Mechanical in vitro fatigue testing of implant materials and components using advanced characterization techniques.

Implants of different material classes have been used for the reconstruction of damaged hard and soft tissue for decades. The aim is to increase and subsequently maintain the patient's quality of life through implantation. In service, most implants are subjected to cyclic loading, which must be taken particularly into consideration, since the fatigue strength is far below the yield and tensile strength. Inaccurate estimation of the structural strength of implants due to the consideration of yield or tensile strength leads to a miscalculation of the implant's fatigue strength and lifetime, and therefore, to its unexpected early fatigue failure. Thus, fatigue failure of an implant based on overestimated performance capability represents acute danger to human health. The determination of fatigue strength by corresponding tests investigating various stress amplitudes is time-consuming and cost-intensive. This study summarizes four investigation series on the fatigue behavior of different implant materials and components, following a standard and an in vitro short-time testing procedure, which evaluates the material reaction in one enhanced test set-up. The test set-up and the applied characterization methods were adapted to the respective application of the implant with the aim to simulate the surrounding of the human body with laboratory in vitro tests only. It could be shown that by using the short-time testing method the number of tests required to determine the fatigue strength can be drastically reduced. In future, therefore it will be possible to exclude unsuitable implant materials or components before further clinical investigations by using a time-efficient and application-oriented testing method.

Vascular Network Formation on Macroporous Polydioxanone Scaffolds.

In this study, microvascular network structures for tissue engineering were generated on newly developed macroporous polydioxanone (PDO) scaffolds. PDO represents a polymer biodegradable within months and offers optimal material properties such as elasticity and nontoxic degradation products. PDO scaffolds prepared by porogen leaching and cryo-dried to achieve pore sizes of 326 ± 149.67 µm remained stable with equivalent values for Young's modulus after 4 weeks. Scaffolds were coated with fibrin for optimal cell adherence. To exclude interindividual differences, autologous fibrin was prepared out of human plasma-derived fibrinogen and proved a comparable quality to nonautologous commercially available fibrinogen. Fibrin-coated scaffolds were seeded with recombinant human umbilical vein endothelial cells expressing GFP (GFP-HUVECs) in coculture with adipose tissue-derived mesenchymal stem cells (AD-hMSCs) to form vascular networks. The growth factor content in culture media was optimized according its effect on network formation, quantified and assessed by AngioTool®. A ratio of 2:3 GFP-HUVECs/AD-hMSCs in medium enriched with 20 ng/mL vascular endothelial growth factor, basic fibroblast growth factor, and hydrocortisone was found to be optimal. Network structures appeared after 2 days of cultivation and stabilized until day 7. The resulting networks were lumenized that could be verified by dextran staining. This new approach might be suitable for microvascular tissue patches as a useful template to be used in diverse vascularized tissue constructs. Impact statement We consider this work as important for the current research in the field of tissue engineering and the development of new and functional tissue. The approach for the production of vascularized tissue patches, consisting of the biodegradable synthetic polymer polydioxanone and of the physiological, autologous, and patient-specific polymer fibrin, and seeded with endothelial cells and mesenchymal stem cells, displayed within this work, could be useful for the sustaining development of diverse and more complex tissue constructs. Therefore, these scaffolds could be used as a cornerstone for future tissue engineering approaches.

Toward Tailoring the Degradation Rate of Magnesium-Based Biomaterials for Various Medical Applications: Assessing Corrosion, Cytocompatibility and Immunological Effects.

Magnesium (Mg)-based biomaterials hold considerable promise for applications in regenerative medicine. However, the degradation of Mg needs to be reduced to control toxicity caused by its rapid natural corrosion. In the process of developing new Mg alloys with various surface modifications, an efficient assessment of the relevant properties is essential. In the present study, a WE43 Mg alloy with a plasma electrolytic oxidation (PEO)-generated surface was investigated. Surface microstructure, hydrogen gas evolution in immersion tests and cytocompatibility were assessed. In addition, a novel in vitro immunological test using primary human lymphocytes was introduced. On PEO-treated WE43, a larger number of pores and microcracks, as well as increased roughness, were observed compared to untreated WE43. Hydrogen gas evolution after two weeks was reduced by 40.7% through PEO treatment, indicating a significantly reduced corrosion rate. In contrast to untreated WE43, PEO-treated WE43 exhibited excellent cytocompatibility. After incubation for three days, untreated WE43 killed over 90% of lymphocytes while more than 80% of the cells were still vital after incubation with the PEO-treated WE43. PEO-treated WE43 slightly stimulated the activation, proliferation and toxin (perforin and granzyme B) expression of CD8+ T cells. This study demonstrates that the combined assessment of corrosion, cytocompatibility and immunological effects on primary human lymphocytes provide a comprehensive and effective procedure for characterizing Mg variants with tailorable degradation and other features. PEO-treated WE43 is a promising candidate for further development as a degradable biomaterial.

Degradation, Bone Regeneration and Tissue Response of an Innovative Volume Stable Magnesium-Supported GBR/GTR Barrier Membrane.

INTRODUCTION: Bioresorbable collagenous barrier membranes are used to prevent premature soft tissue ingrowth and to allow bone regeneration. For volume stable indications, only non-absorbable synthetic materials are available. This study investigates a new bioresorbable hydrofluoric acid (HF)-treated magnesium (Mg) mesh in a native collagen membrane for volume stable situations. MATERIALS AND METHODS: HF-treated and untreated Mg were compared in direct and indirect cytocompatibility assays. In vivo, 18 New Zealand White Rabbits received each four 8 mm calvarial defects and were divided into four groups: (a) HF-treated Mg mesh/collagen membrane, (b) untreated Mg mesh/collagen membrane (c) collagen membrane and (d) sham operation. After 6, 12 and 18 weeks, Mg degradation and bone regeneration was measured using radiological and histological methods. RESULTS: In vitro, HF-treated Mg showed higher cytocompatibility. Histopathologically, HF-Mg prevented gas cavities and was degraded by mononuclear cells via phagocytosis up to 12 weeks. Untreated Mg showed partially significant more gas cavities and a fibrous tissue reaction. Bone regeneration was not significantly different between all groups. DISCUSSION AND CONCLUSIONS: HF-Mg meshes embedded in native collagen membranes represent a volume stable and biocompatible alternative to the non-absorbable synthetic materials. HF-Mg shows less corrosion and is degraded by phagocytosis. However, the application of membranes did not result in higher bone regeneration.

In Vivo Simulation of Magnesium Degradability Using a New Fluid Dynamic Bench Testing Approach.

The degradation rate of magnesium (Mg) alloys is a key parameter to develop Mg-based biomaterials and ensure in vivo-mechanical stability as well as to minimize hydrogen gas production, which otherwise can lead to adverse effects in clinical applications. However, in vitro and in vivo results of the same material often differ largely. In the present study, a dynamic test bench with several single bioreactor cells was constructed to measure the volume of hydrogen gas which evolves during magnesium degradation to indicate the degradation rate in vivo. Degradation medium comparable with human blood plasma was used to simulate body fluids. The media was pumped through the different bioreactor cells under a constant flow rate and 37 °C to simulate physiological conditions. A total of three different Mg groups were successively tested: Mg WE43, and two different WE43 plasma electrolytically oxidized (PEO) variants. The results were compared with other methods to detect magnesium degradation (pH, potentiodynamic polarization (PDP), cytocompatibility, SEM (scanning electron microscopy)). The non-ceramized specimens showed the highest degradation rates and vast standard deviations. In contrast, the two PEO samples demonstrated reduced degradation rates with diminished standard deviation. The pH values showed above-average constant levels between 7.4-7.7, likely due to the constant exchange of the fluids. SEM revealed severe cracks on the surface of WE43 after degradation, whereas the ceramized surfaces showed significantly decreased signs of corrosion. PDP results confirmed the improved corrosion resistance of both PEO samples. While WE43 showed slight toxicity in vitro, satisfactory cytocompatibility was achieved for the PEO test samples. In summary, the dynamic test bench constructed in this study enables reliable and simple measurement of Mg degradation to simulate the in vivo environment. Furthermore, PEO treatment of magnesium is a promising method to adjust magnesium degradation.

Balancing Purification and Ultrastructure of Naturally Derived Bone Blocks for Bone Regeneration: Report of the Purification Effort of Two Bone Blocks.

The present publication reports the purification effort of two natural bone blocks, that is, an allogeneic bone block (maxgraft®, botiss biomaterials GmbH, Zossen, Germany) and a xenogeneic block (SMARTBONE®, IBI S.A., Mezzovico-Vira, Switzerland) in addition to previously published results based on histology. Furthermore, specialized scanning electron microscopy (SEM) and in vitro analyses (XTT, BrdU, LDH) for testing of the cytocompatibility based on ISO 10993-5/-12 have been conducted. The microscopic analyses showed that both bone blocks possess a trabecular structure with a lamellar subarrangement. In the case of the xenogeneic bone block, only minor remnants of collagenous structures were found, while in contrast high amounts of collagen were found associated with the allogeneic bone matrix. Furthermore, only island-like remnants of the polymer coating in case of the xenogeneic bone substitute seemed to be detectable. Finally, no remaining cells or cellular remnants were found in both bone blocks. The in vitro analyses showed that both bone blocks are biocompatible. Altogether, the purification level of both bone blocks seems to be favorable for bone tissue regeneration without the risk for inflammatory responses or graft rejection. Moreover, the analysis of the maxgraft® bone block showed that the underlying purification process allows for preserving not only the calcified bone matrix but also high amounts of the intertrabecular collagen matrix.

Corrosion and Corrosion Fatigue Properties of Additively Manufactured Magnesium Alloy WE43 in Comparison to Titanium Alloy Ti-6Al-4V in Physiological Environment.

Laser powder bed fusion (L-PBF) of metals enables the manufacturing of highly complex geometries which opens new application fields in the medical sector, especially with regard to personalized implants. In comparison to conventional manufacturing techniques, L-PBF causes different microstructures, and thus, new challenges arise. The main objective of this work is to investigate the influence of different manufacturing parameters of the L-PBF process on the microstructure, process-induced porosity, as well as corrosion fatigue properties of the magnesium alloy WE43 and as a reference on the titanium alloy Ti-6Al-4V. In particular, the investigated magnesium alloy WE43 showed a strong process parameter dependence in terms of porosity (size and distribution), microstructure, corrosion rates, and corrosion fatigue properties. Cyclic tests with increased test duration caused an especially high decrease in fatigue strength for magnesium alloy WE43. It can be demonstrated that, due to high process-induced surface roughness, which supports locally intensified corrosion, multiple crack initiation sites are present, which is one of the main reasons for the drastic decrease in fatigue strength.

Improved In Vitro Test Procedure for Full Assessment of the Cytocompatibility of Degradable Magnesium Based on ISO 10993-5/-12.

Magnesium (Mg)-based biomaterials are promising candidates for bone and tissue regeneration. Alloying and surface modifications provide effective strategies for optimizing and tailoring their degradation kinetics. Nevertheless, biocompatibility analyses of Mg-based materials are challenging due to its special degradation mechanism with continuous hydrogen release. In this context, the hydrogen release and the related (micro-) milieu conditions pretend to strictly follow in vitro standards based on ISO 10993-5/-12. Thus, special adaptions for the testing of Mg materials are necessary, which have been described in a previous study from our group. Based on these adaptions, further developments of a test procedure allowing rapid and effective in vitro cytocompatibility analyses of Mg-based materials based on ISO 10993-5/-12 are necessary. The following study introduces a new two-step test scheme for rapid and effective testing of Mg. Specimens with different surface characteristics were produced by means of plasma electrolytic oxidation (PEO) using silicate-based and phosphate-based electrolytes. The test samples were evaluated for corrosion behavior, cytocompatibility and their mechanical and osteogenic properties. Thereby, two PEO ceramics could be identified for further in vivo evaluations.

Development of biomimetic in vitro fatigue assessment for UHMWPE implant materials.

An important research goal in the field of biomaterials lies in the progressive amendment of in vivo tests with suitable in vitro experiments. Such approaches are gaining more significance nowadays because of an increasing demand on life sciences and the ethical issues bound to the sacrifice of animals for the sake of scientific research. Another advantage of transferring the experiments to the in vitro field is the possibility of accurately control the boundary conditions and experimental parameters in order to reduce the need of validation tests involving animals. With the aim to reduce the amount of needed in vivo studies for this cause, a short-time in vitro test procedure using instrumented load increase tests with superimposed environmental loading has been developed at TUD to assess the mechanical long-term durability of ultra-high molecular weight polyethylene (UHMWPE) under fatigue loading in a biological environment.