Ismail Hadisoebroto DİLOGO, Phedy PHEDY, Yoshi Pratama DJAJA, Yuyus KUSNADİ, Lakshmi SANDHOW, Erica KHOLİNNE

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

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.

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

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 fractureMethods: 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.

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Dahabreh Z, Dimitriou R, Giannoudis PV. Health economics: a cost analysis of treatment of persistent fracture non-unions using bone morphogenetic protein-7. Injury 2007;38:371-377.
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.
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.
Megas P. Classification of non-union. Injury 2005;36(Suppl 4):30-37.
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.
Brownlow HC, Reed A, Simpson AH. Growth factor expression during the development of atrophic non- union. Injury 2001;32:519-524.
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.
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.
ominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. Cytotherapy 2006;4:315-317.
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.
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.
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.
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.
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.
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.
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.
Kiernan, JA. Histological and histochemical methods: theory and practice. Butterworth-Heinemann; Boston: 1999. Methods for inorganic ions; p. 267-280.
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.