Local Evaluation of Chitosan and B-Tricalcium Phosphate Alone and Combination in Bone Defect of Rabbit by Histological and Histomorphometric Analysis

Zainab AJ AL-Mashhadi^* and Nada MH AL-Ghaban

^*Correspondence: Zainab AJ AL-Mashhadi, Department of Oral Diagnosis, College of Dentistry, University of Baghdad, Iraq, Email:

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Introduction

Critical bone defect is generally caused by aggressive accident, infection, tumor and congenital malformation. Critical bone defects ranged from too huge to be self-restored through heals spontaneously [1]. Bone is complicated process through overlapping three stages: the inflammatory phase, the repair phase, and the remodelling phase. Early phase of bone healing characterized by systematically and local responses to the defect stimuli. Additionally, mesenchymal stromal cell and immune cells participate in cellular interaction to control bone healing [2].

Chitosan is a polymer-derived from chitin which is manufactured by deacetylation of chitin. Chitin is generally extracted from several types of fungi and exoskeletons of crustaceans [3,4] chitosan has several applications in biomedical field, including wound healing, regulation of hypertension and cholesterol and bilirubin absorption, prevention from dental plaque of skin grafting, hemostasis [5].

On other hand, in spite of advantages uses of chitosan but also has many disadvantages, such as poor mechanical strength, low acid resistance [6]. So to minimize these disadvantages, chitosan should be blended with cross linker polymer to, for example Polyvinyl Alcohol (PVA), cellulose and polyacrylamide [8,9].

PVA regard as a most popular polymer that blended with chitosan to provide mechanical strength due its nontoxic structure and inter molecular hydrogen bonds [10].

Beta tri-calcium regard as most popular bone synthetic due to osteo conductive and ostroinfictive properties make it one of most efficient materials to management bone defect in orthopedic and maxillofacial surgery [11].

Materials and Methods

Preparation of chitosan-Polyvinyl Alcohol (PVA) hydrogels

• In room temperature prepare 0.2 g of chitosan powder (95% deacetylated) in 10 mL of 0.1 M glacial acetic acid solution and stirrer the mixture overnight.

Prepare 1 g of PVA in 10 mL of ultrapure water and stirrer at 90°C for 2 h.

• The two systems mixed in percentage 1:1 in magnetic stirrer to provide homogeneous structure at room temperature, the mixture poured on lab petri dishes. The system left for 1 h at atmospheric pressure.
• Freeze the hydrogels at -20°C for 20 h then thawed in to 8 hours at room temperature, these cycles repeated about 6 times then wash the hydrogels with deionized water, finally the hydrogel kept in ultra-pure water ready to applied in experimental study [12,13].

Animal preparation: All experimental procedures carried out according to the ethical approval of animal experiments of College of Dentistry, University of Baghdad. All animals Supervision and nursing from the stuff of private animal house. 32 males New Zealand rabbits (6-9) months weight (1.5-2 kg) were used in this experimental study, where intrabony defects were achieved 3 mm in depth and 3 mm in width in both right and left femur Figure 1. All animal euthanized in two and four weeks by giving overdose of anesthesia (16 rabbits for each healing periods). Bone specimen was prepared by cutting the bone about 5 mm away from site of operation Figure 1.

Figure 1: Show surgical procedure including, A) bone defect; B) Application of beta tricalcium phosphate; C) Application of chitosan gel; D)Skin suturing; E)Bone cutting.

Histological preparation: Bony specimens were fixed with 10% freshly prepared formalin for 24 hours, then decalcification process started using 10% EDTA (in two months), embedding done using paraffin wax, bone blocks were sectioned by microtome for serial sections of 4 μm was taken then sited on slide. Staining was done by using Hematoxylin and Eosin (H and E) stain. Histomorphometric measured by software using Image J program [14].

Results

Histological finding showed that all histological sections displayed respectable bone repair for groups in the study but there was difference in their rate within time consuming of experiment. In Chitosan Hydrogel (CH) group at 2 weeks showed irregular bone trabeculae with irregular distributer osteocytes with rich count of osteoblast when compared with control group (Figures 2 and 3) whereas within time at 4 weeks chitosan group show mature bone trabeculae with regular distribution of osteocyte and recognizable osteons were observed (Figures 4 and 5).

Figure 2: View of 2 weeks control defect area showed a new fine Bone Trabeculae (BT) rimed by Osteoblast (OB) and Osteocyte (OC), woven bone separated from old bone by reversal line (arrows) also showed osteoclast were seen H and E X10.

Figure 3: Micrograph of control group of 4 weeks showed Osteocytes (OC) entrapped in woven bone (WB) rimed by Osteoblast (OB) and osteoclasts cells also are present (OCL) H and E X40.

Figure 4: View of 2 weeks defect area treated with chitosan shows new bone Trabeculae (T.B) separated from Basal Bone (BB) by reversal line (arrows) H and E X10.

Figure 5: Micrograph of chitosan group of four weeks duration showed mature bone trabeculae which entrapped with regularly arranged Osteocyte (OC), Osteoblasts (OB) seen lined Haversian Canal (HC) H and E X40.

β-tricalcium phosphate groups illustration irregular deposition of osteoid tissues were seen at the site of defect after two weeks duration surrounded by osteoblast and osteoclasts cells also showed osteocyte entrapped, while at 4 weeks indicated maturation of bone by increase trabecular thickness were seen with regular organized osteocytes (Figures 6 and 7).

Figure 6: Micrograph of beta tricalcium phosphate showed woven bone entrapped by Osteocytes (OC), and rimed by Osteoblasts (OB), and also show Osteoclasts (OCL) H and E X40.

Figure 7: Micrograph of TCP group after four weeks revealed regular distribution of Osteocytes (OC) in New Bone (NB) rimed by Osteoblasts (OB) at borders of bone also Osteoclast (OCL) were seen H and EX10.

Histological assessment of after two weeks duration show regular bone trabeculae with entrapped by osteocytes and surrounded by osteoblast, after 4 weeks trabecular area appear more mature and regular distribution of osteocyte were seen (Figures 8 and 9).

Figure 8: Micrograph of combination group of two weeks the bone defect area treated with chitosan hydrogel and β-tricalcium phosphate after 2 weeks showed new Trabecular Bone (BT) also, reversal line that separated the old bone from new one H and E X10.

Figure 9: Micrograph of combination group after of 4 weeks duration of combination group showed mature bone contains Osteocytes (OC), Osteoblast (OB) cells H and EX40.

Statistical analysis: According to the tables below that illustration descriptive statistic of the mean differences and stander error values of trabecular area, bone marrow area, formative bone cell, bone resorping cell, and bone entrapped cell in defected area at 2 and 4 weeks of healing duration for histomorphometric analysis. According to the LSD test Table 1 illustrated highly significant differences of osteocytes between both Chitosan (CH) and Control (CONT) and between Combination (CHCT) and Control (CONT). Table 2 displayed highly significant differences in osteoblast mean value in both Chitosan (CH) and Control (CONT) groups and between Combination (CHCT) and control in 2 weeks duration. Also high significant differences between TCP and Combination (CHCT) in two weeks and significant differences between Chitosan group (CH) and TCP in two weeks duration.

Table 1: Multiple differences in osteocyte mean value in multiple groups in the study by LSD test for the two healing periods.

Table 2: Illustrate descriptive statistic of osteoblasts cell for each healing periods.

Table 3 demonstrate significant differences in osteoclasts mean value between Chitosan (CH) group and control groups in 2 weeks. Table 4 exhibited high significant differences in trabecular area between Combination (CHCT) group and control groups, also high significant differences in both chitosan group and TCP group at two week duration while in 4 weeks show high significant differences between TCP group and Combination (CHCT) group.

Table 3: Show descriptive statistic of osteoclast cell for each healing periods.

Table 4: Illustrate descriptive statistic of the trabecular area in 2, 4 weeks of healing periods.

Table 5 revealed High significant differences between Combination (CHCT) and Control (CONT) and significant differences between Chitosan (CH) group and Control group (CONT), similarly shown significant differences between TCP and combination groups at two week duration.

Table 5: show group differences for bone marrow area between the two and four weeks of healing duration.

Discussion

After 14 days of bone defect histological outcome showed woven bone deposition where the defect are sited, all groups in experimental study provide good healing characteristic with differences rates among them. Microscopic examination after two weeks after treatment with Chitosan (CH) enhanced osteoblast proliferation through increased number of osteoblast significantly when compared with control group this result agree with [15,16] where their finding higher number of osteoblastsat attachment and elevation secretion of ALP when cultured bone marrow stromal cell into plate contain chitosan when compare to control group due to high deacelyation of chitos an which provide favourable environment to osteoblast attachment and spreading and extracellular matrix protein secretion.

Increase thickness of bone trabeculae and reduction in bone marrow area mostly related to increase osteoblasts differentiation and reduction the osteoclastogensis through inhibition to osteoclast activity within progressing time from 2 to 4 weeks. This agree with [17,18]. When drilled tibia of rat after 4 week duration show significantly faster bone healing in group treated with chitosan powder when compare to control groups and inhibition to osteoclastogenis process, moreover show more woven bone and no fibrous tissue in group treated with chitosan.

Histological finding of β-TCP on bone healing revealed increase in osteoblast activity as compared to the control group. This result agrees with [19] augmented tibia of New Zealand rabbits with β-TCP alone who showed β-TCP as optimal materials for inducing significantly provide osteogensis and reducing osteoclast cell activity. After 28 days there were observed increase thickness of trabecular and decrease in bone marrow area when compared with control group, this finding proved with [20]. Who filled the femur of rat by β-TCP who showed new bone formation in experimental group higher when compared to control one [21].

Reveals β-TCP encourage regeneration of by enhance mineralization within progression of time into 4 weeks after scarification which explain that by the interconnecting multi pores system which encourage vascular system to growth to produce nutrition and growth factor to the bone formative cell, moreover biodegradation of β-TCP usually coordinate with bone formation process as a result great amount of water content which enable materials to dissolved in body fluid which resorbed by osteoclast cells.

In combination group there was high count of osteoblast cells after 2 weeks duration when compared with control group, also bone trabecular areas were higher in combination than any of the other groups. This agree with [22] who showed scaffold of β-TCP-chitosan that associated with pulverized human bone which provide excellent mechanical properties and induced significantly higher ALP Within time progression when applied β-TCP and chitosan membrane with different proportions to membrane to guide tissues regeneration in critical sized defect at base of skull of rabbits, who show superior bone tissue regeneration when compared to control group. After 4 weeks noticed increase trabecular area thicknesses compare to control group. This agree with [23] who utilize combination of chitosan and tricalicium phosphate associated with polymehymethacrylate bone cement injected into skull cap of rat which resulted superior osteogenic activity and bone regeneration capacity when compare polymehymethacrylate bone cement alone.

Conclusion

Bone defect treated with chitosan/β-TCP alone or as combination scaffold increase the rate of bone regeneration through enhancement of osteoblast proliferation and decrease osteoclast activity and obtain the process of mineralization process of bone matrix superiorly than control group.

Reference

1. Toogood, Paul, Miclau T. Critical-sized bone defects: sequence and planning. J Orthop Trauma 2017; 23.

   [Crossref][Google Scholar][Indexed]

2. Maruyama, Masahiro. Modulation of the inflammatory response and bone healing. Front Endocrinol 2020; 386.

   [Crossref][Google Scholar][Indexed]

3. Komi EA, Daniel, Hamblin MR. Chitin and chitosan: production and application of versatile biomedical nanomaterials. Int J Adv Res 2016; 4:411.

   [Google Scholar][Indexed]

4. Salehi E, Daraei P, Shamsabadi AA. A review on chitosan-based adsorptive membranes. Carbohydr Polym 2016; 152:419–432.

   [Crossref][Google Scholar][Indexed]

5. Matica, Adina M. Chitosan as a wound dressing starting material: Antimicrobial properties and mode of action. Int J Mol Sci 2019; 20:5889.

   [Crossref][Google Scholar][Indexed]

6. Zhang L, Zeng Y, Cheng Z. Removal of heavy metal ions using chitosan and modified chitosan: a review. J Mol Liquids 2016; 214:175–191.

   [Crossref][Google Scholar]

7. Peng S, Meng H, Ouyang Y, et al. Nanoporous magnetic cellulose–chitosan composite microspheres: preparation, characterization, and application for Cu(II) adsorption. Ind Eng Chem Res 2014; 53:2106–2113.

   [Crossref][Google Scholar]

8. Kamoun, Elbadawy A. Crosslinked poly (vinyl alcohol) hydrogels for wound dressing applications: A review of remarkably blended polymers. Arab J Chem 2015; 8: 1-14.

   [Crossref][Google Scholar]

9. Choo, Kaiwen. Preparation and characterization of polyvinyl alcohol-chitosan composite films reinforced with cellulose nanofiber. Materials 2016; 9:644.

   [Crossref][Google Scholar][Indexed]

10. Kumar, HMP Naveen. Compatibility studies of chitosan/PVA blend in 2% aqueous acetic acid solution at 30°C. Carbohydr Polym 2010; 82:251-255.

    [Crossref][Google Scholar]

11. Marc B, Santoni BLG, Dobelin N. β-tricalcium phosphate for bone substitution: Synthesis and properties. Acta Biomater 2020; 113:23-41.

    [Crossref][Google Scholar][Indexed]

12. Liangquan P, Y Zhou, W Lu, et al. Characterization of a novel polyvinyl alcohol/chitosan porous hydrogel combined with bone marrow mesenchymal stem cells and its application in articular cartilage repair. BMC Musculoskelet Disord 2019; 20:1-12.

    [Crossref][Google Scholar][Indexed]

13. Figueroa-Pizano MD, Dolores M, Itziar Velaz I, et al. A freeze-thawing method to prepare chitosan-poly (Vinyl alcohol) hydrogels without crosslinking agents and diflunisal release studies. J Vis Exp 2020; 155:59636.

    [Crossref][Google Scholar]

14. AL-Ghaban N, Jasem G. Histomorphometric evaluation of the effects of local application of red cloveroil (trifolium pratense) on bone healing in rats. JBCD 2020; 32:26-31.

    [Crossref][Google Scholar][Indexed]

15. Moutzouri, Antonia G, Athanassiou GM. Insights into the alteration of osteoblast mechanical properties upon adhesion on chitosan. Biomed Res Int 2014: 8.

    [Crossref][Google Scholar][Indexed]

16. Sukul M, Sahariah P, Lauzon HL, et al. In vitro biological response of human osteoblasts in 3D chitosan sponges with controlled degree of deacetylation and molecular weight. Carbohydr Polym 2021; 254:117434.

    [Crossref][Google Scholar][Indexed]

17. Ardakani FE, Navabazam A, Fatehi F, et al. Histologic evaluation of chitosan as an accelerator of bone regeneration in micro drilled rat tibias. 2012; 9:694-699.

    [Google Scholar][Indexed]

18. Hendrijantini N, Rostiny N, Kuntjoro M, et al. The Effect of a combination of 12% spirulina and 20% chitosan on macrophage, PMN, and lymphocyte cell expressions in post extraction wound. Dent J (Majalah Kedokteran Gigi) 2017; 50:106-110.

    [Crossref][Google Scholar]

19. Gilev MV, Volokitina EA, Antropova IP, et al. Bone remodeling markers after experimental augmentation of trabecular bone defects with resorbable and non-resorbable osteoplastic materials in rabbits. Genij Ortopedii 2020; 26:222-227.

    [Crossref][Google Scholar]

20. Shiratori K, Matsuzaka K, Koike K, et al. Bone formation in β-tricalcium phosphate-filled bone defects of the rat femur: Morphometric analysis and expression of bone related protein mRNA. Biomed Res 2005; 26:51-59.

    [Crossref][Google Scholar][Indexed]

21. Jehad, Muhanad Tareq. Histological and Histomorphometrical Evaluation of local Application of Melatonin and Beta-Tricalcium Phosphate on Bone Healing in Rats. Diss. University of Baghdad 2018.
22. Kowalczyk P, Podgorski R, Wojasinski M, et al. Chitosan-human bone composite granulates for guided bone regeneration. Int J Mol Sci 2021; 22:2324.

    [Crossref][Google Scholar][Indexed]

23. Fang CH, Lin YW, Sun JS, et al. The chitosan/tri-calcium phosphate bio-composite bone cement promotes better osteo-integration: An in vitro and in vivo study. J Orthop Surg Res 2019; 14:1-9.

    [Crossref][Google Scholar][Indexed]