Functional investigation of bone implant viability using radiotracers in a new model of osteonecrosis

OBJECTIVES: Conventional imaging methods are excellent for the morphological characterization of the consequences of osteonecrosis; however, only specialized techniques have been considered useful for obtaining functional information. To explore the affinity of radiotracers for severely devascularized bone, a new mouse model of isolated femur implanted in a subcutaneous abdominal pocket was devised. To maintain animal mobility and longevity, the femur was harvested from syngeneic donors. Two technetium-99m-labeled tracers targeting angiogenesis and bone matrix were selected. METHODS: Medronic acid and a homodimer peptide conjugated with RGDfK were radiolabeled with technetium-99m, and biodistribution was evaluated in Swiss mice. The grafted and control femurs were evaluated after 15, 30 and 60 days, including computed tomography (CT) and histological analysis. RESULTS: Radiolabeling achieved high (>95%) radiochemical purity. The biodistribution confirmed good blood clearance 1 hour after administration. For 99mTc-hydrazinonicotinic acid (HYNIC)-E-[c(RGDfK)2, remarkable renal excretion was observed compared to 99mTc-methylene diphosphonate (MDP), but the latter, as expected, revealed higher bone uptake. The results obtained in the control femur were equal at all time points. In the implanted femur, 99mTc-HYNIC-E-[c(RGDfK)2 uptake was highest after 15 days, consistent with early angiogenesis. Regarding 99mTc-MDP in the implant, similar uptake was documented at all time points, consistent with sustained bone viability; however, the uptake was lower than that detected in the control femur, as confirmed by histology. CONCLUSIONS: 1) Graft viability was successfully diagnosed using radiotracers in severely ischemic bone at all time points. 2) Analogously, indirect information about angiogenesis could be gathered using 999mTc-HYNIC-E-[c(RGDfK)2. 3) These techniques appear promising and warrant further studies to determine their potential clinical applications.

Avaliação morfológica e dos mecanismos de mobilização de Ca2+ pela glicose e acetilcolina em células pancreáticas humanas / Morphologic evaluation and Ca2+ mobilization by glicose and acetylcholine in human pancreatic cells

OBJETIVOS: Avaliar a morfologia das organelas e do citoesqueleto em células pancreáticas humanas cultivadas e a mobilização de Ca2+ em resposta à glicose e ACh por medidas fluorimétricas. MATERIAL E MÉTODOS: As células foram semeadas em lamínulas, fixadas e marcadas com uma combinação de fluoróforos: o núcleo foi corado com DAPI e as mitocôndrias, com Mytotracker Red. Foram utilizados faloidina e anticorpos secundários conjugados com Alexa Fluor verde e vermelho fluorescentes (488 e 594) para identificar proteína actina F e receptor muscarínico tipo M3, respectivamente. Para estudar a mobilização de Ca2+, as células foram incubadas com fura-2/AM. RESULTADOS: As células pancreáticas humanas apresentaram morfologia preservada com grande quantidade de mitocôndrias. Na região de maior densidade celular, evidenciou-se as pseudo-ilhotas e os receptores muscarínicos M3. Por meio da elevação da [Ca2+]c, devido à ação da glicose e ACh, mostrou-se preservação da capacidade responsiva a esses estímulos e foi dependente de concentração desses agonistas. A glicose promoveu uma resposta sustentada e a ACh induziu uma resposta bifásica. CONCLUSÃO: As células pancreáticas humanas cultivadas conservaram sua morfologia. A mobilização de Ca2+ em resposta à glicose e a ACh confirma a sua funcionalidade. Os receptores muscarínicos M3 estão presentes nessas células.

AIMS: The proposal of this study was to analyze morphology of the organelles and cytoskeleton in human pancreatic cells cultured and the mobilization of the cytosolic calcium ([Ca2+]c) in response to glucose and ACh by fluorimetry method. MATERIAL AND METHODS: The cells were plated on glass coverslips, fixed and stained with a combination of fluorophores: the nuclei were stained with DAPI and mitochondria with Mytotracker Red. It was used phalloidin and the secondary antibodies Alexa Fluor conjugated green and red-fluorescent (488 and 594) to identify the protein cell actin F and type M3 muscarinic receptor respectively. The cells also were loaded with fura-2/AM to study Ca2+ mobilization. RESULTS: The human pancreatic cells show characteristics morphologically preserved with great amount of mitochondria. In region major cell density was evidenced pseudo-islets and type M3 muscarinic receptors. Through increase of [Ca2+]c due to action of glucose and ACh were shown that the cellsÆ capacity to respond to these stimuli were conserved. The elevation of the [Ca2+]c depended on concentration by glucose-induced promoting sustained phase and ACh-induced a biphasic response. CONCLUSION: The morphologic characteristics of human pancreatic cells cultured were preserved. The Ca2+ mobilization in response to glucose and ACh confirmed its functionality. The expression of the M3 muscarinic receptors in human pancreatic cell cultured was demonstrated.