Osteogenic potency of human bone marrow mesenchymal stem cells from femoral atrophic non-union fracture site

Abstract

Objective: Mesenchymal stem cells (MSCs) exist in the site of atrophic non-union fracture. The aim of this study was to evaluate the osteogenic potency of MSCs in order to have a better understanding of the unclear pathophysiology of atrophic non-union fracture Methods: This is an in vitro experimental study. Sample was obtained from the non-union site of a patient with a 6-years-history of atrophic non-union fracture of right femur. The MSCs was isolated from the fracture site and was cultured in the growth medium. Confirmation of the MSCs was performed and then osteogenic differentiation was performed in mono-layered MSC grown in both home-made and commercial osteogenic media. To evaluate the osteogenic differentiation, we performed Alizarin red staining and colorimetric assay for alkaline phosphatase (ALP). Results: From Alizarin red staining, most cells in the osteoblast medium were stained red by the staining. The result of colorimetric assessment of ALP shows that peak concentration was reached after 4 minutes in osteogenic group and control group. Conclusion: The presence of ALP activity and positive Alizarin red staining in our study showed that MSCs stem cells obtained from site of atrophic non-union is capable to be differentiated into osteogenic cells. . J Clin Exp Invest 2014; 5 (2): 159-163

Keywords

• Osteogenic potency, Mesenchymal stem cell, non-union fracture

Femoral atrofik birleşmemiş kırık yerinden alınan insan kemik iliği mezenkimal kök hücresinin osteogenic potansiyeli

Öz

Amaç: Bu çalışmanın amacı atrofik birleşmemiş kırık patofizyolojisini daha iyi anlamak için bu bölgelerdeki mezenkimal kök hücrelerinin (MKH) osteojenik potansiyelini araştırmaktır. Yöntemler: Çalışmamız in vitro deneysel bir çalışmadır. Altı yıllık sağ femurda atrofik birleşmemiş kıık öyküsü bulunan bir hastanın kırık yerinden örnek alındı. Mezenkimal kök hücreler kırık bölgesinden izole edildi ve besiyerine kültüre ekildi. Mezenkimal kök hücrelerinin doğrulanma ve daha sonra osteojenik farklılaşması, tek tabakalı MKH ve ticari osteojeik ortamda yapıldı. Osteojenik farklılaşmayı değerlendirmek için Alizarin kırmızı boya ve alkalen fosfataz için kolorimetrik test yapıldı. Bulgular: Alizarin boyası ile osteoblastik ortamdaki hücrelerin çoğu kırmızıya boyandı. Alkalen fosfatazın kolorimetrtik değerlendirilmesi osteojenik ve kontrol grubunda dört dakika sonra pik konsantrasyona ulaştı. Sonuçlar: Çalışmamızda alkalen fosfataz aktivitesi varlığı ve pozitif Alizarin kırmızı boyanması atrofik birleşmemiş bölgeden alınan MKH\'ların osteojenik hücrelere farklılaşma potansiyeli olduğunu gösterdi.

Anahtar Kelimeler

• Osteojenik potansiyel, mezenkimal kök hücre, birleşmemiş kırık

References

1. Dahabreh Z, Dimitriou R, Giannoudis PV. Health eco- nomics: a cost analysis of treatment of persistent frac- ture non-unions using bone morphogenetic protein-7. Injury 2007;38:371-377. Figure 3. Colori- metric assessment of alkaline phos- phatase. Shows the result of colo- rimetric assess- ment of ALP. Peak concentration was reach after 4 min- utes in osteogenic
2. group and control respectively. DISCUSSION
3. In our previous study, we revealed the existence of
4. mesenchymal stem cells in atrophic non-union frac
5. ture site [8]. However, in our, we did not evaluate
6. the osteogenic potency of those cells in the study.
7. In this study, we evaluated the osteogenic potency
8. of mesenchymal stem cells in the site of atrophic

9. non-union fractures.
10. Characterization of cells obtained from the site
11. of atrophic non-union fracture confirmed the pres
12. ence of mesenchymal stem cells. The cells that we
13. obtained were able to adhere to plastic in standard
14. culture condition, positive expression on CD 105,
15. CD 73, CD 90 for at least 95%, negative expression
16. of CD 45, CD 34, CD 14 or CD 11b, CD 79a, and HLA-DR [9].
17. Despite similar constituents, we found failure of
18. our home-made medium to induce differentiation of
19. MSCs into osteogenic cells. [10] All cells died before
20. 5 weeks and none was left for further evaluation.
21. The failure might due to the imprecise composition
22. of the medium that influence the milieu. Further re
23. searches must be done to evaluate the effect of this
24. homemade osteogenic medium upon its capabil
25. ity in inducing osteogenic differentiation of MSCs.
26. Addition of various chemical substances had been
27. reported to increase the success of osteogenic dif
28. ferentiation [11-13].
29. Osteogenic differentiation of MSCs can be
30. characterized by various technique, depending on
31. the development stage of the cells; cells prolifera
32. tion, matrix maturation, and matrix mineralization [14-15].
33. During cells proliferation, several extracellular
34. matrix proteins can be detected, such as procol
35. Busse JW, Bhandari M, Sprague S, et al. An eco- nomic analysis of management strategies for closed and open grade I tibial shaft fractures. Acta Orthop 2005;76:705-712.
36. Garrison KR, Shemilt I, Donell S, et al. Bone morpho- genetic protein (BMP) for fracture healing in adults. Cochrane Database Syst Rev 2010;6:CD006950.
37. Megas P. Classification of non-union. Injury 2005;36(Suppl 4):30-37.
38. Dickson K, Katzman S, Delgado E, Contreras D. De- layed unions and non-unions of open tibial fractures: correlation with arteriographic results. Clin Orthop Relat Res 2004;302:189-193.
39. Brownlow HC, Reed A, Simpson AH. Growth factor expression during the development of atrophic non- union. Injury 2001;32:519-524.
40. Centeno CJ, Schlutz JR, Cheever M, et al. A case se- ries of percutaneous treatment of nonunion fractures with autologous, culture expanded, bone marrow de- rived mesenchymal stem cells and platelet lysate. J Bioengineer & Biomedical Sci 2011;S2:007.
41. Ismail HD, Phedy P, Kholinne E, et al. Existence of mesenchymal stem cells in sites of atrophic non- union. Bone Joint Res 2013;2:118-121.
42. ominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. Cytotherapy 2006;4:315-317.
43. Jaiswal N, Haynesworth SE, Caplan AI, et al. Osteo- genic Differentiation od Purified, Culture-Expanded Human Mesenchymal Stem Cells In Vitro. J Cell Bio- chem 1997;64:295-312.
44. Cho HH, Park HT, Kim YJ, et al. Induction of Osteo- genic Differentiation of Human Mesenchymal Stem Cells by Histone Deacetylase Inhibitors. J Cell Bio- chem 2005;96:533-542.
45. Chen K, Aenlle KK, Curtis KM, et al. Hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D to- gether stimulate human bone marrow-derived stem cells toward the osteogenic phenotype by HGF-in- duced up-regulation of VDR. Bone 2012;51:69-77.
46. Sammons J, Ahmed N, El-Sheemy M, et al. The Role of BMP-6, IL-6, and BMP-4 in Mesenchymal Stem Cell-Dependent Bone Development: Effects on Os- teoblastic Differentiation Induced by Parathyroid Hor- mone and Vitamin D3. Stem Cells Dev 2004;13:273- 280.
47. Jasiwal RK, Jaiswal N, Bruder SP, et al. Adult human mesenchymal stem cell differentiation to the osteo- genic or adipogenic lineage is mitogen-activated pro- tein kinase. J Biol Chem 2000;275:9645-9652.
48. Stein GS and Lian JB. Molecular mechanisms medi- ating developmental and hormone-regulated expres- sion of genes in osteoblasts:an integrated relationship of cell growth and differentiation. In: Noda M, editor. Cellular and Molecular Biology of Bone. Tokyo: Aca- demic Press. p 47-95,1993.
49. Marom R, Shur I, Solomon R, Benayahy D. Char- acterization of adhesion and differentiation markers of osteogenic marrow stromal cells. J Cell Physiol 2005;202:41-48.
50. Kiernan, JA. Histological and histochemical methods: theory and practice. Butterworth-Heinemann; Boston: 1999. Methods for inorganic ions; p. 267-280.
51. Puchtler H, Meloan SN, Terry MS. On the history and mechanism of alizarin and alizarin red S stains for cal- cium. J Histochem Cytochem 1969;17:110-124.