Gelatin methacrylate hydrogel with drug-loaded polymer microspheres as a new bioink for 3D bioprinting.

3D bioprinted hydrogel constructs are advanced systems of a great drug delivery application potential. One of the bioinks that has recently gained a lot of attention is gelatin methacrylate (GelMA) hydrogel exhibiting specific properties, including UV cross-linking possibility. The present study aimed to develop a new bioink composed of GelMA and gelatin modified by addition of polymer (polycaprolactone or polyethersulfone) microspheres serving as bioactive substance carriers. The prepared microspheres suspension in GelMA/gelatin bioink was successfully bioprinted and subjected to various tests, which showed that the addition of microspheres and their type affects the physicochemical properties of the printouts. The hydrogel stability and structure was examined using scanning electron and optical microscopy, its thermal properties with differential scanning calorimetry and thermogravimetric analysis and its biocompatibility on HaCaT cells using viability assay and electron microscopy. Analyses also included tests of hydrogel equilibrium swelling ratio and release of marker substance. Subsequently, the matrices were loaded with ampicillin and the antibiotic release was validated by monitoring the antibacterial activity on Staphylococcus aureus and Escherichia coli. It was concluded that GelMA/gelatin bioink is a good and satisfying material for potential medical use. Depending on the polymer used, the addition of microspheres improves its structure, thermal and drug delivery properties.

Electrospun UV-cross-linked polyvinylpyrrolidone fibers modified with polycaprolactone/polyethersulfone microspheres for drug delivery.

Electrospun fibers, often used as drug delivery systems, have two drawbacks - in the first stage of their action a sudden active substance burst release occurs and they have a relatively small capacity for a drug. In this work the fibers are modified by the addition of drug-loaded microspheres acting as micro-containers for the drug and increasing the total drug capacity of the system. Its release from such a structure is slowed down by placing the microspheres inside the fibers so they are covered with an outer layer of fiber-forming polymer. The work presents a new method (microsphere suspension electrospinning) of obtaining polyvinylpyrrolidone fibers cross-linked with UV light modified with polycaprolactone/polyethersulphone microspheres loaded with active substance - rhodamine 640 as a marker or ampicillin as a drug example. The influence of UV-cross-linking time and the microspheres addition on the degradation, mechanical strength and transport properties of fibrous mats was investigated. The mats were insoluble in water, in some cases mechanically stronger, their drug capacity was increased and the burst effect was eliminated. The antibacterial properties of ampicillin-loaded mats were confirmed. The product of proposed suspension electrospinning process has application potential as a drug delivery system.

3D printing of cellulose nanocrystals based composites to build robust biomimetic scaffolds for bone tissue engineering.

Cellulose nanocrystals (CNC) are drawing increasing attention in the fields of biomedicine and healthcare owing to their durability, biocompatibility, biodegradability and excellent mechanical properties. Herein, we fabricated using fused deposition modelling technology 3D composite scaffolds from polylactic acid (PLA) and CNC extracted from Ficus thonningii. Scanning electron microscopy revealed that the printed scaffolds exhibit interconnected pores with an estimated average pore size of approximately 400 µm. Incorporating 3% (w/w) of CNC into the composite improved PLA mechanical properties (Young's modulus increased by ~ 30%) and wettability (water contact angle decreased by ~ 17%). The mineralization process of printed scaffolds using simulated body fluid was validated and nucleation of hydroxyapatite confirmed. Additionally, cytocompatibility tests revealed that PLA and CNC-based PLA scaffolds are non-toxic and compatible with bone cells. Our design, based on rapid 3D printing of PLA/CNC composites, combines the ability to control the architecture and provide improved mechanical and biological properties of the scaffolds, which opens perspectives for applications in bone tissue engineering and in regenerative medicine.

Development of a new 3D bioprinted antibiotic delivery system based on a cross-linked gelatin-alginate hydrogel.

3D bioprinting uses bioink deposited directly on a collector to create any previously designed 3D model. One of the most common and the easiest to operate bioinks is gelatin-alginate hydrogel. The present study aimed to combine 3D bioprinting with different cross-linking techniques to develop a new stable and biodegradable gelatin-alginate hydrogel matrix for drug delivery applications. The matrix-building biopolymers were crosslinked by ionotropic gelation with Ca2+ ions, chemical crosslinking with GTA or a combination of the two crosslinkers at various concentrations. The influence of the crosslinking method on the hydrogel properties, stability and structure was examined using scanning electron and optical microscopy, differential scanning calorimetry and thermogravimetric analysis. Analyses included tests of hydrogel equilibrium swelling ratio and release of marker substance. Subsequently, biological properties of the matrices loaded with the antibiotic chlorhexidine were studied, including cytotoxicity on HaCAT cells and antibacterial activity on Staphylococcus aureus and Escherichia coli bacteria. The conducted study confirmed that the 3D bioprinted cross-linked drug-loaded alginate-gelatin hydrogel is a good and satisfying material for potential use as a drug delivery system.

Fabrication of Radio-Opaque and Macroporous Injectable Calcium Phosphate Cement.

The aim of this work was the development of injectable radio-opaque and macroporous calcium phosphate cement (CPC) to be used as a bone substitute for the treatment of pathologic vertebral fractures. A CPC was first rendered radio-opaque by the incorporation of zirconium dioxide (ZrO2). In order to create macroporosity, poly lactic-co-glycolic acid (PLGA) microspheres around 100 µm were homogeneously incorporated into the CPC as observed by scanning electron microscopy. Physicochemical analyses by X-ray diffraction and Fourier transform infrared spectroscopy confirmed the brushite phase of the cement. The mechanical properties of the CPC/PLGA cement containing 30% PLGA (wt/wt) were characterized by a compressive strength of 2 MPa and a Young's modulus of 1 GPa. The CPC/PLGA exhibited initial and final setting times of 7 and 12 min, respectively. Although the incorporation of PLGA microspheres increased the force necessary to inject the cement and decreased the percentage of injected mass as a function of time, the CPC/PLGA appeared fully injectable at 4 min. Moreover, in comparison with CPC, CPC/PLGA showed a full degradation in 6 weeks (with 100% mass loss), and this was associated with an acidification of the medium containing the CPC/PLGA sample (pH of 3.5 after 6 weeks). A cell viability test validated CPC/PLGA biocompatibility, and in vivo analyses using a bone defect assay in the caudal vertebrae of Wistar rats showed the good opacity of the CPC through the tail and a significant increased degradation of the CPC/PLGA cement a month after implantation. In conclusion, this injectable CPC scaffold appears to be an interesting material for bone substitution.

Fabrication of 3D printed antimicrobial polycaprolactone scaffolds for tissue engineering applications.

Synthetic polymers are widely employed for bone tissue engineering due to their tunable physical properties and biocompatibility. Inherently, most of these polymers display poor antimicrobial properties. Infection at the site of implantation is a major cause for failure or delay in bone healing process and the development of antimicrobial polymers is highly desired. In this study, silver nanoparticles (AgNps) were synthesized in polycaprolactone (PCL) solution by in-situ reduction and further extruded into PCL/AgNps filaments. Customized 3D structures were fabricated using the PCL/AgNps filaments through 3D printing technique. As demonstrated by scanning electron microscopy, the 3D printed scaffolds exhibited interconnected porous structures. Furthermore, X-ray photoelectron spectroscopy analysis revealed the reduction of silver ions. Transmission electron microscopy along with energy-dispersive X-ray spectroscopy analysis confirmed the formation of silver nanoparticles throughout the PCL matrix. In vitro enzymatic degradation studies showed that the PCL/AgNps scaffolds displayed 80% degradation in 20 days. The scaffolds were cytocompatible, as assessed using hFOB cells and their antibacterial activity was demonstrated on Escherichia coli. Due to their interconnected porous structure, mechanical and antibacterial properties, these cytocompatible multifunctional 3D printed PCL/AgNps scaffolds appear highly suitable for bone tissue engineering.

Development of new biocompatible 3D printed graphene oxide-based scaffolds.

The aim of this work was to develop a bioresorbable, biodegradable and biocompatible synthetic polymer with good mechanical properties for bone tissue engineering applications. Polylactic acid (PLA) scaffolds were generated by 3D printing using the fused deposition modelling method, and reinforced by incorporation of graphene oxide (GO). Morphological analysis by scanning electron microscopy indicated that the scaffold average pore size was between 400 and 500 µm. Topography imaging revealed a rougher surface upon GO incorporation (Sa = 5.8 µm for PLA scaffolds, and of 9.9 µm for PLA scaffolds with 0.2% GO), and contact angle measurements showed a transition from a hydrophobic surface (pure PLA scaffolds) to a hydrophilic surface after GO incorporation. PLA thermomechanical properties were enhanced by GO incorporation, as shown by the 70 °C increase of the degradation peak (thermal gravimetric analysis). However, GO incorporation did not change significantly the melting point assessed by differential scanning calorimetry. Physicochemical analyses by X-ray diffraction and Raman spectroscopy confirmed the filler presence. Tensile testing demonstrated that the mechanical properties were improved upon GO incorporation (30% increase of the Young's modulus with 0.3% GO). Cell viability, attachment, proliferation and differentiation assays using MG-63 osteosarcoma cells showed that PLA/GO scaffolds were biocompatible and that they promoted cell proliferation and mineralization more efficiently than pure PLA scaffolds. In conclusion, this new 3D printed nanocomposite is a promising scaffold with adequate mechanical properties and cytocompatibility which may allow bone formation.

Boron Nitride Based Nanobiocomposites: Design by 3D Printing for Bone Tissue Engineering.

Here, we produced a synthetic polymer having adequate biocompatibility, biodegradability, and bioresorbability, as well as mechanical properties for applications in bone tissue engineering. We used the fused deposition modeling (FDM) based 3D printing approach in order to produce biomimetic biodegradable scaffolds made of polylactic acid (PLA). We strengthened these scaffolds by addition of exfoliated boron nitride (EBN) as filler. We demonstrated the presence of EBN by physicochemical analysis using Raman spectroscopy and X-ray diffraction (XRD). Using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), we found that EBN incorporation did not influence the transition temperature, but reduced the polymer crystallinity. Scanning electron microscopy for morphology evaluation showed a mean scaffold pore size of 500 µm. EBN incorporation did not affect the scaffold mechanical properties (tensile test), but modified the surface roughness. Moreover, contact angle quantification indicated that the surface of PLA/EBN scaffolds was hydrophilic and that of PLA scaffolds hydrophobic. Finally, the results of the cytotoxicity, cell attachment, and proliferation experiments using MG-63 and MC3T3 cells indicated that PLA scaffolds filled with EBN were nontoxic and compatible with osteoblastic cells and also promoted the scaffold mineralization by MG-63 cells. Altogether, our results suggest that this 3D printed nanocomposite scaffold is suitable for tissue engineering.

Design of Boron Nitride/Gelatin Electrospun Nanofibers for Bone Tissue Engineering.

Gelatin is a biodegradable biopolymer obtained by collagen denaturation, which shows poor mechanical properties. Hence, improving its mechanical properties is very essential toward the fabrication of efficient nontoxic material for biomedical applications. For this aim, various methods are employed using external fillers such as ceramics or bioglass. In this report, we introduce boron nitride (BN)-reinforced gelatin as a new class of two-dimensional biocompatible nanomaterials. The effect of the nanofiller on the mechanical behavior is analyzed. BN is efficiently exfoliated using the biopolymer gelatin as shown through Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The exfoliated BN reinforces gelatin electrospun fibers, which results in an increase in the Young's modulus. The Electrospun Mats (ESM) are stable after the glutaraldehyde cross-linking, and the fibrous morphology is preserved. The cross-linked gelatin/BN ESM is highly bioactive in forming bonelike hydroxyapatite as shown by scanning electron microscopy. Due to their enhanced mineralization ability, the cross-linked ESM have been tested on human bone cells (HOS osteosarcoma cell line). The cell attachment, proliferation, and biocompatibility results show that the ESM are nontoxic and biodegradable. The analysis of osteoblast gene expression and the measurement of alkaline phosphatase activity confirm that these materials are suitable for bone tissue engineering.