Prevalence and associated factors of health anxiety in patients with temporomandibular disorders.

OBJECTIVES: To investigate the prevalence and associated factors of health anxiety (HA) in patients with Temporomandibular Disorders (TMDs) using the 8-item Whiteley Index (WI-8) scale. MATERIALS AND METHODS: Three hundred and twenty-nine TMDs patients completed the Visual Analog Scale (VAS), WI-8, Jaw Functional Limitation Scale-8 (JFLS-8), Patient Health Questionnaire-9 (PHQ-9), and Generalized Anxiety Disorder-7 (GAD-7) scales. Clinical examinations were conducted following the Diagnostic Criteria for TMDs Axis I. RESULTS: The prevalence of HA among TMDs patients was 18.54%. Patients with HA had higher scores of VAS-current (p = 0.026), VAS-maximum (p = 0.024), VAS-average (p = 0.030), JFLS-8 (p < 0.001), GAD-7 (p < 0.001) and PHQ-9 (p < 0.001), lower maximum mouth opening (p = 0.016), lower proportion of structure-related TMDs (p = 0.028), and higher proportion of pain-related TMDs (p < 0.001) compared to those without HA. The correlation coefficient was 0.61 (p < 0.001) between WI-8 and GAD-7 and 0.64 (p < 0.001) between WI-8 and PHQ-9. CONCLUSION: Approximately one-fifth of patients with TMDs experienced HA. HA was associated with pain perception, functional limitations, depressive, and anxiety symptoms in individuals with TMDs. HA may contribute to heightened subjective pain experiences rather than structural changes in the TMJ.

Long-term efficacy and stability of miniscrew-assisted rapid palatal expansion in mid to late adolescents and adults: a systematic review and meta-analysis.

BACKGROUND: The purpose of this study is to investigate the long-term efficacy and stability of Miniscrew-assisted Rapid Palatal Expansion (MARPE), including its primary outcomes, namely the nasomaxillary complex transverse skeletal and dental expansion, and related secondary outcomes. METHODS: Electronic databases and manual literature searches, up to October 31, 2022, were performed. The eligibility criteria were the following: studies on patients with transverse maxillary deficiency treated with MARPE in adults and adolescents over 13.5 years of age. RESULTS: Ultimately, twelve articles were included in the analysis, one prospective and eleven retrospective observational studies. Five studies showed a moderate risk of bias, while the remaining seven studies were at a serious risk of bias. The GRADE quality of evidence was very low. MARPE is an effective treatment modality for transverse maxillary deficiency (mean success rate: 93.87%). Patients showed increased mean in the skeletal and dental transverse expansion. The basal bone composition, mean alveolar bone and mean dental expansion accounted for 48.85, 7.52, and 43.63% of the total expansion, respectively. There was a certain degree of skeletal and dental relapse over time. MARPE could also cause dental, alveolar, and periodontal side effects, and have an impact on other craniofacial bones, upper airway, and facial soft tissue. CONCLUSIONS: MARPE is an effective treatment for transverse maxillary deficiency, with a high success rate and a certain degree of skeletal and dental relapse over time.

Advances in periodontal stem cells and the regulating niche: From in vitro to in vivo.

Periodontium possesses stem cell populations for its self-maintenance and regeneration, and has been proved to be an optimal stem cell source for tissue engineering. In vitro studies have shown that stem cells can be isolated from periodontal ligament, alveolar bone marrow and gingiva. In recent years, more studies have focused on identification of periodontal stem cells in vivo. Multiple genetic markers, including Gli1, Prx1, Axin2, αSMA, and LepR, were identified with the lineage tracing approaches. Characteristics, functions, and regulatory mechanisms of specific populations expressing one of these markers have been investigated. In vivo studies also revealed that periodontal stem cells can be regulafrted by different niche and mechanisms including intercellular interactions, ECM and multiple secreted factors. In this review, we summarized the current knowledge of in vitro characteristics and in vivo markers of periodontal stem cells, and discussed the specific regulating niche.

Psychological status of TMD patients, orthodontic patients and the general population during the COVID-19 pandemic.

Confronting the outbreak of COVID-19, this cross-sectional study was aimed to assess psychological status of temporomandibular disorders (TMD) patients, orthodontic patients and the general population in China during the pandemic. An online anonymous questionnaire was developed in Chinese, including the individual background information, the perception of the epidemic, and level of anxiety and depression through Kessler Psychological Distress Scale (K10). The respondents were divided into ORTHO group, TMD group and Control group. Descriptive analysis and multiple linear regression modelling were performed. In total, 1241 valid questionnaires were collected, covering 587 orthodontic patients and 220 TMD patients. It is shown that the overall mental health is not quite optimistic during the COVID-19 pandemic with the mean score of K10 being 18.65. TMD patients have higher level of anxiety and depression than orthodontic patients as well as the general population. Younger age, female gender, having close contact with individuals from Hubei province, higher self-rated infection possibility, concern about psychological barriers and distrust are negatively affecting patients' psychological status. Mental health care should be emphasized when hospitals and clinics reopen after the COVID-19 pandemic, especially to patients with these relevant characteristics.

Mental distress in orthodontic patients during the coronavirus disease 2019 pandemic.

INTRODUCTION: The ongoing coronavirus disease 2019 (COVID-19) outbreak impacts the mental health of patients, health workers, and the public. The level of impact on the mental health of orthodontic patients in treatment is unknown. The objective of the study was to evaluate the mental health of orthodontic patients in China during the early stage of the pandemic. METHODS: An online survey was conducted on a convenience sample of anonymous participants. The questionnaire, in Chinese (Mandarin), comprised 5 sections. Sections 1-3 included demographic, epidemical, and orthodontic status of the patients. Section 4 assessed mental health-related to orthodontics. Section 5 was the Kessler-10 Mental Distress Scale. A total of 48 orthodontists were invited to distribute the questionnaires to their patients. Descriptive statistics, principal component analysis, K-means cluster analysis, and bivariate logistics regression analysis were performed with significance set at P <0.05. RESULTS: Questionnaires were collected from 558 patients (104 males, 354 females; mean age 24.78 ± 6.33 years). The prevalence of mental distress was 38% (174/458). Higher odds ratios were associated with female participants, missed appointments, and Hubei residence. The type of orthodontic appliance was associated with the anxiety of prolonged treatment duration. The manner of communication with patients regarding the postponement of appointments was associated with patients' concerns of prolonged treatment duration. The frequency of contact from dentists was associated with patients' independence. CONCLUSIONS: Over one-third of orthodontic patients experienced mental distress during the pandemic. Multiple factors affected the level of anxiety of orthodontic patients, such as the type of orthodontic appliance, time since last dental visit, manner of communication with the orthodontist, and the localities of the pandemic progression.

Distribution of Various Maxilla-Mandibular Positions and Cephalometric Comparison in Chinese Skeletal Class II Malocclusions.

AIM: To obtain the distribution of different maxilla-mandibular characteristics in Chinese skeletal class II mixed dentition patients and to compare the differences of cephalometric variables among different maxilla-mandibular types. MATERIALS AND METHODS: A cross-sectional study was conducted among 310 skeletal class II patients in mixed dentition. The patients were divided into 6 groups according to SNA and SNB angle of the cephalogram. A total of 38 cephalometric measurements were measured on their cephalograms. Differences among groups were tested by one-way analysis of variance. RESULTS: There were 34 (10.97%) patients in group I, 10 (3.23%) in group II, 4(1.29%) in group III, 69 (22.26%) in group IV, 133 (42.90%) in group V, and 60 (19.35%) in group VI. In all, 14.19% of the patients exhibited maxillary protrusion (MxP), and 62.26% exhibited mandibular retrusion (MnR) with either normal or retruded maxilla. Groups II and III were excluded for statistical comparison due to a limited sample size. Statistical differences were found in 25 cephalometric measurements among the other 4 groups. Patients with MnR (groups V and VI) exhibited bigger sella angle, gonial angle, Frankfort mandibular plane angle, and smaller mandibular body length and ramus height than patients without MnR (p value < 0.05). CONCLUSION: The most common etiology forming skeletal class II malocclusion in Chinese children was MnR, which was mainly caused by the small size and hyperdivergent growth direction of mandible. CLINICAL SIGNIFICANCE: The study presents various cephalometric characteristics of Chinese skeletal class II malocclusions. The results indicated that for the early orthodontic treatment of Chinese class II children with mixed dentition, orthodontists might emphasize more importance to mandibular length augmentation and growth direction change in mandible.

Pharyngeal Airway Space and Hyoid Bone Positioning After Different Orthognathic Surgeries in Skeletal Class II Patients.

PURPOSE: To obtain good profiles, patients with severe skeletal Class II malocclusion require orthognathic surgery, which might change their airway space and, hence, influence their quality of sleep. The present study aimed to 1) determine the effect of different orthognathic surgeries on pharyngeal airway space and hyoid bone position in patients with skeletal Class II malocclusion and 2) evaluate the stability of changes in the pharyngeal airway space and hyoid bone position after orthognathic surgeries. MATERIALS AND METHODS: Patients with severe skeletal Class II malocclusion who underwent mandibular advancement (MA) or mandibular advancement and maxillary setback (MAMS) were included in this retrospective cohort study. Changes in the pharyngeal airway space and hyoid bone position were evaluated as the primary outcome variables. Measurements were obtained before surgery (T1), 1 month after surgery (T2), and 2 years after surgery (T3) using Dolphin Imaging Software 11.0. Differences in measurements between T2 and T1 and between T3 and T1 were calculated and imported into SPSS 22.0 for data analysis. RESULTS: Fifty patients were included (25 in MA group and 25 in MAMS group). Oropharyngeal and hypopharyngeal airway cross-dimensions and areas were significantly increased in the MA group (P < .05) and the increases were stable at T3. In the MAMS group, the nasopharyngeal airway cross-dimension and area were decreased (P > .05), but the hypopharyngeal cross-dimension and area were significantly increased (P < .05). The hyoid bone moved superiorly and forward after surgery in the MA group (P < .05), and the movement was stable at T3. CONCLUSIONS: MA can widen the oropharyngeal and hypopharyngeal airway space, and maxillary setback can narrow the nasopharyngeal airway space. Some relapse related to the width of the oropharynx and hypopharynx was found at the long-term observation. The hyoid bone moved superiorly and forward in the MA group.

Role of sagittal and oblique smiling profiles in evaluating facial esthetics.

INTRODUCTION: Surgeons and orthodontists used to use a conventional set of facial photographs, composed of front, front smiling, and profile images to evaluate facial esthetics, whereas sagittal and oblique smiling profile images have been largely neglected in practice. The aim of this study was to explore the importance of sagittal and oblique smiling profiles in evaluating facial esthetics. METHODS: Photographs from 80 patients, of whom 40 underwent orthognathic surgery and 40 underwent orthodontic treatment, including front, front smiling, profile, sagittal profile smiling, and oblique profile smiling images before and after treatment, were collected and synthesized into 6 categories. Thirty judges gave scores to these photographs based on their own esthetic conception with a 1-week interval for each category. RESULTS: The results demonstrated that the mean score change of evaluating facial attractiveness of patients who underwent orthognathic surgery was lower when adding sagittal or oblique smiling profiles before the treatment, whereas it was higher after the treatment, which were opposite to the orthodontic treatment group with a higher score before the treatment and a lower score after the treatment when sagittal or oblique smiling profiles were added. The changes have a significant difference in adding both sagittal smiling profiles (P < 0.05) and oblique smiling profiles (P < 0.05) before and after treatment. CONCLUSIONS: Along with oblique smiling profile, sagittal smiling profile is crucial in evaluating facial esthetics for orthodontic treatment and orthognathic surgery. Both of them suggested to be integrated in routine photographic assessment of facial attractiveness evaluation before and after treatment, especially in orthognathic surgery for facial esthetic evaluation.

Anxiety mediates association between sex and jaw function limitation in temporomandibular disorder patients from China.

Aim: The objective of this study is to explore the relationship between sex and jaw function and to test whether anxiety mediates the causal relationship between sex and jaw function in temporomandibular disorders (TMDs) patients. Methods: A total of 488 participants with TMD were included in the analysis. Demographic data were collected. Generalized anxiety symptoms and anxiety severity were initially assessed using the GAD-7 questionnaire. And jaw function limitation was measured using the JFLS-8 scale. A directed acyclic graph (DAG) was used in this study to evaluate the hypotheses. Mediation analysis was conducted to explore causality and to calculate the total effect, natural direct effect (NDE) and natural indirect effect (NIE). Results: In TMD patients, there was a significant association between female and jaw function (r = 0.17, p < 0.001), female and anxiety (r = 0.15, p = 0.002), anxiety and jaw function (r = 0.35, p < 0.001). In addition, sex can directly lead to differences in impaired jaw function (NDE: 3.719, 95% CI: 1.619-5.828, p < 0.001), and can also be causally related to jaw function through anxiety (NIE: 1.146, 95% CI: 0.267-2.024, p = 0.011). And the total effect was 4.865 (95% CI, 2.709-7.029, p < 0.001). Conclusion: A causal mechanism was found that anxiety acts as a mediator of sex effects on jaw function. Therefore, psychological factors need to be taken into account in the treatment of female TMD patients. Further clinical trials are needed to explore whether psychotherapy is more beneficial to improve jaw function in female TMD patients.

Knowledge and Attitudes regarding Temporomandibular Disorders among Postgraduate Dental Students and Practicing Dentists in Western China: A Questionnaire-Based Observational Investigation.

Background: It is necessary for dental students and dentists to apply their temporomandibular disorders (TMDs)-related knowledge to clinical practice. The current study aimed to evaluate the knowledge and awareness of postgraduate dental students and practicing dentists regarding etiology, diagnosis, and treatment of TMD in western China and thus provide suggestions on TMD curricula design to get postgraduate students and dentists better prepared for TMD diagnosis and treatment. Methods: This observational and descriptive cross-sectional study was conducted among postgraduate students and practicing dentists in western China. Twenty-five reorganized knowledge questions in four domains were selected from the published literature and were evaluated with answer options from "strongly agree" to "strongly disagree," and "I don't know." "Consensus" is defined as more than 50% of respondents in a group agree or disagree with a statement. Chi-square tests were performed for comparisons between the two groups. Results: A total of 132 postgraduate dental students and 123 dentists completed the questionnaire. Around 75% of postgraduate students and 85% of dentists claimed that they have never participated in systematic training in TMD. Nine statements in etiology, diagnosis, treatment, and prognosis of TMD had different consensus between the two groups. And the dentist group tended to agree more with 12 statements in the questionnaire. Conclusions: The majority of Chinese dentists and dental students have not taken any TMD courses and possess limited knowledge of TMD. Curriculum reform for predoctoral education, postgraduate education, and continuing education is needed to augment knowledge and skills for TMD diagnosis and treatment.

Advances in 3D printing techniques for cartilage regeneration of temporomandibular joint disc and mandibular condyle.

Temporomandibular joint (TMJ) osteoarthritis causes fibrocartilage damage to the TMJ disc and mandibular condyle, resulting in local pain and functional impairment that further reduces patients' quality of life. Tissue engineering offers a potential treatment for fibrocartilage regeneration of the TMJ disc and mandibular condyle. However, the heterogeneous structure of TMJ fibrocartilage tissue poses significant challenges for the fabrication of biomimetic scaffolds. Over the past two decades, some researchers have attempted to adopt three-dimensional (3D) printing techniques to fabricate biomimetic scaffolds for TMJ fibrocartilage regeneration, but publications on such attempts are limited and rarely report satisfactory results, indicating an urgent need for further development. This review outlines several popular 3D printing techniques and the significant elements of tissue-engineered scaffolds: seed cells, scaffold materials, and bioactive factors. Current research progress on 3D-printed scaffolds for fibrocartilage regeneration of the TMJ disc and mandibular condyle is reviewed. The current challenges in TMJ tissue engineering are mentioned along with some emerging tissue-engineering strategies, such as machine learning, stimuli-responsive delivery systems, and extracellular vesicles, which are considered as potential approaches to improve the performance of 3D-printed scaffolds for TMJ fibrocartilage regeneration. This review is expected to inspire the further development of 3D printing techniques for TMJ fibrocartilage regeneration.

Camouflage treatment guided by facial improvement in hyperdivergent skeletal class II malocclusion.

Background: To detect parameters associated with the craniomaxillofacial system that could be altered during hyperdivergent skeletal class II malocclusion camouflage treatment for better profile improvement. Methods: Forty-two subjects with hyperdivergent skeletal class II malocclusion who had finished their orthodontic treatments and achieved good soft tissue responses were included in this study. Cephalometric analyses of these patients were taken before (T1) and after (T2) treatment. Measurements were made at each treatment stage and analyzed within and between groups. Results: No obvious decrease in the cant of occlusal plane (OP) (the line of point L1 and point L7) and mandibular plane (MP) was observed. However, an obvious decrease in the cant of the posterior occlusal plane (POP) and the angle of plane NA and plane NB (ANB) and a significant increase in the distance from the condylar center (Dc) to OP (DPO) were observed in the subjects (P<0.05). Conclusions: Decreased POP canting and increased DPO values play a vital role in profile improvement. Sagittal discrepancies might be considerably alleviated by a decreasing ANB angle [especially the sella nasion A point (SNA) angle]. Therefore, to attain a successful camouflage treatment of hyperdivergent skeletal class II malocclusion, treatment should be targeted towards finding an alternative to control POP canting, including reducing crowding in the posterior arch, as well as modifying and intruding molars to an upright and lower position.

The Relationship Between Porphyromonas Gingivalis and Rheumatoid Arthritis: A Meta-Analysis.

Rheumatoid arthritis (RA) is a systematical autoimmune disease, characterized by chronic synovial joint inflammation and hurt. Porphyromonas gingivalis(P. gingivalis) can cause life-threatening inflammatory immune responses in humans when the host pathogenic clearance machinery is disordered. Some epidemiological studies have reported that P. gingivalis exposure would increase the prevalence of RA. However, the results remain inconsistent. Therefore, a meta-analysis was done to systematically analyze the relationship between P. gingivalis exposure and the prevalence of rheumatoid arthritis. Database including Cochrane Library, Web of Science, PubMed, and EMBASE were searched for published epidemiological articles assessed the relationship between P. gingivalis and RA. Obtained studies were screened based on the predefined inclusion and exclusion criteria. The overall Odds Ratios (ORs) of incorporated articles were pooled by random-effect model with STATA 15.1 software. The literature search returned a total of 2057 studies. After exclusion, 28 articles were included and analyzed. The pooled ORs showed a significant increase in the risk of RA in individuals with P. gingivalis exposure (OR = 1.86; 95% CI: 1.43-2.43). Subgroup analysis revealed that pooled ORs from populations located in Europe (OR = 2.17; 95% CI: 1.46-3.22) and North America (OR = 2.50; 95% CI: 1.23-5.08) were significantly higher than that from population in Asia (OR = 1.11; 95% CI: 1.03-1.20). Substantial heterogeneity was observed but did not significantly influence the overall outcome. In conclusion, our results indicated P. gingivalis exposure was a risk factor in RA. Prompt diagnosis and management decisions on P. gingivalis antimicrobial therapy would prevent rheumatoid arthritis development and progression.

Response of Gli1+ Suture Stem Cells to Mechanical Force Upon Suture Expansion.

Normal development of craniofacial sutures is crucial for cranial and facial growth in all three dimensions. These sutures provide a unique niche for suture stem cells (SuSCs), which are indispensable for homeostasis, damage repair, as well as stress balance. Expansion appliances are now routinely used to treat underdevelopment of the skull and maxilla, stimulating the craniofacial sutures through distraction osteogenesis. However, various treatment challenges exist due to a lack of full understanding of the mechanism through which mechanical forces stimulate suture and bone remodeling. To address this issue, we first identified crucial steps in the cycle of suture and bone remodeling based on the established standard suture expansion model. Observed spatiotemporal morphological changes revealed that the remodeling cycle is approximately 3 to 4 weeks, with collagen restoration proceeding more rapidly. Next, we traced the fate of the Gli1+ SuSCs lineage upon application of tensile force in three dimensions. SuSCs were rapidly activated and greatly contributed to bone remodeling within 1 month. Furthermore, we confirmed the presence of Wnt activity within Gli1+ SuSCs based on the high co-expression ratio of Gli1+ cells and Axin2+ cells, which also indicated the homogeneity and heterogeneity of two cell groups. Because Wnt signaling in the sutures is highly upregulated upon tensile force loading, conditional knockout of ß-catenin largely restricted the activation of Gli1+ SuSCs and suppressed bone remodeling under physiological and expansion conditions. Thus, we concluded that Gli1+ SuSCs play essential roles in suture and bone remodeling stimulated by mechanical force and that Wnt signaling is crucial to this process. © 2022 American Society for Bone and Mineral Research (ASBMR).

Self-adhesive hydrogels for tissue engineering.

Hydrogels consisting of a three-dimensional hydrophilic network of biocompatible polymers have been widely used in tissue engineering. Owing to their tunable mechanical properties, hydrogels have been applied in both hard and soft tissues. However, most hydrogels lack self-adhesive properties that enable integration with surrounding tissues, which may result in suture or low repair efficacy. Self-adhesive hydrogels (SAHs), an emerging class of hydrogels based on a combination of three-dimensional hydrophilic networks and self-adhesive properties, continue to garner increased attention in recent years. SAHs exhibit reliable and suitable adherence to tissues, and easily integrate into tissues to promote repair efficiency. SAHs are designed either by mimicking the adhesion mechanism of natural organisms, such as mussels and sandcastle worms, or by using supramolecular strategies. This review summarizes the design and processing strategies of SAHs, clarifies underlying adhesive mechanisms, and discusses their applications in tissue engineering, as well as future challenges.

Gli1+ Periodontium Stem Cells Are Regulated by Osteocytes and Occlusal Force.

Teeth are attached to alveolar bone by the periodontal ligament (PDL), which contains stem cells supporting tissue turnover. Here, we identified Gli1+ cells in adult mouse molar PDL as multi-potential stem cells (PDLSCs) giving rise to PDL, alveolar bone, and cementum. They support periodontium tissue turnover and injury repair. Gli1+ PDLSCs are surrounding the neurovascular bundle and more enriched in the apical region. Canonical Wnt signaling is essential for their activation. Alveolar bone osteocytes negatively regulate Gli1+ PDLSCs activity through sclerostin, a Wnt inhibitor. Blockage of sclerostin accelerates the PDLSCs lineage contribution rate in vivo. Sclerostin expression is modulated by physiological occlusal force. Removal of occlusal force upregulates sclerostin and inhibits PDLSCs activation. In summary, Gli1+ cells are the multipotential PDLSCs in vivo. Osteocytes provide negative feedback to PDLSCs and inhibit their activities through sclerostin. Physiological occlusal force indirectly regulates PDLSCs activities by fine-tuning this feedback loop.

Investigation of Postnatal Craniofacial Bone Development with Tissue Clearing-Based Three-Dimensional Imaging.

Traditional two-dimensional histological sections and microcomputed tomography remain to be the major tools for studying craniofacial bones despite the complicated spatial organization of craniofacial organs. Recently, our laboratory developed the Poly(Ethylene Glycol) Associated Solvent System (PEGASOS) tissue clearing method, which can efficiently render hard tissues, including bones and teeth fully transparent without losing endogenous fluorescent signals. Complete tissue transparency enables us to acquire three-dimensional (3D) images of craniofacial bone vasculature, osteogenesis utilizing various labeling strategies, thus to investigate the spatial relationship among different tissues during postnatal craniofacial development. We found out that during the early stage of postnatal development, craniofacial osteogenesis occurs throughout the entire craniofacial bones, including the periosteum, dura, bone marrow, and suture. After 3-4 weeks, craniofacial osteogenesis is gradually restricted to the suture region and remaining bone marrow space. Similarly, craniofacial bone vasculature gradually restricts to the suture region. Osteogenesis is spatially associated with vasculature during the entire postnatal development. Importantly, we demonstrated that in adult calvarial bones, Gli1+ mesenchymal stem cells were also spatially associated with the vasculature. These findings indicate that craniofacial bones share similar osteogenesis mechanism as the long bone despite their distinct osteogenic mechanisms. In addition, the PEGASOS tissue clearing method-based 3D imaging technique is a useful new tool for craniofacial research.

3-dimensional visualization of implant-tissue interface with the polyethylene glycol associated solvent system tissue clearing method.

OBJECTIVES: Dental implants are major treatment options for restoring teeth loss. Biological processes at the implant-tissue interface are critical for implant osseointegration. Superior mechanical properties of the implant constitute a major challenge for traditional histological techniques. It is imperative to develop new technique to investigate the implant-tissue interface. MATERIALS AND METHODS: Our laboratory developed the polyethylene glycol (PEG)-associated solvent system (PEGASOS) tissue clearing method. By immersing samples into various chemical substances, bones and teeth could be turned to transparent with intact internal structures and endogenous fluorescence being preserved. We combined the PEGASOS tissue clearing method with transgenic mouse line and other labelling technique to investigate the angiogenesis and osteogenesis processes occurring at the implant-bone interface. RESULTS: Clearing treatment turned tissue highly transparent and implant could be directly visualized without sectioning. Implant, soft/hard tissues and fluorescent labels were simultaneously imaged in decalcified or non-decalcified mouse mandible samples without disturbing their interfaces. Multi-channel 3-dimensional image stacks at high resolution were acquired and quantified. The processes of angiogenesis and osteogenesis surrounding titanium or stainless steel implants were investigated. CONCLUSIONS: Both titanium and stainless steel implants support angiogenesis at comparable levels. Successful osseointegration and calcium precipitation occurred only surrounding titanium, but not stainless steel implants. PEGASOS tissue clearing method provides a novel approach for investigating the interface between implants and hard tissue.

Tissue clearing of both hard and soft tissue organs with the PEGASOS method.

Tissue clearing technique enables visualization of opaque organs and tissues in 3-dimensions (3-D) by turning tissue transparent. Current tissue clearing methods are restricted by limited types of tissues that can be cleared with each individual protocol, which inevitably led to the presence of blind-spots within whole body or body parts imaging. Hard tissues including bones and teeth are still the most difficult organs to be cleared. In addition, loss of endogenous fluorescence remains a major concern for solvent-based clearing methods. Here, we developed a polyethylene glycol (PEG)-associated solvent system (PEGASOS), which rendered nearly all types of tissues transparent and preserved endogenous fluorescence. Bones and teeth could be turned nearly invisible after clearing. The PEGASOS method turned the whole adult mouse body transparent and we were able to image an adult mouse head composed of bones, teeth, brain, muscles, and other tissues with no blind areas. Hard tissue transparency enabled us to reconstruct intact mandible, teeth, femur, or knee joint in 3-D. In addition, we managed to image intact mouse brain at sub-cellular resolution and to trace individual neurons and axons over a long distance. We also visualized dorsal root ganglions directly through vertebrae. Finally, we revealed the distribution pattern of neural network in 3-D within the marrow space of long bone. These results suggest that the PEGASOS method is a useful tool for general biomedical research.