Titanium-hydroxyapatite composites sintered at low temperature for tissue engineering: in vitro cell support and biocompatibility.

BACKGROUND: In clinical orthopedics, a critical problem is the bone tissue loss produced by a disease or injury. The use of composites from titanium and hydroxyapatite for biomedical applications has increased due to the resulting advantageous combination of hydroxyapatite bioactivity and favorable mechanical properties of titanium. Powder metallurgy is a simple and lower-cost method that uses powder from titanium and hydroxyapatite to obtain composites having hydroxyapatite phases in a metallic matrix. However, this method has certain limitations arising from thermal decomposition of hydroxyapatite in the titanium-hydroxyapatite system above 800°C. We obtained a composite from titanium and bovine hydroxyapatite powders sintered at 800°C and evaluated its bioactivity and cytocompatibility according to the ISO 10993 standard. METHODS: Surface analysis and bioactivity of the composite was evaluated by X-ray diffraction and SEM. MTT assay was carried out to assess cytotoxicity on Vero and NIH3T3 cells. Cell morphology and cell adhesion on the composite surface were analyzed using fluorescence and SEM. RESULTS: We obtained a porous composite with hydroxyapatite particles well integrated in titanium matrix which presented excellent bioactivity. Our data did not reveal any toxicity of titanium-hydroxyapatite composite on Vero or NIH3T3 cells. Moreover, extracts from composite did not affect cell morphology or density. Finally, NIH3T3 cells were capable of adhering to and proliferating on the composite surface. CONCLUSIONS: The composite obtained displayed promising biomedical applications through the simple method of powder metallurgy. Additionally, these findings provide an in vitro proof for adequate biocompatibility of titanium-hydroxyapatite composite sintered at 800°C.

Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers.

INTRODUCTION: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. OBJECTIVE: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. METHODS: a combination of physical, chemical (Triton X-100 and SDS) and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy) and biochemical (DNA quantification) studies. RESULTS: the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde´s staining demonstrated the conservationof reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson´s capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. CONCLUSIONS: the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.

Preparación de una matriz extracelular tridimensional por descelularización de hígados de conejos / Preparación de una matriz extracelular tridimensional por descelularización de hígados de conejos / Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

Introducción: el número de hígados trasplantables es insuficiente para satisfacer las necesidades actuales de la demanda de injerto. La búsqueda de alternativas terapéuticas ha generado diferentes líneas de investigación, una de ellas es la utilización de matrices biológicas tridimensionales descelularizadas y la posterior siembra celular para obtener un órgano funcional. Objetivo: obtención de un protocolo de descelularización de hígado de conejo que genere una matriz hepática tridimensional. Métodos: una combinación de detergentes (Triton X-100 y SDS), agentes físicos y enzimáticos se utilizaron para descelularizar hígados de conejo. Los órganos se pefundieron en forma retrógrada con distintos agentes químicos durante 68 horas. Luego los hígados se examinaron por técnicas morfológicas (microscopía óptica y electrónicade barrido) y bioquímicas (cuantificación de ADN) para evaluar una correcta descelularización así como la obtención de una matriz extracelular preservada. Resultados: la observación macroscópica del órgano permitió inferir la descelularización del mismo. Las tinciones macroscópicas utilizadas mostraron una correcta conservación de los árboles biliar y vascular. Por otro lado, la observación microscópica del hígado permitió corroborar los resultados macroscópicos observados, la tinción de hematoxilina-eosina mostró ausencia de células y de material nuclear así como la presencia de la tríada portal. La tinción de Wilde evidenció la conservación de las fibras de reticulina en la matriz descelularizada. Asimismo, la microscopía electrónica de barrido reveló una cápsula de Glisson conservada y la descelularización de la cuantificación de ADN fue inferior al 10 % en el hígado descelularizado con respecto al hígado control. Finalmente, el tiempo utilizado para la descelularización fue inferior a las 96 horas. Conclusiones: el protocolo de descelularización propuesto fue apropiado ya que se verificó una ausencia de células y una matriz hepática con conductos vasculobiliares conservados y con una arquitectura tridimensional adecuada para una futura siembra celular(AU)

Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. Methods: a combination of physical, chemical (Triton X-100 and SDS) and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy) and biochemical (DNA quantification) studies. Results: the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin- eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde’s staining demonstrated the conservation of reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson’s capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. Conclusions: the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding(AU)

Central ghrelin increases anxiety in the Open Field test and impairs retention memory in a passive avoidance task in neonatal chicks.

Ghrelin (Grh) is an endogenous ligand for the growth hormone secretagogue receptor. Although Ghr stimulates feeding in rats, it inhibits feeding in neonatal chicks. However, little is known about other central behavioral effects of Ghr. Therefore, we investigated the Ghr effects, injected intracerebroventricularly, on anxiety and memory retention of neonatal chicks in an Open Field test and in a one-trial passive avoidance task, respectively. In the Open Field test, the administration of Ghr in a dose-dependent manner increased the latency to ambulate but decreased ambulation activity, indicating an anxiogenic effect. Furthermore, chicks trained on a passive avoidance task and injected with a dose of 30pmol of Ghr immediately after training showed an impairment of memory retention. However, there were no significant effects on the number of pecks during the pretraining, training, retention and discrimination. In addition, different doses of Ghr produced an inhibition in food intake at different times after injection. Our results indicate that Ghr induces anxiogenesis in chicks. Moreover, we have shown for the first time that Ghr can decrease memory retention in a non-mammalian species, suggesting that Ghr may play an important role in the processes of memory retention in birds.

Neonatal acute stress by novelty in the absence of social isolation decreases fearfulness in young chicks.

Two hours after hatching (Day 0), groups of chicks from both sexes were housed either individually (IND) or socially in pairs (SOC) for 24 h. On Day 1, for each of the two conditions, half of the chicks were individually exposed to early novelty for 10 min, which comprised being placed in a novel-cage with small pebbles glued to the floor. The other half (controls) remained in the home-cage (IND-C and SOC-C). Thus, the IND-N group was exposed to early novelty, and the SOC-N+I group was exposed to early novelty and social isolation. Subsequently, all groups were mixed and socially reared until reaching 15 days of age. At this time, chicks were exposed to open field (OF) and tonic immobility (TI) tests. The IND-N group showed a shorter latency to ambulate in the OF test, shorter immobility duration in the TI test, a reduced plasma corticosterone concentration and increased flunitrazepam sensitive-GABA(A) receptor basal forebrain density compared with other groups, indicating that a neonatal novelty induced lower fearfulness in young chicks. In contrast, the effect of neonatal novelty was abolished by a simultaneous effect of social isolation in the SOC-N+I group. Thus, early post-hatch life events such as early novelty could improve a bird's later ability to cope with new stressful events. In addition, it is possible that both novelty and social isolation act on different neurobiological processes.