Jose Viña-Almunia

Activation of p38, p21, and NRF-2 Mediates Decreased Proliferation of Human Dental Pulp Stem Cells Cultured under 21% O2

Summary High rates of stem cell proliferation are important in regenerative medicine and in stem cell banking for clinical use. Ambient oxygen tensions (21% O2) are normally used for in vitro culture, but physiological levels in vivo range between 3% and 6% O2. We compared proliferation of human dental pulp stem cells (hDPSCs) cultured under 21% versus 3% O2. The rate of hDPSC proliferation is significantly lower at 21% O2 compared to physiological oxygen levels due to enhanced oxidative stress. Under 21% O2, increased p38 phosphorylation led to activation of p21. Increased generation of reactive oxygen species and p21 led to activation of the NRF-2 signaling pathway. The upregulation of NRF-2 antioxidant defense genes under 21% O2 may interact with cell-cycle-related proteins involved in regulating cell proliferation. Activation of p38/p21/NRF-2 in hDPSCs cultured under ambient oxygen tension inhibits stem cell proliferation and upregulates NRF-2 antioxidant defenses.

Role of p16INK4a and BMI-1 in oxidative stress-induced premature senescence in human dental pulp stem cells

Human dental pulp stem cells (hDPSCs) are a source for cell therapy. Before implantation, an in vitro expansion step is necessary, with the inconvenience that hDPSCs undergo senescence following a certain number of passages, loosing their stemness properties. Long-term in vitro culture of hDPSCs at 21% (ambient oxygen tension) compared with 3–6% oxygen tension (physiological oxygen tension) caused an oxidative stress-related premature senescence, as evidenced by increased β-galactosidase activity and increased lysil oxidase expression, which is mediated by p16INK4a pathway. Furthermore, hDPSCs cultured at 21% oxygen tension underwent a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16INK4a and BMI-1 might play a role in the oxidative stress-associated premature senescence. We show that it is important for clinical applications to culture cells at physiological pO2 to retain their stemness characteristics and to delay senescence.

Influence of occlusal loading on peri-implant clinical parameters. A pilot study

Objectives: To investigate the relation between occlusal loading and peri-implant clinical parameters (probing depth, bleeding on probing, gingival retraction, width of keratinized mucosa, and crevicular fluid volume) in patients with implant-supported complete fixed prostheses in both arches. Material and Methods: This clinical study took place at the University of Valencia (Spain) dental clinic. It included patients attending the clinic for regular check-ups during at least 12 months after rehabilitation of both arches with implant-supported complete fixed ceramo-metallic prostheses. One study implant and one control implant were established for each patient using the T-Scan®III computerized system (Tesco, South Boston, USA). The maxillary implant closest to the point of maximum occlusal loading was taken as the study implant and the farthest (with least loading) as the control. Occlusal forces were registered with the T-Scan® III and then occlusal adjustment was performed to distribute occlusal forces correctly. Peri-implant clinical parameters were analyzed in both implants before and two and twelve months after occlusal adjustment. Results: Before occlusal adjustment, study group implants presented a higher mean volume of crevicular fluid (51.3±7.4 UP) than the control group (25.8±5.5 UP), with statistically significant difference. Two months after occlusal adjustment, there were no significant differences between groups (24.6±3.8 UP and 26±4.5 UP respectively) (p=0.977). After twelve months, no significant differences were found between groups (24.4±11.1 UP and 22.5±8.9 UP respectively) (p=0.323). For the other clinical parameters, no significant differences were identified between study and control implants at any of the study times (p>0.05). Conclusions: Study group implants receiving higher occlusal loading presented significantly higher volumes of crevicular fluid than control implants. Crevicular fluid volumes were similar in both groups two and twelve months after occlusal adjustment. Key words:Occlusal loading, crevicular fluid, peri-implant clinical parameters, T-Scan®.

Survival of implants placed with the osteotome technique: An update

A literature review is made to analyze the survival of implants placed with the osteotome technique. A PubMed search was made based on the key words “osteotome AND dental implants”, corresponding to publications between 1998-2008. The inclusion criteria were: a) a minimum of 10 patients; b) a minimum follow-up of 6 months; c) implants placed using the osteotome technique with or without indirect sinus lift; and d) specification of the implant number and survival rate. Sixty-four articles were identified, of which 20 met the inclusion criteria. A total of 2006 implants were placed in 1312 patients using the osteotome technique. The duration of follow-up after prosthetic loading ranged from 6-144 months. Indirect sinus lift was carried out in all but one of the studies. The residual crest height ranged from 2.3-11.7 mm. with a mean gain in bone after sinus lift of 2.5-5.5 mm. The time from implant placement to prosthetic loading varied from 1.5-9 months. The percentage implant survival rate was 90.5-100%. The survival rate of implants placed with the osteotome technique is high and does not differ with respect to implant placement with the conventional technique. Key words:Osteotomes, dental implants, indirect sinus lift.

Influence of Partial O₂ Pressure on the Adhesion, Proliferation, and Osteogenic Differentiation of Human Dental Pulp Stem Cells on β-Tricalcium Phosphate Scaffold

PURPOSE To analyze, in vitro, the influence of O₂ pressure on the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (DPSC) on β-tricalcium phosphate (β-TCP) scaffold. MATERIALS AND METHODS DPSC, positive for the molecular markers CD133, Oct4, Nestin, Stro-1, and CD34, and negative for CD45, were isolated from extracted third molars. Experiments were started by seeding 200,000 cells on β-TCP cultured under 3% or 21% O₂ pressure. No osteogenic medium was used. Eight different cultures were performed at each time point under each O₂ pressure condition. Cell adhesion, proliferation, and differentiation over the biomaterial were evaluated at 7, 13, 18, and 23 days of culture. Cell adhesion was determined by light microscopy, proliferation by DNA quantification, and osteogenic differentiation by alkaline phosphatase (ALP) activity analysis. RESULTS DPSC adhered to β-TCP with both O₂ conditions. Cell proliferation was found from day 7 of culture. Higher values were recorded at 3% O₂ in each time point. Statistically significant differences were recorded at 23 days of culture (P = .033). ALP activity was not detectable at 7 days. There was, however, an increase in ALP activity over time in both groups. At 13, 18, and 23 days of culture, higher ALP activity was recorded under 3% O₂ pressure. Statistical differences were found at day 23 (P = .014). CONCLUSION DPSC display capacity of adhering to β-TCP under 3% or 21% O₂ pressure conditions. Cell proliferation on β-TCP phosphate is significantly higher at 3% than at 21% O₂ pressure, the most frequently used O₂ tension. β-TCP can itself promote osteogenic differentiation of DPSC and is enhanced under 3% O₂ compared with 21%.

Soft Tissue Response in Posterior Teeth Adjacent to Interdental Single Implants: A Controlled Randomized Clinical Trial Comparing Intrasulcular vs Trapezoidal Incision.

PURPOSE To evaluate the soft tissue response in posterior teeth adjacent to interdental single implants comparing intrasulcular and trapezoidal incision, and to study their evolution over time. MATERIALS AND METHODS A controlled randomized clinical trial was carried out in the Oral Surgery and Implantology Unit of a University Clinic. All the included patients received an interdental single implant (Frontier 2.45, Ilerimplant; Global Medical Implants). The incision type was randomized by sealed envelopes into two groups using the SPSS statistical package (SPSS): (1) intrasulcular or (2) trapezoidal incision. Probing depth and gingival recession at the mesial and distal teeth adjacent to the implant were measured before implant placement, 1 month after surgery, the day of the abutment connection, and at 6 months and 1 year postloading. Scar formation and papilla index were measured 1 month after surgery, and at 6 months and 1 year postloading. RESULTS Forty patients with one implant per patient were included: 20 in the intrasulcular and 20 in the trapezoidal group. No statistical differences were found between incision types in the measured parameters (probing depth, recession, and interproximal papilla). When analyzing periodontal changes of the total sample, significant differences were found between implant placement and the 1-year follow-up in recession, scar formation, and papilla index. CONCLUSION The incision type used to place a single interdental implant did not significantly influence the periodontal parameters of the adjacent teeth. Considering the whole sample, the values between implant placement and 1 year postloading showed significant differences in recession, scar formation, and papilla index over time.

Buccal bone crest dynamics after immediate implant placement and ridge preservation techniques: review of morphometric studies in animals.

Purpose:To review morphometric studies performed in animals assessing the dynamics of the buccal bone crest after immediate implant placement and ridge preservation techniques. Material and Method:A bibliographic search in PubMed was performed. Studies that analyzed morphometrically in animals the buccal bone crest dynamics after immediate implant placement or ridge preservation techniques were included. Twenty-five studies met the inclusion criteria. Results:Immediate implant placement does not prevent the resorption of the buccal bone crest. To minimize this resorption, 2 mm width of the buccal bone crest, palatal/lingual implant placement, and an adequate implant diameter for the width of the ridge are required. The regeneration of the gap after immediate implant placement limits the resorption of the buccal bone crest. Flap elevation and implant surface showed no relation with this resorption. Ridge preservation techniques associated with mucogingival surgery minimize buccal bone crest resorption. Biomaterials are more effective than autograft. Conclusions:Immediate implant placement does not prevent the resorption of the buccal bone crest after dental extraction. Ridge preservation techniques minimize this resorption.

Clinical parameters of implants placed in healed sites using flapped and flapless techniques: A systematic review

Background Dental implant placement using flapless surgery is a minimally invasive technique that improves blood supply compared with flapped surgery. However, the flapless technique does not provide access to allow bone regeneration. Objectives The aim of this systematic review was to evaluate the clinical parameters following implant surgery in healed sites, using two procedures: flapped vs. flapless surgery. Material and Methods A detailed electronic search was carried out in the PubMed/Medline, Embase and Cochrane Library databases. The focused question was, “How do flapped and flapless surgical techniques affect the clinical parameters of dental implants placed in healed sites?”. All the studies included with a prospective controlled design were considered separately, depending on whether they had been conducted on animals or humans. The following data were recorded in all the included studies: number of implants, failures, location (maxilla, mandible), type of rehabilitation (partial or single), follow-up and flap design. The variables selected for comparison in the animal studies were the following: flap design, gingival index, mucosal height, recession and probing pocket depth. In humans studies the variables were as follows: flap design, plaque index, gingival index, recession, probing pocket depth, papilla index and keratinized gingiva. Results Ten studies were included, six were experimental studies and four were clinical studies. Studies in animals showed better results using the flapless technique in the parameters analyzed. There is no consensus in the clinical parameters analyzed in human studies, but there is a trend to better results using flapless approach. Conclusions The animal studies included in the present review show that implants placed in healed sites with a flapless approach have better clinical parameters than the flapped procedure in a short-term follow-up. In human studies, there is no consensus about which technique offer better results in terms of clinical parameters. Therefore, more research in humans is required in order to overcome the limitations and contrast these results. Key words: Clinical parameters, gingival recession, probing depth, dental implants, flap, flapless.

Influence of different types of pulp treatment during isolation in the obtention of human dental pulp stem cells.

Background Different methods have been used in order to isolate dental pulp stem cells. The aim of this study was to study the effect of different types of pulp treatment during isolation, under 3% O2 conditions, in the time needed and the efficacy for obtaining dental pulp stem cells. Material and Methods One hundred and twenty dental pulps were used to isolate dental pulp stem cells treating the pulp tissue during isolation using 9 different methods, using digestive, disgregation, or mechanical agents, or combining them. The cells were positive for CD133, Oct4, Nestin, Stro-1, CD34 markers, and negative for the hematopoietic cell marker CD-45, thus confirming the presence of mesenchymal stem cells. The efficacy of dental pulp stem cells obtention and the minimum time needed to obtain such cells comparing the 9 different methods was analyzed. Results Dental pulp stem cells were obtained from 97 of the 120 pulps used in the study, i.e. 80.8% of the cases. They were obtained with all the methods used except with mechanical fragmentation of the pulp, where no enzymatic digestion was performed. The minimum time needed to isolate dental pulp stem cells was 8 hours, digesting with 2mg/ml EDTA for 10 minutes, 4mg/ml of type I collagenase, 4mg/ml of type II dispase for 40 minutes, 13ng/ml of thermolysine for 40 minutes and sonicating the culture for one minute. Conclusions Dental pulp stem cells were obtained in 97 cases from a series of 120 pulps. The time for obtaining dental pulp stem cells was reduced maximally, without compromising the obtention of the cells, by combining digestive, disgregation, and mechanical agents. Key words:Dental pulp stem cells, mesenchymal stem cells, isolation method.