Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.

Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.

UCHL1 alleviates apoptosis in chondrocytes via upregulation of HIF­1α­mediated mitophagy.

Stem cell­based tissue engineering has shown significant potential for rapid restoration of injured cartilage tissues. Stem cells frequently undergo apoptosis because of the prevalence of oxidative stress and inflammation in the microenvironment at the sites of injury. Our previous study demonstrated that stabilization of hypoxia­inducible factor 1α (HIF­1α) is key to resisting apoptosis in chondrocytes. Recently, it was reported that Ubiquitin C­terminal hydrolase L1 (UCHL1) can stabilize HIF­1α by abrogating the ubiquitination process. However, the effect of UCHL1 on apoptosis in chondrocytes remains unclear. Herein, adipose­derived stem cells were differentiated into chondrocytes. Next, the CRISPR activation (CRISPRa) system, LDN­57444 (LDM; a specific inhibitor for UCHL1), KC7F2 (a specific inhibitor for HIF­1α), and 3­methyladenine (a specific inhibitor for mitophagy) were used to activate or block UCHL1, HIF­1α, and mitophagy. Mitophagy, apoptosis, and mitochondrial function in chondrocytes were detected using immunofluorescence, TUNEL staining, and flow cytometry. Moreover, the oxygen consumption rate of chondrocytes was measured using the Seahorse XF 96 Extracellular Flux Analyzer. UCHL1 expression was increased in hypoxia, which in turn regulated mitophagy and apoptosis in the chondrocytes. Further studies revealed that UCHL1 mediated hypoxia­regulated mitophagy in the chondrocytes. The CRISPRa module was utilized to activate UCHL1 effectively for 7 days; endogenous activation of UCHL1 accelerated mitophagy, inhibited apoptosis, and maintained mitochondrial function in the chondrocytes, which was mediated by HIF­1α. Taken together, UCHL1 could block apoptosis in chondrocytes via upregulation of HIF­1α-mediated mitophagy and maintain mitochondrial function. These results indicate the potential of UCHL1 activation using the CRISPRa system for the regeneration of cartilage tissue.

Nanoparticles of Cerium-Doped Zeolitic Imidazolate Framework-8 Promote Soft Tissue Integration by Reprogramming the Metabolic Pathways of Macrophages.

Soft tissue integration around the abutment of implants is the basis of long-term retention of implants. Macrophages are an important component involved in the repair of soft tissue due to their crucial role in improving the biological structure of connective tissues by regulating the fiber synthesis, adhesion, and contraction of gingival fibroblasts. Recent studies have illustrated that cerium-doped zeolitic imidazolate framework-8 (Ce@ZIF-8) nanoparticles (NPs) can attenuate periodontitis via both antibacterial and anti-inflammatory effects. However, the effect of Ce@ZIF-8 NPs on soft tissue integration around the abutment is unknown. Herein, we first prepared Ce@ZIF-8 NPs by a one-pot synthesis. Then, we probed the regulatory effect of Ce@ZIF-8 NPs on macrophage polarization, and further experiments were performed to study the changes of fiber synthesis as well as adhesion and contraction of fibroblasts in the M2 macrophage environment stimulated by Ce@ZIF-8 NPs. Strikingly, Ce@ZIF-8 NPs can be internalized by M1 macrophages through macropinocytosis and caveolae-mediated endocytosis in addition to phagocytosis. By catalyzing hydrogen peroxide to produce oxygen, the mitochondrial function was remedied, while hypoxia inducible factor-1α was restrained. Then, macrophages were shifted from the M1 to M2 phenotype via this metabolic reprogramming pathway, provoking soft tissue integration. These results provide innovative insights into facilitating soft tissue integration around implants.

Micro-/nano-topography of selective laser melting titanium enhances adhesion and proliferation and regulates adhesion-related gene expressions of human gingival fibroblasts and human gingival epithelial cells.

BACKGROUND: Selective laser melting (SLM) titanium is an ideal option to manufacture customized implants with suitable surface modification to improve its bioactivity. The peri-implant soft tissues form a protective tissue barrier for the underlying osseointegration. Therefore, original microrough SLM surfaces should be treated for favorable attachment of surrounding soft tissues. MATERIAL AND METHODS: In this study, anodic oxidation (AO) was applied on the microrough SLM titanium substrate to form TiO2 nanotube arrays. After that, calcium phosphate (CaP) nanoparticles were embedded into the nanotubes or the interval of nanotubes by electrochemical deposition (AOC). These two samples were compared to untreated (SLM) samples and accepted mechanically polished (MP) SLM titanium samples. Scanning electron microscopy, energy dispersive spectrometry, X-ray diffraction, surface roughness, and water contact angle measurements were used for surface characterization. The primary human gingival epithelial cells (HGECs) and human gingival fibroblasts (HGFs) were cultured for cell assays to determine adhesion, proliferation, and adhesion-related gene expressions. RESULTS: For HGECs, AOC samples showed significantly higher adhesion, proliferation, and adhesion-related gene expressions than AO and SLM samples (P<0.05) and similar exceptional ability in above aspects to MP samples. At the same time, AOC samples showed the highest adhesion, proliferation, and adhesion-related gene expressions for HGFs (P<0.05). CONCLUSION: By comparison between each sample, we could confirm that both anodic oxidation and CaP nanoparticles had improved bioactivity, and their combined utilization may likely be superior to mechanical polishing, which is most commonly used and widely accepted. Our results indicated that creating appropriate micro-/nano-topographies can be an effective method to affect cell behavior and increase the stability of the peri-implant mucosal barrier on SLM titanium surfaces, which contributes to its application in dental and other biomedical implants.

Enhanced antibacterial efficacy of selective laser melting titanium surface with nanophase calcium phosphate embedded to TiO2 nanotubes.

Selective laser melting (SLM) has promising prospects in manufacturing customized implants, however the rough surface of SLM titanium specimen can facilitate bacterial adherence and biofilm formation, which is a risk to implant success. Therefore, surface modification is required to enhance its antibacterial efficacy. Sandblasting, anodization and electrochemical deposition were applied to construct a novel composite nanostructure of nanophase calcium phosphate embedded to TiO2 nanotubes on microrough SLM titanium substrates (NTN). NTN samples were compared with TiO2 nanotubes (NT) samples, mechanical polished (MP) samples and untreated SLM titanium samples. Surface characterization were analyzed using scanning electron microscope, energy dispersive spectroscopy, x-ray photoelectron spectroscopy, x-ray diffraction, a three dimensional profilometer and a contact angle measuring device. Bacteria adhesion assay for bacteria colony counting and bacteria LIVE/DEAD staining was conducted using Streptococcus mutans and Streptococcus sanguinis. Both S. mutans and S. sanguinis adherence on SLM samples were significantly higher than on NTN, NT and MP samples. The antibacterial efficacy of NTN samples was superior compared to NT and had no significant difference with MP samples, despite the fact that NTN samples had much rougher surface than MP samples. This study elucidates an efficient method to enhance antibacterial efficacy on rough SLM surfaces, which contributes to its application in dental and other biomedical implants.

Clinical Outcomes of Bicortical Engagement Implants in Atrophic Posterior Maxillae: A Retrospective Study with 1 to 5 Years Follow-up.

Bicortical dental implantation technique was regarded as an alternative to internal sinus lift technique with bone grafting in dental implantation in atrophic maxillae. The purpose of this retrospective study was to evaluate the survival rates and bone stability of 6- and 10-mm implants in the posterior maxilla using bicortical dental implantation techniques after 1 to 5 years of prosthetic loading. A total of 53 patients received posterior maxillary implant treatments. Of these, 31 patients received bicortical dental implantation (group A), and 25 patients received traditional implantation (group B). Three patients received an implant with bicortical engagement and another implant without bicortical engagement. The survival rates were calculated, and the marginal bone heights were measured on radiographs at baseline and follow-up. The survival rate of group A was 100%. In contrast, the survival rate of group B was only 74.68%, with survival rates of 51.85% in the 6-mm implant subgroup and 93.33% in the 10-mm implant subgroup. In terms of variations in implant marginal bone heights, a significant difference was found only between the 6-mm subgroups in groups A and B. Significant intrasinus bone gain was found on the mesial and distal sides of the implants in group A. Application of bicortical dental implantation techniques with 6-mm implants could achieve better clinical outcomes in atrophic posterior maxillae.

Influence of Lateral-Medial Sinus Width on No-Grafting Inlay Osteotome Sinus Augmentation Outcomes.

PURPOSE: Intrasinus new bone formation (BF) has been observed after no-grafting osteotome sinus augmentation, and it is hypothesized to be influenced by the dimensions of the maxillary sinus. The aim of this clinical trial is to evaluate the influence of lateral-medial sinus width (SW) on no-grafting osteotome sinus augmentation outcomes using cone-beam computed tomography. PATIENTS AND METHODS: All patients recruited for this prospective study were treated with no-grafting osteotome sinus augmentation with simultaneous implant placement. Cone-beam computed tomography was obtained before, immediately after, and 6 months after the surgical procedure to use for measurements. Descriptive statistics were calculated and univariate, bivariate, and multivariate analysis were conducted to evaluate the influence of average SW and other relevant factors on procedure outcomes, including new BF, residual bone resorption (BR), and change of peri-implant bone height (CPBH). RESULTS: A total of 48 implants placed in 32 elevated sinuses of 29 patients were included. The average SW was 11.3 ± 1.8 mm. Intrasinus BF measured 1.7 ± 0.9 mm at 6 months after surgery. The amount of BR was 0.3 ± 0.9 mm, and CPBH was calculated as 1.3 ± 1.3 mm. Multivariate analysis showed a negative correlation between SW and BF (r = -0.469, P = .001), as well as between SW and CPBH (r = -0.562, P = .001). A positive correlation was discovered between SW and BR (r = 0.311, P = .027) in general. CONCLUSIONS: The lateral-medial SW was observed to have a negative correlation with new BF and CPBH after no-grafting osteotome sinus augmentation.

Mandibular asymmetry: a three-dimensional quantification of bilateral condyles.

INTRODUCTION: The shape and volume of the condyle is considered to play an important role in the pathogenesis of the mandibular deviation. Curvature analysis is informative for objectively assess whether the shape of the condyles matches that of the glenoid fossa. In this study, a three-dimensional (3-D) quantification of bilateral asymmetrical condyles was firstly conducted to identify the specific role of 3-D condylar configuration for mandibular asymmetry. METHODS: 55 adult patients, 26 males (26 ± 5 yrs) and 29 females (26 ± 5 yrs), diagnosed with mandibular asymmetry were included. The examination of deviation of chin point, deviation of dental midlines, inclination of occlusal plane, and depth of the mandibular occlusal plane were conducted. After the clinical investigation, computed tomography images from the patients were used to reconstruct the 3-D mandibular models. Then the condylar volume, surface size, surface curvature and bone mineral density were evaluated independently for each patient on non-deviated and deviated sides of temporomandibular joint. RESULTS: Both the condylar surface size and volume were significantly larger on deviated side (surface size: 1666.14 ± 318.3 mm2, volume: 1981.5 ± 418.3 mm3). The anterior slope of the condyle was flatter (0.12 ± 0.06) and the posterior slope (0.39 ± 0.08) was prominently convex on the deviated side. The corresponding bone mineral density values were 523.01 ±118.1 HU and 549.07 ±120. 6 HU on anterior and posterior slopes. CONCLUSIONS: The incongruence presented on the deviated side resulted in a reduction in contact areas and, thus, an increase in contact stresses and changes of bone density. All aforementioned results suggest that the difference existing between deviated and non-deviated condyles correlates with facial asymmetrical development. In mandibular asymmetry patients, the 3-D morphology of condyle on deviated side differ from the non-deviated side, which indicates the association between asymmetrical jaw function and joint remodeling.