Application In Periodontal Regeneration Research Articles

Evaluation of the biological effects of amelogenin on human oral keratinocytes.

Amelogenins are clinically used in periodontal regeneration as main components of root surface modifying agents, even without specifically preventing the premature colonization of the healing tissue defect by means of a physical barrier membrane. The objective of this study was to investigate the effects of human amelogenin on the proliferation, migration, and morphology of Immortalized Human Oral Keratinocytes (iHOKs). Immortalized Human Oral Keratinocytes were expanded in Keratinocyte Growth Medium-2 (KGM-2). Full-length recombinant amelogenin protein was diluted in KGM-2 in five concentrations (10ng/ml, 100ng/ml, 1.000ng/ml, 5.000ng/ml and 10.000ng/ml). iHOKs were cultured in medium supplemented with the amelogenin dilutions. Samples without amelogenin served as control. Cell metabolism and cell proliferation together with cell migration were evaluated at day 7, 14, 21. At day 7, iHOKs treated with 10,000ng/ml showed a significant decrease in keratinocytes´ proliferation. The metabolic activity at this timepoint was significantly lower for concentrations ≥1000ng/ml. At days 14 and 21, both the addition of 5000ng/ml and even more 10,000ng/ml amelogenin reduced significantly the proliferation of keratinocytes. The effects on the metabolic activity for these timepoints were visible already with 100ng/ml. Treatment of iHOKs with amelogenin of ≥1000ng/ml led to inhibitory effects on cell migration already after 24h. The full-length recombinant amelogenin has a significant biological impact on iHOKs. The increasing dose dependent inhibitory effects of amelogenin shown on iHOKs might explain the disruption of the apical migration of the junctional epithelium during regenerative healing. Amelogenin, presents time- and dose-dependent inhibitory effects on the growth of keratinocytes, which might explain the biological rationale behind its application in periodontal regeneration.

Nanomaterials in Scaffolds for Periodontal Tissue Engineering: Frontiers and Prospects.

The regeneration of periodontium represents important challenges to controlling infection and achieving functional regeneration. It has been recognized that tissue engineering plays a vital role in the treatment of periodontal defects, profiting from scaffolds that create the right microenvironment and deliver signaling molecules. Attributable to the excellent physicochemical and antibacterial properties, nanomaterials show great potential in stimulating tissue regeneration in tissue engineering. This article reviewed the up-to-date development of nanomaterials in scaffolds for periodontal tissue engineering. The paper also represented the merits and defects of different materials, among which the biocompatibility, antibacterial properties, and regeneration ability were discussed in detail. To optimize the project of choosing materials and furthermore lay the foundation for constructing a series of periodontal tissue engineering scaffolds, various nanomaterials and their applications in periodontal regeneration were introduced.

Amniotic Membrane: An Approach to Periodontal Regeneration.

Over the years, various materials have been used for scaffold-based periodontal tissue engineering to regenerate lost periodontal tissues. The use of amniotic membrane (AM) as a scaffold for periodontal regeneration has gained great interest among researchers. This narrative review aims to appraise the properties of AM and its potential clinical applications in periodontal regeneration. PubMed, ScienceDirect, Scopus, and Wiley Online Library databases were searched for relevant articles that highlighted the properties and applications of AM in periodontal regeneration. AM has a unique structure and components contributing to its exceptional properties such as anti-inflammatory (presence of anti-inflammatory factors), low immunogenicity (presence of human leukocyte antigen-G), anti-scarring (downregulation of transforming growth factor-β), antimicrobial (expression of antimicrobial factors), promotion of epithelialization (production of growth factors), and reduction of pain (protection of exposed nerve endings). Its use in the treatment of periodontal tissue defect has shown to be effective. AM showed various beneficial properties as an ideal scaffold. Future studies and long-term clinical trials on the efficacy and survival rate of AM are required to completely understand the potential application of AM in periodontal regeneration.

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications.

Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates such as PL. In this article, the use of extracellular vesicles (EVs) derived from platelet lysate (PL) and their combination with hyaluronic acid biomaterials (HA) in an in vitro wound healing assay is investigated. EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro in gingival fibroblasts and keratinocytes for biocompatibility (LDH activity and metabolic activity) and by an in vitro wound-healing assay and gene expression analysis. EVs and EVs-HA treatments were biocompatible in gingival fibroblasts and keratinocytes and showed an increase in wound healing in vitro compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed after the treatment with EVs. EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet-derived EVs could emerge as a new application for periodontal regeneration in combination with biomaterials in order to enhance their clinical use.

Bioactive Glasses in Periodontal Regeneration: Existing Strategies and Future Prospects-A Literature Review.

The present review deals with bioactive glasses (BGs), a class of biomaterials renowned for their osteoinductive and osteoconductive capabilities, and thus widely used in tissue engineering, i.e., for the repair and replacement of damaged or missing bone. In particular, the paper deals with applications in periodontal regeneration, with a special focus on in vitro, in vivo and clinical studies. The study reviewed eligible publications, identified on the basis of inclusion/exclusion criteria, over a ranged time of fifteen years (from 1 January 2006 to 31 March 2021). While there are many papers dealing with in vitro tests, only a few have reported in vivo (in animal) research, or even clinical trials. Regardless, BGs seem to be an adequate choice as grafts in periodontal regeneration.

Assessment of the Vanillin Anti-Inflammatory and Regenerative Potentials in Inflamed Primary Human Gingival Fibroblast.

Background Inflammatory responses have been associated with delayed oral mucosal wound healing and the pathogenesis of the periodontal disease. The invasion of microbes into the tissues and the establishment of a chronic infection may be due to impaired healing. The protracted inflammatory phase may delay wound healing and probably support tissue fibrosis and reduce tissue regeneration. Vanillin is a well-known natural compound with potential anti-inflammatory capacity. Hence, we hypothesized that Vanillin could accelerate wound healing reducing inflammation and especially cytokine production making the oral tissue repair process easier. Methods Our hypothesis was tested using primary human gingival fibroblast (HGF) cell pretreated with Vanillin and primed with IL-1β, as inductor of proinflammatory environment. After 24 hours of treatments, the gene expression and production of IL-6, TNF-α, IL-8, COX-2, iNOS, and nitric oxide (NO) generation and the wound healing rate were determined. Results In IL-1β-primed cells, preincubation with Vanillin reduced IL-6, IL-8, COX-2, and iNOS expression and NO release, compared to IL-1β-primed cells. Moreover, Vanillin determines the increased gene expression of nAChRα7, leading us to hypothesize a role of Vanillin in the activation of the cholinergic anti-inflammatory pathway. Furthermore, in presence of mechanical injury, the Vanillin preincubation, wound closure may be reducing the expression and release of IL-6 and TNF-α and upregulation of COX-2 and IL-8. Conclusion Together, the results of this study highlight the anti-inflammatory and tissue repair ability of Vanillin in IL-1β-primed HGF. Therefore, Vanillin shows a potential therapeutic interest as an inflammatory modulator molecule with novel application in periodontal regeneration and oral health.

Downregulation of Prolactin-Induced Protein Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells.

BackgroundPeriodontal ligament stem cells (PDLSCs) are promising seed cells for bone tissue engineering and periodontal regeneration applications. However, the mechanism underlying the osteogenic differentiation process remains largely unknown. Previous reports showed that prolactin-induced protein (PIP) was upregulated after PDLSCs osteogenic induction. However, few studies have reported on the function of PIP in osteogenic differentiation. The purpose of the present study was to investigate the effect of PIP on osteogenic differentiation of PDLSCs.Material/MethodsThe expression pattern of PIP during PDLSCs osteogenic differentiation was detected and the effect of each component in the osteogenic induction medium on PIP was also tested by qRT-PCR. Then, the PIP knockdown cells were established using lentivirus. The knockdown efficiency was measured and the proliferation, apoptosis, and osteogenic differentiation ability were examined to determine the functional role of PIP on PDLSCs.ResultsQRT-PCR showed that PIP was sustainedly upregulated during the osteogenic induction process and the phenomenon was mainly caused by the stimulation of dexamethasone in the induction medium. CCK-8 and flow cytometer showed that knocking down PIP had no influence on proliferation and apoptosis of PDLSCs. ALP staining and activity, Alizarin Red staining, and western blot analysis demonstrated PIP knockdown enhanced the osteogenic differentiation and mineralization of PDLSCs.ConclusionsPIP was upregulated after osteogenic induction; however, PIP knockdown promoted PDLSCs osteogenic differentiation. PIP might be a by-product of osteogenic induction, and downregulating of PIP might be a new target in bone tissue engineering applications.

Nanotechnology in Periodontal Regeneration: A Review

Periodontitis involves destruction of the periodontal ligament and supporting structures. It ranges from asimple inflammation of gums to major damage resulting in loss of bone. Multiple conditions like trauma,periodontal disease, reconstructive surgery, ageing, osteoporosis and neoplastic pathology, have a commoncharacteristic feature being loss of bone and tissue. The regenerative procedures that are currently practicedaim to regenerate the lost tissue via means of grafting, placement of membranes and many more, but havebeen accompanied with a set of limitations and major disadvantage being graft rejection. Periodontalregeneration still endures to be a partially unsolved algorithm. In dentistry, a new arena of developmentis being exploited to set a new approach for regeneration that is Nanotechnology. This branch involvestissue engineering which aims to regenerate the lost tissues via manufacturing scaffolds which transportsignaling cells and molecules. These scaffolds possess magnificent physical as well as chemical propertiesand biomimetic features that act as a substitute of the highest standard offering innumerable benefits forpromoting cell growth and tissue regeneration via tissue engineering.Nanomaterials because of their refinedphysiochemical characteristics and biomimetic attributes serve as a scope worthy substitute that offersseveral advancements for stimulation of cell growth and tissue regeneration. This review provides an insightto basic information about nanotechnology, the various nano particles having application in periodontalregeneration, the advanced regenerative procedures fabricated on the principles of nanotechnology, theirapplications, the clinical studies highlighting the achievements so far, supremacy over the conventionalmethod and limitations.

Exploring the potential of human adipocytes in periodontal regeneration: A review

Stem cells, initially identified in embryonic tissues and later in numerous adult tissues, tend to possess the potentiality to differentiate into various cell types. Though most flexible of all stem cell lines, ethical issues restrict the use of embryonic cells. Furthermore, induced pluripotent stem cells (iPS) and adult stem cells (e.g: bone marrow stroma) can also be used. However, procurement of autologous bone marrow has its potential limitations. An alternate source of autologous adult stem cells which can be procured in large quantities, under local anesthesia, with minimal discomfort would be of keen interest. In the present context, human adult adipose tissue may be the best appropriate alternative source of mesenchymal stem cells. Studies have shown that adipose stem cells (ASCs) extracted from subcutaneous human adult adipose tissue tend to contain heterogeneous cell population called stromal vascular fraction (SVF). It may be used directly or cultured in for selection and expansion of an adherent population, and hence, they are called ASCs. The adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), are processed to obtain a fibroblast-like population of cells, also called processed lipoaspirate (PLA). PLA cells has the potentiality to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. This attributable feature of ASCs may be of significant importance in future clinical cell-based therapy for periodontal disease as well. This review describes current knowledge & recent advances in ASCs & their application. This review describes current knowledge and recent advances in ASCs and their application in periodontal regeneration.

A compound of concentrated growth factor and periodontal ligament stem cell-derived conditioned medium

The aim of this study was to determine the in vitro effect of a compound of concentrated growth factor (CGF) and periodontal ligament stem cell-derived conditioned medium (PDLSCs-CM) as a potential product for future applications in periodontal tissue regeneration. Isolated PDLSCs were characterized using flow cytometry and differentiation into osteoblasts and adipocytes cells. PDLSCs-CM and CGF were prepared and lyophilized. To determine the optimal concentration of the CGF-CM compound, the proliferation of PDLSCs after exposure to a wide range of different concentrations of CGF, CM, or their combination (CGF + CM) was investigated by methyl thiazol tetrazolium assay. Successful isolation of PDLSCs was confirmed by high expression of mesenchymal surface markers and differentiation into osteoblasts and adipocytes. PDLSCs showed higher proliferation in the 6.25 mg/mL concentration of CM and 94 μg/mL concentration of CGF. High concentrations of CGF and CM markedly inhibited the proliferation of PDLCs (p < 0.05). The exposure of PDLSCs to the compound of 10% CM + 90% CGF significantly increased the cell proliferation (p < 0.05). The results showed that CGF, CM, or their combination exert a proliferative effect on cells at a certain concentration. Further investigation on the synergistic effect of this compound may approve its application for periodontal regeneration.

Optimization of 3D bioprinting of periodontal ligament cells

Three-dimensional (3D) bioprinting of cells is an emerging area of research but has been not explored yet in the context of periodontal tissue engineering. ObjectiveThis study reports on the optimisation of the 3D bioprinting of periodontal ligament cells for potential application in periodontal regeneration. MethodsWe systematically investigated the printability of various concentrations of gelatin methacryloyl (GelMA) hydrogel precursor using a microextrusion based three-dimensional (3D) printer. The influence of different printing parameters such as photoinitiator concentration, UV exposure, pressure and dispensing needle diameter on the viability of periodontal ligament cells encapsulated within the 3D bioprinted construct were subsequently assessed. ResultsThis systematic evaluation enabled the selection of the most suited printing conditions for achieving high printing resolution, dimensional stability and cell viability for 3D bioprinting of periodontal ligament cells. SignificanceThe optimised bioprinting system is the first step towards to the reproducible manufacturing of cell laden, space maintaining scaffolds for the treatment of periodontal lesions.

Tri-layered functionally graded membrane for potential application in periodontal regeneration

A novel tri-layered, functionally-graded chitosan membrane (FGM) with bioactive glass gradient (50%, 25%, and 0% wt.) was developed by lyophilization. A step-wise grading of chitosan, bioactive glass (BG), and Pluronic F127 was introduced into the membrane in which each layer has separate surface functions that play a role of guided tissue regeneration (GTR) membranes. The lower layer was designed to replicate alveolar bone and contains 50%wt. BG, the middle layer contains 25%wt. BG, while the upper layer was non-porous without BG and it did not support cell growth. Scanning Electron Microscopy (SEM) revealed that the lower FGM surface possessed a porous structure with embedded BG particles, while the upper surface was non-porous with interconnected architecture. The contact angle measurement confirmed that the surface with BG was hydrophilic (≈00), while the opposite surface was hydrophobic (910 ± 3.840). Both osteoblast and fibroblast cells have maximum adhesion at contact angle <80°. Alamar blue assay revealed the biocompatibility of the MC3T3-E1 mouse pre-osteoblasts cells with these membranes in vitro. The cells attachment and proliferation was seen for lower surface, while no cells adhesion was observed for the upper layer. Additionally, the interaction of the tissue with these tri-layered membranes was also investigated in vivo. Hematoxylin and eosin staining revealed the biocompatible nature of these membranes. Altogether, these results indicated that due to the biocompatible nature of these membranes, they will be a good carrier of in vivo implantation.

Thermal annealed silk fibroin membranes for periodontal guided tissue regeneration.

Guided tissue regeneration (GTR) is a surgical procedure applied in the reconstruction of periodontal defects, where an occlusive membrane is used to prevent the fast-growing connective tissue from migrating into the defect. In this work, silk fibroin (SF) membranes were developed for periodontal guided tissue regeneration. Solutions of SF with glycerol (GLY) or polyvinyl alcohol (PVA) where prepared at several weight ratios up to 30%, followed by solvent casting and thermal annealing at 85 °C for periods of 6 and 12 h to produce high flexible and stable membranes. These were characterized in terms of their morphology, physical integrity, chemical structure, mechanical and thermal properties, swelling capability and in vitro degradation behavior. The developed blended membranes exhibited high ductility, which is particular relevant considering the need for physical handling and adaptability to the defect. Moreover, the membranes were cultured with human periodontal ligament fibroblast cells (hPDLs) up to 7 days. Also, the higher hydrophilicity and consequent in vitro proteolytic degradability of these blends was superior to pure silk fibroin membranes. In particular SF/GLY blends demonstrated to support high cell adhesion and viability with an adequate hPDLs' morphology, make them excellent candidates for applications in periodontal regeneration.

Bone morphogenetic proteins: Revivifying periodontium

Bone morphogenetic proteins (BMPs), a family of signaling molecules, are considered to be a central factor present in the bone matrix. Originally discovered by Urist, they have been shown to affect a wide variety of cell types and processes beyond bone and osteogenesis. They are important morphogens in embryogenesis and development and also regulate the maintenance of adult tissue homeostasis. The loss of alveolar bone is a common consequence of periodontal disease. The regeneration of periodontal structures requires an environment consisting of cells, scaffold, and signaling molecules. The emergence of tissue engineering has enabled the mass production of BMPs, to be utilized refining the biomimetic scaffolds, thus facilitating an alternative to treatment approach in bone regeneration lost due to the disease process. This review discusses the critical data on the discovery of BMPs, its structure, mechanisms of action, and potential therapeutic application in periodontal regeneration.

Tooth for a tooth: Tissue engineering made easy at dental chairside.

Background:Stem cells are unspecialized, multipotent cells which can differentiate into different cell types. The human periodontal ligament encloses subpopulation of Periodontal Ligament Stem Cells (PDLSCs) which are able to regenerate the periodontium by differentiating into various cells of PDL. Literature have shown the promising results of periodontal regeneration by exvivo cultured transplantation of PDLSCs into the osseous defects.Methods:Based on current literature on use of ex vivo culture and associated problems, a novel technique is being presented , Stem cell application in periodontal regeneration (SAI-PRT) which uses autologous PDLSCs along with its niche adhering to extracted tooth root for direct application using gelatin sponge as scaffold in regeneration of intrabony periodontal defect bypassing ex-vivo culture.Results:The cases initiated under SAI-PRT protocol, and have shown excellent clinical and radiographic improvement in intrabony defect using direct transplant of autologous PDLSCs .Conclusions:SAI-PRT can be used for the treatment of periodontal osseous defects at chair side.

3D-Printed Scaffolds and Biomaterials: Review of Alveolar Bone Augmentation and Periodontal Regeneration Applications.

To ensure a successful dental implant therapy, the presence of adequate vertical and horizontal alveolar bone is fundamental. However, an insufficient amount of alveolar ridge in both dimensions is often encountered in dental practice due to the consequences of oral diseases and tooth loss. Although postextraction socket preservation has been adopted to lessen the need for such invasive approaches, it utilizes bone grafting materials, which have limitations that could negatively affect the quality of bone formation. To overcome the drawbacks of routinely employed grafting materials, bone graft substitutes such as 3D scaffolds have been recently investigated in the dental field. In this review, we highlight different biomaterials suitable for 3D scaffold fabrication, with a focus on “3D-printed” ones as bone graft substitutes that might be convenient for various applications related to implant therapy. We also briefly discuss their possible adoption for periodontal regeneration.

Dental pulp stem cells. Biology and use for periodontal tissue engineering.

Inflammatory periodontal disease is a major cause of loss of tooth-supporting structures. Novel approaches for regeneration of periodontal apparatus is an area of intensive research. Periodontal tissue engineering implies the use of appropriate regenerative cells, delivered through a suitable scaffold, and guided through signaling molecules. Dental pulp stem cells have been used in an increasing number of studies in dental tissue engineering. Those cells show mesenchymal (stromal) stem cell-like properties including self-renewal and multilineage differentiation potentials, aside from their relative accessibility and pleasant handling properties. The purpose of this article is to review the biological principles of periodontal tissue engineering, along with the challenges facing the development of a consistent and clinically relevant tissue regeneration platform. This article includes an updated review on dental pulp stem cells and their applications in periodontal regeneration, in combination with different scaffolds and growth factors.

Recent Advances in Cell Sheet Technology for Periodontal Regeneration

Tissue engineering has yielded several successes in early clinical trials of regenerative medicine with grafting therapeutic cells seeded into biodegradable scaffolds. However this conventional cell delivery method has limited the field's progress. In recent decades, we have developed a novel cell transferring method, cell sheet technology that allows for controlled attachment and detachment of cells via simple temperature variations of a surface-intelligent temperatureresponsive polymer:poly (N-isopropylacrylamide). It has been widely applied to create functional tissue sheets with cells derived from various tissues to treat a wide range of diseases. Periodontal cell sheets non-invasively harvested from temperature- responsive culture surfaces have been successfully manufactured, resulting in communicative multilayered constructs. Transplantation of cell sheets onto periodontal defects has improved bone and tissue regeneration in animal models and humans and shows low immunogenicity. In this review, we summarize the recent advances of techniques in cell sheet engineering and its application for periodontal regeneration.

Importance of CD44 in the proliferation and mineralization of periodontal ligament cells

CD44 is a transmembrane glycoprotein that exhibits various biological functions. Histological studies have shown that CD44 is expressed in periodontal ligament (PDL). We investigated the role of CD44 in the in vitro biological functions of PDL cells. Immunohistochemistry and immunocytochemistry were used to examine CD44 protein expression in mouse molars and human PDL cells, respectively. PDL cells isolated from human third molars were assayed for their proliferation and mineralization after stably silencing their expression of CD44 using the small hairpin RNA technique. An osteogenesis-associated quantitative polymerase chain reaction array was used to identify downstream molecules of CD44 signaling in osteogenic medium. The PDL cells, transduced with control small hairpin RNA, were used as controls in all assays. PDLs expressed CD44 in vitro and in vivo. When CD44 expression was stably suppressed in PDL cells, their proliferation and mineralization activities in both media were substantially decreased. The quantitative polymerase chain reaction array and Western blotting verified that bone morphogenetic protein-2 (BMP-2), fibroblast growth factor-1 (FGF-1) and intercellular adhesion molecule-1 (ICAM-1) were downregulated after CD44 was knocked down in PDL cells. Exogenous FGF-1 attenuated the proliferative suppression of CD44 knockdown cells. Exogenous BMP-2 rescued the suppressed mineralization of CD44 knockdown cells more than ICAM-1 did. The in vitro assays demonstrated that CD44 is crucial for the proliferation and mineralization of PDL cells and is possibly mediated by BMP-2, FGF-1 and ICAM-1. Further studies are required to verify the mediators and identify the physiological ligands of CD44 for possible application in periodontal regeneration.

Cell sheet engineering and its application for periodontal regeneration

Periodontitis is a inflammation induced by a bacterial infection that causes the destruction of the attachment apparatus of dental roots. Several materials, such as bone graft materials, barrier membranes and protein products have been developed and used to treat periodontal defects clinically; however, it is difficult to regenerate the complete periodontal tissue structure. Recently, cytotherapeutic approaches have been introduced to overcome the limitation of conventional procedures. The in vitro-expanded autologous cells derived from several kinds of tissues have already been used in several clinical trials. These cytotherapeutic treatments have been shown to be safe and effective for the treatment of periodontitis. Our strategy has been to integrate stem cell biology and cell sheet engineering, in which a temperature-responsive intelligent polymer is grafted onto the surface of cell culture dish to create a 'cell sheet', to achieve a novel treatment method for periodontitis. By simple reduction of the temperature to below 32°C, a contiguous cell sheet, which is capable of keeping extracellular matrix proteins and cell-cell interactions intact, can be harvested for transplantation without the use of scaffolds. This technology has already been employed in clinical trials, confirming the safety and efficacy of the treatment. In this review, we introduce recent progress in the engineering of cell sheets and review the potential of cell sheet technology for periodontal regenerative medicine.