Histopathological evaluation of the effect of microspheres with different natural bioactive components (G. lucidum and I. graveolens) on osteoblastic activity in rats with experimental bone wounds

Abstract

This study aimed to analyse the effects of microspheres containing G. lucidum and I. graveolens on bone healing in the alveolar socket after tooth extraction in Wistar rats. In this study, chitosan-coated and uncoated hydrogel microspheres were produced using sodium alginate-gelatine by dispersed phase gelling and crosslinking method in microsphere (MS) production. G. lucidum (GL) and I. graveolens (IG) extracts were entrapped in these microspheres. 126 healthy female rats were randomly divided into 7 different groups. The groups were named according to the microsphere placed in the alveolar socket after extraction. The effects of these microspheres on the healing of the alveolar bone in the groups were evaluated on the 7th day, the 14th day and the 28th day. Immunohistochemical analysis was used to assess bone healing. A statistically significant difference was observed between the negative control group and MS + Chitosan + IG group and between the MS group and MS + Chitosan + IG group in terms of bone formation percentages on the 28th day (p<0.05). The results showed that G. lucidum and I. graveolens in combination with chitosan can enhance the bone healing of the alveolar socket after extraction.

Keywords

• G. lucidum
• I. graveolens
• Bone healing
• Chitosan
• Microsphere

References

1. Afzal, S., Khan, S., Ranjha, N.M., Jalil, A., Riaz, A., Haider, M.S., … Naeem, F. (2018). The structural, crystallinity, and thermal properties of pH-responsive interpenetrating gelatin/sodium alginate-based polymeric composites for the controlled delivery of cetirizine HCl. Turkish Journal of Pharmaceutical Sciences, 15(1), 63 76. https://doi.org/10.4274/tjps.64326
2. Alsmadi, M.M., Obaidat, R.M., Alnaief, M., Albiss, B.A., & Hailat, N. (2020). Development, in vitro characterization, and in vivo toxicity evaluation of chitosan-alginate nanoporous carriers loaded with cisplatin for lung cancer treatment. AAPS PharmSciTech, 21(5), 191. https://doi.org/10.1208/s12249-020-01735-8
3. Al-Snafi, A.E. (2018). Chemical constituents and pharmacological effect of Inula graveolens (Syn: Dittrichia graveolens)-A review. Indo American Journal of Pharmaceutical Sciences, 5(4), 2183-2190.
4. Araújo, M.G., Silva, C.O., Misawa, M., & Sukekava, F. (2015). Alveolar socket healing: what can we learn?. Periodontology 2000, 68(1), 122–134. https://doi.org/10.1111/prd.12082
5. Baby, S., Johnson, A.J., & Govindan, B. (2015). Secondary metabolites from Ganoderma. Phytochemistry, 114, 66–101. https://doi.org/10.1016/j.phytochem.2015.03.010
6. Belgin, E.E., Gönen, H., & Çiçek, H. (2022). Production of Ganoderma lucidum extract loaded gelatin-sodium alginate microspheres, investigation of release kinetics at different pH values and evaluation of kinetic models. Mugla Journal of Science and Technology, 8(1), 41-50.
7. Benzie, I.F.F., & Wachtel-Galor, S. (Eds.). (2011). Herbal medicine: Biomolecular and Clinical Aspects. 2nd ed. CRC Press/Taylor & Francis.
8. Carvalho, T.L., Bombonato, K.F., & Brentegani, L.G. (1997). Histometric analysis of rat alveolar wound healing. Brazilian Dental Journal, 8(1), 9–12.

9. Cohen, N., & Cohen-Lévy, J. (2014). Healing processes following tooth extraction in orthodontic cases. Journal of Dentofacial Anomalies and Orthodontics, 17(3), 304.
10. Cör, D., Knez, Ž., & Knez Hrnčič, M. (2018). Antitumour, antimicrobial, antioxidant and antiacetylcholinesterase effect of ganoderma lucidum terpenoids and polysaccharides: A review. Molecules, 23(3), 649. https://doi.org/10.3390/molecules23030649
11. Darby, I., Chen, S.T., & Buser, D. (2009). Ridge preservation techniques for implant therapy. The International Journal of Oral & Maxillofacial Implants, 24 Suppl, 260–271.
12. Davison, M.J., McMurray, R.J., Smith, C.A., Dalby, M.J., & Meek, R.D. (2016). Nanopit-induced osteoprogenitor cell differentiation: The effect of nanopit depth. Journal of Tissue Engineering, 7, 2041731416652778. https://doi.org/10.1177/2041731416652778
13. Florencio-Silva, R., Sasso, G.R., Sasso-Cerri, E., Simões, M.J., & Cerri, P.S. (2015). Biology of bone tissue: Structure, function, and factors That influence bone cells. BioMed Research International, 2015, 421746. https://doi.org/10.1155/2015/421746
14. Gartner, L.P., Hiatt, J.L., Strum, J.M. (2011). Cell biology and histology. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins.
15. Grawish, M.E., Grawish, L.M., Grawish, H.M., Grawish, M.M., Holiel, A.A., Sultan, N., & El-Negoly, S.A. (2022). Demineralized dentin matrix for dental and alveolar bone tissues regeneration: An innovative scope review. Tissue Engineering and Regenerative Medicine, 19(4), 687–701. https://doi.org/10.1007/s13770-022-00438-4
16. Guillén-Carvajal, K., Valdez-Salas, B., Beltrán-Partida, E., Salomón-Carlos, J., & Cheng, N. (2023). Chitosan, gelatin, and collagen hydrogels for bone regeneration. Polymers, 15(13), 2762. https://doi.org/10.3390/polym15132762
17. Hanafiah, O.A., Hanafiah, D.S., Dohude, G.A., Satria, D., Livita, L., Moudy, N.S., & Rahma, R. (2021). Effects of 3% binahong (Anredera cordifolia) leaf extract gel on alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus). F1000Research, 10, 923. https://doi.org/10.12688/f1000research.72982.2
18. Hassumi, J.S., Mulinari-Santos, G., Fabris, A.L.D.S., Jacob, R.G.M., Gonçalves, A., Rossi, A.C., … Okamoto, R. (2018). Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis. Journal of Applied Oral Science, 26, e20170326. https://doi.org/10.1590/1678-7757-2017-0326
19. Hollý, D., Klein, M., Mazreku, M., Zamborský, R., Polák, Š., Danišovič, Ľ., & Csöbönyeiová, M. (2021). Stem Cells and Their Derivatives-Implications for Alveolar Bone Regeneration: A Comprehensive Review. International Journal of Molecular Sciences, 22(21), 11746. https://doi.org/10.3390/ijms222111746
20. Irinakis T. (2006). Rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. Journal Canadian Dental Association, 72(10), 917–922.
21. Jia, M., Li, Z.B., Chu, H.T., Li, L., & Chen, K.Y. (2015). Alginate-chitosan microspheres for controlled drug delivery of diltiazem hydrochloride in cardiac diseases. Journal of Biomaterials and Tissue Engineering, 5(3), 246-251.
22. Karayürek, F., Kadiroğlu, E.T., Nergiz, Y., Coşkun Akçay, N., Tunik, S., Ersöz Kanay, B., & Uysal, E. (2019). Combining platelet rich fibrin with different bone graft materials: An experimental study on the histopathological and immunohistochemical aspects of bone healing. Journal of Cranio Maxillofacial Surgery, 47(5), 815 825. https://doi.org/10.1016/j.jcms.2019.01.023
23. Kawecki, F., Jann, J., Fortin, M., Auger, F.A., Faucheux, N., & Fradette, J. (2022). Preclinical Evaluation of BMP-9-Treated Human Bone-like Substitutes for Alveolar Ridge Preservation following Tooth Extraction. International Journal of Molecular Sciences, 23(6), 3302. https://doi.org/10.3390/ijms23063302
24. Kim, J.M., Lin, C., Stavre, Z., Greenblatt, M.B., & Shim, J.H. (2020). Osteoblast-Osteoclast Communication and Bone Homeostasis. Cells, 9(9), 2073. https://doi.org/10.3390/cells9092073
25. Kim, S., Cui, Z.K., Fan, J., Fartash, A., Aghaloo, T.L., & Lee, M. (2016). Photocrosslinkable chitosan hydrogels functionalized with the RGD peptide and phosphoserine to enhance osteogenesis. Journal of Materials Chemistry, 4(31), 5289 5298. https://doi.org/10.1039/C6TB01154C
26. Koc, K., Aysin, F., Ozek, N.S., Geyikoglu, F., Taghizadehghalehjoughi, A., Abuc, O.O., Cakmak, O., & Deniz, G.Y. (2021). Inula graveolens induces selective cytotoxicity in glioblastoma and chronic leukemia cells. Revista da Associacao Medica Brasileira, 67(12), 1771–1778. https://doi.org/10.1590/1806-9282.20210614
27. Koh, K.S., Choi, J.W., Park, E.J., & Oh, T.S. (2018). Bone Regeneration using Silk Hydroxyapatite Hybrid Composite in a Rat Alveolar Defect Model. International Journal of Medical Sciences, 15(1), 59–68. https://doi.org/10.7150/ijms.21787
28. Kowalczyk, P., Podgórski, R., Wojasiński, M., Gut, G., Bojar, W., & Ciach, T. (2021). Chitosan-human bone composite granulates for guided bone regeneration. International Journal of Molecular Sciences, 22(5), 2324.
29. Kuo, Y.C., & Wang, C.C. (2013). Guided differentiation of induced pluripotent stem cells into neuronal lineage in alginate-chitosan-gelatin hydrogels with surface neuron growth factor. Colloids and Surfaces B: Biointerfaces, 104, 194 199. https://doi.org/10.1016/j.colsurfb.2013.01.001
30. Laçin, N., İzol, S.B., İpek, F., & Tuncer, M.C. (2019). Ganoderma lucidum, a promising agent possessing antioxidant and anti-inflammatory effects for treating calvarial defects with graft application in rats. Acta Cirurgica Brasileira, 34(9), e201900904. https://doi.org/10.1590/s0102-865020190090000004
31. Lindhe, J., Lang, N.P., Karring, T. (2009). Clinical Periodontology and Implant Dentistry. Wiley.
32. Luvizuto, E.R., Dias, S.M., Queiroz, T.P., Okamoto, T., Garcia, I.R., Jr, Okamoto, R., & Dornelles, R.C. (2010). Osteocalcin immunolabeling during the alveolar healing process in ovariectomized rats treated with estrogen or raloxifene. Bone, 46(4), 1021–1029. https://doi.org/10.1016/j.bone.2009.12.016
33. Ma, H.T., Hsieh, J.F., & Chen, S.T. (2015). Anti-diabetic effects of Ganoderma lucidum. Phytochemistry, 114, 109–113. https://doi.org/10.1016/j.phytochem.2015.02.017
34. Majidinia, M., Sadeghpour, A., & Yousefi, B. (2018). The roles of signaling pathways in bone repair and regeneration. Journal of Cellular Physiology, 233(4), 2937 2948. https://doi.org/10.1002/jcp.26042
35. Menon, J.U., Ravikumar, P., Pise, A., Gyawali, D., Hsia, C.C., & Nguyen, K.T. (2014). Polymeric nanoparticles for pulmonary protein and DNA delivery. Acta Biomaterialia, 10(6), 2643–2652. https://doi.org/10.1016/j.actbio.2014.01.033
36. Min, K.K., Neupane, S., Adhikari, N., Sohn, W.J., An, S.Y., Kim, J.Y., … Suh, J.Y. (2020). Effects of resveratrol on bone-healing capacity in the mouse tooth extraction socket. Journal of Periodontal Research, 55(2), 247–257. https://doi.org/10.1111/jre.12710
37. Miranda, T.S., Napimoga, M.H., De Franco, L., Marins, L.M., Malta, F.S., Pontes, L.A., Morelli, F.M., & Duarte, P.M. (2020). Strontium ranelate improves alveolar bone healing in estrogen deficient rats. Journal of Periodontology, 91(11), 1465 1474. https://doi.org/10.1002/JPER.19-0561
38. Mitic, V., Stankov Jovanovic, V., Ilic, M., Jovanovic, O., Djordjevic, A., & Stojanovic, G. (2016). Dittrichia graveolens (L.) Greuter Essential Oil: Chemical Composition, Multivariate Analysis, and Antimicrobial Activity. Chemistry & Biodiversity, 13(1),85–90. https://doi.org/10.1002/cbdv.201500028
39. Miyamoto, I., Liu, J., Shimizu, K., Sato, M., Kukita, A., Kukita, T., & Kondo, R. (2009). Regulation of osteoclastogenesis by ganoderic acid DM isolated from Ganoderma lucidum. European Journal of Pharmacology, 602(1), 1 7. https://doi.org/10.1016/j.ejphar.2008.11.005
40. Okamoto, T., & de Russo, M.C. (1973). Wound healing following tooth extraction. Histochemical study in rats. Revista da Faculdade de Odontologia de Aracatuba, 2(2), 153–169.
41. Olaitan, O.H., Komolafe, O.A., Owotade, F.J., & Saka, O.S. (2019). Histologic assessment of extraction sockets following tooth extraction: Suitability of a rabbit model. Nigerian Journal of Dental Research, 4(1), 1-4.
42. Ponticelli, M., Lela, L., Russo, D., Faraone, I., Sinisgalli, C., Mustapha, M.B., … Milella, L. (2022). Dittrichia graveolens (L.) Greuter, a Rapidly Spreading Invasive Plant: Chemistry and Bioactivity. Molecules, 27(3), 895. https://doi.org/10.3390/molecules27030895
43. Rodrigues, W.C., Fabris, A. L., Hassumi, J.S., Gonçalves, A., Sonoda, C.K., & Okamoto, R. (2016). Kinetics of gene expression of alkaline phosphatase during healing of alveolar bone in rats. The British Journal of Oral & Maxillofacial Surgery, 54(5), 531–535. https://doi.org/10.1016/j.bjoms.2016.02.018
44. Santos, I.G.B.P., de Santana, C.M.M., Alves, A.T.N.N., de Uzeda, M.J.P.G., Calasans-Maia, M.D., & de Santana, R.B. (2019). Effects of methods of hydration of a biphasic ceramic graft on bone regeneration of extraction socket defects. Journal of Periodontology, 90(4), 425–432. https://doi.org/10.1002/JPER.18-0209
45. Sevindik, E., & Paksoy, M.Y. (2017). Aydın/Türkiye’de yayılış gösteren Dittrichia L. (Asteraceae) cinsinin kimyasal kompozisyonunun değerlendirilmesi [Evaluation of Essential Oil Composition Genus Dittrichia L. (Asteraceae) Plants in Aydın/Türkiye]. Türk Tarım ve Doğa Bilimleri Dergisi, 4(4), 456-460.
46. Shah, F.A., Sayardoust, S., Thomsen, P., & Palmquist, A. (2019). Extracellular matrix composition during bone regeneration in the human dental alveolar socket. Bone, 127, 244–249. https://doi.org/10.1016/j.bone.2019.06.003
47. Shi, G., Yang, C., Wang, Q., Wang, S., Wang, G., Ao, R., & Li, D. (2022). Traditional chinese medicine compound-loaded materials in bone regeneration. Frontiers in Bioengineering and Biotechnology, 10, 851561. https://doi.org/10.3389/fbioe.2022.851561
48. Singh, A., Sharma, A., & Kaur, S. (2014). Micro carrier as colon drug delivery system: a review. International Journal of Research and Development in Pharmacy & Life Sciences, 3(6), 1211-1216.
49. Singh, C., Purohit, S., Singh, M., & Pandey, B.L. (2013). Design and evaluation of microspheres: A review. Journal of Drug Delivery Research, 2(2), 18-27.
50. Sohretoglu, D., & Huang, S. (2018). Ganoderma lucidum polysaccharides as an anti-cancer agent. Anti cancer Agents in Medicinal Chemistry, 18(5), 667 674. https://doi.org/10.2174/1871520617666171113121246
51. Sukpaita, T., Chirachanchai, S., Chanamuangkon, T., Pimkhaokham, A., & Ampornaramveth, R.S. (2024). Alveolar ridge preservation in rat tooth extraction model by chitosan-derived epigenetic modulation scaffold. Journal of Prosthodontic Research, 68(2), 299–309. https://doi.org/10.2186/jpr.JPR_D_23_00006
52. Troiano, G., Zhurakivska, K., Lo Muzio, L., Laino, L., Cicciù, M., & Lo Russo, L. (2018). Combination of bone graft and resorbable membrane for alveolar ridge preservation: A systematic review, meta-analysis, and trial sequential analysis. Journal of Periodontology, 89(1), 46–57. https://doi.org/10.1902/jop.2017.170241
53. Tüylek, Z. (2017). İlaç taşıyıcı sistemler ve nanoteknolojik etkileşim [Drug Delivery Systems and Nanotechnological Interaction]. Bozok Tıp Dergisi, 7(3), 89-98.
54. Tüylek, Z. (2019). İlaç taşıyıcı nanosistemler [Drug Delivery Nanosystems]. Arşiv Kaynak Tarama Dergisi, 28(3), 184-192.
55. Vieira, A.E., Repeke, C.E., Ferreira Junior, S.B., Colavite, P.M., Biguetti, C.C., Oliveira, R.C., … Garlet, G.P. (2015). Intramembranous bone healing process subsequent to tooth extraction in mice: micro-computed tomography, histomorphometric and molecular characterization. PloS One, 10(5), e0128021. https://doi.org/10.1371/journal.pone.0128021
56. Wang, F., Qian, H., Kong, L., Wang, W., Wang, X., Xu, Z., Chai, Y., Xu, J., & Kang, Q. (2021). Accelerated bone regeneration by astragaloside IV through stimulating the coupling of osteogenesis and angiogenesis. International Journal of Biological Sciences, 17(7), 1821–1836. https://doi.org/10.7150/ijbs.57681
57. Yadav, L.R., Chandran, S.V., Lavanya, K., & Selvamurugan, N. (2021). Chitosan-based 3D-printed scaffolds for bone tissue engineering. International Journal of Biological Macromolecules, 183, 1925–1938. https://doi.org/10.1016/j.ijbiomac.2021.05.215
58. Yoshiko, Y., Candeliere, G.A., Maeda, N., & Aubin, J.E. (2007). Osteoblast autonomous Pi regulation via Pit1 plays a role in bone mineralization. Molecular and Cellular Biology, 27(12), 4465–4474. https://doi.org/10.1128/MCB.00104-07