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Tissue engineering approaches using biomaterials have gain important roles in the regeneration of cartilage. This paper describes the production by microfluidics of alginate based microfibers containing both extracellular matrix (ECM)-derived biomaterials and chondrocytes. As ECM components gelatin or decellularized urinary bladder matrix (UBM) were investigated. The effectiveness of the composite microfibers has been tested to modulate the behavior and re-differentiation of de-differentiated chondrocytes. The complete re-differentiation, at the single cell level, of the chondrocytes, without cell aggregate formation, was observed after 14 days of cell culture. Specific chondrogenic markers and high cellular secretory activity was observed in embedded cells. Notably, no sign of collagen type 10 deposition was determined. The obtained data suggest that de-differentiated chondrocytes regain a functional chondrocyte phenotype when embedded in appropriate 3D scaffold based on alginate plus gelatin or UBM. The proposed scaffolds are indeed valuable to form a cellular microenvironment mimicking the in vivo ECM, opening the way to their use in cartilage tissue engineering.

In recent years, advancements in the fields of microfluidic and lab-on-a-chip technologies have provided unique opportunities for the implementation of nanomaterial production processes owing to the miniaturisation of the fluidic environment. It has been demonstrated that microfluidic reactors offer a range of advantages compared to conventional batch reactors, including improved controllability and uniformity of nanomaterial characteristics. In addition, the fast mixing achieved within microchannels, and the predictability of the laminar flow conditions, can be leveraged to investigate the nanomaterial formation dynamics. In this article recent developments in the field of microfluidic production of nanomaterials for drug delivery applications are reviewed. The features that make microfluidic reactors a suitable technological platform are discussed in terms of controllability of nanomaterials production. An overview of the various strategies developed for the production of organic nanoparticles and colloidal assemblies is presented, focusing on those nanomaterials that could have an impact on nanomedicine field such as drug nanoparticles, polymeric micelles, liposomes, polymersomes, polyplexes and hybrid nanoparticles. The effect of microfluidic environment on nanomaterials formation dynamics, as well as the use of microdevices as tools for nanomaterial investigation is also discussed.

The current paper reports the production of polymeric micelles (PMs), based on pluronic block-copolymers, as drug carriers, precisely controlling the cellular delivery of drugs with various physico-chemical characteristics. PMs were produced with a microfluidic platform to exploit further control on the size characteristic of the PMs. PMs were designed for the co-delivery of dexamethasone (Dex) and ascorbyl-palmitate (AP) to in vitro cultured human periodontal ligament mesenchymal stem cells (hPDLSCs) for the combined induction of osteogenic differentiation. Mixtures of block-copolymers and drugs in organic, water miscible solvent, were conveniently converted in PMs within microfluidic channel leveraging the fast mixing at the microscale. Our results demonstrated that the drugs can be efficiently co-encapsulated in PMs and that different production parameters can be adjusted in order to modulate the PM characteristics. The comparative analysis of PM produced by microfluidic and conventional procedures confirmed that the use of microfluidics platforms allowed the production of PMs in a robust manner with improved controllability, reproducibility, smaller size and polydispersity. Finally, the analysis of the effect of PMs, containing Dex and AP, on the osteogenic differentiation of hPDLSCs is reported. The data demonstrated the effectiveness and safety of PM treatment on hPDLSC. In conclusion, this report indicates that microfluidic approach represents an innovative and useful method for PM controlled preparation, warrant further evaluation as general methodology for the production of colloidal systems for the simultaneous drug delivery.

This study describes the use of lecithin organogels (LO) as versatile vehicles for percutaneous administration of methyl nicotinate (MN), fenretinide (4HPR) and curcumin (CUR). LO are able to increase the solubility of poor water-soluble molecules probably due to the formation of a short-range hexagonal organization of the lecithin cylindrical reverse micelles. After six months from production, no phase separation and an almost absence of aggregates was observed. LO exhibited similar viscosities and rheological behaviour both in the absence and in the presence of drug. The in vitro drug diffusion from LO studied by Franz cell showed that higher viscous vehicles result in lower diffusion coefficient. The microscopic investigation of human skin displayed no significant alterations after treatment with LO and with PBS. In vivo topical activity on erythema after cutaneous application of LO showed that LO-MN induce a strong erythema, while LO-CUR inhibit skin erythema due to CUR anti-inflammatory activity. Tape-stripping experiments performed on skin after topical administration of LO showed a decrease in the amount of CUR in the stratum corneum after 1 h from the occlusion. Shelf life stability studies demonstrated the higher stability of LO-HPR as compared to the others drugs.

This article describes the preparation of starch particles, by spray drying, for possible application to a dry powder coating process. Dry powder coating consists of spraying a fine powder and a plasticizer on particles. The efficiency of the coating is linked to the powder morphological and dimensional characteristics. Different experimental parameters of the spray-drying process were analyzed, including type of solvent, starch concentration, rate of polymer feeding, pressure of the atomizing air, drying air flow, and temperature of drying air. An optimization and screening of the experimental parameters by a design of the experiment (DOE) approach have been done. Finally, the produced spray-dried starch particles were conveniently tested in a dry coating process, in comparison to the commercial initial starch. The obtained results, in terms of coating efficiency, demonstrated that the spray-dried particles led to a sharp increase of coating efficiency value.

The description of a microencapsulation procedure for Wharton's jelly mesenchymal stem cells (WJMSCs) is reported. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An ionic alginate encapsulation procedure was utilized for the microbeads hardening. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. The produced barium-alginate microbeads were characterized by excellent morphological characteristics as well as a very narrow size distribution. The microencapsulation procedure did not alter the morphology and viability of the encapsulated WJMSCs. In addition, the current paper reports the functional properties in terms of secretive profiles of both free and encapsulated WJMSCs. The analyzed factors were members of the family of interleukins, chemokines, growth factors, and soluble forms of adhesion molecules. These experiments showed that despite encapsulation, most of the proteins analyzed were secreted both by the free and encapsulated cells, even if in a different extent. In conclusion, the described encapsulation procedure represents a promising strategy to utilize WJMSCs for possible in vivo applications in tissue engineering and biomedicine.

Dielectrophoresis (DEP) is a very valuable approach for designing and developing laboratory-on-a-chip (lab-on-a-chip) devices that are able to manipulate microparticles and cells. Lab-on-a-chip technology will enable laboratory testing to move from laboratories using complex equipment to nonlaboratory settings. We used a lab-on-a-chip device, the SmartSlide, which carries 193 parallel electrodes and generates up to 50 cylinder-shaped DEP cages able to entrap microparticles and cells within DEP cages and move them along the chip. For lab-on-a-chip technology, the characterization of microparticles exhibiting a differential ability to be DEP-caged, levitated, and moved is important for the development of both diagnostic and therapeutic protocols. We determined whether the SmartSlide could be used to levitate and move tripalmitin-based lipospheres carrying increasing concentrations of dihexadecyl dimethyl ammonium bromide (DHDAB) as a cationic surfactant. The data obtained with this DEP-based platform showed that DEP caging, levitation, and movement of the cationic lipospheres depended on the percentage of DHDAB. Tripalmitin lipospheres containing 6% DHDAB could be DEP-caged and manipulated. On the contrary, lipospheres containing 12% DHDAB did not exhibit an efficient ability to be DEP-caged and moved throughout the chip. To our knowledge, this is the first report on the possible use of a DEP-based lab-on-a-chip device for guided manipulation of lipospheres. This information might be of interest in the fields of drug discovery, delivery, and diagnosis. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Background and Objective Bioreactors are devices that efficiently create an environment that enables cell cultures to grow in a three-dimensional (3D) context mimicking in vivo conditions. In this study, we investigate the effect of dynamic fluid flow on the osteogenic potential of human mesenchymal stem cells obtained from periodontal ligament and entrapped in alginate microbeads. Material and Methods After proper immunophenotyping, cells were encapsulated in barium alginate, cultured in 3D static or 3D dynamic conditions represented by a bioreactor system. Calcein-AM/propidium iodide staining was used to assess cellular viability. Quantitative real-time polymerase chain reaction was used to analyze the expression of osteogenic markers (Runx2 and COL1). Alizarin Red S staining and the Fourier transform infrared spectroscopy were used to assess mineral matrix deposition. Results Optimal encapsulation procedure, in terms of polymer pumping rate, distance from droplet generator to the gelling bath and atomizing airflow was assessed. Cell viability was not affected by encapsulation in alginate microbeads. Bioreactor cell exposure was effective in anticipating osteogenic differentiation and improving mineral matrix deposition. Conclusion For the first time human mesenchymal stem cells obtained from periodontal ligaments encapsulated in alginate microbeads were cultured in a bioreactor system. This combination could represent a promising strategy to create a cell-based smart system with enhanced osteogenic potential useful for many different dental applications.

The present paper describes the production of microfluidic chips using an approach based on shrinkable biocompatible polymers (i.e. agarose) for the production of size controlled microfluidic channels. In addition, all steps of chip production were carried out using an inexpensive approach that uses low cost chemicals and equipment. The produced chips were then validated by producing monodisperse polymeric microparticles for drug delivery and hydrogel microfibers for cell embedding.

Among nanoparticle-based drug delivery formulations, lecithin/chitosan liposomes are promising candidates because of their biocompatibility, biodegradability and bioadhesion properties. Lecithin is a mixture of highly biocompatible phospholipids, while chitosan represents one of the most used polymers in pharmaceutical formulations. Their combination results in positively charged complexes that are able to sustain a specific, prolonged and controlled release. Scarce reproducibility and batch-to-batch variation in lecithin/chitosan liposome synthesis, as well as difficult scale-up to industrial production, is a major challenge for their utilization. Here we present a strictly controlled procedure, based on ethanol diffusion in water, which yields to a precise and reproducible self-organization of lecithin and chitosan molecules. We analysed the size, surface charge and stability of chitosan coated liposomes at different lecithin/chitosan ratios. We found that increasing the lecithin/chitosan ratio both mean particle size and surface charge were progressively reduced. A good stability was observed for all formulations, though interactions occurred in the presence of low amount of surface-adsorbed chitosan liposome vesicle. Chitosan coated liposomes interact with A549 and Caco-2 cells inducing low toxic effects only with prolonged incubation times. In conclusion, the proposed procedure provides good reproducibility in the formation of non-toxic and highly stable formulations of chitosan coated liposomes as drug delivery systems.

Norovirus RNA-dependent RNA polymerase (RdRp) is a promising target enzyme for the development of new antiviral drugs. Starting from the crystal structure of norovirus RdRp, we had previously performed an in silico docking search using a library of low-molecular-weight compounds that enabled us to select molecules with predicted enzyme inhibitory activity. Among these, the polysulfonated naphthylurea suramin proved to inhibit in vitro both murine and human norovirus polymerases, with IC50 values in the low micromolar range. The negatively charged inhibitor, however, displayed poor cell permeability in cell-based experiments. Therefore, we produced different suramin-loaded liposome formulations and evaluated their activities in cell-based assays using murine norovirus cultivated in RAW 264.7 macrophages, as a model for norovirus genus. The results obtained show that suramin, when delivered through liposomes, can effectively inhibit murine norovirus replication.

This paper describes the production of living conduits for peripheral nerve regeneration. Different experimental parameters were analyzed to produce biocompatible matrixes for the immobilization of Sertoli cells (SCs). These cells are known to posses a great potential to promote the regeneration and differentiation of different cell types.

The effect of different preparation parameters were analyzed with respect to the rheological and pharmaceutical characteristics of hydrogel blend patches, as transdermal delivery formulation. Mixtures of pectin and gelatin were employed for the production of patches, with adjustable properties, following a two-step gelation procedure. The first gelation, a thermal one, is trigged by the presence of gelatin, whereas, the second gelation, an ionic one, is due to the formation of the typical egg box structure of pectin. In particular, the patch structural properties were assessed by oscillation stress sweep measurements which provided information concerning their viscolelastic properties. In addition, different modalities for drug loading were analyzed with respect to drug homogeneous distribution; testosterone was employed as model drug for transdermal administration. Finally, the performances of the produced transdermal patches were studied, in term of reproducibility and reliability, by determination of in vitro drug release profiles.

The entrapment of cells into hydrogel microdevice in form of microparticles or microfibers is one of the most appealing and useful tools for cell-based therapy and tissue engineering. Cell encapsulation procedures allow the immunoisolation of cells from the surrounding environment, after their transplantation and the maintenance of the normal cellular physiology. Factors affecting the efficacy of microdevices, which include size, size distribution, morphology, and porosity are all highly dependent on the method of preparation. In this respect, microfluidic based methods offer a promising strategy to fabricate highly uniform and morphologically controlled microdevices with tunable chemical and mechanical properties. In the current review, various cell microencapsulation procedures, based on a microfluidics, are critically analyzed with a special focus on the effect of the procedure on the morphology, viability and functions of the embedded cells. Moreover, a brief introduction about the optimal characteristics of microdevice intended for cell encapsulation, together with the currently used materials for the production is reported. A further challenging application of microfluidics for the development of "living microchip" is also presented. Finally, the limitations, challenging and future work on the microfluidic approach are also discussed.

Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts.

The development and the optimization of novel culture systems of mesenchymal osteoprogenitors are some of the most important challenges in the field of bone tissue engineering (TE). A new combination between cells and extracellular matrix (ECM)-scaffold, containing ECM has here been analyzed. As source for osteoprogenitors, mesenchymal stem cells obtained from human umbilical cord Wharton's Jelly (hWJMSCs), were used. As ECM-scaffold, a powder form of isolated and purified porcine urinary bladder matrix (pUBM), was employed. The goals of the current work were: (1) the characterization of the in vitro hWJMSCs behavior, in terms of viability, proliferation, and adhesion to ECM-scaffold; (2) the effectiveness of ECM-scaffold to induce/modulate the osteoblastic differentiation; and (3) the proposal for a possible application of cells/ECM-scaffold construct to the field of cell/TE. In this respect, the properties of the pUBM-scaffold in promoting and guiding the in vitro adhesion, proliferation, and three-dimensional colonization of hWJMSCs, without altering viability and morphological characteristics of the cells, are here described. Finally, we have also demonstrated that pUBM-scaffolds positively affect the expression of typical osteoblastic markers in hWJMSCs.

The entrapment of cells is one of the most promising and usefulness tool in tissue transplantation and regenerative medicine. Cell encapsulation procedures allow the physical isolation of cells from the surrounding environment, after their transplantation and the maintenance of the normal cellular physiology. In this paper, different microencapsulation procedures for Wharton’s Jelly Mesenchymal Stem Cells (WJMSCs) are reported, including coaxial bead generator, vibrating-nozzle procedure and microfluidic based approach. The produced microcapsules were characterized by excellent morphological characteristics and a very narrow size distribution. The experiments demonstrated that the microencapsulation procedure did not alter the morphology, viability and osteogenic differentiation of the enveloped WJMSCs. In conclusion, the encapsulation technologies, here presented, represent a promising strategy for the possible in vivo applications of WJMSCs in tissue engineering and biomedicine.

Entrapment of mammalian cells in natural or synthetic biomaterials represents an important tool for both basic and applied research in tissue engineering. For instance, the encapsulation procedures allow to physically isolate cells from the surrounding environment, after their transplantation maintaining the normal cellular physiology. The first part of the current paper describes different microencapsulation techniques including bulk emulsion technique, vibrating-nozzle procedure, gas driven mono-jet device protocol and microfluidic based approach. In the second part, the application of a microencapsulation procedure to the embedding of IB3-1 cells is also described. IB3-1 is a bronchial epithelial cell line, derived from a cystic fibrosis (CF) patient. Different experimental parameters of the encapsulation process were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of protein secretion, analysing the culture medium by Bio-Plex strategy. The analyzed factors include members of the interleukin family (IL-6), chemokines (IL-8 and MCP-1) and growth factors (G-CSF). The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent.

We have developed a microencapsulation procedure for the entrapment and manipulation of IB3-1 cystic fibrosis cells. The applied method is based on generation of monodisperse droplets by a vibrational nozzle. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of secretomic profile, analyzing the culture medium by Bio-Plex strategy. The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsulated IB3-1 cells could be induced to pro-inflammatory responses, after treatment with TNF-α. In this experimental set-up, encapsulated and free IB3-1 cells were treated with TNF-α, thereafter the culture media from both cell populations were collected. As expected, TNF-αinduced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of interleukin-6 and interleukin-8 occurs also by encapsulated IB3-1 cells.

The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM). Starting from isolated neonatal porcine pancreatic islets (NPIs), we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs) for different time periods (7, 14, 21 days). To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation ofβ-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIsβ-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immatureβ-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.

Polysaccharides (e.g. alginate or agarose) represent a class of polymers commonly employed for the preparation of microparticles for cell entrapment and tissue engineering applications. The present work describes the production and characterization, by a microfluidic approach, of microbeads constituted of alginate and alginate/agarose blends, for the encapsulation of eukaryotic cells. The general production strategy is based on the formation of water-in-oil multiphase flow by a "Y" junction squeezing mechanism. The presented data demonstrate that the gelation step represents the crucial point for the production of morphologically excellent microbeads. In this respect, microfluidic methods appear to be an effective procedure for the production of microbeads intended for cell encapsulation, as proved by the high viability and maintenance of functional capability demonstrated by the encapsulated Sertoli cells.

Aim of this study was to investigate the effect of the preparation parameters on the characteristics of lipospheres that would optimally fit to a lab-on-a-chip platform. Lipospheres were produced by melt dispersion technique using different lipid mixture heated to 70 °C and then emulsified into an external aqueous phase. The initial part of the work was devoted to the selection of the best lipid composition by a classical intuitive approach while the optimization and the screening of the experimental parameters were conducted through a “design of experiments”. Once the best preparation parameters were selected, they were adopted also for the production of cationic lipospheres (CLS). The second part of the study describes the analysis of the lipospheres performances when applied to a DEParray™Chip. The loading, distribution, movement and separation of neutral and cationic lipospheres were investigated. The obtained data show that both neutral and cationic lipospheres can be efficiently used in association with DEParray™Chip.

The optimization, through a Design of Experiments (DoE) approach, of a microencapsulation procedure for isolated neonatal porcine islets (NPI) is described. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An alginate/polyornithine encapsulation procedure, developed and validated in our laboratory for almost a decade, was used to embody pancreatic islets. We analyzed different experimental parameters including frequency of vibration, amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. We produced calcium—alginate gel microbeads with excellent morphological characteristics as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology, viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

The aim of the present paper was to investigate the influence of different formulation parameters on the rheological and functional properties of emulgels (gelified emulsions), intended for the buccal administration of the antiinflammatory drug flurbiprofen. The influence of formulation parameters, such as (a) the amount of gelling polymeric emulsifier (Pemulen® 1621 TR-1) used, (b) the oil to water ratio present in the O/W emulgel and finally (c) the pH of the formulation, was studied by a experimental design (DoE) approach. Formulations were analyzed in term of size and morphology of the internal semi-solid oil droplets as well as in term of rheological properties in the presence or in the absence of flurbiprofen by “shear stress vs. shear rate tests” and “frequency sweep tests”. Emulgels were also characterized in vitro both by bioadhesion tests and release studies. In particular release studies demonstrated that flurbiprofen is released by the emulgels in a controlled manner, the drug release efficacy within the first 100 min was comprised between 50 and 80% of the total amount of the drug. Finally, in vivo tests on healthy volunteers have demonstrated that emulgels were able to remain on buccal mucosa for an average period of 1 h, moreover emulgels did not have bad taste and volunteers referred that were agreeable and pleasant.

A new lab-on-a-chip prototype, recently described, thanks to the use of integrated circuit technology, can generate dielectrophoretic fields that immobilize and allow the control of single biological objects, such as cells, liposomes, or microspheres immersed in a liquid overhanging and in contact with the same chip. With the aim to design and produce polymeric microparticles for specific lab-on-a-chip applications, in the present paper the preparation and characterization of microparticles based on cellulose acetate and cellulose acetate plus other cellulosic polymers is described by a solvent evaporation procedure. In particular the following aspects were investigated: (a) the polymer solubilities, (b) the experimental parameters used for the solvent evaporation procedure, (c) the effect of dyes on the microparticle preparation and morphology, (d) the entrapment efficiency of the vitamins (D3 and E) and, finally, (e) the release kinetics of vitamins from cellulosic microparticles.

The present paper reports the production of microspheres based on alginate/agarose blends by microfluidic chip technology. The general production strategy is based on the formation of water in oil multiphase flow by a “Y” junction squeezing mechanism.

This paper describes a method for the production of alginate microfibres using glass-based microfluidic chips fabricated by a photolithography-wet etching procedure. The main focus of the work is the fabrication of a cell containing multifunctional microfibres which have great potential for applications in drug release formulations and tissue engineering scaffolds (to guide the regeneration of tissues in predefined sizes and shapes) providing cell structural support and immunoisolation. The key parameters, which critically influence the formation of microfibres and their geometries, were identified by a classical intuitive approach COST (Changing One Separate factor a Time). In particular, their effects on the microfibre diameter were investigated, which are directly associated with their functionalities relating to the implantation site, the nutrient availability and diffusion/transport of oxygen, essential nutrients, growth factors, metabolic waste and secretory products. The interplay between the alginate solution concentration, pumping rate and gelling bath concentration in controlling the diameter of the produced microfibres was investigated with a statistical approach by means of a "design of the experiments" (DoEs) optimization and screening. Finally, the processing impacts on cell viability, the cellular effect of wall thickness consistency and the spatial distribution of cells within the alginate microfibre were examined. We provide an approach for the production of alginate microfibres with controlled shape and content, which could be further developed for scaling up and working towards FDA approval.

The present paper describes the production, using a microfluidic approach, of microparticles (constituted of cellulose acetate) and O/ W emulsions (constituted of PEG-6 corn oil droplets). The general production strategy is based on the formation of water-in-oil multiphase flow by a focusing mechanism (X-junction chip). The innovative aspect of the work is represented by the fabrication procedure for the microfluidic chip production. The chips were designed and fabricated by a low cost approach using a shrinkable biocompatible polymer.

This paper describes a method for the production of lipid microparticles (SLM) based on microfluidics using a newly designed modular device constituted of three main parts: a temperature control, a co-flow dripping element and a congealing element. The presented data demonstrated that the microfluidic approach resulted in the production of SLM with narrow size distribution and optimal morphological characteristics in term of sphericity, surface smoothness and absence of defects (i.e. partial coalescence or irregular shape). The optimization of SLM production was performed by screening the effect of different experimental parameters and device configurations by a classical intuitive approach COST (Changing One Separate factor a Time). This process allowed selecting the proper value for a number of parameters including, (i) the congealing element geometry, (ii) the presence and concentration of a stabilizer, (iii) the temperature of water and oil phases and (iv) the water and oil flow rates. In addition, the interplay between oil phase and water phase flow rates, in controlling the size and morphology of SLM, was investigated by a statistical "Design of the Experiments" approach (DoE). The combined use of COST and DoE studies allowed the production of optimized SLM for the encapsulation of dye/drugs. The obtained results demonstrated that the guest molecules did not affect the general characteristics of SLM, confirming the robustness of the microfluidic procedure in view of the production of SLM for biopharmaceutical and biotech protocols.

This paper describes a design of experiments (DoE) approach, of an automatic procedure for the microencapsulation of isolated neonatal porcine islets (NPI). Monodisperse droplets were prepared by a vibrational nozzle, using an alginate/polyornithine encapsulation procedure, that has been developed and validated in our laboratory to encapsulate pancreatic islets. Different experimental parameters were considered such as: frequency of vibration, amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. The produced calcium-alginate gel microbeads displayed an excellent morphology as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology and viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

A novel approach to produce artificial bone composites (microfibers) with distinctive features mimicking natural tissue was investigated. Currently proposed inorganic materials (e.g. apatite matrixes) lack self-assembly and thereby limit interactions between cells and the material. The present work investigates the feasibility of creating "bio-inspired materials" specifically designed to overcome certain limitations inherent to current biomaterials. We examined the dimensions, morphology, and constitutive features of a composite hydrogel which combined an alginate based microfiber with a gelatin solution or a particulate form of urinary bladder matrix (UBM). The effectiveness of the composite microfibers to induce and modulate osteoblastic differentiation in three-dimensional (3D) scaffolds without altering the viability and morphological characteristics of the cells was investigated. The present study describes a novel alginate microfiber production method with the use of microfluidics. The microfluidic procedure allowed for precise tuning of microfibers which resulted in enhanced viability and function of embedded cells.

A method for the production of engineered alginate-based microparticles, containing extracellular matrix and neonatal porcine Sertoli cells (SCs), is described. As a source for extracellular matrix, a powder form of isolated and purified urinary bladder matrix (UBM) was employed. We demonstrated that the incorporation of UBM does not significantly alter the morphological and dimensional characteristics of the microparticles. The alginate microparticles were used for SC encapsulation as an immunoprotective barrier for transplant purposes, while the co-entrapped UBM promoted retention of cell viability and function. These engineered microparticles could represent a novel approach to enhancing immunological acceptance and increasing the functional life-span of the entrapped cells for cell/tissue engineering applications. In this respect, it is noteworthy that isolated neonatal porcine SCs, administered alone in highly biocompatible microparticles, led to diabetes prevention and reversion in nonobese diabetic (NOD) mice.

Lorenzo Capretto1, Stefania Mazzitelli2, Eleonora Brognara2, Ilaria Lampronti2, Dario Carugo1, Martyn Hill1, Xunli Zhang1, Roberto Gambari2, Claudio Nastruzzi31Engineering Sciences, University of Southampton, Southampton, UK; 2Department of Biochemistry and Molecular Biology, 3Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, ItalyAbstract: This report shows that the DNA-binding drug, mithramycin, can be efficiently encapsulated in polymeric micelles (PM-MTH), based on Pluronic® block copolymers, by a new microfluidic approach. The effect of different production parameters has been investigated for their effect on PM-MTH characteristics. The compared analysis of PM-MTH produced by microfluidic and conventional bulk mixing procedures revealed that microfluidics provides a useful platform for the production of PM-MTH with improved controllability, reproducibility, smaller size, and polydispersity. Finally, an investigation of the effects of PM-MTH, produced by microfluidic and conventional bulk mixing procedures, on the erythroid differentiation of both human erythroleukemia and human erythroid precursor cells is reported. It is demonstrated that PM-MTH exhibited a slightly lower toxicity and more pronounced differentiative activity when compared to the free drug. In addition, PM-MTH were able to upregulate preferentially γ-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating α-globin gene expression, which is responsible for the clinical symptoms of ß-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying ß-thalassemia and sickle cell anemia. In conclusion, this report suggests that PM-MTH produced by microfluidic approach warrants further evaluation as a potential therapeutic protocol for ß-thalassemia.Keywords: microfluidics, lab-on-a-chip, design of experiments, erythroid differentiation, human erythroid precursor cells

The paper describes (a) the isolation and characterization of Wharton's Jelly Mesenchymal Stem cells (WJMSCs), (b) the encapsulation of WJMSCs in alginate microbeads by an encapsulation device based on a vibrating-nozzle, (c) the optimization of the procedure for the encapsulation of WJMSCs by a Design of Experiment (DoE) approach, and (d) the evaluation of viability, proliferation and osteoblastic differentiation of the encapsulated WJMSCs.