The zipped folder called “Raw Data for Article” contains 8 files as described below: File cold “Rheology” contains raw data of viscosity study (Fig 1). File “Density” contains raw data of dimensional properties showed in Table 1. File “Amine” contains results of amine group content testing and calculation of the degree of cross-linking (Fig 4 and Table 2). Files “Sigma” and “Devro” contain raw data of degradation studies on collagen (sigma)-and collagen (devro)-based scaffolds, respectively (Fig 5, 6 and 7). Files “Mechanics” and “Art EDC OA mechanics repeated test” contain results of the mechanical testing: the first file shows the raw data for compressive modulus of the scaffolds of different compositions (Fig 8a) and the second shows the data for stress–strain curves (Fig 8b). File “Cells” contains results of static adhesion of platelets and HT1080 cells on collagen (sigma), collagen-gelatin and gelatin films (Fig 9).

Chitosan/collagen (Chit/Col) blends have demonstrated great potential for use in tissue engineering (TE) applications. However, there exists a lack of detailed study on the influence of important design parameters (i.e, component ratio or crosslinking methods) on the essential properties of the scaffolds (morphology, mechanical stiffness, swelling, degradation and cytotoxicity). This work entailed a systematic study of these essential properties of three Chit/Col compositions, covering a wide range of component ratios and using different crosslinking methods. Our results showed the possibility of tailoring these properties by changing component ratios, since different interactions occurred between Chit/Col: samples with Chit-enriched compositions showed a hydrogen-bonding type complex (HC), whereas a self-crosslinking phenomenon was induced in Col-enriched scaffolds. Additionally, material and biological properties of the resultant matrices were further adjusted and tuned by changing crosslinking conditions. In such way, we obtained a wide range of scaffolds whose properties were tailored to meet specific needs of TE applications.

We provide evidence to show that the standard reactant concentrations used in tissue engineering to cross-link collagen-based scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against degradation in an aqueous environment. We demonstrate this with a detailed and systematic study by comparing scaffolds made from (a) collagen from two different suppliers, (b) gelatin (a partially denatured collagen) and (c) 50% collagen-50% gelatin mixtures. The materials were processed, using lyophilisation, to produce homogeneous, highly porous scaffolds with isotropic architectures and pore diameters ranging from 130 to 260 mu m. Scaffolds were cross-linked using a carbodiimide treatment, to establish the effect of the variations in crosslinking conditions (down to very low concentrations) on the morphology, swelling, degradation and mechanical properties of the scaffolds. Carbodiimide concentration of 11.5 mg/ml was defined as the standard (100%) and was progressively diluted down to 0.1%. It was found that 10-fold reduction in the carbodiimide content led to the significant increase (almost 4-fold) in the amount of free amine groups (primarily on collagen lysine residues) without compromising mechanics and stability in water of all resultant scaffolds. The importance of this finding is that, by reducing cross-linking, the corresponding cell-reactive carboxylate anions (collagen glutamate or aspartate residues) that are essential for integrin-mediated binding remain intact. Indeed, a 10-fold reduction in carbodiimide crosslinking resulted in near native-like cell attachment to collagen scaffolds. We have demonstrated that controlling the degree of cross-linking, and hence retaining native scaffold chemistry, offers a major step forward in the biological performance of collagen- and gelatin-based tissue engineering scaffolds.Statement of SignificanceThis work developed collagen and gelatine-based scaffolds with structural, material and biological properties suitable for use in myocardial tissue regeneration. The novelty and significance of this research consist in elucidating the effect of the composition, origin of collagen and crosslinking concentration on the scaffold physical and cell-binding characteristics. We demonstrate that the standard carbodiimide concentrations used to crosslink collagenous scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against dissolution. The importance of this finding is that, by reducing crosslinking, the corresponding cell-reactive carboxylate anions (essential for integrin-mediated binding) remain intact and the native scaffold chemistry is retained. This offers a major step forward in the biological performance of tissue engineered scaffolds. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd.

Polyelectrolyte complexes (PECs) represent promising materials for drug delivery and tissue engineering applications. These substances are obtained in aqueous medium without the need for crosslinking agents. PECs can be produced through the combination of oppositely charged medical grade polymers, which include the stimuli responsive ones. In this work, three-dimensional porous scaffolds were produced through the lyophilization of pH sensitive PECs made of chitosan (CS) and carrageenan (CRG). CS:CRG molar ratios of 1:1 (CSCRG1), 2:1 (CSCRG2), and 3:1 (CSCRG3) were used. The chemical compositions of the PECs, as well as their influence in the final structure of the scaffolds were meticulously studied. In addition, the pH responsiveness of the PECs in a range including the physiological pH values of 7.4 (simulating normal physiological conditions) and 4.5 (simulating inflammatory response) was assessed. Results showed that the PECs produced were stable at pH values of 7.4 and under but dissolved as the pH increased to nonphysiological values of 9 and 11. However, after dissolution, the PEC could be reprecipitated by decreasing the pH to values close to 4.5. The scaffolds obtained presented large and interconnected pores, being equally sensitive to changes in the pH. CSCRG1 scaffolds appeared to have higher hydrophilicity and therefore higher water absorption capacity. The increase in the CS:CRG molar ratios improved the scaffold mechanical properties, with CSCRG3 presenting the higher compressive modulus under wet conditions. Overall, the PEC scaffolds appear promising for tissue engineering related applications that require the use of pH responsive materials stable at physiological conditions.

The aim of the present work was to develop an in situ preparation of chitosan/apatite nanocomposites and evaluate their bioactivity, physiological stability and enzymatic biodegradation. Composites of different chitosan/hydroxyapatite ratios were prepared by wet chemistry using different kinds of chitosan. The method of preparation used to obtain composites in this work could be more attractive with respect to previous procedures because it allows more homogeneous systems and to control the composition and structure of the resulting materials. The bioactivity of the studied material was evidenced by the deposition in its surface of a calcium phosphate layer with apatite morphology after immersion in simulated body fluid (SBF). A higher biodegradation of composites with respect to apatite was obtained due to the presence of chitosan. Also the biodegradability of the composites increased with the chitosan content. Both, biodegradation and bioactivity could be controlled by the molecular weight of chitosan polymer matrix Along the different kinds of chitosan used, a better in vitro biological result was obtained using a chitosan with lower molecular weight. The in vitro biological characteristics of composites indicate that they are promising materials for bone substitution in guided bone regeneration.

Sponge-like matrices with a specific three-dimensional structural design resembling the actual extracellular matrix of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uniaxial and multi-directional temperature gradients across the sample during this process. Scanning electron microscopy analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architectures were obtained where single unidirectional temperature gradients were induced. Altering the freezing conditions by the introduction of multiple temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.

Chitosan-poly(acrylic acid) polyelectrolyte complex nanoparticles were prepared by coacervation under mild experimental conditions Without the use of any organic solvents or surfactants. The influence of some experimental parameters such as the pH of the polyelectrolyte solutions, their concentrations, and the purification procedure on the particle dimensions and their size distribution was studied in detail. The physicochemical properties of the obtained complex were characterized with Fourier transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy, and dynamic light scattering. It was found that for solution concentrations below 0.1 wt %, it was possible to obtain suspensions of nanometer-sized particles. Furthermore, it was established that the pH values of the reactant solutions had a great influence on both the particle size and the yield of the complex that was formed. The most convenient pH values for obtaining chitosan-poly(acrylic acid) particles with a nanometric size and optimum yield (near 90%) were found to be 4.5-5.5 for chitosan and 3.2 for poly(acrylic acid). Additionally, the effects of dialysis and ultrasonic treatment on the stability of complex suspensions, prepared under different experimental conditions, were clarified so that recommendations could be made to bring this system into practical use. (C) 2008 Wiley Periodicals, Inc. J Appl Polym Sci 111: 2362-2371, 2009

The kinetic study of the photopoly-merisation of gels of acrylic acid (AA) and chitosan CHI) prepared by three different methods was accomplished. The kinetic parameters such as conversion and polymerisation rate were determined. The influence of the composition and the preparation method on these parameters was elucidated. The effect of the mixing order on the formation of chitosan-acrylic acid complexes (CHI(+)AA(-)) was determined by measuring the viscosity of non-polymerised gels. The presence of polyacrylic acid (PAA) grafted on to a CHI matrix after photopolymerisation and the influence of the method of preparation and feed composition on the proportion of CHI-PAA interpolymer network were demonstrated by FTIR spectroscopy. The possible mechanism of the graft copolymerisation is discussed in detail.

This work reports on the effect of the amount (0, 10, and 30 wt %) and type of HA powder incorporated into an acrylic bone cement on the tensile properties, compression properties, and fracture toughness. The three different types of HA powders used were synthesized in the laboratory and coated with a silane agent prior to incorporation into the cement powder, and differed in particle size, water content, surface area, and crystallinity. It was found that the inclusion of any type of HA powder led to an increase in the tensile modulus (ET), but all the other mechanical properties of the cement decreased (relative to the values of the unfilled cement). The increase in ET is attributed to the good adhesion between the filler and the cement matrix, which is due to the silane coating agent. The decrease in the other mechanical properties may be a consequence of HA powder agglomeration and porosity. Hydroxyapatite morphology and crack-growth mechanisms were analyzed by scanning electronic microscopy (SEM).

The influence of certain factors [structure and concentration of tertiary amines as coinitiators, monomer composition, presence of inorganic pigments, and incident light intensity (I-0)] on the polymerization rates (R-p), polymerization quantum yields, and conversions of bisphenol A-bis(glycidyl methacrylate) (Bis-GMA)/triethylene glycol dimethacrylate based resins was studied. The initial rate of bulk polymerization increased and the final conversion decreased with the content of Bis-GMA in the mixture. In contrast, it was established that, for all monomer compositions, the R-p, grew when increasing the I-0, the R-p, being directly proportional to the square root of I-0. Such behavior is in agreement with the well-known kinetic expression for the ideal radical photoinitiated polymerization in solution of monofunctional monomers, in spite of the complexity of the dimethacrylate mixtures that were studied. Both the structure and the concentration of reducing amine affected the efficiency of the initiator system and therefore the kinetic behavior of polymerization of these formulations under irradiation. The rate of polymerization increased with the increase of coinitiator concentration over the interval of 0-1%, but later it diminished when increasing the amine content, suggesting that the excess coinitiator retards the polymerization process. The study of the photoreduction of camphorquinone in the presence of different amines showed that the efficiency of the coinitiator depends not only on its ability to photoreduce camphorquinone, forming amine-derived radicals, but also on the reactivity of these radicals toward the initiation of acrylic monomer polymerization. (c) 2005 Wiley Periodicals, Inc.

Acrylic bone cements have been used for about 40 years to fix artificial prosthesis to bone structure. The properties of the acrylic bone cement mostly depend on the characteristics of the beads, which are the main component of the solid part of the cement. In this work beads of poly(methyl methacrylate-co-styrene) were synthesized by suspension polymerization. The objective of this study was to obtain polymeric beads with particle size distributions and molecular weights that will permit the formulation of bone cements according to the international standards. Polyvinylpyrrolidone and polyvinylpyrrolidone-hydroxyapatite mixtures were studied as stabilizers of the system. Benzoyl peroxide (0.1, 1.0, 1.5% wt. in reference to the monomer) was the initiator of the reaction. The copolymeric beads were characterized by different analytical techniques. Polyvinylpyrrolidone alone was the best stabilizer and the bone cements prepared with polymer having low (247,000 g/mol) and high (800,000 g/mol) weight average molecular weights had static mechanical properties according to the requirements of commercial materials.

Experimental data for the photopolymerization of furfuryl acrylate (FA) conformed satisfactorily to the kinetics model proposed for the photopolymerization of furfuryl methacrylate (FM). This model allowed the kinetic constants of the basic steps of the studied mechanism, namely propagation, degradative transfer, re-initiation and cross-termination, to be determined. The calculated values of these constants were in agreement with the chemical nature of FA. For each of these constants, the confidence intervals were determined, and the statistical dependence between some of them was analyzed using the ellipse error method. The equations of moments of the distribution of molecular sizes of the primary chains in the network with order greater than one were developed to describe different molecular averages, such as weight-average chain length and size-average heterogeneity of the primary chains. The results found for the monomer conversion, the cross-link degree and number-average length of the primary chains of the network for FA were compared with those obtained for FM, and it was shown that the process of polymerization of the former monomer was more retarded and produced gels with a greater degree of cross-linking than the latter as expected.

A comparative kinetic study of the polymerization of tetraethyleneglycol dimethacrylate (TEEGDM) under visible light irradiation was carried out in order to determine the effectiveness of titanocene as photoinitiator in this process. The pair camphorquinone/dimethylaminoethyl methacrylate, CQ/DMAEMA (0.5/0.5%, by weight), was used as a reference photoinitiatior system. The reactions were carried out both in the presence and absence of oxygen (in N2) and with variable and constant titanocene concentration and incident light intensity. It was observed that in both atmospheres the polymerization reaction commenced rapidly and its initial rate was comparable with that obtained in the presence of the classic initiation system. However, after reacting for a few seconds this process suffered abrupt deactivation, this tendency being more noticeable at low titanocene concentrations. To explain the observed kinetic behavior, the study of the photofragmentation of titanocene upon irradiation with visible light in both atmospheres was carried out. The quantum yield calculated in the presence of O2 (phifr = 8 at 465 nm) suggests the existence of a chain reaction with participation of oxygen, and this explains, in turn, the high value of the initial rate of polymerization and also the rapid disappearance of the photoinitiator. In an inert atmosphere the photofragmentation occurs in a conventional way, this being a very effective process with quantum yield of 0.7 at 465 nm. However, it seems that only a small part of the active species acts as initiating radicals and these are not sufficient to achieve the appropriate monomer conversion.

In this work, setting kinetics of a poly(methylmethacrylate-co-styrene) bone cement with and without hydroxyapatite filler were studied. Three types of hydroxyapatite with different morphologies were used. Polymerization kinetics were followed by Differential Scanning Calorimetry (DSC). A modification to the Maffezoli kinetic model for bone cement curing process is presented. The proposed kinetic model can predict the whole curing kinetics process of the bone cements studied here. It clearly follows the delay in the "autoacceleration effect" caused by the hydroxyapatites and can predict the behavior of k t , as the system approaches limiting conversion.

The effect of type and amount of hydroxyapatite on the setting kinetics of an experimental bone cement based on poly(methyl methacrylate-co-styrene) was studied. The average molecular weights of the polymeric beads synthesized were determined by SEC and the average particle size was determined by Optical Microscopy. Three types of hydroxyapatites were synthesized in the laboratory and then characterized by ICP, FTIR and X-ray diffraction. To obtain more compatible fillers, the hydroxyapatites were treated with 3-trimethoxysilylpropylmethacrylate. Bone cements formulations filled with 0, 10, 30, and 50 weight % of hydroxyapatite powders were prepared and the kinetics of setting was followed by Differential Scanning Calorimetry. The presence of hydroxyapatite decreased the reaction rate and increased the degree of conversion, which could be beneficial for the long time stability of the implant.

The kinetics modelling of furfuryl methacrylate photopolymerisation using the method of moments has enabled to obtain the concentrations of radicals and pendant groups in the networks formed at different temperatures. The analysis of these results has shown which simple step of the polymerisation mechanism is favoured according to the reaction temperature. For example, at a temperature of 283K, intermolecular degradative transfer to the furan ring plays an important role in network formation. On the other hand, some results obtained by kinetics modelling were used to evaluate the crosslink density of the network. With this aim, the Tobita pseudo-kinetics constants method was applied to the polymerisation at low conversions. The values of crosslink density obtained by means of kinetics modelling and the Tobita method were in good agreement.

A new stochastic model named CORUB has been used to do a phenomenological description of the photopolymerization of furfuryl methacrylate, with the following methodology. Statistical analysis of the model's adequacy shows a good correlation between experimental data and those modelled stochastically. This model was developed using two stochastic variables that represent the random nature of the intermolecular encounters upon which the Collision Theory is sustained. The amounts of random numbers generated simulate time in a new way, based on the standard physical concept, with satisfactory results. Also, calculating the number of times that each individual step of the reaction mechanism takes place, a new possibility for carrying out the sensitivity analysis is presented. In addition to which, the analogy assumed between the frequency of the random number generated and the effective intermolecular collision allows us to model, exclusively and accurately enough, the effect of temperature as well as to estimate the apparent activation energy precisely. The algorithm of CORUB model was coded in Turbo Pascal 6.0, resulting in the CHEMOD-X program, which constitutes a software specifically elaborated as an integrative part of this work, allowing the model to be used.