The effect of continuous long-term illumination with visible light in different spectral ranges on mammalian cells.

One of the biggest challenges in tissue engineering and regenerative medicine is to ensure oxygen supply of cells in the (temporary) absence of vasculature. With the vision to exploit photosynthetic oxygen production by microalgae, co-cultivated in close vicinity to oxygen-consuming mammalian cells, we are searching for culture conditions that are compatible for both sides. Herein, we investigated the impact of long-term illumination on mammalian cells which is essential to enable photosynthesis by microalgae: four different cell types-primary human fibroblasts, dental pulp stem cells, and osteoblasts as well as the murine beta-cell line INS-1-were continuously exposed to warm white light, red or blue light over seven days. We observed that illumination with red light has no adverse effects on viability, metabolic activity and growth of the cells whereas exposure to white light has deleterious effects that can be attributed to its blue light portion. Quantification of intracellular glutathione did not reveal a clear correlation of this effect with an enhanced production of reactive oxygen species. Finally, our data indicate that the cytotoxic effect of short-wavelength light is predominantly a direct effect of cell illumination; photo-induced changes in the cell culture media play only a minor role.

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.

Cellular adhesion and chondrogenic differentiation inside an alginate-based bioink in response to tailorable artificial matrices and tannic acid treatment.

Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.

Combined application of BMP-2 and naturally occurring bioactive factor mixtures for the optimized therapy of segmental bone defects.

Critical bone defects are the result of traumatic, infection- or tumor-induced segmental bone loss and represent a therapeutic problem that has not been solved by current reconstructive or regenerative strategies yet. Scaffolds functionalized with naturally occurring bioactive factor mixtures show a promising chemotactic and angiogenic potential in vitro and therefore might stimulate bone regeneration in vivo. To assess this prospect, the study targets at heparin-modified mineralized collagen scaffolds functionalized with naturally occurring bioactive factor mixtures and/or rhBMP-2. These scaffolds were implanted into a 2-mm segmental femoral defect in mice and analyzed in respect to newly formed bone volume (BV) and bone mineral density (BMD) by micro-computed tomography scans after an observation period of 6 weeks. To rate the degree of defect healing, the number of vessels, and the activity of osteoclasts and osteoblasts were analyzed histologically. The sole application of bioactive factor mixtures is inferior to the use of the recombinant growth factor rhBMP-2 regarding BV and degree of defect healing. A higher rhBMP-2 concentration or the combination with bioactive factor mixtures does not lead to a further enhancement in defect healing. Possibly, a synergistic effect can be achieved by further concentration or a prolonged release of bioactive factor mixtures. STATEMENT OF SIGNIFICANCE: The successful therapy of extended bone defects is still a major challenge in clinical routine. In this study we investigated the bone regenerative potential of naturally occuring bioactive factor mixtures derived from platelet concentrates, adipose tissue and cell secretomes as a cheap and promising alternative to recombinant growth factors in a murine segmental bone defect model. The mixtures alone were not able to induce complete bridging of the bone defect, but in combination with bone morphogenetic protein 2 bone healing seemed to be more physiological. The results show that naturally occuring bioactive factor mixtures are a promising add-on in a clinical setting.

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.

Cell-Material Interactions in Direct Contact Culture of Endothelial Cells on Biodegradable Iron-Based Stents Fabricated by Laser Powder Bed Fusion and Impact of Ion Release.

Additive manufacturing is a promising technology for the fabrication of customized implants with complex geometry. The objective of this study was to investigate the initial cell-material interaction of degradable Fe-30Mn-1C-0.02S stent structures in comparison to conventional 316L as a reference, both processed by laser powder bed fusion. FeMn-based alloys have comparable mechanical properties with clinically applied AISI 316L for a corrosion-resistant stent material. Different corrosion stages of the as-built Fe-30Mn-1C-0.02S stent surfaces were simulated by pre-conditioning in DMEM under cell culture conditions for 2 h, 7 days, and 28 days. Human umbilical vein endothelial cells (HUVECs) were directly seeded onto the pre-conditioned samples, and cell viability, adherence, and morphology were analyzed. These studies were accompanied by measurements of iron and manganese ion release and Auger electron spectroscopy to evaluate the influence of corrosion products and degradation on the cells. In the initial phase (2 h of pre-conditioning), HUVECs were able to attach but the cell number decreased over the cultivation period of 14 days and the CD31 staining pattern of intercellular contacts was disordered. At later time points of corrosion (7 and 28 days of pre-conditioning), CD31 staining was distinctly located at the intercellular contacts, and the cell density increased after seeding and was stable for up to 14 days. Formation of a complex degradation layer, which had a composition and thickness dependent on the pre-conditioning time, led to a reduced ion release and finally showed a positive effect on cell survival. Concluding, our data suggest the suitability of Fe-30Mn-1C-0.02S for in vivo applications.

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration.

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.

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.

Characterization of Naturally Occurring Bioactive Factor Mixtures for Bone Regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.

Engineering considerations on extrusion-based bioprinting: interactions of material behavior, mechanical forces and cells in the printing needle.

Systematic analysis of the extrusion process in 3D bioprinting is mandatory for process optimization concerning production speed, shape fidelity of the 3D construct and cell viability. In this study, we applied numerical and analytical modeling to describe the fluid flow inside the printing head based on a Herschel-Bulkley model. The presented analytical calculation method nicely reproduces the results of Computational Fluid Dynamics simulation concerning pressure drop over the printing head and maximal shear parameters at the outlet. An approach with dimensionless flow parameter enables the user to adapt rheological characteristics of a bioink, the printing pressure and needle diameter with regard to processing time, shear sensitivity of the integrated cells, shape fidelity and strand dimension. Bioinks consist of a blend of polymers and cells, which lead to a complex fluid behavior. In the present study, a bioink containing alginate, methylcellulose and agarose (AMA) was used as experimental model to compare the calculated with the experimental pressure gradient. With cultures of an immortalized human mesenchymal stem cell line and plant cells (basil) it was tested how cells influence the flow and how mechanical forces inside the printing needle affect cell viability. Influences on both sides increased with cell (aggregation) size as well as a less spherical shape. This study contributes to a systematic description of the extrusion-based bioprinting process and introduces a general strategy for process design, transferable to other bioinks.

A Novel Plasma-Based Bioink Stimulates Cell Proliferation and Differentiation in Bioprinted, Mineralized Constructs.

Extrusion-based bioprinting, also known as 3D bioplotting, is a powerful tool for the fabrication of tissue equivalents with spatially defined cell distribution. Even though considerable progress has been made in recent years, there is still a lack of bioinks which enable a tissue-like cell response and are plottable at the same time with good shape fidelity. Herein, we report on the development of a bioink which includes fresh frozen plasma from full human blood and thus a donor/patient-specific protein mixture. By blending of the plasma with 3 w/v% alginate and 9 w/v% methylcellulose, a pasty bioink (plasma-alg-mc) was achieved, which could be plotted with high accuracy and furthermore allowed bioplotted mesenchymal stromal cells (MSC) and primary osteoprogenitor cells to spread within the bioink. In a second step, the novel plasma-based bioink was combined with a plottable self-setting calcium phosphate cement (CPC) to fabricate bone-like tissue constructs. The CPC/plasma-alg-mc biphasic constructs revealed open porosity over the entire time of cell culture (35 d), which is crucial for bone tissue engineered grafts. The biphasic structures could be plotted in volumetric and clinically relevant dimensions and complex shapes could be also generated, as demonstrated for a scaphoid bone model. The plasma bioink potentiated that bioplotted MSC were not harmed by the setting process of the CPC. Latest after 7 days, MSC migrated from the hydrogel to the CPC surface, where they proliferated to 20-fold of the initial cell number covering the entire plotted constructs with a dense cell layer. For bioplotted and osteogenically stimulated osteoprogenitor cells, a significantly increased alkaline phosphatase activity was observed in CPC/plasma-alg-mc constructs in comparison to plasma-free controls. In conclusion, the novel plasma-alg-mc bioink is a promising new ink for several forms of bioprinted tissue equivalents and especially gainful for the combination with CPC for enhanced, biofabricated bone-like constructs.

Electrodeposition of Sr-substituted hydroxyapatite on low modulus beta-type Ti-45Nb and effect on in vitro Sr release and cell response.

Beta-type Ti-based alloys are promising new materials for bone implants owing to their excellent mechanical biofunctionality and biocompatibility. For treatment of fractures in case of systemic diseases like osteoporosis the generation of implant surfaces which actively support the problematic bone healing is a most important aspect. This work aimed at developing suitable approaches for electrodeposition of Sr-substituted hydroxyapatite (Srx-HAp) coatings onto Ti-45Nb. Potentiodynamic polarization measurements in electrolytes with 1.67 mmol/L Ca(NO3)2, which was substituted by 0, 10, 50 and 100% Sr(NO3)2, and 1 mmol/L NH4H2PO4 at 333 K revealed the basic reaction steps for OH- and PO43- formation needed for the chemical precipitation of Srx-HAp. Studies under potentiostatic control confirmed that partial or complete substitution of Ca2+- by Sr2+-ions in solution has a significant effect on the complex reaction process. High Sr2+-ion contents yield intermediate phases and a subsequent growth of more refined Srx-HAp coatings. Upon galvanostatic pulse-deposition higher reaction rates are controlled and in all electrolytes very fine needle-like crystalline coatings are obtained. With XRD the incorporation of Sr-species in the hexagonal HAp lattice is evidenced. Coatings formed in electrolytes with 10 and 50% Sr-nitrate were chemically analyzed with EDX mapping and GD-OES depth profiling. Only a fraction of the Sr-ions in solution is incorporated into the Srx-HAp coatings. Therein, the Sr-distribution is laterally homogeneous but non-homogeneous along the cross-section. Increasing Sr-content retards the coating thickness growth. Most promising coatings formed in the electrolyte with 10% Sr-nitrate were employed for Ca, P and Sr release analysis in Tris-Buffered Saline (150 mM NaCl, pH 7.6) at 310 K. At a sample surface: solution volume ratio of 1:200, after 24 h the amount of released Sr-ions was about 30-35% of that determined in the deposited Srx-HAp coating. In vitro studies with human bone marrow stromal cells (hBMSC) revealed that the released Sr-ions led to a significantly enhanced cell proliferation and osteogenic differentiation and that the Sr-HAp surface supported cell adhesion indicating its excellent cytocompatibility.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

The Secretome of Hypoxia Conditioned hMSC Loaded in a Central Depot Induces Chemotaxis and Angiogenesis in a Biomimetic Mineralized Collagen Bone Replacement Material.

The development of biomaterials with intrinsic potential to stimulate endogenous tissue regeneration at the site of injury is a main demand on future implants in regenerative medicine. For critical-sized bone defects, an in situ tissue engineering concept is devised based on biomimetic mineralized collagen scaffolds. These scaffolds are functionalized with a central depot loaded with a signaling factor cocktail, obtained from secretome of hypoxia-conditioned human mesenchymal stem cells (MSC). Therefore, hypoxia-conditioned medium (HCM)-production is standardized and adapted to achieve high signaling factor-yields; a concentration protocol based on dialysis and freeze-drying is established to enable the integration of sufficient and defined amounts into the depot. In humid milieu-as after implantation-signaling factors are released by forming a chemotactic gradient, inducing a directed migration of human bone marrow stroma cells (hBMSC) into the scaffold. Angiogenic potential, determined by coculturing human umbilical vein endothelial cells (HUVEC) with osteogenically induced hBMSC shows prevascular structures, which sprout throughout the interconnected pores in a HCM-concentration-dependent manner. Retarded release by alginate-based (1 vol%) depots, significantly improves sprouting-depth and morphology of tubular structures. With the intrinsic potential to supply attracted cells with oxygen and nutrients, this bioactive material system has great potential for clinical translation.

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.

Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink.

Due to their characteristic resemblance of the mineral component of bone, calcium phosphates are widely accepted as optimal bone substitute materials. Recent research focused on the development of pasty calcium phosphate cement (CPC) formulations, which can be fabricated into various shapes by low-temperature extrusion-based additive manufacturing, namely 3D plotting. While it could be demonstrated that sensitive substances like growth factors can be integrated in such printed CPC scaffolds without impairment of their biological activity live cells cannot be suspended in CPC as they may not be functional when enclosed in a solid and stiff matrix. In contrast, 3D bioprinting of soft cell-laden hydrogels (bioinks) enables the fabrication of constructs with spatially defined cell distribution, which has the potential to overcome problems of conventional cell seeding techniques-but such objects lack mechanical stability. Herein, we combine 3D plotting of CPC and bioprinting of a cell-laden bioink for the first time. As model bioink, an alginate-methylcellulose blend (alg/mc) was used, previously developed by us. Firstly, a fabrication regime was established, enabling optimal setting of CPC and cell survival inside the bioink. As the cells are exposed to the chemical changes of CPC precursors during setting, we studied the compatibility of the complex system of CPC and cell-laden alg/mc for a combined extrusion process and characterized the cellular behavior of encapsulated human mesenchymal stroma cells within the bioink at the interface and in direct vicinity to the CPC. Furthermore, biphasic scaffolds were mechanically characterized and a model for osteochondral tissue grafts is proposed. The manuscript discusses possible impacts of the CPC setting reaction on cells within the bioink and illustrates the advantages of CPC in bioprinting as alternative to the commonly used thermoplasts for bone tissue engineering.

Endosteal and Perivascular Subniches in a 3D Bone Marrow Model for Multiple Myeloma.

The bone marrow microenvironment is the preferred location of multiple myeloma, supporting tumor growth and development. It is composed of a collection of interacting subniches, including the endosteal and perivascular niche. Current in vitro models mimic either of these subniches. By developing a model combining both niches, this study aims to further enhance the ability to culture primary myeloma cells in vitro. Also, the dependency of myeloma cells on each niche was studied. A 3D bone marrow model containing two subniches was created using 3D bioprinting technology. We used a bioprintable pasty calcium phosphate cement (CPC) scaffold with seeded osteogenic multipotent mesenchymal stromal cells (O-MSCs) to model the endosteal niche, and Matrigel containing both endothelial progenitor cells (EPCs) and MSCs to model the perivascular niche. Within the model containing one or both of the niches, primary CD138+ myeloma cells were cultured and analyzed for both survival and proliferation. The 3D bone marrow model with combined subniches significantly increasing the proliferation of CD138+ myeloma cells compared to both environments separately. The developed model showed an essential role of the perivascular niche over the endosteal niche in supporting myeloma cells. The developed model can be used to study the expansion of primary myeloma cells and their interactions with varying bone marrow subniches.

Novel chitin scaffolds derived from marine sponge Ianthella basta for tissue engineering approaches based on human mesenchymal stromal cells: Biocompatibility and cryopreservation.

The extraordinary biocompatibility and mechanical properties of chitinous scaffolds from marine sponges endows these structures with unique properties that render them ideal for diverse biomedical applications. In the present work, a technological route to produce "ready-to-use" tissue-engineered products based on poriferan chitin is comprehensively investigated for the first time. Three key stages included isolation of scaffolds from the marine demosponge Ianthella basta, confirmation of their biocompatibility with human mesenchymal stromal cells, and cryopreservation of the tissue-like structures grown within these scaffolds using a slow cooling protocol. Biocompatibility of the macroporous, flat chitin scaffolds has been confirmed by cell attachment, high cell viability and the ability to differentiate into the adipogenic lineage. The viability of cells cryopreserved on chitin scaffolds was reduced by about 30% as compared to cells cryopreserved in suspension. However, the surviving cells were able to retain their differentiation potential; and this is demonstrated for the adipogenic lineage. The results suggest that chitin from the marine demosponge I. basta is a promising, highly biocompatible biomaterial for stem cell-based tissue-engineering applications.

* 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.

3D chitinous scaffolds derived from cultivated marine demosponge Aplysina aerophoba for tissue engineering approaches based on human mesenchymal stromal cells.

The recently discovered chitin-based scaffolds derived from poriferans have the necessary prosperities for potential use in tissue engineering. Among the various demosponges of the Verongida order, Aplysina aerophoba is an attractive target for more in-depth investigations, as it is a renewable source of unique 3D microporous chitinous scaffolds. We found these chitinous scaffolds were cytocompatible and supported attachment, growth and proliferation of human mesenchymal stromal cells (hMSCs) in vitro. Cultivation of hMSCs on the scaffolds for 7days resulted in a two-fold increase in their metabolic activity, indicating increased cell numbers. Cells cultured onto chitin scaffolds in differentiation media were able to differentiate into the chondrogenic, adipogenic and osteogenic lineages, respectively. These results indicate A. aerophoba is a novel source of chitin scaffolds to futher hMSCs-based tissue engineering strategies.