Human dental pulp cells modulate CD8+ T cell proliferation and efficiently degrade extracellular ATP to adenosine in vitro.

The pulp of human teeth contains a population of self-renewing stem cells that can regulate the functions of immune cells. When applied to patients, these cells can protect tissues from damage by excessive inflammation. We confirm that dental pulp cells effectively inhibit the proliferation and activation of cytotoxic T cells in vitro, and show that they carry high levels of CD73, a key enzyme in the conversion of pro-inflammatory extracellular ATP to immunosuppressive adenosine. Given their accessibility and abundance, as well as their potential for allogeneic administration, dental pulp cells provide a valuable source for immunomodulatory therapy.

Antibacterial properties of functionalized silk fibroin and sericin membranes for wound healing applications in oral and maxillofacial surgery.

Oral wounds are among the most troublesome injuries which easily affect the patients' quality of life. To date, the development of functional antibacterial dressings for oral wound healing remains a challenge. In this regard, we investigated antibacterial silk protein-based membranes for the application as wound dressings in oral and maxillofacial surgery. The present study includes five variants of casted membranes, i.e., i) membranes-silver nanoparticles (CM-Ag), ii) membranes-gentamicin (CM-G), iii) membranes-control (without functionalization) (CM-C), iv) membranes-silk sericin control (CM-SSC), and v) membranes-silk fibroin/silk sericin (CM-SF/SS), and three variants of nonwovens, i.e., i) silver nanoparticles (NW-Ag), ii) gentamicin (NW-G), iii) control (without functionalization) (NW-C). The surface structure of the samples was visualized with scanning electron microscopy. In addition, antibacterial testing was accomplished using agar diffusion assay, colony forming unit (CFU) analysis, and qrt-PCR. Following antibacterial assays, biocompatibility was evaluated by cell proliferation assay (XTT), cytotoxicity assay (LDH), and live-dead assay on L929 mouse fibroblasts. Findings indicated significantly lower bacterial colony growth and DNA counts for CM-Ag with a reduction of bacterial counts by 3log levels (99.9% reduction) in CFU and qrt-PCR assay compared to untreated control membranes (CM-C and CM-SSC) and membranes functionalized with gentamicin (CM-G and NW-G) (p < 0.001). Similarly, NW-G yielded significantly lower DNA and colony growth counts compared to NW-Ag and NW-C (p < 0.001). In conclusion, CM-Ag represented 1log level better antibacterial activity compared to NW-G, whereas NW-G showed better cytocompatibility for L929 cells. As data suggest, these two membranes have the potential of application in the field of bacteria-free oral wound healing. However, provided that loading strategy and cytocompatibility are adjusted according to the antibacterial agents' characteristic and fabrication technique of the membranes.

Tissue Adhesives in Reconstructive and Aesthetic Surgery-Application of Silk Fibroin-Based Biomaterials.

Tissue adhesives have been successfully used in various kind of surgeries such as oral and maxillofacial surgery for some time. They serve as a substitute for suturing of tissues and shorten treatment time. Besides synthetic-based adhesives, a number of biological-based formulations are finding their way into research and clinical application. In natural adhesives, proteins play a crucial role, mediating adhesion and cohesion at the same time. Silk fibroin, as a natural biomaterial, represents an interesting alternative to conventional medical adhesives. Here, the most commonly used bioadhesives as well as the potential of silk fibroin as natural adhesives will be discussed.

Inversed Ratio of CD39/CD73 Expression on γδ T Cells in HIV Versus Healthy Controls Correlates With Immune Activation and Disease Progression.

Background: γδ T cells are unconventional T cells that have been demonstrated to be crucial for the pathogenesis and potentially for the cure of HIV-1 infection. The ectonucleotidase CD39 is part of the purinergic pathway that regulates immune responses by degradation of pro-inflammatory ATP in concert with CD73. Few studies on the expression of the ectoenzymes CD73 and CD39 on human γδ T cells in HIV have been performed to date. Methods: PBMC of n=86 HIV-1-infected patients were compared to PBMC of n=26 healthy individuals using 16-color flow cytometry determining the surface expression of CD39 and CD73 on Vδ1 and Vδ2 T cells in association with differentiation (CD45RA, CD28, CD27), activation and exhaustion (TIGIT, PD-1, CD38, and HLA-DR), and assessing the intracellular production of pro- and anti-inflammatory cytokines (IL-2, TGF-ß, TNF-α, Granzyme B, IL-10, IFN-γ) after in vitro stimulation with PMA/ionomycin. Results: CD39 and CD73 expression on γδ T cells were inversed in HIV infection which correlated with HIV disease progression and immune activation. CD39, but not CD73 expression on γδ T cells of ART-treated patients returned to levels comparable with those of healthy individuals. Only a small subset (<1%) of γδ T cells co-expressed CD39 and CD73 in healthy or HIV-infected individuals. There were significantly more exhausted and terminally differentiated CD39+ Vδ1 T cells regardless of the disease status. Functionally, IL-10 was only detectable in CD39+ γδ T cells after in vitro stimulation in all groups studied. Viremic HIV-infected patients showed the highest levels of IL-10 production. The highest percentage of IL-10+ cells was found in the small CD39/CD73 co-expressing γδ T-cell population, both in healthy and HIV-infected individuals. Also, CD39+ Vδ2 T cells produced IL-10 more frequently than their CD39+ Vδ1 counterparts in all individuals regardless of the HIV status. Conclusions: Our results point towards a potential immunomodulatory role of CD39+ and CD73+ γδ T cells in the pathogenesis of chronic HIV infection that needs further investigation.

Attachment and Osteogenic Potential of Dental Pulp Stem Cells on Non-Thermal Plasma and UV Light Treated Titanium, Zirconia and Modified PEEK Surfaces.

Ultraviolet (UV) light and non-thermal plasma (NTP) treatment are chairside methods that can efficiently improve the biological aging of implant material surfaces caused by customary storage. However, the behaviors of stem cells on these treated surfaces of the implant are still unclear. This study aimed to investigate the effects of UV light and NTP treated surfaces of titanium, zirconia and modified polyetheretherketone (PEEK, BioHPP) on the attachment and osteogenic potential of human dental pulp stem cells (DPSCs) in vitro. Machined disks were treated using UV light and argon or oxygen NTP for 12 min each. Untreated disks were set as controls. DPSCs were cultured from the wisdom teeth of adults that gave informed consent. After 24 h of incubation, the attachment and viability of cells on surfaces were assessed. Cells were further osteogenically induced, alkaline phosphatase (ALP) activity was detected via a p-Nitrophenyl phosphate assay (day 14 and 21) and mineralization degree was measured using a Calcium Assay kit (day 21). UV light and NTP treated titanium, zirconia and BioHPP surfaces improved the early attachment and viability of DPSCs. ALP activity and mineralization degree of osteoinductive DPSCs were significantly increased on UV light and NTP treated surfaces of titanium, zirconia and also oxygen plasma treated Bio-HPP (p < 0.05). In conclusion, UV light and NTP treatments may improve the attachment of DPSCs on titanium, zirconia and BioHPP surfaces. Osteogenic differentiation of DPSCs can be enhanced on UV light and NTP treated surfaces of titanium and zirconia, as well as on oxygen plasma treated Bio-HPP.

Cytocompatibility of Bone Substitute Materials and Membranes.

BACKGROUND/AIM: With the demographic change and associated chronic bone loss, the need for cytocompatible bone replacement materials arise in modern medicine. The aim of this in vitro study was to investigate the cytocompatibility of eleven different bone substitute materials and membranes. MATERIALS AND METHODS: Seven bone substitute materials and four membranes were assessed in vitro. The specimens were tested based on their interaction with MC3T3 pre-osteoblasts, through the utilization of viability, proliferation, and cytotoxicity assays. Cell vitality was evaluated using live-dead staining. RESULTS: Although we found minor differences in cytocompatibility among the assessed materials, all tested materials can be considered as cytocompatible with a viability of more than 70% of the negative control, which indicates the non-toxic range as defined in current, international standards (DIN EN ISO 10993-5:2009, German Institute for Standardization, Berlin, Germany). Direct live-dead staining assays confirmed satisfactory cytocompatibility of all tested membranes. CONCLUSION: All examined bone substitute materials and membranes were found to be cytocompatible. In order to assess whether the observed minor differences can impact regenerative processes, further in vivo studies need to be conducted.

Risk Stratification for Immediate Postoperative Hearing Loss by Preoperative BAER (Brainstem Auditory Evoked Response) and Audiometry in NF2-Associated Vestibular Schwannomas.

Both brainstem auditory evoked potentials (BAEP) and audiometry play a crucial role in neuro-oncological treatment decisions in Neurofibromatosis Type 2 associated (NF2) vestibular schwannoma (VS) as hearing preservation is the major goal. In this study, we investigated the risk of immediate postoperative hearing deterioration (>15 dB and/or 15% loss in pure-tone average [PTA]/ speech discrimination score [SDS] in a cohort of 100 operated VS (ears) in 72 NF2 patients by retrospective analysis of pre- and postoperative hearing data (PTA, SDS, American Association of Otolaryngology-Head and Neck Surgery [AAO-HNS], and brainstem auditory evoked potential [BAEP] class) taking into account relevant influencing factors, particularly preoperative audiometry and BAEP status and the extent of resection. Immediately after surgery, the hearing was preserved in 73% of ears and approximately ~60% of ears kept their hearing classes. Preoperative BAEP (p = 0.015) and resection amount (p = 0.048) significantly influenced postoperative hearing outcome. The prediction model for postoperative hearing deterioration/loss between preoperative BAEP and AAO-HNS class showed increased risk by increasing BAEP class. Twenty-one tumors/ears were identified with large BAEP and AAO-HNS class discrepancies (≥2 points) and were associated with a high (48-100%) risk of deafness after surgery in ears with preoperative available hearing. Overall, the results were heterogeneous but the better both BAEP and audiometry class before surgery, the higher the chance of hearing maintenance afterwards. Large resection amounts (e.g., 100% risk in near-total resections) exhibit a significant (p < 0.05) higher risk compared to smaller amounts (e.g., 10/20% in laser-coagulated/partially resected tumors). Our results emphasized the indispensable role of both hearing monitoring in form of audiometry and neurophysiology (BAEP) in the pre-and perioperative monitoring of NF2-associated VS. Both BAEP and audiometry are good prognostic markers for the postoperative hearing outcome. The extent of resection should be strictly guided by and adjusted to the intraoperative neurophysiological monitoring.

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.

Marginal Adaptation and Microbial Leakage at Conometric Prosthetic Connections for Implant-Supported Single Crowns: An In Vitro Investigation.

Encouraging clinical results were reported on a novel cone-in-cone coupling for the fixation of dental implant-supported crowns (Acuris, Dentsply Sirona Implants, Mölndal, Sweden). However, the presence or absence of a microgap and a potential bacterial leakage at the conometric joint has not yet been investigated. A misfit and a resulting gap between the conometric components could potentially serve as a bacterial reservoir that promotes plaque formation, which in turn may lead to inflammation of the peri-implant tissues. Thus, a two-fold study set-up was designed in order to evaluate the bidirectional translocation of bacteria along conometrically seated single crowns. On conometric abutments filled with a culture suspension of anaerobic bacteria, the corresponding titanium nitride-coated (TiN) caps were fixed by friction. Each system was sterilized and immersed in culture medium to provide an optimal environment for microbial growth. Positive and negative controls were prepared. Specimens were stored in an anaerobic workstation, and total and viable bacterial counts were determined. Every 48 h, samples were taken from the reaction tubes to inoculate blood agar plates and to isolate bacterial DNA for quantification using qrt-PCR. In addition, one Acuris test system was subjected to scanning electron microscopy (SEM) to evaluate the precision of fit of the conometric coupling and marginal crown opening. Throughout the observational period of one week, blood agar plates of the specimens showed no viable bacterial growth. qrt-PCR, likewise, yielded a result approaching zero with an amount of about 0.53 × 10-4 µg/mL DNA. While the luting gap/marginal opening between the TiN-cap and the ceramic crown was within the clinically acceptable range, the SEM analysis failed to identify a measurable microgap at the cone-in-cone junction. Within the limits of the in-vitro study it can be concluded that the Acuris conometric interface does not allow for bacterial translocation under non-dynamic loading conditions.

Effects of a Novel Cold Atmospheric Plasma Treatment of Titanium on the Proliferation and Adhesion Behavior of Fibroblasts.

Cold plasma treatment increases the hydrophilicity of the surfaces of implants and may enhance their integration with the surrounding tissues. The implaPrep prototype device from Relyon Plasma generates cold atmospheric plasma via dielectric barrier discharge (DBD). In this study, titanium surfaces were treated with the implaPrep device for 20 s and assessed as a cell culture surface for fibroblasts. One day after seeding, significantly more cells were counted on the surfaces treated with cold plasma than on the untreated control titanium surface. Additionally, the viability assay revealed significantly higher viability on the treated surfaces. Morphological observation of the cells showed certain differences between the treated and untreated titanium surfaces. While conventional plasma devices require compressed gas, such as oxygen or argon, the implaPrep device uses atmospheric air as the gas source. It is, therefore, compact in size and simple to handle, and may provide a safe and convenient tool for treating the surfaces of dental implants, which may further improve the implantation outcome.

Two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells in 96-well plates.

BACKGROUND: Although DNA of high quality can be easily prepared from cultured cells with commercially available kits, many studies involve a large number of samples which increases the cost drastically. We optimized two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells directly from wells of 96-well plates. METHODS: Cells (number: 103 -104 ) were lysed with a Direct PCR® lysis buffer or a 10% Chelex100® solution. The lysates were further purified and concentrated by means of DNA precipitation with a blue-colored glycogen as a carrier. PCR and digital PCR were used to evaluate the efficiency of the two methods. RESULTS: For 1000 cells from one primary culture and two tumor cell lines, DNA was reproducible and obtained with recovery rate (obtained/expected amount of DNA) in the range of 50%-90% as measured by the fluorometer dyes instrument Qubit. Using 8 out of a total of 10 µL DNA solution for 1000 cells, both conventional PCR and digital PCR were successful. For digital PCR, more than 1600 positive droplets were obtained for DNA from 1000 cells using the Direct PCR® method, corresponding to a yield efficiency of approximately 80%. Further reducing the number of cells down to 100 would be possible with 160 positive droplets expected. Both reagents are inexpensive (0.08€/sample). CONCLUSIONS: Two methods are efficient, especially the Direct PCR® reagent-based method provides a simple and inexpensive method for preparing DNA suitable for digital PCR from small number of cells.

Time Dependency of Non-Thermal Oxygen Plasma and Ultraviolet Irradiation on Cellular Attachment and mRNA Expression of Growth Factors in Osteoblasts on Titanium and Zirconia Surfaces.

Ultraviolet (UV) light and non-thermal plasma (NTP) are promising chair-side surface treatment methods to overcome the time-dependent aging of dental implant surfaces. After showing the efficiency of UV light and NTP treatment in restoring the biological activity of titanium and zirconia surfaces in vitro, the objective of this study was to define appropriate processing times for clinical use. Titanium and zirconia disks were treated by UV light and non-thermal oxygen plasma with increasing duration. Non-treated disks were set as controls. Murine osteoblast-like cells (MC3T3-E1) were seeded onto the treated or non-treated disks. After 2 and 24 h of incubation, the viability of cells on surfaces was assessed using an MTS assay. mRNA expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed using real-time reverse transcription polymerase chain reaction analysis. Cellular morphology and attachment were observed using confocal microscopy. The viability of MC3T3-E1 was significantly increased in 12 min UV-light treated and 1 min oxygen NTP treated groups. VEGF relative expression reached the highest levels on 12 min UV-light and 1 min NTP treated surfaces of both disks. The highest levels of HGF relative expression were reached on 12 min UV light treated zirconia surfaces. However, cells on 12 and 16 min UV-light and NTP treated surfaces of both materials had a more widely spread cytoskeleton compared to control groups. Twelve min UV-light and one min non-thermal oxygen plasma treatment on titanium and zirconia may be the favored times in terms of increasing the viability, mRNA expression of growth factors and cellular attachment in MC3T3-E1 cells.

In Vitro Feasibility Analysis of a New Sutureless Wound-Closure System Based on a Temperature-Regulated Laser and a Transparent Collagen Membrane for Laser Tissue Soldering (LTS).

For the post-surgical treatment of oral wounds and mucosal defects beyond a certain size, the gold standard is still an autologous skin or mucosal graft in combination with complex suturing techniques. A variety of techniques and biomaterials has been developed for sutureless wound closure including different tissue glues or collagen patches. However, no wound covering that enables for sutureless fixation has yet been introduced. Thus, a new system was developed that allows for sutureless wound covering including a transparent collagen membrane, which can be attached to the mucosa using a specially modified 2λ laser beam with integrated temperature sensors and serum albumin as bio-adhesive. The sutureless wound closure system was tested for its applicability and its cytocompatibility by an established in vitro model in the present study. The feasibility of the laser system was tested ex vivo on a porcine palate. The in vitro cytocompatibility tests excluded the potential release of toxic substances from the laser-irradiated collagen membrane and the bio-adhesive. The results of the ex vivo feasibility study using a porcine palate revealed satisfactory mean tensile strength of 1.2-1.5 N for the bonding of the membrane to the tissue fixed with laser of 980 nm. The results suggest that our newly developed laser-assisted wound closure system is a feasible approach and could be a first step on the way towards a laser based sutureless clinical application in tissue repair and oral surgery.

Defining the CD39/CD73 Axis in SARS-CoV-2 Infection: The CD73- Phenotype Identifies Polyfunctional Cytotoxic Lymphocytes.

The ectonucleotidases CD39 and CD73 regulate immune responses by balancing extracellular ATP and adenosine in inflammation and are likely to be involved in the pathophysiology of COVID-19. Here, we analyzed CD39 and CD73 on different lymphocyte populations in a small cohort of COVID-19 patients and in healthy individuals. We describe a significantly lower level of expression of CD73 on cytotoxic lymphocyte populations, including CD8+ T, natural killer T (NKT), and natural killer (NK) cells, during COVID-19. Interestingly, the decrease of CD73 on CD8+ T cells and NKT cells correlated with serum ferritin levels. Furthermore, we observed distinct functional differences between the CD73+ and CD73- subsets of CD8+ T cells and NKT cells with regard to cytokine/toxin secretion. In COVID-19 patients, the majority of the CD73-CD8+ T cells were capable of secreting granzyme B, perforin, tumor necrosis factor (TNF-α) or interferon-gamma (IFN-γ). To conclude, in this first study of CD39 and CD73 expression of lymphocytes in COVID-19, we show that CD8+ T cells and NKT cells lacking CD73 possess a significantly higher cytotoxic effector functionality compared to their CD73+ counterparts. Future studies should investigate differences of cellular CD39 and CD73 expression in patients at different disease stages and their potential as prognostic markers or targets for immunomodulatory therapies.

Managing NF2-associated vestibular schwannomas in children and young adults: review of an institutional series regarding effects of surgery and bevacizumab on growth rates, tumor volume, and hearing quality.

We reviewed our experience in managing of NF2-associated vestibular schwannoma (VS) in children and young adults regarding the effect of surgery and postoperative bevacizumab treatment. A total of 579 volumetric and hearing data sets were analyzed. The effect of surgery on tumor volume and growth rate was investigated in 46 tumors and on hearing function in 39 tumors. Long-term hearing follow-up behavior was compared with 20 non-operated ears in additional 15 patients. Sixteen operated VS were treated with bevacizumab. Mutation analysis of the NF2 gene was performed in 25 patients. Surgery significantly slowed down VS growth rate. Factors associated with a higher growth rate were increasing patient age, tumor volume, and constitutional truncating mutations. Immediately after surgery, functional hearing was maintained in 82% of ears. Deterioration of hearing was associated with initial hearing quality, larger tumor volumes, and larger resection amounts. Average hearing scores were initially better in the group of non-operated VS. Over time, hearing scores in both groups worsened with a similar dynamic. During bevacizumab treatment of residual tumors, four different patterns of growth were observed. Decompression of the internal auditory canal with various degrees of tumor resection decreases the postoperative tumor growth rates. Carefully tailored BAEP-guided surgery does not cause additional hearing deterioration. Secondary bevacizumab treatment showed heterogenous effects both regarding tumor size and hearing preservation. It seems that postoperative tumor residuals, that grow slower, behave differently to bevacizumab than reported for not-operated faster growing VS.

Cytocompatibility of Titanium, Zirconia and Modified PEEK after Surface Treatment Using UV Light or Non-Thermal Plasma.

A number of modifications have been developed in order to enhance surface cytocompatibility for prosthetic support of dental implants. Among them, ultraviolet (UV) light and non-thermal plasma (NTP) treatment are promising methods. The objective of this study was to compare the effects of UV light and NTP on machined titanium, zirconia and modified polyetheretherketone (PEEK, BioHPP) surfaces in vitro. Machined samples of titanium, zirconia and BioHPP were treated by UV light and NTP of argon or oxygen for 12 min each. Non-treated disks were set as controls. A mouse fibroblast and a human gingival fibroblast cell line were used for in vitro experiments. After 2, 24 and 48 h of incubation, the attachment, viability and cytotoxicity of cells on surfaces were assessed. Results: Titanium, zirconia and BioHPP surfaces treated by UV light and oxygen plasma were more favorable to the early attachment of soft-tissue cells than non-treated surfaces, and the number of cells on those treated surfaces was significantly increased after 2, 24 and 48 h of incubation (p < 0.05). However, the effects of argon plasma treatment on the cytocompatibility of soft tissue cells varied with the type of cells and the treated material. UV light and oxygen plasma treatments may improve the attachment of fibroblast cells on machined titanium, zirconia and PEEK surfaces, that are materials for prosthetic support of dental implants.

A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product.

Introduction: Mesenchymal stromal/stem cells (MSCs) derived from fat tissue are an encouraging tool for regenerative medicine. They share properties similar to the bone marrow-derived MSCs, but the amount of MSCs per gram of fat tissue is 500x higher. The fat tissue can easily be digested by collagenase, releasing a heterogeneous cell fraction called stromal vascular fraction (SVF) which contains a variable amount of stromal/stem cells. In Europe, cell products like the SVF derived from fat tissue are considered advanced therapy medicinal product (ATMPs). As a consequence, the manufacturing process has to be approved via GMP-compliant process validation. The problem of the process validation for SVF is the heterogeneity of this fraction. Methods: Here, we modified existing purification strategies by adding an additional plastic adherence incubation of maximal 20 hours after SVF isolation. The resulting cell fraction was characterized and compared to SVF as well as cultivated adipose-derived stromal/stem cells (ASCs) with respect to viability and cell yield, the expression of surface markers, differentiation potential and cytokine expression. Results: Short-term incubation significantly reduced the heterogeneity of the resulting cell fraction compared to SVF. The cells were able to differentiate into adipocytes, chondrocytes, and osteoblasts. More importantly, they expressed trophic proteins which have been previously associated with the beneficial effects of MSCs. Furthermore, GMP compliance of the production process described herein was acknowledged by the national regulatory agencies (DE_BB_01_GMP_2017_1018). Conclusion: Addition of a short purification-step after the SVF isolation is a cheap and fast strategy to isolate a homogeneous uncultivated GMP-compliant cell fraction of ASCs.

The Influence of an Ultrasonic Cleaning Protocol for CAD/CAM Abutment Surfaces on Cell Viability and Inflammatory Response In Vitro.

BACKGROUND/AIM: To evaluate the effect of an ultrasonic cleaning and disinfection method for CAD/CAM abutment surfaces on cell viability and inflammatory response in vitro. MATERIALS AND METHODS: Untreated and manually polished surfaces of CAD/CAM generated titanium and zirconia disks were randomly assigned, either to a 3-step ultrasonic cleaning and disinfection process (test: TiUF, TiPF, ZrUF, ZrPF) or to 30 sec steam cleaning (control: TiUS, TiPS, ZrUS, ZrPS). Pre-cleaning surface analyses using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and surface profilometry were performed. Human gingival fibroblasts (HGFs) were cultured on test and control specimens and subsequently examined for cell viability and inflammatory response. Expression of acute inflammatory cytokine interleukin (IL)-6 and vascular endothelial growth factor A (VEGFA) were assessed by means of RT-qPCR. RESULTS: Cells on all specimens exhibited a satisfactory viability, indicating firm attachment. Cells on polished zirconia samples, cleaned by means of sonication (ZrPF), exhibited significantly higher viability than cells on the same material cleaned by steam (ZrPS), p=0.019. For all other three material/ surface treatment combinations (TiU, TiP, ZrU), no such difference was observed between the cleaning methods. The messenger ribonucleic acid (mRNA) levels of IL-6 and VEGFA were between 50 and 105% of that of the control cells on the non-toxic control surface. mRNA levels of IL-6 and VEGFA correlated well with each other. CONCLUSION: Except for higher viability of cells cultured on polished zirconia specimens, no universally applicable advantage could be found for the ultrasonic cleaning procedure for zirconia and titanium abutment surfaces regarding cell viability, IL-6 expression or VEGFA expression. The cleaning procedures did not have any negative effect either.

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.

Plasma Electrolytic Oxidation of Titanium Implant Surfaces: Microgroove-Structures Improve Cellular Adhesion and Viability.

BACKGROUND/AIM: Plasma electrolytic oxidation (PEO) is an established electrochemical treatment technique that can be used for surface modifications of metal implants. In this study we to treated titanium implants with PEO, to examine the resulting microstructure and to characterize adhesion and viability of cells on the treated surfaces. Our aim was to identify an optimal surface-modification for titanium implants in order to improve soft-tissue integration. MATERIALS AND METHODS: Three surface-variants were generated on titanium alloy Ti6Al4V by PEO-treatment. The elemental composition and the microstructures of the surfaces were characterized using energy dispersive X-ray spectroscopy, scanning electron microscopy and profilometry. In vitro cytocompatibility of the surfaces was assessed by seeding L929 fibroblasts onto them and measuring the adhesion, viability and cytotoxicity of cells by means of live/dead staining, XTT assay and LDH assay. RESULTS: Electron microscopy and profilometry revealed that the PEO-surface variants differed largely in microstructure/topography, porosity and roughness from the untreated control material as well as from one another. Roughness was generally increased after PEO-treatment. In vitro, PEO-treatment led to improved cellular adhesion and viability of cells accompanied by decreased cytotoxicity. CONCLUSION: PEO-treatment provides a promising strategy to improve the integration of titanium implants with surrounding tissues.