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Mineralized Tissues Repair and Regeneration

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A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Tissues and Organs".

Deadline for manuscript submissions: closed (25 February 2023) | Viewed by 46425

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Special Issue Editors

Prof. Dr. Roberta Di Pietro

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1. Department of Medicine and Aging Sciences, Section of Biomorphology, G. d’Annunzio University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy
2. Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Department of Biology, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA
Interests: aging; cell signaling; cell death; apoptosis; TRAIL; CREB; ionizing radiation; hematological malignancies; pterygium; histology; embryology; biomorphology; rehabilitation; sports medicine
Special Issues, Collections and Topics in MDPI journals

Dr. Florelle Gindraux

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Guest Editor

1. Service de Chirurgie Orthopédique, Traumatologique et Plastique, CHU Besançon, F-25000 Besançon, France
2. Service de Chirurgie Pédiatrique, CHU Besançon, F-25000 Besançon, France
3. Laboratoire de Nanomédecine, Imagerie, Thérapeutique EA 4662, Université Bourgogne Franche-Comté, F-25000 Besançon, France
Interests: advanced therapy medicinal products; regenerative medicine; tissue engineering; adult and perinatal (placenta) mesenchymal stromal cells; perinatal derivates; fetal membranes (amnion and chorion); foreign body membranes: induced membrane and periprosthetic capsules; bone substitutes; biomaterials; medical devices
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Mineralized tissues are hierarchically organized and are temporally and spatially heterogeneous due to continuous (re)modeling. Their mechanical properties depend on macro- and micro-architecture, as well as on material characteristics at the micro-nanoscale. This is particularly important for the understanding of the structure–function relationship in normal, ageing, and diseased bone and for predicting fracture risk—a prerequisite for prevalence and treatment of bone fragility.

Tooth loss is a global health problem representing a burden to society and economy. It affects an individual’s capacity for biting, chewing, smiling, speaking, and psychosocial wellbeing. Complete loss of natural teeth is widespread, particularly affecting older people. Dental caries, periodontal disease, and genetic disorders are major causes of tooth loss. Regenerative dentistry is an emerging field that challenges modern dentistry to improve dental research and translate scientific knowledge into new future clinical treatments.

This Special Issue is focused on the physiological processes of repair and regeneration of mineralized tissues with particular interest in the cellular mechanisms and/or paracrine effects involved in bone and mineralized dental tissues healing during aging/diseases or after injury, infections, and pharmacological or surgical procedures. It will also cover all tissues undergoing a natural or induced mineralization or calcification process under physiological or pathological conditions as well as in diagnostic and therapeutic contexts. Additionally, we are interested in the understanding of the proangiogenic, antimicrobial/bacterial, osteoinductive, osteoconductive, and mechanical effects exerted by therapeutics or different procedures. This Special Issue will cover histology, surgery, biomaterials, cell therapy, and tissue engineering, but also ex vivo/in vitro cell biology experimental models, and in vivo and clinic experimentations.

Prof. Dr. Roberta Di Pietro
Dr. Florelle Gindraux
Guest Editors

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Keywords

bone
tooth
calcifications
cartilage
repair
regeneration
tissue engineering
biomaterials
histology
imaging techniques
experimental models

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Mineralized Tissues Repair and Regeneration 2.0 in Cells (3 articles)

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Research

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26 pages, 5429 KiB

Open AccessArticle

Functionalizing Collagen Membranes with MSC-Conditioned Media Promotes Guided Bone Regeneration in Rat Calvarial Defects

by Siddharth Shanbhag, Carina Kampleitner, Niyaz Al-Sharabi, Samih Mohamed-Ahmed, Karol Ali Apaza Alccayhuaman, Patrick Heimel, Stefan Tangl, Andreas Beinlich, Neha Rana, Mariano Sanz, Einar K. Kristoffersen, Kamal Mustafa and Reinhard Gruber

Cells 2023, 12(5), 767; https://doi.org/10.3390/cells12050767 - 28 Feb 2023

Cited by 11 | Viewed by 2840

Abstract

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel ‘off-the-shelf’ strategy for GBR. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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26 pages, 5945 KiB

Open AccessArticle

Osteogenic Potential of Sheep Mesenchymal Stem Cells Preconditioned with BMP-2 and FGF-2 and Seeded on an nHAP-Coated PCL/HAP/β-TCP Scaffold

by Sandra Stamnitz, Agnieszka Krawczenko, Urszula Szałaj, Żaneta Górecka, Agnieszka Antończyk, Zdzisław Kiełbowicz, Wojciech Święszkowski, Witold Łojkowski and Aleksandra Klimczak

Cells 2022, 11(21), 3446; https://doi.org/10.3390/cells11213446 - 31 Oct 2022

Cited by 10 | Viewed by 2899

Abstract

Mesenchymal stem cells (MSCs) attract interest in regenerative medicine for their potential application in bone regeneration. However, direct transplantation of cells into damaged tissue is not efficient enough to regenerate large bone defects. This problem could be solved with a biocompatible scaffold. Consequently, bone tissue engineering constructs based on biomaterial scaffolds, MSCs, and osteogenic cytokines are promising tools for bone regeneration. The aim of this study was to evaluate the effect of FGF-2 and BMP-2 on the osteogenic potential of ovine bone marrow-derived MSCs seeded onto an nHAP-coated PCL/HAP/β-TCP scaffold in vitro and its in vivo biocompatibility in a sheep model. In vitro analysis revealed that cells preconditioned with FGF-2 and BMP-2 showed a better capacity to adhere and proliferate on the scaffold than untreated cells. BM-MSCs cultured in an osteogenic medium supplemented with FGF-2 and BMP-2 had the highest osteogenic differentiation potential, as assessed based on Alizarin Red S staining and ALP activity. qRT-PCR analysis showed increased expression of osteogenic marker genes in FGF-2- and BMP-2-treated BM-MSCs. Our pilot in vivo research showed that the implantation of an nHAP-coated PCL/HAP/β-TCP scaffold with BM-MSCs preconditioned with FGF-2 and BMP-2 did not have an adverse effect in the sheep mandibular region and induced bone regeneration. The biocompatibility of the implanted scaffold-BM-MSC construct with sheep tissues was confirmed by the expression of early (collagen type I) and late (osteocalcin) osteogenic proteins and a lack of an elevated level of proinflammatory cytokines. These findings suggest that FGF-2 and BMP-2 enhance the osteogenic differentiation potential of MSCs grown on a scaffold, and that such a tissue engineering construct may be used to regenerate large bone defects. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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19 pages, 8751 KiB

Open AccessArticle

The Cytotoxicity and Genotoxicity of Bioactive Dental Materials

by Marta Kunert, Wioletta Rozpedek-Kaminska, Grzegorz Galita, Salvatore Sauro, Rim Bourgi, Louis Hardan, Ireneusz Majsterek and Monika Lukomska-Szymanska

Cells 2022, 11(20), 3238; https://doi.org/10.3390/cells11203238 - 15 Oct 2022

Cited by 21 | Viewed by 5111

Abstract

The promotion of biologically based treatment strategies in restorative dentistry is of paramount importance, as invasive treatments should be avoided to maintain the tooth’s vitality. This study aimed to assess the biocompatibility of commercially available bioactive materials that can be used for dental pulp capping. The study was performed with a monocyte/macrophage peripheral blood SC cell line (ATCC CRL-9855) on the following six specific bioactive materials: ProRoot MTA (Dentsply Sirona), MTA Angelus (Angelus), Biodentine (Septodont), TheraCal LC (Bisco), ACTIVA BioACTIVE (Pulpdent) and Predicta Bioactive Bulk (Parkell). The cytotoxicity of the investigated agents was measured using a resazurin-based cell viability assay, while the genotoxicity was evaluated using an alkaline comet assay. Additionally, flow cytometry (FC) apoptosis detection was conducted with a FITC (fluorescein isothiocyanate) Annexin V Apoptosis Detection Kit I. FC cell-cycle arrest assessment was carried out with propidium iodide staining. The results of this study showed no significant cytotoxicity and genotoxicity (p > 0.05) in ProRoot MTA, MTA Angelus, Biodentine, ACTIVA BioACTIVE and Predicta Bioactive. Conversely, TheraCal LC presented a significant decrease (p < 0.001). In conclusion, due to excellent biocompatibility and low cytotoxicity, MTA, Biodentine, ACTIVA BioACTIVE and Predicta Bioactive may be suitable for pulp capping treatments. On the other hand, due to the high cytotoxicity of TheraCal LC, its use should be avoided in vital pulp therapies. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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16 pages, 5282 KiB

Open AccessArticle

Novel Epigenetic Modulation Chitosan-Based Scaffold as a Promising Bone Regenerative Material

by Teerawat Sukpaita, Suwabun Chirachanchai, Theerapat Chanamuangkon, Katanchalee Nampuksa, Naruporn Monmaturapoj, Piyamas Sumrejkanchanakij, Atiphan Pimkhaokham and Ruchanee Salingcarnboriboon Ampornaramveth

Cells 2022, 11(20), 3217; https://doi.org/10.3390/cells11203217 - 13 Oct 2022

Cited by 9 | Viewed by 3138

Abstract

Bone tissue engineering is a complicated field requiring concerted participation of cells, scaffolds, and osteoactive molecules to replace damaged bone. This study synthesized a chitosan-based (CS) scaffold incorporated with trichostatin A (TSA), an epigenetic modifier molecule, to achieve promising bone regeneration potential. The scaffolds with various biphasic calcium phosphate (BCP) proportions: 0%, 10%, 20%, and 40% were fabricated. The addition of BCP improved the scaffolds’ mechanical properties and delayed the degradation rate, whereas 20% BCP scaffold matched the appropriate scaffold requirements. The proper concentration of TSA was also validated. Our developed scaffold released TSA and sustained them for up to three days. The scaffold with 800 nM of TSA showed excellent biocompatibility and induced robust osteoblast-related gene expression in the primary human periodontal ligament cells (hPDLCs). To evaluate in vivo bone regeneration potential, the scaffolds were implanted in the mice calvarial defect model. The excellent bone regeneration ability was further demonstrated in the micro-CT and histology sections compared to both negative control and commercial bone graft product. New bone formed in the CS/BCP/TSA group revealed a trabeculae-liked characteristic of the mature bone as early as six weeks. The CS/BCP/TSA scaffold is an up-and-coming candidate for the bone tissue engineering scaffold. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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21 pages, 7395 KiB

Open AccessFeature PaperArticle

The Effect of Germanium-Loaded Hydroxyapatite Biomaterials on Bone Marrow Mesenchymal Stem Cells Growth

by Jeevithan Elango, Rodion Bushin, Artiom Lijnev, Piedad N. De Aza, Carlos Pérez-Albacete Martínez, José Manuel Granero Marín, Ana Belen Hernandez, Luis Ramón Meseguer Olmo and José Eduardo Maté Sánchez De Val

Cells 2022, 11(19), 2993; https://doi.org/10.3390/cells11192993 - 26 Sep 2022

Cited by 16 | Viewed by 2588

Abstract

Hydroxyapatite (HA) is a hard mineral component of mineralized tissues, mainly composed of calcium and phosphate. Due to its bioavailability, HA is potentially used for the repair and regeneration of mineralized tissues. For this purpose, the properties of HA are significantly improved by adding natural and synthetic materials. In this sense, the germanium (Ge) mineral was loaded in HA biomaterial by cold isostatic pressure for the first time and characterization and biocompatibility using bone marrow mesenchymal stem cells (BM-MSCs) were investigated. The addition of Ge at 5% improved the solubility (3.32%), stiffness (18.34 MPa), water holding (31.27%) and biodegradation (21.87%) properties of HA, compared to control. Compared to all composite biomaterials, the drug-releasing behavior of HA-3% Ge was higher at pH 1 and 3 and the maximum drug release was obtained at pH 7 and 9 with HA-5% Ge biomaterials. Among the different mediums tested, the DMEM-medium showed a higher drug release rate, especially at 60 min. HA-Ge biomaterials showed better protein adhesion and apatite layer formation, which ultimately proves the compatibility in BM-MSCs culture. Except for higher concentrations of HA (5 and 10 mg/mL), the different concentrations of Ge and HA and wells coated with 1% of HA-1% Ge had higher BM-MSCs growth than control. All these findings concluded that the fabricated HA biomaterials loaded with Ge could be the potential biomaterial for culturing mammalian cells towards mineralized tissue repair and regeneration. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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16 pages, 5004 KiB

Open AccessFeature PaperArticle

Hybrid Mineral/Organic Material Induces Bone Bridging and Bone Volume Augmentation in Rat Calvarial Critical Size Defects

by Marie Dubus, Loïc Scomazzon, Charlotte Ledouble, Julien Braux, Abdelilah Beljebbar, Laurence Van Gulick, Adrien Baldit, Caroline Gorin, Halima Alem, Nicole Bouland, Marissa Britton, Jessica Schiavi, Ted J. Vaughan, Cédric Mauprivez and Halima Kerdjoudj

Cells 2022, 11(18), 2865; https://doi.org/10.3390/cells11182865 - 14 Sep 2022

Cited by 4 | Viewed by 2978

Abstract

In craniofacial bone defects, the promotion of bone volume augmentation remains a challenge. Finding strategies for bone regeneration such as combining resorbable minerals with organic polymers would contribute to solving the bone volume roadblock. Here, dicalcium phosphate dihydrate, chitosan and hyaluronic acid were used to functionalize a bone-side collagen membrane. Despite an increase in the release of inflammatory mediators by human circulating monocytes, the in vivo implantation of the functionalized membrane allowed the repair of a critical-sized defect in a calvaria rat model with de novo bone exhibiting physiological matrix composition and structural organization. Microtomography, histological and Raman analysis combined with nanoindentation testing revealed an increase in bone volume in the presence of the functionalized membrane and the formation of woven bone after eight weeks of implantation; these data showed the potential of dicalcium phosphate dihydrate, chitosan and hyaluronic acid to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in animal models. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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16 pages, 2955 KiB

Open AccessArticle

Osteoblasts in a Perfusion Flow Bioreactor—Tissue Engineered Constructs of TiO₂ Scaffolds and Cells for Improved Clinical Performance

by Maria Schröder, Janne Elin Reseland and Håvard Jostein Haugen

Cells 2022, 11(13), 1995; https://doi.org/10.3390/cells11131995 - 22 Jun 2022

Cited by 9 | Viewed by 3841

Abstract

Combining biomaterial scaffolds with cells serves as a promising strategy for engineering critical size defects; however, homogenous cellular growth within large scaffolds is challenging. Mechanical stimuli can enhance bone regeneration by modulating cellular growth and differentiation. Here, we compare dynamic seeding in a perfusion flow bioreactor with static seeding for a synthetic bone scaffold for up to 21 days using the cell line MC3T3-E1 and primary human osteoblast, confocal laser scanning microscopy, and real-time reverse transcriptase-polymerase chain reaction. The secretion of bone-related proteins was quantified using multiplex immunoassays. Dynamic culture improved cellular distribution through the TiO₂ scaffold and induced a five-fold increase in cell number after 21 days. The relative mRNA expression of osteopontin of MC3T3-E1 was 40-fold enhanced after 7 and 21 days at a flow rate of 0.08 mL/min, and that of collagen type I alpha I expression was 18-fold after 21 days. A flow rate of 0.16 mL/min was 10-fold less effective. Dynamic culture increased the levels of dickkopf-related protein 1 (60-fold), osteoprotegrin (29-fold), interleukin-6 (23-fold), interleukin-8 (36-fold), monocyte chemoattractant protein 1 (28-fold) and vascular endothelial growth factor (6-fold) in the medium of primary human osteoblasts after 21 days compared to static seeding. The proposed method may have clinical potential for bone tissue engineering. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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Review

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24 pages, 3364 KiB

Open AccessReview

Macrophages: From Simple Phagocyte to an Integrative Regulatory Cell for Inflammation and Tissue Regeneration—A Review of the Literature

by Andreas Mamilos, Lina Winter, Volker H. Schmitt, Friedrich Barsch, David Grevenstein, Willi Wagner, Maximilian Babel, Karsten Keller, Christine Schmitt, Florian Gürtler, Stephan Schreml, Tanja Niedermair, Markus Rupp, Volker Alt and Christoph Brochhausen

Cells 2023, 12(2), 276; https://doi.org/10.3390/cells12020276 - 11 Jan 2023

Cited by 34 | Viewed by 6910

Abstract

The understanding of macrophages and their pathophysiological role has dramatically changed within the last decades. Macrophages represent a very interesting cell type with regard to biomaterial-based tissue engineering and regeneration. In this context, macrophages play a crucial role in the biocompatibility and degradation of implanted biomaterials. Furthermore, a better understanding of the functionality of macrophages opens perspectives for potential guidance and modulation to turn inflammation into regeneration. Such knowledge may help to improve not only the biocompatibility of scaffold materials but also the integration, maturation, and preservation of scaffold-cell constructs or induce regeneration. Nowadays, macrophages are classified into two subpopulations, the classically activated macrophages (M1 macrophages) with pro-inflammatory properties and the alternatively activated macrophages (M2 macrophages) with anti-inflammatory properties. The present narrative review gives an overview of the different functions of macrophages and summarizes the recent state of knowledge regarding different types of macrophages and their functions, with special emphasis on tissue engineering and tissue regeneration. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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19 pages, 1122 KiB

Open AccessReview

Reporting Criteria for Clinical Trials on Medication-Related Osteonecrosis of the Jaw (MRONJ): A Review and Recommendations

by Camille Gaudet, Stephane Odet, Christophe Meyer, Brice Chatelain, Elise Weber, Anne-Laure Parmentier, Stéphane Derruau, Sébastien Laurence, Cédric Mauprivez, Esteban Brenet, Halima Kerdjoudj, Mathilde Fenelon, Jean-Christophe Fricain, Narcisse Zwetyenga, David Hoarau, Rémi Curien, Eric Gerard, Aurélien Louvrier and Florelle Gindraux

Cells 2022, 11(24), 4097; https://doi.org/10.3390/cells11244097 - 16 Dec 2022

Cited by 9 | Viewed by 2776

Abstract

Medication-related osteonecrosis of the jaw (MRONJ) is a complication caused by anti-resorptive agents and anti-angiogenesis drugs. Since we wanted to write a protocol for a randomized clinical trial (RCT), we reviewed the literature for the essential information needed to estimate the size of the active patient population and measure the effects of therapeutics. At the same time, we designed a questionnaire intended for clinicians to collect detailed information about their practices. Twelve essential criteria and seven additional items were identified and compiled from 43 selected articles. Some of these criteria were incorporated in the questionnaire coupled with data on clinical practices. Our review found extensive missing data and a lack of consensus. For example, the success rate often combined MRONJ stages, diseases, and drug treatments. The occurrence date and evaluation methods were not harmonized or quantitative enough. The primary and secondary endpoints, failure definition, and date coupled to bone measurements were not well established. This information is critical for writing a RCT protocol. With this review article, we aim to encourage authors to contribute all their findings in the field to bridge the current knowledge gap and provide a stronger database for the coming years. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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34 pages, 6554 KiB

Open AccessReview

Effect of Collagen Crosslinkers on Dentin Bond Strength of Adhesive Systems: A Systematic Review and Meta-Analysis

by Louis Hardan, Umer Daood, Rim Bourgi, Carlos Enrique Cuevas-Suárez, Walter Devoto, Maciej Zarow, Natalia Jakubowicz, Juan Eliezer Zamarripa-Calderón, Mateusz Radwanski, Giovana Orsini and Monika Lukomska-Szymanska

Cells 2022, 11(15), 2417; https://doi.org/10.3390/cells11152417 - 4 Aug 2022

Cited by 41 | Viewed by 5283

Abstract

This study aimed to identify the role of crosslinking agents in the resin–dentin bond strength (BS) when used as modifiers in adhesives or pretreatments to the dentin surface through a systematic review and meta-analysis. This paper was conducted according to the directions of the PRISMA 2020 statement. The research question of this review was: “Would the use of crosslinkers agents improve the BS of resin-based materials to dentin?” The literature search was conducted in the following databases: Embase, PubMed, Scielo, Scopus, and Web of Science. Manuscripts that reported the effect on the BS after the use of crosslinking agents were included. The meta-analyses were performed using Review Manager v5.4.1. The comparisons were performed by comparing the standardized mean difference between the BS values obtained using the crosslinker agent or the control group. The subgroup comparisons were performed based on the adhesive strategy used (total-etch or self-etch). The immediate and long-term data were analyzed separately. A total of 50 articles were included in the qualitative analysis, while 45 articles were considered for the quantitative analysis. The meta-analysis suggested that pretreatment with epigallocatechin-3-gallate (EGCG), carbodiimide, ethylenediaminetetraacetic acid (EDTA), glutaraldehyde, and riboflavin crosslinking agents improved the long-term BS of resin composites to dentin (p ≤ 0.02). On the other hand, the use of proanthocyanidins as a pretreatment improved both the immediate and long-term BS values (p ≤ 0.02). When incorporated within the adhesive formulation, only glutaraldehyde, riboflavin, and EGCG improved the long-term BS to dentin. It could be concluded that the application of different crosslinking agents such as carbodiimide, EDTA, glutaraldehyde, riboflavin, and EGCG improved the long-term BS of adhesive systems to dentin. This effect was observed when these crosslinkers were used as a separate step and when incorporated within the formulation of the adhesive system. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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Other

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22 pages, 6306 KiB

Open AccessSystematic Review

Effect of Different Application Modalities on the Bonding Performance of Adhesive Systems to Dentin: A Systematic Review and Meta-Analysis

by Louis Hardan, Rim Bourgi, Carlos Enrique Cuevas-Suárez, Walter Devoto, Maciej Zarow, Paulo Monteiro, Natalia Jakubowicz, Amine El Zoghbi, Dariusz Skaba, Davide Mancino, Naji Kharouf, Youssef Haïkel and Monika Lukomska-Szymanska

Cells 2023, 12(1), 190; https://doi.org/10.3390/cells12010190 - 3 Jan 2023

Cited by 22 | Viewed by 6717

Abstract

Diverse types of dental adhesives exhibit different cytotoxic outcomes on cells in vitro. Currently, no standard adhesive application technique has so far been decisive for clinicians for better durability of resin–dentin bonds of adhesive systems. The purpose of this study was to systematically review the literature to evaluate the bonding performance of adhesive systems to dentin by using different application modalities. The systematic research strategy was conducted by two reviewers among multiple databases: PubMed, Scopus, Web of Science, Embase, and Scielo. In vitro studies reporting the effects of additional steps for the application of adhesive systems on the bond strength to dentin were selected. Meta-analysis was performed using Review Manager Software version 5.3.5 using the random effects model. The methodological quality of each in vitro study was assessed according to the parameters of a previous systematic review. The electronic research through different databases generated a total of 8318 references. After the examination of titles and abstracts, a total of 106 potentially relevant studies accessed the full-text evaluation phase. After full-text examination, 78 publications were included for the qualitative analysis, and 68 studies were included in the meta-analysis. Regarding the etch-and-rinse adhesive systems, the application modalities that improved the overall bond strength were the application of a hydrophobic resin layer (p = 0.005), an extended application time (p < 0.001), an application assisted by an electric current (p < 0.001), a double-layer application (p = 0.05), the agitation technique (p = 0.02), and the active application of the adhesive (p < 0.001). For self-etch adhesive systems, the techniques that improved the overall bond strength were the application of a hydrophobic resin layer (p < 0.001), an extended application time (p = 0.001), an application assisted by an electric current (p < 0.001), a double-layer application (p < 0.001), the agitation technique (p = 0.01), and the active application of the adhesive (p < 0.001). The in vitro evidence suggests that the application of adhesive systems using alternative techniques or additional strategies may be beneficial for improving their bond strength to dentin. The application modalities that favored the overall bond strength to dentin were an extended application time, a double-layer application, an application assisted by an electric current, the active application of the adhesive, and the application of a hydrophobic resin layer. Worth mentioning is that some techniques are intended to increase the degree of the conversion of the materials, and therefore, improvements in the biocompatibility of the materials can be expected. Full article

(This article belongs to the Special Issue Mineralized Tissues Repair and Regeneration)

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