochemical assaysrevealed strong positive reactions for ALP and calcium and phosphates deposits,mainly in relation t!o cells associated with the clusters. High levels of ALP activity (biochemicaldetermination) were observed. Cells cultured in the absence of Dexa showedsignificantly lower ALP activity and no calcium and phosphates deposits werepresent. Serially passaged cells kept the proliferation rate constant but adecrease in ALP activity was observed either in the presence or in the absenceof Dexa. The ability to form mineralized areas (cultures fed with Dexa) alsodecreased on serial subculture.

Role of monoamine oxidase and cathecol-O-methyltransferase in the metabolism of renal dopamine

Incubation of slices of rat renal cortex with 50 microM L-DOPA during 15 min resulted in the formation of dopamine and of its deaminated (3,4-dihydroxyphenylacetic acid; DOPAC), methylated (3-methoxytyramine; 3-MT) and deaminated plus methylated (homovanillic acid; HVA) metabolites. The presence of pargyline (100 microM) resulted in a 90% reduction in the formation of DOPAC and HVA; levels of dopamine and 3-MT were found to be significantly increased. A concentration dependent decrease in the formation of methylated metabolites was obtained in the presence of (10, 50 and 100 microM) tropolone (10-50% reduction) and (0.1, 0.5, 1.0 and 5.0 microM) Ro 40-7592 (50-95% reduction). Ro 40-7592 was also found to significantly increase DOPAC (20-40%) and dopamine (10-30%) levels, whereas tropolone slightly increased DOPAC (10%) levels. These results show that deamination represents the major pathway in the metabolism of newly formed dopamine under in vitro experimental conditions in the rat kidney. In addition, only when MAO is inhibited does methylation appear to represent an alternative metabolic pathway.

Effect of partial crystallization on the mechanical properties and cytotoxicity of bioactive glass from the 3CaO.P2O5–SiO2–MgO system

The aim of this study is to report on the development and characterization of bioactive glass and glass-ceramics from the 3CaO.P(2)O(5)-SiO(2)-MgO-system, using different degrees of cristallinity for applications as an implant material. A methodology was proposed to induce crystallization of phases. Bioglass samples of the nominal composition (wt %) 57.75 CaO.P(2)O(5)-30 SiO(2)-17.25MgO were heat treated at temperatures ranging from 700 to 1100°C for 4h. The findings from the research illustrate how partial crystallization and phase transformations modified the microstructure of the based glassy material, resulting in improved mechanical properties. The maximum gain was measured for samples treated at 975°C, having a hardness of 6.2GPa, an indentation fracture toughness of 1.7MPam(1/2) and a bending strength of 120MPa, representing an increase of 30, 55 and 70%, respectively, when compared to the nucleated glass. The highest elastic modulus of about 130GPa was determined for samples treated at 1100°C. As a preliminary biological evaluation, in vitro cytotoxicity tests were realized to determine the cytotoxic level of the materials, using the neutral red uptake method with NCTC clones L929 from the American Type Culture Collection (ATCC) bank. On the other hand, no significant influence of the partial crystallization on cytotoxicity was observed. The results provide support for implant materials based on the 3CaO.P(2)O(5)-SiO(2)-MgO-system.

TiO2-induced phase separation and crystallization in SiO2–3CaO·P2O5–MgO glass

Abstract The effect of TiO 2 additions on microstructure and crystallization of a 42.6 wt% SiO 2 , 29.4 wt% 3CaO·P 2 O 5 , 28.0 wt% MgO glass was studied. TiO 2 produced amorphous separation and volume crystallization during ceramming. Heat treatments to 1100°C resulted in a simultaneous crystallization of β-3(Ca, Mg)O·P 2 O 5 and clino-enstatite (MgO·SiO 2 ) in TiO 2 -free glasses and the occurrence of a third phase, forsterite (2MgO·SiO 2 ), in all TiO 2 -containing glasses. Peak crystallization temperatures suggested that crystallization rates increase with TiO 2 contents to 3% and that further changes do not occur.