29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Chair

Poster Talk

Prof. Dr. Dr.

Prof. Dr.

Assessment of corrosion, cytocompatibility and immunological effects in vitro of magnesium-based biomaterials for regenerative applications in oral surgery

Surface modification technologies

Tissue regeneration/regenerated medicine

Abstract text (incl. figure legends and references) Objective: Magnesium-based biomaterials are promising for multiple applications in implant dentistry (including resorbable meshes, pins; screws to fix a bone block). However, the rate of degradation of magnesium needs to be reduced, depending on the indication area, to control toxicity caused by rapid natural corrosion, as well as hydrogen gas evolution and loss of stability. In the development of new magnesium alloys and surface modifications, efficient evaluation of relevant properties is essential. Material &amp; Methodology: In the present study, a WE43 magnesium alloy with a surface generated by plasma electrolytic oxidation (PEO) was investigated. The surface microstructure, hydrogen gas release in immersion tests and cytocompatibility were analyzed. In addition, an in vitro immunological assay was performed using primary human lymphocytes. Results: Hydrogen gas evolution after two weeks of immersion was reduced by 40.7% by the PEO surface, indicating a significantly reduced corrosion rate. In contrast to untreated WE43, PEO-treated WE43 showed good cytocompatibility.After three days of incubation, the former killed more than 90% of lymphocytes, while more than 80% of cells were still vital after incubation with the PEO-treated test specimens. WE43-PEO mildly stimulated the activation, proliferation, and toxin expression of CD8+ T cells. Conclusion: This study demonstrates that the combined assessment of corrosion, cytocompatibility, and immunological effects on primary human lymphocytes is a suitable method for characterizing magnesium variants. PEO-treated WE43 is a promising candidate for further development as a degradable metallic biomaterial for implant/ augmentation indications.

Plasma proteomics to reveal liquid biopsy-accessible indicative biomarkers to impaired bone fracture healing in type 2 diabetes mellitus using personalized 3D-printed scaffolds

Abstract text (incl. figure legends and references) Although Type 2 diabetes mellitus (T2DM) can lead to compromised fracture healing, the extent to which it affects each patient is highly individualized. Yet, there is no biomarker profile available that can identify diabetes patients at risk. Additionally, the molecular mechanisms that lead to impaired fracture healing in diabetic conditions are not fully understood. The SyMBoD project aims to collect, automatically process and share molecular profiles on a digital platform in order to understand the molecular principles in high-risk patients and provide specific treatment. The ultimate goal is to develop image-guided personalized and functionalized polycaprolactone (PCL)-based scaffolds that can address the molecular needs at diabetic conditions. Within this scope, a diabetic rat model was used to investigate PCL-guided bone regeneration in a femur defect. Blood plasma was sampled over 42 days post-surgery. Multiplexed proteomics screening analyses were carried out on a Q Exactive HF mass spectrometer. Proteins, that are specifically altered in the initial fracture response and subsequent regeneration phase were identified by (i) pairwise correlations of disease conditions and time points, (ii) time-aware biclustering and (iii) de novo network enrichment. 667 proteins were monitored in blood plasma over the sampling period of which 26 protein possess significant and diabetes-specific response profiles in comparison to non-diabetic animals. Preliminary integration of the results to tissue proteomics of the same animal cohort in combination with de novo network enrichment revealed proteins and functions that are likewise affected locally (in tissue) and systemically (in plasma). To the best of our knowledge, we report the first list of proteins that include altered proteins that are accessible from liquid biopsies and are directly or functionally linked to local effects. Those proteins may serve as indicative biomarkers to predict bone regeneration capacities.

Surface bio-functionalization for anti-adhesive properties in biotechnological applications

Biofilm formation

Antimicrobial coatings

Abstract text (incl. figure legends and references) Pharmaceutical biotechnology has had a major impact on the treatment of diseases such as diabetes, cancer, and autoimmune disorders. For example, human insulin was the first drug produced through biotechnology and continues to be one of the most widely used biotechnology drugs. Microorganisms such as bacteria, yeast cells, and mammalian cell lines are employed in the production process. Cell-based processes go through growth phases in which the initial cell stock must be cultured to a sufficient number to yield a significant amount of product. Although desired cell growth is necessary for production, unwanted biofilm formation (fouling) can also occur. To address this challenge, a research project has successfully developed an anti-fouling coating for silicon-based glass surfaces. 1 This bio-coating has been validated against different cell types, including yeast cells and animal cell lines.2,3 The reduced adhesion of cells due to the surface coating helps to prevent biofilm formation. Fig. 1.: Images of yeast adhesion Fig. 2: Images of eukaryotic cell line adhesion 1. L. M. Langner, Biofunktionalisierung oxidischer Oberflächen über eine spezifische Bindung mit biotinylierten Wachstumsfaktoren für medizinische Anwendungen, Masterarbeit, Westfälische Hochschule, 2022. 2. V. Ettelt, K. Ekat, P. W. Kämmerer, B. Kreikemeyer, M. Epple, and M. Veith, &quot;Streptavidin-coated surfaces suppress bacterial colonization by inhibiting non-specific protein adsorption,&quot; Journal of biomedical materials research. Part A 106 (3), 758–768 (2018). 3. V. Ettelt, A. Belitsky, M. Lehnert, A. Loidl-Stahlhofen, M. Epple, and M. Veith, &quot;Enhanced selective cellular proliferation by multi-biofunctionalization of medical implant surfaces with heterodimeric BMP-2/6, fibronectin, and FGF-2,&quot; Journal of biomedical materials research. Part A 106 (11), 2910–2922 (2018).

Keratocyte differentiation of bone marrow-derived mesenchymal stromal cells for corneal bioprinting

Clinical applications and translation

Abstract text (incl. figure legends and references) Introduction: Corneal transplantation is the gold standard procedure to cure corneal diseases that can lead to blindness. Fabrication of corneal tissues is a promising solution to overcome the shortage of human donors. Our future goal is to utilize a bioprinter mounted to a robotic arm to print corneal tissue directly onto the eye of the patient. One interesting cell type for in situ and in vivo corneal bioprinting is the use of bone marrow-derived mesenchymal stromal cells (MSCs) in their differentiated state toward the keratocyte lineage. Corneal stromal keratocytes are the main cell type present in the native tissue, and for this reason in this work we investigated the keratocyte differentiation potential of MSCs. Objective: The main goal of this work was to investigate the ability of MSCs to differentiate towards keratocytes by analyzing and comparing the expression of specific markers of these differentiated cells and primary corneal stromal keratocytes (CSKs). Materials &amp; Methods: MSCs were expanded according to previously established protocols1,2. Keratocyte differentiation was initiated by subculturing the MSCs in a differentiation medium supplemented with 10 ng/ml FGF-b and 1 ng/ml TGF-β3 for 14 days. The expression of the keratocyte specific markers keratocan, lumican, and ALDH3A1 were investigated by immunofluorescence staining, Western Blot and qPCR. Results: Keratocyte differentiated cells showed positive immunofluorescent staining of the keratocyte markers keratocan, lumican and ALDH3A1 and the success of differentiation was confirmed by qPCR and Western Blot. Conclusion: We showed successful keratocyte differentiation of MSCs in 2D cultures. After validating our differentiation protocol in 2D, we begin to investigate if the keratocyte differentiation potential is also observed in 3D hydrogel cultures compatible with bioprinting. Hin-Fai Yam G et al. J Cell Mol Med.2018;doi:10.1111/jcmm.13589 Dos Santos et al. IOVS 2019;doi:10.1167/iovs.19-27008

Traumatic fracture treatment: Calcium phosphate bone substitute case-control study in humerus, radius, tibia fractures – assessing efficacy and recovery outcomes

Implant associated

Abstract text (incl. figure legends and references) This study investigates the safety, resorption, healing process, and complications of synthetic bioactive bone substitute materials, specifically calcium phosphate-based bone substitutes (CP), in traumatically induced bone fractures. We hypothesize that CP could improve bone healing. In this retrospective case-control study, over 290 patients who received surgical treatment for acute fractures were examined. CP augmentation was compared with empty defect treatment (ED) between 2011 and 2018. A new scoring system for fracture healing was used in up to six radiological follow-up examinations, and demographic data, concomitant diseases, and complications were analyzed. Data analysis showed significantly fewer postoperative complications in the CP group compared to the ED group (p&lt;0.001). Risks of pseudarthrosis (p&lt;0.001), arthrosis (p=0.01), and neurological diseases (p&lt;0.001) were decreased in the CP group. Intermediate follow-up examinations indicated enhanced fracture edge, gap, and articular surface (p&lt;0.05). Osteosynthesis and general bone density were similar (p&gt;0.05). Subgroup analysis of patients aged over 64 years revealed fewer complications in the CP group (p=0.025). Geriatric patients benefited from CP materials with reduced pseudarthrosis (p=0.059) and improved fracture gap, edge, and articular surface in intermediate follow-ups (p&lt;0.05). In conclusion, calcium phosphate-based bone substitutes demonstrate safety in clinical applications, improved early osseous healing, and fewer severe complications. In vivo advantages were evident for CP bone graft substitutes.

Development of bioresins for volumetric printing for vascular applications

Additive manufacturing (e. g. 3D printing)

Biofabrication

Abstract text (incl. figure legends and references) Volumetric printing (VP) is a novel, cell-friendly 3D printing method capable of in situ encapsulation of cells into CAD-designed models in a one-step process within seconds. This leads to the fast manufacturing of usable sample sizes of complex hydrogel scaffolds. With this method, complex objects can be created that were not able to be printed with conventional biofabrication methods, such as multi-material objects or intricate tubular branching constructs. Models for coronary arteries, especially, are needed for medical training and therapeutic replacement purposes. Even 1:1 copies of 3D-scanned tissue, such as CT scans of blood vessel systems of the coronary tree, can be used. For bioresins, which are a bioink formulation used for vat and vial-based biofabrication techniques, a cytocompatible and photocrosslinkable hydrogel precursor needs to be chosen, with methacrylated gelatin (GelMA) as a prototypical representative. GelMA&quot;s thermal gelling behavior, good optical transparency and its properties can be tuned via the formulation of the precursor solution. This results in different mechanical stiffness, printing resolution, and biological behavior to mimic the extracellular matrix (ECM) for the various cell types in volumetric bioprinting. The tubular models can be post-seeded with endothelial cells and cultivated under physiological flow to further investigate artificial small-diameter blood vessel models. To prevent cell sedimentation, enhance cell distribution, and provide a beneficial physiological environment for cell maturation, special properties for the resin need to be established. By combining chemistry and biofabrication with tight feedback loops to biology, we are testing advanced in vitro vasculature models. However, these properties can only be optimized up to a certain point. Research into alternatives or new polymeric options for photosensitive fabrication needs to be conducted to push the limits of printability and biocompatibility.

Bioprinting of pegylated giant unilamellar lipid vesicles for tissue engineering applications

Abstract text (incl. figure legends and references) Introduction In the field of tissue engineering, 3D bioprinting is used for generating functional 3D tissues that can be exploited for the restoration of damaged tissues. Giant unilamellar lipid vesicles (GUVs) can be synthesized as a micron-sized container surrounded by a lipid bilayer that allows the encapsulation of hydrophilic biomolecules, surface functionalization, and a tailored cargo release. Objectives In this study, we investigate the feasibility of bioprinting GUVs biomedical applications. We evaluated the structural integrity of GUVs during the printing process and characterized the stability of GUVs in hydrogel over time, as well as the interaction of pegylated GUVs with cells (Figure 1a). Materials &amp; methods A protocol for printing GUVs was established, consisting of production, filtration, and printing with a bioprinter. After bioprinting at pressures between 0.2 and 1 bar, the number and size of the GUVs were determined by fluorescence microscopy and the structural integrity was calculated. Results We showed that GUVs formed with 5% pegylated lipids have a 40% increased stability compared to unpegylated GUVs when encapsulated in 1% (w/v) agarose hydrogel for a period of three days (Figure 1b). In addition, up to 65.7 ± 16.1% of unpegylated GUVs and 52.6 ± 15.4% of 5% pegylated GUVs remained intact during the printing process. Therefore, demonstrating that GUVs can be printed using drop-on-demand and extrusion methods. Conclusion Pegylation of GUVs has shown to increase their stability under physiological conditions encapsulated in a hydrogel. However, a trade-off needs to be met between increased physiological stability and reduced mechanical stability. The proposed GUV bioprinting protocol can be utilized in multiple biomedical applications combined with the ability to functionalize the bilayer and the spatiotemporal control of biomolecule release in the fabricated tissue.

Professor

A mechanically homogeneous equivalent of real cells

Abstract text (incl. figure legends and references) Biological cells are built up from many different constituents of varying size and stiffness which all contribute to the global cell's mechanical properties. Despite this heterogeneity, in the analysis of experimental measurements such as atomic force microscopy or high-throughput microfluidic characterisation a strongly simplified homogeneous cell is typically assumed and a single elastic modulus is assigned to the entire cell. This ad-hoc simplification has so far been used mostly without proper justification. Here, we use computer simulations to show that indeed a heterogeneous cell can effectively be replaced by a homogeneous equivalent cell with a volume averaged elastic modulus. To study the validity of this approach, we investigate a hyperelastic cell with an explicitly heterogeneous interior under compression and in shear flow, mimicking atomic force and microfluidic measurements, respectively. We find that the homogeneous equivalent cell reproduces quantitatively the behavior of its inhomogeneous counterpart, and that this equality is largely independent of the stiffness or spatial distribution of the heterogeneity.

Process development for droplet-based bioprinting of cell-laden tissue models in the context of cardiac lesions

Abstract text (incl. figure legends and references) Introduction: Bioprinting provides the ability to adjust factors like cell distribution in biomaterials; cells, growth factors concentration; matrix resolution, polymers combination as well as achieve processing for good cell viability, coculture, 3D cell growth. The capability to load different cell types is other benefit allowing mimicking cell microenvironments to develop functional tissues. Objectives: We aim bioprinting cell-laden hydrogels at high cell density, with the highest precision of bioink deposition into hydrogel patterns, by droplet-based bioprinting DBB. We advance the development of functional tissue models in the context of cardiac lesions, allowing for cardiac cell coculture in target microstructures. Materials &amp; Methods: Flow behavior of inks of alginate at 0.5, 1 and 2%wt and of fibrinogen at 2%wt without and with cells was investigated by rheology. Initially, fibroblast cells were incorporated in bioink at a conc. of 3x10^6 cells/mL. Then, to design cardiac models, 2 types of cells were used: mesenchymal stem cells (BM-hMSC) and immortalized human ventricular fibroblast (HVF) cell line. DBB was performed with a RegenHU 3D bioprinter under minimum pressure 0.05MPa and a valve opening time range: 120-210µs. Cells were fluorescence stained and printed biomaterials were observed at confocal microscope. Results: By increasing valve opening time the droplet size increased and droplet patterns with dosing distance of 1 mm were achieved, preventing the droplets from merging. The smallest alginate droplets were achieved at conc 0.1 %wt by printing with a microvalve of inner diameter 0.2mm and a valve opening time of 120µs. The highest precision in designing cardiac models was achieved with the pattern closest gap of ca. 250µm between fibrinogen/cardiac cell areas. Conclusion: Cell-laden hydrogel patterns of target resolution with high precision and at high cell density were developed, allowing co-culture studies in cardiac functional tissue models.

Short Talks 18
Rapid Fire Session 2

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Short Talks 18Rapid Fire Session 2

Termin

Chair

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Short Talks 18
Rapid Fire Session 2