Final Report Summary - BIODENTISSUE (Design and Synthesis of Novel Bilayered Scaffolds for Periodontal Tissue Regeneration)
Rationale
Periodontitis is one of the most common diseases in humans, affecting in its most severe form, approximately 10-15% of the population [1]. It has been defined as the chronic inflammatory disease of the periodontium triggered by bacterial plaque, and is characterized by progressive destruction of the gingiva (gingivitis), alveolar bone and periodontal ligament [1,2]. The inflammatory response caused by the bacterial plaque results gradually in the apical migration of the epithelial attachment, the further formation of periodontal pockets, and ultimately loosening and tooth resorption [1–3]. Apart from tooth resorption, periodontitis has been linked to many systematic disorders, such as coronary artery disease [4] and stroke [5]. Novel scientific approaches in dental tissue engineering combine the applications of GTR with an artificial Extracellular Matrix (ECM), carried out by scaffolding materials, which are loaded with cells and signaling molecules such as growth factors, cultured in vitro and subsequently implanted into tissue defects to induce and direct the growth of new tissue. Nevertheless, current data concerning the use of scaffolds in PDL regeneration are far from sufficient for tissue-engineering therapeutics in the periodontology field. Consequently, there is need for the development of advanced biomimetic scaffold materials, which are versatile enough to be targeted for tooth-specific applications and capable to drive the growth and functional differentiation of stem/progenitor dental cells into matured organized periodontal tissues in a controlled manner. The aim of this project was the development and optimization of an advanced bilayered scaffold that mimics the complex structure of periodontium, i.e. the hierarchical structure of cementum and alveolar bone, the periodontal ligament and the continuous, fibrous interface between these tissues. A three step procedure, summarized in the following objectives, was implemented towards this goal:
Objective 1. Synthesis and optimization of bioceramic scaffolds for cementum/alveolar bone regeneration.
Objective 2. Synthesis and optimization of fibrous polymer scaffolds for periodontal ligament regeneration.
Objective 3. Design and construction of bilayered scaffolds based on bioceramic foams and electrospun meshes.
Results
In the framework of this project a bilayered scaffold model consisting of a bioceramic and polymeric layer for alveolar bone/cementum and periodontal ligament regeneration, respectively, was established. In the framework of this project, a family of magnesium-containing bioactive silicate glasses varying in both network formers and modifiers were synthesized and optimized according to their microstructural, biological and mechanical characteristics. The optimum composition served as starting material for the successful fabrication of functional bioceramic scaffolds by the typical foam replica technique, which includes the immersion of PU foams in a slurry of the bioceramic powder. Furthermore, the attachment, proliferation and osteogenic differentiation of a bone marrow-derived stromal cell line (ST2) in contact with 3D specimens of the optimum composition were proven. Concerning the polymeric part of the bilayered scaffolds, physically cross-linked gelatin fibrous scaffolds were fabricated, by the electrospinning technique. The selection of gelatin as a candidate scaffold material for periodontal ligament regeneration was based on the fact that gelatin is a self-assembling, nontoxic, biodegradable, inexpensive and nonimmunogenic material. The physical parameters of the synthesized fibrous scaffolds, including fiber diameter, scaffold pore dimension, and degree of scaffold anisotropy were optimized by controlling the composition of the electrospinning solvent, the air gap distance and accelerating voltage. To regulate the degradation behavior, all fibers were cross-linked by immersion in ethanolic solutions of N-(3-Dimethylaminopropyl-N ́-ethylcarbodiimide hydrochloride (EDC) and N- Hydroxysuccinimide (NHS) in different concentrations. The optimum concentration of the cross-linking agent was determined by the morphology, degradation behavior and degree of cross-linking as well as the biocompatibility of the cross-linked fibers in contact with ST2 stem cells. Concerning the bilayered scaffolds, they were fabricated as follows: Firstly, the bioceramic scaffolds were prepared as previously described. These scaffolds, after being coated with gelatin, were fixed using silver paint on a piece of aluminium foil and were used as targets for an electrospun gelatin fiber mesh to be constructed on top of them. The size of the aluminium foil was selected to be as small as possible in order to direct the deposition of the electrospun fibers on the scaffold. The electrospinning solution as well as the conditions was kept the same as for the production of the electrospun fiber mats. The gelatin coating and the gelatin mesh were simultaneously cross-linked by immersion in an EDC/NHS solution as previously described. Both the morphological characterization and the microtensile bond strength evaluation of these bilayered scaffolds revealed the penetration of the fibers in the scaffolds for some micrometers, providing a continuous scaffold/fiber interface, which was strengthened by the simultaneous cross-linking of the gelatin layer and the gelatin mesh.
Conclusion
The final outcome of this study was a bilayered scaffold of improved mechanical and biological properties for potential use in periodontal tissue regeneration.
Potential impact and use and any socio-economic impact of the project
The effective treatment of periodontitis is a medical and socioeconomic challenge, since periodontitis is one of the most common diseases and one of the main causes of tooth loss in adults. Furthermore, periodontal bacteria can enter the bloodstream and travel to major organs; thus contributing to the emergence of heart disease; increase the risk of stroke; increase a woman’s risk of delivering a preterm low-birth-weight baby; and pose a serious threat to people whose health is compromised by diabetes mellitus, respiratory disease or osteoporosis. Major scientific advances in periodontology over the past years have ultimately altered the way clinicians detect and treat periodontal diseases. However, there is still no ideal therapeutic approach to cure periodontitis or to achieve predictable and optimal periodontal tissue regeneration. To restore lost tooth-supporting structures, various regenerative procedures have been proposed, tested, and evaluated in the last two decades. Among them, the regeneration of damaged periodontal structures with various bone graft materials and guided tissue regeneration (GTR) strategies have succeeded in certain ideal clinical scenarios, but the outcomes are controversial, depending upon multiple factors such as defect size and type, patient age and education, genetics, and, indeed, operator skills. Tissue engineering rises as a highly promising approach towards existing treatment regimes for periodontal disease and several efforts have been made for the regeneration of alveolar bone or cementum using scaffolding materials. Research on the periodontal complex as an entity, however, is limited. It is clear that in order to mimic the complex morphology of the periodontium, efforts should be focused on the PDL/cementum and PDL/bone interfaces and the application of a double layer scaffold will provide the appropriate matrix. The present project has provided a step forward towards the establishment of a new clinical technology required for the predictable regeneration of periodontal tissues, providing beneficial impact on the European patients' wellbeing. Consequently, given the significant implications of the research in relieving the considerable effects of periodontitis, it is anticipated that the results of the present project in the long term will have an impact on the European dental healthcare system. Furthermore, considering that the project involves a topic of high interest currently in several research institutions and industry across Europe (synthesis of scaffolds with controlled properties for periodontal tissue regeneration), the successful completion was the first step that will eventually lead to the development of collaborations among European research institutes and industry for achieving a broad application of this new product.
References
[1] Reich BJ, Bandyopadhyay D, Bondell HD. A nonparametric spatial model for periodontal data with non-random missingness A nonparametric spatial model for periodontal data with non-random missingness 1 Introduction 2012.
[2] Loesche WJ, Grossman NS. Periodontal disease as a specific, albeit chronic, infection: diagnosis and treatment. Clin Microbiol Rev 2001;14:727–52, table of contents.
[3] Kim J, Amar S. Periodontal disease and systemic conditions: a bidirectional relationship. Odontology 2006;94:10–21.
[4] DeStefano F, Anda RF, Kahn HS, Williamson DF, Russell CM. Dental disease and risk of coronary heart disease and mortality. BMJ 1993;306.
[5] Yoshida M, Akagawa Y. The relationship between tooth loss and cerebral stroke. Jpn Dent Sci Rev2011;47:157–60.
Periodontitis is one of the most common diseases in humans, affecting in its most severe form, approximately 10-15% of the population [1]. It has been defined as the chronic inflammatory disease of the periodontium triggered by bacterial plaque, and is characterized by progressive destruction of the gingiva (gingivitis), alveolar bone and periodontal ligament [1,2]. The inflammatory response caused by the bacterial plaque results gradually in the apical migration of the epithelial attachment, the further formation of periodontal pockets, and ultimately loosening and tooth resorption [1–3]. Apart from tooth resorption, periodontitis has been linked to many systematic disorders, such as coronary artery disease [4] and stroke [5]. Novel scientific approaches in dental tissue engineering combine the applications of GTR with an artificial Extracellular Matrix (ECM), carried out by scaffolding materials, which are loaded with cells and signaling molecules such as growth factors, cultured in vitro and subsequently implanted into tissue defects to induce and direct the growth of new tissue. Nevertheless, current data concerning the use of scaffolds in PDL regeneration are far from sufficient for tissue-engineering therapeutics in the periodontology field. Consequently, there is need for the development of advanced biomimetic scaffold materials, which are versatile enough to be targeted for tooth-specific applications and capable to drive the growth and functional differentiation of stem/progenitor dental cells into matured organized periodontal tissues in a controlled manner. The aim of this project was the development and optimization of an advanced bilayered scaffold that mimics the complex structure of periodontium, i.e. the hierarchical structure of cementum and alveolar bone, the periodontal ligament and the continuous, fibrous interface between these tissues. A three step procedure, summarized in the following objectives, was implemented towards this goal:
Objective 1. Synthesis and optimization of bioceramic scaffolds for cementum/alveolar bone regeneration.
Objective 2. Synthesis and optimization of fibrous polymer scaffolds for periodontal ligament regeneration.
Objective 3. Design and construction of bilayered scaffolds based on bioceramic foams and electrospun meshes.
Results
In the framework of this project a bilayered scaffold model consisting of a bioceramic and polymeric layer for alveolar bone/cementum and periodontal ligament regeneration, respectively, was established. In the framework of this project, a family of magnesium-containing bioactive silicate glasses varying in both network formers and modifiers were synthesized and optimized according to their microstructural, biological and mechanical characteristics. The optimum composition served as starting material for the successful fabrication of functional bioceramic scaffolds by the typical foam replica technique, which includes the immersion of PU foams in a slurry of the bioceramic powder. Furthermore, the attachment, proliferation and osteogenic differentiation of a bone marrow-derived stromal cell line (ST2) in contact with 3D specimens of the optimum composition were proven. Concerning the polymeric part of the bilayered scaffolds, physically cross-linked gelatin fibrous scaffolds were fabricated, by the electrospinning technique. The selection of gelatin as a candidate scaffold material for periodontal ligament regeneration was based on the fact that gelatin is a self-assembling, nontoxic, biodegradable, inexpensive and nonimmunogenic material. The physical parameters of the synthesized fibrous scaffolds, including fiber diameter, scaffold pore dimension, and degree of scaffold anisotropy were optimized by controlling the composition of the electrospinning solvent, the air gap distance and accelerating voltage. To regulate the degradation behavior, all fibers were cross-linked by immersion in ethanolic solutions of N-(3-Dimethylaminopropyl-N ́-ethylcarbodiimide hydrochloride (EDC) and N- Hydroxysuccinimide (NHS) in different concentrations. The optimum concentration of the cross-linking agent was determined by the morphology, degradation behavior and degree of cross-linking as well as the biocompatibility of the cross-linked fibers in contact with ST2 stem cells. Concerning the bilayered scaffolds, they were fabricated as follows: Firstly, the bioceramic scaffolds were prepared as previously described. These scaffolds, after being coated with gelatin, were fixed using silver paint on a piece of aluminium foil and were used as targets for an electrospun gelatin fiber mesh to be constructed on top of them. The size of the aluminium foil was selected to be as small as possible in order to direct the deposition of the electrospun fibers on the scaffold. The electrospinning solution as well as the conditions was kept the same as for the production of the electrospun fiber mats. The gelatin coating and the gelatin mesh were simultaneously cross-linked by immersion in an EDC/NHS solution as previously described. Both the morphological characterization and the microtensile bond strength evaluation of these bilayered scaffolds revealed the penetration of the fibers in the scaffolds for some micrometers, providing a continuous scaffold/fiber interface, which was strengthened by the simultaneous cross-linking of the gelatin layer and the gelatin mesh.
Conclusion
The final outcome of this study was a bilayered scaffold of improved mechanical and biological properties for potential use in periodontal tissue regeneration.
Potential impact and use and any socio-economic impact of the project
The effective treatment of periodontitis is a medical and socioeconomic challenge, since periodontitis is one of the most common diseases and one of the main causes of tooth loss in adults. Furthermore, periodontal bacteria can enter the bloodstream and travel to major organs; thus contributing to the emergence of heart disease; increase the risk of stroke; increase a woman’s risk of delivering a preterm low-birth-weight baby; and pose a serious threat to people whose health is compromised by diabetes mellitus, respiratory disease or osteoporosis. Major scientific advances in periodontology over the past years have ultimately altered the way clinicians detect and treat periodontal diseases. However, there is still no ideal therapeutic approach to cure periodontitis or to achieve predictable and optimal periodontal tissue regeneration. To restore lost tooth-supporting structures, various regenerative procedures have been proposed, tested, and evaluated in the last two decades. Among them, the regeneration of damaged periodontal structures with various bone graft materials and guided tissue regeneration (GTR) strategies have succeeded in certain ideal clinical scenarios, but the outcomes are controversial, depending upon multiple factors such as defect size and type, patient age and education, genetics, and, indeed, operator skills. Tissue engineering rises as a highly promising approach towards existing treatment regimes for periodontal disease and several efforts have been made for the regeneration of alveolar bone or cementum using scaffolding materials. Research on the periodontal complex as an entity, however, is limited. It is clear that in order to mimic the complex morphology of the periodontium, efforts should be focused on the PDL/cementum and PDL/bone interfaces and the application of a double layer scaffold will provide the appropriate matrix. The present project has provided a step forward towards the establishment of a new clinical technology required for the predictable regeneration of periodontal tissues, providing beneficial impact on the European patients' wellbeing. Consequently, given the significant implications of the research in relieving the considerable effects of periodontitis, it is anticipated that the results of the present project in the long term will have an impact on the European dental healthcare system. Furthermore, considering that the project involves a topic of high interest currently in several research institutions and industry across Europe (synthesis of scaffolds with controlled properties for periodontal tissue regeneration), the successful completion was the first step that will eventually lead to the development of collaborations among European research institutes and industry for achieving a broad application of this new product.
References
[1] Reich BJ, Bandyopadhyay D, Bondell HD. A nonparametric spatial model for periodontal data with non-random missingness A nonparametric spatial model for periodontal data with non-random missingness 1 Introduction 2012.
[2] Loesche WJ, Grossman NS. Periodontal disease as a specific, albeit chronic, infection: diagnosis and treatment. Clin Microbiol Rev 2001;14:727–52, table of contents.
[3] Kim J, Amar S. Periodontal disease and systemic conditions: a bidirectional relationship. Odontology 2006;94:10–21.
[4] DeStefano F, Anda RF, Kahn HS, Williamson DF, Russell CM. Dental disease and risk of coronary heart disease and mortality. BMJ 1993;306.
[5] Yoshida M, Akagawa Y. The relationship between tooth loss and cerebral stroke. Jpn Dent Sci Rev2011;47:157–60.
