Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA.

BACKGROUND: Bone defects remain a challenge today. In addition to osteogenic activation, the crucial role of angiogenesis has also gained attention. In particular, vascular endothelial growth factor (VEGF) is likely to play a significant role in bone regeneration, not only to restore blood supply but also to be directly involved in the osteogenic differentiation of mesenchymal stem cells. In this study, to produce additive angiogenic-osteogenic effects in the process of bone regeneration, VEGF and Runt-related transcription factor 2 (Runx2), an essential transcription factor for osteogenic differentiation, were coadministered with messenger RNAs (mRNAs) to bone defects in the rat mandible. METHODS: The mRNAs encoding VEGF or Runx2 were prepared via in vitro transcription (IVT). Osteogenic differentiation after mRNA transfection was evaluated using primary osteoblast-like cells, followed by an evaluation of the gene expression levels of osteogenic markers. The mRNAs were then administered to a bone defect prepared in the rat mandible using our original cationic polymer-based carrier, the polyplex nanomicelle. The bone regeneration was evaluated by micro-computerized tomography (µCT) imaging, and histologic analyses. RESULTS: Osteogenic markers such as osteocalcin (Ocn) and osteopontin (Opn) were significantly upregulated after mRNA transfection. VEGF mRNA was revealed to have a distinct osteoblastic function similar to that of Runx2 mRNA, and the combined use of the two mRNAs resulted in further upregulation of the markers. After in vivo administration into the bone defect, the two mRNAs induced significant enhancement of bone regeneration with increased bone mineralization. Histological analyses using antibodies against the Cluster of Differentiation 31 protein (CD31), alkaline phosphatase (ALP), or OCN revealed that the mRNAs induced the upregulation of osteogenic markers in the defect, together with increased vessel formation, leading to rapid bone formation. CONCLUSIONS: These results demonstrate the feasibility of using mRNA medicines to introduce various therapeutic factors, including transcription factors, into target sites. This study provides valuable information for the development of mRNA therapeutics for tissue engineering.

Downregulation of DNA methyltransferase-3a ameliorates the osteogenic differentiation ability of adipose-derived stem cells in diabetic osteoporosis via Wnt/ß-catenin signaling pathway.

BACKGROUND: Diabetes-related osteoporosis (DOP) is a chronic disease caused by the high glucose environment that induces a metabolic disorder of osteocytes and osteoblast-associated mesenchymal stem cells. The processes of bone defect repair and regeneration become extremely difficult with DOP. Adipose-derived stem cells (ASCs), as seed cells in bone tissue engineering technology, provide a promising therapeutic approach for bone regeneration in DOP patients. The osteogenic ability of ASCs is lower in a DOP model than that of control ASCs. DNA methylation, as a mechanism of epigenetic regulation, may be involved in DNA methylation of various genes, thereby participating in biological behaviors of various cells. Emerging evidence suggests that increased DNA methylation levels are associated with activation of Wnt/ß-catenin signaling pathway. The purpose of this study was to investigate the influence of the diabetic environment on the osteogenic potential of ASCs, to explore the role of DNA methylation on osteogenic differentiation of DOP-ASCs via Wnt/ß-catenin signaling pathway, and to improve the osteogenic differentiation ability of ASCs with DOP. METHODS: DOP-ASCs and control ASCs were isolated from DOP C57BL/6 and control mice, respectively. The multipotency of DOP-ASCs was confirmed by Alizarin Red-S, Oil Red-O, and Alcian blue staining. Real-time polymerase chain reaction (RT-PCR), immunofluorescence, and western blotting were used to analyze changes in markers of osteogenic differentiation, DNA methylation, and Wnt/ß-catenin signaling. Alizarin Red-S staining was also used to confirm changes in the osteogenic ability. DNMT small interfering RNA (siRNA), shRNA-Dnmt3a, and LVRNA-Dnmt3a were used to assess the role of Dnmt3a in osteogenic differentiation of control ASCs and DOP-ASCs. Micro-computed tomography, hematoxylin and eosin staining, and Masson staining were used to analyze changes in the osteogenic capability while downregulating Dnmt3a with lentivirus in DOP mice in vivo. RESULTS: The proliferative ability of DOP-ASCs was lower than that of control ASCs. DOP-ASCs showed a decrease in osteogenic differentiation capacity, lower Wnt/ß-catenin signaling pathway activity, and a higher level of Dnmt3a than control ASCs. When Dnmt3a was downregulated by siRNA and shRNA, osteogenic-related factors Runt-related transcription factor 2 and osteopontin, and activity of Wnt/ß-catenin signaling pathway were increased, which rescued the poor osteogenic potential of DOP-ASCs. When Dnmt3a was upregulated by LVRNA-Dnmt3a, the osteogenic ability was inhibited. The same results were obtained in vivo. CONCLUSIONS: Dnmt3a silencing rescues the negative effects of DOP on ASCs and provides a possible approach for bone tissue regeneration in patients with diabetic osteoporosis.

Metformin Rescues the Impaired Osteogenesis Differentiation Ability of Rat Adipose-Derived Stem Cells in High Glucose by Activating Autophagy.

The incidence and morbidity of diabetes osteoporosis (DOP) are increasing with each passing year. Patients with DOP have a higher risk of bone fracture and poor healing of bone defects, which make a poor quality of their life. Bone tissue engineering based on autologous adipose-derived stem cells (ASCs) transplantation develops as an effective technique to achieve tissue regeneration for patients with bone defects. With the purpose of promoting auto-ASCs transplantation, this research project explored the effect of metformin on the osteogenic differentiation of ASCs under a high-glucose culture environment. In this study, we found that 40 mM high glucose inhibited the physiological function of ASCs, including cell proliferation, migration, and osteogenic differentiation. Indicators of osteogenic differentiation were all downregulated by 40 mM high glucose, including alkaline phosphatase activity, runt-related transcription factor 2, and osteopontin gene expression, and Wnt signaling pathway. At the same time, the cell autophagy makers BECLIN1 and microtubule-associated protein 1 light chain 3 (LC3 I/II) were decreased. While 0.1 mM metformin upregulated the expression of BECLIN1 and LC3 I/II gene and inhibited the expression of mammalian target of rapamycin (mTOR) and GSK3ß, it contributed to reverse the osteogenesis inhibition of ASCs caused by high glucose. When 3-methyladenine was used to block the activity of metformin, metformin could not exert its protective effect on ASCs. All the findings elaborated the regulatory mechanism of metformin in the high-glucose microenvironment to protect the osteogenic differentiation ability of ASCs. Metformin plays an active role in promoting the osteogenic differentiation of ASCs with DOP, and it may contribute to the application of ASCs transplantation for bone regeneration in DOP.

Accuracy evaluation of partially guided and fully guided templates applied to implant surgery of anterior teeth: A randomized controlled trial.

OBJECTIVES: To study the accuracy of partially guided and fully guided templates applied to implant surgery of anterior teeth. MATERIALS AND METHODS: Sixty patients who were scheduled to receive dental implant treatment in the anterior region were enrolled and randomly assigned to one of the following study groups (n = 20 each): routine implant-supported restoration treatment (control group, 30 implants), implant-supported restoration treatment using a partially guided template (test group 1, 36 implants), and implant-supported restoration treatment using a fully guided template (test group 2, 33 implants). The depth of implant was controlled for fully guided template. After implantation, planned implants and placed implants were superimposed using digital software, and the deviations (angular, coronal, apical, depth) were analyzed. Esthetic parameters were assessed at baseline, 6 months, and 1 year after the final restoration. Pink esthetic score (PES) and white esthetic score (WES) were respectively used to evaluate the soft tissue and restoration esthetic outcome. Each parameter of PES and WES is assessed with a 0-1-2 score with 2 being the best and 0 being the worst score. RESULTS: There were significant differences in all of the deviation parameters between the control group, test group 1, and test group 2 (p < 0.001). Mean angular, coronal, apical and depth deviations were all the highest in the control group (6.61 ± 1.09°, 1.05 ± 0.17 mm, 1.36 ± 0.13 mm, and 1.02 ± 0.13 mm, respectively), and lowest in test group 2 (2.05 ± 0.45°, 0.39 ± 0.12 mm, 0.28 ± 0.09 mm, and 0.24 ± 0.06 mm, respectively). At 1 year after the final restoration, the analysis revealed mean PES values of 7.09 ± 0.56 (control group), 8.39 ± 0.54 (test group 1), and 9.04 ± 0.35 (test group 2). The WES values were 7.24 ± 0.54 (control group), 8.47 ± 0.44 (test group 1), and 8.97 ± 0.38 (test group 2). At all examinations, the mean PES and WES values were both the highest in test group 2 and lowest in the control group. The PES and WES values recorded in the control group at baseline, 6 months, and 1 year after final restoration were significantly lower than those in test groups (p < 0.001). Moreover, the PES and WES values recorded in the test group 1 at baseline, 6 months, and 1 year after final restoration were significantly lower than those in test group 2 (p < 0.05). CONCLUSIONS: Digital surgical guides can improve the accuracy of the three-dimensional position of implants in the maxillary esthetic zone, the fully guided template has higher precision than that of the partially guided template, and plays an important role in obtaining the ideal esthetic outcome for maxillary anterior teeth.

Application of Nanomaterials in Regulating the Fate of Adipose-derived Stem Cells.

Adipose-derived stem cells are adult stem cells which are easy to obtain and multi-potent. Stem-cell therapy has become a promising new treatment for many diseases, and plays an increasingly important role in the field of tissue repair, regeneration and reconstruction. The physicochemical properties of the extracellular microenvironment contribute to the regulation of the fate of stem cells. Nanomaterials have stable particle size, large specific surface area and good biocompatibility, which has led them being recognized as having broad application prospects in the field of biomedicine. In this paper, we review recent developments of nanomaterials in adipose-derived stem cell research. Taken together, the current literature indicates that nanomaterials can regulate the proliferation and differentiation of adipose-derived stem cells. However, the properties and regulatory effects of nanomaterials can vary widely depending on their composition. This review aims to provide a comprehensive guide for future stem-cell research on the use of nanomaterials.

Advanced glycation end products inhibit the osteogenic differentiation potential of adipose-derived stem cells by modulating Wnt/ß-catenin signalling pathway via DNA methylation.

OBJECTIVES: Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose-derived stem cells (ASCs) are widely used in the research of bone regeneration, the mechanisms of the osteogenic differentiation of ASCs from diabetic osteoporosis model remain unclear. This work aimed to investigate the influence and the molecular mechanisms of AGEs on the osteogenic potential of ASCs. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay was used to measure the change of AGEs in diabetic osteoporotic and control C57BL/6 mice. ASCs were obtained from the inguinal fat of C57BL/6 mice. AGEs, 5-aza2'-deoxycytidine (5-aza-dC) and DKK-1 were used to treat ASCs. Real-time cell analysis and cell counting kit-8 were used to monitor the proliferation of ASCs within and without AGEs. Real-time PCR, Western blot and Immunofluorescence were used to analyse the genes and proteins expression of osteogenic factors, DNA methylation factors and Wnt/ß-catenin signalling pathway among the different groups. RESULTS: The AGEs and DNA methylation were increased in the adipose and bone tissue of the diabetic osteoporosis group. Untreated ASCs had higher cell proliferation activity than AGEs-treatment group. The expression levels of osteogenic genes, Opn and Runx2, were lower, and mineralized nodules were less in AGEs-treatment group. Meanwhile, DNA methylation was increased, and the Wnt signalling pathway markers, including ß-Catenin, Lef1 and P-GSK-3ß, were inhibited. After treatment with 5-aza-dC, the osteogenic differentiation capacity of ASCs in the AGEs environment was restored and the Wnt signalling pathway was activated during this process. CONCLUSIONS: Advanced glycation end products inhibit the osteogenic differentiation ability of ASCs by activating DNA methylation and inhibiting Wnt/ß-catenin pathway in vitro. Therefore, DNA methylation may be promising targets for the bone regeneration of ASCs with diabetic osteoporosis.

Modified Tragus Edge and Transmasseteric Anteroparotid Approach for Intracapsular and Condylar Neck Fractures.

The aim of this study was to evaluate the effects of protecting the facial nerve and reducing the scar visibility using a modified tragus edge and transmasseteric anteroparotid approach compared to classic preauricular approach for intracapsular and condylar neck fractures. This retrospective study included 64 patients (78 sides) who underwent surgical treatment for intracapsular or condylar neck fractures from January 2014 to June 2018. Patients were divided into the experimental group (treated via a modified tragus edge and transmasseteric anteroparotid approach), and the control group (treated via the classical preauricular approach). Therapeutic outcome assessment parameters included facial nerve injury, salivary fistulae, wound infection, restricted mouth opening, postoperative occlusion disorders, and scar visibility. In the control group, there were 3 cases of facial nerve injuries and 2 cases of salivary fistulae. One case of temporary facial nerve injury occurred in the experimental group, with complete recovery within 1 month. The scars were less visible in the experimental group than in the control group. These results suggest that a modified tragus edge and transmasseteric anteroparotid approach reduced the incidences of facial nerve injuries, minimized the scar visibility, improved exposure of the operative site and fixation of titanium screws or plates, and did not increase the frequency of other complications.

[Precise investigation of digital guide plates applied to implant surgery of anterior teeth].

OBJECTIVE: To study the precision of digital guide plates applied to the implant surgery of anterior teeth. METHODS: Fifty patients scheduled to receive implant restoration treatment in anterior teeth were enrolled in this study and divided into two groups (n=25, each group): those who were given routine implant restoration treatment (control group, 45 implants) and those who received implant restoration treatment using a digital guide plate (test group, 51 implants). After implantation, planned and placed implants were superimposed using digital software, and deviations (corona, apex, depth, degree) were analyzed. Esthetic parameters were assessed at 1 week (baseline), 6 month, and 1 year post final restoration. Pink esthetic (PES) and white esthetic (WES) scores were respectively used to evaluate the soft tissue and restoration esthetic outcome. RESULTS: The deviation parameters in the test group were significantly lower than those in the control group (P<0.05). PES and WES values recorded for the control group at 1 week, 6 month, and 1 year post final restoration were significantly lower than those in the test group (P<0.05). CONCLUSIONS: The digital guide plate can improve the accuracy of the three-dimensional position of implants in the maxillary esthetic zone. As such, this device may play an important role in obtaining the ideal aesthetic effects of maxillary anterior teeth.

Functional Reconstruction of Mandibular Segment Defects With Individual Preformed Reconstruction Plate and Computed Tomographic Angiography-Aided Iliac Crest Flap.

PURPOSE: With the development of imaging technology and computer-assisted surgery in oral and maxillofacial surgery, digital technology is widely used in vascularized bone flap grafts for mandibular reconstruction. The aim of this study was to use digital technology throughout the treatment process to show that digital techniques can provide a reliable and accurate match between the mandible and the iliac crest flap to achieve functional reconstruction of mandibular segment defects. MATERIALS AND METHODS: Twenty patients underwent 3-dimensional (3D) computed tomography (CT), mirroring technology, 3D model prototyping, and CT angiography (CTA) for treatment planning. Individual preformed reconstruction plates were fabricated and iliac crest flaps were designed preoperatively. After complete resection of the mandibular lesion, the iliac crest flap was shaped to reconstruct the mandibular defects. RESULTS: During follow-up (range, 12 to 36 months), the facial shape, facial symmetry, and mouth opening of all patients recovered well. The 3D CT reconstruction also was evaluated for height, width, length, and bone healing of the iliac crest flap. Postoperative examination showed ideal bone union between the iliac crest flap and the mandible at 6 months. Nine patients received implant-supported fixed dentures to restore dentition. After follow-up, all patients were satisfied with their facial esthetics and function. The new mandible provided a suitable 3D position for implant-supported fixed partial dentures. CONCLUSION: Use of digital techniques throughout the course of treatment improves the predictability and convenience of functional mandibular reconstruction. Individual preformed reconstruction plates and CTA effectively guaranteed the accuracy of iliac flap preparation.

Blockade of receptors of advanced glycation end products ameliorates diabetic osteogenesis of adipose-derived stem cells through DNA methylation and Wnt signalling pathway.

OBJECTIVES: Diabetes mellitus-related osteoporosis is caused by the imbalance between bone absorption and bone formation. Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose-derived stem cells (ASCs) are promising adult stem cells in bone tissue regeneration, the ability of osteogenesis of ASCs in diabetic environment needs to explore. This study aimed to investigate the influence of AGEs on the osteogenic potential of ASCs and to explore the signalling pathways involved in its effect. MATERIALS AND METHODS: ASCs were isolated from inguinal fat and cultured in osteogenic media with or without AGEs and FPS-ZM1, an inhibitor of receptor for AGEs (RAGE). Alizarin red-S, Oil Red-O and Alcian blue staining were used to confirm osteogenic, adipogenic and chondrogenic potential of ASCs, respectively. Immunofluorescence, western blotting and real-time PCR were used to measure changes in markers of osteogenic differentiation, DNA methylation and Wnt signalling. RESULTS: The multipotentiality of ASCs was confirmed. Treated with AGEs, OPN and RUNX2 expressions of ASCs were reduced and there was a noticeable loss of mineralization, concomitant with an increase in the expression of RAGE, 5-MC, DNMT1 and DNMT3a. AGEs treatment also led to a loss of Wnt signalling pathway markers, including ß-Catenin and LEF1, with an increase in GSK-3ß. Treatment with the RAGE inhibitor, FPS-ZM1, rescued AGEs-induced loss of osteogenic potential, modulated DNA methylation and upregulated Wnt signalling in ASCs. CONCLUSIONS: Our results demonstrate that AGEs-RAGE signalling inhibits the osteogenic potential of ASCs under osteoinductive conditions by modulating DNA methylation and Wnt signalling. FPS-ZM1 can rescue the negative effects of AGEs and provide a possible treatment for bone tissue regeneration in patients with diabetic osteoporosis.

Problem and its solution for actuator saturation of integrating process with dead time.

The modified Smith predictor (MSP) for integrating process with dead time (IPDT) has at least one pole at the origin of s-plane. The integral effect leads to large overshoot and slow settling time when the control signal exceeds the saturation limits of the system actuator. The windup problem in MSP is presented and studied. Based on the two degrees of freedom (2DOF) control schemes presented by Normey-Rico and Camacho, an anti-windup controller (AWC) is proposed. By simulation, significant performance improvement demonstrates the effectiveness of the proposed scheme.