29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Chair

Prof. Dr.

siRNA delivery via cross-linked gelatine microparticles for bone tissue regeneration

Tissue regeneration/regenerated medicine

Cell-material interactions

Abstract text (incl. figure legends and references) Objective: Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a promising strategy to support regeneration of bone defects. Cultivation and differentiation in a three-dimensional environment preserves cell-cell interactions and results in enhanced biological functions. Here, we used cross-linked gelatin microparticles (cGM) that served for two purposes for regeneration of bone tissue: 1) as a cell-adhesive biomaterial to increase volume and diffusivity and 2) as a siRNA delivery system to decrease expression of anti-osteogenic factors. Methods: 10,000 hMSCs were aggregated with systematically varied amounts of cGM to form microtissues. We analyzed the impact of different cGM amounts on cell viability and osteogenic differentiation. To load siRNA onto cGM, we used oligomer-stabilized calcium phosphate nanoparticles (CaP-NP). In a previous project, those CaP-NP had been investigated for siRNA transfection and had been shown to provide high loading capacity [1]. We further determined silencing of the BMP-2 antagonist Chordin as a molecular target to improve osteogenic differentiation in presence of BMP-2. Silencing efficiencies were determined by using quantitative real-time PCR. Results: Cultivation of hMSCs with cGM significantly increased cell viability and osteogenic differentiation markers. The loading of cGM with Chordin siRNA-carrying CaP-NP resulted in a successful silencing of the anti-osteogenic target Chordin. This decreased Chordin expression led to a remarkably increased mineralization. Conclusion: Here, we present the concept of microtissues from hMSC and cGM to generate well differentiated bone-like tissues for regenerative purposes. As a cell-adhesive material, cGM increased cell viability and osteogenic differentiation of the microtissues. The loading with a Chordin siRNA via CaP-NP showed a successful silencing of Chordin with a strongly increased mineralization. References: [1] Mitrach, F. et al.: Pharmaceutics 2022, 14(2): 326.

Short Talk

Professor

3D bioprinted constructs supporting the differentiation of primary human osteocytes

Additive manufacturing (e. g. 3D printing)

Abstract text (incl. figure legends and references) Introduction Bioprinting is a versatile method to generate spatially arranged tissue models. Since osteocytes (ocy) are the most abundant bone cell species, in vitro bone models should include not only osteoblasts and their precursors, but also ocy. Objectives The aim of the study was to differentiate primary human ocy in bioprinted constructs. Suitable ink formulations and media supplements needed to be tested to find suitable conditions for cell attachment, spreading and differentiation. Methods Human osteoblasts (hOB) were expanded and bioprinted (Bioscaffolder 3.1. Gesim) using three different alginate-based inks: alginate (alg)/methylcellulose(MC)/human plasma, alg/MC/egg white and alg/MC/gelatin. After printing and cross-linking, constructs were cultivated for up to 14d. Different media with varying serum content were tested. Ocy morphology and gene expression of ocy markers were analyzed. Results Osteoblasts were viable after both printing and cultivation in all examined inks. The cells showed dendritic ocy morphology after cultivation under low serum conditions. While early ocy markers (E11, BGLAP) were already expressed after 7 d, late ocy marker expression was significantly enhanced after 14 d of differentiation. This is in accordance to the more pronounced ocy morphology at d14. Conclusions The application of primary human ocy, directly isolated from bone, is not feasible for bioprinting applications, since very high cell numbers are required. After bioprinting of primary hOB, their differentiation to ocy was achieved for the first time. The applied inks supported ocy differentiation, providing a suitable microenvironment for the formation of dendrites. The generation of a 3D printed ocy network offers the opportunity to establish a biomimetic in vitro bone model with spatially arranged bone cells. Fig.1: Morphology (d14, scale 100 µm) and gene expression of ocy differenciated from hOB bioprinted in scaffolds from alg/MC/human plasma ink

Dental filaments from polycaprolactone-based composites – drug delivery and degradation

Antimicrobial coatings

Biofabrication

Abstract text (incl. figure legends and references) Introduction&amp;Objective As one of the most significant diseases of the periodontium, periodontitis is a major economic and patient-specific health problem. Therefore, sustainable treatment of the inflammation of the gums and the fight against bacteria that lead to the formation of pathological tooth pockets is essential. To provide an easy-to-apply drug delivery system (DDS) while saving the effort of subsequent removal, a filament-based degradable DDS was investigated. For this purpose, a degradable biopolymer should be used. Polycaprolactone (PCL) is particularly suitable for this purpose. Materials&amp;Methods Filaments were made of PCL, which shows usually acidic degradation products. To protect enamel, dentin, and dental cement from demineralization, the PCL was processed to composites with various buffer substances, to compensate the acidification. The comparison of different mineral and organic buffer substances with mass fractions between 20 and 30 wt% showed that the degradation rate of the polymers and the entire composite can be significantly influenced. Only few composites can keep the pH in a physiological range during storage in phosphate-buffered saline (PBS), artificial saliva, and PBS with lipase for up to 28 days. Furthermore, incorporation and release kinetics of different antibiotics (Amoxicillin, Metronidazole, Doxycycline, Rifampicin) were investigated in regards of cytocompatibility and antibacterial effectivity to relevant dental bacteria. Results&amp;Conclusion The PCL/MgCO3 composite can be used as an optimal DDS-basis material. Drug immobilization of antibiotics can be carried out in the same way as the production of composites. The release kinetics of the active ingredients and their efficacy are directly dependent on the antibiotic in question. However, there are indications of a long-range release in parallel with material degradation. This could provide a degradable filament material that can be effectively used for periodontitis treatment.

Fine-tuning the degradation of 3D-printed Calcite/Polycaprolactone scaffolds by mineral morphologies for use as bone graft substitute.

Surface modification technologies

Abstract text (incl. figure legends and references) Introduction&amp;Objective: Gradual degradation of Polycaprolactone (PCL) bone graft substitutes can be adjusted by incorporating calcite particles (CaCO3) of various morphologies to adapt to parallel regeneration of new bone tissue in vivo. The objective was, if either spherical, scalenohedron or rhombohedral CaCO3 particles incorporated in gamma-sterilized PCL scaffolds are most beneficial for bone graft substitutes. Evaluating the results in descending priority on stable buffer buffering of acidic PCL degradation products, mechanical stability after bulk degradation, and continuous calcium release. Materials&amp;Methods: Calcite particles were mixed into melted PCL and afterwards processed to a scaffold via fused deposition modelling (FDM). Gamma-sterilized scaffolds and single strands were incubated in 0.9% NaCl, PBS with Lipase and 2.5 M HCl. Calcium release was measured using a colorimetric calcium kit with UV-VIS, while buffer capacity was proven by pH measurements. Mechanical stability was tested via 3-point tension testing. Scaffolds were incubated over 28 days and single strands over 14 daysd at 37°C in 7% CO2, while performing medium change every 3 to 4 days. Results&amp;Conclusion: While scalenohedron particles cause a very stable calcium release, they are disintegrating the matrix during bulk degradation. In contrast, calcium release of rhombohedron particles decreases after 21 days, while the matrix&quot;s integrity is rather comparable to pure PCL (compare Fig.1 and Fig.2). Furthermore, for comparability to in vitro degradation mechanism, bulk and surface degradation of PCL could be accelerated. It was proven that different morphologies of calcite particles do have a significant impact on the degradation of gamma-sterilized PCL scaffolds, produced by FDM and intended to be used as bone graft substitutes. Trade-off decisions need to be made in order to fine-tune the properties of a suitable bone graft substitute without functional loss.

PD Dr.

Enhancing polymeric implant component visibility with integrated radiopaque markers

Implant associated

Abstract text (incl. figure legends and references) Introduction Due to its low X-Ray attenuation, UHMWPE (Ultra-High Molecular Weight Polyethylene) has limited visibility in radiographs, making it difficult to monitor in-vivo. To tackle this limitation, this project aims to incorporate radiopaque markers into the polymeric components of implants to enable clinical monitoring during routine radiographs. Methods UHMWPE was chosen as the test material and was processed in blocks measuring 100 x 75 x 13 mm. A 5-axis milling machine (Ultrasonic10) and three solid carbide end mills with nominal diameters of 0.4, 0.6, 0.8 mm were used to model the process. Burr formation was detected using a reflecting light microscope (Keyence VHX-5000). Radiopaque composites based on high-density polyethylene (HDPE 76303) and 10, 20 and 30 wt% BaSO4 were chosen for the markers and mixed using a twin-screw extruder. The markers were filled into the microstructures using a pellet extruder. Digital X-Ray radiographs were taken to evaluate the radiopacity. Results 20 wt% BaSO4 was sufficient to create visible contrast. Microstructures with d &gt; 0.4 mm had superior visibility and fill quality. With increasing cutter diameter the proportion of burr-free or acceptable samples increased. A spindle speed between 5,000 rpm and 10,000 rpm was suitable for microstructuring UHMWPE. Conclusion Radiographically detectable markers were successfully integrated into the UHMWPE blocks. Some unfilled voids were detectable which were likely a result of burr occlusion. Burr formation was found to be strongly dependent on the workpiece/tool pairing thus further optimisation of parameters is necessary. References Pegg EC, Gill HS, Zaribaf FP. Characterisation of the physical, chemical and mechanical properties of a radiopaque polyethylene. J Biomater Appl. 2020 Aug;35(2):215-223. doi: 10.1177/0885328220922809. Epub 2020 May 16. PMID: 32419587.

Antibacterial properties of Mg – based alloys studied in a murine model of periprosthetic osteomyelitis

Abstract text (incl. figure legends and references) Introduction: Periprosthetic infections remain a major challenge and a most difficult to treat complication for implants. Different strategies to avoid infections are followed, among them the complete degradation of the implant material. However, during the course of degradation bacteria might still be able to colonize the implant-tissue interface. Here magnesium-based biodegradable alloys are currently under eager attention as alternatives. Objectives: Many in vitro studies reported profound antibacterial potential of pure magnesium and its alloys with improved bactericidal features, and some of the animal models of peri-implant osteomyelitis confirmed these results in vivo. However, to our knowledge, the susceptibility of magnesium and magnesium-silver alloys were previously not assessed in experimental settings for the hematogenous periprosthetic osteomyelitis. Materials &amp; methods: In the present study, we implemented a murine model for acute S.aureus bacteremia – related peri-implant bone infection to compare antibacterial activity of slowly degrading pure Mg and Mg-2Ag with that of medical grade Titanium. Results: Directly on the surface of the explants only for Ti bacteria were detectable while the colonization efficiency of surrounding bone was found to be close between all three materials used as intramedullary implants. Conclusion: Further material modifications e.g. a slightly increased in vivo degradation rate to 1-2 mm / year, which at the same time is not cytotoxic for the surrounding tissue, might be an option to improve the performance of magnesium and magnesium-silver implants for clinical implementation, especially for patients with high risks for S. aureus bacteremia.

Short Talks 11
Bone Substitutes and Regeneration 2

Appointment

Chair

Programme

Short Talks 11Bone Substitutes and Regeneration 2

Appointment

Chair

Programme

Short Talks 11
Bone Substitutes and Regeneration 2