Effects of RNA m6A writer METTL3 and hDPSCs on the peripheral nerve regeneration: In vitro and in vivo study.

PURPOSE: This study aimed to investigate whether RNA m6A participated in the differentiation and proliferation of dental pulp stem cells and improved peripheral nerve regeneration using a rat model of crushed mental nerve injury. MATERIALS AND METHODS: The components of RNA m6A were analyzed through qRT-PCR, while cell proliferation of different groups, including over-expression METTL3 (OE-METTL3) hDPSCs group, knock-down METTL3 (KD-METTL3) hDPSCs group and hDPSCs group in vitro, was clarified by MTT assay. Five groups were designed, namely, Control group, Sham group, hDPSCs group, OE-METTL3 group and KD-METTL3 group. After crushed right mental nerve injury, cells of different groups were transplanted into the lesion area (6 ul in volume). At one, two and three weeks later, histomorphometric analysis and sensory test were conducted in vivo. RESULTS: qRT-PCR results showed that "METTL3" was participated in the differentiation of dental pulp stem cells. There were differences (P < 0.05) between OE-METTL3 group and control group in MTT results in the third, fourth and sixth days. Moreover, the sensory test revealed significant differences (P < 0.05) in difference score and gap score between OE-METTL3 group and KD-METTL3 group in the first and third weeks. The axon counts and retrograde labeled neurons significantly increased in OE-METTL3 group compared with KD-METTL3 group. CONCLUSIONS: These results demonstrated that RNA m6A participated in the differentiation and proliferation of dental pulp stem cells, and that OE-METTL3 group exhibited the greater ability to improve peripheral nerve regeneration than KD-METTL3 group and hDPSCs group.

Rabbit submandibular salivary gland replantation.

OBJECTIVES: To test the feasibility of submandibular salivary gland (SMG) replantation techniques and the survival of the replanted glands. Such a study can provide a rationale for later allotransplantation procedures, along with implementation of conventional and advanced immunosuppression therapy. MATERIALS AND METHODS: Six SMG replantations were performed in New Zealand white rabbits. One week postoperatively, 99mTc scintigraphy was performed and the uptake ratio and salivary excretion fraction were calculated. Two to four weeks later, submandibular glands were excised, fixed, and stained with H&E for histomorphometric evaluation. RESULTS: Intraoperatively, all glands showed patent blood perfusion except gland 5. Positive tracer uptake and saliva excretion were documented by scintigraphy. On excision, all of the glands except glands 4 and 5 looked viable, with a red color and patent pedicles. Gland 4 was infected and filled with creamy pus, while gland 5 looked pale and necrotic. Histologically, glands 1, 2, 3, and 6 had preserved normal glandular tissue with slight variations from the contralateral normal glands, as their parenchyma was composed of mildly atrophic acini. CONCLUSION: Four out of six replanted SMGs successfully survived. The glands maintained good viability and function. Such success depends on safe harvesting, short anastomosis time, and strict control of infection.

Bone-level implants placed in the anterior maxilla: an open-label, single-arm observational study.

PURPOSE: This study assessed marginal bone remodeling and soft tissue esthetics after the loading of single bone-level implants in the anterior maxilla. METHODS: An open, single-arm observational clinical trial with 3 years of follow-up was performed, including 22 implants. The patients presented with a single tooth gap in the anterior maxilla (tooth positions 14-24), with natural or restored adjacent teeth. An implant was placed at least 8 weeks post-extraction and healed submerged for 6 weeks. After the second-stage operation, a fixed provisional prosthesis was provided. The final restoration was placed 6 months after the provisional restoration. The time of the provisional crown connection was considered to be the baseline in this study. Esthetic parameters and the marginal bone level were assessed at 6, 12, 24, and 36 months. RESULTS: All implants were well integrated in the bone. A statistically significant increase was found in the mean implant stability quotient between the time of the provisional prosthesis and the time of the final prosthesis. Most implants (95.5%) revealed marginal bone resorption (<0.5 mm), and just 1 implant (4.5%) showed a change of 2.12 mm from baseline to 36 months (mean 0.07±0.48 mm), while the crestal bone level decreased significantly, from 2.34±0.93 mm at baseline to 1.70±1.10 mm at 36 months. The facial gingival margin and papilla were stable and the esthetic scores indicated high patient and dentist satisfaction. CONCLUSIONS: Platform-switching bone-level implants placed in maxillary single-tooth gaps resulted in successful osseointegration with minimal marginal bone resorption. The peri-implant soft tissue was also esthetically satisfying and stable.

Adenovirus vector-mediated ex vivo gene transfer of brain-derived neurotrophic factor (BDNF) tohuman umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) promotescrush-injured rat sciatic nerve regeneration.

This study was designed toinvestigate the efficacy of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in a rat sciatic nerve crush injury model. BDNF protein and mRNA expression after infection was checked through an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Male Sprague-Dawley rats (200-250g, 6 weeks old) were distributed into threegroups (n=20 each): the control group, UCB-MSC group, and BDNF-adenovirus infected UCB-MSC (BDNF-Ad+UCB-MSC) group. UCB-MSCs (1×106cells/10µl/rat) or BDNF-Ad+UCB-MSCs (1×106cells/10µl/rat)were transplantedinto the rats at the crush site immediately after sciatic nerve injury. Cell tracking was done with PKH26-labeled UCB-MSCs and BDNF-Ad+UCB-MSCs (1×106cells/10µl/rat). The rats were monitored for 4 weeks post-surgery. Results showed that expression of BDNF at both the protein and mRNA levels was higher inthe BDNF-Ad+UCB-MSC group compared to theUCB-MSC group in vitro.Moreover, BDNF mRNA expression was higher in both UCB-MSC group and BDNF-Ad+ UCB-MSC group compared tothe control group, and BDNF mRNA expression in theBDNF-Ad+UCB-MSC group was higher than inboth other groups 5days after surgeryin vivo. Labeled neurons in the dorsal root ganglia (DRG), axon counts, axon density, and sciatic function index were significantly increased in the UCB-MSC and BDNF-Ad+ UCB-MSCgroupscompared to the controlgroup four weeksaftercell transplantation. Importantly,the BDNF-Ad+UCB-MSCgroup exhibited more peripheral nerve regeneration than the other two groups.Our results indicate thatboth UCB-MSCs and BDNF-Ad+UCB-MSCscan improve rat sciatic nerve regeneration, with BDNF-Ad+UCB-MSCsshowing a greater effectthan UCB-MSCs.

Schwann-like cells differentiated from human dental pulp stem cells combined with a pulsed electromagnetic field can improve peripheral nerve regeneration.

The purpose of this study was to investigate the effect of Schwann-like cells combined with pulsed electromagnetic field (PEMF) on peripheral nerve regeneration. Schwann-like cells were derived from human dental pulp stem cells (hDPSCs) and verified with CD104, S100, glial fibrillary acidic protein (GFAP), laminin, and P75NTR immunocytochemistry. Gene expression of P75NTR and S100 were analyzed. Male Sprague-Dawley rats (200-250g, 6-week-old) were divided into seven groups (n = 10 each): control, sham, PEMF, hDPSCs, hDPSCs + PEMF, Schwann-like cells, Schwann-like cells + PEMF. Cells were transplanted (1 × 106 /10µl/rat) at crush-injury site or combined with PEMF (50 Hz, 1 h/day, 1 mT). Nerve regeneration was evaluated with functional test, histomorphometry and retrograde labelled neurons. Schwann-like cells expressed CD104, S100, GFAP, laminin, and p75 neurotrophin receptor (P75NTR ). P75NTR and S100 mRNA expression was highest in Schwann-like cells + PEMF group, which also showed increased Difference and Gap scores. Axons and retrograde labeled neurons increased in all treatment groups. Schwann-like cells, hDPSCs with or without PEMF, and PEMF only improved peripheral nerve regeneration. Schwann-like cells + PEMF showed highest regeneration ability; PEMF has additive effect on hDPSCs, Schwann-like cell in vitro and nerve regeneration ability after transplantation in vivo. Bioelectromagnetics. 37:163-174, 2016. © 2016 Wiley Periodicals, Inc.

Effects of electromagnetic field (PEMF) exposure at different frequency and duration on the peripheral nerve regeneration: in vitro and in vivo study.

PURPOSE: The purpose was to clarify the influence of frequency and exposure time of pulsed electromagnetic fields (PEMF) on the peripheral nerve regeneration. MATERIALS AND METHODS: Immortalized rat Schwann cells (iSCs) (1 × 10(2)/well) were exposed at four different conditions in 1 mT (50 Hz 1 h/d, 50 Hz 12 h/d, 150 Hz 1 h/d and 150 Hz 12h/d). Cell proliferation, mRNA expression of S100 and brain-derived neurotrophic factor (BDNF) were analyzed. Sprague-Dawley rats (200-250 g) were divided into six groups (n = 10 each): control, sham, 50 Hz 1 h/d, 50 Hz 12 h/d, 150 Hz 1 h/d and 150 Hz 12 Hr/d. Mental nerve was crush-injured and exposed at four different conditions in 1 mT (50 Hz 1 Hr/d, 50 Hz 12 Hr/d, 150 Hz 1 h/d and 150 Hz 12 h/d). Nerve regeneration was evaluated with functional test, histomorphometry and retrograde labeling of trigeminal ganglion. RESULTS: iSCs proliferation with 50 Hz, 1 h/d was increased from fourth to seventh day; mRNA expression of S100 and BDNF was significantly increased at the same condition from first week to third week (p < .05 vs. control); difference score was increased at the second and third week, and gap score was increased at the third under 50 Hz 1 h PEMF compared with control while other conditions showed no statistical meaning. Axon counts and retrograde labeled neurons were significantly increased under PEMF of four different conditions compared with control. Although there was no statistical difference, 50 Hz, 1 h PEMF showed highest regeneration ability than other conditions. CONCLUSION: PEMF enhanced peripheral nerve regeneration, and that it may be due to cell proliferation and increase in BDNF and S100 gene expression.

Co-treatment effect of pulsed electromagnetic field (PEMF) with human dental pulp stromal cells and FK506 on the regeneration of crush injured rat sciatic nerve.

PURPOSE: The purpose of this study was to determine whether crush injured rat sciatic nerve could be benefit from pulsed electromagnetic field (PEMF) combined with human dental pulp stromal cells (hDPSCs), with FK506 (Tacrolimus) for immune suppression and neuropromotion. MATERIALS AND METHODS: Male Sprague-Dawley rats (200-250 g, 6 week old) were distributed into 6 groups (n = 18 each): control, PEMF, FK506, PEMF + hDPSCs, PEMF + FK506, and PEMF + hDPSCs + FK506 groups. hDPSCs (cell = 1 × 106/10 µl/rat) were injected at the crush site immediate after injury. FK506 was administered 3 weeks in FK506 group (0.5 mg/kg/d) while pre-op 1 d and post-op 7 d in PEMF + FK506 and PEMF + hDPSCs + FK506 group; cell tracking was done with PKH26-labeled hDPSCs (cell = 1 × 106/10 µl/rat). The rats were follow-up for 3 weeks. RESULTS: PEMF + FK506 and PEMF + hDPSCs + FK506 group showed a sharp increase in sciatic function index (SFI), axon counts, densities, and labeled neurons in dorsal root ganglia (DRG) than control at 3 weeks. Other three treatment groups also showed higher axon counts, densities, and labeled neurons than control. Higher axon counts and densities were found in PEMF + FK506 and PEMF + hDPSCs + FK506 groups comparing with PEMF group. Brain-derived neurotrophic factor (BDNF) mRNA expression pattern in nerve segment and DRG was almost same. Higher expression level in all the treatment groups was discovered in the follow-up period, but there was no significant difference. CONCLUSIONS: All treatment groups can improve regeneration of neurons following crushed injury, PEMF + FK506 and PEMF + hDPSCs + FK506 groups showed higher regeneration ability than other three groups. FK506 plays an important role during hDPSCs transplantation.