Cells Derived from Human Long Bone Appear More Differentiated and More Actively Stimulate Osteoclastogenesis Compared to Alveolar Bone-Derived Cells.

Osteoblasts derived from mouse skulls have increased osteoclastogenic potential compared to long bone osteoblasts when stimulated with 1,25(OH)2 vitamin D3 (vitD3). This indicates that bone cells from specific sites can react differently to biochemical signals, e.g., during inflammation or as emitted by bioactive bone tissue-engineering constructs. Given the high turn-over of alveolar bone, we hypothesized that human alveolar bone-derived osteoblasts have an increased osteogenic and osteoclastogenic potential compared to the osteoblasts derived from long bone. The osteogenic and osteoclastogenic capacity of alveolar bone cells and long bone cells were assessed in the presence and absence of osteotropic agent vitD3. Both cell types were studied in osteogenesis experiments, using an osteogenic medium, and in osteoclastogenesis experiments by co-culturing osteoblasts with peripheral blood mononuclear cells (PBMCs). Both osteogenic and osteoclastic markers were measured. At day 0, long bones seem to have a more late-osteoblastic/preosteocyte-like phenotype compared to the alveolar bone cells as shown by slower proliferation, the higher expression of the matrix molecule Osteopontin (OPN) and the osteocyte-enriched cytoskeletal component Actin alpha 1 (ACTA1). This phenotype was maintained during the osteogenesis assays, where long bone-derived cells still expressed more OPN and ACTA1. Under co-culture conditions with PBMCs, long bone cells also had a higher Tumor necrose factor-alfa (TNF-α) expression and induced the formation of osteoclasts more than alveolar bone cells. Correspondingly, the expression of osteoclast genes dendritic cell specific transmembrane protein (DC-STAMP) and Receptor activator of nuclear factor kappa-Β ligand (RankL) was higher in long bone co-cultures. Together, our results indicate that long bone-derived osteoblasts are more active in bone-remodeling processes, especially in osteoclastogenesis, than alveolar bone-derived cells. This indicates that tissue-engineering solutions need to be specifically designed for the site of application, such as defects in long bones vs. the regeneration of alveolar bone after severe periodontitis.

Non-heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel.

BACKGROUND: Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inactivated allogenic human pooled serum and cannot determine T cell subsets. OBJECTIVE: To develop a radioactive free LPT by using carboxyfluorescein succinimidyl ester (CFSE) and to evaluate the influence of serum source (heat-inactivated human pooled serum [HI HPS] vs autologous serum) on the sensitivity and specificity of the nickel-specific LPT. METHODS: Peripheral blood mononuclear cells derived from nickel-allergic patients and healthy controls were collected, labelled with CFSE and cultured in medium containing 10% HI HPS or 10% autologous serum with or without additional T cell skewing cytokine cocktails (Th1: IL-7/IL-12, Th2: IL-7/IL-4 or Th17: IL-7/IL-23/IL-1ß) in the absence or presence of NiSO4 . The stimulation index (SI) was calculated as the ratio of divided cells, that is the percentage of CFSElow/neg CD3+ CD4+ T-lymphocytes upon nickel stimulation compared to the percentage of CFSElow/neg CD3+ CD4+ T-lymphocytes without antigen. These results were compared with the history of Ni allergy, patch test results and the MELISA test. RESULTS: Autologous serum positively influenced Ni-specific proliferation while HI HPS negatively influenced Ni-specific proliferation. The test protocol analysing CD4+ cells and autologous serum without skewing cytokines scored the best diagnostic values (sensitivity 95%; specificity 93%; and overall accuracy 94%) compared to the parallel test using HI HPS (accuracy 60%). Cytokine supplements did not further improve the test protocol which used autologous serum. The protocol using HI HPS could be further improved by addition of the cytokine skewing cocktails. CONCLUSIONS: Here, we describe an optimized and highly accurate flow cytometric LPT which comprises of CFSE-labelled cells cultured in autologous serum (not heat inactivated) and without the presence of T cell skewing cytokines. CLINICAL RELEVANCE: The sensitivity and specificity of LPT is enhanced, compared to HI HPS, when autologous serum without skewing cytokines is used.

Polymethyl methacrylate does not adversely affect the osteogenic potential of human adipose stem cells or primary osteoblasts.

Custom-made polymethyl methacrylate (PMMA) bone cement is used to treat cranial bone defects but whether it is cytotoxic is still unsure. Possible PMMA-induced adverse effects in vivo affect mesenchymal stem cells and osteoblasts at the implant site. We aimed to investigate whether PMMA affects osteogenic and osteoclast activation potential of human mesenchymal stem cells and/or osteoblasts. Immediately after polymerization, PMMA was added to cultured human adipose stem cells (hASCs) or human osteoblasts (hOBs). Medium lactate dehydrogenase was measured (day 1), metabolic activity, proliferation, osteogenic and osteoclast-activation marker expression (day 1 and 7), and mineralization (day 14). PMMA did not affect lactate dehydrogenase, KI67 gene expression, or metabolic activity in hASCs and hOBs. PMMA transiently decreased DNA content in hOBs only. PMMA increased COL1 gene expression in hASCs, but decreased RUNX2 in hOBs. PMMA did not affect osteocalcin or alkaline phosphatase (ALP) expression, ALP activity, or mineralization. Only in hOBs, PMMA decreased RANKL/OPG ratio. In conclusion, PMMA is not cytotoxic and does not adversely affect the osteogenic potential of hASCs or hOBs. Moreover, PMMA does not enhance production of osteoclast factors by hASCs and hOBs in vitro. Therefore, PMMA bone cement seems highly suitable to treat patients with cranial bone defects.

IL-6 counteracts the inhibitory effect of IL-4 on osteogenic differentiation of human adipose stem cells.

Fracture repair is characterized by cytokine production and hypoxia. To better predict cytokine modulation of mesenchymal stem cell (MSC)-aided bone healing, we investigated whether interleukin 4 (IL-4), IL-6, and their combination, affect osteogenic differentiation, vascular endothelial growth factor (VEGF) production, and/or mammalian target of rapamycin complex 1 (mTORC1) activation by MSCs under normoxia or hypoxia. Human adipose stem cells (hASCs) were cultured with IL-4, IL-6, or their combination for 3 days under normoxia (20% O 2 ) or hypoxia (1% O 2 ), followed by 11 days without cytokines under normoxia or hypoxia. Hypoxia did not alter IL-4 or IL-6-modulated gene or protein expression by hASCs. IL-4 alone decreased runt-related transcription factor 2 (RUNX2) and collagen type 1 (COL1) gene expression, alkaline phosphatase (ALP) activity, and VEGF protein production by hASCs under normoxia and hypoxia, and decreased mineralization of hASCs under hypoxia. In contrast, IL-6 increased mineralization of hASCs under normoxia, and enhanced RUNX2 gene expression under normoxia and hypoxia. Neither IL-4 nor IL-6 affected phosphorylation of the mTORC1 effector protein P70S6K. IL-4 combined with IL-6 diminished the inhibitory effect of IL-4 on ALP activity, bone nodule formation, and VEGF production, and decreased RUNX2 and COL1 expression, similar to IL-4 alone, under normoxia and hypoxia. In conclusion, IL-4 alone, but not in combination with IL-6, inhibits osteogenic differentiation and angiogenic stimulation potential of hASCs under normoxia and hypoxia, likely through pathways other than mTORC1. These results indicate that cytokines may differentially affect bone healing and regeneration when applied in isolation or in combination.

Properties of an In Vivo Fractured Poly(Methyl Methacrylate) Cranioplasty After 15 Years.

BACKGROUND: In 2001, a 27-year-old man was diagnosed with a meningioma with skull bone involvement. A craniectomy was performed and a CMW-3 poly(methyl methacrylate) cranioplasty was manually manufactured to reconstruct the remaining cranial defect. In 2016, he complained about progressive neurologic impairment. A computed tomography scan revealed that the cranioplasty had fractured into 4 dislocated pieces. Removal was indicated, and during the same operation a polyetheretherketone patient-specific implant was inserted. METHODS: The fractured cranioplasty was compared with freshly prepared CMW-3 specimens to determine whether the material properties had changed during 15 years in vivo. Gel permeation chromatography, microcomputed tomography, and flexural strength tests were performed. The fracture itself was analyzed using finite element analysis. RESULTS: The polydispersity index and molecular weight were not significantly different for the fractured cranioplasty and CMW-3. The fractured cranioplasty contained a total porosity of 10.7%, fresh CMW-3 cured at atmospheric pressure contained 4.1%, and 0.06% when cured at 2.2 bar. The flexural strength of the CMW-3 cured at 2.2 bar was significantly higher than both the fractured cranioplasty and CMW-3 cured at atmospheric pressure. Finite element analysis showed stress of 12.2 MPa under a load of 100 N on a weak spot. CONCLUSIONS: This ex vivo study shows that CMW-3 after 15 years in vivo was not influenced in molecular weight or flexural strength. However, the design of the implant and the handling of the poly(methyl methacrylate) seem to be important factors to improve mechanical properties of cranial reconstructions.

Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and CXCL8) was observed followed by complete reepithelialization. Seven days after wounding, tissue integrity, metabolic activity, and cytokine levels had returned to the prewounded state. In conclusion, immortalized human gingiva KC and fibroblasts can be used to make physiologically relevant GE, which resemble either the healthy gingiva or a neoplastic disease model. These organotypic models will provide valuable tools to investigate oral mucosa biology and can also be used as an animal alternative for drug targeting, vaccination studies, microbial biofilm studies, and testing new therapeutics.

Sensitization to palladium and nickel in Europe and the relationship with oral disease and dental alloys.

BACKGROUND: The role of palladium and nickel sensitization in oral disease and dermatitis is not fully understood. OBJECTIVES: To investigate whether sensitization to these metals was associated with exposure to dental alloys and oral and skin complaints/symptoms in a European multicentre study. METHODS: In six dermatology clinics, patch tests with palladium (3% Na2 PdCl4 ; Pd = 102.0 µmol/g) and nickel (5% NiSO4 .6H2 O; Ni = 190.2 µmol/g) were performed in consecutive patients, and patients' characteristics were collected with a questionnaire and a clinical investigation. RESULTS: In total, 906 patients were included, of whom 24.3% reacted to palladium and 25.2% to nickel. The rate of monosensitization was 6-7% for both metals. Palladium sensitization (as opposed to no sensitization to both metals) was associated with exposure to dental crowns [odds ratio (OR) 2.0], skin reactivity to metals (OR 2.8), oral lichenoid lesions (OR 4.7), xerostomia (OR 7.3), and metal taste (OR 20.7), but not with eczema, stomatitis, or oral burning sensation. Additionally, xerostomia (OR 8.7) and metal taste (OR 4.6) were associated with sensitization to both metals. CONCLUSIONS: Clinically, it is important for palladium-sensitized patients to undergo an oral examination, with particular attention to the presence of/exposure to dental crowns. In the case of metal contact allergy, exposure to dental crowns could play a role.

Palladium-based dental alloys are associated with oral disease and palladium-induced immune responses.

BACKGROUND: Palladium (Pd) and gold (Au) based dental alloys have been associated with oral disease. OBJECTIVES: This study was designed to explore possible associations between the presence of Au-based and Pd-based dental alloys, and oral lesions, systemic complaints, and specific in vivo and in vitro immune responses. METHODS: The investigated population consisted of three groups: 26 non-metal-allergic volunteers, 25 metal-allergic patients, and 20 oral disease patients. Medical histories were taken, oral examinations were carried out, and compositions of all dental alloys were determined. Then, Au and Pd patch tests and in vitro assays were performed, revealing cytokine production by peripheral blood mononuclear cells [T helper (Th)1, interferon-γ; Th2, interleukin (IL)-5 and IL-13] and lymphocyte proliferation (LTT-MELISA(®) ). RESULTS: Non-plaque-related gingivitis was associated with the presence of Pd-based dental alloys, and Pd-positive patch tests and in vitro assays. Collectively, participants with Pd-based dental alloys showed increased Pd patch test reactivity (p < 0.05) and lymphoproliferation (p < 0.05). In contrast, oral lichenoid lesions were associated with Au-based alloys (p < 0.05), but this was not reflected by Au-specific immunoreactivity. CONCLUSIONS: Oral lesions and Pd-induced immune responses are associated with the presence of dental alloys. However, most oral disease patients did not show positive patch test results or in vitro signs of specific immunoreactivity, suggesting local toxic reactions or the involvement of innate immune responses.

Effect of long-term repeated deflections on fatigue of preloaded superelastic nickel-titanium archwires.

INTRODUCTION: The aim of this in-vitro study was to investigate the changes in force delivery of superelastic nickel-titanium archwires used in combination with a self-ligating bracket system after dynamic fatigue-loading in a 3-bracket model under controlled temperature. METHODS: Samples of 2 superelastic nickel-titanium (active austenitic) wires, a conventional nickel-titanium wire, and a stainless steel wire, all 0.014-in round, were divided into 2 groups: static deflection and dynamic deflection. The static specimens were under a constant deflection of 3.0 mm. The dynamic specimens had the same constant deflection of 3.0 mm but were subjected to additional repeated deflections of 0.5 mm, applied by a fatigue tester. The test situation simulates a patient's archwire under deflection and subjected to occlusal contact during 1, 100, 10,000, and 100,000 cycles. Fatigue changes were assessed with a 3-point bending test. RESULTS: Type of wire, loading or unloading, and number of cycles as within-subject factors were statistically significantly different. No statistically significant difference between the test condition, static vs dynamic, was found. The repeated deflections of 0.5 mm were not enough to induce an extra effect of fatigue. CONCLUSIONS: Occlusal forces transferred to a considerably deflected archwire, such as in the large malalignments in the early stages of orthodontic treatment, will have no fatigue effect on the unloading force of that archwire.

Shear bond strength of different types of luting cements to an aluminum oxide-reinforced glass ceramic core material.

OBJECTIVE: The aim of this study was to find the optimal choice of luting cement to Synthoceram, an aluminum oxide-reinforced glass ceramic material. The bond strength of five different commercial luting cements to the ceramic material was evaluated. The effect of surface treatments, etching, sandblasting, silanizing, and combinations of these treatments was also investigated. METHODS: Shear bond strength tests were performed using the ceramic material as substrate with each of the luting cements. Cements rods were prepared on pre-treated ceramic surfaces. The shear bond strength was determined 24 h after cementation. The effect of surface treatments: etching, sandblasting, and sandblasting followed by etching, respectively, on the morphology of the material, was investigated with SEM. RESULTS: The shear bond strength increases significantly from Ketac Cem, Rely X Luting, Fuji Plus, Panavia F to Xeno Cem. The surface treatments etching and/or sandblasting followed by silanization generally provide the highest bond strength values. SIGNIFICANCE: Based on the results of this study, the use of resin composite based cements is preferred for cementation of an all-ceramic restoration with an aluminum oxide-reinforced glass ceramic base. Surface treatment of etching and/or sandblasting followed by silanization is recommended.

Coupling of metal-based light-harvesting antennas and electron-donor subunits: trinuclear ruthenium(II) complexes containing tetrathiafulvalene-substituted polypyridine ligands.

Three new tetrathiafulvalene-substituted 2,2'-bipyridine ligands, cis-bpy-TTF(1), trans-bpy-TTF(1), and cis-bpy-TTF(2) have been prepared and characterized. X-ray analysis of trans-bpy-TTF(1) is also reported. Such ligands have been used to prepare two new trinuclear Ru(II) complexes, namely, [[(bpy)(2)Ru(micro-2,3-dpp)](2)Ru(bpy-TTF(1))](PF(6))(6) (9; bpy=2,2'-bipyridine; 2,3-dpp=2,3-bis(2'-pyridyl)pyrazine) and [[(bpy)(2)Ru(micro-2,3-dpp)](2)Ru(bpy-TTF(2))](PF(6))(6) (10). These compounds can be viewed as coupled antennas and charge-separation systems, in which the multichromophoric trinuclear metal subunits act as light-harvesting antennas and the tetrathiafulvalene electron donors can induce charge separation. The absorption spectra, redox behavior, and luminescence properties (both at room temperature in acetonitrile and at 77 K in a rigid matrix of butyronitrile) of the trinuclear metal complexes have been studied. For the sake of completeness, the mononuclear compounds [(bpy)(2)Ru(bpy-TTF(1))](PF(6))(2) (7) and [(bpy)(2)Ru(bpy-TTF(2))](PF(6))(2) (8) were also synthesized and studied. The properties of the tetrathiafulvalene-containing species were compared to those of the model compounds [Ru(bpy)(2)(4,4'-Mebpy)](2+) (4,4'-Mebpy=4,4'-dimethyl-2,2'-bipyridine) and [[(bpy)(2)Ru(micro-2,3-dpp)](2)Ru(bpy)](6+). The absorption spectra and redox behavior of all the new metal compounds can be interpreted by a multicomponent approach, in which specific absorption features and redox processes can be assigned to specific subunits of the structures. The luminescence properties of the complexes in rigid matrices at 77 K are very similar to those of the corresponding model compounds without TTF moieties, whereas the new species are nonluminescent, or exhibit very weak emissions relative to those of the model compounds in fluid solution at room temperature. Time-resolved transient absorption spectroscopy confirmed that the potentially luminescent MLCT states of 7-10 are significantly shorter lived than the corresponding states of the model species. Photoinduced electron-transfer processes from the TTF moieties to the (excited) MLCT chromophore(s) are held responsible for the quenching processes.