Endothelial progenitors increase vascularization and improve fibroblasts function that prevent medication-related osteonecrosis of the jaw.

OBJECTIVES: In a previous rat model, MRONJ occurrence was 50%. Our aim was to investigate the potential of endothelial progenitor cells (EPCs) to improve fibroblasts function and prevent MRONJ. METHODS: Human gingival fibroblasts were cultured with EPC-conditioned media (EPC-CM); endothelial growth media (EGM-2) or DMEM followed by incubation with 10 µM zoledronic (ZOL) and dexamethasone (DEX). Cell proliferation and migration were assessed by XTT and scratch wound healing assays. In vivo, ten Lewis rats were treated weekly with ZOL and DEX for 11 weeks. After a week, EPCs or EGM-2 were injected to the gingiva around the molars. At 3 weeks, bilateral molars were extracted. After 8 weeks, wound healing was assessed, and serum VEGF and blood vessels were quantified. RESULTS: ZOL/DEX significantly reduced fibroblasts proliferation and wound healing. Treatment with EPC-CM before ZOL/DEX improved cell proliferation, and scratch healing (p = .007, p = .023). In vivo, local EPC injection before tooth extraction increased serum VEGF (p = .01) and soft tissue vascularization (p = .05). Normal healing was similar (80%) in EPCs and EGM-2 groups. CONCLUSION: EPC rescued fibroblasts from the cytotoxic effect of ZOL/DEX and elevated serum VEGF and vessel density that might reduce MRONJ occurrence to 20% compared to 50% in a similar model.

Endothelial Progenitor Cells inhibit jaw osteonecrosis in a rat model: A major adverse effect of bisphosphonate therapy.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious adverse effect of antiresorptive and antiangiogenic therapies. MRONJ is identified by chronic wounds in the oral mucosa associated with exposed necrotic bone. We hypothesized that zoledronic acid (ZOL) impairs keratinocyte and fibroblast function and reduces soft tissue vascularization; therefore, treating MRONJ with proangiogenic cells may benefit MRONJ patients. The effect of ZOL and dexamethasone (DEX) on gingival fibroblasts and keratinocytes was investigated. In-vitro, ZOL inhibited fibroblast and keratinocyte proliferation, delaying scratch healing. In-vivo, exposed bone was detected at tooth extraction sites, mainly in ZOL(+)/DEX(+) rats; and was associated with significantly decreased soft tissue vascularization, serum-VEGF, and tissue-VEGF. Local injection of early and late endothelial progenitor cells (EPCs) healed 13 of 14 MRONJ lesions compared with 2/7 lesions in the mesenchymal stem cells, and 2/6, in culture-medium group. The EPCs reduced necrotic bone area, increased serum and tissue VEGF levels. EPCs engraftment was minimal, suggesting their paracrine role in MRONJ healing. The EPC-conditioned medium improved scratch healing of keratinocytes and fibroblasts via VEGF pathway and elevated mRNA of VEGFA and collagen1A1. In conclusion, a novel MRONJ treatment with EPCs, increased vascularization and improved epithelial and fibroblast functions as well as cured the lesion.

Predicting Angiogenesis by Endothelial Progenitor Cells Relying on In-Vitro Function Assays and VEGFR-2 Expression Levels.

Clinical trials have demonstrated the safety and efficacy of autologous endothelial progenitor cell (EPC) therapy in various diseases. Since EPCs' functions are influenced by genetic, systemic and environmental factors, the therapeutic potential of each individual EPCs is unknown and may affect treatment outcome. Therefore, our aim was to compare EPCs function among healthy donors in order to predict blood vessel formation (angiogenesis) before autologous EPC transplantation. Human EPCs were isolated from the blood of ten volunteers. EPCs proliferation rate, chemoattractant ability, and CXCR4 mRNA levels were different among donors (p < 0.0001, p < 0.01, p < 0.001, respectively). A positive correlation was found between SDF-1, CXCR4, and EPCs proliferation (R = 0.736, p < 0.05 and R = 0.8, p < 0.01, respectively). In-vivo, blood vessels were counted ten days after EPCs transplantation in a subcutaneous mouse model. Mean vessel density was different among donors (p = 0.0001); nevertheless, donors with the lowest vessel densities were higher compared to control (p < 0.05). Finally, using a linear regression model, a mathematical equation was generated to predict blood vessel density relying on: (i) EPCs chemoattractivity, and (ii) VEGFR-2 mRNA levels. Results reveal differences in EPCs functions among healthy individuals, emphasizing the need for a potency assay to pave the way for standardized research and clinical use of human EPCs.

Use of transforming growth factor-ß loaded onto ß-tricalcium phosphate scaffold in a bone regeneration rat calvaria model.

BACKGROUND: Transforming growth factor-ß (TGF-ß1 ) enhances mesenchymal stem cell (MSC) differentiation into osteoblasts. PURPOSE: The aim of the study was to assess whether TGF-ß1 loaded onto ß-tricalcium phosphate (ß-TCP) synthetic scaffold enhances bone regeneration in a rat calvaria model. The release kinetics of TGF-ß1 from ß-TCP scaffold was evaluated in vitro. MATERIALS AND METHODS: TGF-ß1 in various concentrations (1-40 ng/mL) was loaded onto the ß-TCP scaffold, and release kinetics was monitored by ELISA. The effect of TGF-ß1 on the proliferation of MSCs was assessed using AlamarBlue, and MSC differentiation was evaluated by Alizarin Red quantification assay.Bone augmentation following transplantation of TGF-ß1 loaded onto ß-TCP in a rat calvaria model was evaluated in vivo. RESULTS: Greater TGF-ß1 release from the 40 ng/mL concentration was found. A suppressive effect of TGF-ß on the MSCs proliferation was observed with maximum inhibition obtained with 40 ng/mL compared to the control group (P = .028). A positive effect on MSCs osteogenic differentiation was found.Bone height and bone area fraction in vivo were similar with or without TGF-ß1 ; however, blood vessel density and degradation of the scaffold were significantly higher in the TGF-ß1 group. CONCLUSION: TGF-ß1 adsorbed to ß-TCP stimulated angiogenesis and scaffold degradation that may enhance bone formation.

Safety profile and long-term engraftment of human CD31+ blood progenitors in bone tissue engineering.

BACKGROUND: Endothelial progenitor cells (EPCs) participate in angiogenesis and induce favorable micro-environments for tissue regeneration. The efficacy of EPCs in regenerative medicine is extensively studied; however, their safety profile remains unknown. Therefore, our aims were to evaluate the safety profile of human peripheral blood-derived EPCs (hEPCs) and to assess the long-term efficacy of hEPCs in bone tissue engineering. METHODS: hEPCs were isolated from peripheral blood, cultured and characterized. ß tricalcium phosphate scaffold (ßTCP, control) or 106 hEPCs loaded onto ßTCP were transplanted in a nude rat calvaria model. New bone formation and blood vessel density were analyzed using histomorphometry and micro-computed tomography (CT). Safety of hEPCs using karyotype analysis, tumorigenecity and biodistribution to target organs was evaluated. RESULTS: On the cellular level, hEPCs retained their karyotype during cell expansion (seven passages). Five months following local hEPC transplantation, on the tissue and organ level, no inflammatory reaction or dysplastic change was evident at the transplanted site or in distant organs. Direct engraftment was evident as CD31 human antigens were detected lining vessel walls in the transplanted site. In distant organs human antigens were absent, negating biodistribution. Bone area fraction and bone height were doubled by hEPC transplantation without affecting mineral density and bone architecture. Additionally, local transplantation of hEPCs increased blood vessel density by nine-fold. CONCLUSIONS: Local transplantation of hEPCs showed a positive safety profile. Furthermore, enhanced angiogenesis and osteogenesis without mineral density change was found. These results bring us one step closer to first-in-human trials using hEPCs for bone regeneration.

VEGF release from a polymeric nanofiber scaffold for improved angiogenesis.

Angiogenesis plays a pivotal role in tissue engineering and regenerative medicine. This study aimed to develop an electrospun fiber scaffold that supports release of recombinant human vascular endothelial growth factor (rhVEGF) to enhance angiogenesis. Scaffolds composed of core-shell fibers were fabricated using co-electrospinning. The core solution was composed of polyethylene oxide and mixed with rhVEGF. The shell solution was composed of polycarpolactone, with 0.25, 1, and 3% of polyethylene glycol (PEG) to manipulate pore size on the shell. Pore size and density increased with higher PEG concentrations. Similarly, rhVEGF release was affected by PEG concentration: initial burst release was found in all scaffolds, followed by continuous 4 h release in 3% PEG and 18 h release in the 0.25 and 1% PEG polymeric scaffolds. Endothelial cell migration toward rhVEGF-incorporated polymeric scaffold was 80-fold higher as compared to VEGF-free polymeric scaffold. In a subcutaneous mouse model, VEGF-incorporated polymeric scaffold stimulated cell migration into the scaffold within three days and significantly enhanced blood vessels formation within 14 days, whereas control scaffolds contained few vessels. In conclusion, the described novel scaffold represents a promising device for vascular tissue engineering, which may be of clinical significance in treating vascular deficient wounds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2712-2721, 2017.

Intraoperative Measurement of the Distance from the Bottom of Osteotomy to the Mandibular Canal Using a Novel Ultrasonic Device.

BACKGROUND: In our previous study, we found that a novel ultrasound (US) device may serve as a useful intraoperative tool to measure the distance from osteotomy to the inferior alveolar canal (IAC). PURPOSE: To validate our previous results in a larger group of osteotomies in the posterior mandible. METHODS: During dental implant placement surgery, osteotomies were created using a standardized 2-mm-diameter pilot drill. The distance from the bottom of the osteotome to the IAC was assessed using an ultrasonic device and compared with a standard panoramic radiograph used to measure the same residual distance. The total distance from the crestal bone to the IAC was measured on a preoperative computed tomography (CT) and compared with total US measurements by summing the drill depth with residual depth measurements. RESULTS: Mean radiographic and US residual distances were 5.19 ± 1.95 mm, 5.01 ± 1.82 mm, p = 0.79 respectively. These measurements presented strong positive correlations (r = 0.61, p = .01). Mean total CT distance was 13.48 ± 2.66 mm; mean total US calculation was 13.69 ± 2.51 mm. No significant difference was found (p > .05). CONCLUSIONS: The results support our previous pilot study and confirm that the tested US device identifies the IAC and measures the distance from the osteotomy to the roof of the mandibular canal.

Periodontal Conditions and Tumor Necrosis Factor-Alpha Level in Gingival Crevicular Fluid of Scleroderma Patients.

BACKGROUND: Systemic sclerosis (SSc) is a chronic disease with prominent vasculopathy, inflammation, production of autoantibodies, and tissue fibrosis. Periodontitis is a chronic inflammatory oral condition manifesting as microbial infection, inflammation and destruction of the alveolar bone. In both conditions tumor necrosis factor-alpha (TNFα) and other proinflammatory cytokines play an important role in pathogenesis. OBJECTIVES: To assess the periodontal status in SSc patients and compare these parameters to TNFα level in gingival crevicular fluid (GCF) of SSc patients and healthy controls. METHODS: Twenty SSc patients and 20 controls underwent periodontal examination, including probing depth (PD), plaque index (PI), gingival-index (GI), bleeding on probing (BOP), and measurement of TNFα levels in collected GCF. RESULTS: SSc patients had a greater PD (3.74 ± 0.32 mm vs. 3.35 ± 0.31 mm, P > 0.003), GI (1.53 ± 0.34 vs. 1.12 ± 0.54, P > 0.049), and non-significantly higher BOP than controls. TNFα levels in GCF were higher in SSc patients (1.63 ± 0.36 vs. 1.15 ± 0.34 pg/ml, P = 0.001). Periodontitis parameters correlated with several SSc variables; PI in particular was higher in patients with longer disease duration, sclerodactyly, more severe skin involvement, and SSc activity score. CONCLUSIONS: Patients with SSc have higher indices of periodontal inflammation and higher TNFα level in GCF than did healthy individuals. These changes probably reflect the complexity of factors that influence oral health in SSc. Common pathologic pathways may be responsible for the association between SSc and periodontitis, which requires further study.

The prevalence of human herpes viruses in the saliva of chronic periodontitis patients compared to oral health providers and healthy controls.

The causative agents in periodontal disease are periopathogenic bacteria; however, viruses have been implicated. The aim of this study was to examine the prevalence of different HHVs in the saliva of chronic periodontitis patients and to compare it to two groups of healthy controls. Three groups were included: chronic periodontitis patients (CP), periodontally healthy patients (NP) and oral health providers with a healthy periodontium (NPOHP). For each subject, 1 ml of unstimulated whole saliva was collected and mixed with 2 ml lysis buffer. HHVs assays were performed using real-time PCR. Fifteen percent of the subjects in the CP group tested positive for CMV compared to none in the NP and NPOHP groups (p = 0.04). Recurrent herpes was more frequent in females (51.7 %) than in males (33.3 %), and this was statistically significant (p = 0.038). The higher prevalence of CMV in the unstimulated saliva of CP patients suggests that CMV may play a role in the pathogenesis of chronic periodontitis.

Periodontal condition of patients with autoimmune diseases and the effect of anti-tumor necrosis factor-α therapy.

BACKGROUND: The aim of this study is to evaluate the effect of autoimmune diseases (AIs), as well as anti-tumor necrosis factor-α (TNF-α) therapy on the clinical and immunologic parameters of the periodontium. METHODS: Thirty-six AI patients (12 rheumatoid arthritis [RA], 12 psoriatic arthritis, and 12 systemic sclerosis) were recruited together with 12 healthy (H) and 10 RA patients receiving anti-TNF-α therapy (RA+). Periodontal indices including plaque index, gingival index (GI), probing depth (PD), and bleeding on probing (BOP) were measured, and gingival crevicular fluid (GCF) was collected from five deepest pockets using papers strips. The TNF-α level was analyzed using enzyme-linked immunosorbent assay. Analysis of variance test was used for statistical comparison between groups, whereas Pearson linear correlation coefficient test was used to examine the association between TNF-α and periodontal status indices. RESULTS: The three AI subgroups were very similar in clinical and immunologic parameters. GI was greater in the AI patients compared to the H and RA+ groups (1.91 ± 0.54, 1.21 ± 0.67, and 1.45 ± 0.30, respectively, P = 0.0005). AI patients exhibited significantly more BOP than H and RA+ (46.45% ± 17.08%, 30.08% ± 16.86%, and 21.13% ± 9.51%, respectively, P = 0.0002). PD in H and RA+ groups were lower than in the AI (3.47 ± 0.33, 3.22 ± 0.41, and 3.91 ± 0.49 mm, P = 0.0001). Number of sites with PD >4 mm was higher in AI patients compared to H and RA+ (42.44 ± 17.5 versus 24.33 ± 15.62 versus 33.3 ± 6.6, P = 0.0002). GCF TNF-α was higher among the AI patients (1.67 ± 0.58 ng/site) compared to 1.07 ± 0.33 ng/site for the H group and 0.97 ± 0.52 ng/site for the RA+ group (P = 0.0002). A significant positive correlation was found between PD and TNF-α levels in the GCF (r = 0.4672, P = 0.0002), BOP (r = 0.7491, P = 0.0001), and GI (r = 0.5420, P = 0.0001). CONCLUSIONS: Patients with AI diseases have higher periodontal indices and higher TNF-α levels in GCF than H controls. Anti-TNF-α treatment appears to reverse this phenomenon.

Treatment of peri-implantitis using multiple applications of chlorhexidine chips: a double-blind, randomized multi-centre clinical trial.

BACKGROUND: Universal strategies for managing peri-implantitis are yet to be adopted. The aim of this study is to examine a protocol of intensive application of chlorhexidine containing chips in sites with peri-implantitis. MATERIALS AND METHODS: This multi-centre, randomized, double-blind, parallel, two-arm clinical trial included 60 patients (77 implants) with probing depth (PD) 6-10 mm and bone loss ≥2 mm around 1-2 implants. One to two weeks following SRP, baseline measurements were made followed by implants' debridement. Patients were randomized to receive matrix chips (MatrixC) or chlorhexidine Chips (PerioC). Measurements and chips placement were repeated at weeks 2, 4, 6, 8, 12 and 18. At 6 months, patients returned for final examination. RESULTS: Probing depth reduction was greater in the PerioC (2.19 ± 0.24 mm) compared with MatrixC (1.59 ± 0.23 mm), p = 0.07. Seventy percentage of the implants in the PerioC and 54% in the MatrixC had PD reduction ≥ 2 mm. Likewise, 40% of the sites (PerioC) and 24% (MatrixC) had PD reduction ≥ 3 mm. Clinical attachment level gains for both groups were significant; however, the changes in the PerioC group were significantly greater than in MatrixC [2.21 ± 0.23 mm. and 1.56 ± 0.25 mm respectively, p = 0.05]. Bleeding on probing was reduced by half in both groups. CONCLUSION: Frequent placement of PerioC and MatrixC together with implants debridement resulted in a substantial improvement in sites with peri-implantitis. Further studies will be required to fully appreciate the mechanism of this treatment.