Periodic Reporting for period 1 - MultiphaseGTR (Bioactive multiphase membrane for periodontal regeneration)
Periodo di rendicontazione: 2023-07-01 al 2025-06-30
Periodontitis is one of the world's most prevalent chronic dental and maxillofacial diseases that will ultimately lead to unwanted tooth loss. This disease was responsible for a significant loss of 158.64 billion in Europe in 2018. Autograft and allograft-based methods have been long used for restoring the lost supportive periodontal tissues. However, these methods also have significant limitations, such as morbidity at the donor site, painful secondary infections, rejection of the donor tissue. Due to the limitations, the efficiency of these procedures is notably affected. Different polymeric hydrogels have been reported beneficial for periodontal tissue regeneration application. However, the application of the polymeric biomaterials/membranes have also limitations, such as inadequate physiochemical properties, low porosity, low stiffness, and lack of stability. In this context, multiphasic scaffold with different non-mineralized and mineralized layers have been advised for its efficiency found in periodontal tissue regeneration. Till now, most of the reported hydrogels for periodontal regeneration lack the specific physico-mechanical and biological properties that are highly desirable. MultiphaseGTR focuses on the development of bioactive multiphase hydrogel-based bio-membrane with high porosity, mechanical property, antimicrobial and cell biological property for periodontal regeneration. To achieve this, microcrystalline cellulose (MCC) was biofunctionalized by lactic acid (MCC-LA). The physico-chemical characterization of MCC-LA indicated that LA was successfully grafted on the MCC. MCC-LA reinforced multiphasic hydrogels were developed. The hydrogel based nonmineralized bio-membranes were prepared with biosafe crosslinking agents. The developed non-mineralized hydrogels were mineralized to achieve calcium carbonate mineralized multiphasic hydrogel. The multiphasic hydrogels showed a compressive modulus like periodontal tissue. Moreover, hydrogels were found antimicrobial in nature. The cell viability was also found higher for multiphasic hydrogels. Hence, based on these results, it can be concluded that, these multiphasic hydrogels have the potentiality to utilize in periodontal tissue regeneration application. However, further studies like detailed gene expression analysis, proteomics analysis are required to explore its efficiency in guided tissue regeneration application.
MultiphaseGTR involves 3 important work packages which involves the development and characterization of biofunctionalized multiphaseGTR bio-membrane. At first, lactic acid biofunctionalization was performed to microcrystalline cellulose crystals (MCC- LA). Thereafter, this MCC-LA was utilized to prepare nonmineralized hydrogel-based bio-membrane. Following this, nonmineralized hydrogels were template mediated mineralized to develop multiphase GTR hydrogel-based bio-membrane. The results of MultiphaseGTR work focussing each work packages are described below: WP1. Development of the components of the multiphase GTR hydrogel membranes. Task 1.1. involved biofunctionalization of MCC. The MCC is biofunctionalized with DL-lactic acid. The biofunctionalized MCC (MCC-LA) were then characterized on the basis of physico-chemical properties: Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and molecular weight distribution analysis indicated successful grafting of lactic acid on MCC. Task 1.2. involved the preparation of non-mineralized hydrogel based biomembrane preparation. Two groups of samples were prepared. Then the non-mineralized hydrogels were analysed on the basis of their physio-chemical properties, which are discussed below: FTIR and Energy dispersive X-ray analysis (EDX) indicated the presence of important ingredients like chitosan, MCC, lactic acid etc; Scanning Electron Microscopy (SEM): SEM indicated the non-mineralized hydrogels contains rough surface structure due to the presence of MCC-LA. On the other hand, the hydrogels exhibited significant porous structures; Micro CT (µ-CT) analysis indicated that the non-mineralized hydrogels have significant porosity. Differential pore size distribution of the non-mineralized hydrogel bio-membranes was also observed. The non-mineralized hydrogel membranes were characterized on the basis of compression analysis. Compressive modulus of the non-mineralized hydrogels was found to be similar to the periodontal tissue. Thereafter, the non-mineralized hydrogel biomembranes were selected on the basis of their physio-chemical property for the preparation of mineralized and nonmineralized-multiphasic hydrogel based biomembrane. The major achievements of WP-1. are Preparation of lactic acid grafted MCC and development of nonmineralized hydrogel biomembrane. WP2. Preparation of multiphase GTR hydrogel membrane: In Task 2.1. the freshly prepared non-mineralized hydrogel membranes were mineralized for CaCO3 mineralization. The freshly prepared mineralized multiphasic membranes were then freeze dried for further evaluation. Thereafter, in Task 2.2. the freeze-dried mineralized multiphasic hydrogels were characterized on the basis of physical, morphological, chemical, mechanical properties, as follows: SEM, EDX and XRD study of the multiphasic hydrogels demonstrated the significant presence of CaCO3 crystals. Micro-CT (µ-CT) analysis demonstrated that the mineralized multiphasic hydrogels showed notable porosity (%) and pore distribution. Then the mechanical property of multiphasic hydrogels was analysed on the basis of rheology. Then the swelling property of the multiphasic hydrogels was analysed. In vitro degradation of the multiphasic hydrogels was also studied The major achievements of WP-2. is development of nonmineralized and mineralized multiphasic hydrogel biomembrane. WP3. Biological characterization of multiphase GTR hydrogel membrane: In Task 3.1. Antimicrobial analysis of the multiphasic hydrogels was performed through direct biofilm exposure with Staphylococcus epidermidis Xen43. Both the genipin and tannic acid crosslinked multiphasic hydrogels have shown notable antimicrobial property. In Task 3.2. the cell viability analysis was performed with alamar blue study. Cell viability was found notable. The notable metabolic activity of the cells indicated that, the hydrogels are favorable to the cell. The major achievements of WP-3. are characterization of multiphasic hydrogel biomembrane for antimicrobial property and cytocompatibility.
The primary objective of MultiphaseGTR is to develop novel multiphase bio-membrane with significant pore structures/porosity and mechanical properties that will facilitate the periodontal regeneration. The MultiphaseGTR focuses the development of novel multiphase GTR hydrogel through sustainable approach by utilizing biopolymers like cellulose (MCC) and chitosan, which has considerably less ethical issues as they do not originate from vertebrates. The developed bio-membrane will fulfil the current research/product gap with improved mechanical property (i.e. high compressive modulus), porosity and biocompatibility. From the project results, it can be understood that, the physio-mechanical, cell biological property of the developed multiphasic hydrogels are promising in terms of its applicability for periodontal tissue regeneration. Results of MultiphasicGTR will provide an in-depth insight to the future researchers regarding the advantages and limitations of nonmineralized and mineralized multiphasic hydrogel-based biomaterials in terms of periodontal tissue regeneration. The multiphasic hydrogel based biomembrane have elaborated the importance and significance of biomimetic biomaterials. However, further study and adjustments are needed in terms of coherent material property to increase its biocompatibility. To achieve this, study related to surface treatment and other analysis like proteomics are needed to strongly support the applicability of these multiphasic hydrogel biomembrane.