A Standardized Rat Model to Study Peri-implantitis of Transmucosal Osseointegrated Implants.

With the high incidence rate, distinctive implant characteristic and unique infection pattern, peri-implantitis (PI) requires a specially designed implant animal model for the researches on the pathogenesis and treatments. Previous small-animal PI models exhibit variability in implant site selection, design, and surgical procedures resulting in unnecessary tissue damage and less effectivity. Herein, a quantitative-analysis-based standardized rat model for transmucosal PI-related research was proposed. After dissecting the anatomic structures of the rat maxilla, we determined that placing the implant anterior to the molars in the rat maxilla streamlined the experimental period and enhanced animal welfare. We standardized the model by controlling the rat strain, gender, and size. The customized implant and a series of matched surgical instruments were appropriately designed. A clear, step-by-step surgical process was established. These designs ensured the success rate, stability, and replicability of the model. Each validation method confirmed the successful construction of the model. This study proposed a quantitative-analysis-based standardized transmucosal PI rat model with improved animal welfare and reliable procedures. This model could provide efficient in vivo insights to study the pathogenesis and treatments of PI and preliminary screening data for further large-animal and clinical trials.

The influence of guide stabilizers and their application sequences on trueness and precision of surgical guides in free end situations: An in vitro analysis.

OBJECTIVES: To evaluate the impact of guide stabilizers and their application sequences on implant placement accuracy of guided implant surgery in multiple teeth loss at free end. MATERIALS AND METHODS: In this study, 96 implants were placed in the regions of #34, #36, and #37 of 32 identical mandibular models. The influence of using guide stabilizers or not (group A and group B) and various guide stabilizers application sequences (group B: #34 → #36 → #37; group C: #36 → #34 → #37; group D: #37 → #34 → #36) on implant placement trueness and precision was investigated. Data were analyzed using T-tests and one-way ANOVA. RESULTS: Group B showed significant benefits in enhancing implant placement precision. Compared to group A, it resulted in reducing 3D-deviation at crest and 2D deviation in vestibular-oral direction at both crest and apex. Furthermore, group D demonstrated greater improvement in global implant placement precision by reducing 2D deviation in mesial-distal direction at both crest and apex. Among the three different stabilizer application sequences, group D exhibited the highest level of implant placement precision. CONCLUSIONS: In cases of missing teeth at distal free end, the use of guide stabilizers and their application sequences does not have a significant impact on implant placement trueness. However, they do improve implant placement precision compared to methods that do not utilize guide stabilizers. Specifically, applying a guide stabilizer first at the furthest implant site to change teeth loss classification from free end to edentulous space with posterior support is the most reliable sequence.

Placement accuracy and primary stability of implants in the esthetic zone using dynamic and static computer-assisted navigation: A retrospective case-control study.

STATEMENT OF PROBLEM: Both the placement accuracy and primary stability of implants are important to implant therapy in the esthetic zone. The effect of dynamic and static computer-assisted navigation on the primary stability of implants in the esthetic zone remains uncertain. PURPOSE: The purpose of this case-control study was to investigate the effect of dynamic and static computer-assisted navigation on the placement accuracy and primary stability of implants in the esthetic zone. MATERIAL AND METHODS: Partially edentulous participants who received at least 1 implant in the anterior maxilla using either fully guided static or dynamic computer-assisted implant surgery (s-CAIS, d-CAIS) from January 2020 to February 2022 were screened. Participant demographic information, timing of implant placement, primary stability represented by the insertion torque value (ITV) in Ncm, and implant survival were collected from the treatment record. Bone quality at the implant sites was determined according to the Lekholm and Zarb classification. The accuracy of implant placement represented by the linear (platform: Dpl, mm; apex: Dap, mm) and angular deviations (axis: Dan, degree) between the planned and placed implants was evaluated based on the preoperative surgical plan and postoperative cone beam computed tomography (CBCT) data. A statistical analysis of the data was completed by using the chi-square, Fisher exact, Student t, and Mann-Whitney U tests (α=.05). RESULTS: A total of 32 study participants (38 implants) were included. The groups of s-CAIS (16 participants, 18 implants) and d-CAIS (16 participants, 20 implants) were statistically comparable in sex (P=.072), age (P=.548), bone quality (P=.671), and timing of implant placement (P=.719). All implants survived during an average follow-up period of 13 months. The d-CAIS group showed close linear deviations (Dpl 1.07 ±0.57 mm, Dap 1.26 ±0.53 mm) but lower angular deviation (Dan 2.14 ±1.20 degrees) and primary stability (ITV 25.25 ±7.52 Ncm) than the s-CAIS group (Dpl 0.92 ±0.46 mm, Dap 1.31 ±0.43 mm, Dan 3.31 ±1.61 degrees, ITV 30.56 ±11.23 Ncm, PDpl=.613, PDap=.743, PDan=.016, PITV=.028). CONCLUSIONS: Comparable linear positioning accuracy and higher angular deviation were found for implants placed in the esthetic zone by using s-CAIS than when using d-CAIS. Higher primary stability of implants may be achieved by using s-CAIS, as s-CAIS seemed to have higher osteotomy accuracy than d-CAIS.

Plasma polymerized bio-interface directs fibronectin adsorption and functionalization to enhance "epithelial barrier structure" formation via FN-ITG ß1-FAK-mTOR signaling cascade.

BACKGROUND: Transepithelial medical devices are increasing utilized in clinical practices. However, the damage of continuous natural epithelial barrier has become a major risk factor for the failure of epithelium-penetrating implants. How to increase the "epithelial barrier structures" (focal adhesions, hemidesmosomes, etc.) becomes one key research aim in overcoming this difficulty. Directly targeting the in situ "epithelial barrier structures" related proteins (such as fibronectin) absorption and functionalization can be a promising way to enhance interface-epithelial integration. METHODS: Herein, we fabricated three plasma polymerized bio-interfaces possessing controllable surface chemistry. Their capacity to adsorb and functionalize fibronectin (FN) from serum protein was compared by Liquid Chromatography-Tandem Mass Spectrometry. The underlying mechanisms were revealed by molecular dynamics simulation. The response of gingival epithelial cells regarding the formation of epithelial barrier structures was tested. RESULTS: Plasma polymerized surfaces successfully directed distinguished protein adsorption profiles from serum protein pool, in which plasma polymerized allylamine (ppAA) surface favored adsorbing adhesion related proteins and could promote FN absorption and functionalization via electrostatic interactions and hydrogen bonds, thus subsequently activating the ITG ß1-FAK-mTOR signaling and promoting gingival epithelial cells adhesion. CONCLUSION: This study offers an effective perspective to overcome the current dilemma of the inferior interface-epithelial integration by in situ protein absorption and functionalization, which may advance the development of functional transepithelial biointerfaces. Tuning the surface chemistry by plasma polymerization can control the adsorption of fibronectin and functionalize it by exposing functional protein domains. The functionalized fibronectin can bind to human gingival epithelial cell membrane integrins to activate epithelial barrier structure related signaling pathway, which eventually enhances the formation of epithelial barrier structure.

Cell-free immunomodulatory biomaterials mediated in situ periodontal multi-tissue regeneration and their immunopathophysiological processes.

Cell-free biomaterials-inducing endogenous in situ multi-tissue regeneration is very challenging and applying advanced immunomodulatory biomaterials can be an effective strategy to overcome it. In-depth knowledge of the immunopathophysiological mechanisms should be acquired before applying such an immunomodulation strategy. In this study, we implanted different immunoregulatory cell-free biomaterials into periodontal multi-tissue defects and showed that the outcome of multi-tissue regeneration is closely regulated by the immune reaction. The underlying immunopathophysiological processes, including the blood clotting response and fibrinoid necrosis, innate and adaptive immune response, local and systemic immune reaction, growth factors release, and stem cells recruitment, were revealed. The implantation of biomaterials with anti-inflammatory properties could direct the immunopathophysiological process and make it more favorable for in situ multi-tissue regeneration, ultimately enabling the regeneration of the periodontal ligament, the acellular cementum matrix, and the alveolar bone in the periodontium. These findings further confirm the effectiveness of immunomodulatory based strategy and the unveiling of their immunopathophysiological processes could provide some favorable theoretical bases for the development of advanced cell-free immunomodulatory multi-tissue regenerative biomaterials.

Optimizing the bio-degradability and biocompatibility of a biogenic collagen membrane through cross-linking and zinc-doped hydroxyapatite.

Biogenic collagen membranes have been widely used as soft tissue barriers in guided bone regeneration (GBR) and guided tissue regeneration (GTR). Nevertheless, their clinical performance remains unsatisfactory because of their low mechanical strength and fast degradation rate in vivo. Although cross-linking with chemical agents is effective and reliable for prolonging the degradation time of collagen membranes, some adverse effects including potential cytotoxicity and undesirable tissue integration have been observed during this process. As a fundamental nutritional trace element, zinc plays an active role in promoting the growth of cells and regulating the degradation of the collagen matrix. Herein, a biogenic collagen membrane was cross-linked with glutaraldehyde-alendronate to prolong its degradation time. The physiochemical and biological properties were enhanced by the incorporation of zinc-doped nanohydroxyapatite (nZnHA), with the native structure of collagen preserved. Specifically, the cross-linking combined with the incorporation of 1% and 2% nZnHA seemed to endow the membrane with the most appropriate biocompatibility and tissue integration capability among the cross-linked membranes, as well as offering a degradation period of six weeks in a rat subcutaneous model. Thus, improving the clinical performance of biogenic collagen membranes by cross-linking together with the incorporation of nZnHA is a promising strategy for the improvement of biogenic collagen membranes. STATEMENT OF SIGNIFICANCE: The significance of this research includes.

Tuning the immune reaction to manipulate the cell-mediated degradation of a collagen barrier membrane.

In order to elicit a desired barrier function in guided bone regeneration (GBR) or guided tissue regeneration (GTR), a barrier membrane has to maintain its integrity for a certain period of time to guarantee the regeneration of target tissue. Due to the complexity and variety of clinical conditions, the healing time required for tissue regeneration varies from one case to another, which implies the need for tailoring the barrier membranes to diverse conditions via manipulating their degradation property. As a "non-self" biomaterial, a barrier membrane will inevitably trigger host-membrane immune response after implantation, which entails the activation of phagocytic cells. In the degradation process of a barrier membrane, the cell-mediated degradation may play a more vital role than enzymatic and physicochemical dissolution; however, limited studies have been carried out on this topic. In this context, we investigated the cell-mediated degradation and illustrated the possible key cells and mediators for immunomodulation via in vivo and in vitro studies. We discovered that IL-13, a key cytokine mainly released by T helper 2 cells (Th2), induced the formation of foreign body giant cells (FBGCs), thus resulting in membrane degradation. Neutralizing IL-13 could suppress membrane degradation and formation of FBGC. The contributions of this study are (1) unveiling the immune mechanisms underlying the cell-mediated collagen membrane degradation; (2) allowing the formation of an "immunodegradation" strategy to develop an "immune-smart" barrier membrane to manipulate its degradation; (3) providing the key regulatory immune cells and cytokines for the immunomodulation target in collagen membrane degradation. STATEMENT OF SIGNIFICANCE: The significance of this research includes.

Suppressing MicroRNA-30b by Estrogen Promotes Osteogenesis in Bone Marrow Mesenchymal Stem Cells.

MicroRNAs (miRNAs) have been widely demonstrated to interact with multiple cellular signaling pathways and to participate in a wide range of physiological processes. Estradiol-17ß (E2) is the most potent and prevalent endogenous estrogen that plays a vital role in promoting bone formation and reducing bone resorption. Currently, little is known about the regulation of miRNAs in E2-induced osteogenic differentiation. In the present study, the primary bone marrow mesenchymal stem cells from rats (rBMSCs) were isolated and incubated with E2, followed by miRNA profiling. The microarray showed that 29 miRNAs were differentially expressed in response to E2 stimulation. Further verification by real-time reverse-transcriptase polymerase chain reaction revealed that E2 enhanced the expression of let-7b and miR-25 but suppressed the miR-30b expression. Moreover, a gain-of-function experiment confirmed that miR-30b negatively regulated the E2-induced osteogenic differentiation. These data suggest an important role of miRNAs in osteogenic differentiation.

A standardized rat burr hole defect model to study maxillofacial bone regeneration.

With high incidence rate and unique regeneration features, maxillofacial burr hole bone defects require a specially designed bone defect animal model for the evaluation of related bone regenerative approaches. Although some burr hole defect models have been developed in long bones or calvarial bones, the mandible has unique tissue development origins and regenerative environments. This suggests that the defect model should be prepared in the maxillofacial bone area. After dissecting the anatomic structures of rat mandibles, we found that creating defects in the anterior tooth area avoided damaging important organs and improved animal welfare. Furthermore, the available bone volume at the anterior tooth area was superior to that of the posterior tooth and ascending ramus areas. We then managed to standardize the model by controlling the age, weight and gender of the animal, creating standardized measurement instruments and reducing the variations derived from various operators. We also succeeded in deterring the self-rehabilitation of the proposed model by increasing the defect size. The 6 × 2 mm and 8 × 2 mm defects were found to meet the requirements of bone regenerative studies. This study provided a step-by-step standardized burr hole bone defect model with minimal tissue damage in small animals. The evaluations resulting from this model testify to the in vitro outcomes of the proposed regenerative approaches and provide preliminary screening data for further large animal and clinical trials. Therefore, the inclusion of this model may optimize the evaluation systems for maxillofacial burr hole bone defect regenerative approaches. STATEMENT OF SIGNIFICANCE: Unremitting effort has been devoted to the development of bone regenerative materials to restore maxillofacial burr hole bone defects because of their high clinical incidence rate. In the development of these biomaterials, in vivo testing in small animals is necessary to evaluate the effects of candidate biomaterials. However, little has been done to develop such defect models in small animals. In this study, we developed a standardized rat mandible burr hole bone defect model with minimal injury to the animals. A detailed description and supplementary video were provided to guide the preparation. The development of this model optimizes the maxillofacial bone regenerative approach evaluation system.

Sodium Fluoride under Dose Range of 2.4-24 µM, a Promising Osteoimmunomodulatory Agent for Vascularized Bone Formation.

Fluoride has essential effects on bone physiological activity and is widely used in bone biomaterials modification. However, this beneficial effect is highly related to the dose range and improper dosing can lead to pathological conditions such as fluorosis of bone. Therefore, this study first investigated the dose dependent effect of fluoride on bone regeneration. In the range of 0.24-240 µM, in vivo vascularized bone formation can be achieved via fine-tuning the fluoride concentration, and the peak osteogenic effect was found at 2.4-24 µM. The underlying mechanism is related to the modulation of the osteoimmune environment. Fluoride elicited significant osteoimmunomodulatory effect in modulation of the inflammatory cytokines and expression of osteogenic factors (BMP2, OSM, spermine/spermidine) and angiogenic factor (VEGF, IGF-1) during the early response. Fluorine with the doses of 2.4 and 24 µM could increase polyamines and IGF-1 production in macrophages, thus promoting osteogenesis of BMSCs and angiogenesis of HUVECs. These doses could also inhibit the inflammatory response of macrophages. In vitro osteogenesis and angiogenesis were both improved by the fluorine (2.4 and 24 µM)/macrophage conditioned medium, which is consistent with the in vivo results. These results collectively imply that fluoride is an effective osteoimmunomodulatory agent that can regulate both osteogenesis and angiogenesis. "Osteoimmune-smart" bone biomaterials can be developed via incorporating fluorine, and the release concentration should be controlled within the range of 2.4-24 µM for improved bone formation.

Contribution of the in situ release of endogenous cations from xenograft bone driven by fluoride incorporation toward enhanced bone regeneration.

Xenograft, namely bone-derived biological apatite (BAp), is widely recognized as a favorable biomaterial in bone tissue engineering owing to its biodegradability, biocompatibility, and osteoconductive properties. Substitutions of endogenous trace ions are thought to improve the osteogenic capacity of xenograft compared with synthetic hydroxyapatite (HAp). In order to modify the physicochemical and biological properties of apatite, different approaches to induce trace ion incorporation have been widely considered. In this study, we demonstrated that the incorporation of fluoride ions into porcine bone-derived biological apatite (pBAp) contributes to altered crystal morphology of the apatite, the sustained release of fluoride, and the in situ release of endogenous trace ions (e.g., magnesium and calcium) into the peripheral tissue microenvironment. This ionic balanced perimaterial microenvironment not only led to superior proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs), but also accelerated new bone formation of the calvarial defect on a rat model via the activation of Wnt/ß-catenin signaling. These promising observations may be attributed to the controlled release of endogenous trace ions from the xenograft to the peripheral tissue microenvironment driven by fluoride ion incorporation. Lastly, this study may provide a new insight to strengthen the osteogenicity of xenografts for clinical applications in the future.

Tuning surface properties of bone biomaterials to manipulate osteoblastic cell adhesion and the signaling pathways for the enhancement of early osseointegration.

Osteoblast cell adhesion is the initial step of early osseointegration responding to bone material implants. Enhancing the osteoblastic cell adhesion has become one of the prime aims when optimizing the surface properties of bone biomaterials. The traditional strategy focuses in improving the physical attachment of osteoblastic cells onto the surfaces of biomaterials. However, instead of a simple cell physical attachment, the osteoblastic cell adhesion has been revealed to be a sophisticated system. Despite the well-documented effect of bone biomaterial surface modifications on adhesion, few studies have focused on the underlying molecular mechanisms. Physicochemical signals from biomaterials can be transduced into intracellular signaling network and further initiate the early response cascade towards the implants, which includes cell survival, migration, proliferation, and differentiation. Adhesion is vital in determining the early osseointegration between host bone tissue and implanted bone biomaterials via regulating involving signaling pathways. Therefore, the modulation of early adhesion behavior should not simply target in physical attachment, but emphasize in the manipulation of downstream signaling pathways, to regulate early osseointegration. This review firstly summarized the basic biological principles of osteoblastic cell adhesion process and the activated downstream cell signaling pathways. The effects of different biomaterial physicochemical properties on osteoblastic cell adhesion were then reviewed. This review provided up-to-date research outcomes in the adhesion behavior of osteoblastic cells on bone biomaterials with different physicochemical properties. The strategy is optimised from traditionally focusing in physical cell adhesion to the proposed strategy that manipulating cell adhesion and the downstream signaling network for the enhancement of early osseointegration.

The osteoimmunomodulatory property of a barrier collagen membrane and its manipulation via coating nanometer-sized bioactive glass to improve guided bone regeneration.

A barrier membrane is a major component of guided bone regeneration (GBR), which is traditionally viewed as a physical barrier. Due to its "foreign body" nature, the implantation of a barrier membrane would inevitably modulate immune response and subsequently affect bone dynamics, which has long been neglected. To bridge this knowledge gap, we investigated the osteoimmunomodulatory effects of barrier collagen membranes. It is found that barrier collagen membranes elicit significant effects on modulating the osteoimmune response of macrophages, by upregulating the expression of pro-inflammatory cytokines (TNFα, IL-1ß, IL-6, and IL-18) and osteogenic factors (BMP2/6, WNT10b, OSM). The modulated-osteoimmune environment was beneficial for the osteogenic differentiation of BMSCs, due to the activation of BMP, canonical WNT/ß-catenin, and OSM signalling pathways. The membrane-mediated osteoimmunomodulation was further modulated to show whether osteogenesis could be enhanced via manipulating the membrane-mediated osteoimmunomodulation. The membrane-mediated osteoimmune response was successfully tuned through coating the collagen membranes with nanometer-sized bioactive glass Ca2ZnSi2O7 by pulsed laser deposition, which is indicated from the change in the expression profile of inflammatory cytokines and the upregulated expression of osteogenic factors. The modulated osteoimmune environment enhanced the osteogenic differentiation of BMSCs, suggesting that collagen membranes with nanometer-sized Ca2ZnSi2O7 coating can be promising for GBR applications. These results collectively imply that barrier membranes are bioactive barriers with an osteoimmunomodulatory effect and not just physical barriers. New generation barrier membranes should be designed with a favourable osteoimmunomodulatory property.

Fluorination Enhances the Osteogenic Capacity of Porcine Hydroxyapatite.

In a previous study, we successfully prepared fluorinated porcine hydroxyapatite (FPHA) by immersing porcine hydroxyapatite (PHA) in an aqueous solution of 0.25 M sodium fluoride (NaF) under thermal treatment, and the resulting FPHA showed better physicochemical and biological properties than PHA. The purpose of this study was to further investigate how fluorine incorporation influenced the biocompatibility and osteogenic capacity of PHA. The concentrations of Ca, P, F, and Mg ions in PHA and FPHA extracts were detected by inductively coupled plasma optical emission spectrometry. Rat bone marrow stromal cells (rBMSCs) were treated with PHA and FPHA extracts, and the effects of these extracts on cell proliferation and osteoblastic differentiation were evaluated via Cell Counting Kit-8 assay, alkaline phosphatase assay, and real time-quantitative polymerase chain reaction. For the in vivo assessment, PHA and FPHA were implanted into subcutaneous pockets (n = 6) and rat calvarial defects (diameter = 5 mm, n = 14) for 12 weeks to determine their biocompatibility and osteogenic capacity by using micro-computed tomography (CT) and histological analysis. FPHA extracts, which release higher concentrations of F and Mg ions, better promoted the osteoblastic differentiation of rBMSCs in vitro. The result of biocompatibility evaluation confirmed that the host response and chronic inflammation cells infiltration degree around PHA and FPHA granules were similar. Micro-CT and histological analysis showed newer mineralized bone formation in rats with FPHA-treated defects than in rats with PHA-treated defects. The results of in vitro and in vivo tests consistently indicate that fluorine incorporation effectively enhanced the osteogenic capacity of PHA.

Blood Prefabrication Subcutaneous Small Animal Model for the Evaluation of Bone Substitute Materials.

Because of the size of bone substitute material particles, large animal bone defect models are usually required for the assessment of these materials. However, these models have several disadvantages including high cost, complicated operation procedures, ethical issues, and difficulties in sample analysis. In addition, for mimicking the bone environment, conventional subcutaneous models require the addition of osteogenic factors and stem cells, resulting in an expensive model with a complex experimental procedure. To avoid these issues, in this study, we proposed a convenient and effective blood prefabrication subcutaneous small animal model that could be applied to assess bone substitute materials. Our results demonstrated that blood prefabrication could be an economical, convenient, and useful "adhesive" for handling bone substitute particles. This process provided porcine hydroxyapatite (PHA) with a microenvironment enriched with mesenchymal stem cells and growth factors. Using this strategy, a bonelike structure could form in a rat subcutaneous pocket. Furthermore, the optimized subcutaneous model was used to evaluate the PHA's osteoinductivity, producing results similar to those of the calvarial bone defect in terms of osteogenesis, osteoclastogenesis, and blood vessel formation. These results collectively imply that the blood prefabrication subcutaneous small animal model is convenient and effective for the assessment of osteoinductivity of bone substitute materials.