Effect of high content nanohydroxyapatite composite scaffolds prepared via melt extrusion additive manufacturing on the osteogenic differentiation of human mesenchymal stromal cells.

The field of bone tissue engineering seeks to mimic the bone extracellular matrix composition, balancing the organic and inorganic components. In this regard, additive manufacturing (AM) of high content calcium phosphate (CaP)-polymer composites holds great promise towards the design of bioactive scaffolds. Yet, the biological performance of such scaffolds is still poorly characterized. In this study, melt extrusion AM (ME-AM) was used to fabricate poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT)-nanohydroxyapatite (nHA) scaffolds with up to 45 wt% nHA, which presented significantly enhanced compressive mechanical properties, to evaluate their in vitro osteogenic potential as a function of nHA content. While osteogenic gene upregulation and matrix mineralization were observed on all scaffold types when cultured in osteogenic media, human mesenchymal stromal cells did not present an explicitly clear osteogenic phenotype, within the evaluated timeframe, in basic media cultures (i.e. without osteogenic factors). Yet, due to the adsorption of calcium and inorganic phosphate ions from cell culture media and simulated body fluid, the formation of a CaP layer was observed on PEOT/PBT-nHA 45 wt% scaffolds, which is hypothesized to account for their bone forming ability in the long term in vitro, and osteoconductivity in vivo.

Tuning Cell Behavior on 3D Scaffolds Fabricated by Atmospheric Plasma-Assisted Additive Manufacturing.

Three-dimensional (3D) scaffolds with optimum physicochemical properties are able to elicit specific cellular behaviors and guide tissue formation. However, cell-material interactions are limited in scaffolds fabricated by melt extrusion additive manufacturing (ME-AM) of synthetic polymers, and plasma treatment can be used to render the surface of the scaffolds more cell adhesive. In this study, a hybrid AM technology, which combines a ME-AM technique with an atmospheric pressure plasma jet, was employed to fabricate and plasma treat scaffolds in a single process. The organosilane monomer (3-aminopropyl)trimethoxysilane (APTMS) and a mixture of maleic anhydride and vinyltrimethoxysilane (MA-VTMOS) were used for the first time to plasma treat 3D scaffolds. APTMS treatment deposited plasma-polymerized films containing positively charged amine functional groups, while MA-VTMOS introduced negatively charged carboxyl groups on the 3D scaffolds' surface. Argon plasma activation was used as a control. All plasma treatments increased the surface wettability and protein adsorption to the surface of the scaffolds and improved cell distribution and proliferation. Notably, APTMS-treated scaffolds also allowed cell attachment by electrostatic interactions in the absence of serum. Interestingly, cell attachment and proliferation were not significantly affected by plasma treatment-induced aging. Also, while no significant differences were observed between plasma treatments in terms of gene expression, human mesenchymal stromal cells (hMSCs) could undergo osteogenic differentiation on aged scaffolds. This is probably because osteogenic differentiation is rather dependent on initial cell confluency and surface chemistry might play a secondary role.

3D additive manufactured composite scaffolds with antibiotic-loaded lamellar fillers for bone infection prevention and tissue regeneration.

Bone infections following open bone fracture or implant surgery remain a challenge in the orthopedics field. In order to avoid high doses of systemic drug administration, optimized local antibiotic release from scaffolds is required. 3D additive manufactured (AM) scaffolds made with biodegradable polymers are ideal to support bone healing in non-union scenarios and can be given antimicrobial properties by the incorporation of antibiotics. In this study, ciprofloxacin and gentamicin intercalated in the interlamellar spaces of magnesium aluminum layered double hydroxides (MgAl) and α-zirconium phosphates (ZrP), respectively, are dispersed within a thermoplastic polymer by melt compounding and subsequently processed via high temperature melt extrusion AM (~190 °C) into 3D scaffolds. The inorganic fillers enable a sustained antibiotics release through the polymer matrix, controlled by antibiotics counterions exchange or pH conditions. Importantly, both antibiotics retain their functionality after the manufacturing process at high temperatures, as verified by their activity against both Gram + and Gram - bacterial strains. Moreover, scaffolds loaded with filler-antibiotic do not impair human mesenchymal stromal cells osteogenic differentiation, allowing matrix mineralization and the expression of relevant osteogenic markers. Overall, these results suggest the possibility of fabricating dual functionality 3D scaffolds via high temperature melt extrusion for bone regeneration and infection prevention.

Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity.

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the biocompatible macromolecules dextran and Ficoll (Ficoll-Paque) were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran and Ficoll-Paque -based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration. STATEMENT OF SIGNIFICANCE: Additive manufacturing has emerged as one of the desired technologies to fabricate complex and patient-specific 3D scaffolds for bone regeneration. Along with the technology, new synthetic polymeric materials have been developed to meet processability requirements, as well as the mechanical properties and biocompatibility necessary for the application. Yet, there is still lack of methodology for a universal cell seeding method applicable to all additive manufactured 3D scaffolds regardless of their characteristics. We believe that our simple and reliable method, which is based on adjusting the cell settling velocity to aid cell attachment, could potentially help to maximize the efficiency, and therefore, functionality of cell-seeded constructs. This is of great importance when aiming for both in vitro and future clinical applications.

Nerve Cells Decide to Orient inside an Injectable Hydrogel with Minimal Structural Guidance.

Injectable biomaterials provide the advantage of a minimally invasive application but mostly lack the required structural complexity to regenerate aligned tissues. Here, we report a new class of tissue regenerative materials that can be injected and form an anisotropic matrix with controlled dimensions using rod-shaped, magnetoceptive microgel objects. Microgels are doped with small quantities of superparamagnetic iron oxide nanoparticles (0.0046 vol %), allowing alignment by external magnetic fields in the millitesla order. The microgels are dispersed in a biocompatible gel precursor and after injection and orientation are fixed inside the matrix hydrogel. Regardless of the low volume concentration of the microgels below 3%, at which the geometrical constrain for orientation is still minimum, the generated macroscopic unidirectional orientation is strongly sensed by the cells resulting in parallel nerve extension. This finding opens a new, minimal invasive route for therapy after spinal cord injury.