Gingival fibroblast response to (hybrid) ceramic implant reconstruction surfaces is modulated by biomaterial type and surface treatment.

OBJECTIVES: Surface characteristics of implant reconstructions determine the gingival fibroblast (GF) response and thus soft tissue integration (STI). However, for monolithic implant reconstructions it is unknown whether the (hybrid) ceramic biomaterial type and its surface treatment affect GF response. Therefore, this investigation examined the influence of the implant reconstruction biomaterials hybrid ceramic (HC), lithium disilicate ceramic (LS), 4 and 5 mol% yttria partially stabilized zirconiumdioxide ceramics (4/5Y-PSZ) and their surface treatment - machining, polishing or glazing - on surface characteristics and GF response. METHODS: After characterization of surface topography and wettability by scanning electron microscopy, interferometry and contact angle measurement, the adhesion, morphology, metabolic activity and proliferation of GFs from six donors was investigated by fluorescent staining and a resazurin-based assay at days 1, 3 and 7. Titanium (Ti) served as control. RESULTS: Biomaterial type and surface treatment affected the GF response in a topography-dependent manner. Smooth polished and glazed surfaces demonstrated enhanced GF adhesion and earlier proliferation onset compared to rough machined surfaces. Due to minor differences in surface topography of polished and glazed surfaces, however, the GF response was similar for polished and glazed HC, LS, 4- and 5Y-PSZ as well as Ti. SIGNIFICANCE: Within the limits of the present investigation, polishing and glazing of machined HC, LS and 4/5Y-PSZ can be recommended to support STI-relevant cell functions in GF. Since the GF response on polished and glazed HC, LS, 4- and 5Y-PSZ surfaces and the Ti control was comparable, this investigation proofed equal cytocompatibility of these surfaces in vitro.

Analysis of soft tissue integration-supportive cell functions in gingival fibroblasts cultured on 3D printed biomaterials for oral implant-supported prostheses.

To date, it is unknown whether 3D printed fixed oral implant-supported prostheses can achieve comparable soft tissue integration (STI) to clinically established subtractively manufactured counterparts. STI is mediated among others by gingival fibroblasts (GFs) and is modulated by biomaterial surface characteristics. Therefore, the aim of the present work was to investigate the GF response of a 3D printed methacrylate photopolymer and a hybrid ceramic-filled methacrylate photopolymer for fixed implant-supported prostheses in the sense of supporting an STI. Subtractively manufactured samples made from methacrylate polymer and hybrid ceramic were evaluated for comparison and samples from yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP), comprising well documented biocompatibility, served as control. Surface topography was analyzed by scanning electron microscopy and interferometry, elemental composition by energy-dispersive x-ray spectroscopy, and wettability by contact angle measurement. The response of GFs obtained from five donors was examined in terms of membrane integrity, adhesion, morphogenesis, metabolic activity, and proliferation behavior by a lactate-dehydrogenase assay, fluorescent staining, a resazurin-based assay, and DNA quantification. The results revealed all surfaces were smooth and hydrophilic. GF adhesion, metabolic activity and proliferation were impaired by 3D printed biomaterials compared to subtractively manufactured comparison surfaces and the 3Y-TZP control, whereas membrane integrity was comparable. Within the limits of the present investigation, it was concluded that subtractively manufactured surfaces are superior compared to 3D printed surfaces to support STI. For the development of biologically optimized 3D printable biomaterials, consecutive studies will focus on the improvement of cytocompatibility and the synthesis of STI-relevant extracellular matrix constituents.

Effect of Hydrogen Peroxide on the Surface and Attractiveness of Various Zirconia Implant Materials on Human Osteoblasts: An In Vitro Study.

The aim of this in vitro study was to investigate the effect of hydrogen peroxide (H2O2) on the surface properties of various zirconia-based dental implant materials and the response of human alveolar bone osteoblasts. For this purpose, discs of two zirconia-based materials with smooth and roughened surfaces were immersed in 20% H2O2 for two hours. Scanning electron and atomic force microscopy showed no topographic changes after H2O2-treatment. Contact angle measurements (1), X-ray photoelectron spectroscopy (2) and X-ray diffraction (3) indicated that H2O2-treated surfaces (1) increased in hydrophilicity (p < 0.05) and (2) on three surfaces the carbon content decreased (33-60%), while (3) the monoclinic phase increased on all surfaces. Immunofluorescence analysis of the cell area and DNA-quantification and alkaline phosphatase activity revealed no effect of H2O2-treatment on cell behavior. Proliferation activity was significantly higher on three of the four untreated surfaces, especially on the smooth surfaces (p < 0.05). Within the limitations of this study, it can be concluded that exposure of zirconia surfaces to 20% H2O2 for 2 h increases the wettability of the surfaces, but also seems to increase the monoclinic phase, especially on roughened surfaces, which can be considered detrimental to material stability. Moreover, the H2O2-treatment has no influence on osteoblast behavior.

Identification of Zirconia Particle Uptake in Human Osteoblasts by ToF-SIMS Analysis and Particle-Size Effects on Cell Metabolism.

As the use of zirconia-based nano-ceramics is rising in dentistry, the examination of possible biological effects caused by released nanoparticles on oral target tissues, such as bone, is gaining importance. The aim of this investigation was to identify a possible internalization of differently sized zirconia nanoparticles (ZrNP) into human osteoblasts applying Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), and to examine whether ZrNP exposure affected the metabolic activity of the cells. Since ToF-SIMS has a low probing depth (about 5 nm), visualizing the ZrNP required the controlled erosion of the sample by oxygen bombardment. This procedure removed organic matter, uncovering the internalized ZrNP and leaving the hard particles practically unaffected. It was demonstrated that osteoblasts internalized ZrNP within 24 h in a size-dependent manner. Regarding the cellular metabolic activity, metabolization of alamarBlue by osteoblasts revealed a size- and time-dependent unfavorable effect of ZrNP, with the smallest ZrNP exerting the most pronounced effect. These findings point to different uptake efficiencies of the differently sized ZrNP by human osteoblasts. Furthermore, it was proven that ToF-SIMS is a powerful technique for the detection of zirconia-based nano/microparticles that can be applied for the cell-based validation of clinically relevant materials at the nano/micro scale.

A matter of origin - identification of SEMA3A, BGLAP, SPP1 and PHEX as distinctive molecular features between bone site-specific human osteoblasts on transcription level.

In oral and maxillofacial bone reconstruction, autografts from the iliac crest represent the gold standard due to their superior clinical performance, compared to autografts derived from other extraoral regions. Thus, the aim of our study was to identify putative differences between osteoblasts derived from alveolar (hOB-A) and iliac crest (hOB-IC) bone of the same donor (nine donors) by means of their molecular properties in 2D and 3D culture. We thereby focused on the gene expression of biomarkers involved in osteogenic differentiation, matrix formation and osteoclast modulation. Furthermore, we examined the transcriptional response to Vit.D3 in hOB-A and hOB-IC. Our results revealed different modulation modes of the biomarker expression in osteoblasts, namely cell origin/bone entity-dependent, and culture configuration- and/or time-dependent modulations. SEMA3A, SPP1, BGLAP and PHEX demonstrated the strongest dependence on cell origin. With respect to Vit.D3-effects, BGLAP, SPP1 and ALPL displayed the highest Vit.D3-responsiveness. In this context we demonstrated that the transcriptional Vit.D3-response concerning SPP1 and ALPL in human osteoblasts depended on the cell origin. The results indicate a higher bone remodeling activity of iliac crest than alveolar osteoblasts and support the growing evidence that a high osteoclast activity at the host-/donor bone interface may support graft integration.

3D printed zirconia dental implants with integrated directional surface pores combine mechanical strength with favorable osteoblast response.

Dental implants need to combine mechanical strength with promoted osseointegration. Currently used subtractive manufacturing techniques require a multi-step process to obtain a rough surface topography that stimulates osseointegration. Advantageously, additive manufacturing (AM) enables direct implant shaping with unique geometries and surface topographies. In this study, zirconia implants with integrated lamellar surface topography were additively manufactured by nano-particle ink-jetting. The ISO-14801 fracture load of as-sintered implants (516±39 N) resisted fatigue in 5-55 °C water thermo-cycling (631±134 N). Remarkably, simultaneous mechanical fatigue and hydrothermal aging at 90 °C significantly increased the implant strength to 909±280 N due to compressive stress generated at the seamless transition of the 30-40 µm thick, rough and porous surface layer to the dense implant core. This unique surface structure induced an elongated osteoblast morphology with uniform cell orientation and allowed for osteoblast proliferation, long-term attachment and matrix mineralization. In conclusion, the developed AM zirconia implants not only provided high long-term mechanical resistance thanks to the dense core along with compressive stress induced at the transition zone, but also generated a favorable osteoblast response owing to the integrated directional surface pores. STATEMENT OF SIGNIFICANCE: Zirconia ceramics are becoming the material of choice for metal-free dental implants, however significant efforts are required to obtain a rough/porous surface for enhanced osseointegration, along with the risk of surface delamination and/or microstructure variation. In this study, we addressed the challenge by additively manufacturing implants that seamlessly combine dense core with a porous surface layer. For the first time, a unique surface with a directional lamellar pore morphology was additively obtained. This AM implant also provided strength as strong as conventionally manufactured zirconia implants before and after long-term fatigue. Favorable osteoblast response was proved by in-vitro cell investigation. This work demonstrated the opportunity to AM fabricate novel ceramic implants that can simultaneously meet the mechanical and biological functionality requirements.

Proteomic profiling of human bone from different anatomical sites - A pilot study.

PURPOSE: The study aim is a comparative proteome-based analysis of different autologous bone entities (alveolar bone [AB], iliac cortical [IC] bone, and iliac spongiosa [IS]) used for alveolar onlay grafting. EXPERIMENTAL DESIGN: Site-matched bone samples of AB, IC, and IS were harvested during alveolar onlay grafting. Proteins were extracted using a detergent-based (sodium dodecyl sulfate) strategy and trypsinized. Proteome analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used for peptide-to-spectrum matching, peak detection, and quantitation. Linear models for microarray analysis (LIMMA) were used to detect differentially abundant peptides and proteins. RESULTS: A total of 1730 different proteins were identified across the 15 samples at a false discovery rate of 1%. Partial least-squares discriminant analysis approved segregation of AB, IC, and IS protein profiles. LIMMA statistics highlighted 66 proteins that were more abundant in AB then in IC (vs. 92 proteins were enriched in IC over AB). Gene Ontology enrichment analysis revealed a matrisomal versus an immune-related proteome fingerprint in AB versus IC. CONCLUSION AND CLINICAL RELEVANCE: This pilot study demonstrates an ECM protein-related proteome fingerprint in AB and an immune-related proteome fingerprint in IS and IC.

Human osteoblast and fibroblast response to oral implant biomaterials functionalized with non-thermal oxygen plasma.

Plasma-treatment of oral implant biomaterials prior to clinical insertion is envisaged as a potential surface modification method for enhanced implant healing. To investigate a putative effect of plasma-functionalized implant biomaterials on oral tissue cells, this investigation examined the response of alveolar bone osteoblasts and gingival fibroblasts to clinically established zirconia- and titanium-based implant surfaces for bone and soft tissue integration. The biomaterials were either functionalized with oxygen-plasma in a plasma-cleaner or left untreated as controls, and were characterized in terms of topography and wettability. For the biological evaluation, the cell adhesion, morphogenesis, metabolic activity and proliferation were examined, since these parameters are closely interconnected during cell-biomaterial interaction. The results revealed that plasma-functionalization increased implant surface wettability. The magnitude of this effect thereby depended on surface topography parameters and initial wettability of the biomaterials. Concerning the cell response, plasma-functionalization of smooth surfaces affected initial fibroblast morphogenesis, whereas osteoblast morphology on rough surfaces was mainly influenced by topography. The plasma- and topography-induced differential cell morphologies were however not strong enough to trigger a change in proliferation behaviour. Hence, the results indicate that oxygen plasma-functionalization represents a possible cytocompatible implant surface modification method which can be applied for tailoring implant surface wettability.

Controlling osteoblast morphology and proliferation via surface micro-topographies of implant biomaterials.

Current research on surface modifications has yielded advanced implant biomaterials. Various implant surface modifications have been shown to be promising in improving bone target cell response, but more comprehensive studies whether certain implant surface modifications can directly target cell behavioural features such as morphogenesis and proliferation are needed. Here, we studied the response of primary alveolar bone cells on various implant surface modifications in terms of osteoblast morphology and proliferation in vitro. Analyses of surface modifications led to surface-related test parameters including the topographical parameters micro-roughness, texture aspect and surface enlargement as well as the physicochemical parameter surface wettability. We compared osteoblast morphology and proliferation towards the above-mentioned parameters and found that texture aspect and surface enlargement but not surface roughness or wettability exhibited significant impact on osteoblast morphology and proliferation. Detailed analysis revealed osteoblast proliferation as a function of cell morphology, substantiated by an osteoblast size- and morphology-dependent increase in mitotic activity. These findings show that implant surface topography controls cell behavioural morphology and subsequently cell proliferation, thereby opening the road for cell instructive biomaterials.

Differential osteopontin expression in human osteoblasts derived from iliac crest and alveolar bone and its role in early stages of angiogenesis.

In our previous study, we revealed significant differences of osteopontin (OPN) gene expression in primary human osteoblasts (HOBs) derived from iliac crest bone (iHOBs) and alveolar bone (aHOBs). The present study aims at assigning this discriminative expression to a possible biologic function. OPN is known to be involved in several pathologic and physiologic processes, among others angiogenesis. Therefore, we studied the reaction of human umbilical vein endothelial cells (HUVECs) to HOB-derived OPN regarding angiogenesis. To this end, human primary explant cultures of both bone entities from ten donors were established. Subsequent transcription analysis detected higher gene expression of OPN in iHOBs compared to aHOBs, thereby confirming the results of our previous study. This difference was particularly apparent when cultures were derived from female donors. Hence, OPN protein expression as well as the angiogenic potential of OPN was analyzed, originating from HOBs of one female donor. In accordance to the gene expression level, secreted OPN was more abundant in the supernatant of iHOBs than in aHOBs. Moreover, secreted OPN was found to stimulate migration of HUVECs, but not proliferation or tube formation. These results indicate an involvement in very early stages of angiogenesis and a functional distinction of OPN from HOBs derived from different bone entities.