Functionalization of a zirconia surface by covalently immobilized fibronectin and its effects on resistance to thermal, acid, and mechanical exposure.

Silane chemistry has emerged as a powerful tool for surface modification, offering a versatile means to enhance the properties of various substrates, such as dental implant abutment materials. In this study, we investigated the stability of the 3-aminopropyldiisopropylethoxysilane (APDS) layer on yttria-partially stabilized zirconia (Y-TZP) surfaces after mechanical, acid, and thermal treatment in order to simulate fluctuations within the oral cavity. To accomplish that, the viability of human gingival fibroblasts on APDS-modified surfaces after applied treatment strategies was assessed by live/dead staining. Moreover, the hydrolysis stability and enzymatic degradation resistance of crosslinked fibronectin to the APDS layer was examined by immunostaining and western blot. The results revealed that the applied modifications were not affected by the different treatment conditions and could withstand the fluctuations in the oral cavity. Furthermore, crosslinked fibronectin on silanized Y-TZP was stable against hydrolysis over 21 days and enzymatic degradation. We thus can conclude that the proposed functionalization method has high potential to tolerate harmful effects within the oral cavity and remains unchanged on the surface.

A Fibrin-Based Human Multicellular Gingival 3D Model Provides Biomimicry and Enables Long-Term In Vitro Studies.

Collagen-type I gels are widely used for the fabrication of 3D in vitro gingival models. Unfortunately, their long-term stability is low, which limits the variety of in vitro applications. To overcome this problem and achieve better hydrolytic stability of 3D gingival models, fibrin-based hydrogel blends with increased long-term stability in vitro are investigated. Two different fibrin-based hydrogels are tested: fibrin 2.5% (w/v) and fibrin 1% (w/v)/gelatin 5% (w/v). Appropriate numbers of primary human gingival fibroblasts (HGFs) and OKG4/bmi1/TERT (OKG) keratinocytes are optimized to achieve a homogeneous distribution of cells under the assumed 3D conditions. Both hydrogels support the viability of HGFs and the stability of the hydrogel over 28 days. In vitro cultivation at the air-liquid interface triggers keratinization of the epithelium and increases its thickness, allowing the formation of multiple tissue-like layers. The presence of HGFs in the hydrogel further enhances epithelial differentiation. In conclusion, a fibrin-based 3D gingival model mimics the histology of native gingiva in vitro and ensures its long-term stability in comparison with the previously reported collagen paralogs. These results open new perspectives for extending the period within which specific biological or pathological conditions of artificial gingival tissue can be evaluated.

Transglutaminase enables highly hydrolytically and proteolytically stable crosslinking of collagen on titanium surfaces and promotes osteogenic differentiation of human mesenchymal stem cells.

Collagen with its bioactive ligand motives would be predestined as coating on bone implant surfaces like titanium hip stems to facilitate receptor-mediated cell adhesion and thereby improve early osseointegration. Unfortunately, collagen as coating exhibits very low proteolytic resistance in vivo. To overcome this limitation, different crosslinking methods of collagen (transglutaminase, GTA, EDC/NHS, riboflavin, and lysyl oxidase) with silanized titanium alloy (Ti6Al4V) were investigated in terms of degradation resistance, hydrolysis stability, tensile strength, and metabolic cell activity. The in vitro osteogenic differentiation ability of human mesenchymal stem cells (hMSCs) induced by the surface modification was evaluated by immunofluorescence of early osteogenic markers, Alizarin red staining, and energy dispersive X-ray spectroscopy. The expression of the adhesion-related protein vinculin was analyzed on the different functionalized surfaces. The results revealed that the enzymatic crosslinker transglutaminase offered high degradation resistance, tensile strength, and hydrolysis stability compared to the other crosslinking reagents tested. Remarkably, the adhesion sequences within the collagen were accessible to the hMSCs despite the transglutaminase crosslinking procedure. In conclusion, the organochemical functionalization of Ti6Al4V surfaces with collagen using transglutaminase holds great potential to facilitate an enhanced interaction with attached bone cells and thereby could potentially improve and accelerate osseointegration of a titanium-based bone implant in vivo.

Bioprinting-associated pulsatile hydrostatic pressure elicits a mild proinflammatory response in epi- and endothelial cells.

During nozzle-based bioprinting, like inkjet and microextrusion, cells are subjected to hydrostatic pressure for up to several minutes. The modality of the bioprinting-related hydrostatic pressure is either constant or pulsatile depending on the technique. We hypothesized that the difference in the modality of hydrostatic pressure affects the biological response of the processed cells differently. To test this, we used a custom-made setup to apply either controlled constant or pulsatile hydrostatic pressure on endothelial and epithelial cells. Neither bioprinting procedure visibly altered the distribution of selected cytoskeletal filaments, cell-substrate adhesions, and cell-cell contacts in either cell type. In addition, pulsatile hydrostatic pressure led to an immediate increase of intracellular ATP in both cell types. However, the bioprinting-associated hydrostatic pressure triggered a pro-inflammatory response in only the endothelial cells, with an increase of interleukin 8 (IL-8) and a decrease of thrombomodulin (THBD) transcripts. These findings demonstrate that the settings adopted during nozzle-based bioprinting cause hydrostatic pressure that can trigger a pro-inflammatory response in different barrier-forming cell types. This response is cell-type and pressure-modality dependent. The immediate interaction of the printed cells with native tissue and the immune system in vivo might potentially trigger a cascade of events. Our findings, therefore, are of major relevance in particular for novel intra-operative, multicellular bioprinting approaches.

A defined heat pretreatment of gelatin enables control of hydrolytic stability, stiffness, and microstructural architecture of fibrin-gelatin hydrogel blends.

Fibrin-gelatin hydrogel blends exhibit high potential for tissue engineering in vitro applications. However, the means to tailor these blends in order to control their properties, thus opening up a broad range of new target applications, have been insufficiently explored. We hypothesized that a controlled heat treatment of gelatin prior to blend synthesis enables control of hydrolytic swelling and shrinking, stiffness, and microstructural architecture of fibrin-gelatin based hydrogel blends while providing tremendous long-term stability. We investigated these hydrogel blends' compressive strength, in vitro degradation stability, and microstructure in order to test this hypothesis. In addition, we examined the gel's ability to support endothelial cell proliferation and stretching of encapsulated smooth muscle cells. This research showed that a controlled heat pretreatment of the gelatin component strongly influenced the stiffness, swelling, shrinking, and microstructural architecture of the final blends regardless of identical gelatin mass fractions. All blends offered high long-term hydrolytic stability. In conclusion, the results of this study open the possibility to use this technique in order to tune low-concentrated, open-porous fibrin-based hydrogels, even in long-term tissue engineering in vitro experiments.

Biofunctionalization of Dental Abutment Surfaces by Crosslinked ECM Proteins Strongly Enhances Adhesion and Proliferation of Gingival Fibroblasts.

To ensure the long-term success of dental implants, a functional attachment of the soft tissue to the surface of the implant abutment is decisively important in order to prevent the penetration of bacteria into the implant-bone interface, which can trigger peri-implant disease. Here a surface modification approach is described that includes the covalent immobilization of the extracellular matrix (ECM) proteins fibronectin and laminin via a crosslinker to silanized Ti6Al4V and Y-TZP surfaces. The surface properties are evaluated using static contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The interaction of human gingival fibroblasts (HGFs) with the immobilized ECM proteins is verified by analyzing the localization of focal contacts, cell area, cell morphology, proliferation rate, and integrin expression. It is observed in the presence of fibronectin and laminin an increased cellular attachment, proliferation, and integrin expression of HGFs accompanied by a significantly higher number of focal adhesions. The presented approach holds great potential to enable a stronger bond between soft tissue and implant abutment surface. This could potentially help to prevent the penetration of bacteria in an in vivo application and thus reduce the risk of periimplant disease.