Poly(dl-lactide) Polymer Blended with Mineral Phases for Extrusion 3D Printing-Studies on Degradation and Biocompatibility.

A promising therapeutic option for the treatment of critical-size mandibular defects is the implantation of biodegradable, porous structures that are produced patient-specifically by using additive manufacturing techniques. In this work, degradable poly(DL-lactide) polymer (PDLLA) was blended with different mineral phases with the aim of buffering its acidic degradation products, which can cause inflammation and stimulate bone regeneration. Microparticles of CaCO3, SrCO3, tricalcium phosphates (α-TCP, ß-TCP), or strontium-modified hydroxyapatite (SrHAp) were mixed with the polymer powder following processing the blends into scaffolds with the Arburg Plastic Freeforming 3D-printing method. An in vitro degradation study over 24 weeks revealed a buffer effect for all mineral phases, with the buffering capacity of CaCO3 and SrCO3 being the highest. Analysis of conductivity, swelling, microstructure, viscosity, and glass transition temperature evidenced that the mineral phases influence the degradation behavior of the scaffolds. Cytocompatibility of all polymer blends was proven in cell experiments with SaOS-2 cells. Patient-specific implants consisting of PDLLA + CaCO3, which were tested in a pilot in vivo study in a segmental mandibular defect in minipigs, exhibited strong swelling. Based on these results, an in vitro swelling prediction model was developed that simulates the conditions of anisotropic swelling after implantation.

Biofabrication's Contribution to the Evolution of Cultured Meat.

Cultured Meat (CM) is a growing field in cellular agriculture, driven by the environmental impact of conventional meat production, which contributes to climate change and occupies ≈70% of arable land. As demand for meat alternatives rises, research in this area expands. CM production relies on tissue engineering techniques, where a limited number of animal cells are cultured in vitro and processed to create meat-like tissue comprising muscle and adipose components. Currently, CM is primarily produced on a small scale in pilot facilities. Producing a large cell mass based on suitable cell sources and bioreactors remains challenging. Advanced manufacturing methods and innovative materials are required to subsequently process this cell mass into CM products on a large scale. Consequently, CM is closely linked with biofabrication, a suite of technologies for precisely arranging cellular aggregates and cell-material composites to construct specific structures, often using robotics. This review provides insights into contemporary biomedical biofabrication technologies, focusing on significant advancements in muscle and adipose tissue biofabrication for CM production. Novel materials for biofabricating CM are also discussed, emphasizing their edibility and incorporation of healthful components. Finally, initial studies on biofabricated CM are examined, addressing current limitations and future challenges for large-scale production.

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Multimodal additive manufacturing of biomimetic tympanic membrane replacements with near tissue-like acousto-mechanical and biological properties.

The three additive manufacturing techniques fused deposition modeling, gel plotting and melt electrowriting were combined to develop a mimicry of the tympanic membrane (TM) to tackle large TM perforations caused by chronic otitis media. The mimicry of the collagen fiber orientation of the TM was accompanied by a study of multiple funnel-shaped mimics of the TM morphology, resulting in mechanical and acoustic properties similar to those of the eardrum. For the different 3D printing techniques used, the process parameters were optimized to allow reasonable microfiber arrangements within the melt electrowriting setup. Interestingly, the fiber pattern was less important for the acousto-mechanical properties than the overall morphology. Furthermore, the behavior of keratinocytes and fibroblasts is crucial for the repair of the TM, and an in vitro study showed a high biocompatibility of both primary cell types while mimicking the respective cell layers of the TM. A simulation of the in vivo ingrowth of both cell types resulted in a cell growth orientation similar to the original collagen fiber orientation of the TM. Overall, the combined approach showed all the necessary parameters to support the growth of a neo-epithelial layer with a similar structure and morphology to the original membrane. It therefore offers a suitable alternative to autologous materials for the treatment of chronic otitis media. STATEMENT OF SIGNIFICANCE: Millions of people worldwide suffer from chronic middle ear infections. Although the tympanic membrane (TM) can be reconstructed with autologous materials, the grafts used for this purpose require extensive manual preparation during surgery. This affects not only the hearing ability but also the stability of the reconstructed TM, especially in the case of full TM reconstruction. The synthetic alternative presented here mimicked not only the fibrous structure of the TM but also its morphology, resulting in similar acousto-mechanical properties. Furthermore, its high biocompatibility supported the migration of keratinocytes and fibroblasts to form a neo-epithelial layer. Overall, this completely new TM replacement was achieved by combining three different additive manufacturing processes.

A comparative analysis of 3D printed scaffolds consisting of poly(lactic-co-glycolic) acid and different bioactive mineral fillers: aspects of degradation and cytocompatibility.

Their excellent mechanical properties, degradability and suitability for processing by 3D printing technologies make the thermoplastic polylactic acid and its derivatives favourable candidates for biomaterial-based bone regeneration therapies. In this study, we investigated whether bioactive mineral fillers, which are known to promote bone healing based on their dissolution products, can be integrated into a poly(L-lactic-co-glycolic) acid (PLLA-PGA) matrix and how key characteristics of degradation and cytocompatibility are influenced. The polymer powder was mixed with particles of CaCO3, SrCO3, strontium-modified hydroxyapatite (SrHAp) or tricalcium phosphates (α-TCP, ß-TCP) in a mass ratio of 90 : 10; the resulting composite materials have been successfully processed into scaffolds by the additive manufacturing method Arburg Plastic Freeforming (APF). Degradation of the composite scaffolds was investigated in terms of dimensional change, bioactivity, ion (calcium, phosphate, strontium) release/uptake and pH development during long-term (70 days) incubation. The mineral fillers influenced the degradation behavior of the scaffolds to varying degrees, with the calcium phosphate phases showing a clear buffer effect and an acceptable dimensional increase. The amount of 10 wt% SrCO3 or SrHAp particles did not appear to be appropriate to release a sufficient amount of strontium ions to exert a biological effect in vitro. Cell culture experiments with the human osteosarcoma cell line SAOS-2 and human dental pulp stem cells (hDPSC) indicated the high cytocompatibility of the composites: For all material groups cell spreading and complete colonization of the scaffolds over the culture period of 14 days as well as an increase of the specific alkaline phosphatase activity, typical for osteogenic differentiation, were observed.

Treatment of critical bone defects using calcium phosphate cement and mesoporous bioactive glass providing spatiotemporal drug delivery.

Calcium phosphate cements (CPC) are currently widely used bone replacement materials with excellent bioactivity, but have considerable disadvantages like slow degradation. For critical-sized defects, however, an improved degradation is essential to match the tissue regeneration, especially in younger patients who are still growing. We demonstrate that a combination of CPC with mesoporous bioactive glass (MBG) particles led to an enhanced degradation in vitro and in a critical alveolar cleft defect in rats. Additionally, to support new bone formation the MBG was functionalized with hypoxia conditioned medium (HCM) derived from rat bone marrow stromal cells. HCM-functionalized scaffolds showed an improved cell proliferation and the highest formation of new bone volume. This highly flexible material system together with the drug delivery capacity is adaptable to patient specific needs and has great potential for clinical translation.

Egg white improves the biological properties of an alginate-methylcellulose bioink for 3D bioprinting of volumetric bone constructs.

Three-dimensional microextrusion bioprinting has attracted great interest for fabrication of hierarchically structured, functional tissue substitutes with spatially defined cell distribution. Despite considerable progress, several significant limitations remain such as a lack of suitable bioinks which combine favorable cell response with high shape fidelity. Therefore, in this work a novel bioink of alginate-methylcellulose (AlgMC) blend functionalized with egg white (EW) was developed with the aim of solving this limitation. In this regard, a stepwise strategy was proposed to improve and examine the cell response in low-viscosity alginate inks (3%, w/v) with different EW concentrations, and in high-viscosity inks after gradual methylcellulose addition for enhancing printability. The rheological properties and printability of these cell-responsive bioinks were characterized to obtain an optimized formulation eliciting balanced physicochemical and biological properties for fabrication of volumetric scaffolds. The bioprinted AlgMC + EW constructs exhibited excellent shape fidelity while encapsulated human mesenchymal stem cells showed high post-printing viability as well as adhesion and spreading within the matrix. In a proof-of-concept experiment, the impact of these EW-mediated effects on osteogenesis of bioprinted primary human pre-osteoblasts (hOB) was evaluated. Results confirmed a high viability of hOB (93.7 ± 0.15%) post-fabrication in an EW-supported AlgMC bioink allowing cell adhesion, proliferation and migration. EW even promoted the expression of osteogenic genes, coding for bone sialoprotein (integrin binding sialoprotein/bone sialoprotein precursor (IBSP)) and osteocalcin (BGLAP) on mRNA level. To demonstrate the suitability of the novel ink for future fabrication of multi-zonal bone substitutes, AlgMC + EW was successfully co-printed together with a pasty calcium phosphate bone cement biomaterial ink to achieve a partly mineralized 3D volumetric environment with good cell viability and spreading. Along with the EW-mediated positive effects within bioprinted AlgMC-based scaffolds, this highlighted the promising potential of this novel ink for biofabrication of bone tissue substitutes in clinically relevant dimensions.

Composites consisting of calcium phosphate cements and mesoporous bioactive glasses as a 3D plottable drug delivery system.

Calcium phosphate cements (CPC) and mesoporous bioactive glasses (MBG) are two well studied biomaterial groups widely under investigation on their applicability to treat bone defects in orthopaedics and maxillofacial surgery. Recently the extrusion properties of CPC-MBG composites using a pasty CPC based on a hydrophobic carrier-liquid were studied in our group demonstrating that such composites are suitable for low temperature 3D plotting. Based on this work, we show in this study that by variation of the MBG content in the composite the degradation of the final scaffolds can be influenced. Furthermore, by modifying the cement phase and/or the MBG with therapeutically active ions like strontium, the released ion concentration can be varied over a wide range. In a second step the MBG was functionalized exploiting the high specific surface area of the glass as a carrier system for proteins like lysozyme or grow factors. We developed a protocol that allows the incorporation of protein-laden MBG in CPC pastes without impairing the extrudability of the CPC-MBG composites. Additionally, we found that released proteins from pure MBG or 3D plotted composite-scaffolds maintained their biological activity. Therefore, the combination of CPC and MBG allows the creation of a highly flexible composite system making it a promising candidate for bone tissue engineering. STATEMENT OF SIGNIFICANCE: Calcium phosphate cements and mesoporous bioactive glasses are two promising degradable biomaterials for the regenerative treatment of bone defects. The combination of both materials to a 3D printable composite enables the creation of implants with patient specific geometry. By varying the composition of the composite, the degradation behaviour can be influenced and especially the release of therapeutically active ions is tailorable over a wide range. We demonstrated this for strontium, as it has been shown to stimulate bone formation. Moreover, the bioactive glass can be used as a carrier system for drugs or growth factors and we show the successful combination of such functionalised glass particles and a cement paste without affecting the printability.

Evaluation of an Injectable Biphasic Calcium Sulfate/Hydroxyapatite Cement for the Augmentation of Fenestrated Pedicle Screws in Osteoporotic Vertebrae: A Biomechanical Cadaver Study.

Cement augmentation of pedicle screws is one of the most promising approaches to enhance the anchoring of screws in the osteoporotic spine. To date, there is no ideal cement for pedicle screw augmentation. The purpose of this study was to investigate whether an injectable, bioactive, and degradable calcium sulfate/hydroxyapatite (CaS/HA) cement could increase the maximum pull-out force of pedicle screws in osteoporotic vertebrae. Herein, 17 osteoporotic thoracic and lumbar vertebrae were obtained from a single fresh-frozen human cadaver and instrumented with fenestrated pedicle screws. The right screw in each vertebra was augmented with CaS/HA cement and the un-augmented left side served as a paired control. The cement distribution, interdigitation ability, and cement leakage were evaluated using radiographs. Furthermore, pull-out testing was used to evaluate the immediate mechanical effect of CaS/HA augmentation on the pedicle screws. The CaS/HA cement presented good distribution and interdigitation ability without leakage into the spinal canal. Augmentation significantly enhanced the maximum pull-out force of the pedicle screw in which the augmented side was 39.0% higher than the pedicle-screw-alone side. Therefore, the novel biodegradable biphasic CaS/HA cement could be a promising material for pedicle screw augmentation in the osteoporotic spine.

Treatment of Critical Size Femoral Bone Defects with Biomimetic Hybrid Scaffolds of 3D Plotted Calcium Phosphate Cement and Mineralized Collagen Matrix.

To treat critical-size bone defects, composite materials and tissue-engineered bone grafts play important roles in bone repair materials. The purpose of this study was to investigate the bone regenerative potential of hybrid scaffolds consisting of macroporous calcium phosphate cement (CPC) and microporous mineralized collagen matrix (MCM). Hybrid scaffolds were synthetized by 3D plotting CPC and then filling with MCM (MCM-CPC group) and implanted into a 5 mm critical size femoral defect in rats. Defects left empty (control group) as well as defects treated with scaffolds made of CPC only (CPC group) and MCM only (MCM group) served as controls. Eight weeks after surgery, micro-computed tomography scans and histological analysis were performed to analyze the newly formed bone, the degree of defect healing and the activity of osteoclasts. Mechanical stability was tested by 3-point-bending of the explanted femora. Compared with the other groups, more newly formed bone was found within MCM-CPC scaffolds. The new bone tissue had a clamp-like structure which was fully connected to the hybrid scaffolds and thereby enhanced the biomechanical strength. Together, the biomimetic hybrid MCM-CPC scaffolds enhanced bone defect healing by improved osseointegration and their differentiated degradation provides spatial effects in the process of critical-bone defect healing.

Core-shell bioprinting as a strategy to apply differentiation factors in a spatially defined manner inside osteochondral tissue substitutes.

One of the key challenges in osteochondral tissue engineering is to define specified zones with varying material properties, cell types and biochemical factors supporting locally adjusted differentiation into the osteogenic and chondrogenic lineage, respectively. Herein, extrusion-based core-shell bioprinting is introduced as a potent tool allowing a spatially defined delivery of cell types and differentiation factors TGF-ß3 and BMP-2 in separated compartments of hydrogel strands, and, therefore, a local supply of matching factors for chondrocytes and osteoblasts. Ink development was based on blends of alginate and methylcellulose, in combination with varying concentrations of the nanoclay Laponite whose high affinity binding capacity for various molecules was exploited. Release kinetics of model molecules was successfully tuned by Laponite addition. Core-shell bioprinting was proven to generate well-oriented compartments within one strand as monitored by optical coherence tomography in a non-invasive manner. Chondrocytes and osteoblasts were applied each in the shell while the respective differentiation factors (TGF-ß3, BMP-2) were provided by a Laponite-supported core serving as central factor depot within the strand, allowing directed differentiation of cells in close contact to the core. Experiments with bi-zonal constructs, comprising an osteogenic and a chondrogenic zone, revealed that the local delivery of the factors from the core reduces effects of these factors on the cells in the other scaffold zone. These observations prove the general suitability of the suggested system for co-differentiation of different cell types within a zonal construct.

Homogeneous and Reproducible Mixing of Highly Viscous Biomaterial Inks and Cell Suspensions to Create Bioinks.

Highly viscous bioinks offer great advantages for the three-dimensional fabrication of cell-laden constructs by microextrusion printing. However, no standardised method of mixing a high viscosity biomaterial ink and a cell suspension has been established so far, leading to non-reproducible printing results. A novel method for the homogeneous and reproducible mixing of the two components using a mixing unit connecting two syringes is developed and investigated. Several static mixing units, based on established mixing designs, were adapted and their functionality was determined by analysing specific features of the resulting bioink. As a model system, we selected a highly viscous ink consisting of fresh frozen human blood plasma, alginate, and methylcellulose, and a cell suspension containing immortalized human mesenchymal stem cells. This bioink is crosslinked after fabrication. A pre-crosslinked gellan gum-based bioink providing a different extrusion behaviour was introduced to validate the conclusions drawn from the model system. For characterisation, bioink from different zones within the mixing device was analysed by measurement of its viscosity, shape fidelity after printing and visual homogeneity. When taking all three parameters into account, a comprehensive and reliable comparison of the mixing quality was possible. In comparison to the established method of manual mixing inside a beaker using a spatula, a significantly higher proportion of viable cells was detected directly after mixing and plotting for both bioinks when the mixing unit was used. A screw-like mixing unit, termed "HighVisc", was found to result in a homogenous bioink after a low number of mixing cycles while achieving high cell viability rates.

3D bioprinting of hepatocytes: core-shell structured co-cultures with fibroblasts for enhanced functionality.

With the aim of understanding and recapitulating cellular interactions of hepatocytes in their physiological microenvironment and to generate an artificial 3D in vitro model, a co-culture system using 3D extrusion bioprinting was developed. A bioink based on alginate and methylcellulose (algMC) was first shown to be suitable for bioprinting of hepatocytes; the addition of Matrigel to algMC enhanced proliferation and morphology of them in monophasic scaffolds. Towards a more complex system that allows studying cellular interactions, we applied core-shell bioprinting to establish tailored 3D co-culture models for hepatocytes. The bioinks were specifically functionalized with natural matrix components (based on human plasma, fibrin or Matrigel) and used to co-print fibroblasts and hepatocytes in a spatially defined, coaxial manner. Fibroblasts acted as supportive cells for co-cultured hepatocytes, stimulating the expression of certain biomarkers of hepatocytes like albumin. Furthermore, matrix functionalization positively influenced both cell types in their respective compartments by enhancing their adhesion, viability, proliferation and function. In conclusion, we established a functional co-culture model with independently tunable compartments for different cell types via core-shell bioprinting. This provides the basis for more complex in vitro models allowing co-cultivation of hepatocytes with other liver-specific cell types to closely resemble the liver microenvironment.

Toward Biofabrication of Resorbable Implants Consisting of a Calcium Phosphate Cement and Fibrin-A Characterization In Vitro and In Vivo.

Cleft alveolar bone defects can be treated potentially with tissue engineered bone grafts. Herein, we developed novel biphasic bone constructs consisting of two clinically certified materials, a calcium phosphate cement (CPC) and a fibrin gel that were biofabricated using 3D plotting. The fibrin gel was loaded with mesenchymal stromal cells (MSC) derived from bone marrow. Firstly, the degradation of fibrin as well as the behavior of cells in the biphasic system were evaluated in vitro. Fibrin degraded quickly in presence of MSC. Our results showed that the plotted CPC structure acted slightly stabilizing for the fibrin gel. However, with passing time and fibrin degradation, MSC migrated to the CPC surface. Thus, the fibrin gel could be identified as cell delivery system. A pilot study in vivo was conducted in artificial craniofacial defects in Lewis rats. Ongoing bone formation could be evidenced over 12 weeks but the biphasic constructs were not completely osseous integrated. Nevertheless, our results show that the combination of 3D plotted CPC constructs and fibrin as suitable cell delivery system enables the fabrication of novel regenerative implants for the treatment of alveolar bone defects.

Biomimetic Tympanic Membrane Replacement Made by Melt Electrowriting.

The tympanic membrane (TM) transfers sound waves from the air into mechanical motion for the ossicular chain. This requires a high sensitivity to small dynamic pressure changes and resistance to large quasi-static pressure differences. The TM achieves this by providing a layered structure of about 100µm in thickness, a low flexural stiffness, and a high tensile strength. Chronically infected middle ears require reconstruction of a large area of the TM. However, current clinical treatment can cause a reduction in hearing. With the novel additive manufacturing technique of melt electrowriting (MEW), it is for the first time possible to fabricate highly organized and biodegradable membranes within the dimensions of the TM. Scaffold designs of various fiber composition are analyzed mechanically and acoustically. It can be demonstrated that by customizing fiber orientation, fiber diameter, and number of layers the desired properties of the TM can be met. An applied thin collagen layer seals the micropores of the MEW-printed membrane while keeping the favorable mechanical and acoustical characteristics. The determined properties are beneficial for implantation, closely match those of the human TM, and support the growth of a neo-epithelial layer. This proves the possibilities to create a biomimimetic TM replacement using MEW.

Bioprinting of Magnetically Deformable Scaffolds.

Mechanical stimulation of cells embedded in scaffolds is known to increase the cellular performance toward osteogenic or chondrogenic differentiation and tissue development. Three-dimensional bioplotting of magnetically deformable scaffolds enables the spatially defined distribution of magnetically inducible scaffold regions. In this study, a magnetic bioink based on alginate (alg, 3%) and methylcellulose (MC, 9%) with incorporated magnetite microparticles (25% w/w) was developed and characterized. The size and shape of particles were monitored via scanning electron microscopy and X-ray micro-computed tomography. Shear-thinning properties of the algMC ink were maintained after the addition of different concentrations of magnetite microparticles to the ink. Its viscosity proportionally increased with the added amount of magnetite, and so did the level of saturation magnetization as determined via vibrating sample magnetometry. The printability and shape fidelity of various shapes were evaluated, so that the final composition of algMC + 25% w/w magnetite was chosen. With application of this ink, cytocompatibility was proven in indirect cell culture and bioplotting experiments using a human mesenchymal stem cell line. Toward the deformation of cell-laden scaffolds to support cell differentiation in the future, radiography allowed the real-time monitoring of magnetically induced deformation of scaffolds of different pore architectures and scaffold orientations inside the magnetic field. Varying the strand distance and scaffold design will allow fine-tuning the degree of deformation in stimulatory experiments.

Composite Bioinks With Mesoporous Bioactive Glasses-A Critical Evaluation of Results Obtained by In Vitro Experiments.

Besides osteoconductivity and a high degradation rate, mesoporous bioactive glasses (MBGs) are specific for their highly ordered channel structure and high specific surface area, making them suitable as drug and/or growth factor delivery systems. On the other hand, the mesoporous channel structure and MBG composition can have an effect on common cell evaluation assays, leading to inconclusive results. This effect is especially important when MBG is mixed in composite bioinks, together with cells. Additionally, the hydrogel component of the ink can influence the degradation of MBG, leading to different ion releases, which can additionally affect the analyses. Hence, our aim here was to show how the MBG structure and composition influence common cell viability and differentiation assays when calcium (Ca)- or magnesium (Mg)-containing glass is part of an alginate-based composite bioink. We suggested pre-labeling of cells with DiI prior to bioprinting and staining with calcein-AM to allow identification of metabolically active cells expressing signals in both green and red channels, allowing the use of fluorescence imaging for cell viability evaluations in the presence of high amounts (7 wt %) of MBGs. The release and uptake of ions during degradation of CaMBG and MgMBG were significantly changed by alginate in the composite bioinks, as confirmed by higher release and uptake from bulk glasses. Additionally, we detected a burst release of Mg2+ from composites only after 24 h of incubation. Furthermore, we demonstrated that released ions and the mesoporous channel structure affect the measurement of lactate dehydrogenase (LDH) and alkaline phosphatase activity (ALP) in bioprinted composite scaffolds. Measured LDH activity was significantly decreased in the presence of CaMBG. On the other hand, the presence of MgMBG induced increased signal measured for the ALP. Taken together, our findings show how composite bioinks containing MBGs can interfere with common analyses, obtaining misleading results.

Uniaxial compression testing and Cauchy stress modeling to design anatomical silicone replicas.

Anatomically realistic organ replicas or phantoms allow for accurate studies and reproducible research. To recreate a human kidney, mimicry of the elastic properties of the human kidney is crucial. However, none of the related work addressed the design and development of a kidney phantom using only silicone as material. In contrast to paraffin and hydrogel, silicone is an ideal variant for its extended shelf life, soft-tissue-like feeling, and viscoelastic modularity. To this end, we conducted Uniaxial Compression testing and Cauchy stress modeling. Results indicate that none of the available manufacturer silicone brands are suitable for the task of creating a realistic kidney phantom. Indeed, the tested silicone mixtures in low and high strain fall outside the required approximate target compressive moduli of 20 kPa and 500 kPa, respectively. This work provides a frame of reference for future work by avoiding the pitfalls of the selected ready-made silicones and reusing the reported theoretical and experimental setup to design a realistic replica of the kidney organ.

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 Printing of Bone Grafts for Cleft Alveolar Osteoplasty - In vivo Evaluation in a Preclinical Model.

One of the most common hereditary craniofacial anomalies in humans are cleft lip and cleft alveolar bone with or without cleft palate. Current clinical practice, the augmentation of the persisting alveolar bone defect by using autologous bone grafts, has considerable disadvantages motivating to an intensive search for alternatives. We developed a novel therapy concept based on 3D printing of biodegradable calcium phosphate-based materials and integration of osteogenic cells allowing fabrication of patient-specific, tissue-engineered bone grafts. Objective of the present study was the in vivo evaluation of implants in a rat alveolar cleft model. Scaffolds were designed according to the defect's geometry with two different pore designs (60° and 30° rotated layer orientation) and produced by extrusion-based 3D plotting of a pasty calcium phosphate cement. The scaffolds filled into the artificial bone defect in the palate of adult Lewis rats, showing a good support. Half of the scaffolds were colonized with rat mesenchymal stromal cells (rMSC) prior to implantation. After 6 and 12 weeks, remaining defect width and bone formation were quantified histologically and by microCT. The results revealed excellent osteoconductive properties of the scaffolds, a significant influence of the pore geometry (60° > 30°), but no enhanced defect healing by pre-colonization with rMSC.