Microcirculation and Neutrophil-related cytokine concentrations are not altered around narrow diameter implants in T2DM patients during wound healing

Microcirculation and Neutrophil-related cytokine concentrations are not altered around narrow diameter implants in T2DM patients during wound healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Microcirculation and Neutrophil-related cytokine concentrations are not altered around narrow diameter implants in T2DM patients during wound healing Daniel Diehl, Doğan Kaner, Amelie Bockholt, Hakan Bilhan, Anton Friedmann This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1614566/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Objectives The aim of this study was to assess the microcirculation and the expression patterns of wound-healing related cytokines around narrow-diameter implants in type 2 diabetes mellitus (T2DM) and normo-glycemic patients. Materials and methods A total of 31 patients, 15 of which diagnosed with T2DM (HbA1c > 6.5) and 16 normo-glycemic patients received narrow diameter implants in the posterior mandible or maxilla. During the 3-month healing period, soft-tissue perfusion was monitored via Laser-Doppler flowmetry. Peri-implant fluid (PICF) was harvested and analyzed for concentrations of interleukin-1ß (IL-1ß), interleukin-23 (IL-23), interleukin-17 (IL-17) and granulocyte colony-stimulating factor (G-CSF) by a multiplex, bead-based immunoassay. Results Microcirculatory perfusion patterns during wound healing exhibited no significant differences throughout the observation period. IL-1ß concentrations were expectedly elevated during the early phases of wound healing. At the first visit after surgery, IL-23 concentrations were significantly higher in implants of diabetic patients. This difference was diminished over the course of the observation period. For the other tested analytes, no differences were observable between both groups. Conclusion Wound healing after implant surgery was similar in T2DM and healthy patients. Hydrophilic-surface titanium-zirconium implants with reduced diameter may be considered for implant therapy of Diabetes mellitus type II patients. type 2 diabetes gingival perfusion gingival vascularization wound healing cytokines PMN Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Type-2 diabetes mellitus (T2DM) is a metabolic disorder characterized by high serum glycemic levels either due to insufficient insulin levels, defective function, or both [ 1 ]. In the last decade, the number of diabetic patients has nearly doubled and is now estimated to be at 285 million worldwide, 90% of which suffering from T2DM [ 2 ]. T2DM patients exhibit impaired wound healing and microcirculation, causing an increased risk of wound infection and an advanced susceptibility for peri-implantitis as well [ 3 – 5 ]. Known underlying mechanisms include decreased growth factor and cytokine production [ 6 ], impaired macrophage and neutrophil (PMN) function [ 7 , 8 ], and accumulation of matrix-metalloproteases [ 9 ]. PMN constitute the first line of innate defense from the cellular immune system. Immediately after surgery-related injury inflicted on the tissue, an inflammatory reaction is initiated and a variety of pro-inflammatory cytokines and chemokines mediate PMN chemotaxis to the affected area. One of the most abundant cytokines is interleukin 1ß (IL-1ß), which has emerged as a key regulator of pro-inflammatory tissue reaction and associated disorders [ 10 ]. Interleukin-17 is a cytokine family derived from T helper 17 (Th17) cells, which is known for its pro-inflammatory effects on both adaptive and innate host response, especially on PMN [ 11 ]. Moreover, the activated PMN themselves were shown to upregulate IL-17 expression in order to enhance the phagocytotic activity and the inflammatory phenotype differentiation [ 12 ]. Vice versa, the Il-17 mediated PMN activation upregulates the secretion of other chemotactic agents like CXC chemokines and the granulocyte colony stimulating factor (G-CSF) [ 13 ]. The activating cytokine repertoire needs to be maintained during the first phases of wound healing, in order for the PMN to function properly. This is achieved by PMN and macrophage-derived IL-23 [ 14 ]. Thus, IL-23 is known to be the main driver for Th17 polarization by CD4 + T-cells, which on the opposite constitutes the most sustainable source for IL-17 [ 15 ]. Under hyperglycemic conditions, chemotactic PMN trafficking and phagocytosis are significantly debased, resulting in exacerbated and prolonged inflammation [ 16 , 17 ]. With regard to dental implant therapy, the T2DM-related biological alteration in wound healing may account for increased complication rates and even implant failures. Accordingly, attempts to reduce the complexity and invasiveness of surgical procedures and to simplify the treatment regimen appears desirable. Narrow-diameter implants (NDI) were developed for sites with diminished alveolar ridge dimensions and are a viable treatment option [ 18 , 19 ]. In T2DM patients, NDI may represent a less invasive treatment option by reducing the need for lateral ridge augmentation procedures and preventing an extended wound healing burden. Recently, in a one-year clinical case and control pilot study, we were able to demonstrate the positive outcomes of NDI in T2DM patients. However, the significant role of PMN in diabetic wound healing justifies the wish to better understand the role of PMN-related cytokines in this context. Here, we aimed to further substantiate the clinical performance of NDI in T2DM and non-diabetic patients by characterizing the process wound healing in both study groups. Material And Methods Study population The study protocol was nested inside a one-year prospective case-control trial [ 20 ]. The Witten/Herdecke University Ethics Committee approved the study protocol (108/2012) and the study is in accordance with the Declaration of Helsinki. All participants provided written informed consent and were compliant with the study protocol. In brief, 31 patients with a mean age of 67 and one or more missing teeth posterior to the canine area of the maxilla or mandible and an associated diminished alveolar ridge dimension were enclosed (Table 1). Sixteen patients diagnosed with T2DM (HbA1C > 6.5%) were assigned to the test group and 16 normo-glycemic patients were assigned to the control group (HbA1C ≤ 6.0%). Exclusion criteria have been described previously [ 20 ]. A STROBE checklist for this study was provided for review of this manuscript. Therapeutic intervention All participants received hydrophilic-surface reduced-diameter tissue level implants (3.3mm; RN Standard plus, SLActive, Institut Straumann AG, Basel, CH). By protocol, additional surgical steps aiming at the augmentation of bone volume at the site of interest were not intended. Placement of all implants was carried out under local anesthesia (3.4ml, Ultracain DS forte, Sanofi-Aventis, Frankfurt, Germany) strictly following the standard transmucosal healing protocol in both groups. All surgeries were carried out by two experienced periodontists in a standardized manner. After midcrestal incision, a buccal and lingual flap was reflected, strictly omitting vertical releasing incisions. Care was taken to maintain at least 2mm of keratinized mucosa on both lingual and buccal flaps. The osteotomy was performed according to the manufacturer’s instructions. In cases of two adjacent implants, the posterior one served as the study implant. The post-op regimen included instructions to abstain from mechanical plaque control in the treated area for 1 week and to use a 0.2% chlorhexidine mouth rinse twice a day (Chlorhexamed, GlaxoSmithKline Consumer Healthcare GmbH & Co. KG, Munich, Germany). The administration of systemic antibiotics was restricted to individual needs, there was no prescribing policy by protocol; analgesic medication (Ibuprofen 600mg/3x daily) on demand was recommended. Sutures were removed after 7–10 days. Laser Doppler flowmetry A Laser Doppler flowmeter (Periflux 5010, Perimed AB, Jarfalla, Sweden) equipped with a PF 416 probe (outside diameter 1.0 mm, fibre separation 0.25 mm; wavelength 780 nm) was used for the assessment of microcirculation. The scores were recorded in perfusion units (PU) and monitored by the Perisoft software (Perisoft 2.10, Perimed AB, Jarfalla, Sweden). To standardize the reproducibility of assessments, a customized acrylic stent fitting the retained teeth determined the positioning of the probe tip. The stent carried a perforation on the buccal aspect above and beneath the muco-gingival junction (MGJ). The perforation fitted the diameter of the LDF probe tip and coordinated both, the perpendicular position of the tip at the mucosa surface and the distance of 0.5 mm above its surface. The stent was extended to the contralateral side and at the contralateral tooth the perforation was prepared according to the position at the implant. The microcirculation in peri-implant tissues was assessed above and beneath the MGJ before treatment (V1a), 2 min after local anaesthesia (V1b), directly after completion of surgery (V1c), 3 days (V2), 7–10 days (V3), 4 weeks (V4), 8 weeks (V5) and 3 months (V6) post-surgery for 1 minute each. One calibrated investigator performed all measurements. Statistical analysis of perfusion units (PU) was carried out for the delta (Δ) between the baseline and following LDF measurements PICF sampling Sampling of peri-implant crevicular fluid (PICF) was performed three days after surgery (V2), after suture removal (V3), and after four weeks (V4). In brief, a paper strip (PerioCol collection paper; Oraflow; Smithtown; NY; USA) was inserted to the sulcus on the buccal aspect of each study implant and kept in place for 10s. Strips contaminated with blood were discarded. Fluid volume was determined immediately with a micro-moisture metre by means of a standard calibration curve (Periotron 8000, Oraflow, USA). Samples were stored in 200 µL sterile phosphate-buffered saline at -80°C until further processing. For elution, thawed samples were vortexed for 1 minute and subsequently centrifuged at 3000xg. Microsphere-based multiplex immunoassay Reagent sets for Luminex immunoassay were customized by Merck Millipore and included four analytes (IL-1ß, GM-CSF, IL-17a, IL23). Immunoassays were performed according to the manufacturer’s Luminex magnetic screening protocol. The magnetic beads were dispensed into a 96-well microplate after blocking with washing buffer for 15 minutes. Fluid samples were normalized to a PICF volume equivalent of 0.1ml (Table 2) and incubated with the mixed beads overnight at 4°C. Beads were washed and incubated with biotinylated antibodies against the analytes for 1 hour. Plates were washed again and streptavidin-phycoerythrin (PE) was added for 30 minutes with another subsequent washing step. Plates were read immediately on the MAGPIX (Luminex Corp., Austin, Texas) instrument. Absolute protein concentrations were calculated in pg/ml from a standard curve derived from a 7-fold (3.2-10000pg/ml) serial dilution of manufacturer’s analyte standard. Statistical Analysis For all data obtained, mean and standard deviation were calculated. Raw data from luminex immunoassay were acquired from xPONENT (Luminex Corp., Austin, Texas) software. All statistical analyses were performed with GraphPad Prism 8 (GraphPad, San Diego, California). Censored values below the limit of detection (LOD) were replaced by a value between zero and the analyte-specific detection limit. In order to avoid overestimation of the statistical mean, half of the detectable LOD was employed using the formula (½)*(LOD − 0) [ 21 ]. Further statistical analysis included the Shapiro-Wilk, Kolmogorov-Smirnov and D’Agostino-Pearson test to assess data distribution. Respectively, comparisons between independent samples were calculated by Mann-Whitney U test and intragroup comparisons were analyzed by Wilcoxon signed-rank test. LDF data were analyzed by Two-way ANOVA and Sidaks multiple comparisons post-hoc analysis. The level of significance was set at p = 0.05. Results The clinical outcome after the surgical intervention was reported previously [ 20 ]. In brief, thirty-two patients with a mean age of 67 years were enrolled. Mean HbA1c value for the hyperglycemic test group was 7.34% (± 0.73). Thirty-one patients were eligible for wound healing assessments, as one patient from the control group was treated with systemic antibiotics for endocarditis prophylaxis (Table 1). Microcirculation As shown by the Friedman test and consecutive Dunnet’s post hoc analysis, the LDF values in the control group experienced a significant reduction in perfusion rate immediately after completion of implant surgery (p = 0.0019) with a mean ∆PU (perfusion unit) of -108.5 ± 142.5 (V1c, Fig. 2). This was followed by a consecutive increase in perfusion rate three days (V2) post-surgery (∆PU -14.76 ± 185.0). However, this was statistically non-significant (p > 0.99). The consecutive LDF measurements revealed a non-significant diminution over the observation period (V3-V6, Fig. 2). In the T2DM group, ∆PU values exhibited a significant decrease at completion of the implant surgery (-92.36 ± 87.40, p = 0.0019). At all further measurements, the development of site perfusion exhibited a pattern like the control group (Fig. 2c). Consequently, the intergroup comparison via Student’s t-test failed to show statistically significant differences at any visit and assessment, respectively (Fig. 2c). Cytokine quantities in peri-implant crevicular fluid IL-1ß – At baseline, cytokine concentrations were significantly elevated compared with the subsequent visits in both group (Fig. 3A), indicating a significant time-dependent reduction of IL-1ß in the PICF. Furthermore, the mean analyte concentration was substantially higher at implants of the T2DM group (178.05 ± 173.77) than in the control group (64.22 ± 77.42, Fig. 4A). However, these differences were not statistically significant (Table 3). IL23 – Stratified by study group, the baseline cytokine concentrations exhibited significantly higher amounts (p = 0.003) in the T2DM group (55.16 ± 33.05) than in normo-glycemic group implants (34.98 ± 80.54) (Fig. 4B). In relation to the process of wound healing, both groups exhibited a similar (p = 0.82) increase 10 days after surgery (V3, Table 3). These changes were statistically significant in the control group. IL-17 – In total, the cytokine concentrations of IL-17 were lower compared to the other analytes, ranging from 0.73 to 6.44 pg/ml. No significant differences were detectable, neither stratified by time nor by study group (Fig. 3 + 4, Table 3). GM-CSF – No significant changes in cytokine concentration were found during the entire observation period. Moreover, test and control group implants displayed no different levels of GM-CSF. Discussion The present prospective clinical study was conducted to monitor the wound healing after placing narrow-diameter implants in T2DM and normo-glycemic patients by assessment of microcirculation and PMN-related cytokine expression. No significant differences between T2DM and healthy patients were observed, with the exception of pro-inflammatory cytokines IL-23 and Il-1ß during initial healing a few days after implant surgery. In our study, the perfusion of the peri-implant tissues was evaluated by Laser Doppler Flowmetry (LDF), a technique which allows for the detection of blood flow disturbances after surgical injury [ 22 – 24 ]. Both groups displayed significant reduction of microcirculation activity directly after surgery and one day post-op, when compared to baseline. However, microcirculation activity appeared to recover rapidly, returning to levels assessed before injury. Moreover, there was no significant difference regarding reduction of blood flow found between T2DM and healthy patients. The rationale behind this observation may be the similarly minimal-invasive surgical approach in both groups, since LDF measurements of post-surgical perfusion impairment were shown to correlate well with the extent of tissue trauma [ 24 , 25 ]. The transmucosal healing pattern may have contributed to this positive outcome, as there was no need in flap closure above the implant shoulder, which regularly requires a coronal advancement of the flap. The perfusion rates altered at similar altitude during the observation period closely reflecting the healing progress in both groups. Statistically significant impairment of perfusion was only found before and after application of local anesthesia containing the vasoconstrictor in both groups. Moreover, the perfusion rate showed statistically significant improvement at day 3 after the surgery. Regardless of systemic background, the recorded microcirculation was apparently related to uneventful healing progress. Furthermore, the plotted perfusion units per time were in accordance with those observed earlier in a beagle model, that aimed at monitoring the changes of microcirculation in the context of bone augmentation surgery [ 22 ]. These results, however, contradicted our expectations. As suggested by recent studies on the microvascular skin perfusion in diabetic patients, we anticipated a significantly lower perfusion rate in the wounds of diabetic patients [ 26 , 27 ]. The Hba1c values in the T2DM patient cohort with 7.34% might have been estimated to cause a bigger discrepancy. Nevertheless, the results highlight our hypothesis that T2DM patients may benefit from the NDI design because of the reduced wound healing burden associated with their use [ 28 ]. In this study, the concentrations of interleukin-1ß, interleukin-23, interleukin17A and GM-CSF were measured in PICF of NDI recently placed in the posterior jaw. The sampling of the PICF for analyzing the molecular content is considered a viable method for studying the wound healing biology around teeth and implants in humans [ 29 – 31 ]. In particular, the bead-based immunoassay applied in this study allows for a multiplex analysis of up to 96 analytes per sample. Interleukin1ß is a pro-inflammatory cytokine which upregulates a plethora of inflammatory effector molecules such as chemokines and prostaglandins. Due to its abundance in inflamed and injured tissues of all kind, it is considered a ubiquitous biomarker for acute tissue inflammation [ 32 , 10 ]. This study revealed upregulated concentrations of Il-1ß immediately after surgery presenting with significantly reduced levels up to the end of the observation period. Interestingly, the T2DM patients presented with non-significantly altered concentrations to the control group at any of the visits, indicating both, an innate initialization and resolution of the inflammatory process, respectively. The pattern of IL-1ß secretion to PICF is in line with a similar study conducted by [ 33 ], who reported similar results for IL-1ß concentrations in PIF of healthy patients. According to a recent experimental study in diabetic rats, IL-23 levels are significantly increased under diabetic conditions [ 34 ]. IL-23 is considered the main inducer of CD4 + T-lymphocyte differentiation towards IL-17 producing Th17 cells [ 15 ]. In context of wound healing, large amounts of Th17 helper cells are associated with a delay in wound closure. The inhibition of the backbone IL-23 / Il-17 axis may even promote wound healing under diabetic conditions [ 35 , 36 ]. Our results demonstrated significantly higher levels of IL-23 at implants of T2DM patients, when compared to the control group. Over the course of the observation period (V2-V4), however, the IL-23 and IL-17 concentrations equalized to insignificant differences. These results are corroborated further by the works of Santos et al. and Vieira Ribeiro et al., who reported no significant increase in IL-23 expression among diabetic and healthy patients suffering from periodontitis [ 37 , 38 ]. The last investigated analyte, GM-CSF, did not show any timing- or group-related differences. GM-CSF is a multipotent growth factor responsible for granulopoiesis and keratinocyte-related proliferation and re-epithelialization, and a variety of studies has verified its necessity for complication-free wound healing [ 39 – 41 ]. Therefore, unchanged GM-CSF levels in T2DM patients may have been associated with the uneventful healing period observed in this trial. This analysis was nested in a larger pilot study [ 20 ]. Thus, the presented results have some obvious limitations. As indicated by the large variability among individuals, selected datasets may not be adequately powered. Nevertheless, we were able to elucidate The measured effector molecules have a pivotal role in regulating the PMN-mediated early wound healing, which did not appear significantly affected by the T2DM condition. However, while diabetic patients showed a more pro-inflammatory initial cytokine profile, wound healing and PMN response appeared similar in diabetic and non-diabetic patients on a longer term. Moreover, these results corroborate our previously reported findings of similar clinical success placing reduced-diameter implants in these patients [ 20 ]. In conclusion, hydrophilic-surface titanium-zirconium implants with reduced diameter exhibit no molecular alterations regarding wound healing in T2DM patients and may thus be a viable option for this group of patients. Declarations figures; D.K..: conceptualization of the study, proofreading; A.H.: experimental procedures and measurements; H.B.: manuscript drafting and proof reading; A.F.: conceptualization of the study; implant treatments, proofreading the manuscript. Acknowledgements The authors thank Dr. Kai R. Fischer for conducting implant surgeries during the study. The authors declare that they do not have any commercial, proprietary or financial interest in the products or companies described in this article. The study was partly funded by a restricted grant and donation of the narrow diameter implants from Institut Straumann AG, Basel, Switzerland Registration number: NCT04630691 (clinicaltrials.gov) Conflicts of Interest The authors declare that they do not have any commercial, proprietary or financial interest in the products or companies described in this article. Funding The study was supported by a restricted grant including the donation of the NDI’s by the Institute Straumann AG, Basel, Switzerland Ethics approval This study was performed in line with the Declaration of Helsinki. Approval was granted by the Witten/Herdecke University Ethics Committee (108/2012). Consent to participate Written informed consent was obtained from all individual participants involved in the study. References Zimmet P, Alberti KG, Magliano DJ, Bennett PH (2016) Diabetes mellitus statistics on prevalence and mortality: facts and fallacies. 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Elsevier, pp 87–92 Schirmacher P, Mann A, Breuhahn K, Blessing M (2001) Keratinocyte-Derived Granulocyte-Macrophage Colony Stimulating Factor Accelerates Wound Healing: Stimulation of Keratinocyte Proliferation, Granulation Tissue Formation, and Vascularization. Journal of Investigative Dermatology 117 (6):1382–1390 Castro-Dopico T, Fleming A, Dennison TW, Ferdinand JR, Harcourt K, Stewart BJ, Cader Z, Tuong ZK, Jing C, Lok LS (2020) GM-CSF calibrates macrophage defense and wound healing programs during intestinal infection and inflammation. Cell reports 32 (1):107857 Tables Table 1-3 are available in the Supplementary Files section. Supplementary The STROBE checklist is not available with this version Additional Declarations No competing interests reported. Supplementary Files Table1.pptx Table 1: Patient demographics. ✢ = Student’s t-test; ✥ = Fisher’s exact test. Tbl2PMN.pptx Table 2: Mean PICF volumes at different time points stratified by group. Tbl3PMN.pptx  Table 3: Mean analyte concentrations at different time points stratified by group and analyte. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 31 Jul, 2022 Reviews received at journal 13 Jun, 2022 Reviewers agreed at journal 22 May, 2022 Reviewers invited by journal 22 May, 2022 Editor assigned by journal 10 May, 2022 Submission checks completed at journal 10 May, 2022 First submitted to journal 01 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1614566","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":104852342,"identity":"47908df4-7216-42cd-a11b-7665493b0e8b","order_by":0,"name":"Daniel Diehl","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIie3PL2vDQBjH8d8xUXNl9qArewtPqCq05K1kPilVo2Ii6ma26ohC38odB5tJWzvYxFRVRGRFGXuS/YGJXCcr7ksSnsB97hIgFDrDYr4N5hAaUpgakD9XZ1HeEGoJbPEfQqZ9QuS8zH2tPEWeN9bUhOGy92jcVL9dQWUG1cJDylliC8JIy23iMr2XULNErErfh6XkJOFGKx4y7WT8mtJFX3vIriJ3bMg1D2MmGDTkw0NeeHO0p0hy4pfk3SQqKrIPpPhfUh62TPg4u3rqJvFlOqoPi8lwfV9G74dbF0PyUN11k+/U31dzEoRCoVDI2ye07lK1q7iaQwAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Periodontology, Witten/Herdecke University","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Diehl","suffix":""},{"id":104852343,"identity":"a3ef077b-78b7-4a73-94d4-1b6ea7993b27","order_by":1,"name":"Doğan Kaner","email":"","orcid":"","institution":"Department of Periodontology, Witten/Herdecke University","correspondingAuthor":false,"prefix":"","firstName":"Doğan","middleName":"","lastName":"Kaner","suffix":""},{"id":104852344,"identity":"352cdfc5-65f5-47ea-925a-3457ab30cf8c","order_by":2,"name":"Amelie Bockholt","email":"","orcid":"","institution":"Department of Periodontology, Witten/Herdecke University","correspondingAuthor":false,"prefix":"","firstName":"Amelie","middleName":"","lastName":"Bockholt","suffix":""},{"id":104852345,"identity":"64151bf8-c62a-42af-8fa9-ce4e3a990cb6","order_by":3,"name":"Hakan Bilhan","email":"","orcid":"","institution":"Department of Periodontology, Witten/Herdecke University","correspondingAuthor":false,"prefix":"","firstName":"Hakan","middleName":"","lastName":"Bilhan","suffix":""},{"id":104852346,"identity":"43dec57e-6a0d-4d2e-a758-a32ea6f1df37","order_by":4,"name":"Anton Friedmann","email":"","orcid":"","institution":"Department of Periodontology, Witten/Herdecke University","correspondingAuthor":false,"prefix":"","firstName":"Anton","middleName":"","lastName":"Friedmann","suffix":""}],"badges":[],"createdAt":"2022-05-01 19:29:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1614566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1614566/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":21521103,"identity":"1e58b185-7480-4967-a0bf-71fd355c9952","added_by":"auto","created_at":"2022-05-16 15:46:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30874,"visible":true,"origin":"","legend":"\u003cp\u003eFlow Chart of the study protocol \u003c/p\u003e\u003cp\u003ePICF= Peri-implant crevicular fluid, LDF=Laser Doppler flowmetry.\u003c/p\u003e\u003cp\u003e Visit 2=3 days; visit 3=7-10 days; visit 4=4 Weeks; Visit 5=8 weeks; Visit 6=3 months \u003c/p\u003e\u003cp\u003epost op.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e \u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/1516af982cdfabd129135994.png"},{"id":21520035,"identity":"40b05f11-8cc6-44ad-8f32-9dc30e1e352c","added_by":"auto","created_at":"2022-05-16 15:41:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25135,"visible":true,"origin":"","legend":"\u003cp\u003eLDF measurements at different visits \u003c/p\u003e\u003cp\u003eValues represent the Δ of PU and the baseline measurement at V1a. Whiskers represent minimum and maximum.\u0026nbsp;\u003c/p\u003e\u003cp\u003e \u003cstrong\u003ea) \u003c/strong\u003ePerfusion units at different visits in healthy control. \u003cstrong\u003eb) \u003c/strong\u003ePerfusion units at different visits in T2DM patients. \u003cstrong\u003ec) \u003c/strong\u003eIntergroup differences.\u003c/p\u003e\u003cp\u003e V=Visit.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/68b689467235ab9464ce4569.png"},{"id":21520037,"identity":"99e716b7-df91-49de-add0-fa918e13330e","added_by":"auto","created_at":"2022-05-16 15:41:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13740,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyte concentrations of IL-1ß (A), IL-23(B), IL-17a(C) and GM-CSF(D)\u003c/p\u003e\u003cp\u003e stratified by study groups. Whiskers represent minimum and maximum. Black dots signify outliers.\u003c/p\u003e\u003cp\u003e***p\u0026lt;0.0001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/20bb07907924526c6a7d2859.png"},{"id":21521614,"identity":"546b2e67-7481-4086-8a40-7ec86649c920","added_by":"auto","created_at":"2022-05-16 15:51:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25519,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyte concentrations IL-1ß (A), IL-23(B), IL-17a(C) and GM-CSF(D)\u003c/p\u003e\u003cp\u003estratified by time. Whiskers represent minimum and maximum.\u003c/p\u003e\u003cp\u003e***p\u0026lt;0.0001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/ffb1ab01e54296e7b0c910f6.png"},{"id":21521618,"identity":"98f960e9-ad00-4278-8a5a-afbb6c461145","added_by":"auto","created_at":"2022-05-16 15:51:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":400139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/2ffce2fc-ecc8-4a35-a5d3-edd3795239c9.pdf"},{"id":21520041,"identity":"e8bc80bd-c7bc-4a27-93fb-9f9f03b9a8f5","added_by":"auto","created_at":"2022-05-16 15:41:19","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":41047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: Patient demographics. ✢ = Student’s t-test; \u003csup\u003e✥ \u003c/sup\u003e= Fisher’s exact test.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Table1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/38baf16792458040c061a552.pptx"},{"id":21521104,"identity":"f2e0a807-c559-4fc4-b1ca-bb19ccfef39e","added_by":"auto","created_at":"2022-05-16 15:46:19","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":41158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e: Mean PICF volumes at different time points stratified by group.\u003c/p\u003e","description":"","filename":"Tbl2PMN.pptx","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/a5391731bfb93098b97fc31b.pptx"},{"id":21521108,"identity":"36ad9121-057e-422d-9c0c-6246f497fdf0","added_by":"auto","created_at":"2022-05-16 15:46:19","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":47452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cspan class=\"ql-cursor\"\u003e\u003c/span\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e: Mean analyte concentrations at different time points stratified by group and analyte.\u003c/p\u003e","description":"","filename":"Tbl3PMN.pptx","url":"https://assets-eu.researchsquare.com/files/rs-1614566/v1/e2c37be11f35a687a1781816.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microcirculation and Neutrophil-related cytokine concentrations are not altered around narrow diameter implants in T2DM patients during wound healing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eType-2 diabetes mellitus (T2DM) is a metabolic disorder characterized by high serum glycemic levels either due to insufficient insulin levels, defective function, or both [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the last decade, the number of diabetic patients has nearly doubled and is now estimated to be at 285\u0026nbsp;million worldwide, 90% of which suffering from T2DM [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eT2DM patients exhibit impaired wound healing and microcirculation, causing an increased risk of wound infection and an advanced susceptibility for peri-implantitis as well [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Known underlying mechanisms include decreased growth factor and cytokine production [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], impaired macrophage and neutrophil (PMN) function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and accumulation of matrix-metalloproteases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePMN constitute the first line of innate defense from the cellular immune system. Immediately after surgery-related injury inflicted on the tissue, an inflammatory reaction is initiated and a variety of pro-inflammatory cytokines and chemokines mediate PMN chemotaxis to the affected area. One of the most abundant cytokines is interleukin 1\u0026szlig; (IL-1\u0026szlig;), which has emerged as a key regulator of pro-inflammatory tissue reaction and associated disorders [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterleukin-17 is a cytokine family derived from T helper 17 (Th17) cells, which is known for its pro-inflammatory effects on both adaptive and innate host response, especially on PMN [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, the activated PMN themselves were shown to upregulate IL-17 expression in order to enhance the phagocytotic activity and the inflammatory phenotype differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Vice versa, the Il-17 mediated PMN activation upregulates the secretion of other chemotactic agents like CXC chemokines and the granulocyte colony stimulating factor (G-CSF) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe activating cytokine repertoire needs to be maintained during the first phases of wound healing, in order for the PMN to function properly. This is achieved by PMN and macrophage-derived IL-23 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, IL-23 is known to be the main driver for Th17 polarization by CD4\u0026thinsp;+\u0026thinsp;T-cells, which on the opposite constitutes the most sustainable source for IL-17 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder hyperglycemic conditions, chemotactic PMN trafficking and phagocytosis are significantly debased, resulting in exacerbated and prolonged inflammation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith regard to dental implant therapy, the T2DM-related biological alteration in wound healing may account for increased complication rates and even implant failures. Accordingly, attempts to reduce the complexity and invasiveness of surgical procedures and to simplify the treatment regimen appears desirable.\u003c/p\u003e \u003cp\u003eNarrow-diameter implants (NDI) were developed for sites with diminished alveolar ridge dimensions and are a viable treatment option [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In T2DM patients, NDI may represent a less invasive treatment option by reducing the need for lateral ridge augmentation procedures and preventing an extended wound healing burden. Recently, in a one-year clinical case and control pilot study, we were able to demonstrate the positive outcomes of NDI in T2DM patients.\u003c/p\u003e \u003cp\u003eHowever, the significant role of PMN in diabetic wound healing justifies the wish to better understand the role of PMN-related cytokines in this context. Here, we aimed to further substantiate the clinical performance of NDI in T2DM and non-diabetic patients by characterizing the process wound healing in both study groups.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eThe study protocol was nested inside a one-year prospective case-control trial [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The Witten/Herdecke University Ethics Committee approved the study protocol (108/2012) and the study is in accordance with the Declaration of Helsinki. All participants provided written informed consent and were compliant with the study protocol.\u003c/p\u003e \u003cp\u003eIn brief, 31 patients with a mean age of 67 and one or more missing teeth posterior to the canine area of the maxilla or mandible and an associated diminished alveolar ridge dimension were enclosed (Table\u0026nbsp;1). Sixteen patients diagnosed with T2DM (HbA1C\u0026thinsp;\u0026gt;\u0026thinsp;6.5%) were assigned to the test group and 16 normo-glycemic patients were assigned to the control group (HbA1C\u0026thinsp;\u0026le;\u0026thinsp;6.0%). Exclusion criteria have been described previously [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A STROBE checklist for this study was provided for review of this manuscript.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic intervention\u003c/h2\u003e \u003cp\u003e All participants received hydrophilic-surface reduced-diameter tissue level implants (3.3mm; RN Standard plus, SLActive, Institut Straumann AG, Basel, CH). By protocol, additional surgical steps aiming at the augmentation of bone volume at the site of interest were not intended. Placement of all implants was carried out under local anesthesia (3.4ml, Ultracain DS forte, Sanofi-Aventis, Frankfurt, Germany) strictly following the standard transmucosal healing protocol in both groups. All surgeries were carried out by two experienced periodontists in a standardized manner. After midcrestal incision, a buccal and lingual flap was reflected, strictly omitting vertical releasing incisions. Care was taken to maintain at least 2mm of keratinized mucosa on both lingual and buccal flaps. The osteotomy was performed according to the manufacturer\u0026rsquo;s instructions. In cases of two adjacent implants, the posterior one served as the study implant.\u003c/p\u003e \u003cp\u003eThe post-op regimen included instructions to abstain from mechanical plaque control in the treated area for 1 week and to use a 0.2% chlorhexidine mouth rinse twice a day (Chlorhexamed, GlaxoSmithKline Consumer Healthcare GmbH \u0026amp; Co. KG, Munich, Germany). The administration of systemic antibiotics was restricted to individual needs, there was no prescribing policy by protocol; analgesic medication (Ibuprofen 600mg/3x daily) on demand was recommended. Sutures were removed after 7\u0026ndash;10 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLaser Doppler flowmetry\u003c/h2\u003e \u003cp\u003eA Laser Doppler flowmeter (Periflux 5010, Perimed AB, Jarfalla, Sweden) equipped with a PF 416 probe (outside diameter 1.0 mm, fibre separation 0.25 mm; wavelength 780 nm) was used for the assessment of microcirculation. The scores were recorded in perfusion units (PU) and monitored by the Perisoft software (Perisoft 2.10, Perimed AB, Jarfalla, Sweden). To standardize the reproducibility of assessments, a customized acrylic stent fitting the retained teeth determined the positioning of the probe tip. The stent carried a perforation on the buccal aspect above and beneath the muco-gingival junction (MGJ). The perforation fitted the diameter of the LDF probe tip and coordinated both, the perpendicular position of the tip at the mucosa surface and the distance of 0.5 mm above its surface. The stent was extended to the contralateral side and at the contralateral tooth the perforation was prepared according to the position at the implant. The microcirculation in peri-implant tissues was assessed above and beneath the MGJ before treatment (V1a), 2 min after local anaesthesia (V1b), directly after completion of surgery (V1c), 3 days (V2), 7\u0026ndash;10 days (V3), 4 weeks (V4), 8 weeks (V5) and 3 months (V6) post-surgery for 1 minute each. One calibrated investigator performed all measurements. Statistical analysis of perfusion units (PU) was carried out for the delta (Δ) between the baseline and following LDF measurements\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePICF sampling\u003c/h2\u003e \u003cp\u003eSampling of peri-implant crevicular fluid (PICF) was performed three days after surgery (V2), after suture removal (V3), and after four weeks (V4). In brief, a paper strip (PerioCol collection paper; Oraflow; Smithtown; NY; USA) was inserted to the sulcus on the buccal aspect of each study implant and kept in place for 10s. Strips contaminated with blood were discarded. Fluid volume was determined immediately with a micro-moisture metre by means of a standard calibration curve (Periotron 8000, Oraflow, USA). Samples were stored in 200 \u0026micro;L sterile phosphate-buffered saline at -80\u0026deg;C until further processing. For elution, thawed samples were vortexed for 1 minute and subsequently centrifuged at 3000xg.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMicrosphere-based multiplex immunoassay\u003c/h2\u003e \u003cp\u003eReagent sets for Luminex immunoassay were customized by Merck Millipore and included four analytes (IL-1\u0026szlig;, GM-CSF, IL-17a, IL23). Immunoassays were performed according to the manufacturer\u0026rsquo;s Luminex magnetic screening protocol. The magnetic beads were dispensed into a 96-well microplate after blocking with washing buffer for 15 minutes. Fluid samples were normalized to a PICF volume equivalent of 0.1ml (Table\u0026nbsp;2) and incubated with the mixed beads overnight at 4\u0026deg;C. Beads were washed and incubated with biotinylated antibodies against the analytes for 1 hour. Plates were washed again and streptavidin-phycoerythrin (PE) was added for 30 minutes with another subsequent washing step. Plates were read immediately on the MAGPIX (Luminex Corp., Austin, Texas) instrument. Absolute protein concentrations were calculated in pg/ml from a standard curve derived from a 7-fold (3.2-10000pg/ml) serial dilution of manufacturer\u0026rsquo;s analyte standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eFor all data obtained, mean and standard deviation were calculated. Raw data from luminex immunoassay were acquired from xPONENT (Luminex Corp., Austin, Texas) software. All statistical analyses were performed with GraphPad Prism 8 (GraphPad, San Diego, California). Censored values below the limit of detection (LOD) were replaced by a value between zero and the analyte-specific detection limit. In order to avoid overestimation of the statistical mean, half of the detectable LOD was employed using the formula (\u0026frac12;)*(LOD \u0026minus;\u0026thinsp;0) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Further statistical analysis included the Shapiro-Wilk, Kolmogorov-Smirnov and D\u0026rsquo;Agostino-Pearson test to assess data distribution. Respectively, comparisons between independent samples were calculated by Mann-Whitney U test and intragroup comparisons were analyzed by Wilcoxon signed-rank test. LDF data were analyzed by Two-way ANOVA and Sidaks multiple comparisons post-hoc analysis. The level of significance was set at p\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe clinical outcome after the surgical intervention was reported previously [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In brief, thirty-two patients with a mean age of 67 years were enrolled. Mean HbA1c value for the hyperglycemic test group was 7.34% (\u0026plusmn;\u0026thinsp;0.73). Thirty-one patients were eligible for wound healing assessments, as one patient from the control group was treated with systemic antibiotics for endocarditis prophylaxis (Table\u0026nbsp;1).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicrocirculation\u003c/h2\u003e \u003cp\u003eAs shown by the Friedman test and consecutive Dunnet\u0026rsquo;s post hoc analysis, the LDF values in the control group experienced a significant reduction in perfusion rate immediately after completion of implant surgery (p\u0026thinsp;=\u0026thinsp;0.0019) with a mean ∆PU (perfusion unit) of -108.5\u0026thinsp;\u0026plusmn;\u0026thinsp;142.5 (V1c, Fig.\u0026nbsp;2). This was followed by a consecutive increase in perfusion rate three days (V2) post-surgery (∆PU -14.76\u0026thinsp;\u0026plusmn;\u0026thinsp;185.0). However, this was statistically non-significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.99). The consecutive LDF measurements revealed a non-significant diminution over the observation period (V3-V6, Fig.\u0026nbsp;2). In the T2DM group, ∆PU values exhibited a significant decrease at completion of the implant surgery (-92.36\u0026thinsp;\u0026plusmn;\u0026thinsp;87.40, p\u0026thinsp;=\u0026thinsp;0.0019). At all further measurements, the development of site perfusion exhibited a pattern like the control group (Fig.\u0026nbsp;2c). Consequently, the intergroup comparison via Student\u0026rsquo;s t-test failed to show statistically significant differences at any visit and assessment, respectively (Fig.\u0026nbsp;2c).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCytokine quantities in peri-implant crevicular fluid\u003c/h2\u003e \u003cp\u003e \u003cb\u003eIL-1\u0026szlig; \u0026ndash;\u003c/b\u003e At baseline, cytokine concentrations were significantly elevated compared with the subsequent visits in both group (Fig.\u0026nbsp;3A), indicating a significant time-dependent reduction of IL-1\u0026szlig; in the PICF. Furthermore, the mean analyte concentration was substantially higher at implants of the T2DM group (178.05\u0026thinsp;\u0026plusmn;\u0026thinsp;173.77) than in the control group (64.22\u0026thinsp;\u0026plusmn;\u0026thinsp;77.42, Fig.\u0026nbsp;4A). However, these differences were not statistically significant (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIL23 \u0026ndash;\u003c/b\u003e Stratified by study group, the baseline cytokine concentrations exhibited significantly higher amounts (p\u0026thinsp;=\u0026thinsp;0.003) in the T2DM group (55.16\u0026thinsp;\u0026plusmn;\u0026thinsp;33.05) than in normo-glycemic group implants (34.98\u0026thinsp;\u0026plusmn;\u0026thinsp;80.54) (Fig.\u0026nbsp;4B). In relation to the process of wound healing, both groups exhibited a similar (p\u0026thinsp;=\u0026thinsp;0.82) increase 10 days after surgery (V3, Table\u0026nbsp;3). These changes were statistically significant in the control group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIL-17 \u0026ndash;\u003c/b\u003e In total, the cytokine concentrations of IL-17 were lower compared to the other analytes, ranging from 0.73 to 6.44 pg/ml. No significant differences were detectable, neither stratified by time nor by study group (Fig.\u0026nbsp;3\u0026thinsp;+\u0026thinsp;4, Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGM-CSF \u0026ndash;\u003c/b\u003e No significant changes in cytokine concentration were found during the entire observation period. Moreover, test and control group implants displayed no different levels of GM-CSF.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present prospective clinical study was conducted to monitor the wound healing after placing narrow-diameter implants in T2DM and normo-glycemic patients by assessment of microcirculation and PMN-related cytokine expression. No significant differences between T2DM and healthy patients were observed, with the exception of pro-inflammatory cytokines IL-23 and Il-1\u0026szlig; during initial healing a few days after implant surgery.\u003c/p\u003e \u003cp\u003eIn our study, the perfusion of the peri-implant tissues was evaluated by Laser Doppler Flowmetry (LDF), a technique which allows for the detection of blood flow disturbances after surgical injury [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Both groups displayed significant reduction of microcirculation activity directly after surgery and one day post-op, when compared to baseline. However, microcirculation activity appeared to recover rapidly, returning to levels assessed before injury. Moreover, there was no significant difference regarding reduction of blood flow found between T2DM and healthy patients. The rationale behind this observation may be the similarly minimal-invasive surgical approach in both groups, since LDF measurements of post-surgical perfusion impairment were shown to correlate well with the extent of tissue trauma [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The transmucosal healing pattern may have contributed to this positive outcome, as there was no need in flap closure above the implant shoulder, which regularly requires a coronal advancement of the flap. The perfusion rates altered at similar altitude during the observation period closely reflecting the healing progress in both groups. Statistically significant impairment of perfusion was only found before and after application of local anesthesia containing the vasoconstrictor in both groups. Moreover, the perfusion rate showed statistically significant improvement at day 3 after the surgery. Regardless of systemic background, the recorded microcirculation was apparently related to uneventful healing progress. Furthermore, the plotted perfusion units per time were in accordance with those observed earlier in a beagle model, that aimed at monitoring the changes of microcirculation in the context of bone augmentation surgery [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese results, however, contradicted our expectations. As suggested by recent studies on the microvascular skin perfusion in diabetic patients, we anticipated a significantly lower perfusion rate in the wounds of diabetic patients [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The Hba1c values in the T2DM patient cohort with 7.34% might have been estimated to cause a bigger discrepancy. Nevertheless, the results highlight our hypothesis that T2DM patients may benefit from the NDI design because of the reduced wound healing burden associated with their use [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the concentrations of interleukin-1\u0026szlig;, interleukin-23, interleukin17A and GM-CSF were measured in PICF of NDI recently placed in the posterior jaw. The sampling of the PICF for analyzing the molecular content is considered a viable method for studying the wound healing biology around teeth and implants in humans [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In particular, the bead-based immunoassay applied in this study allows for a multiplex analysis of up to 96 analytes per sample.\u003c/p\u003e \u003cp\u003eInterleukin1\u0026szlig; is a pro-inflammatory cytokine which upregulates a plethora of inflammatory effector molecules such as chemokines and prostaglandins. Due to its abundance in inflamed and injured tissues of all kind, it is considered a ubiquitous biomarker for acute tissue inflammation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This study revealed upregulated concentrations of Il-1\u0026szlig; immediately after surgery presenting with significantly reduced levels up to the end of the observation period. Interestingly, the T2DM patients presented with non-significantly altered concentrations to the control group at any of the visits, indicating both, an innate initialization and resolution of the inflammatory process, respectively. The pattern of IL-1\u0026szlig; secretion to PICF is in line with a similar study conducted by [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], who reported similar results for IL-1\u0026szlig; concentrations in PIF of healthy patients.\u003c/p\u003e \u003cp\u003eAccording to a recent experimental study in diabetic rats, IL-23 levels are significantly increased under diabetic conditions [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. IL-23 is considered the main inducer of CD4\u0026thinsp;+\u0026thinsp;T-lymphocyte differentiation towards IL-17 producing Th17 cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In context of wound healing, large amounts of Th17 helper cells are associated with a delay in wound closure. The inhibition of the backbone IL-23 / Il-17 axis may even promote wound healing under diabetic conditions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our results demonstrated significantly higher levels of IL-23 at implants of T2DM patients, when compared to the control group. Over the course of the observation period (V2-V4), however, the IL-23 and IL-17 concentrations equalized to insignificant differences. These results are corroborated further by the works of Santos et al. and Vieira Ribeiro et al., who reported no significant increase in IL-23 expression among diabetic and healthy patients suffering from periodontitis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe last investigated analyte, GM-CSF, did not show any timing- or group-related differences. GM-CSF is a multipotent growth factor responsible for granulopoiesis and keratinocyte-related proliferation and re-epithelialization, and a variety of studies has verified its necessity for complication-free wound healing [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, unchanged GM-CSF levels in T2DM patients may have been associated with the uneventful healing period observed in this trial.\u003c/p\u003e \u003cp\u003eThis analysis was nested in a larger pilot study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, the presented results have some obvious limitations. As indicated by the large variability among individuals, selected datasets may not be adequately powered. Nevertheless, we were able to elucidate\u003c/p\u003e \u003cp\u003eThe measured effector molecules have a pivotal role in regulating the PMN-mediated early wound healing, which did not appear significantly affected by the T2DM condition. However, while diabetic patients showed a more pro-inflammatory initial cytokine profile, wound healing and PMN response appeared similar in diabetic and non-diabetic patients on a longer term. Moreover, these results corroborate our previously reported findings of similar clinical success placing reduced-diameter implants in these patients [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In conclusion, hydrophilic-surface titanium-zirconium implants with reduced diameter exhibit no molecular alterations regarding wound healing in T2DM patients and may thus be a viable option for this group of patients.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003efigures; D.K..: conceptualization of the study, proofreading; A.H.: experimental procedures and measurements; H.B.: manuscript drafting and proof reading; A.F.: conceptualization of the study; implant treatments, proofreading the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr. Kai R. Fischer for conducting implant surgeries during the study.\u003c/p\u003e\n\u003cp\u003eThe authors declare that they do not have any commercial, proprietary or financial interest in the products or companies described in this article.\u003cbr\u003e\u0026nbsp;The study was partly funded by a restricted grant and donation of the narrow diameter implants from Institut Straumann AG, Basel, Switzerland \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegistration number: NCT04630691 (clinicaltrials.gov)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they do not have any commercial, proprietary or financial interest in the products or companies described in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by a restricted grant including the donation of the NDI’s by the Institute Straumann AG, Basel, Switzerland\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the Declaration of Helsinki. Approval was granted by the Witten/Herdecke University Ethics Committee (108/2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all individual participants involved in the study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZimmet P, Alberti KG, Magliano DJ, Bennett PH (2016) Diabetes mellitus statistics on prevalence and mortality: facts and fallacies. Nature Reviews Endocrinology 12 (10):616\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaw JE, Sicree RA, Zimmet PZ (2010) Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes research and clinical practice 87 (1):4\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrain WD, Pald\u0026aacute;nius P (2018) Diabetes, cardiovascular disease and the microcirculation. 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Microvascular Research 124:91\u0026ndash;96\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomanos GE, Delgado-Ruiz R, Sculean A (2019) Concepts for prevention of complications in implant therapy. Periodontology 2000 81 (1):7\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaner D, Zhao H, Terheyden H, Friedmann A (2014) Submucosal implantation of soft tissue expanders does not affect microcirculation. Clinical oral implants research 25 (7):867\u0026ndash;870\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedmann A, Ozmeric N, Bernimoulin J-P, Kleber B-M, Ayhan E, Aykan T, G\u0026ouml;kmenoğlu C (2014) Receptor activator of NF-kappaB ligand (RANKL) and CD 31 expressions in chronic periodontitis patients before and after surgery. Central-European journal of immunology 39 (4):508\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedmann A, Friedrichs M, Kaner D, Kleber BM, Bernimoulin JP (2006) Calprotectin and cross-linked N‐terminal telopeptides in peri‐implant and gingival crevicular fluid. Clinical oral implants research 17 (5):527\u0026ndash;532\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraves DT, Nooh N, Gillen T, Davey M, Patel S, Cottrell D, Amar S (2001) IL-1 plays a critical role in oral, but not dermal, wound healing. The Journal of Immunology 167 (9):5316\u0026ndash;5320\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuarnieri R, Miccoli G, Reda R, Mazzoni A, Di Nardo D, Testarelli L (2021) Sulcus fluid volume, IL-6 and Il‐1b concentrations in periodontal and peri‐implant tissues comparing machined and laser‐microtextured collar/abutment surfaces during 12 weeks of healing: a split‐mouth RCT. 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The FASEB Journal 32 (4):2086\u0026ndash;2094\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParis I, Charreau S, Guignouard E, Garnier M, Favot-Laforge L, Huguier V, Bernard F-X, Morel F, Lecron J-C (2012) O014 Critical role of Th17 pro-inflammatory cytokines to delay skin wound healing. Cytokine 59 (3):503\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVieira Ribeiro F, de Mendon\u0026ccedil;a AC, Santos VR, Bastos MF, Figueiredo LC, Duarte PM (2011) Cytokines and bone-related factors in systemically healthy patients with chronic periodontitis and patients with type 2 diabetes and chronic periodontitis. Journal of periodontology 82 (8):1187\u0026ndash;1196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantos VR, Ribeiro FV, Lima JA, Napimoga MH, Bastos MF, Duarte PM (2010) Cytokine levels in sites of chronic periodontitis of poorly controlled and well-controlled type 2 diabetic subjects. Journal of clinical periodontology 37 (12):1049\u0026ndash;1058\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMann A, Niekisch K, Schirmacher P, Blessing M Granulocyte\u0026ndash;macrophage colony-stimulating factor is essential for normal wound healing. In: Journal of Investigative Dermatology Symposium Proceedings, 2006. vol\u0026nbsp;1. Elsevier, pp\u0026nbsp;87\u0026ndash;92\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchirmacher P, Mann A, Breuhahn K, Blessing M (2001) Keratinocyte-Derived Granulocyte-Macrophage Colony Stimulating Factor Accelerates Wound Healing: Stimulation of Keratinocyte Proliferation, Granulation Tissue Formation, and Vascularization. Journal of Investigative Dermatology 117 (6):1382\u0026ndash;1390\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastro-Dopico T, Fleming A, Dennison TW, Ferdinand JR, Harcourt K, Stewart BJ, Cader Z, Tuong ZK, Jing C, Lok LS (2020) GM-CSF calibrates macrophage defense and wound healing programs during intestinal infection and inflammation. Cell reports 32 (1):107857\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1-3 are available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Supplementary","content":"\u003cp\u003eThe STROBE checklist is not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"type 2 diabetes, gingival perfusion, gingival vascularization, wound healing, cytokines, PMN","lastPublishedDoi":"10.21203/rs.3.rs-1614566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1614566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThe aim of this study was to assess the microcirculation and the expression patterns of wound-healing related cytokines around narrow-diameter implants in type 2 diabetes mellitus (T2DM) and normo-glycemic patients.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eA total of 31 patients, 15 of which diagnosed with T2DM (HbA1c\u0026thinsp;\u0026gt;\u0026thinsp;6.5) and 16 normo-glycemic patients received narrow diameter implants in the posterior mandible or maxilla. During the 3-month healing period, soft-tissue perfusion was monitored via Laser-Doppler flowmetry. Peri-implant fluid (PICF) was harvested and analyzed for concentrations of interleukin-1\u0026szlig; (IL-1\u0026szlig;), interleukin-23 (IL-23), interleukin-17 (IL-17) and granulocyte colony-stimulating factor (G-CSF) by a multiplex, bead-based immunoassay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMicrocirculatory perfusion patterns during wound healing exhibited no significant differences throughout the observation period. IL-1\u0026szlig; concentrations were expectedly elevated during the early phases of wound healing. At the first visit after surgery, IL-23 concentrations were significantly higher in implants of diabetic patients. This difference was diminished over the course of the observation period. For the other tested analytes, no differences were observable between both groups.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWound healing after implant surgery was similar in T2DM and healthy patients. Hydrophilic-surface titanium-zirconium implants with reduced diameter may be considered for implant therapy of Diabetes mellitus type II patients.\u003c/p\u003e","manuscriptTitle":"Microcirculation and Neutrophil-related cytokine concentrations are not altered around narrow diameter implants in T2DM patients during wound healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-16 15:41:17","doi":"10.21203/rs.3.rs-1614566/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-07-31T10:28:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-13T09:30:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9264beb6-a28f-497e-a07b-167d54408062","date":"2022-05-22T11:31:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-22T08:05:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-10T09:34:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-05-10T08:44:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Oral Investigations","date":"2022-05-01T19:27:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"248062ac-c753-4959-9151-94c758cbd0df","owner":[],"postedDate":"May 16th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-01T13:44:14+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-16 15:41:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1614566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1614566","identity":"rs-1614566","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}