Electrochemical quartz crystal microbalance with dissipation investigation of fibronectin adsorption dynamics driven by electrical stimulation onto a conducting and partially biodegradable copolymer.

Functional surface coatings are a key option for biomedical applications, from polymeric supports for tissue engineering to smart matrices for controlled drug delivery. Therefore, the synthesis of new materials for biological applications and developments is promising. Hence, biocompatible and stimuli-responsive polymers are interesting materials, especially when they present conductive properties. PEDOT-co-PDLLA graft copolymer exhibits physicochemical and mechanical characteristics required for biomedical purposes, associated with electroactive, biocompatible, and partially biodegradable properties. Herein, the study of fibronectin (FN) adsorption onto PEDOT-co-PDLLA carried out by an electrochemical quartz crystal microbalance with dissipation is reported. The amount of FN adsorbed onto PEDOT-co-PDLLA was higher than that adsorbed onto the Au surface, with a significant increase when electrical stimulation was applied (either at +0.5 or -0.125 V). Additionally, FN binds to the copolymer interface in an unfolded conformation, which can promote better NIH-3T3 fibroblast cell adhesion and later cell development.

The Worldwide Graphene Flake Production.

There are hundreds of companies worldwide claiming to produce "graphene," showing a large variation in its properties. A systematic and reliable protocol is developed to test graphene quality using electron microscopy, atomic force microscopy, Raman spectroscopy, elemental analysis, X-ray photoelectron spectrometry, and scanning and transmission electron microscopy, which is used to study graphene from 60 producers. The statistical nature of the liquid-phase exfoliation of graphite is established. It is shown that the current classification of graphene flakes used in the market is erroneous. A new classification is proposed in terms of distribution functions for number of layers and flake size. It is shown unequivocally that the quality of the graphene produced in the world today is rather poor, not optimal for most applications, and most companies are producing graphite microplatelets. This is possibly the main reason for the slow development of graphene applications, which usually require a customized solution in terms of graphene properties. It is argued that the creation of stringent standards for graphene characterization and production, taking into account both the physical properties, as well as the requirements from the particular application, is the only way forward to create a healthy and reliable worldwide graphene market.

CVD-grown monolayer graphene induces osteogenic but not odontoblastic differentiation of dental pulp stem cells.

OBJECTIVE: The objective was to investigate the potential of graphene (Gp) to induce odontogenic and osteogenic differentiation in dental pulp stem cells (DPSC). METHODS: Gp was produced by chemical vapor deposition. DPSC were seeded on Gp or glass (Gl). Cells were maintained in culture medium for 28 days. Every two days, culture medium from Gp was used to treat cells on Gl and vice versa. Mineralization and differentiation of DPSC on all substrates were evaluated after 14 and 28 days by alizarin red S staining, qPCR, immunofluorescence and FACS. Statistics were performed with two-way ANOVA and multiple comparisons were performed using Tukey's post hoc test at a pre-set significance level of 5%. RESULTS: After 14 and 28 days, Gp induced higher levels of mineralization as compared to Gl. Odontoblastic genes (MSX-1, PAX and DMP) were down-regulated and osteogenic genes and proteins (RUNX2, COL and OCN) were significantly upregulated on Gp comparing to Gl (p<0.05 for all cases). Medium from Gp induced downregulation of odontoblastic genes and increased bone-related gene and protein on Gl. SIGNIFICANCE: Graphene induced osteogenic and not odontoblastic differentiation of DPSC without the use of chemical inducers for osteogenesis. Graphene has the potential to be used as a substrate for craniofacial bone tissue engineering research.

The performance of bone tissue engineering scaffolds in in vivo animal models: A systematic review.

Bone tissue engineering is an excellent alternative for the regeneration of large bone defects caused by trauma or bone pathologies. Scaffolds, stem cells, and bioactive molecules are the three key components of bone regeneration. Although a wide range of biomaterials of various compositions and structures has been proposed in the literature, these materials are rarely used in clinical applications. Therefore, more standardized studies are required to design scaffolds that enable better bone regeneration and are suitable for clinical use. The aim of this systematic review was to compare the performance of scaffolds used in preclinical animal studies to determine which class of materials has achieved a higher rate of bone neoformation (osteoinduction and osteoconduction). The selected studies were divided into three groups according to the following experimental models: studies that used subcutaneous models, bone defects in calvaria, and bone defects in long bones. Despite the large number of parameters in the included studies, we generally concluded that biomaterials containing calcium phosphates had important osteoinductive effects and were essential for better performance of the materials. Furthermore, natural polymers generally had better performance than synthetic polymers did, especially when the materials were associated with stem cells. The combination of materials from different classes was the most promising strategy for bone tissue regeneration.

Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses.

Regeneration of periodontal tissues requires a concerted effort to obtain consistent and predictable results in vivo. The aim of the present study was to test a new family of bioactive polymeric membranes in combination with stem cell therapy for periodontal regeneration. In particular, the novel polyester poly(isosorbide succinate-co-L-lactide) (PisPLLA) was compared with poly(L-lactide) (PLLA). Both polymers were combined with collagen (COL), hydroxyapatite (HA) and the growth factor bone morphogenetic protein-7 (BMP7), and their osteoinductive capacity was evaluated via in vitro and in vivo experiments. Membranes composed of PLLA/COL/HA or PisPLLA/COL/HA were able to promote periodontal regeneration and new bone formation in fenestration defects in rat jaws. According to quantitative real-time polymerase chain reaction (qRT-PCR) and Alizarin Red assays, better osteoconductive capacity and increased extracellular mineralization were observed for PLLA/COL/HA, whereas better osteoinductive properties were associated with PisPLLA/COL/HA. We concluded that membranes composed of either PisPLLA/COL/HA or PLLA/COL/HA present promising results in vitro as well as in vivo and that these materials could be potentially applied in periodontal regeneration.

Graphene: A Versatile Carbon-Based Material for Bone Tissue Engineering.

The development of materials and strategies that can influence stem cell attachment, proliferation, and differentiation towards osteoblasts is of high interest to promote faster healing and reconstructions of large bone defects. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have received increasing attention for biomedical applications as they present remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. These biocompatible carbon-based materials can induce and sustain stem cell growth and differentiation into various lineages. Furthermore, graphene has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research. This paper will review the important advances in the ability of graphene and its related forms to induce stem cells differentiation into osteogenic lineages.

Hybrid Membranes of PLLA/Collagen for Bone Tissue Engineering: A Comparative Study of Scaffold Production Techniques for Optimal Mechanical Properties and Osteoinduction Ability.

Synthetic and natural polymer association is a promising tool in tissue engineering. The aim of this study was to compare five methodologies for producing hybrid scaffolds for cell culture using poly-l-lactide (PLLA) and collagen: functionalization of PLLA electrospun by (1) dialkylamine and collagen immobilization with glutaraldehyde and by (2) hydrolysis and collagen immobilization with carbodiimide chemistry; (3) co-electrospinning of PLLA/chloroform and collagen/hexafluoropropanol (HFP) solutions; (4) co-electrospinning of PLLA/chloroform and collagen/acetic acid solutions and (5) electrospinning of a co-solution of PLLA and collagen using HFP. These materials were evaluated based on their morphology, mechanical properties, ability to induce cell proliferation and alkaline phosphatase activity upon submission of mesenchymal stem cells to basal or osteoblastic differentiation medium (ODM). Methods (1) and (2) resulted in a decrease in mechanical properties, whereas methods (3), (4) and (5) resulted in materials of higher tensile strength and osteogenic differentiation. Materials yielded by methods (2), (3) and (5) promoted osteoinduction even in the absence of ODM. The results indicate that the scaffold based on the PLLA/collagen blend exhibited optimal mechanical properties and the highest capacity for osteodifferentiation and was the best choice for collagen incorporation into PLLA in bone repair applications.

Synthesis and characterization of xanthan-hydroxyapatite nanocomposites for cellular uptake.

In this work xanthan-nanohydroxyapatite (XnHAp) and its equivalent strontium substituted (XnHApSr) were synthesized by the precipitation of nanohydroxyapatite in xanthan aqueous solution, characterized and compared to conventional hydroxyapatite particles (HAp). XnHAp and XnHApSr were less crystalline than HAp, as revealed by X-ray diffraction. Xanthan chains enriched the surface of XnHAp and XnHApSr particles, increasing the zeta potential values from -(7±1)mV, determined for HAp, to -(17±3)mV and -(25±3)mV, respectively. This effect led to high colloidal stability of XnHAp and XnHApSr dispersions and acicular particles (140±10)nm long and (8±2)nm wide, as determined by scanning electron microscopy and atomic force microscopy. XnHAp and XnHApSr particles were added to xanthan hydrogels to produce compatible nanocomposites (XCA/XnHAp and XCA/XnHApSr). Dried nanocomposites presented surface energy, Young's modulus and stress at break values comparable to those determined for bare xanthan matrix. Moreover, adding XnHAp or XnHApSr nanoparticles to xanthan hydrogel did not influence its porous morphology, gel content and swelling ratio. XCA/XnHAp and XCA/XnHApSr composites proved to be suitable for osteoblast growth and particularly XCA/XnHapSr composites induced higher alkaline phosphatase activity.

Synthesis and swelling behavior of xanthan-based hydrogels.

In this work xanthan chains were crosslinked by esterification reaction at 165 °C either in the absence or in the presence of citric acid. Higher crosslinking density was obtained using citric acid, as evidenced by its lower swelling degree. Tensiometry, a very precise and sensitive technique, was applied to study swelling rates and diffusion mechanisms of water, which was initially quasi-Fickian, controlled by wicking properties, changing to Fickian or Anomalous, depending on hydrogel composition. Hydrogels swelling degree increased at high pH values, due to electrostatic repulsion and ester linkages rupture. Equilibrium swelling degree was affected by salts, depending on gel composition and kind of salt. Effects could be explained by interaction between ions and polymeric chains, EPA/EPD ability of water or osmotic gradient.

Produção de suportes poliméricos para o crescimento de células-tronco mesenquimais e sua aplicação em regeneração óssea / Polymer scaffolds production for stem cell growing and their application in bone regeneration

Um novo método de modificação da superfície de nanopartículas de hidroxiapatita (HAP) por reação com cloreto de lauroíla foi desenvolvido, gerando a nanopartícula funcionalizada por laurato (HAP-CL). A superfície modificada da HAP foi confirmada por infravermelho, termogravimetria, ressonância magnética nuclear e análise elementar. Provamos por testes mecânicos a capacidade de criar compósitos com alto teor de HAP-CL em matrizes poliméricas de poli(L-acido láctico) (PLLA) e poli(succinato de isosorbídeo-b-L-lactídeo) (PLLA-co-PIS) sem perda significativa de propriedades mecânicas. Diferentes quantidades de HAP-CL, HAP enxertado com PLLA (PLLA-g-HAP) e HAP puro foram dispersadas em soluções de PLLA para formar fibras eletrofiadas. Para comparar a dispersão destas nanopartículas nas fibras e a sua morfologia, análise de microscopia eletrônica de varredura e de transmissão foram empregadas. A HAP-CL exibiu melhor dispersão na matriz polimérica do que o PLLA-g-HAP e HAP, e permitiu a produção de fibras com grande quantidade de HAP-CL (até 30% massa da fase mineral em relação à massa do polímero(mm/mp)), tanto para o PLLA como para o PLLA-co-PIS. Células tronco mesenquimais derivadas de polpa de dente foram cultivadas em fibras de PLLA com alto teor de HAP-CL, resultando em um aumento significativo da atividade de fosfatase alcalina (ALP), nos dias 14 e 21 (p <0,001) quando comparados com conteúdos mais baixos de HAP-CL, assim como um melhor processo de mineralização apresentado pelos teste de vermelho de alizarina depois de 21 dias (p <0,001). As células cultivadas nas fibras de PLLA-co-PIS contendo 30% de HAP-CL (mm/mp) apresentaram maior atividade de ALP após 21 dias (p <0,05), e melhor processo de mineralização, depois de 14 e 21 dias (p <0,05) do que as fibras de PLLA com 30% de HAP-CL (mm/mp). PLLA e PLLA-co-PIS, ambos contendo 30% de HAP-CL (mm/mp) induziram uma maior expressão de osteocalcina e osteopontina, dois marcadores de diferenciação osteoblástica, quando comparados ao PLLA e PLLA-co-PIS (controle). Finalmente, em experimentos in vivo, as fibras de PLLA-co-PIS contendo 30% de HAP-CL (mm/mp) apresentaram um desempenho superior no processo de neoformação óssea do que as fibras de PLLA com o mesmo conteúdo de nanopartículas. Em conclusão, os nossos resultados in vitro demonstraram que os suportes construídos de compósitos de PLLA-co-PIS contendo 30% de HAP-CL (mm/mp) se mostraram superiores tanto na adesão e proliferação quanto na diferenciação de células mesenquimais de polpa de dente em osteoblastos. Além disso, experimentos in vivo confirmaram estes resultados, demonstrando que estes nanocompósitos são excelentes modelos para implantes destinados a regeneração óssea

A new method of surface modification of hydroxyapatite nanoparticles (HAP) by reaction with lauroyl chloride was developed, producing the laurate functionalized nanoparticle (HAP-CL). The surface modified HAP was confirmed by infrared, thermogravimetric analysis, nuclear magnetic resonance and elemental analysis. We proved by mechanical tests the ability to create composites with high HAP-CL content in poly(L-lactic acid) (PLLA) and poly(isosorbide succinate-b-L-lactide) (PLLA-co-PIS) polymeric matrixes without significant loss of mechanical properties. Different amounts of HAP-CL, HAP grafted with PLLA (PLLA-g-HAP) and HAP were dispersed in pure PLLA solutions to form nanofibers. To compare the dispersion of these nanoparticles in the fibers and their morphology, scanning and transmission electron microscopies were employed. HAP-CL showed better dispersion in the polymer matrix than the PLLA-g-HAP and HAP, and allowed fiber production with large amounts of HAP-CL (up to 30% mineral to polymer weight (wm/wp)) for both PLLA and PLLA-co-PIS. Mesenchymal stem cells derived from dental pulp were cultured in PLLA fibers with high levels of HAP-CL, resulting in a significant increase in alkaline phosphatase activity (ALP), on days 14 and 21 (p <0.001) as compared to those with lower content HAP-CL, as well as a better mineralization process shown by alizarin red test after 21 days (p <0.001). Cells grown in PLLA-co-PIS fibers containing 30% of HAP-CL showed higher ALP activity after 21 days (p <0.05) and a better mineralization process, after 14 and 21 days (p <0.05 ) than fibers of PLLA with 30% HAP-CL. PLLA and PLLA-co-PIS, both containing 30% of HAP-CL (wm/wp) induced a higher expression of osteocalcin and osteopontin, two osteoblast differentiation markers when compared with PLLA and PLLA-co-PIS (control). Finally, in the in vivo experiments, the PLLA-co-PIS fibers containing 30% HAP-CL (wm/wp) outperformed the process of bone formation than PLLA fibers with the same content of nanoparticles. In conclusion, our in vitro results demonstrated that scaffolds from composites of PLLA-co-PIS containing 30% HAP-CL (wm/wp) were superior both in adhesion and in the differentiation and proliferation of dental pulp stem cells in osteoblasts. Furthermore, in vivo experiments confirmed these results, demonstrating that these nanocomposites are excellent models for implants for bone regeneration