Dental implant surface temperatures following double wavelength (2780/940 nm) laser irradiation in vitro

Objective: To estimate the implant surface temperature at titanium dental implants during calibrated irradiation using double wavelength laser. Material and methods: A double wavelength laser, 2780 nm Er,Cr:YSGG and 940 nm diode, was calibrated and used to irradiate pristine titanium dental implants, OsseoSpeed, TiUnite and Roxolid SLActive, representing different surface modifications. Initial calibration (21 implants; 7 implants/group) intended to identify optimal wavelength/specific output power/energy that not critically increased the temperature or altered the micro-texture of the implant surface. Subsequent experimental study (30 implants; 10 implants/group) evaluated implant surface temperature changes over 190 s. Irradiation using a computerized robotic setup. Results: Based on the initial calibration, the following output powers/energies were employed: Er,Cr:YSGG laser 18.4 mJ/pulse (7.3 J/cm2)–36.2 mJ/pulse (14.4 J/cm2) depending on implant surface; diode laser 3.3 W (1321.0 W/cm2). During double wavelength irradiation, implant surface temperatures dropped over the first 20 s from baseline 37°C to mean temperatures ranging between 25.7 and 26.3°C. Differences in mean temperatures between OsseoSpeed and TiUnite implants were statistically significant (p < 0.001). After the initial 20 s, mean temperatures continued to decrease for all implant surfaces. The decrease was significantly greater for TiUnite and Roxolid SLActive compared with OsseoSpeed implants (p < 0.001). Conclusion: Calibrated double wavelength laser irradiation did not critically influence the implant surface temperature. During laser irradiation the temperature decreased rapidly to steady-state levels, close to the water/air-spray temperature.publishedVersio

Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells

To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM) was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering

Mesenchymal stem cells induce endothelial cell quiescence and promote capillary formation

Introduction: Rapid establishment of functional blood vessels is a prerequisite for successful tissue engineering. During vascular development, endothelial cells (ECs) and perivascular cells assemble into a complex regulating proliferation of ECs, vessel diameter and production of extracellular matrix proteins. The aim of this study was to evaluate the ability of mesenchymal stem cells (MSCs) to establish an endothelial-perivascular complex in tissue-engineered constructs comprising ECs and MSCs. Methods: Primary human ECs and MSCs were seeded onto poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) scaffolds and grown in dynamic culture before subcutaneous implantation in immunocompromised mice for 1 and 3 weeks. Cellular activity, angiogenic stimulation and vascular assembly in cell/scaffold constructs seeded with ECs or ECs/MSCs in a 5:1 ratio was monitored with real-time RT-PCR, ELISA and immunohistochemical microscopy analysis. Results: A quiescent phenotype of ECs was generated, by adding MSCs to the culture system. Decreased proliferation of ECs, in addition to up-regulation of selected markers for vascular maturation was demonstrated. Baseline expression of VEGFa was higher for MSCs compared with EC (P <0.001), with subsequent up-regulated VEGFa-expression for EC/MSC constructs before (P <0.05) and after implantation (P <0.01). Furthermore, an inflammatory response with CD11b + cells was generated from implantation of human cells. At the end of the 3 week experimental period, a higher vascular density was shown for both cellular constructs compared with empty control scaffolds (P <0.01), with the highest density of capillaries being generated in constructs comprising both ECs and MSCs. Conclusions: Induction of a quiescent phenotype of ECs associated with vascular maturation can be achieved by co-seeding with MSCs. Hence, MSCs can be appropriate perivascular cells for tissue-engineered constructs

The effect of smoking on inflammatory and bone remodeling markers in gingival crevicular fluid and subgingival microbiota following periodontal therapy

Background and Objective: Periodontal health is mediated by suppressing microorganisms inducing a local inflammatory host response. Smoking may impair this process. This study compares gingival crevicular fluid levels of inflammatory and bone remodeling markers in heavy smokers and non-smokers following active and supportive periodontal therapy in patients with chronic periodontitis. Material and Methods: Gingival crevicular fluid and subgingival plaque were collected from the deepest periodontal pocket in 50 patients, 25 smokers and 25 non-smokers, at baseline (T0), following active (T1) and 12 mo of supportive periodontal therapy (T2). Smoking status was validated measuring serum cotinine levels. Gingival crevicular fluid levels of 27 inflammatory and two bone remodeling markers were analyzed using multiplex and singleplex micro-bed immunoassays, and subgingival plaque samples using checkerboard DNA–DNA hybridization. Amounts of markers in smokers and non-smokers were compared calculating the effect size. Results: Expression of inflammatory and bone-remodeling markers in smokers demonstrated an overall reduced effect size at T0 and T2 (p < 0.001). In particular, proinflammatory markers (p < 0.001), chemokines (p = 0.007) and growth factors (p = 0.003) at T0, osteoprotegerin (p = 0.003) at T1, proinflammatory markers (p = 0.019) and chemokines (p = 0.005) at T2. At T2, interleukin-8 was detected in significantly higher levels in smokers. Ten different markers in non-smokers and none in smokers responded to periodontal therapy (p < 0.05). An overall negative association was revealed between smoking and subgroups of markers at sites presenting ≥ 105 red complex periodontal microbial species. Conclusion: Except for an upregulation of interleukin-8, smokers exhibited reduced gingival crevicular fluid levels of several inflammatory markers at baseline and following active and supportive periodontal therapy. Only inflammatory responses in non-smokers adapted to periodontal therapy. Apparently, there seems to be an immunosuppressant effect of smoking regulating the local inflammatory response and bone remodeling markers captured in gingival crevicular fluid following periodontal therapy

Serum biomarker levels in smokers and non-smokers following periodontal therapy. A prospective cohort study

Abstract Background To compare presence and levels of serum cytokines in smokers and non-smokers with periodontitis following periodontal therapy. Methods Thirty heavy smokers and 30 non-smokers with stage III or IV periodontitis were included in this prospective cohort study. Clinical data and blood serum were collected at baseline (T0), after step I-III (T1), and after 12 months step IV periodontal therapy (T2). Cytokine IL-1β, IL-6, IL-8, TNF-α, IL-10, and IP-10 levels were measured using multiplex kit Bio-Plex Human Pro™ Assay. Linear regression models with cluster robust variance estimates to adjust for repeated observations were used to test intra- and intergroup levels for each marker, IL-6 and IL-8 defined as primary outcomes. Results Clinical outcomes improved in both groups following therapy (p < 0.05). IL-6 levels increased with 75.0% from T0-T2 among smokers (p = 0.004). No significant intra- or intergroup differences were observed for IL-8. Higher levels of TNF-α (44.1%) and IL-10 (50.6%) were detected in smokers compared with non-smokers at T1 (p = 0.007 and p = 0.037, respectively). From T1-T2, differences in mean change over time for levels of TNF-α and IL-10 were observed in smokers compared with non-smokers (p = 0.005 and p = 0.008, respectively). Conclusion Upregulated levels of serum cytokines in smokers indicate a systemic effect of smoking following periodontal therapy. Differences in cytokine levels between smokers and non-smokers demonstrate a smoking induced modulation of specific systemic immunological responses in patients with severe periodontitis

Non-surgical treatment of peri-implantitis with and without erythritol air-polishing a 12-month randomized controlled trial

Abstract Background A variety of interventions have been explored in the non-surgical management of peri-implantitis. In spite of extensive testing of various study protocols, effective treatments largely remain unavailable. The objective of the present 12-month single-centre, examiner-masked, randomized controlled clinical trial was to explore whether a low-abrasive erythritol air-polishing system produces added clinical benefit when used adjunctive to conventional non-surgical management of peri-implantitis and to record any associated patient-centered outcomes. Methods Forty-three patients with mild to severe peri-implantitis including at least one implant either received ultrasonic/curette subgingival instrumentation and erythritol air-polishing (test) or ultrasonic/curette instrumentation only (control) at baseline and at 3, 6, 9, and 12 months. Probing depth (PD), bleeding on probing (BoP), dental plaque, suppuration (SUP), crestal bone level (CBL), and peri-implant crevicular fluid (PCF) were recorded at baseline, 6 and 12 months. Visual Analogue Scale (VAS) scores were collected immediately following subgingival interventions at all time-points. Results A reduction in PD was observed from baseline to 6 months for the test (p = 0.006) and control (p  0.05). At 6 months, a intergroup difference in PCF was observed in favor of the test (p = 0.042). Moreover, a reduction in SUP from baseline to 6 and 12 months was observed in the test (p = 0.019). Overall, patients in the control group experienced less pain/discomfort compared with the test (p < 0.05), females reporting more pain/discomfort than males (p = 0.005). Conclusions This study confirms that conventional non-surgical management of peri-implantitis produces limited clinical improvement. It is shown that an erythritol air-polishing system may not produce added clinical benefits when used adjunctive to conventional non-surgical management. In other words, neither approach effectively resolved peri-implantitis. Moreover, the erythritol air-polishing system produced added pain/discomfort particularly in female patients. Trial registration The clinical trial was prospectively registered in ClinicalTrials.gov with registration NCT04152668 (05/11/2019)

Cell seeding density is a critical determinant for copolymer scaffolds-induced bone regeneration

Constructs intended for bone tissue engineering (TE) are influenced by the initial cell seeding density. Therefore, the objective of this study was to determine the effect of bone marrow stromal stem cells (BMSCs) density loaded onto copolymer scaffolds on bone regeneration. BMSCs were harvested from rat's bone marrow and cultured in media with or without osteogenic supplements. Cells were seeded onto poly(l-lactide-co-ε-caprolactone) [poly(LLA-co-CL)] scaffolds at two different densities: low density (1 × 106 cells/scaffold) or high density (2 × 106 cells/scaffold) using spinner modified flasks and examined after 1 and 3 weeks. Initial attachment and spread of BMSC onto the scaffolds was recorded by scanning electron microscopy. Cell proliferation was assessed by DNA quantification and cell differentiation by quantitative real-time reverse transcriptase-polymerized chain reaction analysis (qRT-PCR). Five-millimeter rat calvarial defects (24 defects in 12 rats) were implanted with scaffolds seeded with either low or high density expanded with or without osteogenic supplements. Osteogenic supplements significantly increased cell proliferation (p < 0.001). Scaffolds seeded at high cell density exhibited higher mRNA expressions of Runx2 p = 0.001, Col1 p = 0.001, BMP2 p < 0.001, BSP p < 0.001, and OC p = 0.013. More bone was formed in response to high cell seeding density (p = 0.023) and high seeding density with osteogenic medium (p = 0.038). Poly (LLA-co-CL) scaffolds could be appropriate candidates for bone TE. The optimal number of cells to be loaded onto scaffolds is critical for promoting Extracellular matrix synthesis and bone formation. Cell seeding density and osteogenic supplements may have a synergistic effect on the induction of new bone

A 12-month randomized controlled trial evaluating erythritol air-polishing versus curette/ultrasonic debridement of mandibular furcations in supportive periodontal therapy

Background Due to complex morphology and limited access, the cleaning of the furcation area is extremely challenging. Therefore, novel therapeutic approaches need to be tested to potentially overcome debridement limitations. The aim of the present prospective 12-month study was to compare clinical and microbiological effects following erythritol air-polishing versus conventional mechanical debridement of furcation defects in a cohort of periodontal maintenance patients. Methods Twenty patients with grade II mandibular molar furcation defects volunteered to enroll in this single-centre, examiner masked, randomized controlled trial. In a split-mouth study design, two furcation sites in each patient were randomly assigned to either receive subgingival debridement using erythritol air-polishing (test) or conventional ultrasonic/curette debridement (control) at baseline, and at 3, 6, 9 and 12 months. Probing depth, clinical attachment level and bleeding on probing were recorded at 3-month intervals. Subgingival microbiological samples obtained at baseline, 6 and 12 months were analyzed using checkerboard DNA–DNA hybridization. Discomfort from treatment was scored at 12 months using a visual analogue scale. The differences between treatments, and time-points, were tested using multilevel analysis (mixed effect models and robust variance estimates). Results A significant reduction in probing depth took place following both treatments (p < 0.001). Control sites experienced a significant mean gain in clinical attachment level of 0.5 mm (± 0.2) (p = 0.004), whereas a non-significant gain of 0.4 mm (± 0.3) was observed at test sites (p = 0.119). At 6 months, a significant between-treatment difference of 0.8 mm (± 0.4) was observed in favor of the control (p = 0.032). No significant between-treatment differences were observed in microbial load or composition. Notably, at 12 months patients experienced significantly less discomfort following air-polishing compared with control (p = 0.001). Conclusions The 12-month observations indicate that erythritol air-polishing and conventional mechanical debridement both support clinical improvements. A significant between-treatment difference in clinical attachment level was, however, detected in favour of control debridement at 6 months. In terms of patient comfort, erythritol air-polishing is superior. Trial Registration: The clinical trial was retrospectively registered in ClinicalTrial.gov with registration NCT04493398 (07/28/2020)

Site-specific treatment outcome in smokers following 12 months of supportive periodontal therapy

Aim: To evaluate the effect of cigarette smoking on periodontal health at patient, tooth, and site levels following supportive therapy. Materials and Methods: Eighty chronic periodontitis patients, 40 smokers and 40 non-smokers, were recruited to a single-arm clinical trial. Periodontal examinations were performed at baseline (T0), 3 months following active periodontal therapy (T1), and 12 months following supportive periodontal therapy (T2). Smoking status was validated measuring serum cotinine levels. Probing depth (PD) ≥ 5 mm with bleeding on probing (BoP) was defined as the primary outcome. Logistic regression analyses adjusted for clustered observations of patients, teeth, and sites and mixed effects models were employed to analyse the data. Results: All clinical parameters improved from T0 to T2 (p < 0.001), whereas PD, bleeding index (BI), and plaque index (PI) increased from T1 to T2 in smokers and non-smokers (p < 0.001). An overall negative effect of smoking was revealed at T2 (OR = 2.78, CI: 1.49, 5.18, p < 0.001), with the most pronounced effect at maxillary single-rooted teeth (OR = 5.08, CI: 2.01, 12.78, p < 0.001). At the patient level, less variation in treatment outcome was detected within smokers (ICC = 0.137) compared with non-smokers (ICC = 0.051). Conclusion: Smoking has a negative effect on periodontal health following 12 months of supportive therapy, in particular at maxillary single-rooted teeth

Endothelial microvascular networks affect gene-expression profiles and osteogenic potential of tissue-engineered constructs

Introduction: A major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene- expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation. Methods: MSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining. Results: The results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks. Conclusions: This study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs