Assembled/Disassembled Modular Scaffolds for Multicellular Tissue Engineering.

The behavior of tissue resident cells can be influenced by the spatial arrangement of cellular interactions. Therefore, it is of significance to precisely control the spatial organization of various cells within multicellular constructs. It remains challenging to construct a versatile multicellular scaffold with ordered spatial organization of multiple cell types. Herein, a modular multicellular tissue engineering scaffold with ordered spatial distribution of different cell types is constructed by assembling varying cell-laden modules. Interestingly, the modular scaffolds can be disassembled into individual modules to evaluate the specific contribution of each cell type in the system. Through assembling cell-laden modules, the macrophage-mesenchymal stem cell (MSC), endothelial cell-MSC, and chondrocyte-MSC co-culture models are successfully established. The in vitro results indicate that the intercellular cross-talk can promote the proliferation and differentiation of each cell type in the system. Moreover, MSCs in the modular scaffolds may regulate the behavior of chondrocytes through the nuclear factor of activated T-Cells (NFAT) signaling pathway. Furthermore, the modular scaffolds loaded with co-cultured chondrocyte-MSC exhibit enhanced regeneration ability of osteochondral tissue, compared with other groups. Overall, this work offers a promising strategy to construct a multicellular tissue engineering scaffold for the systematic investigation of intercellular cross-talk and complex tissue engineering.

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.

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.

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.

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.

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.

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.

Tailoring Materials for Modulation of Macrophage Fate.

Human immune system acts as a pivotal role in the tissue homeostasis and disease progression. Immunomodulatory biomaterials that can manipulate innate immunity and adaptive immunity hold great promise for a broad range of prophylactic and therapeutic purposes. This review is focused on the design strategies and principles of immunomodulatory biomaterials from the standpoint of materials science to regulate macrophage fate, such as activation, polarization, adhesion, migration, proliferation, and secretion. It offers a comprehensive survey and discussion on the tunability of material designs regarding physical, chemical, biological, and dynamic cues for modulating macrophage immune response. The range of such tailorable cues encompasses surface properties, surface topography, materials mechanics, materials composition, and materials dynamics. The representative immunoengineering applications selected herein demonstrate how macrophage-immunomodulating biomaterials are being exploited for cancer immunotherapy, infection immunotherapy, tissue regeneration, inflammation resolution, and vaccination. A perspective on the future research directions of immunoregulatory biomaterials is also provided.

Black Bioceramics: Combining Regeneration with Therapy.

Bioceramics have been developed from bioinert to bioactive or biodegradable materials in the past few decades. However, at present, traditional bioceramics are still mainly used in bone tissue regeneration and dental restoration. In this work, a new generation of "black bioceramics," extending the applications from tissue regeneration to disease therapy, is presented. Black bioceramics, through magnesium thermal reduction of traditional white ceramics, including silicate-based (e.g., CaSiO3 , MgSiO3 ) and phosphate-based (e.g., Ca3 (PO4 )2 , Ca5 (PO4 )3 (OH)), are successfully synthesized. Due to the presence of oxygen vacancies and structural defects, the black bioceramics possess photothermal functionality while maintaining their initial high bioactivity and regenerative capacity. These black bioceramics show excellent photothermal antitumor effects for both skin and bone tumors. At the same time, they have significantly improved bioactivity for skin/bone tissue repair in vitro and in vivo. These fascinating properties award the black bioceramics with profound applications in both tumor therapy and tissue regeneration, which should greatly promote the scientific relevance and clinical application of bioceramics, representing a promising new direction of cell-instructive biomaterials.

Catechol Containing Polyelectrolyte Complex Nanoparticles as Local Drug Delivery System for Bortezomib at Bone Substitute Materials.

The proteasome inhibitor bortezomib (BZM) is one of the most potent anti-cancer drugs in the therapy of multiple myeloma. In this study, an adhesive drug delivery system (DDS) for BZM was developed. Therefore, we extended the present DDS concept of polyelectrolyte complex (PEC) nanoparticle (NP) based on electrostatic interactions between charged drug and polyelectrolyte (PEL) to a DDS concept involving covalent bonding between PEL and uncharged drugs. For this purpose, 3,4-dihydroxyphenyl acetic acid (DOPAC) was polymerized via an oxidatively induced coupling reaction. This novel chemo-reactive polyanion PDOPAC is able to temporarily bind boronic acid groups of BZM via its catechol groups, through esterification. PDOPAC was admixed to poly(l-glutamic acid) (PLG) and poly(l-lysine) (PLL) forming a redispersible PEC NP system after centrifugation, which is advantageous for further colloid and BZM loading processing. It was found that the loading capacity (LC) strongly depends on the PDOPAC and catechol content in the PEC NP. Furthermore, the type of loading and the net charge of the PEC NP affect LC and the residual content (RC) after release. Release experiments of PDOPAC/PEC coatings were performed at medically relevant bone substitute materials (calcium phosphate cement and titanium niobium alloy) whereby the DDS worked independently of the surface properties. Additionally, in contrast to electrostatically based drug loading the release behavior of covalently bound, uncharged BZM is independent of the ionic strength (salt content) in the release medium.

IL-11 and soluble VCAM-1 are important components of Hypoxia Conditioned Media and crucial for Mesenchymal Stromal Cells attraction.

INTRODUCTION: Bone marrow stromal cells (BMSC) are highly attractive for tissue engineering due to their ability to differentiate into different cell types, to expand extensively in vitro and to release paracrine soluble factors with a high regenerative potential. They were observed to migrate towards the sites of injury in response to chemotactic signals in vivo. During the last years hypoxia has become a proven method to control proliferation, differentiation and multipotency of BMSC. Conditioned medium from hypoxia-treated BMSC (Hypoxia-conditioned Medium; HCM) has been shown to have various favorable properties on tissue regeneration - such as on cell recruitment, wound healing, angiogenesis and revascularization. Due to this regenerative potential many studies attempt to further characterize HCM and its main functional components. In this study we used HCM generated from umbilical cord mesenchymal stem cells (UC-MSC) instead of BMSC, because GMP-verified methods were used to isolate and cultivate the cells and ensure their constant quality. UC-MSC have a high regenerative potential and are still immunologically naive and therefore highly unlikely to cause an immune reaction. In our article we took the first steps to closer investigate the role of umbilical cord MSC-derived HCM components, namely stromal cell-derived factor 1 (SDF-1α), interleukin 11 (IL-11) and soluble vascular cell adhesion molecule 1 (sVCAM-1). RESULTS: Our results show previously unknown roles of IL-11 and sVCAM-1 in the attraction of BMSC. The synergistic effect of the investigated protein mixture consisting of IL-11, sVCAM-1 and SDF-1α as well as those recombinant proteins alone revealed a significantly higher chemoattractive capacity towards human BMSC compared to normoxic control medium. Both, the protein mixtures and proteins alone as well as UC-HCM showed an angiogenic effect by promoting the formation of significantly longer tubule structures and higher amounts of junctions and tubules compared to normoxic control medium. CONCLUSIONS: By showing the prominent upregulation of IL-11, sVCAM-1 and SDF-1α under hypoxic conditions compared to normoxic control and revealing their crucial role in migration of human BMSC we took a further step forward in characterization of the chemoattractive components of HCM.

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.

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.

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.

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.

Strontium enhances BMP-2 mediated bone regeneration in a femoral murine bone defect model.

The application of strontium is one option for the clinical treatment of osteoporosis-a disease characterized by reduced bone density and quality-in order to reduce the risk of vertebral and nonvertebral fractures. Unlike other drugs used in osteoporosis therapy, strontium shows a dual effect on bone metabolism by attenuating cellular resorption and simultaneously enhancing new bone tissue formation. Current concerns regarding the systemic application of highly dosed strontium ranelate led to the development of strontium-modified scaffolds based on mineralized collagen (MCM) capable to release biologically active Sr2+ ions directly at the fracture site. In this study, we investigated the regenerative potential of these scaffolds. For in vitro investigations, human mesenchymal stromal cells were cultivated on the scaffolds for 21 days (w/ and w/o osteogenic supplements). Biochemical analysis revealed a significant promoting effect on proliferation rate and osteogenic differentiation on strontium-modified scaffolds. In vivo, scaffolds were implanted in a murine segmental bone defect model-partly additionally functionalized with the osteogenic growth factor bone morphogenetic protein 2 (BMP-2). After 6 weeks, bridging calluses were obtained in BMP-2 functionalized scaffolds; the quality of the newly formed bone tissue by means of morphological scores was clearly enhanced in strontium-modified scaffolds. Histological analysis revealed increased numbers of osteoblasts and blood vessels, decreased numbers of osteoclasts, and significantly enhanced mechanical properties. These results indicate that the combined release of Sr2+ ions and BMP-2 from the biomimetic scaffolds is a promising strategy to enhance bone regeneration, especially in patients suffering from osteoporosis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:174-182, 2020.