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Journal of Hard Tissue Biology 30[3] (2021) 303-3082021 The Hard Tissue Biology Network AssociationPrinted in Japan, All rights reserved.CODEN-JHTBFF, ISSN 1341-7649

OriginalPreclinical Evaluation of Sol-gel Synthesized Modulated 45S5-Bioglass Based

Biodegradable Bone Graft Intended for Alveolar Bone Regeneration

Nebu George Thomas1,2), Anand Manoharan3) and Anand Anbarasu1)

1) School of Bio-Sciences and Technology, VIT, Vellore, India2) Pushpagiri College of Dental Sciences, Kerala, India3) The Childs’ Trust Medical Research Foundation, Chennai, India(Accepted for publication, June 24, 2021)

Abstract: Reconstruction and augmentation of the alveolar bone defects pose a challenge for the dental surgeons due to its complex structure. The primary objective of tissue engineering is to regenerate or replace damaged tissues or organs includ-ing damaged bone tissues with bone grafts, cells, and biological molecules. 45S5-bioglass (45S5-BG), with its superior os-teoconductive and osteoinductive abilities, has been at the forefront of tissue engineering, alveolar bone regeneration, and periodontal regenerative surgical procedures for the past several years. With the aim of regenerating supporting alveolar bone, 45S5-BG was synthesized via sol-gel technique. 45S5-BG was characterized by X-ray Diffraction (XRD) and Trans-mission Electron Microscopy (TEM) analysis. In vitro bioactivity study was validated in simulated body fluid (SBF) and analysed by Fourier-Transform Infrared Spectroscopy (FTIR). In vitro cell compatibility was assessed by 3-(4,5-dimeth-ylthyazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using L929 cells. Further, in vivo alveolar bone regenerative potential of 45S5-BG bone graft was evaluated. XRD spectrum confirmed the formation of combeite crystalline phase after sintering. TEM images imparted ultra-structural features of the sample and proved the presence of a major crystalline phase embedded in a glassy matrix. In vitro bioactivity study proved the formation of hydroxy carbonate apatite (HCA) as con-firmed by FTIR analysis. The in vitro MTT assay results confirmed the cell compatibility of 45S5-BG and histological anal-ysis proved new bone formation. Within the limitations of this study, the results demonstrated that in addition to the ob-served bioactive and cell compatible properties, sol-gel synthesized 45S5-BG bone graft exhibited notable alveolar bone regenerative potential.

Key words: 45S5-bioglass, Alveolar bone regeneration, Biomaterial, Cell compatibility, Sol-gel

IntroductionThe need for regenerating alveolar bone that has been lost due to

chronic diseases, trauma or malignancy, has been on the rise in perio-dontal and maxillofacial surgery. This increasing need may be attributed to the patients seeking to prolong the longevity of their natural teeth or implant assisted prosthesis. Literature testifies several approaches that facilitate predictable alveolar bone regeneration with the aid of bone grafts1).

Autografts are considered as the gold standard for bone grafts in re-generative procedures due to their good osteogenic and osteoinductive properties2). Due to limitations like the insufficient volume of graft, the need for a second surgical site and associated morbidity, alloplastic ma-terials like synthetic hydroxyapatite (HA) replaced autografts for a wide range of regenerative procedures. However, HA assisted procedures are associated with disadvantages like reduced resorption rate and healing by fibrous encapsulation3). Various studies showed that synthetic bioac-tive glasses can be effectively used as a bone graft material in periodon-tal regeneration and for augmentation of the edentulous ridge4).

45S5-bioglass, hereafter denoted as 45S5-BG, (45% SiO2, 24.5% Na2O, 24.5% CaO and 6% P2O5) was invented by L.L. Hench in 1971.

45S5-bioglass was originally synthesized through conventional melt-quenching technique, where melting oxides of precursors above 1300°C was done followed by the quenching process5). Sol-gel based technique enabled synthesis of bioglass with enhanced bioactivity, greater bone-bonding properties, higher purity, homogeneity, higher dis-solution rates and positive gene expression leading to accelerated osteo-genesis as compared to melt derived glass6-9).

The synthesis protocol for sol-gel bioglass is well documented and researched whereas studies involving sol-gel application for synthesis of Na2O-containing bioactive glasses or glass ceramics are few, mainly due to high hydrolytic reactivity of sodium alkoxide in water10,11). In vitro cell compatibility and the preclinical in vivo regenerative potential of sol-gel synthesized 45S5-BG and its role in osteogenesis has not been studied well. The objectives of this present study are to evaluate the bio-activity, in vitro cell compatibility and in vivo alveolar bone regenera-tion capacity of 45S5-BG synthesized via modified sol-gel method.

Materials and MethodsMaterials

Chemicals used as precursors for the synthesis of the sol-gel 45S5 materials: tetraethyl orthosilicate (TEOS) (Sigma Aldrich, St Louis, MO, USA), triethyl phosphate (TEP) (Sigma Aldrich, St Louis, MO, USA, 99.8%), sodium nitrate (Sigma Aldrich, 99%) and calcium nitrate

Correspondence to: Dr. Anand Anbarasu, Medical and Biological Computing Laboratory, School of Bio Sciences and Technology, VIT, Vellore, India; Tel: +91-416-2202547; Fax: +91-416-2243092; E-mail: aanand@vit.ac.in

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tetrahydrate Sigma Aldrich, St Louis, MO, USA, 99%).

Sol-gel processThe molar ratios of TEOS, TEP, NaNO3 and Ca (NO3)2 · 4H2O were

prepared as per the molar ratio of SiO2, P2O5, Na2O and CaO in 45S5. To attain a clear sol the molar ratio between water and the four precur-sor chemicals was set at 10. Each chemical was added at a slow rate into the HNO3 aqueous solution of 0.3 molar at room temperature. Each compound was added only when the previous solution became clear, and was then stirred for at least 1 h. The resulting gel was dried at 60 and 200°C for 72 and 40 h, respectively, aged at 500°C for 5 h and sin-tered at 680°C for 2 h.

Characterization of the prepared 45S5-BG was performed using powder X-ray diffraction (XRD) analysis and high-resolution transmis-sion electron microscopy (TEM).

XRD analysis The phase composition of the 45S5-BG after setting was analysed

by X-ray diffraction (XRD), (GE, 3003TT, Germany), with CuK α radi-ation source (λ=1.54059 Å) operated at 40 kV. XRD patterns were re-corded from 20° to 60° (2θ) with a step size of 0.04° and a counting time of 2s/step. The size of bioglass crystals in the cement samples were calculated from the characteristic peak fitting (2θ = 34.09°), according to the Scherrer equation, (eq.-1).

　   

=KDCos

(eq.-1)

where, D = size of crystal, K = crystallite constant = 0.9, λ = radia-tion wavelength = 1.54 Å, β = full width half maximum of the (002) peak.

TEM analysis TEM investigation was employed for morphological evaluation and

size distribution of the 45S5-BG with JEM-2010F (JEOL, Tokyo, Ja-pan) and image J technical software (version 1.53).

Evaluation of in vitro bioactivity In vitro bioactivity of the 45S5-BG in simulated body fluid (SBF) at

pH 7.4 was evaluated using Fourier transform infrared spectrophotome-try (FTIR), (Shimadzu, Tokyo, Japan) analysis frequency range 400 - 4000 cm-1 12). BG powder (3.75 mg) was immersed in 50 ml SBF at 37°C for 7, 14 and 21 days. Samples were then dried at 60°C. FTIR was used to evaluate formation of hydroxy carbonate apatite (HCA) layer.

In vitro cytotoxicity analysisThe 3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) assay was used for the evaluation of cell proliferation (L929 fi-broblasts). For the in vitro cytotoxic analysis of 45S5-BG, cells were seeded in 96-well plate at a density of 3×104 cells/well and incubated for 24 hours in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich, St Louis, MO, USA) supplemented with 10% Fetal Bovine Se-rum, (FBS) , (Gibco, Carlsbad, CA, USA) and 1% Penicillin streptomy-cin (Gibco) solution in 5% CO2 at 37°C. 45S5-BG stock (25 mg/10 ml) was prepared in DMEM, after incubation for 24 hrs, suspension was centrifuged at 2,000 rpm for 10 min and the supernatant was used for preparing serial working dilutions at 10, 20, 40, 60, 80%, and 100% added to cells and further incubated for 24 hrs. Dimethyl Sulfoxide 10% (DMSO) (Gibco) was used as the positive control for cytotoxicity, whereas cell alone served as the control. MTT (5 mg/ml) reagent was

added to the wells and incubated for 4 hrs. Then the reaction was stopped, and formazan crystals were solubilized using 100μl sodium do-decyl sulfate and absorbance was read at 570 nm in microplate reader (Model550, Bio-Rad, Tokyo, Japan).

In vivo experimentsAmerican satin guinea pigs (male) were purchased from Kerala Vet-

erinary College, Mannuthy, Thrissur, Kerala, India, housed under uni-form husbandry condition of temperature (25 ± 5 ºC), humidity ranging between 50 % - 60 % and normal photoperiod (12: 12 h dark: light cy-cle). Commercial pellet (guinea pigs specific) diet (Krish Scientist’s Shoppe Pvt. Ltd., Bangalore, Karnataka, India) and UV sterilized double filter water (Aquafresh, Austinroz India Pvt. Ltd., Kochin, Kerala, India) were provided ad libitum in Laboratory Animal Facility (LAF) of Push-pagiri Institute of Medical Sciences and Research Centre.

The in vivo experiments were carried out with sixteen, six months old male American satin guinea pigs (350-450 g, 8 animals / group) af-ter obtaining approval from the institutional animal ethics committee (602/P0/Re/ S/2002 CPCSEA) and conducted strictly adhering to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA) as constituted by the Animal Wel-fare division of Government of India.

Surgical proceduresAll the in vivo experiments were performed after intramuscular (IM)

administration of ketamine hydrochloride (35 to 50 mg/kg) (Neon Labo-ratories Limited, Mumbai, Maharashtra, India) along with xylazine hy-drochloride (5 to 10 mg/kg) (Indian Immunologicals Limited, Hydera-bad, Telangana, India) to accomplish adequate anaesthesia. Using a sterile bone trephine (6 mm) attached to a surgical physiodispensor (No-bel Biocare, Göteborg, Sweden), an intrabony defect extending through cortical bone and cancellous bone with a 6 mm diameter and 4 mm depth was created on the mandibular symphysis at the mesial surface of lower left incisor in both the groups. In the test group (B) the created in-trabony defect was filled with 45S5-BG bone graft and in the control group (A) the defect was left unfilled.

Post-surgical care All animals involved in the experiment were monitored after the sur-

gical procedures for postoperative complications. Xylazine hydrochlo-ride, 5 mg/kg administered IM, was used when needed for post-surgical analgesia.

Sample procurement and analysisOn the 42nd day after surgical procedures, the animals were sacri-

ficed in carbon dioxide chamber and the mandible was removed from which the block of bone containing intrabony defect was obtained by using a surgical micro motor. Specimens were labelled and fixed with 10% formalin for 72 hours.

Histologic analysisAll specimens were demineralized using 0.003 M solution of ethyl-

enediaminetetraacetic acid (EDTA) and 1.35N hydrochloric acid (Sig-ma) and stained using haematoxylin and eosin (H and E staining). Sec-tions were cut at 5 to 6 µm thickness and at 500 µm intervals for test and control sites.

All specimens were examined for features of bone regeneration (shown by osteoblastic activity and osteoid formation) and foreign body reaction (by the presence of giant cells).

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Statistical analysis In cytotoxicity analysis of 45S5-BG, the data was represented as

mean± SD (n=3) with P value of 0.05 being significant using Graph Pad prism. The results were analysed by two-way ANOVA with Dunnett’s multiple comparison post-test. Comparisons were performed as Positive control vs other groups (ns-nonsignificant, *p<0.05, **p<0.01, ***p<0.001).

ResultsXRD analysis

The wide-angle X-ray diffractogram of the sintered 45S5-BG is de-picted in Fig. 1. Major crystalline phase was identified as Na2Ca2Si3O9

(Combeite crystalline phase) (PDF #22.1455). The crystallite size of the main phase in the 45S5-BG found out using Scherrer equation was

24.84 nm at full width half maximum (FWHM) 0.3198 corresponding to the 2θ angle of 34.09°.

TEM analysis Morphological evaluation of 45S5-BG performed using TEM, is

represented in Fig. 2. The ultra-structures of the 45S5-BG clearly indi-cated the presence of crystalline particles embedded in an amorphous glassy matrix after sintering with each particle having an average size of 150 nm. Crystallinity was confirmed from the fringe patterns observed in the TEM images as well as from the selected-area electron diffraction (SAED) pattern.

In vitro bioactivityFTIR spectra taken on the powder before and after SBF exposure (7,

Figure 1. Wide angle XRD pattern of 45S5 - bioglass.

Figure 2. TEM - Microstructure of the 45S5 - bioglass (i, ii) fringe pattern (iii, iv) and inset (in iii) showed SAED patterns.

Figure 3. FTIR spectra of Bioglass scaffolds before SBF exposure (BG1) and after SBF exposure 7, 14 and 21 days (BG7, BG14 and BG 21).

Figure 4. MTT analysis of 45S5. The data was represented as mean± SD (n=3). The results were analysed by two-way ANOVA with Dunnett’s mul-tiple comparison post-test. Comparisons were performed as Positive con-trol vs other groups (ns-nonsignificant, *p<0.05, **p<0.01, ***p<0.001).

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14 and 21 days) to analyse its in vitro bioactivity is shown in Fig. 3. Bi-oglass before SBF exposure (BG1) exhibited characteristic peaks at 460.58 cm-1 attributed to Si-O-Si bending vibrations peak at 1020 cm-1 is due to Si-O-Si stretching and absorption peaks at 513 cm-1 and 584 cm-1 belong to the respective Si-O-Si and P-O bending, originating due to the presence of crystalline phase. SBF treated bioglass exhibited peaks at 558 and 604cm-1 attributing to P-O bending vibration. Intensification of peaks at 558 and 604 cm-1 are in accordance with the increase in time of SBF exposure to the bioglass.

In vitro cytotoxicity analysis In the present study, in vitro toxicity profiling of 45S5-BG was per-

formed and is represented in Fig. 4. Among the tested concentrations of 10%, 20% and 40%, exhibited similar cell viability to that of the con-trol. The highest tested concentration of 45S5-BG (100%) exhibited 32.60% of cell viability in comparison to that of the positive control, 10% DMSO (22.33% viability). The cell viability associated with 10%, 20%, 40%, 60% and 80% of the 45S5-BG test concentration was found to be 90.33%, 73.46%, 62.39%, 56.92%, and 52.25% respectively (p < 0.001).

Figure 5. Group A - Figure A and B - retained intrabony defect where black arrow indicates tooth structure, blue arrow indicates periodontal liga-ment and white arrow indicate bone margin of retained defect. Group B - Figure C - indicate completely restored defects by the regeneration of periodontal ligament with new bone formation, black arrow indicates tooth structure, blue arrow indicates fully regenerated periodontal ligament and green arrow indicates fully restored intrabony defect with new bone formation. Figure D - yellow arrow indicates resting and reversal lines and red arrow indicates formation of new blood vessels. Figure E - water blue arrows indicate new bone formation with osteoid, yellow arrow in-dicates resting and reversal lines, red arrow indicates formation of new blood vessels and violet arrow indicates osteocytes.

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In vivo bone regenerative potential of 45S5-BG Subjective observations - 45S5-BG grafts were easily packed into

the intrabony defect. Test and control sites healed uneventfully without any postoperative complications

Histologic evaluation At 42nd day, histological analysis by H and E staining of test and

control group animals were devoid of inflammatory infiltrates. There was a significant difference between test and control groups in terms of lamellar and trabecular bone pattern. In Group B, Fig. 5C the osteotomy defect was fully restored with new bone formation with complete resto-ration of periodontal ligament. Bioglass particles had undergone cellular mediated excavation with the resulting space being filled by newly formed bone and osteoid tissues as presented in Fig. 5D, E. Group A an-imals retained the intrabony defect with minimal periodontal ligament regeneration and new bone formation depicted in Fig. 5A, B.

DiscussionBiomaterials capable of inducing specific cellular responses when

they come into contact with tissue fluids are considered to be bioac-tive13). Alloplastic graft materials should possess biocompatibility, bio-activity, osteoconductive, osteoinductive properties and provide space for new bone formation. Bioactive glass has the ability to elicit intra- and extracellular responses at graft and host bone interface and hence has been categorized as Class A material14).

Higher the specific surface area, leading to increase in the contact surface between the material and biologic fluid, greater will be the glass bioactivity15). Larry Hench in 1982, clinically used bioglass for first time to reconstruct the ossicles of middle ear16). US Food and Drug Adminis-tration (FDA) approved melt-derived compositions of 45S5-BG for clinical application in regenerative surgical procedures17).

Sol-gel synthesized bioglass has bioactivity and degradation rate significantly higher than conventional melt-derived bioglass of the same composition9,18). Sol-gel technique enables synthesis of bioglass by alter-ing processing parameters in a highly versatile way19). Sol-gel derived bioglass particles possess an increase in pore volume and specific sur-face area twice magnitude higher than the melt-derived ones20). Due to its bone regeneration potential and antimicrobial properties, bioglass is the material of choice for alveolar bone regeneration.

In the present work, we have synthesized, characterized and investi-gated the preclinical properties of 45S5-BG. XRD analysis of bioglass samples revealed specific crystalline phase for 45S5-BG identified as Na2Ca2Si3O9 (Combeite crystalline phase) (PDF #22.1455). The same was identified by the other studies of sintered bioactive glass of same composition21,22). There are reports which claim that the crystallinity of the sintered bioglass cannot be 100%21). Fringe and SAED pattern con-firm presence of crystalline phase.

Bioglass before SBF exposure (BG1) exhibited characteristic peaks at 460.58 cm-1 attributed to Si-O-Si bending vibrations. Peak at 1020 cm-1 is due to Si-O-Si stretching corroborating with the earlier report23). In addition, absorption peaks at 513 cm-1 and 584 cm-1 belong to the re-spective Si-O-Si and P-O bending, originating due to the crystalline phase24). SBF treated bioglass exhibited peaks at 558 and 604 cm-1 attrib-uting to P-O bending vibration arising due to surface minerals and amorphous calcium phosphate crystallized to HCA. Intensification of peaks at 558 and 604 cm-1 are in accordance with the increase in time of SBF exposure to the bioglass, highlighting the bioactive characteristics of synthesized 45S5-BG25).

Toxicity of biodegradable scaffolds are mainly attributed to release

of their degradation end products, which in turn will stimulate or inhibit metabolic activities in the cells26).

In vitro cell compatibility was assessed by exposing L929 cell lines to 45S5-BG to analyse for cytotoxicity. L929 Cell lines treated with the 45S5-BG extract (10, 20 and 40%) did not show any significant change in their viability even after 24 h of exposure, in comparison with the control. As a matter of fact, after treating with the highest test concen-tration (100%), 32.61% of the cells remained viable in comparison to the positive control. As per ISO 10993-5 guideline, a reduction in cell viability greater than 30% is considered a cytotoxic effect27).

At 42nd day, histological examination of mandibular bone of Group B animals exhibited complete restoration of the intrabony defect with new bone formation in Fig. 5C. Implanted 45S5-BG grafts were com-pletely resorbed, providing space for new bone formation. In group B animals, the intrabony defect showed new bone formation with osteoid, regeneration of periodontal ligament, newly formed blood vessels and increased number of osteocytes. In addition, resting and reversal lines were also noted in Fig. 5D, E indicating remodelling of bone28,29). These observations confirm the bone regenerative potential of 45S5-BG. Group A animals, at 42nd day retained the intrabony defect with minimal periodontal ligament regeneration and new bone formation in Fig. 5A, B.

Studies have shown that dissolution products of 45S5-BG will pro-mote differentiation of stem cells into osteoblast and also accelerate gene expression of osteoblast leading to bone regeneration, and influ-ence neovascularization effect promoting the formation of blood vessels in vitro30,31). As indicated in our in vitro bioactivity studies, 45S5-BG graft had reacted with body fluid to form a HCA layer leading to a tight bond-ing between the bone graft, bone and the soft tissue providing a suitable surface for osteogenic cell attachment and proliferation32). It is proposed that this might be the possible mechanism for the observations associat-ed with bioglass assisted bone regeneration.

In the present study, 45S5-BG has been synthesized through modi-fied sol-gel method to investigate its role in alveolar bone regeneration. FTIR analysis confirmed the in vitro bioactivity and MTT results proved the in vitro cell compatibility of 45S5-BG. Histological analysis proved new bone formation and bone maturation validating its osteoinductive behaviour, demonstrating the potential flair of 45S5-BG bone graft to be used for alveolar bone regeneration material.

AcknowledgementsWe wish to acknowledge the administrative support from Rev. Fr.

Aby Vadakkumthala Director of Medicity, Pushpagiri group of institu-tions and Rev. Dr. Mathew Mazhavancheril, Director and Head of Re-search, Pushpagiri Institute of Medical Sciences, Pushpagiri group of institutions.

Conflicts of InterestThe authors have declared that no conflicts of interest exist.

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