Hannu Larjava

Background:Leucocyte and platelet rich fibrin (L-PRF) is a membrane containing fibrin matrix with platelets and leucocytes made via blood centrifugation. Molecules released from L-PRF have been shown to regulate wound healing-related gene expression in human gingival fibroblasts. It is not well-understood how the length of time elapsed between blood collection and centrifugation affects the final L-PRF. We hypothesized that greater elapsed time would be associated with smaller L-PRF membranes, containing fewer leucocytes and platelets, and altered expression of gingival fibroblast wound healing-related genes. Methods:Blood samples, collected from 18 healthy individuals, were centrifuged immediately (T-0) or after 1-, 2-, 4-, and 6-minute storage. Each L-PRF was measured. L-PRF from T-0 and T-6 from 11 of the individuals were each incubated 48 hours in cell culture medium at 37°C to create L-PRF “releaseate”. Human gingival fibroblasts were incubated 48 hours with releaseate, followed by RNA isolation and real-time polymerase chain reaction to measure the expression of select genes. Additional pairs of membranes from T-0 and T-6 were used for visualization of leucocyte nuclei for counting. Platelets were additionally identified by immunostaining. Results:Immediate centrifugation (T-0) resulted in the largest membrane for each participant, on average 26% larger than T-6 membranes, with individual variation in membrane mass. The mean mass for the T-0 membranes was 1.805 g (1.252 g to 2.322 g) compared to 1.283 g (0.570 g to 1.858 g) for the T-6 membranes (p
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Background: About 20% of subjects receiving implants develop peri-implantitis (PI) that associates with progressive inflammation and bone loss around implants, often leading to implant failure. PI is caused by bacteria that accumulate in peri-implant space but the consensus on microbial profile is still lacking. Microbial sampling of PI lesions has largely focused on analyzing bacterial species that have been shed from implant surface and captured in the pocket fluid. The purpose of the present study was to investigate the morphotypes of bacteria in microbial ecosystem that covers the implant threads and explore whether different brands of implants favor different morphotypes and whether certain morphotypes were associated with more advanced disease.Methods: The implants (N=14) that were determined to have failed by the clinician were removed and instantly processed for scanning electron microscope analysis. The implants were imaged at three equally divided levels of the exposed area due to diseased bone loss. Bacterial morphotypes [cocci, rods, filaments, spirilla/spirochetes] in each level were further analyzed at higher magnification to enable identification and quantification by three examiners. The different types of surfaces, mobility and years in functions were correlated to the presence of specific morphotypes.Results: Implants removed due to PI demonstrated the presence of variable bacterial morphotypes that did not correlate to disease progression in our preliminary study. Some implants were dominated by filaments and others showed the presence of combinations of cocci and rods or mixed morphotypes of spirilles/spirochetes. Rods and filaments were dominant species throughout the surfaces and cocci showed increased presence towards the apical third compared to coronal and middle thirds. There were significant differences in the morphotypes in the implants with TiUnite and SLA surfaces (except for cocci), with mobility and with more than 10 years of function.Conclusions: The profiles of morphotypes in biofilms of different implants with similar clinical presentation of PI were highly variable and did not clearly associate with implant brand. While there were significant differences between implants, interestingly, similar morphotypes on individual implants were found throughout the entire implant surface.

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Objectives: The prevalence of peri-implantitis and subsequent failure of implants continues to rise. The majority of cases are bacterial in etiology. In peri-implantitis, the subsequent bone loss around dental implants exposes their micro-roughened surfaces to bacterial colonization, further aggravating inflammation, bone loss and progression of disease. The challenge of peri-implantitis treatment is the complete decontamination of roughened implant surfaces once colonized with a biofilm. Many chemical agents are not effective in decontamination of the rough-surface implants with mature biofilms. Therefore, we examined if air-flow with glycine (AFG) would be effective in decontamination of implants with mature subgingival mature biofilms. Methods: SLA® (sand-blasted acid-etched) titanium discs were inoculated with dental plaque and anaerobically incubated at 37°C for 21 days to allow for the formation of a structurally mature oral biofilm on the disc surface. Discs were then separated into different treatment groups: AFG with or without the powder and with or without prior rinsing with 0.9% sterile saline. Control groups included no saline rinse, single rinse and double rinse controls. For assessment of decontamination, discs were imaged, and bacterial cells counted, under a scanning electron microscope (SEM) at 5000X magnification.Results: The no-rinse control group contained multi-species undisrupted biofilm. Saline rinsing removed most of the biofilm except the rough-surface-associated microorganisms. Numerous cocci and some rods remaining on the pits of the SLA® surface. AFG without the powder was not effective in decontamination of the rough SLA® surface. However, AFG with the powder produced practically clean SLA® surfaces. In addition, prior rinsing with saline was not necessary for this effect.Conclusions: AFG appears be an effective method to decontaminate mature biofilm contaminated implant surfaces when used directly on the contaminated implant surface.

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Objectives: Peri-implantitis is a frequent and serious clinical problem affecting between 1 and 47% of implants. Bacterial contamination of the roughened implant surface plays a major role in the etiology and progression of the disease. Successful treatment of peri-implantitis requires disinfection of the rough implant surface. There is no generally accepted protocol for implant disinfection. Autologous leukocyte and platelet-rich fibrin (L-PRF) membranes can be produced from autologous human blood via a one-step centrifugation procedure. It was hypothesized that the antimicrobial defense system of L-PRF may decontaminate the SLAⓇ implant surface. The objective of this study was to test the efficacy of L-PRF for SLAⓇ implant surface disinfection.Methods: Collagen-coated SLAⓇ (sand blasted, large grit acid etched) titanium discs were inoculated with dispersed dental plaque with a minimum bacterial cell concentration of 3.2 × 10⁷ CFU/ml. After 21 days of anaerobic incubation at 37C, discs were rinsed with 12 ml 0.9% NaCl to remove unattached biofilm, and exposed for 48 hours to Leukocyte-Platelet Rich Fibrin (L-PRF) in DMEM. Disks with or without rinsing with 12 ml of 0.9 % NaCl were fixed for SEM. Bacterial counts and perforations in bacteria were quantified from standardized scanning electron micrographs of the implant surface. The rinsing solution was collected and Western blot analysis was performed. L-PRF disks were compared with the control group (rinse). Results: Difference in presence of bacteria displaying perforation of the cell wall between cell–rich L-PRF treated samples and rinsed control group was statistically significant (p
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Objectives: A recent meta-analysis reported that 18.8% of patients treated with dental implants are affected by peri-implantitis. Chemotherapeutic agents are often used during surgical decontamination of the dental implant despite limited evidence to support their efficacy. It is also known that mature biofilms are more resistant to antimicrobial agents. No studies have tested disinfectants on mature multispecies oral biofilms on titanium substrata. The aim of this study is to develop a multispecies oral biofilm implant model and to determine its susceptibility to antimicrobial agents. The null hypothesis is that no chemical agent is more effective than saline rinse to decontaminate sandblasted acid etched (SLA) titanium dental implant.Methods: Collagen-coated SLA titanium discs were inoculated with dispersed dental plaque with minimum bacterial cell concentration of 3.2 × 10⁷ CFU/ml. After 21 days of anaerobic incubation, discs were rinsed with 0.9% NaCl to remove unattached biofilm, and exposed for 2 minutes to tetracycline paste, 1% chlorhexidine (CHX) gel, 35% phosphoric acid gel. Discs were rinsed again to remove the chemical agents. Bacterial counts were quantified from standardized scanning electron micrographs of the implant surface. Disinfectants were compared within each other and with the control groups (rinse and double-rinse).Results: After three weeks, the biofilm thickness on SLA discs was approximately 30 µm and showed the presence of multitude of rod and coccoid organisms. Rinsing the surfaces with 0.9% NaCl removed the majority of the biofilm. However, bacteria persisted in all specimens regardless of the treatment and none of the disinfectants was superior to the saline double-rinse group. CLSM analysis showed that CHX and Etch groups had a statistically significant reduction of viable bacteria within the biofilms, although small. New chemical and peeling-off techniques were also tested but did not remove significantly more bacteria than the double-rinse group.Conclusions: This mature multispecies biofilm model may be useful for the evaluation of decontamination of SLA implant surface. The tested chemical agents and the peeling-off techniques did not improve the decontamination effect when compared with the 0.9% NaCl rinse. CHX and Etch may provide a slight advantage in killing some of the remaining bacteria.

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Kindler syndrome (KS) is an autosomal recessive skin disorder of unknown etiology resulting in congenital skin blisters, photosensitivity, generalized progressive poikiloderma, and mucosal alterations. Early onset and severe periodontal disease has been noted as a common clinical finding. Kindlin-1 is an intracellular focal adhesion protein that functions in cellular adhesion in the epidermis and mucosal tissues through interaction and activation of integrins. Kindlin-1 deficiency, as occurs in KS, results in impaired adhesion of the junctional epithelium to the basement membrane and may contribute to severe periodontal disease. We hypothesize that kindlin-1 deficiency may affect other aspects of keratinocyte behavior, including expression of cytokines and mediators involved in inflammation and repair. Expression of kindlin-1 was down-regulated in human gingival keratinocytes (HGK) and confirmed via Western blot analysis and RT-PCR. HGKs were exposed to varying concentrations of a native and heat-inactivated oral biofilm extract as well as a 3-week live oral biofilm. mRNA was isolated from control and kindlin-1 deficient HGKs and RT-PCR was used to assess relative gene expression of the following gene products: β6 integrin, fibronectin ED-A and ED-B, IL-1α, IL-1β, IL-6, IL-8, TNFα, TGFβ-1, TGFβ-3, MMP-1, MMP-2, MMP-9, and tenascin C. Kindlin-1 deficiency in HGKs was found to significantly up-regulate the expression of genes encoding for β6 integrin, IL-1α, and IL-1β. β6 integrin expression was increased in K1-deficient cells in the absence of biofilm treatment and when exposed to native biofilm extract. The pro-inflammatory cytokines IL-1α and IL-1β were significantly up-regulated in K1-deficient cells as compared to control cells in keratinocytes exposed to native oral biofilm extract, and in control cells exposed to heat- inactivated biofilm.iiMMP-2, MMP-9, IL-8, tenascin C, ED-B fibronectin, and TGFβ-3 were differentially regulated by the presence of biofilm extract or live biofilm in the gingival keratinocytes. TGFβ-3 and ED- B fibronectin expression were down-regulated in cell exposed to biofilm, while IL-1α, IL-1β, MMP-2, MMP-9, and tenascin C were up-regulated. Our findings suggest that the severe and early-onset periodontal disease found in patients with KS may be a result of enhanced expression of certain pro-inflammatory cytokines.

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Objectives: The histologic, clinical, and radiographic findings together indicated infection as a major etiology of implant failure. Antibiotics have been prescribed following implant surgery to control infection. Inability to take penicillin seems to be a determining factor in implants failure in particular, with certain types of implant-related procedures. The aim of this study was to investigate retrospectively whether self-reported allergy to penicillin contributes to higher rate of implant failure. Methods: The survival of 5576 implants (985 Nobel Biocare and 4591 Straumann) placed surgically by an experienced periodontist was assessed in patients with the age range of 20-89 (mean: 60 years old) and with the follow-up period of up to 10 years. 4132 implants followed for at least one year. The survival was defined as the implant remaining in the jaw. Pearson χ2 test and Logistic regression were applied to examine the relation between pairs of variables. All tests were 2-tailed with a significance level of 0.05. Results: Out of 5106 implants placed for patients taking penicillin, 0.8% failed, while out of 470 implants placed for patients with self-reported allergy to penicillin, 2.1% failed with statistically significant difference (P= 0.002). Odds of failure for implants placed in patients allergic to penicillin were 3.2 times higher than those for non-allergic patients while controlling for the other variables. Kaplan Meier test revealed statistically significant difference between survivals of implants placed in penicillin-allergic group compared with non-allergic group. 54% of implant failures in non-allergic group have occurred during the first 6 months of implant insertion, while in penicillin allergic group this amount was 80% with statistically significant difference. Immediate implant placement in fresh extraction socket had 10-times higher rate of failure in patients with self-reported allergy to penicillin. Significant association between smoking and implant failure was found (p=0.005). Implants in the area of second molars have the highest failure rate with more frequency in maxilla relative to mandible. Conclusion: Inability to take penicillin may contribute to higher rate of implant failure and thus, penicillin allergy test could be implemented in clinical settings to increase the likelihood of prescribing penicillin relative to its alternates.

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Objective: A multicenter clinical trial was conducted to characterize the surface topography of the peri-abutment epithelium after removal of the dental implant healing abutment. Methods: The study included a total of 10 Straumann (S) and 19 Nobel Biocare (N) dental implants. All dental implants were placed by either a certified periodontist (private practice) or residents of the Graduate Periodontics clinic at the University of British Columbia (UBC). Photographs of the peri-abutment epithelium were taken and analyzed using Image J software for % area of redness. Silicone impressions of the peri-abutment epithelium were taken and used to fabricate epoxy replicas that were imaged under SEM. Student T-tests were used to analyze differences between groups (p 0.05). There was a positive correlation between the peri-abutment soft tissue height and % area of redness that was found to be statistically significant (p
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Scar formation is a frequent consequence of wound healing and has widespread negative effects on individuals’ quality of life, both physically and psychologically. For most people, scars are unsightly, but in addition to this, they can result in serious morbidities such as pruritus, pain, contracture, and decreased heat tolerance in severe situations. The association between degree of scarring and depth of dermal injury has been recognized by surgeons for many years, however the cellular and molecular basis for these observations remains poorly understood. Interestingly, oral wounds have been shown to heal faster and with less clinical and histological scar formation than similar skin wounds. It was hypothesized that palatal wounds in general show relatively little scarring and also that there is increased scar formation of the palatal mucosa following a connective tissue graft (CTG) harvest (deep wound) than a free gingival graft (FGG) harvest (superficial wound). This was a retrospective clinical study carried out at the University of British Columbia, Faculty of Dentistry. Intraoral photographs were taken of the palate in 37 subjects. Each subject had undergone a CTG and/or FGG harvest by a Graduate Periodontics resident more than six months prior to the study. 23 FGG and 23 CTG scars were assessed. Two independent calibrated blinded examiners assessed the photographs using a modified version of the Manchester Scar Proforma. A value of zero, one, or two was given for each parameter, with no difference from normal tissue scored as zero and gross mismatch scored as two. The values for each parameter were summed to produce a total scar score, zero to six, for each site. The component parameters were also examined individually so that color, contour, and distortion could be evaluated independently. The results of this study demonstrated that scar formation in the palatal mucosa is minimal and in many cases, non-existent. CTG donor sites did not have more severe scar formation than FGG sites.

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Objective: A prospective multi-centre clinical trial was conducted to assess the biofilm inhibitory potential of 1% chlorhexidine (CHX) gel in the internal cavity of implant screw holes, when utilized at the time of surgical implant placement. Methods: The study included a total of 40 Straumann (S) and Nobel Biocare (N) implants, divided into test (ST or NT) and control (SC or NC) based on the implant system. The implants were placed by two periodontists (private practice) as well as by senior Graduate Periodontics residents at the University of British Columbia. Total colony forming units (CFUs/ml) were assessed 3 months post surgery by means of aerobic and anaerobic culturing and Gram staining was conducted on 15 of the samples. Univariate Mann-Whitney U tests were used to analyze differences between groups (p 0.05). No statistical differences in term of CFUs/ml were evident when comparing aerobic and anaerobic culturing methods (p > 0.05). The use of 1% CHX gel significantly reduced biofilm formation in both the ST and NT samples when compared with controls (SC and NC) (p 0.05). Overall, 13 implants from the test population demonstrated CFUs/ml below a threshold of 1000 compared to only 1 implant from the control population. Microscopic analysis revealed the presence of mainly Gram-positive coccoid species in 14/15 samples; one sample consisted mainly of rod shaped bacteria. Conclusion: The application of 1% CHX gel in the internal implant cavity at the time of initial implant surgery greatly reduces the biofilm formation over a 3-month period.

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Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. Thus, we hypothesized that αvβ6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both β6 integrin mRNA and protein. The maxillary incisors of Itgb6-/- mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6-/- mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6-/- enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6-/- mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, which did not use αvβ6 integrin as an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvβ6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition/turnover and subsequent enamel biomineralization.

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Objective: A retrospective review of charts was conducted to assess the survival rate and marginal bone loss around Nobel Active implants after one year of loading. Methods: The study included 124 NobelActive dental implants that were placed by experienced practitioners in two private clinics and by senior Graduate Periodontics residents at the University of British Columbia. The effect of patient, medical condition, site, implant, surgeon’s experience and timing related risk factors on implant survival and marginal bone loss was evaluated. Implant failure was defined as the loss or removal of an implant. Radiographic measurements of marginal bone loss (mm) were made using Image J 1.42 software and Planmeca Romexis 2.2.7R software. Bivariate analyses were used to identify variables associated with implant failure and marginal bone loss. Risk factors that were shown to be significant (p
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Objectives: Dental implants have predictable outcomes and high survival rates. However, a small subset of patients experience implant failure. A retrospective review of charts at UBC was conducted to determine how patient-, disease-, site-, surgeon- and implant design-centered risk factors affect the survival of implants.Methods: A review of implants placed between 1989-2006 was completed. Inclusion criteria required a one-year post-placement diagnostic radiograph. Implant failure was defined as the loss or removal of an implant at any time. Bivariate analyses were used to identify variables associated with implant failure. Risk factors with p-values
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