Fiber reinforced hydrated networks recapitulate the poroelastic mechanics of articular cartilage.

The role of poroelasticity on the functional performance of articular cartilage has been established in the scientific literature since the 1960s. Despite the extensive knowledge on this topic there remain few attempts to design for poroelasticity and to our knowledge no demonstration of an engineered poroelastic material that approaches the physiological performance. In this paper, we report on the development of an engineered material that begins to approach physiological poroelasticity. We quantify poroelasticity using the fluid load fraction, apply mixture theory to model the material system, and determine cytocompatibility using primary human mesenchymal stem cells. The design approach is based on a fiber reinforced hydrated network and uses routine fabrication methods (electrohydrodynamic deposition) and materials (poly[ɛ-caprolactone] and gelatin) to develop the engineered poroelastic material. This composite material achieved a mean peak fluid load fraction of 68%, displayed consistency with mixture theory, and demonstrated cytocompatibility. This work creates a foundation for designing poroelastic cartilage implants and developing scaffold systems to study chondrocyte mechanobiology and tissue engineering. STATEMENT OF SIGNIFICANCE: Poroelasticity drives the functional mechanics of articular cartilage (load bearing and lubrication). In this work we develop the design rationale and approach to produce a poroelastic material, known as a fiber reinforced hydrated network (FiHy™), that begins to approach the native performance of articular cartilage. This is the first engineered material system capable of exceeding isotropic linear poroelastic theory. The framework developed here enables fundamental studies of poroelasticity and the development of translational materials for cartilage repair.

Contrast-enhanced nano-CT reveals soft dental tissues and cellular layers.

AIM: To introduce a methodology designed to simultaneously visualize dental ultrastructures, including cellular and soft tissue components, by utilizing phosphotungstic acid (PTA) as a contrast-enhancement agent. METHODOLOGY: Sound third molars were collected from healthy human adults and fixed in 4% buffered paraformaldehyde. To evaluate the impact of PTA in concentrations of 0.3%, 0.7% and 1% on dental soft and hard tissues for CT imaging, cementum and dentine-pulp sections were cut, dehydrated and stained with immersion periods of 12, 24 h, 2 days or 5 days. The samples were scanned in a high-resolution nano-CT device using pixel sizes down to 0.5 µm to examine both the cementum and pulpal regions. RESULTS: Dental cementum and periodontium as well as odontoblasts and predentine were made visible through PTA staining in high-resolution three-dimensional nano-CT scans. Different segments of the tooth required different staining protocols. The thickness of the cementum could be computed over the length of the tooth once it was made visible by the PTA-enhanced contrast, and the attached soft tissue components of the interior of the tooth could be shown on the dentine-pulp interface in greater detail. Three-dimensional illustrations allowed a histology-like visualization of the sections in all orientations with a single scan and easy sample preparation. The segmentation of the sigmoidal dentinal tubules and the surrounding dentine allowed a three-dimensional investigation and quantitative of the dentine composition, such as the tubular lumen or the ratio of the tubular lumen area to the dentinal surface. CONCLUSION: The staining protocol made it possible to visualize hard tissues along with cellular layers and soft tissues in teeth using a laboratory-based nano-CT technique. The protocol depended on both tissue type and size. This methodology offers enhanced possibilities for the concomitant visualization of soft and hard dental tissues.

Hard tissue volumetric and soft tissue contour linear changes at implants with different surface characteristics after experimentally induced peri-implantitis: an experimental in vivo investigation.

OBJECTIVE: To evaluate the hard tissue volumetric and soft tissue contour linear changes in implants with two different implant surface characteristics after a ligature-induced peri-implantitis. MATERIAL AND METHODS: In eight beagle dogs, implants with the same size and diameter but distinct surface characteristics were placed in the healed mandibular sites. Test implants had an external monolayer of multi-phosphonate molecules (B+), while control implants were identical but without the phosphonate-rich surface. Once the implants were osseointegrated, oral hygiene was interrupted and peri-implantitis was induced by placing subgingival ligatures. After 16 weeks, the ligatures were removed and peri-implantitis progressed spontaneously. Bone to implant contact (BIC) and bone loss (BL) were assessed three-dimensionally with Micro-Ct (µCT). Dental casts were optically scanned and the obtained digitalized standard tessellation language (STL) images were used to assess the soft tissue vertical and horizontal contour linear changes. RESULTS: Reduction of the three-dimensional BIC percentage during the induction and progression phases of the experimental peri-implantitis was similar for both the experimental and control implants, without statistically significant differences between them. Soft tissue analysis revealed for both implant groups an increase in horizontal dimension after the induction of peri-implantitis, followed by a decrease after the spontaneous progression period. In the vertical dimension, a soft tissue dehiscence was observed in both groups, being more pronounced at the buccal aspect. CONCLUSIONS: The added phosphonate-rich surface did not provide a more resistant environment against experimental peri-implantitis, when assessed by the changes in bone volume and soft tissue contours. CLINICAL RELEVANCE: Ligature-induced peri-implantitis is a validated model to study the tissue changes occurring during peri-implantitis. It was hypothesized that a stronger osseointegration mediated by the chemical bond of a phosphonate-rich implant surface would develop an environment more resistant to the inflammatory changes occurring after experimental peri-implantitis. These results, however, indicate that the hard and soft tissue destructive changes occurring at both the induction and progression phases of experimental peri-implantitis were not influenced by the quality of osseointegration.

Creation of well-balanced experimental groups for comparative endodontic laboratory studies: a new proposal based on micro-CT and in silico methods.

AIM: To introduce a new method to select anatomically matched teeth using micro-computed tomographic (micro-CT) technology. METHODOLOGY: Single-rooted mandibular incisors with a single root canal (n = 60) were selected and distributed into three experimental groups according to the method used for matching 10 pairs of teeth in each group. In group 1, the pairs of mandibular incisors were randomly selected from a pool of teeth. In group 2, teeth were paired based on the measurement of canal width 5 mm from the root apex using radiographs taken from buccolingual and mesiodistal directions. In group 3, teeth were scanned (pixel size of 14.25 µm) and pair-matched based on the anatomical aspects of the root canal, named aspect ratio (AR), volume and three-dimensional canal geometry. After allocating the specimens into groups 1 and 2, the teeth were scanned and the canal morphology evaluated as in group 3. A bivariate Pearson's regression analysis was performed correlating the individual AR values of each pair, and the correlation coefficient was used to estimate the strength of the pair-matching process. One-way anova post hoc Tukey's tests were applied for pairwise comparisons at a significance level of 5%. RESULTS: The micro-CT revealed that 100% of the samples had strong (80%) or very strong (20%) correlations with respect to AR values. Analysis of the radiographic method revealed strong correlation in two pairs (20%), but most of the samples had weak (30%) or negligible (30%) correlation coefficients. The randomization method resulted in three pairs (30%) with very strong correlations, whilst 50% had weak or negligible rates. A significant difference in correlation coefficients was observed in the micro-CT method compared to the other groups (P < 0.05), whilst no difference was detected between radiographic and randomized methods (P > 0.05). Eta-squared (η2 ) calculations demonstrated a very high effect size in the micro-CT group for selecting pairs (0.99) and lower effect sizes in the radiographic (0.67) and randomized (0.66) groups. CONCLUSIONS: Use of Micro-CT was able to provide better control of the confounding effect that anatomical variances in tooth morphology may have on the results in experiments with matched-pair design.

Melatonin expression in periodontitis and obesity: An experimental in-vivo investigation.

BACKGROUND AND OBJECTIVE: Melatonin deficiency has been associated with obesity and systemic inflammation. This study aims to evaluate whether melatonin could interfere with the mechanisms of co-morbidity linking obesity and periodontitis. MATERIAL AND METHODS: Twenty-eight male Wistar rats were randomly divided in 4 groups: control group (Con) (fed with standard diet); high-fat diet group (HFD) (fed with a diet containing 35.2% fat); Con group with induced periodontitis (Con-Perio) and HFD group with induced periodontitis (HFD-Perio). To induce periodontitis, the method of oral gavages with Porphyromonas gingivalis ATCC W83K1 and Fusobacterium nucleatum DMSZ 20482 was used. Circulating melatonin levels were analyzed by multiplex immunoassays. Periodontitis was assessed by alveolar bone loss (micro-computed tomography and histology) and by surrogate inflammatory outcomes (periodontal pocket depth, modified gingival index and plaque dental index). RESULTS: Plasma melatonin levels were significantly decreased (P < .05) in the obese rats with periodontitis when compared with controls or with either obese or periodontitis rats. Alveolar bone loss increased 27.71% (2.28 µm) in HFD-Perio group compared with the Con group. The histological analysis showed marked periodontal tissue destruction with osteoclast activity, particularly in the HFD-Perio group. A significant negative correlation (P < .05) was found between periodontal pocket depth, modified gingival index and circulating melatonin levels. CONCLUSION: Obese and periodontitis demonstrated significantly lower melatonin concentrations when compared with controls, but in obese rats with periodontitis these concentrations were even significantly lower when compared with either periodontitis or obese rats. These results may indicate that melatonin deficiency could be a key mechanism explaining the co-morbidity effect in the association between obesity and periodontitis.

Validation of contralateral premolars as the substrate for endodontic comparison studies.

AIM: To use micro-CT technology and metrology software to validate the use of contralateral premolars as samples in endodontic comparison studies by comparing them before and after canal instrumentation with one instrumentation system. Furthermore, to determine whether contralateral premolar roots (CPRs) will yield non-significantly different outcomes regarding shaping ability (volume), degree of twisting and three-dimensional shape changes. The null-hypothesis (H0 ) is that there are no differences between the CPRs pre- or post-instrumentation. METHODOLOGY: Twenty-eight extracted human contralateral premolars (n = 44 contralateral roots) from 12 donor patients were scanned with microcomputed tomography before and after instrumentation. Root canal lengths (RCLs) were measured visually using a dental-operating microscope, electronic apex locator and micro-CT scans. Data were analysed statistically for differences between pre- and post-instrumentation. RESULTS: Instrumentation increased the volume of the canals significantly (P < 0.05). Degree of twisting for a majority (83%) of the contralateral roots pairs did not change significantly (P > 0.05). There was no significant difference (P > 0.05) in the shape deviation analysis between contralateral pairs. There was no significant difference (P > 0.05) for RCL between the contralateral pairs for any of the three endometric methods. CONCLUSION: Contralateral premolar root canals were associated with similar changes in terms of volume, three-dimensional shape and degree of twisting from pre- to post-instrumentation. There was no difference between the CPR pairs pre- and post-instrumentation, and the study validates contralateral premolars as samples for root canal comparison studies. The null-hypothesis (H0 ) could not be rejected.

Gelatin-poly(vinyl alcohol) porous biocomposites reinforced with graphene oxide as biomaterials.

The present work aims to develop new biocomposites based on gelatin (Gel) and poly(vinyl alcohol) (PVA) reinforced with graphene oxide (GO). On the one hand, the model is designed with consideration of the high performance of the aforementioned biopolymers as biomaterials; on the other hand, the original component of the system, GO, is expected to improve structural stability and boost mechanical strength. Porous Gel-PVA/GO materials with GO content ranging from 0.5 to 3 wt% are obtained by freeze-drying. Structural analysis by Fourier transform infrared spectrometry (FT-IR), X-ray diffraction (XRD) and transmission electron microscopy (TEM) revealed the ability of well-dispersed GO nanosheets to form interactions with the polymers, leading to a unique molecular structuration. 3D analysis by X-ray microtomography (microCT) and scanning electron microscopy (SEM) suggests that GO has an influence on pore adjustment. According to mechanical tests, GO undoubtedly exhibits a beneficial effect on the polymer resistance against compressive stress, improving their compressive strengths by 97-100% with the addition of 0.5-3 wt% GO. Moreover, biological assessment using the MC3T3-E1 preosteoblast murine cell line indicated the fabrication of a cytocompatible composite formula, with potential for further in vivo testing and tissue engineering applications.

Bioactive implant surface with electrochemically bound doxycycline promotes bone formation markers in vitro and in vivo.

OBJECTIVES: The objective of this study was to demonstrate a successful binding of Doxy hyclate onto a titanium zirconium alloy surface. METHODS: The coating was done on titanium zirconium coins in a cathodic polarization setup. The surface binding was analyzed by SEM, SIMS, UV-vis, FTIR and XPS. The in vitro biological response was tested with MC3T3-E1 murine pre-osteoblast cells after 14 days of cultivation and analyzed in RT-PCR. A rabbit tibial model was also used to confirm its bioactivity in vivo after 4 and 8 weeks healing by means of microCT. RESULTS: A mean of 141 µg/cm(2) of Doxy was found firmly attached and undamaged on the coin. Inclusion of Doxy was documented up to a depth of approximately 0.44 µm by tracing the (12)C carbon isotope. The bioactivity of the coating was documented by an in vitro study with murine osteoblasts, which showed significantly increased alkaline phosphatase and osteocalcin gene expression levels after 14 days of cell culture along with low cytotoxicity. Doxy coated surfaces showed increased bone formation markers at 8 weeks of healing in a rabbit tibial model. SIGNIFICANCE: The present work demonstrates a method of binding the broad spectrum antibiotic doxycycline (Doxy) to an implant surface to improve bone formation and reduce the risk of infection around the implant. We have demonstrated that TiZr implants with electrochemically bound Doxy promote bone formation markers in vitro and in vivo.

Human gingival fibroblasts function is stimulated on machined hydrided titanium zirconium dental implants.

OBJECTIVES: The aim of this study was to evaluate the influence of different titanium zirconium (TiZr) alloy surfaces on primary human gingival fibroblasts (HGF) for improved soft tissue integration of dental implants. METHODS: TiZr polished, machined and machined+HCl/H2SO4 acid-etched surfaces were modified by cathodic polarization and/or HNO3/HF acid etching. Contact angle of surfaces was measured. The influence of modified TiZr surfaces on HGF was evaluated through the analysis of cell number, morphology, recovery after a wound (wound healing assay) and the expression of several genes, including matrix metalloproteinase-1 (MMP1) and metallopeptidase inhibitor-1 (TIMP1). RESULTS: Modification of TiZr surfaces decreased its hydrophilicity. Hydride implementation on TiZr surfaces via cathodic polarization increased TIMP1 expression and decreased MMP1/TIMP1 mRNA ratio. Cathodic polarization of machined surfaces promoted cell attachment. Cells on machined and machined+cathodic polarization surfaces grew aligned to the microgrooves whereas on all polished surfaces they grew randomly. Acid etching of polished and machined surfaces did not improve HGF function. CONCLUSIONS: Hydride implementation on TiZr machined surfaces may be used as new dental implant material for improved soft tissue integration. CLINICAL SIGNIFICANCE: Enhancing dental implant surfaces' bioactivity by hydride implementation may promote soft tissue attachment and sealing around the implant and reduce peri-implantitis related to ECM-destruction compared with conventional machined surfaces.

Differential response of human gingival fibroblasts to titanium- and titanium-zirconium-modified surfaces.

BACKGROUND AND OBJECTIVE: Gingival fibroblasts are responsible for the constant adaptation, wound healing and regeneration of gingival connective tissue. New titanium-zirconium (TiZr) abutment surfaces have been designed to improve soft tissue integration and reduce implant failure compared with titanium (Ti). The aim of the present study was first to characterize a primary human gingival fibroblast (HGF) model and secondly to evaluate their differential response to Ti and TiZr polished (P), machined (M) and machined + acid-etched (modMA) surfaces, respectively. MATERIAL AND METHODS: HGF were cultured on tissue culture plastic or on the different Ti and TiZr surfaces. Cell morphology was evaluated through confocal and scanning electron microscopy. A wound healing assay was performed to evaluate the capacity of HGF to close a scratch. The expression of genes was evaluated by real-time RT-PCR, addressing: (i) extracellular matrix organization and turnover; (ii) inflammation; (iii) cell adhesion and structure; and (iv) wound healing. Finally, cells on Ti/TiZr surfaces were immunostained with anti-ITGB3 antibodies to analyze integrin ß3 production. Matrix metalloproteinase-1 (MMP1) and inhibitor of metallopeptidases-1 (TIMP1) production were analyzed by enzyme-linked immunosorbent assays. RESULTS: On tissue culture plastic, HGF showed no differences between donors on cell proliferation and on the ability for wound closure; α-smooth muscle actin was overexpressed on scratched monolayers. The differentiation profile showed increased production of extracellular matrix components. Ti and TiZr showed similar biocompatibility with HGF. TiZr increased integrin-ß3 mRNA and protein levels, compared with Ti. Cells on TiZr surfaces showed higher MMP1 protein than Ti surfaces, although similar TIMP1 protein production. In this in vitro experiment, P and M surfaces from both Ti and TiZr showed better HGF growth than modMA. CONCLUSION: Taking into account the better mechanical properties and bioactivity of TiZr compared with Ti, the results of the present study show that TiZr is a potential clinical candidate for soft tissue integration and implant success.

Increased reactivity and in vitro cell response of titanium based implant surfaces after anodic oxidation.

In the quest for improved bone growth and attachment around dental implants, chemical surface modifications are one possibility for future developments. The biological properties of titanium based materials can be further enhanced with methods like anodic polarization to produce an active rather than a passive titanium oxide surface. Here we investigate the formation of hydroxide groups on sand blasted and acid etched titanium and titanium-zirconium alloy surfaces after anodic polarization in an alkaline solution. X-ray photoelectron spectroscopy shows that the activated surfaces had increased reactivity. Furthermore the activated surfaces show up to threefold increase in OH(-) concentration in comparison to the original surface. The surface parameters Sa, Sku, Sdr and Ssk were more closely correlated to time and current density for titanium than for titanium-zirconium. Studies with MC3T3-E1 osteoblastic cells showed that OH(-) activated surfaces increased mRNA levels of osteocalcin and collagen-I.

Effect of TiO2 scaffolds coated with alginate hydrogel containing a proline-rich peptide on osteoblast growth and differentiation in vitro.

The aim of this study was to investigate the effect of TiO2 scaffold (SC) coated with an alginate hydrogel containing a proline-rich peptide (P2) on osteoblast proliferation and differentiation in vitro. Peptide release was evaluated and a burst release was observed during the first hours of incubation, and then progressively released overtime. No changes were observed in the cytotoxicity after 48 h of seeding MC3T3-E1 cells on the coated and uncoated TiO2 SC. The amount of cells after 7 days was higher on uncoated TiO2 SC than on alginate-coated TiO2 SC, measured by DNA content and scanning electron microscope imaging. In addition, while lower expression of integrin beta1 was detected for alginate-coated TiO2 SC at this time point, similar gene expression was observed for other integrins, fibronectin-1, and several osteoblast differentiation markers. After 21 days, gene expression of integrin beta3, fibronectin-1, osterix, and collagen-I was increased in alginate-coated compared to TiO2 SC. Moreover, increased gene expression of integrin alpha8, bone morphogenetic protein 2, interleukin-6, and collagen-I was found on P2 alginate-coated TiO2 SC compared to alginate-coated TiO2 SC. In conclusion, our results indicate that alginate-coated TiO2 SC can act as a matrix for delivery of proline-rich peptides increasing osteoblast differentiation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

The effect of fluoride surface modification of ceramic TiO2 on the surface properties and biological response of osteoblastic cells in vitro.

This study investigates the effect of fluoride surface modification on the surface properties of polycrystalline ceramic TiO(2) and the biological response of murine osteoblast cells to fluoride-modified TiO(2) in vitro. Fluoride concentrations up to 9 at.% were detected and the fluoride was found to bind to the surface in a ligand exchange reaction between surface hydroxyl groups and the fluoride anions from the HF. No significant changes in the surface topography were detected. In vitro experiments were performed in order to evaluate the biological response of the MC3T3-E1 cells to the fluoride-modified ceramic TiO(2) surfaces. No difference in the lactate dehydrogenase (LDH) activity was seen in comparison to unmodified samples, apart from the highest fluoride concentration (∼9 at.%) which was found to be more toxic to the cells. Real-time PCR analysis showed no conclusive evidence for the fluoride-induced promotion of osteoblast differentiation as no significant increase in the collagen-1, osteocalcin, or BMP-2 mRNA levels was detected on the fluoride-modified ceramic TiO(2) surfaces apart from one group, which showed an elevated osteocalcin level and higher number of cells. Since the observed grain boundary corrosion is also anticipated to reduce the mechanical properties of ceramic TiO(2), this surface modification method may not be an ideal method for improving the osteogenic response of ceramic TiO(2) scaffolds.

Impact of trace elements on biocompatibility of titanium scaffolds.

A titanium oxide scaffold has recently been reported with high compressive strength (>2 MPa) which may allow its use in bone. However, would it be possible to enhance the scaffolds' performance by selecting a titanium oxide raw material without elemental contamination? Elements in implant surfaces have been reported to provoke implant failure. Thus, this study aims to compare different commercial titanium dioxide powders in order to choose the appropriate powder for scaffold making. The x-ray photoelectron spectroscopy (XPS) analysis identified the trace elements, mainly Al, Si, C, Ca and P. Cellular response was measured by cytotoxic effect, cell growth and cytokine secretion from murine preosteoblasts (MC3T3-E1) in vitro. The XPS data showed that traces of carbon-based molecules, silicon, nitrogen and aluminium in the powder were greatly reduced after cleaning in 1 M NaOH. As a result, reduction in cytotoxicity and inflammatory response was observed. Carbon contamination seemed to have a minor effect on the cellular response. Strong correlations were found between Al and Si contamination levels and the inflammatory response and cytotoxic effect. Thus, it is suggested that the concentration of these elements should be reduced in order to enhance the scaffolds' biocompatibility.

Ability of polyurethane foams to support placenta-derived cell adhesion and osteogenic differentiation: preliminary results.

In bone tissue reconstruction, the use of engineered constructs created by mesenchymal stem cells (MSCs) that differentiate and proliferate into 3D porous scaffolds is an appealing alternative to clinical therapies. Human placenta represents a possible source of MSCs, as it is readily available without invasive procedures and because of the phenotypic plasticity of many of the cell types isolated from this tissue. The scaffold considered in this work is a slowly degradable polyurethane foam (EF PU foam), synthesized and characterized for morphology and in vitro interaction with chorion mesenchymal cells (CMCs). These cells were isolated from human term placenta and cultured onto the EF PU foam using two different culture media (EMEM and NH osteogenic differentiation medium). Synthesized EF PU foam showed homogeneous pore size and distribution, with 89% open porosity. In vitro tests showed CMCs scaffold colonization, as confirmed by Scanning Electron Microscopy (SEM) observations and hematoxylin-eosin staining. Alizarin Red staining revealed the presence of a small amount of calcium deposition for the samples treated with the osteogenic differentiation medium. Therefore, the proposed EF PU foam appears to stimulate cell adhesion in vitro, sustaining CMCs growth and differentiation into the osteogenic lineage.

Effect of different gamma-irradiation doses on cytotoxicity and material properties of porous polyether-urethane polymer.

Biomaterials respond to sterilization methods differently. Steam sterilization might decrease the performance of thermoplastic polyether-urethane (TPU); however, the effect of different gamma-radiation doses on this polymer is contradictory in present literature. The purpose of this study was to investigate the differences between irradiative doses in comparison with steam sterilization on a porous TPU scaffold produced by a new processing method. No significant differences in the surface chemical structure were found with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis when comparing with the sterilization methods. The molecular weight (M(w)) had a net increase from 11.5 +/- 0.039 to 13.2 +/- 0.072 kDa by gamma-sterilization from 10 to 60 kGy. The samples that were irradiated (>60 kGy) had also an increase in polydispersity index (PDI; 1.45 +/- 0.007) in comparison with the nonsterile ones (1.31 +/- 0.017), which indicate branching. Liquid chromatography/mass spectroscopy (LC/MS) analysis showed that there was a correlation between the concentration of the breakdown product, methyl dianiline, and cytotoxicity. The concentration of this compound was found to be four times higher in steam-sterilized sample (1.3 +/- 0.01 ppb) compared with that of the polymer sample gamma-sterilized at 10 kGy (0.3 +/- 0.01 ppb). The cytotoxicity of TPU was found to decrease with higher radiation doses, and was significantly higher for the steam-sterilized samples. It is recommended that TPU produced with the described foaming method should be sterilized by gamma-irradiation at 25 kGy or higher doses.