Periodontitis isa widely prevalent inflammatory disease resulting in the destruction of tooth-supporting structures. It occurs due to the extension of inflammation from the marginal gingiva to the underlying tooth-supporting structures including the attached gingiva, alveolar bone, and periodontal ligament. If left untreated, it results in tooth mobility, loss of chewing function, esthetic disturbances, and ultimately tooth loss [1], [2], [3], [4]. The incidence of periodontal disease varies with individual conditions, such as lifestyle and genetics, and it presents in diverse distinct clinical forms at several ages of onset, and at variable progression rates [4], [5], [6]. Moreover, recent longitudinal studies have revealed positive associations between chronic periodontitis and about fifty systemic conditions and diseases, such as cardiovascular disease, osteoporosis, respiratory diseases, metabolic deficiencies, Alzheimer’s disease etc. [7], [8]. Thus, it is necessary to cease the progression of periodontitis by initiating treatment at early stages of diagnosis.

The progression of periodontitis can be ceased by clinical procedures such as scaling and root planing. However, for the treatment of periodontitis and to promote regeneration of the destroyed tooth-supporting structures, surgical regenerative techniques including either guided tissue regeneration (GTR) or tissue engineering approach, are essential [4], [9], [10]. The tissue engineering approach utilizes stem/progenitor cells such as bone marrow-derived mesenchymal stem cells [11], [12], adipose-derived stem cells [13], periodontal ligament stem cells [14], [15], and dental pulp stem cells [16], scaffolds, and bioactive molecules to facilitate periodontal tissue regeneration. [17]. Recently, antimicrobials laden 3D printed tissue engineering scaffolds were investigated for periodontal regeneration [18], [19], [20]. Tissue engineering approach is still in its early stage of development, and its efficacy in in vivo and clinical evaluation of periodontal defect models is currently uncertain due to limited data availability [18]. On the other hand, the GTR technique has been widely employed in the treatment of periodontal defects with successful clinical outcomes [21]. The key element of the GTR technique involves the temporary positioning of a barrier membrane, also known as the GTR membrane, beneath the gingiva [4], [22]. The basis for the placement of a GTR membrane beneath the gingiva is to isolate the periodontal defect by preventing the infiltration of rapidly proliferating gingival epithelial cells and gingival epithelium and thus protecting the periodontal defect space for regeneration of periodontium and alveolar bone by periodontal ligament cells and osteoblasts, respectively [4], [9], [21]. Several clinical studies [22], [23], [24], [25] have substantiated the positive outcomes of GTR procedures, which include greater clinical attachment level gain, reduction in probing pocket depth, and effective alveolar bone regeneration. At present, commercially successful GTR membranes are largely tissue-derived collagen-based products. Despite promising clinical results of the GTR technique for treating periodontal defects, the high cost, inadequate availability in large quantities, and improper degradation kinetics of collagen membranes limit the widespread clinical applicability of the GTR technique, especially in cases of moderate and severe periodontitis with more than one tooth involved, where larger membranes are required [9], [17], [21], [26], [27]. To overcome the limitations of collagen-based GTR membranes, synthetic polyester-based GTR membranes were introduced. But it has been reported that the synthetic membranes showed unsatisfactory cell response [17], [21]. Thus, there is a need for the development of novel cost-effective GTR membranes that could assimilate the advantages of natural as well as synthetic biomaterials and could promote effective periodontal regeneration.

One such naturally available cost-effective bioactive material is the chicken eggshell membrane (ESM), which is a thin, bilayered microporous membrane present between eggshell and egg white. It is made of highly cross-linked interwoven protein fibers comprising of type I, V, and X collagen, osteopontin, and sialoprotein [28], [29]. ESM has a unique fibrous structure on the outer and inner surfaces which facilitates the mineralization of eggshell on the outer side while preventing the mineralization of egg yolk on the inner side [30]. Owing to the unique properties of ESM, two studies were done to investigate the potential applicability of natural avian and ostrich ESM as GTR membranes in animal models. However, the results were unsatisfactory due to the poor mechanical properties, structural properties, and dimensional instability of ESM [31], [32]. Also, raw ESM is insoluble and poses the risk of pathogen transmission. Thus, to overcome these limitations, soluble eggshell protein (SEP), a soluble form of ESM, has been extracted from raw ESM and it broadened ESM applications in regenerative medicine. However, SEP also is a low molecular weight biodegradable polymer with poor mechanical properties, which is a major limitation for its application as a GTR membrane.

To improve the mechanical properties of SEP for GTR applications, we are blend-electrospinning SEP, a natural biomimetic polymer with a synthetic FDA-approved additive polymer, PLGA, and a bioceramic nano-hydroxyapatite (HAp). Among synthetic polymers, PLGA attracts wide attention as its degradation rate and mechanical properties are tunable by altering the ratio of lactic to glycolic acids [33], [34], [35]. Moreover, blend electrospinning of PLGA with SEP has improved the cyto-compatibility and mechanical properties of SEP/PLGA blend [36]. However, PLGA poses certain limitations that include lack of osteoconductivity and local inflammatory response at the site of implantation caused by their acidic degradation products. To solve these limitations of PLGA, some researchers prepared HAp particles-loaded polymer membrane with excellent biocompatibility and osteoconductivity, which can enhance cell activity and neutralize acidic degradation products of PLGA polymer by ionic interactions. Furthermore, due to its structural and chemical similarities to natural bone mineral, HAp is relatively easier to be identified by cells or bio macromolecules, and thus can improve the bioactivity and osteoconductivity of implanted membranes [37], [38], [39], [40]. Electrospinning is the direct and continuous preparation of nano-scale fibers to mimic the natural microscopic structure of ESM. Also, electrospun nanofibrous membranes were reported to have a small pore diameter, high specific surface area, and easy surface modification, which is highly suitable for use in periodontal barrier membranes. Furthermore, the electrospinning process is advantageous as it includes a basic apparatus, simple operation, and cost-effectiveness [35], [39], [41], [42], [43], [44]. Thus, this novel biomimetic SEP-based fibrous GTR membrane was designed to assimilate the advantages of natural as well as synthetic biomaterials for promoting effective periodontal regeneration and is economical, facilitating the widespread clinical applicability of the GTR technique. This SEP-based fibrous GTR membrane is a promising, environment-friendly, cruelty-free, and cost-effective alternative for commercial collagen-based GTR membrane products as ESM is abundantly available as a waste product from the poultry and food industry and is usually disposed of through landfill facilities [45]. Additionally, in this study, we explored the feasibility of reutilizing a valuable natural, biodegradable, and cost-effective industrial waste, i.e., ESM and its derivatives for potential cost-effective dental material applications.

Apart from SEP wt%, other components of this novel membrane including the PLGA wt% and HAp wt% could affect the mechanical properties of the final product. Also, the electrospinning parameters such as the flow rate of the polymer solution, the spinneret-collector distance, and the applied voltage might influence the mechanical properties of this PLGA/SEP/HAp membrane. The research objective of this study is to prepare a novel cost-effective electrospun PLGA/SEP/HAp membrane with mechanical and biocompatible properties appropriate to be used in GTR procedures. Taguchi orthogonal arrays for designing experiments will aid in determining the effect of several different parameters on the performance characteristic in a condensed set of experiments [43], [46]. A response surface design methodology is a set of advanced design of experiments (DOE) techniques that help in better understanding the effect of independent variables on a performance characteristic and optimization of the response. It is often used to refine models after assessing the significant parameters using screening designs such as Taguchi orthogonal arrays; especially if a curvature in the response surface is suspected [47], [48], [49]. Thus, we utilized DOE strategies such as Taguchi orthogonal arrays and response surface design to screen and optimize the electrospinning parameters as well as the membrane composition for preparing an electrospun PLGA/SEP/HAp membrane with maximum mechanical properties. Furthermore, the morphological and physicochemical characterization of this novel optimized PLGA/SEP/HAp membrane and the electrospun membrane prepared with pristine PLGA polymer (control group) were carried out. Finally, we performed the biocompatibility evaluation and compared the proliferation activity of Human periodontal ligament fibroblasts on this optimized PLGA/SEP/HAp membrane electrospun using static collector, optimized PLGA/SEP/HAp membrane electrospun using rotating collector at 1500 rpm, a commercial collagen membrane, and TCPS.