3D bioprinting of mouse pre-osteoblasts and human MSCs using bioinks consisting of gelatin and decellularized bone particles.

One of the key challenges in biofabrication applications is to obtain bioinks that provide a balance between printability, shape fidelity, cell viability, and tissue maturation. Decellularization methods allow the extraction of natural extracellular matrix, preserving tissue-specific matrix proteins. However, the critical challenge in bone decellularization is to preserve both organic (collagen, proteoglycans) and inorganic components (hydroxyapatite) to maintain the natural composition and functionality of bone. Besides, there is a need to investigate the effects of decellularized bone (DB) particles as a tissue-based additive in bioink formulation to develop functional bioinks. Here we evaluated the effect of incorporating DB particles of different sizes (≤45 and ≤100µm) and concentrations (1%, 5%, 10% (wt %)) into bioink formulations containing gelatin (GEL) and pre-osteoblasts (MC3T3-E1) or human mesenchymal stem cells (hTERT-MSCs). In addition, we propose a minimalistic bioink formulation using GEL, DB particles and cells with an easy preparation process resulting in a high cell viability. The printability properties of the inks were evaluated. Additionally, rheological properties were determined with shear thinning and thixotropy tests. The bioprinted constructs were cultured for 28 days. The viability, proliferation, and osteogenic differentiation capacity of cells were evaluated using biochemical assays and fluorescence microscopy. The incorporation of DB particles enhanced cell proliferation and osteogenic differentiation capacity which might be due to the natural collagen and hydroxyapatite content of DB particles. Alkaline phosphatase activity is increased significantly by using DB particles, notably, without an osteogenic induction of the cells. Moreover, fluorescence images display pronounced cell-material interaction and cell attachment inside the constructs. With these promising results, the present minimalistic bioink formulation is envisioned as a potential candidate for bone tissue engineering as a clinically translatable material with straightforward preparation and high cell activity.

Addition of High Acyl Gellan Gum to Low Acyl Gellan Gum Enables the Blends 3D Bioprintable.

Long-term stability of gellan gum (GG) at physiological conditions is expected, as very low concentration of divalent ions are required for crosslinking, as compared to alginate­which is extensively used for tissue engineering (TE) applications. Hence, GG is proposed as an ideal candidate to substitute alginate for TE. Deacylated (low acyl; LA) GG forms brittle gels, thus only low concentrations were used for cell encapsulation, whereas acylated (high acyl; HA) GG forms weak/soft gels. 3D bioprinting using pure LAGG or HAGG is not possible owing to their rheological properties. Here, we report development and characterization of bioprintable blends of LAGG and HAGG. Increase in HAGG in the blends improved shear recovery and shape fidelity of printed scaffolds. Low volumetric swelling observed in cell culture conditions over 14 days indicates stability. Volumetric scaffolds were successfully printed and their mechanical properties were determined by uniaxial compressive testing. Mesenchymal stem cells bioprinted in blends of 3% LAGG and 3% HAGG survived the printing process showing >80% viability; a gradual decrease in cell numbers was observed over 21 days of culture. However, exploiting intrinsic advantages of 3D bioprinting, LAGG/HAGG blends open up numerous possibilities to improve and/or tailor various aspects required for TE.

Controlled and Local Delivery of Antibiotics by 3D Core/Shell Printed Hydrogel Scaffolds to Treat Soft Tissue Infections.

Soft tissue infections in open fractures or burns are major cause for high morbidity in trauma patients. Sustained, long-term and localized delivery of antimicrobial agents is needed for early eradication of these infections. Traditional (topical or systemic) antibiotic delivery methods are associated with a variety of problems, including their long-term unavailability and possible low local concentration. Novel approaches for antibiotic delivery via wound coverage/healing scaffolds are constantly being developed. Many of these approaches are associated with burst release and thus seldom maintain long-term inhibitory concentrations. Using 3D core/shell extrusion printing, scaffolds consisting of antibiotic depot (in the core composed of low concentrated biomaterial ink 3% alginate) surrounded by a denser biomaterial ink (shell) were fabricated. Denser biomaterial ink (composed of alginate and methylcellulose or alginate, methylcellulose and Laponite) retained scaffold shape and modulated antibiotic release kinetics. Release of antibiotics was observed over seven days, indicating sustained release characteristics and maintenance of potency. Inclusion of Laponite in shell, significantly reduced burst release of antibiotics. Additionally, the effect of shell thickness on release kinetics was demonstrated. Amalgamation of such a modular delivery system with other biofabrication methods could potentially open new strategies to simultaneously treat soft tissue infections and aid wound regeneration.

3D Bioprinting of osteochondral tissue substitutes - in vitro-chondrogenesis in multi-layered mineralized constructs.

For the generation of multi-layered full thickness osteochondral tissue substitutes with an individual geometry based on clinical imaging data, combined extrusion-based 3D printing (3D plotting) of a bioink laden with primary chondrocytes and a mineralized biomaterial phase was introduced. A pasty calcium phosphate cement (CPC) and a bioink based on alginate-methylcellulose (algMC) - both are biocompatible and allow 3D plotting with high shape fidelity - were applied in monophasic and combinatory design to recreate osteochondral tissue layers. The capability of cells reacting to chondrogenic biochemical stimuli inside the algMC-based 3D hydrogel matrix was assessed. Towards combined osteochondral constructs, the chondrogenic fate in the presence of CPC in co-fabricated and biphasic mineralized pattern was evaluated. Majority of expanded and algMC-encapsulated cells survived the plotting process and the cultivation period, and were able to undergo redifferentiation in the provided environment to produce their respective extracellular matrix (ECM) components (i.e. sulphated glycosaminoglycans, collagen type II), examined after 3 weeks. The presence of a mineralized zone as located in the physiological calcified cartilage region suspected to interfere with chondrogenesis, was found to support chondrogenic ECM production by altering the ionic concentrations of calcium and phosphorus in in vitro culture conditions.

3D Plotted Biphasic Bone Scaffolds for Growth Factor Delivery: Biological Characterization In Vitro and In Vivo.

Bioprinting enables the integration of biological components into scaffolds during fabrication that has the advantage of high loading efficiency and better control of release and/or spatial positioning. In this study, a biphasic scaffold fabricated by extrusion-based 3D multichannel plotting of a calcium phosphate cement (CPC) paste and an alginate/gellan gum (AlgGG) hydrogel paste laden with the angiogenic factor VEGF (vascular endothelial growth factor) is investigated with regard to biological response in vitro and in vivo. Rat mesenchymal stromal cells are able to adhere and grow on both CPC and AlgGG strands, and differentiate toward osteoblasts. A sustained VEGF release is observed, which is able to stimulate endothelial cell proliferation as well as angiogenesis in vitro that indicates maintenance of its biological activity. After implantation into a segmental bone defect in the femur diaphysis of rats, a clear reduction of the defect size by newly formed bone tissue occurs from the distal and proximal ends of the host bone within 12 weeks. The CPC component shows excellent osteoconductivity whereas the local VEGF release from the AlgGG hydrogel gives rise to an enhanced vascularization of the defect region. This work contributes to the development of novel therapeutic concepts for improved bone regeneration which are based on 3D bioprinting.

* Central Growth Factor Loaded Depots in Bone Tissue Engineering Scaffolds for Enhanced Cell Attraction.

Tissue engineering, the application of stem and progenitor cells in combination with an engineered extracellular matrix, is a promising strategy for bone regeneration. However, its success is limited by the lack of vascularization after implantation. The concept of in situ tissue engineering envisages the recruitment of cells necessary for tissue regeneration from the host environment foregoing ex vivo cell seeding of the scaffold. In this study, we developed a novel scaffold system for enhanced cell attraction, which is based on biomimetic mineralized collagen scaffolds equipped with a central biopolymer depot loaded with chemotactic agents. In humid milieu, as after implantation, the signaling factors are expected to slowly diffuse out of the central depot forming a gradient that stimulates directed cell migration toward the scaffold center. Heparin, hyaluronic acid, and alginate have been shown to be capable of depot formation. By using vascular endothelial growth factor (VEGF) as model factor, it was demonstrated that the release kinetics can be adjusted by varying the depot composition. While alginate and hyaluronic acid are able to reduce the initial burst and prolong the release of VEGF, the addition of heparin led to a much stronger retention that resulted in an almost linear release over 28 days. The biological activity of released VEGF was proven for all variants using an endothelial cell proliferation assay. Furthermore, migration experiments with endothelial cells revealed a relationship between the degree of VEGF retention and migration distance: cells invaded deepest in scaffolds containing a heparin-based depot indicating that the formation of a steep gradient is crucial for cell attraction. In conclusion, this novel in situ tissue engineering approach, specifically designed to recruit and accommodate endogenous cells upon implantation, appeared highly promising to stimulate cell invasion, which in turn would promote vascularization and finally new bone formation.

Design and Fabrication of Complex Scaffolds for Bone Defect Healing: Combined 3D Plotting of a Calcium Phosphate Cement and a Growth Factor-Loaded Hydrogel.

Additive manufacturing enables the fabrication of scaffolds with defined architecture. Versatile printing technologies such as extrusion-based 3D plotting allow in addition the incorporation of biological components increasing the capability to restore functional tissues. We have recently described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of an oil-based CPC paste under mild conditions. In the present study, we have developed a strategy for growth factor loading based on multichannel plotting: a biphasic scaffold design was realised combining CPC with VEGF-laden, highly concentrated hydrogel strands. As hydrogel component, alginate and an alginate-gellan gum blend were evaluated; the blend exhibited a more favourable VEGF release profile and was chosen for biphasic scaffold fabrication. After plotting, two-step post-processing was performed for both, hydrogel crosslinking and CPC setting, which was shown to be compatible with both materials. Finally, a scaffold was designed and fabricated which can be applied for testing in a rat critical size femur defect. Optimization of CPC plotting enabled the fabrication of highly resolved structures with strand diameters of only 200 µm. Micro-computed tomography revealed a precise strand arrangement and an interconnected pore space within the biphasic scaffold even in swollen state of the hydrogel strands.

A versatile method for combining different biopolymers in a core/shell fashion by 3D plotting to achieve mechanically robust constructs.

Three-dimensional extrusion of two different biomaterials in a core/shell (c/s) fashion has gained much interest in the last couple of years as it allows for fabricating constructs with novel and interesting properties. We now demonstrate that combining high concentrated (16.7 wt%) alginate hydrogels as shell material with low concentrated, soft biopolymer hydrogels as core leads to mechanically stable and robust 3D scaffolds. Alginate, chitosan, gellan gum, gelatin and collagen hydrogels were utilized successfully as core materials-hydrogels which are too soft for 3D plotting of open-porous structures without an additional mechanical support. The respective c/s scaffolds were characterized concerning their morphology, mechanical properties and swelling behavior. It could be shown that core as well as shell part can be loaded with growth factors and that the release depends on core composition and shell thickness. Neither the plotting process nor the crosslinking with 1M CaCl2 denatured the proteins. When core and shell were loaded with different growth factors (VEGF and BMP-2, respectively) a dual release was achieved. Finally, live human endothelial cells were integrated in the core material, demonstrating that this new strategy can be used for bioprinting purposes as well.

Highly Concentrated Alginate-Gellan Gum Composites for 3D Plotting of Complex Tissue Engineering Scaffolds.

In tissue engineering, additive manufacturing (AM) technologies have brought considerable progress as they allow the fabrication of three-dimensional (3D) structures with defined architecture. 3D plotting is a versatile, extrusion-based AM technology suitable for processing a wide range of biomaterials including hydrogels. In this study, composites of highly concentrated alginate and gellan gum were prepared in order to combine the excellent printing properties of alginate with the favorable gelling characteristics of gellan gum. Mixtures of 16.7 wt % alginate and 2 or 3 wt % gellan gum were found applicable for 3D plotting. Characterization of the resulting composite scaffolds revealed an increased stiffness in the wet state (15%⁻20% higher Young's modulus) and significantly lower volume swelling in cell culture medium compared to pure alginate scaffolds (~10% vs. ~23%). Cytocompatibility experiments with human mesenchymal stem cells (hMSC) revealed that cell attachment was improved-the seeding efficiency was ~2.5⁻3.5 times higher on the composites than on pure alginate. Additionally, the composites were shown to support hMSC proliferation and early osteogenic differentiation. In conclusion, print fidelity of highly concentrated alginate-gellan gum composites was comparable to those of pure alginate; after plotting and crosslinking, the scaffolds possessed improved qualities regarding shape fidelity, mechanical strength, and initial cell attachment making them attractive for tissue engineering applications.

A Hydrogel Model Incorporating 3D-Plotted Hydroxyapatite for Osteochondral Tissue Engineering.

The concept of biphasic or multi-layered compound scaffolds has been explored within numerous studies in the context of cartilage and osteochondral regeneration. To date, no system has been identified that stands out in terms of superior chondrogenesis, osteogenesis or the formation of a zone of calcified cartilage (ZCC). Herein we present a 3D plotted scaffold, comprising an alginate and hydroxyapatite paste, cast within a photocrosslinkable hydrogel made of gelatin methacrylamide (GelMA), or GelMA with hyaluronic acid methacrylate (HAMA). We hypothesized that this combination of 3D plotting and hydrogel crosslinking would form a high fidelity, cell supporting structure that would allow localization of hydroxyapatite to the deepest regions of the structure whilst taking advantage of hydrogel photocrosslinking. We assessed this preliminary design in terms of chondrogenesis in culture with human articular chondrocytes, and verified whether the inclusion of hydroxyapatite in the form presented had any influence on the formation of the ZCC. Whilst the inclusion of HAMA resulted in a better chondrogenic outcome, the effect of HAP was limited. We overall demonstrated that formation of such compound structures is possible, providing a foundation for future work. The development of cohesive biphasic systems is highly relevant for current and future cartilage tissue engineering.

3D plotting of growth factor loaded calcium phosphate cement scaffolds.

Additive manufacturing allows to widely control the geometrical features of implants. Recently, we described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of a storable CPC paste based on water-immiscible carrier liquid. Plotting and hardening is conducted under mild conditions allowing the (precise and local) integration of biological components. In this study, we have developed a procedure for efficient loading of growth factors in the CPC scaffolds during plotting and demonstrated the feasibility of this approach. Bovine serum albumin (BSA) or vascular endothelial growth factor (VEGF), used as model proteins, were encapsulated in chitosan/dextran sulphate microparticles which could be easily mixed into the CPC paste in freeze-dried state. In order to prevent leaching of the proteins during cement setting, usually carried out by immersion in aqueous solutions, the plotted scaffolds were aged in water-saturated atmosphere (humidity). Setting in humidity avoided early loss of loaded proteins but provided sufficient amount of water to allow cement setting, as indicated by XRD analysis and mechanical testing in comparison to scaffolds set in water. Moreover, humidity-set scaffolds were characterised by altered, even improved properties: no swelling or crack formation was observed and accordingly, surface topography, total porosity and compressive modulus of the humidity-set scaffolds differed from those of the water-set counterparts. Direct cultivation of mesenchymal stem cells on the humidity-set scaffolds over 21days revealed their cytocompatibility. Maintenance of the bioactivity of VEGF during the fabrication procedure was proven in indirect and direct culture experiments with endothelial cells. STATEMENT OF SIGNIFICANCE: Additive manufacturing techniques allow the fabrication of implants with defined architecture (inner pore structure and outer shape). Especially printing technologies conducted under mild conditions allow additionally the (spatially controlled) integration of biological components such as drugs or growth factors. That enables the generation of individualized implants which can better meet the requirements of a patient and of tissue engineering constructs. To our knowledge, simultaneous printing of biological components was up to now only described for hydrogel/biopolymer-based materials which suffer from poor mechanical properties. In contrast, we have developed a procedure (based on 3D plotting of a calcium phosphate cement paste) for the fabrication of designed and growth factor loaded calcium-phosphate-based scaffolds applicable for bone regeneration.

Three-dimensional plotting is a versatile rapid prototyping method for the customized manufacturing of complex scaffolds and tissue engineering constructs.

OBJECTIVE: To review recent literature on three-dimensional (3-D) plotting as a rapid prototyping method for the manufacturing of patient specific biomaterial scaffolds and tissue engineering constructs. METHODS: Literature review and description of own recent work. RESULTS: In contrast to many other rapid prototyping technologies which can be used only for the processing of distinct materials, 3-D plotting can be utilized for all pasty biomaterials and therefore opens up many new options for the manufacturing of bi- or multiphasic scaffolds or even tissue engineering constructs, containing e. g. living cells. CONCLUSION: 3-D plotting is a rapid prototyping technology of growing importance which provides flexibility concerning choice of material and allows integration of sensitive biological components.