Biofunctionalization of 3D-printed silicone implants with immunomodulatory hydrogels for controlling the innate immune response: An in vivo model of tracheal defect repair.

The recent advances in 3D-printed silicone (PDMS: polydimethylsiloxane) implants present prospects for personalized implants with highly accurate anatomical conformity. However, a potential adverse effect, such as granuloma formation due to immune reactions, still exists. One potential way to overcome this problem is to control the implant/host interface using immunomodulatory coatings. In this study, a new cytokine cocktail composed of interleukin-10 and prostaglandin-E2 was designed to decrease adverse immune reactions and promote tissue integration by fixing macrophages into M2 pro-healing phenotype for an extended period of time. In vitro, the cytokine cocktail maintained low levels of pro-inflammatory cytokine (TNF-α and IL-6) secretions and induced the secretion of IL-10 and the upregulation of multifunctional scavenging and sorting receptor stabilin-1, expressed by M2 macrophages. This cocktail was then loaded in a gelatine-based hydrogel to develop an immunomodulatory material that could be used as a coating for medical devices. The efficacy of this coating was demonstrated in an in vivo rat model during the reconstruction of a tracheal defect by 3D-printed silicone implants. The coating was stable on the silicone implants for over 2 weeks, and the controlled release of the cocktail components was achieved for at least 14 days. In vivo, only 33% of the animals with bare silicone implants survived, whereas 100% of the animals survived with the implant equipped with the immunomodulatory hydrogel. The presence of the hydrogel and the cytokine cocktail diminished the thickness of the inflammatory tissue, the intensity of both acute and chronic inflammation, the overall fibroblastic reaction, the presence of oedema and the formation of fibrinoid (assessed by histology) and led to a 100% survival rate. At the systemic level, the presence of immunomodulatory hydrogels significantly decreased pro-inflammatory cytokines such as TNF-α, IFN-γ, CXCL1 and MCP-1 levels at day 7 and significantly decreased IL-1α, IL-1ß, CXCL1 and MCP-1 levels at day 21. The ability of this new immunomodulatory hydrogel to control the level of inflammation once applied to a 3D-printed silicone implant has been demonstrated. Such thin coatings can be applied to any implants or scaffolds used in tissue engineering to diminish the initial immune response, improve the integration and functionality of these materials and decrease potential complications related to their presence.

Improving the colonization and functions of Wharton's Jelly-derived mesenchymal stem cells by a synergetic combination of porous polyurethane scaffold with an albumin-derived hydrogel.

The development of neo-tissues assisted by artificial scaffolds is continually progressing, but the reproduction of the extracellular environment surrounding cells is quite complex. While synthetic scaffolds can support cell growth, they lack biochemical cues that can prompt cell proliferation or differentiation. In this study, Wharton's Jelly-derived mesenchymal stem cells are seeded on a polyurethane (PU) scaffold combined with a hydrogel based on bovine serum albumin (BSA). BSA hydrogel is obtained through thermal treatment. While such treatment leads to partial unfolding of the protein, we show that the extent of denaturation is small enough to maintain its bioactivity, such as protein binding. Therefore, BSA provides a suitable playground for cells inside the scaffold, allowing higher spreading, proliferation and matrix secretions. Furthermore, the poor mechanical properties of the hydrogel are compensated for by the porous PU scaffold, whose architecture is well controlled. We show that even though PU by itself can allow cell adhesion and protein secretion, cell proliferation is 3.5 times higher in the PU + BSA scaffolds as compared to pure PU after 21 d, along with the non-collagenous protein secretions (389 versus 134 µmmg -1). Conversely, the secretion of sulphated glycosaminoglycans is 12.3-fold higher in the scaffold made solely of PU. Thereby, we propose a simple approach to generating a hybrid material composed of a combination of PU and BSA hydrogel as a promising scaffold for tissue regeneration.

Modulation of Cellular Colonization of Porous Polyurethane Scaffolds via the Control of Pore Interconnection Size and Nanoscale Surface Modifications.

Full-scale cell penetration within porous scaffolds is required to obtain functional connective tissue components in tissue engineering applications. For this aim, we produced porous polyurethane structures with well-controlled pore and interconnection sizes. Although the influence of the pore size on cellular behavior is widely studied, we focused on the impact of the size of the interconnections on the colonization by NIH 3T3 fibroblasts and Wharton's jelly-derived mesenchymal stem cells (WJMSCs). To render the material hydrophilic and allow good material wettability, we treated the material either by plasma or by polydopamine (PDA) coating. We show that cells weakly adhere on these surfaces. Keeping the average pore diameter constant at 133 µm, we compare two structures, one with LARGE (52 µm) and one with SMALL (27 µm) interconnection diameters. DNA quantification and extracellular matrix (ECM) production reveal that larger interconnections is more suitable for cells to move across the scaffold and form a three-dimensional cellular network. We argue that LARGE interconnections favor cell communication between different pores, which then favors the production of the ECM. Moreover, PDA treatment shows a truly beneficial effect on fibroblast viability and on matrix production, whereas plasma treatment shows the same effect for WJMSCs. We, therefore, claim that both pore interconnection size and surface treatment play a significant role to improve the quality of integration of tissue engineering scaffolds.

Impact of surface chemistry and topography on the function of antigen presenting cells.

Antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs) play a crucial role in orchestrating immune responses against foreign materials. The activation status of APCs can determine the outcome of an immune response following implantation of synthetic materials, towards either healing or inflammation. A large range of biomaterials are used in the fabrication of implantable devices and drug delivery systems. These materials will be in close contact with APCs and characteristics such as surface chemistry and topography may have a critical role in initiating pro- or anti-inflammatory immune responses. Controlling biomaterial surface attributes provides a powerful tool for modulating the phenotype and function of immune cells with the aim of reducing detrimental pro-inflammatory responses and promoting beneficial healing responses. In this article, we review recent literature on how biomaterial surface topography and chemistry can modulate APC populations towards distinct pro- or anti-inflammatory phenotypes with specific examples of how these properties can be used to control host response in vivo. Topographical and/or chemical design of biomaterial surfaces with respect to the APC responses can pave the way for a new generation of 'cell instructive' materials with immunomodulatory properties with a wide range of clinical applications.

Double entrapment of growth factors by nanoparticles loaded into polyelectrolyte multilayer films.

Delivery of growth factors and control of vascularization are prominent problems in regenerative medicine. Vascular endothelial growth factor (VEGF) has been used both in vitro and in vivo to promote angiogenesis but due to its short half-life its controlled delivery is a sought after method. In this study we present a new concept of degradable drug loaded nanoparticles entrapped into exponentially growing multilayer films. Through hydrolysis of the nanoparticles, the drug can be delivered over long periods in a controlled manner. Poly(ε-caprolactone) nanoparticles were loaded with VEGF and in turn the release of VEGF from a surface is controlled by a thick layer-by-layer polyelectrolyte film. Direct loading of VEGF inside the film was not efficient for long-term applications. When VEGF loaded nanoparticles were introduced into the film, the particles were equally distributed inside and were stable after several washes. Moreover, the presence of the film sustained the release of VEGF for 7 days. Addition of the nanoparticles to the film promoted endothelial cell proliferation, mainly due to the presence of VEGF. Mechanical properties of the film (Young's moduli) were also improved by the presence of nanoparticles. However, in the presence of the film loaded with nanoparticles and without any direct contact with this film, endothelial cell growth was also enhanced on polystyrene and on Transwell insert surfaces which demonstrates the effectiveness of the nanoparticles not only to improve the mechanical properties of the film but also to deliver active VEGF. An increase in nitric oxide levels as an indicator of endothelial cell activity was monitored and was correlated with the release of VEGF from the nanoparticle/film platform. Finally, such a system can be used as an auxiliary delivery body within implants to finely control the release of bioactive agent containing nanoparticles.

Cell encapsulation within PVA-based hydrogels via freeze-thawing: a one-step scaffold formation and cell storage technique.

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA-based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue-engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA-gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze-thaw process can be utilized to prepare and store cell-laden hydrogels with adjustable mechanical properties.

Physically crosslinked composite hydrogels of PVA with natural macromolecules: structure, mechanical properties, and endothelial cell compatibility.

Polyvinyl alcohol (PVA) hydrogels have been considered potentially suitable for applications as engineered blood vessels because of their structure and mechanical properties. However, PVA's hydrophilicity hinders its capacity to act as a substrate for cell attachment. As a remedy, PVA was blended with chitosan, gelatin, or starch, and hydrogels were formed by subjecting the solutions to freeze-thaw cycles followed by coagulation bath immersion. The structure-property relationships for these hydrogels were examined by measurement of their swelling, rehydration, degradation, and mechanical properties. For the case of pure PVA hydrogels, the equilibrium swelling ratio was used to predict the effect of freeze thaw cycles and coagulation bath on average molecular weights between crosslinks and on mesh size. For all hydrogels, trends for the reswelling ratio, which is indicative of the crosslinked polymer fraction, were consistent with relative tensile properties. The coagulation bath treatment increased the degradation resistance of the hydrogels significantly. The suitability of each hydrogel for cell attachment and proliferation was examined by protein adsorption and bovine vascular endothelial cell culture experiments. Protein adsorption and cell proliferation was highest on the PVA/gelatin hydrogels. This study demonstrates that the potential of PVA hydrogels for artificial blood vessel applications can be improved by the addition of natural polymers, and that freeze-thawing and coagulation bath treatment can be utilized for fine adjustment of the physical characteristics.

EDC/NHS cross-linked collagen foams as scaffolds for artificial corneal stroma.

In this study, a highly porous collagen-based biodegradable scaffold was developed as an alternative to synthetic, non-degradable corneal implants. The developed method involved lyophilization and subsequent stabilization through N-ethyl-N'-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) cross-linking to yield longer lasting, porous scaffolds with a thickness similar to that of native cornea (500 microm). For collagen-based scaffolds, cross-linking is essential; however, it has direct effects on physical characteristics crucial for optimum cell behavior. Hence, the effect of cross-linking was studied by examining the influence of cross-linking on pore size distribution, bulk porosity and average pore size. After seeding the foam with human corneal keratocytes, cell proliferation, cell penetration into the scaffold and ECM production within the scaffold were studied. After a month of culture microscopical and immunohistochemical examinations showed that the foam structure did not undergo any significant loss of integrity, and the human corneal keratocytes populated the scaffold with cells migrating both longitudinally and laterally, and secreted some of the main constituents of the corneal ECM, namely collagen types I, V and VI. The foams had a layer of lower porosity (skin layer) both at the top and the bottom. Foams had an optimal porosity (93.6%), average pore size (67.7 microm), and chemistry for cell attachment and proliferation. They also had a sufficiently rapid degradation rate (73.6+/-1.1% in 4 weeks) and could be produced at a thickness close to that of the natural corneal stroma. Cells were seeded at the top surface of the foams and their numbers there was higher than the rest, basically due to the presence of the skin layer. This is considered to be an advantage when epithelial cells need to be seeded for the construction of hemi or full thickness cornea.