Isolation, culture and biological characteristics of high-purity orofacial-bone-derived mesenchymal stem cells of the rats

doi:10.3969/j.issn.2095-4344.2151

Abstract

Abstract: BACKGROUND: It is widely accepted to recruit mesenchymal stem cells from the jaws to repair the defects of the jaws. However, there are relatively few researches on orofacial-bone-derived mesenchymal stem cells, mainly due to the difficulty in separating mesenchymal stem cells from the jaws.
OBJECTIVE: To establish the methods of in vitro isolation and culture of rat orofacial-bone-derived mesenchymal stem cells and observe and study its related biological characteristics.
METHODS: The mandibles of rats were dissected. The attached muscles were stripped and cut into pieces. Cortical bone was loosened by digesting with collagenase II. The migration and adherent growth ability of mesenchymal stem cells was used to isolate orofacial-bone-derived mesenchymal stem cells. Cell morphology was observed by inverted microscope. Surface markers of the cell were detected by flow cytometry. Cell proliferation was detected by MTT assay and cell growth curve was drawn. Fibroblast colony forming rate was calculated by colony formation. Osteogenic and lipogenic induction experiments were conducted to study the multi-directional differentiation potential of cells.
RESULTS AND CONCLUSION: Cells isolated by collagenase digestion and bone slice culture were positive for CD29, CD44 and Sca-1, and negative for CD31, CD34 and CD45. Cell proliferation test showed that the growth curves of orofacial-bone-derived mesenchymal stem cells exhibited incubation period, logarithmic phase and platform period. In addition, the cells had a strong ability of proliferation, and the cell clone formation rate was 20% and the cells in DNA synthesis stage accounted for 52.5%. Alizarin red and oil red O staining showed positive reaction after osteogenic and lipogenic induction, indicating that the cells have the potential of multi-directional differentiation. It is concluded that the method of bone fragment culture after digestion with collagenase II could separate orofacial-bone-derived mesenchymal stem cells sufficiently and purely. Besides, the orofacial-bone-derived mesenchymal stem cells show strong proliferative and osteogenic differentiation capacities. Thus, it provides abundant source of seed cells for bone tissue engineering of maxillofacial represented by bone defects repairing of implants.

Key words: stem cells, mesenchymal stem cells, mandible, type II collagenase, proliferation, differentiation, bone, rat

CLC Number:

Cheng Bingkun, Liang Jianfei, Qin Dongze, Cheng Zixu, Zhao Yunzhuan. Isolation, culture and biological characteristics of high-purity orofacial-bone-derived mesenchymal stem cells of the rats[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(1): 67-72.

Figures/Tables 7

References

[1] CHAN CK, SEO EY, CHEN JY, et al. Identification and specification of the mouse skeletal stem cell. Cell. 2015;160(1-2):285-298.
[2] CASIRAGHI F, PERICO N, CORTINOVIS M, et al. Mesenchymal stromal cells in renal transplantation: opportunities and challenges. Nat Rev Nephrol. 2016;12(4):241-253.
[3] LU MM, WU PS, GUO XJ, et al. Osteoinductive effects of tantalum and titanium on bone mesenchymal stromal cells and bone formation in ovariectomized rats. Eur Rev Med Pharmacol Sci. 2018;22(21): 7087-7104.
[4] MASTROLIA I, FOPPIANI EM, MURGIA A, et al. Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review. Stem Cells Transl Med. 2019;8(11):1135-1148.
[5] LEUCHT P, KIM JB, AMASHA R, et al. Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration. Development. 2008;135(17):2845-2854.
[6] LLOYD B, TEE BC, HEADLEY C, et al. Similarities and differences between porcine mandibular and limb bone marrow mesenchymal stem cells. Arch Oral Biol. 2017;77:1-11.
[7] PARK JB, CHO SH, KIM I, et al. Evaluation of the bisphosphonate effect on stem cells derived from jaw bone and long bone rabbit models: A pilot study. Arch Oral Biol. 2018;85:178-182.
[8] DAI J, KUANG Y, FANG B, et al. The effect of overexpression of Dlx2 on the migration, proliferation and osteogenic differentiation of cranial neural crest stem cells. Biomaterials. 2013;34(8):1898-1910.
[9] YAMAZA T, REN G, AKIYAMA K, et al. Mouse mandible contains distinctive mesenchymal stem cells. J Dent Res. 2011;90(3):317-324.
[10] NAKAJIMA R, ONO M, HARA ES, et al. Mesenchymal stem/progenitor cell isolation from tooth extraction sockets. J Dent Res. 2014;93(11):1133-1140.
[11] SHORT BJ, BROUARD N, SIMMONS PJ. Prospective isolation of mesenchymal stem cells from mouse compact bone. Methods Mol Biol. 2009;482:259-268.
[12] ZHU H, GUO ZK, JIANG XX, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc. 2010;5(3):550-560.
[13] LEE DJ, KWON J, CURRENT L, et al. Osteogenic potential of mesenchymal stem cells from rat mandible to regenerate critical sized calvarial defect. J Tissue Eng. 2019;10:2041731419830427.
[14] GUO Z, LI H, LI X, et al. In vitro characteristics and in vivo immunosuppressive activity of compact bone-derived murine mesenchymal progenitor cells. Stem Cells. 2006;24(4):992-1000.
[15] ANDRZEJEWSKA A, LUKOMSKA B, JANOWSKI M. Concise Review: Mesenchymal Stem Cells: From Roots to Boost. Stem Cells. 2019;37(7): 855-864.
[16] MUSHAHARY D, SPITTLER A, KASPER C, et al. Isolation, cultivation, and characterization of human mesenchymal stem cells. Cytometry A. 2018;93(1):19-31.
[17] DOMINICI M, LE BLANC K, MUELLER I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317.
[18] GALIPEAU J, SENSÉBÉ L. Mesenchymal Stromal Cells: Clinical Challenges and Therapeutic Opportunities. Cell Stem Cell. 2018;22(6):824-833.
[19] WIATER J, NIEDZIELA M, POSMYSZ A, et al. Identification of perivascular and stromal mesenchymal stem/progenitor cells in porcine endometrium. Reprod Domest Anim. 2018;53(2):333-343.
[20] GOODISON S, URQUIDI V, TARIN D. CD44 cell adhesion molecules. Mol Pathol. 1999;52(4):189-196.
[21] DONG Q, WANG Y, MOHABATPOUR F, et al. Dental Pulp Stem Cells: Isolation, Characterization, Expansion, and Odontoblast Differentiation for Tissue Engineering. Methods Mol Biol. 2019;1922:91-101.
[22] VAN PHAM P, TRUONG NC, LE PT, et al. Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications. Cell Tissue Bank. 2016;17(2):289-302.
[23] BORZABADI-FARAHANI A. Effect of low-level laser irradiation on proliferation of human dental mesenchymal stem cells; a systemic review. J Photochem Photobiol B. 2016;162:577-582.
[24] COURTNEY JM, SUTHERLAND BA. Harnessing the stem cell properties of pericytes to repair the brain. Neural Regen Res. 2020;15(6): 1021-1022.
[25] CORRADETTI B, TARABALLI F, POWELL S, et al. Osteoprogenitor cells from bone marrow and cortical bone: understanding how the environment affects their fate. Stem Cells Dev. 2015;24(9):1112-1123.
[26] REISSIS D, TANG QO, COOPER NC, et al. Current clinical evidence for the use of mesenchymal stem cells in articular cartilage repair. Expert Opin Biol Ther. 2016;16(4):535-557.
[27] MUKHAMEDSHINA YO, GRACHEVA OA, MUKHUTDINOVA DM, et al. Mesenchymal stem cells and the neuronal microenvironment in the area of spinal cord injury. Neural Regen Res. 2019;14(2):227-237.
[28] PITTENGER MF, MACKAY AM, BECK SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411): 143-147.
[29] LEE BK, CHOI SJ, MACK D, et al. Isolation of mesenchymal stem cells from the mandibular marrow aspirates. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;112(6):e86-93.
[30] JEON YJ, KIM J, CHO JH, et al. Comparative Analysis of Human Mesenchymal Stem Cells Derived From Bone Marrow, Placenta, and Adipose Tissue as Sources of Cell Therapy. J Cell Biochem. 2016; 117(5): 1112-1125.
[31] NANCARROW-LEI R, MAFI P, MAFI R, et al. A Systemic Review of Adult Mesenchymal Stem Cell Sources and their Multilineage Differentiation Potential Relevant to Musculoskeletal Tissue Repair and Regeneration. Curr Stem Cell Res Ther. 2017;12(8):601-610.