Biomechanical and cellular segmental characterization of human meniscus: building the basis for Tissue Engineering therapies.

OBJECTIVE: To overcome current limitations of Tissue Engineering (TE) strategies, deeper comprehension on meniscus biology is required. This study aims to combine biomechanical segmental analysis of fresh human meniscus tissues and its correlation with architectural and cellular characterization. METHOD: Morphologically intact menisci, from 44 live donors were studied after division into three radial segments. Dynamic mechanical analysis (DMA) was performed at physiological-like conditions. Micro-computed tomography (CT) analysis of freeze-dried samples assessed micro-structure. Flow cytometry, histology and histomorphometry were used for cellular study and quantification. RESULTS: Anterior segments present significantly higher damping properties. Mid body fresh medial meniscus presents higher values of E' compared to lateral. Cyclic loads influence the viscoelastic behavior of menisci. By increasing the frequency leads to an increase in stiffness. Conversely, with increasing frequencies, the capacity to dissipate energy and damping properties initially decrease and then rise again. Age and gender directly correlate with higher E' and tan δ. Micro-CT analysis revealed that mean porosity was 55.5 (21.2-89.8)% and 64.7 (47.7-81.8)% for freeze-dried lateral and medial meniscus, respectively. Predominant cells are positive for CD44, CD73, CD90 and CD105, and lack CD31, CD34 and CD45 (present in smaller populations). Histomorphometry revealed that cellularity decreases from vascular zone 1 to zone 3. Anterior segments of lateral and medial meniscus have inferior cellularity as compared to mid body and posterior ones. CONCLUSION: Menisci are not uniform structures. Anterior segments have lower cellularity and higher damping. Cyclic loads influence viscoelastic characteristics. Future TE therapies should consider segmental architecture, cellularity and biomechanics of fresh tissue.

Photopatterned antibodies for selective cell attachment.

We present a phototriggerable system that allows for the spatiotemporal controlled attachment of selected cell types to a biomaterial using immobilized antibodies that specifically target individual cell phenotypes. o-Nitrobenzyl caged biotin was used to functionalize chitosan membranes and mediate site-specific coupling of streptavidin and biotinylated antibodies after light activation. The ability of this system to capture and immobilize specific cells on a surface was tested using endothelial-specific biotinylated antibodies and nonspecific ones as controls. Homogeneous patterned monolayers of human umbilical vein endothelial cells were obtained on CD31-functionalized surfaces. This is a simple and generic approach that is applicable to other ligands, materials, and cell types and shows the flexibility of caged ligands to trigger and control the interaction between cells and biomaterials.

Gelatin microparticles aggregates as three-dimensional scaffolding system in cartilage engineering.

A three-dimensional (3D) scaffolding system for chondrocytes culture has been produced by agglomeration of cells and gelatin microparticles with a mild centrifuging process. The diameter of the microparticles, around 10 µ, was selected to be in the order of magnitude of the chondrocytes. No gel was used to stabilize the construct that maintained consistency just because of cell and extracellular matrix (ECM) adhesion to the substrate. In one series of samples the microparticles were charged with transforming growth factor, TGF-ß1. The kinetics of growth factor delivery was assessed. The initial delivery was approximately 48 % of the total amount delivered up to day 14. Chondrocytes that had been previously expanded in monolayer culture, and thus dedifferentiated, adopted in this 3D environment a round morphology, both with presence or absence of growth factor delivery, with production of ECM that intermingles with gelatin particles. The pellet was stable from the first day of culture. Cell viability was assessed by MTS assay, showing higher absorption values in the cell/unloaded gelatin microparticle pellets than in cell pellets up to day 7. Nevertheless the absorption drops in the following culture times. On the contrary the cell viability of cell/TGF-ß1 loaded gelatin microparticle pellets was constant during the 21 days of culture. The formation of actin stress fibres in the cytoskeleton and type I collagen expression was significantly reduced in both cell/gelatin microparticle pellets (with and without TGF-ß1) with respect to cell pellet controls. Total type II collagen and sulphated glycosaminoglycans quantification show an enhancement of the production of ECM when TGF-ß1 is delivered, as expected because this growth factor stimulate the chondrocyte proliferation and improve the functionality of the tissue.

Platelet lysates-based hydrogels incorporating bioactive mesoporous silica nanoparticles for stem cell osteogenic differentiation.

Scaffolds for bone tissue regeneration should provide the right cues for stem cell adhesion and proliferation, but also lead to their osteogenic differentiation. Hydrogels of modified platelet lysates (PLMA) show the proper mechanical stability for cell encapsulation and contain essential bioactive molecules required for cell maintenance. We prepared a novel PLMA-based nanocomposite for bone repair and regeneration capable of releasing biofactors to induce osteogenic differentiation. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were encapsulated in PLMA hydrogels containing bioactive mesoporous silica nanoparticles previously loaded with dexamethasone and functionalized with calcium and phosphate ions. After 21 d of culture, hBM-MSCs remained viable, presented a stretched morphology, and showed signs of osteogenic differentiation, namely the presence of significant amounts of alkaline phosphatase, bone morphogenic protein-2 and osteopontin, hydroxyapatite, and calcium nodules. Developed for the first time, PLMA/MSNCaPDex nanocomposites were able to guide the differentiation of hBM-MSCs without any other osteogenic supplementation.

Modeling of Cell-Mediated Self-Assembled Colloidal Scaffolds.

A critical step in tissue engineering is the design and synthesis of 3D biocompatible matrices (scaffolds) to support and guide the proliferation of cells and tissue growth. The most existing techniques rely on the processing of scaffolds under controlled conditions and then implanting them in vivo, with questions related to biocompatibility and implantation that are still challenging. As an alternative, it was proposed to assemble the scaffolds in loco through the self-organization of colloidal particles mediated by cells. To overcome the difficulty to test experimentally all the relevant parameters, we propose the use of large-scale numerical simulation as a tool to reach useful predictive information and to interpret experimental results. Thus, in this study, we combine experiments, particle-based simulations, and mean-field calculations to show that, in general, the size of the self-assembled scaffold scales with the cell-to-particle ratio. However, we have found an optimal value of this ratio, for which the size of the scaffold is maximal when the cell-cell adhesion is suppressed. These results suggest that the size and structure of the self-assembled scaffolds may be designed by tuning the adhesion between cells in the colloidal suspension.

Role of active nanoliposomes in the surface and bulk mechanical properties of hybrid hydrogels.

Nanoliposomes are widely used as delivery vehicles for active compounds. Nanoliposomes from rapeseed phospholipids were incorporated into interpenetrating polymer network hydrogels of gelatin methacryloyl and alginate. The multiscale physicochemical properties of the hydrogels are studied both on the surface and through the thickness of the 3D network. The obtained composite hydrogels exhibited strong mechanical properties and a highly porous surface. The blend ratio, as well as the concentration of nanoliposomes, affects the properties of the hydrogels. Nanofunctionalized hydrogels induced keratinocyte â€‹growth. These advantageous characteristics may open up many applications of the developed hydrogels in drug delivery and tissue engineering.

Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications.

Chitosan is a natural based polymer, obtained by alkaline deacetylation of chitin, which presents excellent biological properties such as biodegradability and immunological, antibacterial and wound-healing activity. Recently, there has been a growing interest in the chemical modification of chitosan in order to improve its solubility and widen its applications. The main chemical modifications of chitosan that have been proposed in the literature are reviewed in this paper. Moreover, these chemical modifications lead to a wide range of derivatives with a broad range of applications. Recent and relevant examples of the distinct applications, with particular emphasis on tissue engineering, drug delivery and environmental applications, are presented.

Design of spherically structured 3D in vitro tumor models -Advances and prospects.

Three-dimensional multicellular tumor models are receiving an ever-growing focus as preclinical drug-screening platforms due to their potential to recapitulate major physiological features of human tumors in vitro. In line with this momentum, the technologies for assembly of 3D microtumors are rapidly evolving towards a comprehensive inclusion of tumor microenvironment elements. Customized spherically structured platforms, including microparticles and microcapsules, provide a robust and scalable technology to imprint unique biomolecular tumor microenvironment hallmarks into 3D in vitro models. Herein, a comprehensive overview of novel advances on the integration of tumor-ECM components and biomechanical cues into 3D in vitro models assembled in spherical shaped platforms is provided. Future improvements regarding spatiotemporal/mechanical adaptability, and degradability, during microtumors in vitro 3D culture are also critically discussed considering the realistic potential of these platforms to mimic the dynamic tumor microenvironment. From a global perspective, the production of 3D multicellular spheroids with tumor ECM components included in spherical models will unlock their potential to be used in high-throughput screening of therapeutic compounds. It is envisioned, in a near future, that a combination of spherically structured 3D microtumor models with other advanced microfluidic technologies will properly recapitulate the flow dynamics of human tumors in vitro. STATEMENT OF SIGNIFICANCE: The ability to correctly mimic the complexity of the tumor microenvironment in vitro is a key aspect for the development of evermore realistic in vitro models for drug-screening and fundamental cancer biology studies. In this regard, conventional spheroid-based 3D tumor models, combined with spherically structured biomaterials, opens the opportunity to precisely recapitulate complex cell-extracellular matrix interactions and tumor compartmentalization. This review provides an in-depth focus on current developments regarding spherically structured scaffolds engineered into in vitro 3D tumor models, and discusses future advances toward all-encompassing platforms that may provide an improved in vitro/in vivo correlation in a foreseeable future.

Bioinstructive microparticles for self-assembly of mesenchymal stem Cell-3D tumor spheroids.

3D multicellular tumor spheroids (3D-MCTS) that closely mimic in vitro the complex lung tumor microenvironment (TME) are highly desirable for screening innovative anti-cancer therapeutics. Despite significant improvements in mimicking lung TME, few models have combined tumor-infiltrating mesenchymal stem cells from bone marrow (hBM-MSCs) with heterotypic 3D tumor spheroid models containing ECM mimetic components. Herein, we engineered hybrid 3D-MCTS that combine, for the first time, A549:fibroblasts:hBM-MSCs in heterotypic tri-culture, with bioinstructive hyaluronan microparticles that act as tumor-ECM mimetics and as cell-anchoring hotspots. The obtained results indicated that 3D microspheres provided proper support for cells to self-assemble into compact 3D microtissues and promoted an increase in CD44 expression, emulating the presence of native-ECM hyaluronan. 3D-MCTS size and sphere-like morphology was reproducible and tri-culture models presented the characteristic solid tumors necrotic core. Mesenchymal stem cells tracking demonstrated that hBM-MSCs migrate to different regions in 3D microtumors mass exhibiting dynamic interactions with cancer cells and stromal fibroblasts, alike in human tumors. Importantly, doxorubicin administration revealed hBM-MSCs effect on cytotoxic responses in 3D tri-culture models and in dual cultures of hBM-MSCs:A549 at 10:1 ratio. Such findings evidence the relevance of including hBM-MSCs in combination with cancer-stromal fibroblasts in 3D in vitro tumor models and the importance to test different cell-to-cell ratios to mimic tumor heterogeneity. In addition, bioinstructive hyaluronan-microparticles were also effective as cell-agglomerating scaffolds and showed potential to be used as an enabling technology for including different ECM components in 3D in vitro models in the future.

Multifunctional laminarin microparticles for cell adhesion and expansion.

Microfabrication technologies have been widely explored to produce microgels that can be assembled in functional constructs for tissue engineering and regenerative medicine applications. Here, we propose microfluidics coupled to a source of UV light to produce multifunctional methacrylated laminarin microparticles with narrow distribution of sizes using photopolymerization. The multifunctional microparticles were loaded with platelet lysates and further conjugated with an adhesive peptide. The adhesive peptides dictated cell adhesiveness to the laminarin microparticles, the incorporation of platelet lysates have resulted in improved cell expansion compared to clear microparticles. Overall, our findings demonstrate that multifunctional methacrylated laminarin microparticles provide an effective support for cell attachment and expansion. Moreover, expanded cells provide the link for microparticles aggregation resulting in robust 3D structures. This suggest the potential for using the methacrylated laminarin microplatforms capable to be assembled by the action of cells to rapidly produce large tissue engineered constructs.

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes.

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.

Microhardness of starch based biomaterials in simulated physiological conditions.

In this work the variation of the surface mechanical properties of starch-based biomaterials with immersion time was followed using microhardness measurements. Two blends with very distinct water uptake capabilities, starch/cellulose acetate (SCA) and starch/poly(epsilon-caprolactone) (SPCL), were immersed in a phosphate buffer solution (PBS) at 37.5 degrees C for various times. The microhardness of the blends decreased significantly ( approximately 50% for SPCL and approximately 94% for SCA), within a time period of 30 days of immersion, reflecting the different hydrophilic character of the synthetic components of the blends. The dependence of microhardness on the applied loading time and load was also analysed and showed a power law dependency for SCA. Water uptake and weight loss measurements were performed for the same immersion times used in the microhardness experiments. The different swelling/degradation behaviour presented by the blends was related to the respective variation in microhardness. Moreover, complementary characterization of the mechanical properties of SCA and SPCL was accomplished by dynamic mechanical analysis (DMA) and creep measurements. Microhardness measurements proved to be a useful technique for characterizing the mechanical behaviour near the surface of polymeric biomaterials, including in simulated physiological conditions.

Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends.

The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing failing or malfunctioning organs, by means of combining a scaffold/support material, adequate cells and bioactive molecules. Different materials have been proposed to be used as both three-dimensional porous scaffolds and hydrogel matrices for distinct tissue engineering strategies. Among them, polymers of natural origin are one of the most attractive options, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this review, the most studied and promising and recently proposed naturally derived polymers that have been suggested for tissue engineering applications are described. Different classes of such type of polymers and their blends with synthetic polymers are analysed, with special focus on polysaccharides and proteins, the systems that are more inspired by the ECM. The adaptation of conventional methods or non-conventional processing techniques for processing scaffolds from natural origin based polymers is reviewed. The use of particles, membranes and injectable systems from such kind of materials is also overviewed, especially what concerns the present status of the research that should lead towards their final application. Finally, the biological performance of tissue engineering constructs based on natural-based polymers is discussed, using several examples for different clinically relevant applications.

Preparation and characterization of poly(L-lactic acid)-chitosan hybrid scaffolds with drug release capability.

Novel poly(L-lactic acid) (PLLA)-chitosan hybrid scaffolds were developed in order to be used as tissue-engineering scaffolds and drug release carriers. The incorporation of chitosan into the PLLA porous structure allows for producing chitosan-based scaffold devices with interesting damping and stiffness aimed at being used in tissue engineering of bone or cartilage. The pore structure of the hybrid scaffolds was influenced by the concentration of the chitosan solution introduced into the PLLA scaffold. For lower concentrations, chitosan was mainly deposited onto the PLLA surface, whereas for higher concentration chitosan formed also microfibrilar structures within the pore walls of the PLLA foam that may act as additional soft anchorage sites for cells. Equilibrium water uptakes up to about 110% were achieved in 24 h. An anti-inflammatory drug, ketoprofen, was loaded within the chitosan component of the hybrid scaffolds by immersing the scaffolds in a drug-ethanol solution. The drug was released sharply within the initial periods ( approximately 2-4 h), but the rate decreased further, showing a sustained release. The drug release rate can be controlled by the chitosan content and cross-link densities, suggesting the effectiveness of the hybrid scaffold as a drug delivery system.

Synthesis and characterization of pH-sensitive thiol-containing chitosan beads for controlled drug delivery applications.

The aim of this study was to develop chitosan-based materials in drug delivery systems possessing covalent attachment of thiol moieties. Thiol-containing chitosan (TCS), found to be soluble in water, was synthesized by graft copolymerization technique. The TCS beads were prepared by using tripolyphoshate, at pH 4.0. The morphology of TCS beads was examined by scanning electron microscopy. The in vitro drug release behavior was studied in phosphate buffer solution at various pH, using indomethacin as a model drug at two different concentrations (0.3 and 0.6% w/w). The release amounts of indomethacin from TCS beads were higher increasing pHs in the dissolution medium. The release rate of indomethacin at pH 7.4 was higher than the release rate at pH 1.4 due to ionization of thiol groups and high solubility of indomethacin in an alkaline medium. These results indicated that the TCS beads may become a delivery system for the controlled release of different drugs wherever pH sensitive mechanics might be useful. This is especially applicable in cases when it is important to minimize drug release in acidic sites, such as in the stomach.

Biomedical applications of natural-based polymers combined with bioactive glass nanoparticles.

In recent years, the combination of natural polymers with nanoparticles has permitted the development of sophisticated and efficient bioinspired constructs. In this regard, the incorporation of bioactive glass nanoparticles (BGNPs) confers a bioactive nature to these constructs, which can then induce the formation of a bone-like apatite layer upon immersion in a physiological environment. Moreover, the incorporation of bioactive glass nanoparticles has been found to be beneficial; the constructs proved to be biocompatible, promote cell adhesion and spreading, and regulate osteogenic commitment. This review provides a summary and discussion of the composition, design, and applications of bioinspired nanocomposite constructs based on BGNPs. Examples of nanocomposite systems will be highlighted with relevance to biomedical applications. It is expected that understanding the principles and the state-of-the-art of natural nanocomposites may lead to breakthroughs in many research areas, including tissue engineering and orthopaedic devices. The challenges regarding the future translation of these nanostructured composites into clinical use are also summarized.

Development of a bioactive glass-polymer composite for wound healing applications.

This study reports the production and characterization of a composite material for wound healing applications. A bioactive glass obtained by sol-gel process and doped with two different metal ions was investigated. Silver (Ag) and cobalt (Co) were chosen due to their antibacterial and angiogenic properties, respectively, very beneficial in the wound healing process. Poly(ε-caprolactone) (PCL) fibers were produced by electrospinning (ES) from a polymeric solution using acetone as a solvent. After optimization of the ES parameters, two main suspensions were prepared, namely: PCL containing bioactive glass nanoparticles (BG-NP) and PCL with Ag2O and CoO doped BG-NP (DP BG-NP), which were processed with different concentrations of BG-NP (0.25%, 0.5% and 0.75wt%). The composite membranes were characterized in terms of morphology, fiber diameter, weight loss, mineralization potential and mechanical performance.

Extracellular vesicles, exosomes and shedding vesicles in regenerative medicine - a new paradigm for tissue repair.

Tissue regeneration by stem cells is driven by the paracrine activity of shedding vesicles and exosomes, which deliver specific cargoes to the recipient cells. Proteins, RNA, cytokines and subsequent gene expression, orchestrate the regeneration process by improving the microenvironment to promote cell survival, controlling inflammation, repairing injury and enhancing the healing process. The action of microRNA is widely accepted as an essential driver of the regenerative process through its impact on multiple downstream biological pathways, and its ability to regulate the host immune response. Here, we present an overview of the recent potential uses of exosomes for regenerative medicine and tissue engineering. We also highlight the differences in composition between shedding vesicles and exosomes that depend on the various types of stem cells from which they are derived. The conditions that affect the production of exosomes in different cell types are deliberated. This review also presents the current status of candidate exosomal microRNAs for potential therapeutic use in regenerative medicine, and in applications involving widely studied organs and tissues such as heart, lung, cartilage and bone.

Elastic chitosan/chondroitin sulfate multilayer membranes.

Freestanding multilayered films were obtained using layer-by-layer (LbL) technology from the assembly of natural polyelectrolytes, namely chitosan (CHT) and chondroitin sulfate (CS). The morphology and the transparency of the membranes were evaluated. The influence of genipin (1 and 2 mg ml(-1)), a naturally-derived crosslinker agent, was also investigated in the control of the mechanical properties of the CHT/CS membranes. The water uptake ability can be tailored by changing the crosslinker concentration that also controls the Young's modulus and ultimate tensile strength. The maximum extension tends to decrease upon crosslinking with the highest genipin concentration, compromising the elastic properties of CHT/CS membranes: nevertheless, when using a lower genipin concentration, the ultimate tensile stress is similar to the non-crosslinked one, but exhibits a significantly higher modulus. Moreover, the crosslinked multilayer membranes exhibited shape memory properties, through a simple hydration action. The in vitro biological assays showed better L929 cell adhesion and proliferation when using the crosslinked membranes and confirmed the non-cytotoxicity of the developed CHT/CS membranes. Within this research work, we were able to construct freestanding biomimetic multilayer structures with tailored swelling, mechanical and biological properties that could find applicability in a variety of biomedical applications.

Antibacterial bioadhesive layer-by-layer coatings for orthopedic applications.

In this study, thin LbL films were produced by combining the adhesive properties of the hyaluronic acid-dopamine conjugate with the bioactivity and bactericidal properties of silver doped bioactive glass nanoparticles. The build-up of these films was investigated by quartz crystal microbalance with dissipation monitoring. LbL coatings were then constructed on a glass substrate for further characterization. We found that these antimicrobial bioinspired films display enhanced adhesive strength. In vitro bioactivity tests were performed by immersing them in simulated body fluid solution for 14 days where the constructed films promoted the formation of a bone-like apatite layer. From microbiological assays, it was found that coatings containing silver doped nanoparticles exhibited a remarkable antibacterial effect against Staphylococcus aureus and Escherichia coli cultures. Finally, in vitro cellular behavior tests showed enhanced cell adhesion, proliferation and viability for these antibacterial bioadhesive films. Therefore, the constructed thin films showed promising properties and evidenced great potential to be used as coatings for orthopedic implants.