New prospects in skin regeneration and repair using nanophased hydroxyapatite embedded in collagen nanofibers.

This study reflects an exploitation of a composite matrix produced by electrospinning of collagen and electrospraying of nanophased hydroxyapatite (nanoHA), for skin regeneration applications. The main goal was to evaluate the effect of nanoHA, as source of localized calcium delivery, on human dermal fibroblasts, keratinocytes, and human mesenchymal stem cells (hMSCs) growth, proliferation, differentiation, and extracellular matrix production. This study revealed that calcium ions provided by nanoHA significantly enhanced cellular growth and proliferation rates and prevented adhesion of pathogenic bacteria strains typically found in human skin flora. Moreover, hMSCs were able to differentiate in both osteogenic and adipogenic lineages. Rat subcutaneous implantation of the membranes also revealed that no adverse reaction occurred. Therefore, the mechanically fit composite membrane presents a great potential to be used either as cell transplantation scaffold for skin wound regeneration or as wound dressing material in plastic surgery, burns treatment or skin diseases.

In situ Enabling Approaches for Tissue Regeneration: Current Challenges and New Developments.

In situ tissue regeneration can be defined as the implantation of tissue-specific biomaterials (by itself or in combination with cells and/or biomolecules) at the tissue defect, taking advantage of the surrounding microenvironment as a natural bioreactor. Up to now, the structures used were based on particles or gels. However, with the technological progress, the materials' manipulation and processing has become possible, mimicking the damaged tissue directly at the defect site. This paper presents a comprehensive review of current and advanced in situ strategies for tissue regeneration. Recent advances to put in practice the in situ regeneration concept have been mainly focused on bioinks and bioprinting techniques rather than the combination of different technologies to make the real in situ regeneration. The limitation of conventional approaches (e.g., stem cell recruitment) and their poor ability to mimic native tissue are discussed. Moreover, the way of advanced strategies such as 3D/4D bioprinting and hybrid approaches may contribute to overcome the limitations of conventional strategies are highlighted. Finally, the future trends and main research challenges of in situ enabling approaches are discussed considering in vitro and in vivo evidence.

A new era for sterilization based on supercritical CO2 technology.

The increasing complexity in morphology and composition of modern biomedical materials (e.g., soft and hard biological tissues, synthetic and natural-based scaffolds, technical textiles) and the high sensitivity to the processing environment requires the development of innovative but benign technologies for processing and treatment. This scenario is particularly applicable where current conventional techniques (steam/dry heat, ethylene oxide, and gamma irradiation) may not be able to preserve the functionality and integrity of the treated material. Sterilization using supercritical carbon dioxide emerges as a green and sustainable technology able to reach the sterility levels required by regulation without altering the original properties of even highly sensitive materials. In this review article, an updated survey of experimental protocols based on supercritical sterilization and of the efficacy results sorted by microbial strains and treated materials was carried out. The application of the supercritical sterilization process in materials used for biomedical, pharmaceutical, and food applications is assessed. The opportunity of supercritical sterilization of not only replace the above mentioned conventional techniques, but also of reach unmet needs for sterilization in highly sensitive materials (e.g., single-use medical devices, the next-generation biomaterials, and medical devices and graft tissues) is herein unveiled.

Influence of PLLA/PCL/HA Scaffold Fiber Orientation on Mechanical Properties and Osteoblast Behavior.

Scaffolds based on aligned and non-aligned poly (L-lactic acid) (PLLA)/polycaprolactone (PCL) fibers obtained by electrospinning, associated to electrosprayed hydroxyapatite (HA) for tissue engineering applications were developed and their performance was compared in terms of their morphology and biological and mechanical behaviors. The morphological results assessed by scanning electron microscopy showed a mesh of PLLA/PCL fibers (random and perfectly aligned) associated with aggregates of nanophased HA. Fourier transform infrared spectrometry confirmed the homogeneity in the blends and the presence of nanoHA in the scaffold. As a result of fiber alignment a 15-fold increase in Young's Modulus and an 8-fold increase in tensile strength were observed when compared to non-aligned fibers. In PLLA/PCL/HA scaffolds, the introduction of nanoHA caused a remarkable improvement of the mechanical strength of this material acting as a reinforcement, enhancing the response of these constructs to tensile stress. In vitro testing was evaluated using osteoblast (MC3T3-E1) cells. The results showed that both fibrous scaffolds were able to support osteoblast cell adhesion and proliferation and that fiber alignment induced increased cellular metabolic activity. In addition, the adhesion and proliferation of Staphylococcus aureus were evaluated and a lower number of colony forming units (CFUs) was obtained in the scaffolds with aligned fibers.

Spindly and Bub3 expression in oral cancer: Prognostic and therapeutic implications.

OBJECTIVES: Bub3 and Spindly are essential proteins required for the activation and inactivation of the spindle assembly checkpoint, respectively. Here, we explored the clinicopathological significance and the therapeutic potential of the opposing roles of the two proteins in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Bub3 and Spindly expression was evaluated by immunohistochemistry in 62 tissue microarrays from OSCC and by real-time PCR in OSCC cell lines and in normal human oral keratinocytes. The results were analyzed as to their clinicopathological significance. RNA interference-mediated Spindly or Bub3 inhibition was combined with cisplatin treatment, and the effect on the viability of OSCC cells was assessed. RESULTS: Overexpression of Bub3 and Spindly was detected in OSCC patients. High expression of Spindly, Bub3, or both was an independent prognostic indicator for cancer-specific survival and was associated with increased cellular proliferation. Accordingly, Bub3 and Spindly were upregulated in OSCC cells comparatively to their normal counterpart. Inhibition of Bub3 or Spindly was cytotoxic to OSCC cells and enhanced their chemosensitivity to cisplatin. CONCLUSIONS: The data point out Bub3 and Spindly as potential markers of proliferation and prognosis, and highlight the potential therapeutic benefit of combining their inhibition with cisplatin.

Sterile and Dual-Porous Aerogels Scaffolds Obtained through a Multistep Supercritical CO2-Based Approach.

Aerogels from natural polymers are endowed with attractive textural and biological properties for biomedical applications due to their high open mesoporosity, low density, and reduced toxicity. Nevertheless, the lack of macroporosity in the aerogel structure and of a sterilization method suitable for these materials restrict their use for regenerative medicine purposes and prompt the research on getting ready-to-implant dual (macro + meso)porous aerogels. In this work, zein, a family of proteins present in materials for tissue engineering, was evaluated as a sacrificial porogen to obtain macroporous starch aerogels. This approach was particularly advantageous since it could be integrated in the conventional aerogel processing method without extra leaching steps. Physicochemical, morphological, and mechanical characterization were performed to study the effect of porogen zein at various proportions (0:1, 1:2, and 1:1 zein:starch weight ratio) on the properties of the obtained starch-based aerogels. From a forward-looking perspective for its clinical application, a supercritical CO2 sterilization treatment was implemented for these aerogels. The sterilization efficacy and the influence of the treatment on the aerogel final properties were evaluated mainly in terms of absence of microbial growth, cytocompatibility, as well as physicochemical, structural, and mechanical modifications.

Behavior of prostate cancer cells in a nanohydroxyapatite/collagen bone scaffold.

Prostate cancer (PCa) is the second leading cause of death among men in Europe and U.S. The metastatic dissemination pattern of PCa is unique, developing bone metastasis as the only site of progression, consequently with a prognosis very poor. The cancer cells interactions within the surrounding bone environment are critical for tumor growth and progression. Secreted protein, acidic and rich in cysteine (SPARC) is described to be involved in PCa cells migration and invasion into bone. Three-dimensional (3D) in vitro systems that are able to closely resemble the in vivo microenvironment are recently taking importance in cancer research. Original nanohydroxyapatite/collagen scaffolds were designed to resemble bone microenvironment in order to be applied as substitutes in bone defects and as potential biomaterials to mimic skeletal tumors. In fact, these 3D structures were cytocompatible and able to support osteoblast (MC3T3-E1) colonization and to promote bone ingrowth. Additionally, SPARC adsorption onto the scaffolds affected PC3 and LNCaP PCa cell lines behavior. PC3 cells were found to adapt and colonize the scaffolds, differing from LNCaP where cells underwent morphogenic changes and grew as clusters. Furthermore, for the tested SPARC concentration, SPARC plays a role in retaining LNCaP cells at the latter time points while with PC3 cells no significant differences were observed. This characterization study is required to establish a bone model to provide new insights into the poorly understood PCa mechanisms of metastasis to bone and the generation of improved therapies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2035-2046, 2017.

Suppression of spindly delays mitotic exit and exacerbates cell death response of cancer cells treated with low doses of paclitaxel.

Microtubule-targeting agents (MTAs) are used extensively for the treatment of diverse types of cancer. They block cancer cells in mitosis through the activation of the spindle assembly checkpoint (SAC), the surveillance mechanism that ensures accurate chromosome segregation at the onset of anaphase. However, the cytotoxic activity of MTAs is limited by premature mitotic exit (mitotic slippage) due to SAC silencing. Here we have explored the dual role of the protein Spindly in chromosome attachments and SAC silencing to analyze the consequences of its depletion on the viability of tumor cells treated with clinically relevant doses of paclitaxel. As expected, siRNA-mediated Spindly suppression induced chromosome misalignment and accumulation of cells in mitosis. Remarkably, these cells were more sensitive to low-doses of paclitaxel. Sensitization was due to an increase in the length of mitotic arrest and high frequency of multinucleated cells, both correlated with an exacerbated post-mitotic cell death response as determined by cell fate profiling. Thus, by affecting both SAC silencing and chromosome attachment, Spindly targeting offers a double-edged sword that potentiates tumor cell killing by clinically relevant doses of paclitaxel, providing a rationale for combination chemotherapy against cancer.

Comprehensive Analysis of Secreted Protein, Acidic and Rich in Cysteine in Prostate Carcinogenesis: Development of a 3D Nanostructured Bone-Like Model.

Most aggressive prostate cancer (PCa) types tend to metastasize frequently to bone and SPARC, a matricellular protein, might participate in such biological processes. The objective of this study was to evaluate the effect of SPARC in prostate carcinogenesis and bone metastization. This was explored assessing the morphology, metabolic activity and SPARC expression of different PCa cell lines resembling different stages of carcinogenesis, using a 3D bone-biomimetic model (collagen nanofibers/nanohydroxyapatite) grafted with SPARC. Our findings highlight distinct cellular behavior depending on cell type and presence of exogenous SPARC. In fact, SPARC addition contributed to the survival and significant growth of a non-bone metastatic PCa cell line (LNCaP) on bone-like biomaterial. Moreover, SPARC expression levels were evaluated in a series of prostatic tissues, comparing normal prostate, pre-neoplastic prostate intraepithelial neoplasias and overtly malignant tumors, and also metastasis to its correspondent primary prostate tumors, ascertaining potential association between SPARC and clinicopathological data. Remarkably, SPARC was overexpressed in patients with higher Gleason Score, indicating tumors with poor prognosis, as well as in metastasis, particularly from bone sites, compared with their respective primary tumors. The results suggest a potential role of SPARC as a clinical target on PCa, due to its association with bone metastization.

A biocomposite of collagen nanofibers and nanohydroxyapatite for bone regeneration.

This work aims to design a synthetic construct that mimics the natural bone extracellular matrix through innovative approaches based on simultaneous type I collagen electrospinning and nanophased hydroxyapatite (nanoHA) electrospraying using non-denaturating conditions and non-toxic reagents. The morphological results, assessed using scanning electron microscopy and atomic force microscopy (AFM), showed a mesh of collagen nanofibers embedded with crystals of HA with fiber diameters within the nanometer range (30 nm), thus significantly lower than those reported in the literature, over 200 nm. The mechanical properties, assessed by nanoindentation using AFM, exhibited elastic moduli between 0.3 and 2 GPa. Fourier transformed infrared spectrometry confirmed the collagenous integrity as well as the presence of nanoHA in the composite. The network architecture allows cell access to both collagen nanofibers and HA crystals as in the natural bone environment. The inclusion of nanoHA agglomerates by electrospraying in type I collagen nanofibers improved the adhesion and metabolic activity of MC3T3-E1 osteoblasts. This new nanostructured collagen-nanoHA composite holds great potential for healing bone defects or as a functional membrane for guided bone tissue regeneration and in treating bone diseases.

Role of SPARC in bone remodeling and cancer-related bone metastasis.

There is a growing socioeconomic recognition that clinical bone diseases such as bone infections, bone tumors and osteoporotic bone loss mainly associated with ageing, are major issues in today's society. SPARC (secreted protein, acidic and rich in cysteine), a matricellular glycoprotein, may be a promising therapeutic target for preventing or treating bone-related diseases. In fact, SPARC is associated with tissue remodeling, repair, development, cell turnover, bone mineralization and may also participate in growth and progression of tumors, namely cancer-related bone metastasis. Yet, the function of SPARC in such biological processes is poorly understood and controversial. The main objective of this work is to review the current knowledge related to the activity of SPARC in bone remodeling, tumorigenesis, and bone metastasis. Progress in understanding SPARC biology may provide novel strategies for bone regeneration and the development of anti-angiogenic, anti-proliferative, or counter-adhesive treatments specifically against bone metastasis.

Comparação de técnicas não invasivas para medir a pressão arterial em recém-nascidos / Comparison of noninvasive techniques to measure blood pressure in newborns

OBJETIVO: Comparar a pressão arterial pelos métodos do flush, da oximetria de pulso e da oscilometria utilizando o Doppler em neonatos. MÉTODOS: Foram realizadas medidas de pressão arterial não invasiva utilizando três métodos distintos (flush, oximetria de pulso e oscilometria automática) em três grupos de recém-nascidos selecionados por sorteio: 15 a termo e normais, 16 prematuros estáveis e 14 doentes. Todas as medidas foram filmadas, editadas separadamente, codificadas e analisadas independentemente por três neonatologistas. RESULTADOS: Realizaram-se 57 medidas por cada método. Os métodos do flush e da oximetria de pulso mostraram melhor correlação com o Doppler do que a oscilometria (coeficiente de correlação 0,89, 0,85 e 0,71, respectivamente, p < 0,01). A diferença entre as médias das medidas, seus respectivos desvios padrão e o intervalo de confiança de 95 por cento quando comparados com Doppler foram: -5,2±7,9 (-21,1:10,7) mmHg com o método do flush; 0,4±8,9 (-17,5:18,2) mmHg com a oximetria de pulso; e 6,4±16,1 (-25,8:8,6) mmHg com a oscilometria. O método do flush mostrou melhor concordância com o Doppler para diagnóstico de hipotensão do que os métodos da oximetria e da oscilometria. CONCLUSÕES: Os métodos do flush e da oximetria de pulso mostraram-se úteis para medir a pressão arterial sistólica de recém-nascidos, sendo que o método oscilométrico mostrou-se o menos concordante com o Doppler para detectar hipotensão.

OBJECTIVE: To compare blood pressure measurements in newborn infants using the flush method, pulse oximetry and oscillometry according to Doppler findings. METHODS: Noninvasive blood pressure measurements were made using three techniques (flush method, pulse oximetry and oscillometry) in three groups of newborns: 15 normal term infants, 16 stable preterm infants, and 14 critically ill infants. All measurements were video recorded, edited separately, coded and analyzed independently by three neonatologists. RESULTS: Fifty-seven measurements were made using each method. The flush method and pulse oximetry had a better correlation with Doppler findings than oscillometry (correlation coefficients: 0.89, 0.85, 0.71; p < 0.01). The difference between measurement means, their standard deviations and the 95 percent confidence intervals when compared with Doppler findings were: -5.2±7.9 (-21.1:10.7) mmHg for the flush method; 0.4±8.9 (-17.5:18.2) mmHg for pulse oximetry; and 6.4±16.1 (-25.8:8.6) mmHg for oscillometry. The flush method had a better agreement with Doppler findings for the diagnosis of hypotension than oximetry and oscillometry. CONCLUSIONS: The flush method and pulse oximetry seem to be useful techniques to measure systolic blood pressure in newborn infants; oscillometry had the poorest agreement with Doppler findings to detect hypotension.

Comparison of noninvasive techniques to measure blood pressure in newborns.

OBJECTIVE: To compare blood pressure measurements in newborn infants using the flush method, pulse oximetry and oscillometry according to Doppler findings. METHODS: Noninvasive blood pressure measurements were made using three techniques (flush method, pulse oximetry and oscillometry) in three groups of newborns: 15 normal term infants, 16 stable preterm infants, and 14 critically ill infants. All measurements were video recorded, edited separately, coded and analyzed independently by three neonatologists. RESULTS: Fifty-seven measurements were made using each method. The flush method and pulse oximetry had a better correlation with Doppler findings than oscillometry (correlation coefficients: 0.89, 0.85, 0.71; p < 0.01). The difference between measurement means, their standard deviations and the 95% confidence intervals when compared with Doppler findings were: -5.2 ± 7.9 (-21.1:10.7) mmHg for the flush method; 0.4 ± 8.9 (-17.5:18.2) mmHg for pulse oximetry; and 6.4 ± 16.1 (-25.8:8.6) mmHg for oscillometry. The flush method had a better agreement with Doppler findings for the diagnosis of hypotension than oximetry and oscillometry. CONCLUSIONS: The flush method and pulse oximetry seem to be useful techniques to measure systolic blood pressure in newborn infants; oscillometry had the poorest agreement with Doppler findings to detect hypotension.

[Breastfeeding and early childhood caries: a critical review]. / Aleitamento materno e cárie do lactente e do pré-escolar: uma revisão crítica.

OBJECTIVE: To find scientific evidence that can prove or refute the assumption that nocturnal and on demand breastfeeding are associated with caries in infants and preschool children. SOURCES OF DATA: MEDLINE, Lilacs, and SciELO articles were searched, as well as important internet sites, technical books and consensus publications of national and international organisms. The following keywords were used: "early childhood caries", "dental caries", "dental decay" and "breastfeeding". References cited in the articles selected were also included. SUMMARY OF THE FINDINGS: Studies associating caries with breastfeeding invariably observe factors associated with how this disease develops, letting aside those associated with breastfeeding. Many of these factors act as confusing variables because in the same way as they interfere in breastfeeding, they also influence the development of caries. Besides, current studies have already demonstrated the cariogenic potential of some types of aliments given to children against the non-cariogenic potential of the human milk. CONCLUSIONS: There is no scientific evidence proving that human milk can be associated with the development of caries. This is a complex relation to be established, as it is often blurred by too many variables.

Aleitamento materno e cárie do lactente e do pré-escolar: uma revisão crítica / Breastfeeding and early childhood caries: a critical review

Objetivo: Buscar evidências científicas que comprovem ou refutem a afirmação de que o aleitamento materno noturno e em livre demanda está associado com cárie do lactente e do pré-escolar. Fontes dos dados: Foi realizada busca de artigos científicos utilizando-se as bases de dados MEDLINE, Lilacs e SciELO, páginas de internet relevantes, livros técnicos e publicações de consenso de organismos nacionais e internacionais. As palavras-chave utilizadas foram: early childhood caries, dental caries, dental decay e breastfeeding. Percebida a relevância, também se buscou diretamente as referências indicadas nos artigos encontrados. Síntese dos dados: Os estudos que relacionam a cárie com o aleitamento materno invariavelmente só observam os fatores inter-relacionados com o surgimento dessa doença, deixando de lado aqueles associados à amamentação. Muitos desses fatores atuam como variáveis de confusão porque, do mesmo modo que interferem no aleitamento materno, também têm influência no surgimento da cárie. Além disso, estudos atuais têm demonstrado a cariogenicidade de vários alimentos dados às crianças e a não-cariogenicidade do leite materno. Conclusões: Não há evidências científicas que comprovem que o leite materno possa estar associado com o surgimento de cárie, sendo essa relação complexa e confundida por muitas variáveis.

Cirurgia orovalvar na criança: experiência de 135 casos / Valvular surgery in children: experience with 135 cases

Foram analisados 135 pacientes levados a cirurgia do aparelho valvar, com idade inferior a quinze anos, no período de 11 anos (fevereiro de 1975 a fevereiro de 1986). Os pacientes foram divididos em dois grupos, de acordo com o tipo de cirurgia realizada. No grupo I, formado por 60 crianças, foi preservado o aparelho valvar por técnicas cirúrgicas reparadoras da anatomia e funçäo valvar. Tivemos uma mortalidade cirúrgica de 7%, 34% estäo sem seguimento. Dos pacientes acompanhados, 72% encontram-se assintomáticos, ou pouco sintomáticos; 2 pacientes (5%) foram a óbito tardio e 22% foram reoperados para implante de prótese. No grupo II, 75 pacientes foram levados a cirurgia, para implante de prótese. A mortalidade hospitalar foi de 12% e 13 pacientes (20%) estäo sem seguimento. Dos pacientes acompanhados, 71% estäo assintomáticos, ou pouco sintomáticos; 4% voltaram a apresentar sintomas; 13% (7 pacientes) foram a óbito tardio e 23% (12 pacientes) foram reoperados. A análise mais detalhada dos dois grupos, as mortalidades hospitalar e tardia nos induzem a concluir que se deve fazer todo o empenho na preservaçäo do aparelho valvar lesado. O baixo nível sócio-econômico, responsável pela baixa faixa etária em que säo atingidos os pacientes em suas lesöes valvares, é também responsável pela extrema dificuldade em se manter o acompanhamento clínico pós-operatório