Towards Complex Tissues Replication: Multilayer Scaffold Integrating Biomimetic Nanohydroxyapatite/Chitosan Composites.

This study explores an approach to design and prepare a multilayer scaffold mimicking interstratified natural tissue. This multilayer construct, composed of chitosan matrices with graded nanohydroxyapatite concentrations, was achieved through an in situ biomineralization process applied to individual layers. Three distinct precursor concentrations were considered, resulting in 10, 20, and 30 wt% nanohydroxyapatite content in each layer. The resulting chitosan/nanohydroxyapatite (Cs/n-HAp) scaffolds, created via freeze-drying, exhibited nanohydroxyapatite nucleation, homogeneous distribution, improved mechanical properties, and good cytocompatibility. The cytocompatibility analysis revealed that the Cs/n-HAp layers presented cell proliferation similar to the control in pure Cs for the samples with 10% n-HAp, indicating good cytocompatibility at this concentration, while no induction of apoptotic death pathways was demonstrated up to a 20 wt% n-Hap concentration. Successful multilayer assembly of Cs and Cs/n-HAp layers highlighted that the proposed approach represents a promising strategy for mimicking multifaceted tissues, such as osteochondral ones.

The Evolution of Technology-Driven In Vitro Models for Neurodegenerative Diseases.

The alteration in the neural circuits of both central and peripheral nervous systems is closely related to the onset of neurodegenerative disorders (NDDs). Despite significant research efforts, the knowledge regarding NDD pathological processes, and the development of efficacious drugs are still limited due to the inability to access and reproduce the components of the nervous system and its intricate microenvironment. 2D culture systems are too simplistic to accurately represent the more complex and dynamic situation of cells in vivo and have therefore been surpassed by 3D systems. However, both models suffer from various limitations that can be overcome by employing two innovative technologies: organ-on-chip and 3D printing. In this review, an overview of the advantages and shortcomings of both microfluidic platforms and extracellular matrix-like biomaterials will be given. Then, the combination of microfluidics and hydrogels as a new synergistic approach to study neural disorders by analyzing the latest advances in 3D brain-on-chip for neurodegenerative research will be explored.

In Vitro and In Vivo Biocompatibility Assessment of a Thermosensitive Injectable Chitosan-Based Hydrogel for Musculoskeletal Tissue Engineering.

Musculoskeletal impairments, especially cartilage and meniscus lesions, are some of the major contributors to disabilities. Thus, novel tissue engineering strategies are being developed to overcome these issues. In this study, the aim was to investigate the biocompatibility, in vitro and in vivo, of a thermosensitive, injectable chitosan-based hydrogel loaded with three different primary mesenchymal stromal cells. The cell types were human adipose-derived mesenchymal stromal cells (hASCs), human bone marrow stem cells (hBMSCs), and neonatal porcine infrapatellar fat-derived cells (IFPCs). For the in vitro study, the cells were encapsulated in sol-phase hydrogel, and then, analyzed via live/dead assay at 1, 4, 7, and 14 days to compare their capacity to survive in the hydrogel. To assess biocompatibility in vivo, cellularized scaffolds were subcutaneously implanted in the dorsal pouches of nude mice and analyzed at 4 and 12 weeks. Our data showed that all the different cell types survived (the live cell percentages were between 60 and 80 at all time points in vitro) and proliferated in the hydrogel (from very few at 4 weeks to up to 30% at 12 weeks in vivo); moreover, the cell-laden hydrogels did not trigger an immune response in vivo. Hence, our hydrogel formulation showed a favorable profile in terms of safety and biocompatibility, and it may be applied in tissue engineering strategies for cartilage and meniscus repair.

pH-sensing hybrid hydrogels for non-invasive metabolism monitoring in tumor spheroids.

The constant increase in cancer incidence and mortality pushes biomedical research towards the development of in vitro 3D systems able to faithfully reproduce and effectively probe the tumor microenvironment. Cancer cells interact with this complex and dynamic architecture, leading to peculiar tumor-associated phenomena, such as acidic pH conditions, rigid extracellular matrix, altered vasculature, hypoxic condition. Acidification of extracellular pH, in particular, is a well-known feature of solid tumors, correlated to cancer initiation, progression, and resistance to therapies. Monitoring local pH variations, non-invasively, during cancer growth and in response to drug treatment becomes extremely important for understanding cancer mechanisms. Here, we describe a simple and reliable pH-sensing hybrid system, based on a thermoresponsive hydrogel embedding optical pH sensors, that we specifically apply for non-invasive and accurate metabolism monitoring in colorectal cancer (CRC) spheroids. First, the physico-chemical properties of the hybrid sensing platform, in terms of stability, rheological and mechanical properties, morphology and pH sensitivity, were fully characterized. Then, the proton gradient distribution in the spheroids proximity, in the presence or absence of drug treatment, was quantified over time by time lapse confocal light scanning microscopy and automated segmentation pipeline, highlighting the effects of the drug treatment in the extracellular pH. In particular, in the treated CRC spheroids the acidification of the microenvironment resulted faster and more pronounced over time. Moreover, a pH gradient distribution was detected in the untreated spheroids, with more acidic values in proximity of the spheroids, resembling the cell metabolic features observed in vivo in the tumor microenvironment. These findings promise to shed light on mechanisms of regulation of proton exchanges by cellular metabolism being essential for the study of solid tumors in 3D in vitro models and the development of personalized medicine approaches.

Chitosan and Pectin Hydrogels for Tissue Engineering and In Vitro Modeling.

Hydrogels are fascinating biomaterials that can act as a support for cells, i.e., a scaffold, in which they can organize themselves spatially in a similar way to what occurs in vivo. Hydrogel use is therefore essential for the development of 3D systems and allows to recreate the cellular microenvironment in physiological and pathological conditions. This makes them ideal candidates for biological tissue analogues for application in the field of both tissue engineering and 3D in vitro models, as they have the ability to closely mimic the extracellular matrix (ECM) of a specific organ or tissue. Polysaccharide-based hydrogels, because of their remarkable biocompatibility related to their polymeric constituents, have the ability to interact beneficially with the cellular components. Although the growing interest in the use of polysaccharide-based hydrogels in the biomedical field is evidenced by a conspicuous number of reviews on the topic, none of them have focused on the combined use of two important polysaccharides, chitosan and pectin. Therefore, the present review will discuss the biomedical applications of polysaccharide-based hydrogels containing the two aforementioned natural polymers, chitosan and pectin, in the fields of tissue engineering and 3D in vitro modeling.

Genipin-crosslinked collagen scaffolds inducing chondrogenesis: a mechanical and biological characterization.

Articular cartilage degeneration is still an unsolved issue owing to its weak repairing capabilities, which usually result in fibrocartilage tissue formation. This fibrous tissue lacks of structural and bio-mechanical properties, degrading over time. Currently, arthroscopic techniques and autologous transplantation are the most used clinical procedures. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Therefore, tissue engineering strategies aimed at selecting scaffolds that remarkably support the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) could represent a promising solution, but they are still challenging for researchers. In this study, the influence of two different genipin (Gp) crosslinking routes on collagen (Coll)-based scaffolds in terms of hMSCs chondrogenic differentiation and biomechanical performances was investigated. Three-dimensional (3D) porous Coll scaffolds were fabricated by freeze-drying techniques and were crosslinked with Gp following a "two-step" and an in "bulk" procedure, in order to increase the physico-mechanical stability of the structure. Chondrogenic differentiation efficacy of hMSCs and biomechanical behavior under compression forces through unconfined stress-strain tests were assessed. Coll/Gp scaffolds revealed an isotropic and highly homogeneous pore distribution along with an increase in the stiffness, also supported by the increase in the Coll denaturation temperature (Td  = 57-63°C) and a significant amount of Coll and GAG deposition during the 3 weeks of chondrogenic culture. In particular, the presence of Gp in "bulk" led to a more uniform and homogenous chondral-like matrix deposition by hMSCs if compared to the results obtained from the Gp "two-step" functionalization procedure.

Thermosensitive chitosan-based hydrogels supporting motor neuron-like NSC-34 cell differentiation.

Motor neuron diseases are neurodegenerative diseases that predominantly affect the neuromuscular system. To date, there are no valid therapeutic treatments for such diseases, and the classical experimental models fail in faithfully reproducing the pathological mechanisms behind them. In this regard, the use of three-dimensional (3D) culture systems, which more closely reproduce the native in vivo environment, can be a promising approach. Hydrogel-based systems are among the most used materials to reproduce the extracellular matrix, featuring an intrinsic similarity with its physiological characteristics. In this study, we developed a thermosensitive chitosan-based hydrogel combined with ß-glycerophosphate (ßGP) and sodium hydrogen carbonate (SHC), which give the system optimal mechanical properties and injectability, inducing the hydrogel sol-gel transition at 37 °C. An ad hoc protocol for the preparation of the hydrogel was established in order to obtain a highly homogeneous system, leading to reproducible physicochemical characteristics and easy cell encapsulation. All formulations supported the viability of a neuroblastoma/spinal cord hybrid cell line (NSC-34) beyond two weeks of culture and enabled cell differentiation towards a motor neuron-like morphology, characterized by the presence of extended neurites. Based on our results, these hydrogels represent excellent candidates for establishing 3D in vitro models of motor neuron diseases.

A thermo-sensitive chitosan/pectin hydrogel for long-term tumor spheroid culture.

Hydrogels represent a key element in the development of in vitro tumor models, by mimicking the typical 3D tumor architecture in a physicochemical manner and allowing the study of tumor mechanisms. Here we developed a thermo-sensitive, natural polymer-based hydrogel, where chitosan and pectin were mixed and, after a weak base-induced chitosan gelation, a stable semi-Interpenetrating Polymer Network formed. This resulted thermo-responsive at 37 °C, injectable at room temperature, stable up to 6 weeks in vitro, permeable to small/medium-sized molecules (3 to 70 kDa) and suitable for cell-encapsulation. Tunable mechanical and permeability properties were obtained by varying the polymer content. Optimized formulations successfully supported the formation and growth of human colorectal cancer spheroids up to 44 days of culture. The spheroid dimension and density were influenced by the semi-IPN stiffness and permeability. These encouraging results would allow the implementation of faithful tumor models for the study and development of personalized oncological treatments.

Preparation and Characterization of Salt-Mediated Injectable Thermosensitive Chitosan/Pectin Hydrogels for Cell Embedding and Culturing.

In recent years, growing attention has been directed to the development of 3D in vitro tissue models for the study of the physiopathological mechanisms behind organ functioning and diseases. Hydrogels, acting as 3D supporting architectures, allow cells to organize spatially more closely to what they physiologically experience in vivo. In this scenario, natural polymer hybrid hydrogels display marked biocompatibility and versatility, representing valid biomaterials for 3D in vitro studies. Here, thermosensitive injectable hydrogels constituted by chitosan and pectin were designed. We exploited the feature of chitosan to thermally undergo sol-gel transition upon the addition of salts, forming a compound that incorporates pectin into a semi-interpenetrating polymer network (semi-IPN). Three salt solutions were tested, namely, beta-glycerophosphate (ßGP), phosphate buffer (PB) and sodium hydrogen carbonate (SHC). The hydrogel formulations (i) were injectable at room temperature, (ii) gelled at 37 °C and (iii) presented a physiological pH, suitable for cell encapsulation. Hydrogels were stable in culture conditions, were able to retain a high water amount and displayed an open and highly interconnected porosity and suitable mechanical properties, with Young's modulus values in the range of soft biological tissues. The developed chitosan/pectin system can be successfully used as a 3D in vitro platform for studying tissue physiopathology.

A microfabricated multi-compartment device for neuron and Schwann cell differentiation.

Understanding the complex communication between different cell populations and their interaction with the microenvironment in the central and peripheral nervous systems is fundamental in neuroscience research. The development of appropriate in vitro approaches and tools, able to selectively analyze and/or probe specific cells and cell portions (e.g., axons and cell bodies in neurons), driving their differentiation into specific cell phenotypes, has become therefore crucial in this direction. Here we report a multi-compartment microfluidic device where up to three different cell populations can be cultured in a fluidically independent circuit. The device allows cell migration across the compartments and their differentiation. We showed that an accurate choice of the device geometrical features and cell culture parameters allows to (1) maximize cell adhesion and proliferation of neuron-like human cells (SH-SY5Y cells), (2) control the inter-compartment cell migration of neuron and Schwann cells, (3) perform long-term cell culture studies in which both SH-SY5Y cells and primary rat Schwann cells can be differentiated towards specific phenotypes. These results can lead to a plethora of in vitro co-culture studies in the neuroscience research field, where tuning and investigating cell-cell and cell-microenvironment interactions are essential.

Selenium-doped hydroxyapatite nanoparticles for potential application in bone tumor therapy.

In the present study we have studied the incorporation and release of selenite ions (SeO32-) in hydroxyapatite nanoparticles for the treatment of bone tumors. Two types of selenium-doped hydroxyapatite (HASe) nanoparticles (NPs) with a nominal Se/(P + Se) molar ratio ranging from 0.01 up to 0.40 have been synthesized by a new and mild wet method. The two series of samples were thoroughly characterized and resulted to be slightly different in chemical composition, but they had similar properties in terms of morphology and degree of crystallinity. Selenium release from HASe was investigated under neutral and acidic conditions to simulate both healthy tissues and the low-pH environment surrounding a tumor mass, respectively. The comparison of the release profiles at two pH values clearly showed the possibility of modulating the Se release by simply changing the amount of Se in the HASe particles. The correlation between the physicochemical properties of HASe and their dissolution as a function of pH has been also investigated to facilitate future application of the NPs as chemotherapeutic adjuvant agents. Finally, the cytotoxic activity of HASe was evaluated using prostate (PC3) and breast (MDA-MB-231) cancer cells as well as healthy human bone marrow stem cells (hBMSc). HASe NPs exerted a good cytocompatibility at low concentration of Se but, with high Se doping concentration, they displayed strong cytotoxicity.

Evidence of Modular Responsiveness of Osteoblast-Like Cells Exposed to Hydroxyapatite-Containing Magnetic Nanostructures.

The development of nanocomposites with tailored physical-chemical properties, such as nanoparticles containing magnetic iron oxides for manipulating cellular events at distance, implies exciting prospects in biomedical applications for bone tissue regeneration. In this context, this study aims to emphasize the occurrence of differential responsiveness in osteoblast-like cells to different nanocomposites with diverse features: dextran-grafted iron oxide (DM) nanoparticles and their hybrid nano-hydroxyapatite (DM/n-HA) counterpart. Here, responsiveness of cells in the presence of DMs or DM/n-HAs was evaluated in terms of cytoskeletal features. We observed that effects triggered by the DM are no more retained when DM is embedded onto the DM/n-HA nanocomposites. Also, analysis of mRNA level variations of the focal adhesion kinase (FAK), P53 and SLC11A2/DMT1 human genes showed that the DM/n-HA-treated cells retain tracts of physiological responsiveness compared to the DM-treated cells. Overall, a shielding effect by the n-HA component can be assumed, masking the DM's cytotoxic potential, also hinting a modular biomimicry of the nanocomposites respect to the physiological responses of osteoblast-like cells. In this view, the biocompatibility of n-HA together with the magnetic responsiveness of DMs represent an optimized combination of structural with functional features of the DM/n-HA nano-tools for bone tissue engineering, for finely acting within physiological ranges.

Sintering of magnesium-strontium doped hydroxyapatite nanocrystals: Towards the production of 3D biomimetic bone scaffolds.

The development of biomimetic scaffolds is a challenging aim in the field of bone repair for fabrication of osteoconductive and osteoinductive scaffolds. Biogenic hydroxyapatite (HA) is not stoichiometric but is substituted by several ions. An approach to improve synthetic scaffolds biomimetism can be the doping with osteoinductive ions. To this aim, herein thermally stable magnesium-strontium hydroxyapatite (HAMgSr) nanocrystals were synthesized and used for the fabrication of sintered highly porous scaffolds. The chemical and physical properties of the obtained scaffolds were analyzed by X-ray diffraction, scanning electron microscopy, mechanical testing. Three different substituting ions percentage were analyzed and among these, the copresence of Mg and Sr at 0.5 wt% has shown the best results in terms of thermal stability and mechanical properties. The potential utilization of these materials for bone regeneration purposes was preliminarily evaluated in vitro, by assaying proliferation of viable osteoblast-like cells; the experimental evidences suggest that the scaffolds can be exploited as bone-mimicking substrates suitable to support cell growth and proliferation. These observations underline the importance of the presence of Mg and Sr in scaffolds for bone remodeling as well as the good potential of the newly developed scaffolds for clinical use in tissue engineering.

Evaluation of in Vivo Response of Three Biphasic Scaffolds for Osteochondral Tissue Regeneration in a Sheep Model.

Osteochondral defects are a common problem in both human medicine and veterinary practice although with important limits concerning the cartilaginous tissue regeneration. Interest in the subchondral bone has grown, as it is now considered a key element in the osteochondral defect healing. The aim of this work was to generate and to evaluate the architecture of three cell-free scaffolds made of collagen, magnesium/hydroxyapatite and collagen hydroxyapatite/wollastonite to be implanted in a sheep animal model. Scaffolds were designed in a bilayer configuration and a novel "Honey" configuration, where columns of hydroxyapatite were inserted within the collagen matrix. The use of different types of scaffolds allowed us to identify the best scaffold in terms of integration and tissue regeneration. The animals included were divided into four groups: three were treated using different types of scaffold while one was left untreated and represented the control group. Evaluations were made at 3 months through CT analysis. The novel "Honey" configuration of the scaffold with hydroxyapatite seems to allow for a better reparative process, although we are still far from obtaining a complete restoration of the defect at this time point of follow-up.

Bioactive chitosan-based scaffolds with improved properties induced by dextran-grafted nano-maghemite and l-arginine amino acid.

Over the past years, fundamentals of magnetism opened a wide research area of interest, in the field of tissue engineering and regenerative medicine. The integration of magnetic nanoarchitectures into synthetic/natural scaffold formulations allowed obtaining "on demand" responsive structures able to guide the regeneration process. The aim of this work was the design and characterization of three-dimensional (3D) chitosan-based scaffolds containing dextran-grafted maghemite nanoarchitectures (DM) and functionalized with l-arginine (l-Arg) amino acid as bioactive agent. A homogeneous pore distribution and a high degree of interconnection were obtained for all the structures with DMs, which resulted well distributed inside the polymer matrix. All the results suggest that the simultaneous presence of DMs and l-Arg conferred interesting mechano-structural and bioactive properties toward osteoblast-like and human mesenchymal stem cells, differentially stimulating their proliferation both in the absence and in the presence of a time-dependent magnetic field. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1244-1252, 2019.

Influence of Nanofiber Orientation on Morphological and Mechanical Properties of Electrospun Chitosan Mats.

This work explored the use of chitosan (Cs) and poly(ethylene oxide) (PEO) blends for the fabrication of electrospun fiber-orientated meshes potentially suitable for engineering fiber-reinforced soft tissues such as tendons, ligaments, or meniscus. To mimic the fiber alignment present in native tissue, the CS/PEO blend solution was electrospun using a traditional static plate, a rotating drum collector, and a rotating disk collector to get, respectively, random, parallel, circumferential-oriented fibers. The effects of the different orientations (parallel or circumferential) and high-speed rotating collector influenced fiber morphology, leading to a reduction in nanofiber diameters and an improvement in mechanical properties.

Biomechanical evaluation of hMSCs-based engineered cartilage for chondral tissue regeneration.

Articular cartilage regeneration is still an open challenge in the field of tissue engineering. Although autologous chondrocytes seeded on collagen scaffolds (CSs) have already showed interesting results in the long-term repair of chondral lesions, they are not exempt from disadvantages that could be overcome using mesenchymal stem cells (MSCs). The ability of polymeric scaffolds to support MSCs proliferation and differentiation has been widely documented. However, few studies assessed their mechanical performances and additionally performing a single mechanical test, i.e. stress-strain or stress-relaxation in compression. Articular cartilage, though, possesses unique and multifaceted mechanical properties that can be exhaustively described only implementing a complete set of mechanical tests. Hence, the final aim of this study was to in depth assess the mechanical properties of human MSCs-cultured collagen scaffolds applying unconfined stress-strain, stress-relaxation and dynamic compression tests and identify key mechanical parameters. Firstly, plain CSs were fabricated and cultured under chondrogenic conditions with human MSCs (hMSCs). CSs displayed a high-interconnected porosity permitting uniform hMSCs distribution along the scaffold depth. Within CSs, hMSCs differentiated in chondroblasts, characterized by the presence of the lacunae and by a pericellular matrix positive for GAGs and for type 2 collagen deposition. The deep implemented mechanical characterization highlighted that the engineered constructs display (i) higher resistance to compression, (ii) more marked viscoelastic behavior over time and (iii) increased dynamic properties compared to naked CSs. In particular, stress-strain testes showed significant increase in the engineered constructs' stiffness that can be related to the proteoglycan deposition, observed by histology at the end of culture. Stress-relaxation and dynamic tests pointed out a substantial increase of peak and equilibrium stresses, relaxation time and dynamic modulus in the engineered constructs compared to empty CSs, suggesting a considerable decrease in scaffold permeability due to a strong chondral matrix deposition. Overall, the obtained results indicate a significant improvement of cell/CS mechanical performance toward a cartilage-like mechanical behavior.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons.

INTRODUCTION: An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. METHODS: The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. RESULTS: The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. CONCLUSION: We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.

Simplified preparation and characterization of magnetic hydroxyapatite-based nanocomposites.

Authors aimed to provide a magnetic responsiveness to bone-mimicking nano-hydroxyapatite (n-HA). For this purpose, dextran-grafted iron oxide nanoarchitectures (DM) were synthesized by a green-friendly and scalable alkaline co-precipitation method at room temperature and used to functionalize n-HA crystals. Different amounts of DM hybrid structures were added into the nanocomposites (DM/n-HA 1:1, 2:1 and 3:1weight ratio) which were investigated through extensive physicochemical (XRD, ICP, TGA and Zeta-potential), microstructural (TEM and DLS), magnetic (VSM) and biological analyses (MTT proliferation assay). X-ray diffraction patterns have confirmed the n-HA formation in the presence of DM as a co-reagent. Furthermore, the addition of DM during the synthesis does not affect the primary crystallite domains of DM/n-HA nanocomposites. DM/n-HAs have shown a rising of the magnetic moment values by increasing DM content up to 2:1 ratio. However, the magnetic moment value recorded in the DM/n-HA 3:1 do not further increase showing a saturation behaviour. The cytocompatibility of the DM/n-HA was evaluated with respect to the MG63 osteoblast-like cell line. Proliferation assays revealed that viability, carried out in the absence of external magnetic field, was not affected by the amount of DM employed. Interestingly, assays also suggested that the DM/n-HA nanocomposites exhibit a possible shielding effect with respect to the anti-proliferative activity induced by the DM particles alone.

Design and characterization of microcapsules-integrated collagen matrixes as multifunctional three-dimensional scaffolds for soft tissue engineering.

Three-dimensional (3D) porous scaffolds based on collagen are promising candidates for soft tissue engineering applications. The addition of stimuli-responsive carriers (nano- and microparticles) in the current approaches to tissue reconstruction and repair brings about novel challenges in the design and conception of carrier-integrated polymer scaffolds. In this study, a facile method was developed to functionalize 3D collagen porous scaffolds with biodegradable multilayer microcapsules. The effects of the capsule charge as well as the influence of the functionalization methods on the binding efficiency to the scaffolds were studied. It was found that the binding of cationic microcapsules was higher than that of anionic ones, and application of vacuum during scaffolds functionalization significantly hindered the attachment of the microcapsules to the collagen matrix. The physical properties of microcapsules-integrated scaffolds were compared to pristine scaffolds. The modified scaffolds showed swelling ratios, weight losses and mechanical properties similar to those of unmodified scaffolds. Finally, in vitro diffusional tests proved that the collagen scaffolds could stably retain the microcapsules over long incubation time in Tris-HCl buffer at 37°C without undergoing morphological changes, thus confirming their suitability for tissue engineering applications. The obtained results indicate that by tuning the charge of the microcapsules and by varying the fabrication conditions, collagen scaffolds patterned with high or low number of microcapsules can be obtained, and that the microcapsules-integrated scaffolds fully retain their original physical properties.