29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Prof. Dr.

Chair

Prof. Dr

Enhancing Collagen Properties for Tissue Engineering with UVA/Riboflavin Crosslinking: A Promising Alternative to Chemical Crosslinking

Tissue regeneration/regenerated medicine

Abstract text (incl. figure legends and references) Crosslinked collagen is widely used in clinical treatments. A promising technique is UVA/riboflavin crosslinking which has been approved for clinical ophthalmological therapy since the early 1980. However, the crosslinking mechanism is still not fully understood, and a well-validated manufacturing process with definable composition and reaction kinetics is needed to comply with regulations. In this study, we investigated the mechanism using physical and physicochemical methods, with a focus on stability and mechanical properties. The UVA/riboflavin crosslinked collagen showed improved visco-elasticity, and comparable thermal stability and resistance to degradation compared with EDC/NHS or genipin crosslinking. In vivo studies demonstrated excellent biocompatibility, reduced tissue inflammation, and promoted tissue regeneration effects, indicating the applicability of UVA/riboflavin crosslinked collagen as a biomaterial for wound care and tissue engineering. This study provides valuable insights into the crosslinking mechanism and physical properties of UVA/riboflavin crosslinked collagen, supporting the development of collagen-based materials for biomedical applications with improved mechanical properties and reduced risks of adverse events. Funding: This work was supported by the Federal Ministry of Education and Research (BMBF, Germany, FKZ: 13GW0400A and C); and the State Ministry of Baden–Württemberg for Economic Affairs, Labor, and Tourism (FKZ: 35-4223.10/22; Project MIK)

Short Talk

Generation and characterization of a blue light crosslinkable bioink for corneal tissue engineering

Biofabrication

Abstract text (incl. figure legends and references) Introduction: Corneal damage is among the leading causes of blindness1 and is currently treated by corneal transplantations. To address the associated donor shortage2, in vivo corneal tissue engineering seeks to replace the damaged tissue by fabricating an artificial cornea directly on the patient"s eye. For this purpose, we aim at developing visible light crosslinkable bioinks. Objectives: A blue light crosslinkable system based on riboflavin and methacryloylated gelatin (Gel-MA) was studied with regards to cytotoxicity and crosslinking behavior. Furthermore, keratocytes encapsulated in the hydrogels were studied after one and three days in culture. Materials and Methods: The influence of the co-initiator arginine on the gelation point of a 30 wt. % Gel-MA solution was examined via photorheology. The MTT assay was used to test the cytotoxicity of different riboflavin and arginine concentrations. Additionally, the encapsulation of keratocytes in 30 wt. % Gel-MA hydrogels was studied via a gel-casting process and the viability was determined via live/dead staining. Results: Upon increasing the arginine concentration from 15 mM to 610 mM, the gelation point decreased from 4 min to 1 min 20 s. However, the metabolic activity of keratocytes continuously decreased with increasing arginine concentration. Different concentrations of riboflavin had no effect on the metabolic activity. Furthermore, keratocytes have a viability of about 90 % on Day 1 and 78 % on Day 3 after encapsulation. Conclusion: The studied system is a good candidate for the generation of Gel-MA hydrogels via blue light. A compromise must be met on the concentration of the co-initiator to ensure a fast gelation and minimize cell toxicity. In further experiments, the printability of the system will be tested. References: J. M. J. Roodhooft. Bull. Soc. belge Ophtalmol. 2002, 283, 19. Gain, P.; Jullienne, R.; He, Z.; Aldossary, M.; Acquart, S.; Cognasse, F.; Thuret, G. JAMA Ophthalmol 2016, 134 (2), 167.

Professor

Influence of Mg-Li biomaterials on schwann cell response in an in vitro nerve injury model

Cell-material interactions

Implant associated

Abstract text (incl. figure legends and references) Peripheral nerve injury leading to long nerve gaps can result in incomplete recovery and require support to enable complete nerve regeneration. Wires made of biodegradable metals, like Magnesium (Mg), can potentially be used to bridge this nerve gap. In the nerve, Schwann cells play an important role in initiating and promoting successful nerve regeneration after injury. Hence, we tested the effect of Mg and Mg-1.8Li metal foils on Schwann cell functions such as MCP-1 secretion (macrophage recruitment), gene expression of neurotrophins and myelin. In order to study the effects in a nerve injury-like microenvironment, we developed an in vitro injury model. We tested two models for RT4 Schwannoma cells - freeze-killed Schwannoma cells and rat sciatic nerves. MCP-1 release was used as an injury marker, since injured Schwann cells release this chemokine to recruit macrophages and clear the injured debris. There was a dose-dependent increase in MCP-1 release due to both injury stimulants. However, the freeze-killed nerve extracts induced a quicker release of MCP-1, indicating that there are factors in addition to the cellular damage-associated molecules that contribute to the response. Using the freeze-killed cell extracts, we studied the influence of Mg and Mg-1.8Li foils in indirect contact with injury-stimulated Schwannoma cells. Both materials reduced MCP-1 release after 48 h of exposure. The presence of the metals along with the injury stimulant significantly downregulated Gdnf expression that was upregulated by injury. Furthermore, they siginificantly upregulated p75 expression which plays a positive role in the remyelination process. Both materials upregulated the expression of Mpz, the most abundant myelin protein. In conclusion, Mg-based materials when used as a support in nerve regeneration could reduce the pro-inflammatory response. The results also suggest that Mg could positively influence the remyelination and further in vivo studies could be useful.

A novel dynamic bioreactor system enables the investigation of mechanobiological cues in periodontal ligament fibroblasts in vitro

Abstract text (incl. figure legends and references) Introduction The periodontal ligament (PDL) is a connective tissue that supports and surrounds the teeth and is vital for their proper function and movement. Orthodontic tooth movement (OTM) applies mechanical forces to the PDL, resulting in tissue remodeling. However, the intracellular signaling mechanisms involved in PDL regeneration in response to mechanical stimuli are not well understood due to a lack of suitable in vitro models. Objectives We developed a custom bioreactor for 3D dynamic cultivation of PDL-derived fibroblast (PDLF) to mimic native periodontal tissue during OTM. This 3D in vitro PDL model exposed to cyclic mechanical loading is a potential tool for investigating mechanotransduction effect and may provide insights into the molecular mechanisms underlying tissue remodeling during OTM. Methods Using a customized bioreactor, we provided cyclic stretching to PDLF cells embedded in a collagen-based hydrogel. Cell proliferation, morphology, and behavior were investigated and compared between static and dynamic conditions. Results We examined hydrogel stability, permeability, and biocompatibility to establish a proper PDLF cultivation model. We optimized stretching conditions using our dynamic bioreactor and observed increased cell proliferation (1.2-fold) and Periostin expression (3-fold). Glycosaminoglycan production analysis revealed extensive ECM remodeling, as evidenced by Collagen type I and Collagen type III expression. The dynamic cultivation period also increased hydrogel stiffness due to collagen fiber orientation and ECM remodeling. Conclusion With our customized bioreactor, we are able to apply mechanical stretching on PDLF cells embedded in collagen-based hydrogels. Therefore, the introduced dynamic bioreactor system is a highly effective device to investigate mechanobiological cues induced in the PDL during OTM.

Mechanical testing of a nanocomposite based on alginate and calcium phosphate nanoparticles for application as bone glue

Abstract text (incl. figure legends and references) Introduction Every year, millions of people suffer from bone fractures. Typically, proper fixation is achieved with plates, pins and screws. In addition, calcium phosphate (CaP) cements and pastes are successfully used in dental and orthopedic applications to fill bone defects. They are biocompatible, biodegradable, osteoconductive, and to some extent osteoinductive. Objectives The aim of this project is to develop a synthetic paste based on chemically modified calcium phosphate nanoparticles which is capable of gluing bone fragments. Materials &amp; Methods Calcium phosphate nanoparticles were synthesized by co-precipitation of calcium lactate and di-ammonium hydrogen carbonate. They were stabilized with carboxy&shy;methylcellulose (CMC) and a silica shell (SiO2). CMC-stabilized nanoparticles represented the inorganic part of the paste which was thoroughly chemically and biologically characterized. The particles were mixed with an alginate solution in order to obtain a bone glue capable of binding under wet conditions. This was tested by submerging glued bone fragment in water. Osteoblast differentiation from SCP1 cells was investigated on the surface of this compound. Results The combination of CaP/CMC/SiO2 nanoparticles with alginate resulted in a nanocomposite capable of gluing bone fragments. The bond was stable for at least 24h in water. Osteoblast differentiation from SCP1 cells was evaluated by quantifying collagen deposition with Sirius red. The staining showed no difference in collagen deposition compared to the control group after 28 days. Conclusion Calcium phosphate-alginate nanocomposites can be used to glue bone fragments. This bond is stable for at least 24h, even if submerged in fluids. Osteoblast differentiation from SCP1 cells on the bone glue indicated no adverse effects on collagen production, which is an important requirement for bone fracture remodeling.

PD Dr.

High hydrostatic pressure as a novel method for devitalization and quality enhancement of cartilage tissue grafts in head and neck surgery

Abstract text (incl. figure legends and references) The increasing importance of regenerative medicine, especially in the context of demographic change, has resulted in a growing need for tissue replacement materials. Compared to constantly improving alloplastic materials, tissue donations preserve the native extracellular matrix (ECM). However, the reprocessing is time-consuming and can damage the ECM. Therefore, new methods are needed to provide tissue that is both native and biomechanically stable. High hydrostatic pressure (HHP) could allow specific devitalization and quality enhancement of the grafts without significant damage to the ECM. In this study, thyroid cartilage and chondrocytes were treated with HHP at 150 - 600 MPa for 10 min. Analysis of devitalization and decellularization of the tissue (flow cytometry, histology, DNA content) were performed. In addition, the ECM was examined using histology, scanning electron microscopy, and biomechanical testing. Furthermore, the biocompatibility of the high pressure treated tissue was evaluated in vivo using a dorsal skinfold chamber in a mouse model. Finally, the ability of the tissue to revitalize with stem cells (hMSCs) was investigated in vitro. HHP causes devitalization above 300 MPa without detectable decellularization. Morphologically, no changes in the ECM were identified, but the e-modulus significantly increased in cartilage after pressure treatment of 450 MPa or higher. Animal experiments confirmed the biocompatibility of the prepared tissue. Moreover, hMSCs were successfully cultured on pressure-treated tissue in vitro. HHP devitalizes cartilage tissue quickly and specifically while retaining the essential properties of the ECM. In addition, the biocompatibility of treated samples was demonstrated, and their ability to be revitalized with hMSCs was established. Prospectively, HHP could simplify the preparation of allo- and xenogeneic tissue replacements and increase the availability of grafts in head and neck surgery.

Short Talks 13
Tissue Regeneration 2

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Short Talks 13Tissue Regeneration 2

Appointment

Chair

Topic

Programme

Short Talks 13
Tissue Regeneration 2