INTRODUCTION:

Periodontitis is a chronic inflammatory disease and it causes destruction of connective tissue and alveolar bone primarily due to infection by periodontal bacteria followed by inflammatory host response.^1,2

Periodontitis is a serious disease which may cause redness and swelling in gingiva, bleeding gums with severe pain, persistent metallic taste in mouth, deep pockets between the teeth and the gums, bone destruction and gradual loosening and falling of teeth.^3,4

Periodontitis which can lead to alveolar bone resorption and in some more severe condition becomes an alveolar bone defect.⁵ Alveolar damage in one tooth tends to be accompanied by injury in the contiguous tooth.⁶

Periodontitis is the second most prevalent oral disease after dental caries.⁷ Together with dental caries, periodontitis is the main reason for tooth loss in adults.^7,8 Approximately 743 million people globally suffer from periodontitis, and this figure has increased by 57.3% over the last ten years.^9,10 Globally, the losses that are due to reduced productivity caused by severe periodontitis are estimated to be 53.99 million United States (US) dollars annually.^10,11

Based on the previous study, there are about 80% patients suffering from chronic periodontitis experience alveolar bone defect.¹²This indicates that alveolar bone defect therapy is quite challenging and it still requires a potent and effective treatment.¹³ Severe damage requires a bone grafting procedure to fill in the defect and to promote bone regeneration.¹⁴ However, bone grafts are still not effective, therefore an alternative tissue engineering approaches are required.¹⁵

The increase of tissue regeneration is created to establish tissue constructs consisting of biological mediators and cells (growth factors and adesine) in a biological matrix or synthetic that can be implanted in the patient to promote specific tissues regeneration. The success of new bone formation is affected by several important components that must be fulfilled, such as scaffold/bone graft, signaling molecules (cytokines, growth factors), and cells/progenitor cells or better known as the tissue engineering triad.¹⁶

Bone graft is the material used during periodontal flap action, which is expected to help bone growth through the process of osteogenesis, osteoinduction or osteoconduction. The use of bone graft as a filler and scaffolds in bone defects to support alveolar bone regeneration and help accelerate the healing process, providing a place for cells to promote migration and differentiation of osteoblasts.¹⁴

Osteogenesis is a compact postpartum bone formation process. Osteogenesis process is conducted by osteoblast. Meanwhile, bone remodeling and bone resorption are organized by osteoclast. On extracellular bone matrix formation, osteoblast and osteoclast have important role during bone remodeling. Osteogenesis and bone remodeling processes are crucial mechanism for compact bone development, structure repairment on fracture bone and bone defect, and maintenance for the bone as a whole.^17,18

Osteoblasts play an important role in bone metabolism by regulating matrix mineralization, producing various bone matrix proteins, controlling osteoclastic activity and differentiating in the formation of new bone. At the end of this process, the mature osteoblasts undergo apoptosis to differentiate into osteocytes or to become bone lining cells.¹⁹ When differentiated, osteoblasts secrete substances such as alkaline phosphatase, osteocalcin, osteopontin and osteonectin.²⁰

Osteocalcin is a bone extracellular protein matrix secreted by osteoblasts during the bone remodeling phase.²¹ Excreted during the final stage of differentiation, in the early stages of mineralization, is one of the markers of specific bone metabolic activity produced by osteoblasts contained in the bone matrix and is used as a markers of bone formation activity.²²

Osteopontin is a secretory protein derived from osteoblasts that modulates the process of bone remodeling, the chemotaxis of inflammatory cells, and the response of osteoblasts to extracellular stimuli.²³ Osteopontin-hydroxyapatite bonds will inhibit the growth of hydroxyapatite crystals.²⁴

There are several types of growth factors that play a role in the regeneration process of bone and soft tissue.²⁵ Growth factors stimulate cell migration to defects and increase cell proliferation and mitogenesis.²⁶ Insulin-like growth factor type 1 (IGF-1) is a growth factor that plays a role to stimulate growth, differentiation and inhibit apoptosis in many tissues, increase proliferation and play a major role in stimulation of mature osteoblast function.²⁷ IGF-1 stimulates osteoblast replication and synthesis of bone matrix. Tao Qiu et al. (2018) stated that IGF-1 stimulates osteoblasts by carboxylation of osteocalcin, binding to insulin receptors (IR) on osteoblasts and osteoclasts and insulin receptor substrate (IRS) which only exists in osteoblasts.²⁸ Insulin growth factor has a role in local regulation of formation. bone and can help treat periodontal defects and tissue healing.²⁹

In this study, we would like to know the role of IGF-1 added to bone graft in the osteogenesis process by looking at the expression of osteoblasts, osteoclasts, osteopontin and osteocalcin in bone defects.

MATERIALS AND METHODS:

We were starting this study after obtained ethical eligibility approval from the Health Research Ethics Feasibility Commission, Faculty of Dentistry, Airlangga University.

The study was conducted on 4-5 months old New Zealand male white rabbits weighing 1500-2000grams. All rabbits were adjusted during the week and were kept in experimental animal cages with a size of 60cm x 60 cm x 90cm. Each treatment group consisted of 9 rabbits. 18 rabbits were randomized and divided into 2 groups. The first group was treated with xenograft alone. The second group was treated with xenograft and IGF-1.

Rabbits were anesthetized with intramuscular administration of ketamine. Then the hair on the rabbit's right tibial area is removed to facilitate incision. Added 1cc lidocaine local anesthetic to strengthen ketamine anesthesia prior to incision. With Rasparatorium, the separation of muscle tissue was carried out to obtain the structure of the rabbit's tibia. Bone defects were made using a low speed bur with a diameter of 5 mm and a depth of 3mm.

In the first group, the defects were given xenograft only, while in the second group, the defects were given xenograft and IGF-1 as much as 0.3cc (500ng/ml). Then the wound was sewn back together.

Observations were done after 21 days, then the animal was sacrificed and surgery was carried out to take the tibones. The tissue is immersed in 70% formalin buffer solution to prevent tissue changes, tissue hardening, increase the refractive index of the tissue components and increase the affinity of the tissue with the coating.

After the first 48 hours, change the fixation solution, and then cut the tissue into smaller sizes so that the fixation can evenly penetrate into the tissue. In the second stage, it was left in the solution for 48 hours. After fixation, the tissue was rinsed with running water for 6-9 hours, and then placed in a 5% HNO3 decalcification solution for 1 hour. After the decalcification process is carried out the process of making a paraffin block which is then carried out cutting the slides for examination.

Quantitative observation of osteoblast, osteoclast, osteopontin and osteocalcin expression was done by using immunohistochemical techniques. This technique uses the principle of antigen-antibody reaction to determine the location of an antigen (target protein) in a tissue or cell.

STUDY RESULTS:

From the study that has been done, it is obtained the standard deviation and mean of the number of expressions of osteoblasts, osteoclasts, osteopontin and osteocalcin in each group as shown in the following table:

Table 1. Independent test of osteoblast sample t-test in the study groups

Groups	Sig. (2-tailed)	Mean	SD
Xenograft	0.000	16.22	3.801
Xenograft + IGF-1	0.000	27.22	2.048

Table 2. Independent test of osteoclast sample t-test in the study groups

Groups	Sig. (2-tailed)	Mean	SD
Xenograft	0.000	11.89	2.315
Xenograft + IGF-1	0.000	6.89	1.764

Table 3. Independent test of the sample osteopontin t-test in the study group

Groups	Sig. (2-tailed)	Mean	SD
Xenograft	0.000	7.56	2.261
Xenograft + IGF-1	0.000	16.67

Table 4. Independent test of the osteocalcin sample t-test in the study groups

Groups	Sig. (2-tailed)	Mean	SD
Xenograft	0.000	8.89	2.261
Xenograft + IGF-1	0.000	21.78	4.738

Figure 1: Bar chart of the control group (xenograft) and the treatment group (xenograft + IGF-1)

In the observations, the number of osteoblasts, osteopontin and osteocalcin expressions in the xenograft + IGF-1 group was higher than the xenograft group. Meanwhile, for the number of osteoclasts expression, the xenograft + IGF-1 group was lower than the xenograft group.

In the independent sample t-test as shown in Tables 1, 2, 3 and 4, a significance value of 0.000 was obtained for the variables osteoblast, osteoclast, osteopontin and osteocalcin. The significance value p <0.05 indicates that there are significant differences between the treatment group and the control group. This shows that the administration of IGF-1 to the defect has an influence on the expression of osteoblasts, osteoclasts, osteopontin and osteocalcin.

The observation of osteopontin and osteocalcin using a microscope showed the following histological features:

Figure 2a. The appearance of osteopontin in the control group (xenograft)

Figure 2b. The appearance of osteopontin in the treatment group (xenograft + IGF-1).

Figure 3a. The appearance of osteocalcin in the control group (xenograft)

Figure 3b. The appearance of osteocalcin in the treatment group (xenograft + IGF-1)

DISCUSSION:

This study was carried out on rabbit tibial bone which was deformed in such a way for the xenograft material application which was added with IGF-1 growth factor. The healing process for bone defects was compared by looking at two study groups, such as group 1 (control) and was only given xenograft, and group 2 (treatment), bone defect which was given xenograft + IGF-1. Observations were carried out on the 21st day with immunohistochemical examination to see the number of expressions of osteoblasts, osteoclasts, osteopontin and osteocalcin which are markers on bone formation.

Xenografts were used with the purpose of helping bone regeneration through the osteoconduction process. Xenograft as a scaffold will guide the migration of osteogenic cells into its axis to bind and react directly with growth factors to initiate cell proliferation and differentiation. Initially, the cells were only distributed on the surface of the scaffold, over time the cells were distributed until they reached the central bone defect.³⁰ The addition of growth factors can increase the scaffold's osteoinductive and angioinductive abilities so that it can accelerate the regeneration process. This was supported by the role of growth factors that distribute capillaries to supply nutrients in the scaffold matrix.³¹

Growth factors in the blood and inflammatory mediators cause premesenchymal cells to differentiate into osteoblasts resulting in bone formation, then fusion of bone graft with the host bone.³² IGF-1 is the foremost abundant growth factor put away within the bone matrix and stimulates cell proliferation and function, as well as osteoblast survival. The nearness of IGF-1 in osteoclasts and osteoblasts shows its inclusion in growth and bone remodeling.³³

In this study, independent t test was carried out in the xenograft and xenograft + IGF-1 groups against osteoblasts with significant results (p <0.05). The mean osteoblast observation results in the xenograft + IGF-1 group were higher than the xenograft group. This is in accordance with Xian L's (2012) study that IGF-1 can affect the growth and development of osteoblasts and accelerate bone regeneration.³⁴Zhang X (2020) reports that IGF-1 plays a role in bone remodeling by increasing osteoblast activity in-vivo. 15 Insulin-like growth factor type 1 receptor (IGF-1R), which binds to and is activated by IGF-1, mediates the signaling pathway required to stimulate osteoblast proliferation, thereby increasing bone matrix production and osteoblast differentiation.³⁵IGF-1R signaling plays an important role in cell growth and development, as well as in bone formation via osteoblasts for bone mineralization.³⁶

Mature osteoblasts produce regulators for matrix mineralization such as osteopontin, osteocalcin and osteonectin and RANKL which are important for osteoclast differentiation.³⁷In this study, there was a significant difference in osteoclast expression in the xenograft and xenograft + IGF-1 groups (P <0.05). The mean value of osteoclasts in the xenograft group was higher than that of the xenograft + IGF-1 group. These results indicate that the addition of IGF-1 suppresses osteoclast expression which causes bone resorption. This finding was consistent with in vitro studies using bone tissue cultures in which IGF-I was reported to interfere with osteoblast-derived factors that stimulate existing osteoclasts and inhibit osteoclasts.³⁸

Osteopontin and osteocalcin are two of the foremost common non-collagen proteins within the bone matrix, representing to 10% to 20% of non-collagen proteins.³⁹Chen's research (2016) mentions the role of IGF-1 in osteogenesis, one of which is the activation of transcription factors that regulate cell differentiation. bone and bone biomarkers, such as osteopontin, osteocalcin.⁴⁰

Osteopontin regulates many physiological processes such as cell adhesion, collagen organization, cell viability, angiogenesis, calcification and cell migration.⁴¹ The most part of osteopontin during inflammation is to trigger distinct leukocytes, initiate cytokine secretion and build up an immune respone. At the site of injury, osteopontin can stimulate the migration, accumulation, and persistence of macrophages and control their cytokine production promoting Th1 cell-mediated immunity.⁴²

In this study, the mean value of osteopontin expression in the xenograft group was lower than that of the xenograft + IGF-1 group. These results are in accordance with Paulo's research (2015) which states that IGF-1 can activate second mesenger's which acts as a nuclear potential transcription factor which will eventually lead to osteopontin expression through osteoblasts during the bone remodeling process.⁴³The level of osteopontin expression plays an important role in osteoblast differentiation as mineralization mediators,⁴⁴and its level of expression gradually increases from day 7 to day 21.⁴⁵Osteopontin can be used as an indicator of intermediate and late stages of differentiation.

Apart from osteoblasts, osteopontin is also secreted by human osteoclasts during bone resorption, which can be used as chemokines for bone formation and resorption.^[46] Osteopontin can inhibit the osteoblast osteogenesis process by inhibiting BMP-2, and acts as an inhibitor mineralization osteoblast in a phosphate-dependent manner.⁴⁷

Osteocalcin is defined as a marker for bone breakdown and bone formation, undergoes carboxylation to bind to hydroxyapatite in bone and has a higher affinity for calcium, thereby facilitating bone mineralization.⁴⁸

From the results of this study, data was obtained as in table 4 which exposed that the mean number of osteocalcin expressions in the treatment group was higher, when compared to the mean of the control group. This explains that the xenograft + IGF-1 group was able to produce more osteocalcin expression than the xenograft group without IGF-1 administration. These results are consistent with research conducted by Crane and Cao (2014) which states that increasing IGF-1 expression can increase bone formation rate and bone volume at weeks 3 and 6.⁴⁹. The increase in osteocalcin levels on IGF-1 administration indicates a role of IGF-1 on osteocalcin regulation as reported by Johansson et al.⁴⁸ Activation of IGF-IR stimulates type I collagen synthesis, alkaline-phosphatase activity, and osteocalcin secretion.⁵⁰

CONCLUSIONS:

The addition of IGF-1 to the xenograft for the bone damage treatment can increase the osteoblasts, osteopontin and osteocalcin expression and reduce osteoclast expression as an indicator of the bone regeneration process (osteogenesis).

The combination of xenograft and IGF-1, increased the rate of bone formation and bone repair, and will be useful in the future in dental therapy.

REFERENCES:

1. Christeena Abraham, Sankari Malaiappan, Savitha G. Association of Hematological and Periodontal Parameters in Healthy, Chronic and Aggressive Periodontitis Patients – A Cross Sectional Study. Research J. Pharm. and Tech 2019; 12(1): 74-78. doi: 10.5958/0974-360X.2019.00014.3 Available on: https://rjptonline.org/AbstractView.aspx?PID=2019-12-1-14;

2. S. Mounika, Gopinath. Periodontitis as a Risk Factor of Atherosclerosis. Research J. Pharm. and Tech 2016; 9(11):2017-2019.

3. S. Shreya, Gheena. The General Awareness among People about the Prevalence of Periodontitis. Research J. Pharm. and Tech. 8(8): August, 2015; Page 1119-1124. doi: 10.5958/0974-360X.2015.00196.1 Available on: https://rjptonline.org/AbstractView.aspx?PID=2015-8-8-29

4. Sahar H. Al-Hindawi, Noori M. Luaibi, Batool H. Al-Ghurabi. Estimation of Alkaline Phosphatase level in the Serum and Saliva of Hypothyroid Patients with and without Periodontitis. Research J. Pharm. and Tech 2018; 11(7): 2993-2996. doi: 10.5958/0974-360X.2018.00551.6 Available on: https://rjptonline.org/AbstractView.aspx?PID=2018-11-7-50

5. Rahendra Wira Hermawan, Ida Bagus Narmada, Irwadi Djaharu’ddin, Alexander Patera Nugraha, Dwi Rahmawati. The Influence of Epigallocatechin Gallate on the Nuclear Factor Associated T Cell-1 and Sclerostin Expression in Wistar Rats (Rattus novergicus) during the Orthodontic Tooth Movement. Research J. Pharm. and Tech. 2020; 13(4):1730-1734. doi: 10.5958/0974-360X.2020.00312.1 Available on: https://rjptonline.org/AbstractView.aspx?PID=2020-13-4-22)

6. Fatima Malik Abood1, Ghassan A. Abbas HD. Conserv, Luma Jasim Witwit, Nada Khazal Kadhim Hindi, Halah Khaleel Ahmed Abu Khmra, Mohmmed R. Abid Ali. The occurrence of alveolar bone resorption with oral bacterial infection. Research J. Pharm. and Tech. 2017; 10(6): 1997-2000. doi: 10.5958/0974-360X.2017.00349.3 Available on: https://rjptonline.org/AbstractView.aspx?PID=2017-10-6-73

7. Edman K, Öhrn K, NordstrÖm B, et al.: Trends over 30 years in the prevalence and severity of alveolar bone loss and the influence of smoking and socioeconomic factors–based on epidemiological surveys in Sweden 1983 – 2013. Int J Dent Hyg. 2015; 13(4): 283–291.

8. Shaddox LM, Walker CB: Treating chronic periodontitis: Current status, challenges, and future directions. Clin Cosmet Investig Dent. 2010; 2: 79–91.

9. Frencken JE, Sharma P, Stenhouse L, et al.: Global epidemiology of dental caries and severe periodontitis–a comprehensive review. J Clin Periodontol. 2017; 44 Suppl 18: S94–S105.

10. Tonetti MS, Jepsen S, Corgel JO: Impact of the global burden of periodontal diseases on health, nutrition and wellbeing of mankind: A call for global action. J Clin Periodontol. 2017; 44(5): 456–462.

11. Listl S, Galloway JS, Mossey PA, et al.: Global economic impact of dental diseases. J Dent Res. 2015; 94(10): 1355–6

12. Divyadharsini V, Sankari M. Effect of Smoking on Interleukin- 1 and Reactive Oxygen Species in Periodontitis. Research J. Pharm. and Tech. 2018; 11(3): 1247-1250. doi: 10.5958/0974-360X.2018.00232.9 Available on: https://rjptonline.org/AbstractView.aspx?PID=2018-11-3-79

13. Fianza Rezkita, Kadek G. P. Wibawa, Alexander P. Nugraha. Curcumin loaded Chitosan Nanoparticle for Accelerating the Post Extraction Wound Healing in Diabetes Mellitus Patient: A Review. Research J. Pharm. and Tech 2020; 13(2):1039-1042. doi: 10.5958/0974-360X.2020.00191.2 Available on: https://rjptonline.org/AbstractView.aspx?PID=2020-13-2-95

14. Kumar P, Vinitha B, Fathima G. 2013. Bone grafts in dentistry. Bone Graft Dent.;5(1): 125-128. doi:10.4103/0975-7406.113312

15. Saskianti T, Yualiartanti W, Ernawati DS, et al.: BMP4 expression following stem cells from human exfoliated deciduous and carbonate apatite transplantation on Rattus norvegicus. J Khrishna Institute Med Sci Uni. 2018; 7(2): 56–61.

16. Jangid, M. R., Rakhewar, P. S., Nayyar, A. S., and Cholepatil, A. Bone Grafts in Periodontal Regeneration : Factors Impacting Treatment Outcome. Basic Research Journal of Medicine and Clinical Science. August 2, 2016. 106–109. Retrieved from http//www.basicresearchjournals.org

17. Alexander Patera Nugraha, Fianza Rezkita, Martining Shoffa Puspitaningrum, Mahela Sefrian Luthfimaidah, Ida Bagus Narmada, Chiquita Prahasanti, Diah Savitri Ernawati, Fedik Abdul Rantam. Gingival Mesenchymal Stem Cells and Chitosan Scaffold to Accelerate Alveolar Bone Remodelling in Periodontitis: A Narrative Review. Research J. Pharm. and Tech 2020; 13(5):2502-2506. doi: 10.5958/0974-360X.2020.00446.1 Available on: https://rjptonline.org/AbstractView.aspx?PID=2020-13-5-76)

18. Ari Triwardhani, Intan Oktaviona, Ida Bagus Narmada, Alexander Patera Nugraha. The Effect of Bifidobacterium Probiotic on Heat Shock Protein-70 Expression and Osteoclast Number during Orthodontic Tooth Movement in Rats (Rattus novergicus). Research J. Pharm. and Tech 2021; 14(3):1477-1481. doi: 10.5958/0974-360X.2021.00262.6 Available on: https://rjptonline.org/AbstractView.aspx?PID=2021-14-3-51

19. Staines, K.A., MacRae, V.E., Farquharson, C. The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling. J. Endocrinol. 2012; 214 (3), 241–255.

20. Kim I, Song Y, Lee B, Hwang S. Human mesenchymal stromal cells are mechanosensitive to vibration stimuli. J Dent Res. 2012; 91: 1135–1140. doi: 10.1177/0022034512465291.

21. Singh, S., Kumar, D. and Lal, A. K. ‘Serum osteocalcin as a diagnostic biomarker for primary osteoporosis in women’, Journal of Clinical and Diagnostic Research, 2015; 9(8): pp. RC04–RC07. doi: 10.7860/JCDR/2015/14857.6318.

22. Yodthong, T., et al. l-Quebrachitol Promotes the Proliferation, Differentiation, and Mineralization of MC3T3-E1 Cells: Involvement of the BMP-2/Runx2/MAPK/Wnt/-Catenin Signaling Pathway. Mol. 2018; 23(12). http:// https://doi.org/10.3390/molecules23123086

23. Kusuyama, J., Bandow, K., Ohnishi, T., Hisadome, M., Shima, K., Semba, I., Matsuguchi, T. Osteopontin inhibits osteoblast responsiveness through the down-regulation of focal adhesion kinase mediated by the induction of low-molecular weight protein tyrosine phosphatase. Mol. Biol. Cell. 2017; 28(10): 1326–1336.

24. Quan Yuan, Yan Jiang, Xuefeng Zhao, Tadatoshi Sato, Michael Densmore, Christiane Schüler, Reinhold G Erben, Marc D McKee, Beate Lanske Increased Osteopontin Contributes to Inhibition of Bone Mineralization in FGF23‐Deficient Mice. 2013. https://doi.org/10.1002/jbmr.2079

25. Shipra Gupta, Shaveta Sood, and Aneet Mahendra. Gene therapy with growth factors for periodontal tissue engineering–A review, Med Oral Patol Oral Cir Bucal. 2011 Mar; 17(2): e301–e310. doi: 10.4317/medoral.17472

26. Puspito Ratih Hardhani, Sri Pramestri Lastianny dan Dahlia Herawati. Pengaruh penambahan platelet-rich plasma pada cangkok tulang terhadap kadar osteocalcin cairan sulkus gingiva pada terapi poket infraboni. 2013. Jurnal PDGI. Vol. 62, No. 3, September-Desember l 2013, Hal. 75-82 | ISSN 0024-9548

27. Amer Youssef, Doaa Aboalola and Victor K. M. Han. The Roles of Insulin-Like Growth Factors in Mesenchymal Stem Cell Niche. Stem Cells Int. 2017: 9453108. doi: 10.1155/2017/9453108

28. Tao Qiu, Janet L. Crane, Liang Xie, Lingling Xian, Hui Xie and Xu Cao. IGF-I induced phosphorylation of PTH receptor enhances osteoblast to osteocyte transition. Bone Research 2018; 6: 5 https://doi.org/10.1038/s41413-017-0002-7

29. Zhang Xiaoxuan, Helin Xing, Feng Qi, Hongchen Liu, Lizeng Gao and Xing Wang. Local delivery of insulin/IGF-1 for bone regeneration:carriers, strategies, and effects. Nanotheranostic. 2020 Sep 8; 4(4): 242-255. doi: 10.7150/ntno.46408

30. Bi F, Shi Z, Liu A, Guo P, Yan S. Anterior cruciate ligament reconstruction in a rabbit model using silkcollagen scaffold and comparison with autograft. Plos ONE. 2015; 10(5): 1-15.

31. Zhang Q, Yan S, Li M. Silk fibroin based porous materials. Materials 2009; 2: 2276-95.

32. Saima S, Jan SM, Shah AF, Yousuf A, Batra M. Bone grafts and bone substitutes in dentistry. J Oral Res Rev. 2016; 8: 36-8

33. Shoshana Yakar, Olle Isaksson. Regulation of skeletal growth and mineral acquisition by the GH/IGF-1 axis: Lessons from mouse models. 2016; 26-32

34. Xian L, Wu X, Pang L, Lou M, Rosen CJ, Qiu T, et al. Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells. Nat Med. 2012; 18: 1095-101.

35. Wang, T., Wang, Y., Menendez, A., Fong, C., Babey, M., Tahimic, C. G., et al. Osteoblast-specific loss of IGF1R signaling results in impaired endochondral bone formation during fracture healing. J. Bone. Miner. Res. 2015; 30: 1572–1584. doi: 10.1002/jbmr.2510

36. Fang, Y., Xue, Z., Zhao, L., Yang, X., Yang, Y., Zhou, X., et al. Calycosin stimulates the osteogenic differentiation of rat calvarial osteoblasts by activating the IGF1R/PI3K/Akt signaling pathway. Cell Biol. Int. 2019; 43: 323–332. doi: 10. 1002/cbin.11102

37. Eriksen, E. F. ‘Cellular mechanisms of bone remodeling’, Reviews in Endocrine and Metabolic Disorders, 2010; 11(4): pp. 219–227. doi: 10.1007/s11154-010-9153-1.

38. Lucia Guerra-Menéndez, Maria C Sádaba, Juan E Puche, Jose L Lavandera, Luis F de Castro, Arancha R de Gortázar and Inma Castilla-Cortázar. J Transl Med. 2013; 11: 271. doi: 10.1186/1479-5876-11-271

39. Sroga GE, Karim L, Colón W, Vashishth D. Biochemical characterization of major bone‐matrix proteins using nanoscale‐ size bone samples and proteomics methodology. Mol Cel Proteom. 2011; 11: M110.006718‐M110.006718. https://doi.org/ 10.1074/mcp.M110.006718

40. Chen L, Zou X, Zhang RX, Pi CJ, Wu N, Yin LJ, Deng ZL. IGF1 potentiates BMP9-induced osteogenic differentiation in mesenchymal stem cells through the enhancement of BMP/Smad signaling. BMB Rep 2016; 49(2): 122–127.

41. Carvalho, M. S., Cabral, J. M. S., da Silva, C. L., and Vashishth, D. Synergistic effect of extracellularly supplemented osteopontin and osteocalcinon stem cell proliferation, osteogenic differentiation, and angiogenicproperties. J. Cell Biochem. 2019; 120, 6555–6569. doi: 10.1002/jcb.27948

42. Kahles, F.; Findeisen, H.M.; Bruemmer, D. Osteopontin: A novel regulator at the cross roads of inflammation, obesity and diabetes. Mol. Metab. 2014, 3, 384–393

43. Paulo Roberto Camati and Allan Fernando Giovanini and Hugo Eduardo de Miranda Peixoto and Cassiana Majewski Schuanka and Maria Cecília Giacomel and Melissa Rodrigues de Araújo and João César Zielak and Rafaela Scariot and Tatiana Miranda Deliberador. Immunoexpression of IGF1, IGF2, and osteopontin in craniofacial bone repair associated with autogenous grafting in rat models treated with alendronate sodium. Clin Oral Invest 2015. DOI 10.1007/s00784-016-1975-0.

44. Chai X., Zhang W., Chang B., Feng X., Song J., Li L., Yu C., Zhao J., Si H. GPR39 agonist TC-G 1008 promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Artif. Cells Nanomed. Biotechnol. 2019; 47: 3569–3576. doi: 10.1080/21691401.2019.1649270.

45. Wei, W.;Liu, S.; Song, J.; Feng, T.; Yang, R.; Cheng, Y.; Li, H.; Hao, L. MGF-19E peptide promoted proliferation, diﬀerentiation and mineralization of MC3T3-E1 cell and promoted bone defect healing. Gene 2020; 749: 144703.

46. Luukkonen, J., Hilli, M., Nakamura, M., Ritamo, I., Valmu, L., Kauppinen, K., et al. Osteoclasts secrete osteopontin into resorption lacunae during bone resorption. Histochem. Cell Biol. 2019; 151: 475–487. doi: 10.1007/s00418-019- 01770-y

47. Singh, A., Gill, G., Kaur, H., Amhmed, M., and Jakhu, H. Role of osteopontin in bone remodeling and orthodontic tooth movement: a review. Prog. Orthod. 2018; 19: 18. doi: 10.1186/s40510-018-0216-2

48. Tulika Tripathi, Prateek Gupta, Priyank Rai, Jitender Sharma, Vinod Kumar Gupta and Navneet Singh. Osteocalcin and serum insulin-like growth factor-1 as biochemical skeletal maturity indicators. Progress in Orthodontics volume 18. 2017. Article number: 30

49. Crane Janet L. and Cao Xu. Function of Matrix IGF-1 in Coupling Bone Resorption and Formation. J Mol Med (Berl). February 2014; 92(2): 107–115. doi:10.1007/s00109-013-1084-3.

50. Yakar Shoshana, Haim Werner and Clifford J Rosen. Insulin-like growth factors: actions on the skeleton. J Mol Endocrinol. 2018; 61(1): T115–T137. doi:10.1530/JME-17-0298.

Received on 22.04.2021 Modified on 05.06.2021

Research J. Pharm.and Tech 2022; 15(4):1737-1742.

DOI: 10.52711/0974-360X.2022.00291