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.

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.

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.

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.

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.

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.

Two-step stem cell therapy improves bone regeneration compared to concentrated bone marrow therapy.

Adult stem cells are a promising tool to positively influence bone regeneration. Concentrated bone marrow therapy entails isolating osteoprogenitor cells during surgery with, however, only low cells yield. Two step stem cell therapy requires an additional harvesting procedure but generates high numbers of progenitor cells that facilitate osteogenic pre-differentiation. To further improve bone regeneration, stem cell therapy can be combined with growth factors from platelet rich plasma (PRP) or its lysate (PL) to potentially fostering vascularization. The aim of this study was to investigate the effects of bone marrow concentrate (BMC), osteogenic pre-differentiation of mesenchymal stromal cells (MSCs), and PL on bone regeneration and vascularization. Bone marrow from four different healthy human donors was used for either generation of BMC or for isolation of MSCs. Seventy-two mice were randomized to six groups (Control, PL, BMC, BMC + PL, pre-differentiated MSCs, pre-differentiated MSCs + PL). The influence of PL, BMC, and pre-differentiated MSCs was investigated systematically in a 2 mm femoral bone defect model. After a 6-week follow-up, the pre-differentiated MSCs + PL group showed the highest bone volume, highest grade of histological defect healing and highest number of bridged defects with measurable biomechanical stiffness. Using expanded and osteogenically pre-differentiated MSCs for treatment of a critical-size bone defect was favorable with regards to bone regeneration compared to treatment with cells from BMC. The addition of PL alone had no significant influence; therefore the role of PL for bone regeneration remains unclear. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1318-1328, 2019.

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.

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

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

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

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