Swelling and Mechanical Characterization of Polyelectrolyte Hydrogels as Potential Synthetic Cartilage Substitute Materials.

Hydrogels have become an increasingly interesting topic in numerous fields of application. In addition to their use as immobilization matrixes in (bio)catalysis, they are widely used in the medical sector, e.g., in drug delivery systems, contact lenses, biosensors, electrodes, and tissue engineering. Cartilage tissue engineering hydrogels from natural origins, such as collagen, hyaluronic acid, and gelatin, are widely known for their good biocompatibility. However, they often lack stability, reproducibility, and mechanical strength. Synthetic hydrogels, on the other hand, can have the advantage of tunable swelling and mechanical properties, as well as good reproducibility and lower costs. In this study, we investigated the swelling and mechanical properties of synthetic polyelectrolyte hydrogels. The resulting characteristics such as swelling degree, stiffness, stress, as well as stress-relaxation and cyclic loading behavior, were compared to a commercially available biomaterial, the ChondroFiller® liquid, which is already used to treat articular cartilage lesions. Worth mentioning are the observed good reproducibility and high mechanical strength of the synthetic hydrogels. We managed to synthesize hydrogels with a wide range of compressive moduli from 2.5 ± 0.1 to 1708.7 ± 67.7 kPa, which addresses the span of human articular cartilage.

Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro.

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.

Re-Differentiation Capacity of Human Chondrocytes in Vitro Following Electrical Stimulation with Capacitively Coupled Fields.

Treatment of cartilage lesions remains a clinical challenge. Therefore, biophysical stimuli like electric fields seem to be a promising tool for chondrocytic differentiation and treatment of cartilage lesions. In this in vitro study, we evaluated the effects of low intensity capacitively coupled electric fields with an alternating voltage of 100 mVRMS (corresponds to 5.2 × 10-5 mV/cm) or 1 VRMS (corresponds to 5.2 × 10-4 mV/cm) with 1 kHz, on human chondrocytes derived from osteoarthritic (OA) and non-degenerative hyaline cartilage. A reduction of metabolic activity after electrical stimulation was more pronounced in non-degenerative cells. In contrast, DNA contents in OA cells were significantly decreased after electrical stimulation. A difference between 100 mVRMS and 1 VRMS was not detected. However, a voltage-dependent influence on gene and protein expression was observed. Both cell types showed increased synthesis rates of collagen (Col) II, glycosaminoglycans (GAG), and Col I protein following stimulation with 100 mVRMS, whereas this increase was clearly higher in OA cells. Our results demonstrated the sensitization of chondrocytes by alternating electric fields, especially at 100 mVRMS, which has an impact on chondrocytic differentiation capacity. However, analysis of further electrical stimulation parameters should be done to induce optimal hyaline characteristics of ex vivo expanded human chondrocytes.

Effect of electric stimulation on human chondrocytes and mesenchymal stem cells under normoxia and hypoxia.

During joint movement and mechanical loading, electric potentials occur within cartilage tissue guiding cell development and regeneration. Exposure of cartilage exogenous electric stimulation (ES) may imitate these endogenous electric fields and promote healing processes. Therefore, the present study investigated the influence of electric fields on human chondrocytes, mesenchymal stem cells and the co­culture of the two. Human chondrocytes isolated from articular cartilage obtained post­mortally and human mesenchymal stem cells derived from bone marrow (BM­MSCs) were seeded onto a collagen­based scaffold separately or as co­culture. Following incubation with the growth factors over 3 days, ES was performed using titanium electrodes applying an alternating electric field (700 mV, 1 kHz). Cells were exposed to an electric field over 7 days under either hypoxic or normoxic culture conditions. Following this, metabolic activity was investigated and synthesis rates of extracellular matrix proteins were analyzed. ES did not influence metabolic activity of chondrocytes or BM­MSCs. Gene expression analyses demonstrated that ES increased the expression of collagen type II mRNA and aggrecan mRNA in human chondrocytes under hypoxic culture conditions. Likewise, collagen type II synthesis was significantly increased following exposure to electric fields under hypoxia. BM­MSCs and the co­culture of chondrocytes and BM­MSCs revealed a similar though weaker response regarding the expression of cartilage matrix proteins. The electrode setup may be a valuable tool to investigate the influence of ES on human chondrocytes and BM­MSCs contributing to fundamental knowledge including future applications of ES in cartilage repair.

The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes.

Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs). Thus, we exposed cells with a mixture of 200 µg/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-α, MCP-1, MMP1, TIMP1) were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions.