Assessing peri-implant bacterial community structure: the effect of microbiome sample collection method.

BACKGROUND: Oral microbiota comprises polymicrobial communities shaped by mutualistic coevolution with the host, contributing to homeostasis and regulating immune function. Nevertheless, dysbiosis of oral bacterial communities is associated with a number of clinical symptoms that ranges from infections to oral cancer. Peri-implant diseases are biofilm-associated inflammatory conditions affecting the soft and hard tissues around dental implants. Characterization and identification of the biofilm community are essential for the understanding of the pathophysiology of such diseases. For that sampling methods should be representative of the biofilm communities Therefore, there is a need to know the effect of different sampling strategies on the biofilm characterization by next generation sequencing. METHODS: With the aim of selecting an appropriate microbiome sampling procedure for periimplant biofilms, next generation sequencing was used for characterizing the bacterial communities obtained by three different sampling strategies two months after transepithelial abutment placement: adjacent periodontal crevicular fluid (ToCF), crevicular fluid from transepithelial abutment (TACF) and transepithelial abutment (TA). RESULTS: Significant differences in multiple alpha diversity indices were detected at both the OTU and the genus level between different sampling procedures. Differentially abundant taxa were detected between sample collection strategies, including peri-implant health and disease related taxa. At the community level significant differences were also detected between TACF and TA and also between TA and ToCF. Moreover, differential network properties and association patterns were identified. CONCLUSIONS: The selection of sample collection strategy can significantly affect the community composition and structure. TRIAL REGISTRATION: This research is part of a randomized clinical trial that was designed to assess the effect of transepithelial abutment surface on the biofilm formation. The trial was registered at Trial Registration ClinicalTrials.gov under the number NCT03554876.

Protein adsorption/desorption dynamics on Ca-enriched titanium surfaces: biological implications.

Calcium ions are used in the development of biomaterials for the promotion of coagulation, bone regeneration, and implant osseointegration. Upon implantation, the time-dependent release of calcium ions from titanium implant surfaces modifies the physicochemical characteristics at the implant-tissue interface and thus, the biological responses. The aim of this study is to examine how the dynamics of protein adsorption on these surfaces change over time. Titanium discs with and without Ca were incubated with human serum for 2 min, 180 min, and 960 min. The layer of proteins attached to the surface was characterised using nLC-MS/MS. The adsorption kinetics was different between materials, revealing an increased adsorption of proteins associated with coagulation and immune responses prior to Ca release. Implant-blood contact experiments confirmed the strong coagulatory effect for Ca surfaces. We employed primary human alveolar osteoblasts and THP-1 monocytes to study the osteogenic and inflammatory responses. In agreement with the proteomic results, Ca-enriched surfaces showed a significant initial inflammation that disappeared once the calcium was released. The distinct protein adsorption/desorption dynamics found in this work demonstrated to be useful to explain the differential biological responses between the titanium and Ca-ion modified implant surfaces.

Enhancing the correlation between in vitro and in vivo experiments in dental implant osseointegration: investigating the role of Ca ions.

This study delves into the osteogenic potential of a calcium-ion modified titanium implant surface, unicCa, employing state-of-the-art proteomics techniques both in vitro (utilizing osteoblasts and macrophage cell cultures) and in vivo (in a rabbit condyle model). When human osteoblasts (Hobs) were cultured on unicCa surfaces, they displayed a marked improvement in cell adhesion and differentiation compared to their unmodified counterparts. The proteomic analysis also revealed enrichment in functions associated with cell migration, adhesion, extracellular matrix organization, and proliferation. The analysis also underscored the involvement of key signalling pathways such as PI3K-Akt and mTOR. In the presence of macrophages, unicCa initially exhibited improvement in immune-related functions and calcium channel activities at the outset (1 day), gradually tapering off over time (3 days). Following a 5-day implantation in rabbits, unicCa demonstrated distinctive protein expression profiles compared to unmodified surfaces. The proteomic analysis highlighted shifts in adhesion, immune response, and bone healing-related proteins. unicCa appeared to influence the coagulation cascade and immune regulatory proteins within the implant site. In summary, this study provides a comprehensive proteomic analysis of the unicCa surface, drawing correlations between in vitro and in vivo results. It emphasizes the considerable potential of unicCa surfaces in enhancing osteogenic behavior and immunomodulation. These findings significantly contribute to our understanding of the intricate molecular mechanisms governing the interplay between biomaterials and bone cells, thereby facilitating the development of improved implant surfaces for applications in bone tissue engineering.

Time-of-flight secondary ion mass spectrometry with principal component analysis of titania-blood plasma interfaces.

Treatment of osseoimplant surfaces with autologous platelet-rich plasma prepared according to the plasma rich in growth factors (PRGF-Endoret) protocol prior to implantation yields promising results in the clinic. Our objective is to understand the organization of complex interfaces between blood plasma preparations of various compositions and model titania surfaces. Here we present the results of the morphological and chemical characterization of TiO(2) surfaces incubated with four types of blood plasma preparations devoid of leukocytes and red blood cells: either enriched in platelets (PRGF-Endoret) or platelet-depleted, and either activated with CaCl(2) to induce clotting, or not. Chemical characterization was done by time-of-flight secondary ion mass spectrometry with principal component analysis (ToF-SIMS/PCA). The interface morphology was studied with scanning electron and atomic force microscopy. Immunofluorescence microscopy was used to identify platelets and infer their activation state. We observe clear differences among the four types of interfaces by ToF-SIMS/PCA. Some of these could be straightforwardly related to the differences in the sample morphology and known effects of platelet activation, but others are more subtle. Strikingly, it was possible to differentiate between these samples by ToF-SIMS/PCA of the protein species alone. This clearly indicates that the composition, orientation, and/or conformation of the proteins in these specimens depend both on the platelets' presence and on their activation. The ToF-SIMS imaging functionality furthermore provides unique insight into the distribution of phospholipid species in these samples.

Coarse Surface Microcavities Permit Bone Ingrowth and Improve Implant Osseointegration.

PURPOSE: To evaluate the effects of coarse microcavities added to micron and submicron rough implant surfaces in the implant-bone anchorage in rabbits. MATERIALS AND METHODS: Confocal interferometry was used to quantify roughness. Electron microscopy and energy-dispersive x-rays characterized the surfaces prior to and after implantation. X-ray photoelectron spectroscopy and contact angle determined the surface chemistry and energy, respectively. Fifteen New Zealand White rabbits received, respectively, one cavity-less (C-) and one cavity-rich (C+) implant per femoral condyle and were allowed to integrate for 2 and 8 weeks. The bone-to-implant contact (BIC), bone volume density (BVD), and removal torque (RTQ) were then analyzed. RESULTS: The cavities produced on the surfaces were 48.4 ± 16.8 µm in diameter and 37.8 ± 36.5 µm deep (5.9% ± 1.1% surface coverage). C+ did not alter the surface chemistry or energy. In vivo, C+ implants produced more BIC and RTQ at 8 weeks (P = .002 and P = .059, respectively) and more BVD at 2 and 8 weeks postimplantation (P = .031 and P = .078, respectively). Bone tissue was observed inside the cavities of C+ both histologically and by scanning electron microscopy after implant removal. CONCLUSION: Unevenly distributed coarse cavities within a micron and submicron rough surface allow bone ingrowth and increase implant stability and bone-surface unions in rabbits. These results encourage the design of implants with multilevel surface topographies to improve implant-based regeneration.

Provisional Matrix Formation at Implant Surfaces-The Bridging Role of Calcium Ions.

The success of dental implants lies in their strong and lasting integration into the patient's receiving bone. The first biological interactions at the implant surface determine the subsequent evolution of the integration process. In this study we set our objective to analyze the mechanistic interaction of the early regenerative matrix at implant surfaces modified with calcium ions (Ca) as compared to standard implant surfaces (NoCa). We put the surfaces in a Quartz Crystal Microbalance with Dissipation (QCM-D) to monitor the frequency shift (f) and the viscoelastic properties of the adsorbed biofilms and used Scanning Electron Microscopy (SEM) to visualize the resulting interfaces. Upon the addition of human blood plasma, Ca surfaces formed an adsorbed three-dimensional film attached to the surface (∆f = -40 Hz), while with NoCa, the biofilm formed but was not attached to the surface (∆f = 0 Hz). After 20 min in blood, two representative commercial implants with Ca and NoCa surfaces showed also distinct interfaces: Ca implants formed a visible clot attached to the implant which was composed mainly of platelets (Surface Coverage: 40 ± 20%) and some red blood cells (SC: 9 ± 3%) entrapped within a fibrin network (SC: 93 ± 5%). The NoCa implants were largely populated by red blood cells (SC: 67 ± 12%) with scarce fibrin remnants (SC: 3 ± 2%), and the implants showed no clot on their surfaces macroscopically. The pre-clinical and clinical results discussed in this work encourage the modification of titanium implant surfaces with calcium ions to improve the bone regenerative process. Taken together, these results add more information about the roles of Ca ions in bridging the formation of the provisional matrix at implant surfaces and their effects on implant osseointegration.

Open reduction and internal fixation of metacarpal fractures using a thermoplastic splint as a surgical instrument.

Adequate positioning of the hand is a critical step in hand fracture operative repair that can impact both the clinical outcome and the efficiency of the operation. In this paper, we introduce the use of a thermoplastic splint with an added thumb stabilizing component as a means to increase the surgeon's autonomy and to streamline the patient care pathway. The thermoplastic splint is custom fabricated preoperatively by the specialist hand therapist. The splint is used prior, during, and post operation with minimal modification. The thumb component assists maintaining the forearm in a stable pronated position whilst drilling and affixing metal work. This is demonstrated in the video of removal of metal work and open reduction and internal fixation of a metacarpal fracture.

Influence of calcium ion-modified implant surfaces in protein adsorption and implant integration.

BACKGROUND: Calcium (Ca) is a well-known element in bone metabolism and blood coagulation. Here, we investigate the link between the protein adsorption pattern and the in vivo responses of surfaces modified with calcium ions (Ca-ion) as compared to standard titanium implant surfaces (control). We used LC-MS/MS to identify the proteins adhered to the surfaces after incubation with human serum and performed bilateral surgeries in the medial section of the femoral condyles of 18 New Zealand white rabbits to test osseointegration at 2 and 8 weeks post-implantation (n=9). RESULTS: Ca-ion surfaces adsorbed 181.42 times more FA10 and 3.85 times less FA12 (p<0.001), which are factors of the common and the intrinsic coagulation pathways respectively. We also detected differences in A1AT, PLMN, FA12, KNG1, HEP2, LYSC, PIP, SAMP, VTNC, SAA4, and CFAH (p<0.01). At 2 and 8 weeks post-implantation, the mean bone implant contact (BIC) with Ca-ion surfaces was respectively 1.52 and 1.25 times higher, and the mean bone volume density (BVD) was respectively 1.35 and 1.13 times higher. Differences were statistically significant for BIC at 2 and 8 weeks and for BVD at 2 weeks (p<0.05). CONCLUSIONS: The strong thrombogenic protein adsorption pattern at Ca-ion surfaces correlated with significantly higher levels of implant osseointegration. More effective implant surfaces combined with smaller implants enable less invasive surgeries, shorter healing times, and overall lower intervention costs, especially in cases of low quantity or quality of bone.

Glucuronic acid and phosphoserine act as mineralization mediators of collagen I based biomimetic substrates.

Glucuronic acid (GlcA) and phosphoserine (pS) carrying acidic functional groups were used as model molecules for glycosaminoglycans and phosphoproteins, respectively to mimic effects of native biomolecules and influence the mineralization behaviour of collagen I. Collagen substrates modified with GlcA showed a stable interaction between GlcA and collagen fibrils. Substrates were mineralized using the electrochemically assisted deposition (ECAD) in a Ca(2+)/H( x )PO (4) ((3-x)) electrolyte at physiological pH and temperature. During mineralization of collagen-GlcA matrices, crystalline hydroxyapatite (HA) formed earlier with increasing GlcA content of the collagen matrix, while the addition of pS to the electrolyte succeeded in inhibiting the transformation of preformed amorphous calcium phosphate (ACP) to HA. The lower density of the resulting mineralization and the coalesced aggregates formed at a certain pS concentration suggest an interaction between calcium and the phosphate groups of pS involving the formation of complexes. Combining GlcA-modified collagen and pS-modified electrolyte showed dose-dependent cooperative effects.

Relevance of Topographic Parameters on the Adhesion and Proliferation of Human Gingival Fibroblasts and Oral Bacterial Strains.

Dental implantology allows replacement of failing teeth providing the patient with a general improvement of health. Unfortunately not all reconstructions succeed, as a consequence of the development of infections of bacterial origin on the implant surface. Surface topography is known to modulate a differential response to bacterial and mammalian cells but topographical measurements are often limited to vertical parameters. In this work we have extended the topographical measurements also to lateral and hybrid parameters of the five most representative implant and prosthetic component surfaces and correlated the results with bacterial and mammalian cell adhesion and proliferation outcomes. Primary human oral gingival fibroblast (gum cells) and the bacterial strains: Streptococcus mutans, Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans, implicated in infectious processes in the oral/implant environment were employed in the presence or absence of human saliva. The results confirm that even though not all the measured surface is available for bacteria to adhere, the overall race for the surface between cells and bacteria is more favourable to the smoother surfaces (nitrided, as machined or lightly acid etched) than to the rougher ones (strong acid etched or sandblasted/acid etched).

Corrosion behavior of surface modifications on titanium dental implant. In situ bacteria monitoring by electrochemical techniques.

The effects of surface modifications and bacteria on the corrosion behavior of titanium have been studied. Five surface modifications were analyzed: two acid etchings (op V, op N), acid etching + anodic oxidation (op NT), sandblasting + acid etching (SLA), and machined surfaces (mach). The corrosion behavior of the surface modifications was evaluated by following the standard ANSI/AAMI/ISO 10993-15:2000. Cyclic potentiodynamic and potentiostatic anodic polarization tests and ion release by ICP-OES after immersion for 7 days in 0.9% NaCl were carried out. Microbiologically induced corrosion (MIC) of low and high roughness (mach, op V) was assessed in situ by electrochemical techniques. Streptococcus mutans bacteria were resuspended in PBS at a concentration of 3 × 108 bacteria mL-1 and maintained at 37°C. MIC was measured through the open circuit potential, Eoc , and electrochemical impedance spectroscopy from 2 to 28 days. Potentiodynamic curves showed the typical passive behavior for all the surface modifications. The titanium ion release after immersion was below 3 ppb. In situ bacteria monitoring showed the decrease in Eoc from -0.065 (SD 0.067) Vvs. Ag/AgCl in mach and -0.115 (SD 0.084) Vvs. Ag/AgCl in op V, to -0.333 (SD 0.147) Vvs. Ag/AgCl in mach and -0.263 (SD 0.005) Vvs. Ag/AgCl in op V, after 2 and 28 days, respectively. A reduction of the oxide film resistance, especially in op V (54 MΩ cm2 and 6 MΩ cm2 , after 2 and 28 days, respectively) could be seen. Streptococcus mutans negatively affected the corrosion resistance of titanium. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 997-1009, 2018.

Balancing microbial and mammalian cell functions on calcium ion-modified implant surfaces.

Implant integration is a complex process mediated by the interaction of the implant surface with the surrounding ions, proteins, bacteria, and tissue cells. Although most implants achieve long-term bone-tissue integration, preventing pervasive implant-centered infections demands further advances, particularly in surfaces design. In this work, we analyzed classical microrough implant surfaces (only acid etched, AE; sandblasted then acid etching, SB + AE) and a new calcium-ion-modified implant surface (AE + Ca) in terms of soft- and hard-tissue integration, bacterial adhesion, and biofilm formation. We cultured on the surfaces primary oral cells from gingiva and alveolar bone, and three representative bacterial strains of the oral cavity, emulating oral conditions of natural saliva and blood plasma. With respect to gingiva and bone cells and in the presence of platelets and plasma proteins, AE + Ca surfaces yielded in average 86% higher adhesion, 44% more proliferation, and triggered 246% more synthesis of extracellular matrix biomolecules than AE-unmodified controls. Concomitantly, AE + Ca surfaces regardless of conditioning with saliva and/or blood plasma showed significantly less bacterial adhesion (67% reduction in average) and biofilm formation (40% reduction in average) than unmodified surfaces. These results highlight the importance of a calcium-rich hydrated interface to favor mammalian cell functions over microbial colonization at implant surfaces. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 421-432, 2018.

Progress in the Use of Autologous Regenerative Platelet-based Therapies in Implant Dentistry.

The field of medicine is rapidly moving towards the development of personalized treatments and non-invasive tools to achieve a more predictable and optimal tissue regeneration. In this sense, the goal of periodontal healing is to arrest disease progression and functionally regenerate all the tissues that comprise the periodontium. The latter implies a well-orchestrated interaction among oral cells, growth factors and extracellular matrix. Although several procedures are performed in an attempt to regenerate lost periodontal tissue, outcomes are not always predictable. Growth factors represent a class of biologically active polypeptides that have a critical role in the healing process. Their use provides a new paradigm to understand the regenerative medicine. The use of platelet- rich plasma (PRP) products as a local source and delivery system of autologous growth factors has emerged recently. Among them, PRGF stands for its remarkable stimulatory effect on oral tissue regeneration, making it a very safe and successful tool with a great value in Dentistry.

Nontraumatic Implant Explantation: A Biomechanical and Biological Analysis in Sheep Tibia.

Preclinical research in a sheep tibia model has been conducted to evaluate the underlying mechanisms of the nontraumatic implant explantation of failed implants, which allow placing a new one in the bone bed. Twelve dental implants were placed in sheep diaphysis tibia and once osseointegrated they were explanted using a nontraumatic implant explantation approach. Implant osseointegration and explantation were monitored by means of frequency resonance, removal torque, and angle of rotation measurement. The host bone bed and the explanted implant surface were analyzed by conventional microscopy and scanning electron microscope. Results show that osseointegration was broken with an angular displacement of less than 20°. In this situation the implant returns to implant stability quotient values in the same range of their primary stability. Moreover, the explantation technique causes minimal damage to the surrounding bone structure and cellularity. This nontraumatic approach allows the straightforward replacement of failed implants and emerges as a promising strategy to resolve clinically challenging situations.

Effects of calcium-modified titanium implant surfaces on platelet activation, clot formation, and osseointegration.

The clinical success of load bearing dental and orthopedic implants relies on adequate osseointegration. Because of its favorable properties, titanium is generally considered as the material of choice. Following implant placement, titanium surfaces establish an ionic equilibrium with the surrounding tissues in which calcium plays major roles. Calcium is a cofactor of the coagulation cascade that mediates plasma protein adsorption and intervenes in a number of other intra and extracellular processes relevant for bone regeneration. In this study, titanium surfaces were modified with calcium ions (Ca(2+) surfaces) and their responses to in vitro and in vivo models were analyzed. Unlike unmodified surfaces, Ca(2+) surfaces were superhydrophilic and induced surface clot formation, platelet adsorption and activation when exposed to blood plasma. Interestingly, in vivo osseointegration using a peri-implant gap model in rabbit demonstrated that Ca(2+) surfaces significantly improved peri-implant bone volume and density at 2 weeks and bone implant contact at 8 weeks as compared to the unmodified controls. The combination of Ca(2+) surfaces with plasma rich in growth factors produced significantly more bone contact already at 2 weeks of implantation. These findings suggest the importance of the provisional matrix formation on tissue integration and highlight the clinical potential of Ca(2+) titanium surfaces as efficient stimulators of implant osseointegration.

Effects of calcium ions on titanium surfaces for bone regeneration.

The chemistry and topography of implant surfaces are of paramount importance for the successful tissue integration of load-bearing dental and orthopedic implants. Here we evaluate in vitro and in vivo titanium implant surfaces modified with calcium ions (Ca(2+) surfaces). Calcium ions produce a durable chemical and nano-topographical modification of the titanium oxide interface. Time of flight secondary ion mass spectrometry examination of the outermost surface composition, shows that calcium ions in Ca(2+) surfaces effectively prevent adventitious hydrocarbon passivation of the oxide layer. In aqueous solutions Ca(2+) surfaces release within the first minute, 2/3 of the total measured Ca(2+), the rest is released over the following 85 days. Additionally, Ca(2+) surfaces significantly increase human fetal osteoblasts-like cell adhesion, proliferation and differentiation, as measured by the autocrine synthesis of osteopontin. Relevant for clinical application, after 12 weeks of healing in sheep tibia, microcomputer tomography and histomorphometric analysis show that Ca(2+) surfaces develop significantly more bone contacts and higher bone density in the 1mm region around the implant. Consequently, titanium implants modified with calcium ions represent a valuable tool to improve endosseous integration in the clinical practice.

Plasma rich in growth factors promotes bone tissue regeneration by stimulating proliferation, migration, and autocrine secretion in primary human osteoblasts.

BACKGROUND: Alveolar bone loss can be a major clinical concern affecting both functionality and esthetics. Osteoblasts are the main cells charged with the repair and regeneration of missing bone tissue. Plasma rich in growth factors (PRGF) allows delivery of a cocktail of proteins and growth factors that promote wound healing and tissue regeneration to the site of injury. This study tests the effect of this endogenous regenerative technology to stimulate alveolar osteoblast bone-forming potential. METHODS: Primary human osteoblasts were retrieved from alveolar bone of patients undergoing oral surgery. Cell proliferation was evaluated, and culture inserts and permeable transwell supports were used to assess cell migration and chemotaxis. The expression of differentiation markers was quantified by enzyme-linked immunosorbent assay. RESULTS: PRGF succeeded in increasing proliferation, migration, and chemotaxis of osteoblasts. Also, PRGF significantly enhanced the autocrine expression of two relevant proangiogenic factors, vascular endothelial growth factor and hepatocyte growth factor, and three markers of osteoblastic activity, procollagen I, osteocalcin, and alkaline phosphatase. CONCLUSION: The results indicate that PRGF can stimulate some of the biologic processes of the main cells responsible for bone regeneration and help support the positive clinical outcomes that have been reported with this technology.

Platelet-rich plasma to improve the bio-functionality of biomaterials.

Growth factors and cytokines are active players in controlling the different stages of wound healing and tissue regeneration. Recent trends in personalized regenerative medicine involve using patient's own platelet-rich plasma for stimulating wound healing and tissue regeneration. This technology provides a complex cocktail of growth factors and even a fibrin scaffold with multiple biologic effects. In the last few years, an increasing number of studies provide evidence of the potential of combining platelet-rich plasma with different biomaterials in order to improve their properties, including handling, administration, bioactivity, and level of osseointegration, among others. In this review, we discuss the use of platelet-rich plasma as an alternative, easy, cost-effective, and controllable strategy for the release of high concentrations of many endogenous growth factors. Additionally, we provide an overview of the current progress and future directions of research combining different types of biomaterials with platelet-rich plasma in tissue engineering and regenerative medicine.

Time-dependent release of growth factors from implant surfaces treated with plasma rich in growth factors.

Plasma rich in growth factors (PRGFs) technology is an autologous platelet-rich plasma approach that provides a pool of growth factors and cytokines that have been shown to increase tissue regeneration and accelerate dental implant osseointegration. In this framework, the spatiotemporal release of growth factors and the establishment of a provisional fibrin matrix are likely to be key aspects governing the stimulation of the early phases of tissue regeneration around implants. We investigated the kinetics of growth factor release at implant surfaces functionalized either with PRGFs or platelet-poor plasma and correlated the results obtained with the morphology of the resulting interfaces. Our main finding is that activation and clot formation favors longer residence times of the growth factors at the interfaces studied, probably due to their retention in the adsorbed fibrin matrix. The concentration of the platelet-derived growth factors above the interfaces becomes negligible after 2-4 days and is significantly higher in the case of activated interfaces than in the case of nonactivated ones, whereas that of the plasmatic hepatocyte growth factor is independent of platelet concentration and activation, and remains significant for up to 9 days. Platelet-rich plasma preparations should be activated to permit growth factor release and thereby facilitate implant surface osseointegration.