New Nano-Crystalline Hydroxyapatite-Polycarboxy/Sulfo Betaine Hybrid Materials: Synthesis and Characterization.

Hybrid materials based on calcium phosphates and synthetic polymers can potentially be used for caries protection due to their similarity to hard tissues in terms of composition, structure and a number of properties. This study is focused on the biomimetic synthesis of hybrid materials consisting of hydroxiapatite and the zwitterionic polymers polysulfobetaine (PSB) and polycarboxybetaine (PCB) using controlled media conditions with a constant pH of 8.0-8.2 and Ca/P = 1.67. The results show that pH control is a dominant factor in the crystal phase formation, so nano-crystalline hydroxyapatite with a Ca/P ratio of 1.63-1.71 was observed as the mineral phase in all the materials prepared. The final polymer content measured for the synthesized hybrid materials was 48-52%. The polymer type affects the final microstructure, and the mineral particle size is thinner and smaller in the synthesis performed using PCB than using PSB. The final intermolecular interaction of the nano-crystallized hydroxyapatite was demonstrated to be stronger with PCB than with PSB as shown by our IR and Raman spectroscopy analyses. The higher remineralization potential of the PCB-containing synthesized material was demonstrated by in vitro testing using artificial saliva.

Sol-Gel Technologies to Obtain Advanced Bioceramics for Dental Therapeutics.

The aim of this work is to review the application of bioceramic materials in the context of current regenerative dentistry therapies, focusing on the latest advances in the synthesis of advanced materials using the sol-gel methodology. Chemical synthesis, processing and therapeutic possibilities are discussed in a structured way, according to the three main types of ceramic materials used in regenerative dentistry: bioactive glasses and glass ceramics, calcium phosphates and calcium silicates. The morphology and chemical composition of these bioceramics play a crucial role in their biological properties and effectiveness in dental therapeutics. The goal is to understand their chemical, surface, mechanical and biological properties better and develop strategies to control their pore structure, shape, size and compositions. Over the past decades, bioceramic materials have provided excellent results in a wide variety of clinical applications related to hard tissue repair and regeneration. Characteristics, such as their similarity to the chemical composition of the mineral phase of bones and teeth, as well as the possibilities offered by the advances in nanotechnology, are driving the development of new biomimetic materials that are required in regenerative dentistry. The sol-gel technique is a method for producing synthetic bioceramics with high purity and homogeneity at the molecular scale and to control the surfaces, interfaces and porosity at the nanometric scale. The intrinsic nanoporosity of materials produced by the sol-gel technique correlates with the high specific surface area, reactivity and bioactivity of advanced bioceramics.

Sol-Gel Synthesis of Endodontic Cements: Post-Synthesis Treatment to Improve Setting Performance and Bioactivity.

The sol-gel process is a wet chemical technique that allows very fine control of the composition, microstructure, and final textural properties of materials, and has great potential for the synthesis of endodontic cements with improved properties. In this work, the influence of different sol-gel synthesis variables on the preparation of endodontic cement based on calcium silicate with Ca/Si stoichiometry equal to 3 was studied. Starting from the most optimal hydraulic composition selected, a novel second post-synthesis treatment using ethanol was essayed. The effects of the tested variables were analyzed by X-ray diffraction, infrared spectroscopy, scanning electron microscopy, nitrogen physisorption, and Gillmore needles to determine the setting time and simulated body fluid (SBF) immersion to measure the bioactive response in vitro. The results indicated that the sol-gel technique is effective in obtaining bioactive endodontic cements (BECs) with high content of the hydraulic compound tricalcium silicate (C3S) in its triclinic polymorph. The implementation of a novel post-synthesis treatment at room temperature using ethanol allows obtaining a final BEC product with a finer particle size and a higher CaCO3 content, which results in an improved material in terms of setting time and bioactive response.

Mesoporous Silica-Based Nanoparticles as Non-Viral Gene Delivery Platform for Treating Retinitis Pigmentosa.

BACKGROUND: Gene therapy is a therapeutic possibility for retinitis pigmentosa (RP), in which therapeutic transgenes are currently delivered to the retina by adeno-associated viral vectors (AAVs). Although their safety and efficacy have been demonstrated in both clinical and preclinical settings, AAVs present some technical handicaps, such as limited cargo capacity and possible immunogenicity in repetitive doses. The development of alternative, non-viral delivery platforms like nanoparticles is of great interest to extend the application of gene therapy for RP. METHODS: Amino-functionalized mesoporous silica-based nanoparticles (N-MSiNPs) were synthesized, physico-chemically characterized, and evaluated as gene delivery systems for human cells in vitro and for retinal cells in vivo. Transgene expression was evaluated by WB and immunofluorescence. The safety evaluation of mice subjected to subretinal injection was assessed by ophthalmological tests (electroretinogram, funduscopy, tomography, and optokinetic test). RESULTS: N-MSiNPs delivered transgenes to human cells in vitro and to retinal cells in vivo. No adverse effects were detected for the integrity of the retinal tissue or the visual function of treated eyes. N-MSiNPs were able to deliver a therapeutic transgene candidate for RP, PRPF31, both in vitro and in vivo. CONCLUSIONS: N-MSiNPs are safe for retinal delivery and thus a potential alternative to viral vectors.

Nanofibrous Gelatin-Based Biomaterial with Improved Biomimicry Using D-Periodic Self-Assembled Atelocollagen.

Design of bioinspired materials that mimic the extracellular matrix (ECM) at the nanoscale is a challenge in tissue engineering. While nanofibrillar gelatin materials mimic chemical composition and nano-architecture of natural ECM collagen components, it lacks the characteristic D-staggered array (D-periodicity) of 67 nm, which is an important cue in terms of cell recognition and adhesion properties. In this study, a nanofibrous gelatin matrix with improved biomimicry is achieved using a formulation including a minimal content of D-periodic self-assembled atelocollagen. We suggest a processing route approach consisting of the thermally induced phase separation of the gelatin based biopolymeric mixture precursor followed by chemical-free material cross-linking. The matrix nanostructure is characterized using field emission gun scanning electron microscopy (FEG-SEM), transmission electron microscopy (TEM), wide angle X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR). The cell culture assays indicate that incorporation of 2.6 wt.% content of D-periodic atelocollagen to the gelatin material, produces a significant increase of MC3T3-E1 mouse preosteoblast cells attachment and human mesenchymal stem cells (hMSCs) proliferation, in comparison with related bare gelatin matrices. The presented results demonstrate the achievement of an efficient route to produce a cost-effective, compositionally defined and low immunogenic "collagen-like" instructive biomaterial, based on gelatin.

Sponge-like processed D-periodic self-assembled atelocollagen supports bone formation in vivo.

Fibrous biopolymeric collagen extracted from animal tissues has been widely used for fabricating matrices for bone tissue engineering (BTE). However, animal extracted collagens can trigger immune reactions when implanted in vivo and the presence of native crosslinks leads to batch-to-batch variability. Atelocollagen, a monomeric form of collagen, is free of telopeptides, which are mainly responsible for the immunogenicity of collagen, and can self-assemble in vitro to obtain fibrils with the characteristic D-periodic staining pattern of native collagen. However, atelocollagen-based biomaterials have not extensively been studied and, hence, their suitability for BTE remains relatively unexplored. Besides, to stabilize collagen biomaterials, chemical and physical crosslinking are used, although chemical agents are cytotoxic while the physical methods yield a less effective crosslinking. A combination of physical and chemical crosslinking is a suitable alternative that has rarely been tested in BTE programs. In this work, a sponge-like biomaterial (DCol-S) was processed from D-periodic self-assembled atelocollagen and its stabilization was studied using the combination of a dehydrothermal treatment (DHT) and minimal glutaraldehyde (GTA) exposition crosslinking, to increase the resistance to degradation of the scaffold without a major effect on the biomaterial structure. The microstructural features of the final sponges were characterised and compared to a commercial biomaterial processed from native bovine collagen (Helistat®, Integra Lifesciences, NJ, USA), demonstrating that a D-periodic nanostructure was obtained and maintained after processing of the sponges. MC3T3-E1 preosteoblast adhesion, proliferation and differentiation assays in vitro showed that DCol-S is biocompatible. Furthermore, intramuscular implantation of the biomaterials loaded with rhBMP-2 revealed that the double-crosslinked sponges were able to support ectopic bone formation, while sponges stabilised only with the DHT treatment were not. Altogether, these findings show that atelocollagen-based sponges stabilised with a DHT treatment followed by a mild GTA crosslinking are a suitable alternative to polymeric extracted collagen for BTE applications.

Nanofibrous Matrix of Defined Composition Sustains Human Induced Pluripotent Stem Cell Culture.

Human induced pluripotent stem cells (hiPSCs) represent the most promising biological material for regenerative medicine applications. In this work, a 3D solid nanofibrous matrix of defined composition (Colamigel-S) consisting of 97 wt % gelatin, 2.6 wt % atelocollagen, and 0.4 wt % laminin has been reproducibly processed and characterized and exhibits a homogeneous nanofibrillar network of high surface area, interconnected microcavities, and typical D-periodic collagen fibril nanostructural features. The purpose of the study was to test the performance of Colamigel-S as substrate for in vitro hiPSCs culture, finding that these cells efficiently attach and grow keeping their characteristic stem morphology and undifferentiated state.

A Microstructure Insight of MTA Repair HP of Rapid Setting Capacity and Bioactive Response.

Mineral trioxide aggregate (MTA) is considered a bioactive endodontic material, which promotes natural mineralization at the material-tooth tissue interface. MTA Repair HP stands out because of the short setting time and the quick and effective bioactive response in vitro. The bioactivity, depens on material composition and microstructure. This work is devoted to analyze MTA Repair HP microstructural features, of both the powder precursor and set material, to get insights into the material physicochemical parameters-functionality performance relationships. Transmission electron microscopy (TEM), and field emission gun scanning electron microscopy (FEG-SEM) coupled with energy-dispersive X-ray (EDX) analyses were performed. X-ray diffraction (XRD) measurements were carried out at different times to investigate setting process. Bioactivity evaluation in vitro was carried out by soaking the processed cement disk in simulated body fluid (SBF). The presented results point out those MTA Repair HP precursor material characteristics of tricalcium silicate particles of nanometric size and high aspect ratio, which provide an elevated surface area and maximized components dispersion of calcium silicate and very reactive calcium aluminate. The MTA Repair HP precursor powder nanostructure and formulation, allows a hydration process comprising silicate hydrate structures, which are very effective to achieve both fast setting and efficient bioactive response.

Physicochemical parameters - hydration performance relationship of the new endodontic cement MTA Repair HP.

BACKGROUND: To characterize the chemical composition and textural parameters of the MTA Repair HP precursor powder and their influence to hydration performance. MATERIAL AND METHODS: Un-hydrated precursor material was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), X-ray fluorescence (XRF), laser diffraction (LD), N2 physisorption and field emission gun scanning electron microscopy (FEG-SEM). Setting time was assessed according to ASTM specification C 266. Hydrated material was analysed by XRD, FT-IR, energy dispersive X-ray (EDX) analysis and FEG-SEM. RESULTS: Ca3SiO5 and Ca2SiO4, in addition to CaWO4 as radiopacifier are the main compositional phases. Other measured parameter indicates high specific surface area of 4.8 m2 g-1, high aluminium content of 1.7 wt.% and low initial and final setting times of 12 and 199 min, respectively. Singular microstructural features consisting of high aspect ratio nanoparticles are main constituents of un-hydrated precursor. Besides, FEM-SEM observation shows notably growth of hexagonal shaped plate-like morphologies homogeneously distributed along the sample during hydration process. CONCLUSIONS: The short setting time measured for HP Repair, is correlated with high surface area of precursor powder, high Al content and the absence of compositional sulphate phases. Key words:Bioactive endodontic cements, hydration performance, MTA HP Repair, physicochemical parameters.

MTA HP Repair stimulates in vitro an homogeneous calcium phosphate phase coating deposition.

BACKGROUND: To study the mineralization capacity in vitro of the bioceramic endodontic material MTA HP Repair. MATERIAL AND METHODS: Bioactivity evaluation in vitro was carried out, by soaking processed cement disk in simulated body fluid (SBF) during 168 h. The cement surface was studied by Fourier transform infrared spectroscopy (FT-IR), field emission gun scanning electron microscopy (FEG-SEM) and energy dispersive X-ray analysis (EDX). Release to the SBF media of ionic degradation products was monitored using inductively coupled plasma atomic emission spectroscopy (ICP-AES). RESULTS: FT-IR showed increasing formation of phosphate phase bands at 1097, 960, 607 and 570 cm-1 with prolonged SBF soaking. FEG-SEM analysis reveals that HP produces a effectively surface covering consisting in homogeneous spherical phosphate phase aggregates with an average diameter of 0.5-1.0 µm. EDX analysis comparing un-treated (hydrated), 24 h and 72 h SBF treated surfaces of MTA HP Repair revealed phosphate deposition after 24 h, with high phosphorous/silicon element ratio signal measured after 24 h, indicating a very high phosphate phase deposition for this material. CONCLUSIONS: The study shows that MTA HP Repair produces a quick and effective bioactive response in vitro in terms of crystalline calcium phosphate surface coating formation. The high bioactive response of MTA HP Repair makes it an interesting candidate for endodontic use as repair cement. Key words:Bioactive endodontic cements, bioactive response, MTA HP Repair.

Higher hydration performance and bioactive response of the new endodontic bioactive cement MTA HP repair compared with ProRoot MTA white and NeoMTA plus.

The aim of this study was to characterize the hydration performance and the bioactive response of the new bioactive endodontic cement MTA HP repair (HP), comparing its physicochemical parameters with those of ProRoot MTA White (Pro) and NeoMTA Plus (Neo). Un-hydrated precursor materials were characterized by X-ray fluorescence, laser diffraction, N2 physisorption and field emission gun scanning electron microscopy (FEG-SEM). Setting time was assessed according to ASTM specification C 266. Hydrated materials were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy (FT-IR) and (FEG-SEM). Bioactivity evaluation in vitro was carried out, by soaking processed cement disk in simulated body fluid (SBF) during 168 h. The cements surface was studied by FT-IR, FEG-SEM, and energy dispersive X-ray. Release to the SBF media of ionic degradation products was monitored using inductively coupled plasma atomic emission spectroscopy. HP showed shorter initial setting time compared to Pro and Neo and produce a quick and effective bioactive response in vitro in terms of phosphate phase surface coating formation. This higher bioactive response for HP is correlated with increasing calcium aluminate content, increasing surface area of un-hydrated powder precursor and the increasing release capacity of Si ionic products of the final hydrated product. The higher bioactive response of MTA HP repair highlights this material, as very interesting to further investigate its performance to improve the outcome of vital pulp therapy procedures. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2109-2120, 2019.

Copper-containing mesoporous bioactive glass promotes angiogenesis in an in vivo zebrafish model.

The osteogenic and angiogenic responses of organisms to the ionic products of degradation of bioactive glasses (BGs) are being intensively investigated. The promotion of angiogenesis by copper (Cu) has been known for more than three decades. This element can be incorporated to delivery carriers, such as BGs, and the materials used in biological assays. In this work, Cu-containing mesoporous bioactive glass (MBG) in the SiO2-CaO-P2O5 compositional system was prepared incorporating 5% mol Cu (MBG-5Cu) by replacement of the corresponding amount of Ca. The biological effects of the ionic products of MBG biodegradation were evaluated on a well-known endothelial cell line, the bovine aorta endothelial cells (BAEC), as well as in an in vivo zebrafish (Danio rerio) embryo assay. The results suggest that ionic products of both MBG (Cu free) and MBG-5Cu materials promote angiogenesis. In vitro cell cultures show that the ionic dissolution products of these materials are not toxic and promote BAEC viability and migration. In addition, the in vivo assay indicates that both exposition and microinjection of zebrafish embryos with Cu free MBG material increase vessel number and thickness of the subintestinal venous plexus (SIVP), whereas assays using MBG-5Cu enhance this effect. STATEMENT OF SIGNIFICANCE: Mesoporous bioactive glasses (MBGs) with high specific surface area, well-ordered pores, large pore volumes and controllable amount of ions are interesting to develop controlled drug delivery systems for bone tissue regeneration. Copper (Cu) incorporation to the basic SiO2-CaO-P2O5 composition has attracted high interest due to its multifunctional biological properties. Promotion of angiogenesis is one of these properties, which can be integrated to the biomaterial with lower cost and higher stability when compared with growth factors. This work reports the synthesis and characterization of Cu-containing MBG evaluating its angiogenic properties in the subintestinal vessel zebrafish assay. This transgenic in vivo assay is merging as an alternative model providing short-time consuming protocols and facilities during pro-angiogenic drug screenings. The report shows that the ionic products of this MBG material delivered to the zebrafish incubation media significantly enhance angiogenesis in comparison with control groups. Besides, results indicate Cu ions may exhibit a synergic effect with Si, Ca, and P ions in angiogenesis stimulation both in vitro and in vivo. To our knowledge, this is the first time that zebrafish in vivo assays are used to evaluate angiogenic activity of ionic dissolution products from MBG materials.

Regenerative Endodontic Procedures: A Perspective from Stem Cell Niche Biology.

INTRODUCTION: Endodontics uses cell therapy strategies to treat pulpal and periapical diseases. During these therapies, surgeons aim to reconstruct the natural microenvironments that regulate the activity of dental stem cells. METHODS: We searched for more than 400 articles in PubMed using key words from regenerative endodontics and dental stem cell biology. In 268 articles, we reviewed what factors may influence histologic results after preclinical dental treatments that use regenerative endodontic procedures after pulpectomy. RESULTS: Several factors, such as the origin of stem cells, the biomimicry of scaffolds used, and the size of lesions, are considered to influence the histologic appearance of the regenerated pulp-dentin complex after treatments. Information is accumulating on transcription factors that generate the pulp-dentin complex and survival/trophic factors that would benefit niche recovery and histologic results. CONCLUSIONS: In this article, we discuss the noninterchangeability of stem cells, the influence of dentin-entrapped molecule release on pulp regeneration and survival of stem cells, and the need of positional markers to assess treatments histologically. The ex vivo amplification of appropriate dental stem cells, the search for scaffolds storing the molecular diversity entrapped in the dentin, and the use of positional transcription factors as histologic markers are necessary to improve future preclinical experiments.

Reticulated bioactive scaffolds with improved textural properties for bone tissue engineering: nanostructured surfaces and porosity.

Organised nanoporous SBA-15 type silica precursor (SP) particulate material has been processed into three-dimensional macroporous, reticulated structures using a novel strategy consisting of blending increasing percentages of SP with a SiO2 -CaO-P2 O5 (80Si15Ca5P) mesoporous bioactive glass (MBG) sol. The procedure successfully produced consolidated and functionally competent open-cell scaffolds while preserving the nanoporous order of the SP. Scaffolds were prepared using four different (MBG)/(SP) ratios. These structures were then characterized using field emission gun scanning electron microscopy, X-ray diffraction (XRD), nitrogen adsorption-desorption measurements, and compressive strength testing. Open-cell interconnected structures with dual macro (150-500 µm) and nano (4-6 nm)-organised porosity were produced. Both the textural and mechanical properties were found to improve with increasing SBA-15 content. The in vitro bioactive response using simulated body fluid confirmed high reactivity for all prepared scaffolds. In addition, the SBA-15 containing scaffolds exhibited a superior ability to delay the pH-triggered lysozyme release with antibiotic activity.

Tuning of cell-biomaterial anchorage for tissue regeneration.

Which mechanisms mediate cell attachment to biomaterials? What role does the surface charge or wettability play on cell-material anchorage? What are the currently investigated strategies to modify cell-matrix adherence spatiotemporally? Considering the development of scaffolds made of biocompatible materials to temporarily replace the structure and/or function of the extracellular matrix, focus is given to the analysis of the specific (i.e., cell adhesive peptide sequences) and unspecific (i.e., surface charge, wettability) mechanisms mediating cell-matrix interactions. Furthermore, because natural tissue regeneration is characterized by the dynamic attachment/detachment of different cell populations, the design of advanced scaffolds for tissue engineering, based in the spatiotemporal tuning of cell-matrix anchorage is discussed.

Light induced hydrophilicity and osteoblast adhesion promotion on amorphous TiO2.

We have studied the effect of the UV induced superhydrophilic wetting of TiO(2) thin films on the osteoblasts cell adhesion and cytoskeletal organization on its surface. To assess any effect of the photo-catalytic removal of adventitious carbon as a factor for the enhancement of the osteoblast development, 100 nm amorphous TiO(2) thin layers were deposited on polyethylene terephthalate (PET), a substrate well known for its poor adhesion and limited wettability and biocompatibility. The TiO(2) /PET materials were characterized by X-ray photoelectron spectroscopy, and atomic force microscopy and their wetting behavior under light illumination studied by the sessile drop method. The amorphous TiO(2) thin films showed a very poor photo-catalytic activity even if becoming superhydrophilic after illumination. The illuminated samples recovered partially its initial hydrophobic state only after their storage in the dark for more than 20 days. Osteoblasts (HOB) were seeded both on bare PET and on TiO(2) /PET samples immediately after illumination and also after four weeks storage in darkness. Cell attachment was much more efficient on the immediately illuminated TiO(2)/PET samples, with development of focal adhesions and cell traction forces. Although we cannot completely discard some photo-catalytic carbon removal as a factor contributing to this cell enhanced attachment, our photodegradation experiments on amorphous TiO(2) are conclusive to dismiss this effect as the major cause for this behavior.

Tuning liver stiffness against tumours: an in vitro study using entrapped cells in tumour-like microcapsules.

Liver fibrosis is a reversible pathology characterized by the up-regulated secretion and deposition of ECM proteins and inhibitors of metalloproteinases, which increase the stiffness and viscosity of this organ. Since recent studies have shown that fibrosis preceded the generation of hepatocellular carcinomas, we hypothesize that liver fibrosis could play a role as a mechanism for restricting uncontrolled cell proliferation, inducing the mortality of cancer cells and subsequent development of primary tumours. With this purpose, in this work we analysed in vitro how the modulation of stiffness can influence proliferation, viability and aggregation of hepatocarcinoma cells (HepG(2)) embedded in 3D micromilieus mimicking values of elasticity of fibrotic liver tissues. Experiments were performed by immobilizing up to 10 HepG(2) cells within microcapsules made of 0.8%, 1.0% and 1.4% w/v alginate which, besides having values of elasticity from the lower-healthy to the upper-fibrotic range liver tissues, lacked domains for proteases, mimicking the micromilieu existing in hepatic primary tumours. Our results show that entrapped cells exhibited a short duplication phase followed by an irreversible decay stage, in which cell mortality could be mediated by two mechanisms: mechanical stress, in the case of cells entrapped in a stiffer micromilieu; and mass transfer limitations produced by pore coarsening at the interface cell-matrix, in softer micromilieus. According to the authors' knowledge, this work represents the first attempt to elucidate the role of liver fibrosis during Hepatocarcinoma pathologies, suggesting that the generation of a non-biodegradable and mechanically unfavourable environment surrounding cancer cells could control the proliferation, migration of metastatic cells and the subsequent development of primary tumours.

Determination of pore size distribution at the cell-hydrogel interface.

BACKGROUND: Analyses of the pore size distribution in 3D matrices such as the cell-hydrogel interface are very useful when studying changes and modifications produced as a result of cellular growth and proliferation within the matrix, as pore size distribution plays an important role in the signaling and microenvironment stimuli imparted to the cells. However, the majority of the methods for the assessment of the porosity in biomaterials are not suitable to give quantitative information about the textural properties of these nano-interfaces. FINDINGS: Here, we report a methodology for determining pore size distribution at the cell-hydrogel interface, and the depth of the matrix modified by cell growth by entrapped HepG(2) cells in microcapsules made of 0.8% and 1.4% w/v alginate. The method is based on the estimation of the shortest distance between two points of the fibril-like network hydrogel structures using image analysis of TEM pictures. Values of pore size distribution determined using the presented method and those obtained by nitrogen physisorption measurements were compared, showing good agreement. A combination of these methodologies and a study of the cell-hydrogel interface at various cell culture times showed that after three days of culture, HepG(2) cells growing in hydrogels composed of 0.8% w/v alginate had more coarse of pores at depths up to 40 nm inwards (a phenomenon most notable in the first 20 nm from the interface). This coarsening phenomenon was weakly observed in the case of cells cultured in hydrogels composed of 1.4% w/v alginate. CONCLUSIONS: The method purposed in this paper allows us to obtain information about the radial deformation of the hydrogel matrix due to cell growth, and the consequent modification of the pore size distribution pattern surrounding the cells, which are extremely important for a wide spectrum of biotechnological, pharmaceutical and biomedical applications.

Determination of the decay rate constant for hepatocytes immobilized in alginate microcapsules.

Primary mouse hepatocytes (between 10-250 cells per capsule) were immobilized within 1.0% w/v alginate microbeads. The textural properties of the alginate matrix were characterized and a full protocol based upon the measurement of the initial rate of Resazurin reduction was studied and standardized. Using this method, the decay rate constant (K(d) = 0.45 +/- 0.01 days(-1)) and the time in which the cell viability decreases in half (VI(50) = 37 +/- 0.7 h) have been measured. The method was compared with the analysis of cell vitality using Calcein A/M and Ethidium Homodimer I. Differences between the two methods were found in the viability profile due to the significant presence of double stained cells along the culture time. According to the author's knowledge, this is the first report of a systematic study and determination of the K(d) value for immobilized hepatocytes, incorporating a wide range of cell concentrations within the alginate matrix.