Osseointegration of Anodized vs. Sandblasted Implant Surfaces in a Guided Bone Regeneration Acute Dehiscence-Type Defect: An In Vivo Experimental Mandibular Minipig Model.

OBJECTIVES: This controlled preclinical study analyzed the effect of implant surface characteristics on osseointegration and crestal bone formation in a grafted dehiscence defect minipig model. MATERIAL AND METHODS: A standardized 3 mm × 3 mm acute-type buccal dehiscence minipig model grafted with deproteinized bovine bone mineral and covered with a porcine collagen membrane after 2 and 8 weeks of healing was utilized. Crestal bone formation was analyzed histologically and histomorphometrically to compare three implant groups: (1) a novel, commercially available, gradient anodized (NGA) implant, to two custom-made geometric replicas of implant "1," (2) a superhydrophilic micro-rough large-grit sandblasted and acid-etched surface, and (3) a relatively hydrophobic micro-rough large-grit sandblasted and acid-etched surface. RESULTS: At 2 and 8 weeks, there was no difference between the amount and height of newly formed bone (NBH, new bone height; BATA, bone area to total area) for any of the groups (p > 0.05). First bone-to-implant contact (fBIC) and vertical bone creep (VBC) at 2 and 8 weeks were significantly increased for Groups 2 and 3 compared to Group 1 (p < 0.05). At 8 weeks, osseointegration in the dehiscence (dehiscence bone-implant-contact; dBIC) was significantly higher for Groups 2 and 3 compared to Group 1 (p < 0.05). CONCLUSIONS: The amount of newly formed bone (BATA) and NBH was not influenced by surface type. However, moderately rough surfaces demonstrated significantly superior levels of osseointegration (dBIC) and coronal bone apposition (fBIC) in the dehiscence defect compared to the NGA surface at 2 and 8 weeks. TRIAL REGISTRATION: For this type of study, clinical trial registration is not required. This study was conducted at the Biomedical Department of Lund University (Lund, Sweden) and approved by the local Ethics Committee of the University (M-192-14).

Primary stability and osseointegration comparing a novel tapered design tissue-level implant with a parallel design tissue-level implant. An experimental in vivo study.

OBJECTIVES: The aim of the present study was to compare a novel tapered, double-threaded self-tapping tissue-Level design implant (TLC) to a well-established parallel walled tissue-level (TL) implant in terms of primary and secondary stability over time. MATERIALS AND METHODS: Test TLC (n = 10/per timepoint) and control TL (n = 10/per timepoint) implants were placed in the mandible of minipigs and left for submerged healing for 3, 6, and 12 weeks. Maximum insertion torque and implant stability quotient (ISQ) were measured for each implant at placement. Osseointegration and cortical bone maintenance were histologically evaluated by measuring total bone-to-implant contact (BIC) and first bone-to-implant contact (fBIC). RESULTS: A significantly higher maximum insertion torque was measured for the test implant TLC compared to the control TL implant (57.83 ± 24.73 Ncm and 22.62 ± 23.16 Ncm, respectively; p < .001). The mean ISQ values were comparable between the two implant types (75.00 ± 6.70 for TL compared to 75.40 ± 3.20 for TLC, p = .988). BIC was comparable between both implant types at each of the evaluated time points. The fBIC was found to be significantly more coronal at 12 weeks for the TLC implant compared to the TL implant (0.31 ± 0.83 mm for TLC compared to -0.22 ± 0.85 for TL, p = .027). CONCLUSION: The novel tapered tissue level design implant showed improved primary stability and an overall improved crestal bone height maintenance compared to the parallel walled design at 12 weeks.

A novel bioactive glass/graphene oxide composite coating for a polyether ether ketone-based dental implant.

Polyether ether ketone (PEEK) is a biocompatible material that lacks antimicrobial activity and bioactivity; therefore, is not appropriate for use as a dental implant. To overcome these deficiencies, a novel composite coating of bioactive glass and graphene oxide was prepared. PEEK discs were polished, cleaned, and the surface treated with sulfuric acid for 15 min. The composite coating consisted of bioactive glass produced by the sol-gel route and doped with 0.75 wt% graphene oxide. X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy-energy dispersive spectroscopy analyses were employed to characterize the composite coating, and the coating adhesion strength quantified using a pull-off test. Cytotoxicity was assessed using osteoblast-like cells and gingival fibroblasts. The wettability of the coated and non-coated samples was determined by optical contact angle assessment, and bioactivity was assessed by immersion in simulated body fluid. The results revealed that the bioactive glass/graphene oxide composite coating, approximately 7 µm thick, was transparent, homogenous with few microcracks and microporosities, but adhered strongly and was not cytotoxic to either osteoblast-like cells or gingival fibroblasts. The wettability of the PEEK sample was increased to <20° after coating with the composite, and apatite formation was detectable after 14 days of immersion in simulated body fluid.

Polyether ether ketone coated with nanohydroxyapatite/graphene oxide composite promotes bioactivity and antibacterial activity at the surface of the material.

Polyether ether ketone (PEEK) is considered an alternative material for manufacturing dental implants. However, PEEK lacks bioactivity and antibacterial action. In a series of experiments designed to enhance the surface properties of PEEK, we present a nanohydroxyapatite (nHA) and graphene oxide (GO) composite as a coating for PEEK-based dental implants to improve biological properties and antibacterial action. PEEK discs were polished, cleaned, and coated with the composite consisting of nHA particles doped with 0.75 wt% graphene oxide by a micro-emulsion technique according to patent US8,206,813. X-ray diffraction, field emission scanning electron microscopy-energy dispersive spectroscopy, and atomic force microscopy were utilized to characterize the composite coating. The wettability of the coated and non-coated samples was assessed by optical contact angle measurement. Antibacterial action of the composite coating was explored against S. aureus and E. coli and cytotoxicity determined utilizing osteoblast-like cells and gingival fibroblasts. The findings showed that the nHA/GO composite coating, approximately 1.3 µm thick, was homogenous with few micro-cracks and adhered to the PEEK surface. The surface roughness was reduced to 21.26 nm and the wettability was improved to 54.6° after coating with the composite coating. Antibacterial activity was moderate, killing 99% of S. aureus and E. coli, with acceptable levels of cytotoxicity to mammalian osteoblast-like cells and gingival fibroblasts.

A bioactive and anti-bacterial nano-sized zirconium phosphate/GO (nZrP/GO) composite: Potential use as a coating for dental implants?

OBJECTIVE: Dental implants fabricated from titanium have several limitations and therefore, alternative materials that fulfil the criteria of successful dental implant (bioactivity and anti-bacterial activity) need to be considered. Polyether ether ketone (PEEK) has been suggested to replace titanium implants. However, this material needs surface modification to meet the appropriate criteria. A nano-sized zirconium phosphate/GO (nZrP/GO) composite coating was prepared to improve PEEK's biological qualities. METHODS: Polished and cleaned PEEK discs were coated with the composite of nZrP doped with 1.25 wt% GO by the soft-template method. To analyze the composite coating, X-ray, atomic force microscopy, and field emission scanning electron microscopy-energy dispersive spectroscopy were used. The adhesion of the coating to PEEK was measured by adhesive tape test. By measuring the optical contact angle, the coated and non-coated samples' differences in wettability were evaluated. Antimicrobial activity was evaluated against S. aureus and E. coli and cytotoxicity tested employing gingival fibroblasts and osteoblast-like cells. RESULTS: The nZrP/GO composite coating was 23.45 µm thick, was irregular and attached strongly to the PEEK surface. Following coating, the water contact angle dropped to 34° and surface roughness to 13 nm. The coating reduced the count of bacteria two-fold and was non-cytotoxic to mammalian osteoblast-like cells and fibroblasts. A precipitation of nano-calcium-deficient apatite was observed on the surface of the nZrP/GO coating following a 28-day immersion in SBF. SIGNIFICANCE: PEEK-coated with nZr/GO coating is a good candidate as dental implant.

MgF2- containing glasses as a coating for titanium dental implant. I- Glass powder.

Bioactive glasses can be used to coat titanium implants to promote osseointegration. However, incorporating elements such as magnesium, zinc and fluoride into bioactive glasses might have a negative effect on bioactivity or the coefficient of thermal expansion of the glass. In this study, the impact of substituting MgO for CaO on physical properties and bioactivity of glass containing 1 mol % MgF2 was assessed. Seven glasses were produced by melt-quenched route. The glasses comprise (SiO2, CaO, Na2O, MgO, MgF2, K2O and P2O5) and were characterized utilizing XRD, DSC, FTIR and dilatometry analyses. The bioactivity of these glasses was investigated in biological fluids. The results showed that these glasses have wide sintering windows, low TECs and low glass transition and softening temperatures. The bioactive glasses containing up to 13.3 mol% MgO were able to form surface apatite within a short time period; whereas glasses containing ≥16.13 mol% demonstrated only structural variations with no clear sign of apatite precipitation.

Zinc bioglasses regulate mineralization in human dental pulp stem cells.

OBJECTIVE: A promising strategy in regenerative endodontics is the combination of human dental pulp stem cells (hDPSCs) with an appropriate biomaterial substrate. The effects of zinc and zinc containing bioactive glasses (ZnBGs) on hDPSCs have been characterized in this study. METHODS: ZnBGs were designed and produced. Then the odontogenic differentiation and mineralization potential of hDPSCs upon ZnBGs treatment were investigated. RESULTS: Free Zn ions (0-5ppm) enhanced proliferation and alkaline phosphatase (ALP) activity of hDPSCs. Further, ZnBGs conditioned medium (ZnBG-CM) increased the production and secretion of odontogenic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1). In addition, we identified that mRNA expression of the osteogenic markers RUNX2, OCN, BSP, BMP-2, MEPE and ON was increased following treatment with ZnBG-CM. Long term treatment with ZnBG-CM increases the formation rate of mineralized nodules (similar to hydroxyapatite, Ca:P=1.6), as confirmed by scanning electron microscopy combined with energy dispersive X-ray spectroscopy (SEM-EDX). Lastly, the administration of ZnBG-CM induces VEGF expression. SIGNIFICANCE: These findings implicate that ZnBG would be beneficial in regenerative endodontics and could influence the way present Zn containing clinical products are used.

Strontium (Sr) elicits odontogenic differentiation of human dental pulp stem cells (hDPSCs): A therapeutic role for Sr in dentine repair?

UNLABELLED: Strontium (Sr) forms a significant component of dental restorative materials and although it is widely used in toothpastes, the biological effects of Sr on the dentine-pulp complex have not been investigated. In this first study, we characterise the Sr elicited effects on human dental pulp stem cells (hDPSC) in vitro using exogenously Sr added to culture medium, and bioavailable Sr derived from a novel bioactive glass (BG). The related mechanisms were also investigated. Our results indicate that low dose Sr (between 0.1 and 2.5mM) induces proliferation and alkaline phosphatase (ALP) activity of hDPSCs, but has no effect on colony formation or cell migration. Sr at specific concentrations (1 and 2.5mM) stimulated collagen formation and mineralisation of the hDPSC generated matrix. In addition, qRT-PCR, Western blotting and immunocytochemistry revealed that Sr regulates gene expression and the protein secretion of the odontogenic markers: dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP-1) and protein localisation (DSPP was localised to the Golgi, while no apparent changes occur in DMP-1 distribution which remains in both cytosol and the nucleus). Additionally, the calcium sensing receptor (CaSR) and downstream pathway MAPK/ERK signalling pathway in hDPSCs were activated by Sr. Bioavailable Sr from the BG revealed novel biological insights of regulating metabolic and ALP activities in hDPSCs. Taken together, these results suggest that Sr at specific doses significantly influences proliferation, odontogenic differentiation and mineralisation of hDPSCs in vitro via the CaSR using a pathway with similarities to osteoblast differentiation. These are the first such studies and indicate that Sr treatment of hDPSCs could be a promising therapeutic agent in dental applications. In conclusion, we propose that Sr from a substituted BG could be used more effectively in biomaterials designed for dental applications. STATEMENT OF SIGNIFICANCE: Despite the fact that strontium (Sr) is used widely in dental practise, its potential effects on odontoblasts have been ignored. Our study provides the first evidence that Sr (exogenous and that derived from a bioglass (BG)) can stimulate dentinogenesis in human dental pulp stem cells (hDPSCs) by promoting their proliferation, differentiation and mineralisation in vitro. Therefore, while previously unrecognised, Sr BG is likely to be beneficial in atraumatic dentistry practise and maintenance of a competent tooth in conditions such as caries. Repair of defected dentine is still one of the main challenges in dental research and annually untreated caries results in the loss of productivity equivalent to US$ 27 billion. Advances in tissue engineering technology, alongside the use of dental pulp stem cells provide an approach to achieve dentine regeneration. Understanding the actions of Sr will permit a more controlled application of Sr in the clinic. These data are thus likely to be of great interest to the material scientists, biological researchers, clinicians and manufacturers of dental products.

Fluoride incorporation in high phosphate containing bioactive glasses and in vitro osteogenic, angiogenic and antibacterial effects.

OBJECTIVES: To manufacture and assess bioactivity of low fluoride/high phosphate (low F(-)/high P2O5) bioglasses (BGs). Then the effects of BG-conditioned medium on osteoblast-like cell behavior and BG particles on bactericidal activity were investigated. METHODS: BGs (0-7% F(-) content, constant 6.33% P2O5 in mol%) were designed and produced. BG particles was immersed in Tris Buffer solution or α-MEM to determine apatite formation and ion (Ca, P, Si and F) release. Osteoblast-like cells MC3T3-E1 were treated with BG-conditioned medium and assessed for cytotoxicity, pre-osteogenic and pro-angiogenic responses. Antibacterial ability was explored by incubating sub-gingival bacteria with BG particulates. RESULTS: Rapid apatite formation was observed in F(-) containing BGs after only 2-8h immersion in Tris buffer solution. In the F(-) free group, apatite was not detectable until 72h. Peak Ca, P and F release into Tris buffer was at 2h immersion, and then the levels decreased. In α-MEM, apatite formation in all the BGs was undetectable until 72h immersion. Alkaline phosphatase activity, cell number, collagen formation, bone-like mineral nodules and osteogenic gene expression of MC3T3-E1 cells were significantly promoted in low F(-) BG (P6.33F1) conditioned medium. MC3T3-E1 VEGF gene expression was increased, and protein production was dose-dependently promoted with F(-) BG-conditioned medium. After incubation with BG particulates, the growth of sub-gingival bacteria, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, was significantly inhibited; the antibacterial activity being dependent on the F(-) content of the BGs. SIGNIFICANCE: These results show that low F(-)/high P2O5 BGs significantly accelerated apatite formation and promoted both pre-osteogenic and pro-angiogenic responses of MC3T3-E1 osteoblast-like cells and inhibited the growth of periodontal pathogens in vitro. These BGs may prove useful as bone graft substitutes.

Strontium-substituted bioactive glasses in vitro osteogenic and antibacterial effects.

OBJECTIVES: Bioactive glass forms a bone mineral apatite interface and can be engineered to promote optimal bone regeneration. Strontium (Sr(2+)) stimulates osteoblast and inhibits osteoclast activities in vitro, and is used clinically as a treatment for osteoporosis. Dental bone defect repair requires rapid bone formation for early osseointegration but, can be subject to infection. The aim of this study was to investigate the osteogenic and antibacterial effects of strontium-substituted bioactive glasses in vitro. METHODS: Strontium-substituted bioactive glasses were designed and produced. Then the osteogenic potential and antibacterial effects of bioactive glass particulates were explored. RESULTS: Alkaline phosphatase activity, cell number, Type I collagen and mineral nodule formation of MC3T3-E1 cells were significantly promoted by the 5% strontium-substituted glass (5Sr). Furthermore, after incubation with 0.001g and 0.01g glass particulates, the growth of sub-gingival bacteria, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis was significantly inhibited; the antibacterial activity being dependent on the percentage of strontium in the glasses. SIGNIFICANCE: These results show that strontium-substituted bioactive glasses significantly promote osteogenic responses of MC3T3-E1 osteoblast-like cells and inhibit the growth of A. actinomycetemcomitans and P. gingivalis.

Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants.

Orthopedic and dental implants are prone to infection. In this study, we describe a novel system using zinc oxide nanoparticles (nZnO) as a coating material to inhibit bacterial adhesion and promote osteoblast growth. Electrohydrodynamic atomisation (EHDA) was employed to deposit mixtures of nZnO and nanohydroxyapatite (nHA) onto the surface of glass substrates. Nano-coated substrates were exposed to Staphylococcus aureus suspended in buffered saline or bovine serum to determine antimicrobial activity. Our results indicate that 100% nZnO and 75% nZnO/25% nHA composite-coated substrates have significant antimicrobial activity. Furthermore, osteoblast function was explored by exposing cells to nZnO. UMR-106 cells exposed to nZnO supernatants showed minimal toxicity. Similarly, MG-63 cells cultured on nZnO substrates did not show release of TNF-α and IL-6 cytokines. These results were reinforced by both proliferation and differentiation studies which revealed that a substrate coated with exclusively nZnO is more efficient than composite surface coatings. Finally, electron and light microscopy, together with immunofluorescence staining, revealed that all cell types tested, including human mesenchymal cell (hMSC), were able to maintain normal cell morphology when adhered onto the surface of the nano-coated substrates. Collectively, these findings indicate that nZnO can, on its own, provide an optimal coating for future bone implants that are both antimicrobial and biocompatible.

3D imaging of cell interactions with electrospun PLGA nanofiber membranes for bone regeneration.

The interaction between resident cells and electrospun nanofibers is critical in determining resultant osteoblast proliferation and activity in orthopedic tissue scaffolds. The use of techniques to evaluate cell-nanofiber interactions is critical in understanding scaffold function, with visualization promising unparalleled access to spatial information on such interactions. 3D tomography exploiting focused ion beam (FIB)-scanning electron microscopy (SEM) was used to examine electrospun nanofiber scaffolds to understand the features responsible for (osteoblast-like MC3T3-E1 and UMR106) cell behavior and resultant scaffold function. 3D imaging of cell-nanofiber interactions within a range of electrospun poly(d,l-lactide-co-glycolide acid) (PLGA) nanofiber scaffold architectures indicated a coherent interface between osteoblasts and nanofiber surfaces, promoting osteoblast filopodia formation for successful cell growth. Coherent cell-nanofiber interfaces were demonstrated throughout a randomly organized and aligned nanofiber network. Gene expression of UMR106 cells grown on PLGA fibers did not deviate significantly from those grown on plastic, suggesting maintenance of phenotype. However, considerably lower expression of Ibsp and Alpl on PLGA fibers might indicate that these cells are still in the proliferative phase compared with a more differentiated cell on plastic. This work demonstrates the synergy between designing electrospun tissue scaffolds and providing comprehensive evaluation through high resolution imaging of resultant 3-dimensional cell growth within the scaffold. STATEMENT OF SIGNIFICANCE: Membranes made from electrospun nanofibers are potentially excellent for promoting bone growth for next-generation tissue scaffolds. The effectiveness of an electrospun membrane is shown here using high resolution 3D imaging to visualize the interaction between cells and the nanofibers within the membrane. Nanofibers that are aligned in one direction control cell growth at the surface of the membrane whereas random nanofibers cause cell growth into the membrane. Such observations are important and indicate that lateral cell growth at the membrane surface using aligned nanofibers could be used for rapid tissue repair whereas slower but more extensive tissue production is promoted by membranes containing random nanofibers.

Gene expression profiles of mandible reveal features of both calvarial and ulnar bones in the adult rat.

OBJECTIVES: Limb and mandibular alveolar bone of the mandible are susceptible to disuse osteopenia, whilst skull and mandibular basal bone appear to resist excessive generalised bone loss. We wanted to compare the site-specific transcriptome of anatomically and functionally distinct bones to confirm the composite nature of the mandible at the molecular level. METHODS: Gene expression profiles were obtained for the mandible, ulna, and calvaria of adult male rats using Affymetrix Rat Genome 230 2.0 GeneChips. Ingenuity Pathways Assist generated association maps, and RGD database software identified site-specific pathways. RESULTS: The majority of expressed transcripts (84%) are common to all three sites. The mandible expressed 873 transcripts in common with ulna but not calvaria, and 1014 transcripts in common with calvaria but not ulna. Transcripts in these groups were excluded if they showed significant differential expression (>2-fold) and the remaining mapped genes were filtered for those related to modulation of gene transcription. Analysis of these genes revealed common pathways shared by the mandible and ulna, or mandible and calvaria, which were not shared by the calvaria and ulna. CONCLUSIONS: There were relatively few differences in the expression of genes responsible for the bone formation process per se in different functional skeletal sites. Differential transcription factor expression suggests that it is the regulation of bone formation and not the mechanism of bone formation itself that differs between the skeletal sites. CLINICAL SIGNIFICANCE: The mandible has areas both prone to, and resistant to, resorption whilst skull and limb bone differ in their susceptibilities to osteopenia. This report reveals that the mandible shares some genetic pathways in common with calvaria and others in common with ulna. Study of these pathways could identify novel treatment strategies for bone preservation.