The Analysis of MMP-13 Expression on Hydroxyapatite Tooth Graft Application Compared to Hydroxyapatite Xenograft

 

Sonny Perdana1, Chiquita Prahasanti2*, Lambang Bargowo2, Sista Prasetyo1, Wibi Riawan3

1Student of Periodontic Residency Program, Faculty of Dental Medicine,

Airlangga University, Surabaya, Indonesia.

2Department of Periodontology, Faculty of Dental Medicine, Airlangga University, Surabaya, Indonesia.

{Faculty of Dental Medicine - Airlangga University. Mayjen.

Prof. Dr. Moestopo Street 47 Surabaya 60132, Indonesia}

3Department - Laboratory of Biochemistry-Biomolecular, Faculty of Medicine,

Brawijaya University, Malang, Indonesia.

*Corresponding Author E-mail: chiquita-p-s@fkg.unair.ac.id

 

ABSTRACT:

Background: Bone graft materials are widely used to support the success of periodontal tissue treatments. Graft material has been used extensively however it has some deficiencies such as limited availability, high levels of resorption, immunological reactions and the risk of contamination as well as high costs. Therefore recent research is needed to explore potential usage of the tooth graft. Objective(s): The objective of this research is to comparing the MMP-13 expression in bone healing after grafting the incisor’s extraction sockets with hydroxyapatite tooth graft and hydroxyapatite xenograft. Research Methods: 33 adult male Cavia cobayas were divided into three groups: hydroxyapatite xenograft, hydroxyapatite tooth graft, and control (without treatment). Bone tissue sections were stained with diaminobenzidine and then dripping with anti-MMP-13 monoclonal antibody. The number of MMP-13 expression by osteoblast cells was carried out quantitatively based on the colourintensity in each group under the OLYMPUS microscope with 400x magnification. Results: The value of MMP-13 expression was higher on the tooth graft and xenograft group than the control group. The ability of osteoconduction of hydroxyapatite derived from porous hydroxyapatite structure will facilitate angiogenesis so that osteogenic cells can migrate and attach to the graft to induce the new bone growth. The tooth graft group had higher MMP-13 value than xenograft group. High solubility of graft material could affect bone remodeling and mineralization processes because of the presence of inflammatory cells is detrimental to the healing process. Conclusion:  As a new graft material, tooth graft has a capability to induce osteoconductive and osteoinductive that similar with the xenograft.

 

KEYWORDS: Xenograft, Tooth graft, Hydroxyapatite, MMP-13.

 

 


INTRODUCTION: 

Rapid technological development has resulted in many new methods in modern periodontal treatment. The main purpose of this treatment is to maintain the teeth and supporting the tissue structure.1 Many materials are used to support the success of periodontal treatment, one of which is bone graft.2

 

 

Bone graft is widely used to repair bone defects caused by trauma, carcinoma cell, degeneration due to pathological processes and congenital bone defects, as well as to reconstruct bone defects due to periodontitis. The using of bone graft after the periodontal flap treatment is expected to accelerate the regeneration of periodontal tissue.3,4,5,6

 

Recent research in bone graft is directed in the use of human dentin as a bone graft material, because the structure of the teeth is similar to bone so that teeth can also be considered as a bio-inorganic material. Dentin contains inorganic and organic components which are same with components in the human bones. It is expected that graft sourced from teeth can stimulate new bone formation. Dentin and bone has a difference in the content of minor ions, such as Mg, N, CO3, HPO4. The differences in the content of minor ions affect the crystal size and solubility of dentin and bone. Mg and CO3 ions when reacting in apatite biogenic cause the growth of apatite crystals that are smaller and more soluble.

 

Research conducted by Pan and Brian (2010) found that the replacement of PO4 ions by CO3 ions causes the shift in the balance of hydroxyapatite so that it dissolves more easily.7,8 The high solubility of graft material can affect the process of bone remodelling and mineralization due to the number of inflammatory cells so that it is detrimental to the healing process, the ability of osteoconduction of graft material is not persistent because it is absorbed too quickly by the body, and the apposition of bones is unstable due to graft disintegration. Recent studies report that bone apatite solubility is higher than dentinal apatite.7

 

Bone quality can be seen from the number of MMP-13 expression, as one of the non-collagenous proteins produced by osteoblasts in the bone mineralization phase with standard graft material.9 Hydroxyapatite is an excellent filler for bone imperfections and as a covering material to advance in growth of bone tissue into frameworks or prosthetic inserts.10 So far there has been no research on bone quality resulted from the hydroxyapatite tooth graft content. The researchers want to see the expression of MMP-13 in the hydroxyapatite tooth graft applications compared to hydroxyapatite xenograft.

 

MATERIALS AND METHODS:

Tooth graft:

·       Tooth graft material derived from teeth that have been extracted.

·       The crown and roots were separated, then the dentin powder was taken from the tooth root.

·       The dentin was washed with 3% H2O2 solution until all fat solution was lost.

·       The deproteinization process was heat-treated leaving 100% hydroxyapatite in crystalline form. Tooth graft was made with particle sizes of 150-355 µm and 355-710 µm.

 

This research was an experimental laboratory with the post-test only control group design using 33 male guinea pigs (Cavia cobaya), 90-120 days age, and 250-300 grams body weight. All guinea pigs were adjusted during the week and were kept in experimental animal cages with 60 cm x 60 cm x 90 cm size. Each treatment group consisted of 11 guinea pigs.

The experimental animals were divided into 3 groups randomly, guinea pigs were anesthetized by injection of ketamine + xylazine 5-10 mg + 0.5 mg/head. The mandibular left incisor was extracted, then the socket was irrigated with saline solution, has been grafted with hydroxyapatite xenograft for group 1, hydroxyapatite tooth graft for group 2, and blood as a control for group 3.

 

The graft was left for 14 days then guinea pigs were sacrificed under 10% ether inhalation anaesthesia and preparations taken from the bone tissue in the socket were fixed in 70% buffered formalin solution. Decalcification using EDTA solution and making preparations with a 4 mm thickness and immunohistochemical staining. Staining was carried out on the preparation using DAB (Diamino benzidine) and then dripping with anti-MMP-13 monoclonal antibody (MoAb). The number of MMP-13 expression by osteoblast cells was carried out quantitatively based on the colour intensity in each group under the OLYMPUS microscope with 400x magnification.

 

Statistical analysis of the research data using the Kruskal-Wallis non parametric test followed by the Mann-Whitney test with a value of p < 0.05 for the statistical significance.

 

RESULTS:

 

Figure 1. Immunohistochemical Expression of MMP-13. The number of MMP-13 expression is indicated by the color intensity shown by the arrows (a) the xenograft treatment group, (b) the tooth graft treatment group and (c) the control group.

 

The number of MMP-13 expression from 3 treatment groups is shown in Figure 2.

 

Figure 2. Average Number of MMP-13 Expressions

 

Table 1. The Kruskal-Wallis test result

 

Group

N

Mean Rank

Asymp. Sig.

 

Control

11

2.64

 

MMP-13

Xenograft

11

6.91

0.000

 

Tooth graft

11

11.45

 

 

The analysis of Kruskal-Wallis test is shown in Table 1, with a significance value of 0.000 (p < 0.05) which means the mean values of the three treatment groups had significant differences.

 

Table 2. Mean, Minimum, Maximum Number of MMP-13 Expression in The Xenograft Group Compared to The Control Group

Variable

Xenograft n = 11

Control n = 11

p*

Mean ± SD

Min - Max

Mean ± SD

Min - Max

MMP-13

11.82 ± 2.040

8 – 15

6.09 ± 1.578

4 – 9

.000

Note: Mean = average; Min = Minimum; Max = Maximum; SD = Standard Deviation; p* = significance value, Mann-Whitney test

 

Table 2 shows the number of MMP-13 from the xenograft group with an average number was 11.82, that more than the number of MMP-13 from the control group which the average 6.09. From the two treatment groups, there were significant differences in the number of MMP-13 with p < 0.05 (p = 0.000) where the xenograft group was higher than the control group.

 

Table 3. Mean, Minimum, Maximum Number of MMP-13 Expressions in The Tooth Graft Group Compared to The Control Group

Variable

Tooth graft n = 11

Control n = 11

p*

Mean ± SD

Min - Max

Mean ± SD

Min - Max

MMP-13

15,36 ± 2,838

12 – 19

6.09 ± 1.578

4 – 9

.000

Note: Mean = average; Min = Minimum; Max = Maximum; SD = Standard Deviation; p* = significance value, Mann-Whitney test

 

Table 3 shows that the number of MMP-13 from the tooth graft group with an average number of 15.36 is more than the number of MMP-13 from the control group with an average was 6.09. From the two treatment groups, there were significant differences in the number of MMP-13 with p < 0.05 (p = 0.000) where the tooth graft group was higher than the control group.

 

Table 4. Mean, Minimum, Maximum Number of MMP-13 Expressions in The Xenograft Group Compared to The Tooth Graft Group

Variable

Xenograft n = 11

Tooth graft n = 11

p*

Mean ± SD

Min - Max

Mean ± SD

Min - Max

MMP-13

11.82 ± 2.040

8 – 15

15.36 ± 2.838

12 – 19

.009

Note: Mean = average; Min = Minimum; Max = Maximum; SD = Standard Deviation; p* = significance value, Mann-Whitney test

 

 

The comparison the number of MMP-13 from the xenograft group to the tooth graft group is shown in Table 4, which the average number of xenograft groups (11.82) was less than the number of MMP-13 from the tooth graft group with an average of 15.36. The difference between the two groups was also significant with p < 0.05 (p = 0.009).                   

 

DISCUSSION:

The primary haemostatic process begins with the formation of blood clots that will contact the collagen connective tissue to form a platelet aggregation (blood clot). This blood clot will fill the socket cavity in a few minutes. Platelet activation will release a number of cytokines, such as IL-1, IL-6, IL-8, and TNF-α which the function is to stimulate inflammatory cells out of the blood vessels to begin the healing process.11,12 Inflammatory cells, especially neutrophils and macrophages, will produce pro-inflammatory cytokines and growth factors, such as VEGF, FGF, TGF-β1, BMP, which are important as tissue repair regulators.13 Blood clot fibrils will be replaced with fibrous tissue and blood vessels within 4 weeks.14 Various cytokines and growth factors released by platelets and inflammatory cells that will induce the differentiation of mesenchymal stem cells and bone marrow-derived cells into osteogenic cells locally in the socket. Cytokines and growth factors in the socket healing process can be derived from the cytokines and growth factors released by pro-inflammatory cells, and the cytokines and growth factors from the mineralized bone deposits.15,16,17

 

The results showed that at the end of the second week the xenograft and tooth graft treatment groups showed MMP-13 expression which were very high compared to the control group, so it can be concluded that the healing socket process had reached the bone mineralization phase. Research conducted by Araujo et al (2010) on the extraction sockets that were grafted with hydroxyapatite, showed that in the second week the osteoclasts had been replaced by osteoblasts and the process of remineralizing the trabecular bone (mature bone) had begun.18 MMP-13 expression in the group of MMP-13 xenograft and tooth graft were higher than the control group with a significant difference in number (p < 0.05). The hydroxyapatite graft material will stimulate the remodelling process faster than the normal healing process without graft because hydroxyapatite has osteoconduction and osteoinduction capabilities. The ability of osteoconduction of hydroxyapatite derived from porous hydroxyapatite structure will facilitate angiogenesis so that osteogenic cells can migrate and attach to the graft to induce new bone growth.19,20,22,22

 

The metalloproteinase-13 matrix (MMP-13 or collagenase-3) is a member of the metalloproteinase matrix family (MMPs) produced in high numbers by cells that have potential for mineralization. This indicates that the higher value of MMP-13 expression, the higher the potential for mineralization to occur also. Research conducted by Sury (2008) reported that human dental pulp has been shown to express high levels of MMP-13 RNA.23 This showed that cells from dental pulp have excellent mineralization potential. Research conducted by Ohkubo (2003) proved that MMP-13 plays a role in creating intercellular space for cell enlargement by cartilage matrix degradation and the onset of mineralization during the early stages of development.24

 

MMP-13 expression in the tooth graft treatment group was higher than the xenograft group with a significant difference in number (p < 0.05). Dentin and bone have different Mg ion concentrations which are 1.23 in dentin and 0.72 in bone. The concentration of CO3 ions in dentin was 5.6 and in bone was 7.4. The higher the concentration of Mg and CO3 ions, the smaller the size of the apatite crystals formed and the higher the solubility of the particles. The size of bone apatite crystals ± 2.5x0.3 nm is almost the same as the size of the dentin apatite crystals which is ± 2x0.4 nm, but the bone apatite solubility is higher than the dentin apatite.23 In biogenic apatite CO3 ions can replace PO4 or OH- ions, causing the balance shift in bone apatite of hydroxyapatite to Ca10-2x/3(PO4)6-x(CO3)x(OH)2-x/3 so it is more soluble.7,26 High solubility of graft material can affect the process of bone remodelling and mineralization due to the presence of Inflammatory cells are too numerous so that it harms the healing process, the ability of osteoconduction of graft material is not persistent because it is absorbed too quickly by the body, and the apposition of the bones is unstable due to the disintegration of graft material.27,28 This study used a hydroxyapatite graft with particle sizes of 150-355 µm and 355-750 µm which is mixed manually to get the maximum effect from the graft material. If the graft particle size is too small, the graft will be quickly absorbed by macrophages so that the ability to induce the bone will not optimal. In contrast, the graft with too large particle, the resorption by macrophages will be too slow so it can prevent the process of new bone formation.16,29

 

Hydroxyapatite osteoinduction originates from the surface microporous. The existence of microporous provides interconnection between macroporous graft resulting interstitial fluid circulation along the matrix. After the graft is placed in the bone defect, the hydroxyapatite granule will dissolve into a biological apatite layer (calcium phosphate) on the surface of the hydroxyapatite. Calcium phosphate has a high affinity for endogenous proteins and growth factors including BMP, so it can induce the differentiation of pluripotent stem cells into cells that play a role in osteogenesis.30,31 Calcium (Ca2+) extracellular will increase the production of autocrinal growth factors such as BMP-1, BMP-2, IGF-I, and IGF-II which are effective stimulators for osteoblast differentiation. Calcium ions also have an important role in the bone mineralization phase because MMP-13 requires free calcium ions to be able to bind hydroxyapatite. MMP-13 consists of 3-carboxylated glutamic acid or bone gla protein (BGP) residues, these residues that have an affinity for hydroxyapatite.9,31 Research conducted by Hoang (2003) found that to bind hydroxyapatite, MMP-13 requires free extracellular calcium ions (Ca2+).18 Calcium that interacts with MMP-13 will direct the surface of MMP-13 to the surface of hydroxyapatite so that MMP-13 can bind with hydroxyapatite.32 Phosphate ions ((PO4)3-) will enter the cell and then induce bone mineralization process and MMP-13 expression as mature osteoblast marker.21,32

 

The ability of osteoinduction hydroxyapatite is actually still under debate until now. Research conducted by Lin et al (2008) showed hydroxyapatite particles that cultured in human mesenchymal stem cells (hMSC) without bone morphogenetic protein have the perfect ability to regulate the four osteoblast markers, namely alkaline phosphatase (ALP), Runx2, type 1 collagen, and MMP-13.21 Sun et al (2008) also proved the ability of hydroxyapatite osteoinduction and found expression of osteoblast markers, namely Runx2, collagen type 1, osteopontin and MMP-13, in human mesenchymal stem cells (hMSC) cultured in hydroxyapatite particles.30

 

CONCLUSIONS:

The conclusion of this study that hydroxyapatite tooth graft was shown to be higher in inducing MMP-13 expression compared to hydroxyapatite xenograft. Tooth graft is a candidate for alternative graft material that can be considered for the use in dentistry, especially in the treatment of periodontal abnormalities so the further research needed to be done on the physical and chemical characteristics of hydroxyapatite tooth graft so this graft material can be developed for the use in humans.

 

REFERENCES:

1.     Smriti Agarwal, Vinayak Jhunjhunwala, G. Priya. Fabrication and Morphological Analysis of Gelatin-Alginate Scaffolds. Research J. Pharm. and Tech 2018; 11(9): 3816-3818. doi: 10.5958/0974-360X.2018.00699.6

2.     Michael Josef Kridanto Kamadjaja, Sherman Salim, Birgitta Dwitya Swastyayana Subiakto. Application of Hydroxyapatite scaffold from Portunus pelagicus on OPG and RANKL expression after tooth extraction of Cavia cobaya. Research Journal of Pharmacy and Technology. 2021; 14(9):4647-1. doi: 10.52711/0974-360X.2021.00807

3.     Sukumar & Ivo D. Bone graft in periodontal. Acta Medica (Hradec Králové) 2008;51(4): 203-7

4.     Grupta R, Nymphea P, Rajvir  M, Shaveta S. Clinical and radiological evaluation of an osseous xenografts for The Treatment of Infrabony Defect. J Clin Dent American 2007; 73(6): 513-9

5.     Sushma Singh, Abhisek Pal, Sangeeta Mohanty. Nano Structure of Hydroxyapatite and its modern approach in Pharmaceutical Science. Research J. Pharm. and Tech. 2019; 12(3): 1463-1472. doi: 10.5958/0974-360X.2019.00243.9   Available on: https://rjptonline.org/AbstractView.aspx?PID=2019-12-3-82

6.     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

7.     Pan H, Brian WD. 2010. Effect Of Carbonate On Hydroxyapatite Solubility. Crystal Growth & Design 10(2):845-50 DOI: 10.1021/cg901199h

8.     R.Panneerselvam, N.Anandhan,K.P.Ganesan, T. Marimuthu, I. Joseph Paneerdoss. Effect of Concentration on Nano Hydroxyapatite Powder by Wet Chemical Precipitation Route. Asian J. Research Chem. 2018; 11(3):545-550. doi: 10.5958/0974-4150.2018.00097.4   Available on: https://ajrconline.org/AbstractView.aspx?PID=2018-11-3-7

9.     Chaves MD, Leandro S, Renato V, Leandro A, Hugo N, Mariza A, Daniel A. 2012.Bovine Hydroxyapatite (Bio-Oss) Induces MMP-13, RANK-L, And Osteoprotegerin Expression In Sinus Lift Of Rabbits. Journal of Cranio-Maxillo-Facial Surgery 40:315-20

10.  C. Girija, M. N. Sivakumar. Amalgamation and Characterization of Hydroxyapatite Powders from Eggshell for Functional Biomedical Application. Research J. Pharm. and Tech 2018; 11(10): 4242-4244. doi: 10.5958/0974-360X.2018.00777.1

11.  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

12.  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

13.  Keerthic Aswin S, Jothishwar S, Visvavela Chellaih Nayagam P, G. Priya. Scaffolds for Biomolecule Delivery and Controlled Release–A Review. Research J. Pharm. and Tech 2018; 11(10): 4719-4730. doi: 10.5958/0974-360X.2018.00861.2

14.  Marei, Mona. 2010. Regenerative Dentistry. Morgan & Claypool Publisher:32-3

15.  Larjava, Hannu (ed). 2012. Oral Wound Healing. Wiley-Balckwell:1-8, 195-223

16.  Cohen, N & Cohen-Lévy. 2014. Healing Processes Following Tooth Extraction In Orthodontic Cases. J Dentofacial Anom Orthod 17:304. Available at http://www.jdao-journal.orgatauhttp://dx.doi.org/10.1051/odfen/2014006

17.  Zhang X, Wei C, Paul L, Yuhao W, Min Y, Jun L, Sangamesh GK, Xiaojun Y. 2014. Polymer-Ceramics Spiral Structured Scaffolds For Bone Tissue Engineering : Effect of Hydroxyapatite On Human Fetal Osteoblast. Plos One 9(1):1-10. Available at www.plosone.org

18.  Hoang QQ. 2003. Crystal Structure And Hydroxyapatite Binding Of Porcine MMP-13. Thesis. McMaster University, Ontario:1-134

19.  Sotto-Maior BS, Plinio MS, Beatriz J, Rosangela AR, Neuza M, Altair A. 2011. Effect Of Bovine Hydroxyapatite On Early Stages Of Bone Formation. Rev Odonto Cienc 26(3):198-202. E-mail: psenna3@fop.unicamp.br

20.  Woodard JR, Amanda H, Sheeny K, C.J. Park, Abby W, Jo A, Sherrie G, Matthew B, Russell D, Amy J. 2007. The Mechanical Properties And Osteoconductivity Of Hydroxyapatite Bone Scaffolds With Multi-scale Porosity. Biomaterials 28:45–54

21.  Lin L, King LC, Yang L. 2008. Study of Hydroxyapatite Osteoinductivity With An Osteogenic Differentiation Of Mesenchymal Stem Cells. Available at www.interscience.wiley.com. Doi: 10.1002/jbm.a.31994

22.  Michael Josef Kridanto Kamadjaja, Sherman Salim, Birgitta Dwitya Swastyayana Subiakto. Application of Hydroxyapatite scaffold from Portunus pelagicus on OPG and RANKL expression after tooth extraction of Cavia cobaya. Research Journal of Pharmacy and Technology. 2021; 14(9):4647-1. doi: 10.52711/0974-360X.2021.00807

23.  Suri L, Damoulis P, Le T, Gagari E. 2008. Expression of MMP-13 (Collagenase-3) in Long-erm Cultures of Human Dental Pulp Cells. Archives of Oral Biology 53: 791–799

24.  Ohkubo K, Shimokawa H, Ogawa T, Suzuki S, Fukada K, Ohya K, Ohyama K. 2003. Immunohistochemical Localization of Matrix Metalloproteinase-13 (MMP-13) in Mouse Mandibular Condylar Cartilage. J Med Dent Sci 50: 203–211

25.  Patti A, Luigi Gennari, Daniela Merlotti, Francesco Dotta, Ranuccio Nuti. 2013. Endocrine Actions Of MMP-13. International Journal of Endocrinology http://dx.doi.org/10.1155/2013/846480

26.  Elena K, Danilović A, Vesna, Teodorović N. 2005. Histological Evaluation Of Bone Response To Bioactive Ceramics As Graft Material In Rats. Acta Veterinaria (Beograd) 55(5-6):461-70

27.  Hing K, Lester FW, Thomas B. 2007.Comparative Performance Of Three Ceramic Bone Graft Subtitutes. The Spine Journal 7(4):475-90

28.  Ramesh S, C.Y. Tanb, M.Hamdib, I.Sopyanc, WD.Teng. 2007. The Influence Of Ca/P Ratio On The Properties Of Hydroxyapatite Bioceramics. Proceddings Of International Conference On Smart Materials And Nanotechnology In Engineering. Proc. of SPIE 6423:1-6

29.  Cruz A, Márcia TP, Josélia DB, José C, Gibson L, Fábio A. 2006. Physico-chemical Characterization And Biocompatibility Evaluation Of Hydroxyapatites. Journal of Oral Science 48(4):219-26

30.  Sun H, Ye, J Wang, Y Shi, Z Tu, J Bao, M Qin, H Bu, Y Li. 2008. The Upregulation Of Osteoblast Marker Genes In Mesenchymal Stem Cells Prove The Osteoinductivity Of Hydroxyapatite/Tricalcium Phosphate Biomaterial. Elsevier Inc.:2645-9

31.  Wu X, Norio Itoh, Takashi Taniguchi, Tsuyoshi Nakanishi, Keiichi Tanaka. 2003. Requirement Of Calcium And Phosphate Ions In Expression Of Sodium-Dependent Vitamin C Transporter 2 And Osteopontin In MC3T3-E1 Osteoblastic Cells. Biochimica et Biophysica Acta 1641:65–70

32.  Contante FP, Crivellato E, Nico B, Ribatti O. 2005. MMP-13 Is Angiogenic In Vivo. Cell Biol Int 29:583-5

 

 

 

Received on 23.10.2021             Modified on 11.03.2022

Accepted on 25.07.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):261-265.

DOI: 10.52711/0974-360X.2023.00048