Osseous Regeneration of Periapical Lesions using Rice Straw Nanofibers-Nano Amorphous Calcium Phosphate Composite Hydrogel (In-Vivo Study)
Published date: March 06, 2020
Osseous Regeneration of Periapical Lesions using Rice Straw Nanofibers-Nano Amorphous Calcium Phosphate Composite Hydrogel (In-Vivo Study)
Engie M. Safwat1, Nehal F. Sharaf1, Abdel Razik H. Farrag2, Mohammad L. Hassan3*, Dalia Y. Zaki1
1Restorative and Dental Materials Department, National Research Centre, Egypt
2Pathology Department, National Research Centre, Egypt
3Cellulose and Paper Department, National Research Centre, Egypt
*Corresponding author: Mohamed L. Hassan, Cellulose and Paper Department, National Research Centre, Egypt, E-mail: email@example.com
Citations: Safwat EM, Sharaf NF, Farrag ARH, Hassan ML, Zaki DY (2020). Osseous Regeneration of Periapical Lesions using Rice Straw Nanofibers-Nano Amorphous Calcium Phosphate Composite Hydrogel (In-Vivo Study). J Mol Nanot Nanom 2(1):109.
The objective of this study was to investigate the ability of rice straw extracted cellulose nanofibers (CNF) / nano amorphous calcium phosphate (nACP) composite hydrogel, to induce bone regenerationin the periapical lesions of endodontically treated teeth. The in-vivo study was performed through drilling surgical cavities, periapical to endodontically treated molars of experimental dog. Three materials were tested during this study including; CNF hydrogel, nACP powder, and CNF-nACP nanocomposite in 2:1 weight ratio. Periapical bone density analysis was done using Dual-energy X-ray absorptiometry (DXA), in addition, histopathological investigations of the filled periapical bone defect were obtained using H&E and Masson’s trichrome (MT) staining. Areas of newly formed collagenous bone, MT stained in green-blue, were measured in multiple fields for each group. The analysis showed that nACP and CNF-nACP nanocomposite hydrogel could form the normal bone density, meanwhile, only CNF-nACP composite presented matured bone and collagen formation (P < 0.05). The DXA and histological results indicated that CNF-nACP nanocomposite could have promising osteoconductive potential.
Keywords: Cellulose nanofibers; Nano amorphous calcium phosphate; Osseous regeneration
Infection of dental pulp tissue may be a consequence of untreated caries or trauma, causing tissue necrosis . Bacterial infection with its released by-product causes a series of inflammatory and immunological reactions in the periapical area. According to the host's immune response together with the virulence of microbial infection, various grades of periapical lesions could develop. However, no self-healing could occur, where periapical abscess, radicular cysts or periapical granuloma could develop in improperly treated teeth. For the treatment of these cases, elimination or decreasing microbial infection is to be done through efficient chemo-mechanical preparation of the teeth root canals.
On the other hand, large periapical lesions may require surgical approach especially when the lesion is covered by an intact epithelium, separating it from the apical foramen and preventing the drainage of pus through the canal. Recent studies revealed that following regenerative techniques (RT) using bone graft; produce a superior outcome when compared with that without using RT . Bone graft substitutes are used to accelerate periapical healing, to allow healing in compromised clinical situations, and to serve as scaffold for new bone formation .
Synthetic biomaterials have been developed as bone graft substitutes, including ceramics e.g., hydroxyapatite and various calcium phosphate compositions . The use of polymeric materials also allowed; better handling, favorable vascularization and provide the capability to carry inorganic bioactive particles into their matrix forming composites . Examples of tested polymers for bone regeneration applications are poly (lactic-co-glycolic acid) , gelatin , chitosan , and collagen .
In bone tissue regeneration applications natural polymers proved to have several advantages when compared with synthetic polymers. Natural polymers provide higher biocompatibility, sufficient hydrophilicity, and biodegradability. Cellulose, starch, chitosan, chitin, collagen, gelatin are examples of natural polymers that have been investigated and used for the development of 3D scaffold . The unique properties of cellulose regarding its biocompatibility, non-toxicity, stability to moderate temperature and pH variations and adequate surface chemistry profile were the driving force for its biological applications .
Many literatures have focused on injectable gels for bone tissue engineering applications, prepared from different materials such as calcium phosphates combined with methylcellulose [12, 13], carboxymethyl cellulose [14, 15], hydroxyl propylmethylcellulose [16, 17], silanized-hydroxypropyl methylcellulose [18, 19] and bisphosphonated-nanocellulose . Nanocellulose has been also used for bone tissue engineering but the in-vivo studies on experimental animals to test bone tissue regeneration; were rarely done [21, 22].
Previous in-vitro study was done to investigate the use of cellulose nanofibers (CNF), isolated from rice straw for the preparation of an injectable hydrogel for bone regeneration . In that study, Tetramethyl pyridine oxyl (TEMPO) oxidized CNF, were mineralized through the incorporation of 50 wt% biphasic calcium phosphate. Cytotoxicity and alkaline phosphatase level tests on osteoblast like-cells were investigated. Results proved that the isolated CNF is a nanocomposite of cellulose nanofibers and silica nanoparticles. The tested materials showed a nontoxic effect on osteoblast-like cells. However, CNF-biphasic calcium phosphate composite hydrogel showed higher cytotoxicity and lower bioactivity when compared to that of neat CNF. For that reason, nACP was used in this study due to its promising bioactivity as revealed in many kinds of literature [24-29].
Therefore, the aim of the present study was to assess the in-vivo biocompatibility and the osteogenic potential of the experimental CNF hydrogel prepared according to Safwat, et al., 2018 , loaded by nACP for treatment of periapical lesions of endodontically treated teeth.
Materials and Methods
Rice straw agricultural wastes were collected from local farms in Giza, Egypt and used for the preparation of CNF hydrogel according to Safwat et al, 2018 .
The prepared rice straw CNF had dimensions in the range of 4 nm to 8 nm in width and reached up to several microns in length . The isolated CNF was found to be loaded with nano-sized silica (~ 50 nm in diameter) which is naturally intimately embedded in rice straw . Nano Amorphous calcium phosphate (nACP) was purchased from Sigma Aldrich Chemical Company St. Louis, USA. CNF and nACP were mixed such that each 2cm3 volume of CNF hydrogel (2% dry weight) was added to 1cm3 in volume (0.2304 gm weight) of nACP.
Grouping of the Tested Materials
Three group samples were tested in this study as follows: group 1: CNF hydrogel, group 2: nACP powder and group 3: CNF-nACPnanocomposite hydrogel.
Sterilization of the Tested materials
Samples were sterilized by gamma-ray irradiation at 25 kGy, using cobalt-60 as irradiation source, with an irradiation dose rate of 1 kGy per hour.
The surgical procedures were conducted according to the protocol approved by the Ethical Committee of the National Research Centre, Cairo, Egypt with the registration number of (18079), and was done in accordance with ISO 10993-6:2016 . The study was done on adult mongrel dogs, weighed 25±1 kg body weight, and aged 1±0.5 years. Dogs were examined thoroughly before the selection and were kept under observation for 2 weeks.
Surgical procedures were carried out under general intramuscular anesthesia injection of ketamine (6.5-13 mg/kg IM, midazolam). Routine infiltration anesthesia of Lindocaina 2% was administered at the surgical sites.
In this study, three molars in the mandibular right side of the dog were endodontically treated. After rubber dam application, the teeth surfaces were disinfected with 2% chlorohexidine gluconate. Root canals were instrumented to the full working length up to size 40 using k-files (Maillefer instruments, Switzerland). 2.6% Sodium hypochlorite (NaOCl) was used as a root canal irrigating solution. The prepared canals were then dried and obturated using guttapercha cones and Ad Seal™ resin sealer (Metabiomed OK, Chungbuk, Korea). A full thickness flap was then elevated in the alveolar ridge to expose the periapical areas of the root canal treated teeth. Seven surgical cavities were drilled periapical to the roots of the endodontically treated teeth using 3-mm diameter surgical drills (Neobiotech Co., Seoul, Korea) with a 30: 1 reduction handpiece at a speed of 1000 RPM. The final torque value was 35 Ncm. For standardization and ethical considerations, all the tested materials were presented in the same dog.
The first two cavities received group 1 samples, the second two cavities received group 2 samples, and the third two cavities received group 3 samples. A fourth cavity was left empty as a control. The flap was repositioned and sutured over the bony cavities (figure 1). The dogs were fed with a soft diet for the first week postoperatively followed by a regular diet. Sacrifice was done after 15 weeks using high doses of anesthesia and then mandible was dissected for DXA scanning followed by histological examination.
Figure 1: Experimental samples insertion in the surgical cavities periapical to the endodontically treated roots.
Bone mineral density (BMD)
DXA Norland densitometer (XR-46, USA) Rev. 4.6.4/2.3.1 was used to evaluate bone regeneration and remodeling in each particular periapical region containing one of the three experimental groups. The DXA scanner was adapted for the analysis, such that the entered parameters were set up to measure the human child age of near weight . Measures of bone mineral density (BMD) expressed in g/cm2 were performed for each particular periapical region containing one of the three experimental groups in the separated mandibular right side. All analyses were conducted using high-precision mode DXA at 1-mm scan pitch. Results were expressed as mean BMD of periapical areas in the same group and were compared to the normal bone in the periapical region of the same size.
Histological sample preparation: Mandible was fixed in 10% formaldehyde for 24 h, rinsed in running tap water for 24 h to remove formaldehyde, then incubated with EDTA (pH 7.4) as a decalcifying solution. The decalcification process was performed under continuous shaking and the decalcifying solution was changed weakly. The decalcification procedure was ended when a needle without any force easily penetrated through the bone. Subsequently, samples were washed in running tap water for 24 h, followed by dehydration in a series of ethanol and then embedded in paraffin. 5 µm sections were cut using a Leica microtome (Leica, Germany) and placed on glass slides .
Staining methods: Hematoxylin and Eosin staining were performed according to Go, et al, . Briefly, after deparaffinization and rehydration, the 5 µm longitudinal sections were stained with hematoxylin solution for 5 min followed by 5 dips in 1% acid ethanol (1% HCl in 70% ethanol) and then rinsed in distilled water. The sections were then stained with eosin solution for 3 minutes and followed by dehydration with graded alcohol and clearing in xylene. The mounted slides were then examined and photographed using an Olympus BX51 light microscope (Tokyo, Japan).
Masson’s trichrome (MT) staining was also used to identify bone tissue formation and maturation stage [35, 36]. Sections were stained in Biebrich scarlet-acid fuchsine solution for 15 min and rinsed in distilled water. Sections were then incubated in phosphomolybdic–phosphotungstic acid solution for 15 minutes, transferred directly to aniline blue solution and then stained for 5–10 minutes. Sections were rinsed in distilled water, washed in 1% acetic acid solution for 2–5 minutes, and then rinsed in distilled water. Stained sections were dehydrated; coverslipped with DPX as a mounting medium, to be examined using an Olympus BX51 light microscope. MT dye stains the new collagenous bone in green-blue and the mature osteoid in red; six fields in each groupwere photographed using a digital camera, analyzed to indicate the area percentage of newly mineralized bone formation.
The statistical analyses were carried out using SPSS version 13.0. The significance between means was calculated by the single factor ANOVA. The difference was regarded as significant if the ‘P’ value was < 0.05.
Results and Discussion
CNF with a diameter close to that of elementary cellulose fibers (~4nm) could be isolated from TEMPO-oxidized cellulose pulp prepared from rice straw. The isolated CNF form injectable gel at 2 wt.% in water . The addition of nACP to CNF gel at the studied concentration of the former did not affect the ease of injectability.
DXA has been demonstrated to measure skeletal maturity  and was used to evaluate the effects of pharmaceutical therapy on bone density . BMD of all groups was presented in Table (1). A significant decrease in BMD of bone received group 1 was found, as compared with non-cavitated normal bone (P < 0.05). However, statistically insignificant differences were found in the results of group 2, group 3 and the non-cavitated normal bone (P < 0.05). Results indicate that; both nACP and CNF-nACP composite stimulated the formation of new calcified bone.
Table 1: Mean BMD of the tested periapical areas in all groups. Data presented as mean ± standard error. * Significant decrease at P < 0.05 as compared with non-cavitated normal bone. NS non-significant decrease at P < 0.05 as compared with non-cavitated normal bone.
These primary results were confirmed by the histological examination of H&E and Masson’s trichrome dyes. As for the H&E stained sections, the control grouprevealed the formation of collagen-rich connective tissue. Tissue formation was detected mainly in the intramedular space, with no new bone formation (Fig. 2A). Regarding cavities of group 1, almost no regenerated bone in the defect areas were detected, only bundles of collagenous are found (Fig. 2B).On the other hand,new regenerated bone volumes filling the defect were detected in cavities of group 2. In this group, osteoblasts were found around the newly formed bone, while few numbers of osteocytes were found inside the newly formed bone indicating its immaturity, hollow areas were detected in the defect area in some samples demonstrating incomplete bone formation (Fig. 2C). On the other hand, group 3 showed almost total bone repairing in the defected area. Osteoblasts were found around the newly formed bone, while high numbers of osteocytes were found inside the new bone. New blood vessels were found around the new bone area, indicating full maturity of the formed bone (Fig. 2D).
Figure 2: A section in dog’s mandibular bone in the periapical region, showing: (A) section of the control group, (B) section of group 1, (C) section of group 2, (D) section of group 3 (H and E, Scale bar 5 µm).
A representative sample of histological sections of different groups stained using Masson’s trichrome staining was shown in figure 2. Sections of the control group showed no evidence of new bone formation (Fig. 3A). Regarding cavities of group 1, only bundles of collagenous were found as revealed by the green-blue color but diminutive regenerated bone (red) was formed (Fig. 3B).Cavities of group 2 showed newly regenerated bone filling the defect but of few osteocytes indicating immature bone formation (Fig. 3C).On the other hand, cavities of group 3 (Fig. 3D) showed both reddish-colored regenerated boneand green-blue collagenous bone formation, with osteocytes inside the newly formed bone indicating bone maturity.
Figure 3: Histological section in the periapical region of dog’s mandibular bone showing; (A) section of the control group, (B) section of group 1, (C) section of group 2, (D) section of group 3 (Masson’s trichrome staining, Scale bar 30 µm).
Quantitative measurement of the areas of newly formed collagenous bone, stained in the green-blue stains showed a significant increase at P < 0.05 in all groups as compared with the control group, though, a significant decrease of bone formed areas compared with group 1 (Table 2). Although group 1 had the highest collagenous bone formed, yet no regenerated bone has formed, indicating its low osteogenic potential. On the other hand, the lowest green-blue areas revealed by group 2 is due to the regenerated bone formation (red) though not fully matured as indicated by scarce osteocytes. Meanwhile, group 3 had both collagenous and regenerated bone with osteocytes and osteoblasts, indicating favorable conditions for high quality mature bone formation.
Table 2: Areas of newly formed bone of different groups. All data are presented as mean ± SE. *Significant increase at P< 0.05 as compared with control group. •Significant decrease at P< 0.05 as compared with group 1.
Such findings indicate the biocompatibility, the osteoconductive potential and the bioactivity of both nACP and CNF-nACP composite hydrogel with the preeminence of CNF-nACP composite. This is probably due to the known role of nACP as a bioapatite forming precursor. Dissolution and precipitation take place at the particle surface in body fluid. Under physiological conditions dissolution process is affected by particle composition, the body fluid mineral supersaturation level, and the implantation site. The nanoscale of the nACP particles and their amorphous structure enhance the dissolution process through their higher total surface area exposed to the surrounding fluids together with the increased reactivity due to their amorphous structure improving their in-vivo osteoconductivity .
The presence of well-formed bone filling the defect cavity after using CNF-nACP hydrogel composite may indicate the more favorable effect of using CNF hydrogel together with nACP on bone formation. Such finding is in agreement with results obtained by the study done by Safwat et al ,where nanocellulose hydrogel prepared from rice straw proved to have the ability to induce ossification as indicated by the positive alkaline phosphate assay. The presence CNF with its bioactive role and the known ability of the hydrogel to confine and maintain a high concentration of the loaded active ingredient at the implantation site over an extended period of time; may play a role in increasing the level of bone formation .
Within the limitations of the present study, it could be concluded thatCNF-nACPnanocomposite hydrogel is a biocompatible material with osteoconductive potential and bioactivity. CNF-nACPnanocomposite hydrogel proved to have a more favorable bone forming effect when compared to nACP powder.
The authors would like to acknowledge the financial support of “National Research Centre, Egypt”, grant number; AR111402.
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