Seda VATANSEVER, Feyzan OZDAL KURT, Remziye KENDİRCİ, Gorkem SAY, Emil MAMMADOV

3D Baskılı PLA-İskelelerin Üzerinde Farklılaştırılmış Mezenkimal Kök Hücrelerin In Vitro ve In Vivo Uyumları

Üç boyutlu baskı, doku mühendisliği uygulamaları için biyolojik olarak uyumlu ve biyolojik olarak parçalanabilen malzemelerin üretim aracı olarak hızlı bir giriş yaptı. Üç boyutlu baskı, özel biyobozunur implantlar oluşturmayı mümkün kıldı ve polilaktik asit (PLA) en umut verici polimerlerden biridir. Bu çalışmada, 3D baskı PLA polimerleri üzerinde kemik iliği stromal mezenkimal kök hücrelerinden (BMSC) hem kemik hem de kıkırdağa farklılaştırılan hücreler oluşturmayı amaçladık. PLA iskeleleri tasarlanmış, Solidworks Yazılımı kullanılarak 3D olarak basılmış ve Yakın Doğu Üniversitesi'nin özel tesislerinde sterilize edilmiştir. Mezenkimal kök hücreler sıçan kemik iliğinden toplandı ve daha sonra osteoblast veya kondroblastta farklılaştırıldı. Hücrelerin karakterizasyonu Alizarin kırmızısı, Alcian mavisi boyama, osteonektin ve kollajen II kullanılarak indirekt immünositokimya ile analiz edildi. Farklılaştırılmış hücreler, 3D yapı iskelesine ekilerek 2 hafta boyunca kültüre edildi. İn vivo test için, farklılaştırılmış BMSC'leri olan veya olmayan 3D yapı iskelesi, subkutan alanın bağ dokusuna implante edildi. Dört aylık implantasyondan sonra sıçanlar sakrifiye edildi ve tüm numuneler histokimyasal ve immünohistokimyasal olarak incelendi. BMSC'den osteojenik ve kondrojenik farklılaşma 2 haftalık kültür koşulundan sonra gözlendi. Hücreler hem histokimyasal hem de immünohistokimyasal olarak pozitif boyandılar. Hücreler 3D PLA iskelesine aktarıldıktan sonra farklılaşmaları devam etmiş ve hem in vitro hem de in vivo koşullarda histokimyasal ve immünohistokimyasal analizlerden sonra hem kemik hem de kıkırdak oluşumu gözlemlenmiştir. 3D baskılı PLA iskeleleri hem kemik hem de kıkırdak oluşumunu desteklediği ve bu nedenle bu hücrelerin in vivo çalışmalar için rahatlıkla kullanılabileceği gözlenmiştir.

Anahtar Kelimeler:

Kemik, Kıkırdak, 3D baskı, Mezenkimal Kök Hücre

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[1]. Huayu T, Tang Z, Zhuang X, Chen X, Jing X. Biodegradable synthetic polymers: preparation, functionalization, and biomedical application. Progress in Polymer Science. 2012; 37: 237-80.
[2]. Beresford JN, Bennett JH, Devlin C, Leboy PS, Owen ME. Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. Journal of Cell Science. 1992; 102: 341-50.
[3]. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake,J, Schwager C, Eckstein V, Ansorge W, Ho AD. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Experimental. Hematology. 2005; 3: 1402-16.
[4]. Caplan AI. Mesenchymal stem cells. Journal of Orthopaedic Research. 1991; 9: 641-50.
[5]. Pérez-Silos V, Camacho-Morales A, Fuentes-Mera L. Mesenchymal Stem Cells Subpopulations: Application for Orthopedic Regenerative Medicine. Stem Cells International. 2016; 3187491.
[6]. Ronzière MC, Perrier E, Mallein-Gerin F, Freyria AM. Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells. Biomedical. Materials and Engineering; 2010; 20: 145-58.
[7]. Morelli S, Salerno S, Holopainen J, Ritala M, De Bartolo, L. Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid-nanohydroxyapatite fiber scaffolds. Journal of. Biotechnology. 2015; 204: 53-62.
[8]. Lasprilla AJ, Martinez GA, Lunelli BH, Jardini AL, Filho RM. Poly-lactic acid synthesis for application in biomedical devices - a review. Biotechnology. Advances. 2012; 30: 321-8.
[9]. Daniels AU, Chang MK, Andrian, KP. Mechanical properties of biodegradable polymers and composites proposed for internal fixation of bone. Journal of Applied Biomaterials. 1990; 1: 57-78.
[10]. Rasal RM, Janorkar AV, Hirt DE. Poly (lactic acid) modifications. Progress in Polymer Science. 2010; 35: 338-56.
[11]. Athanasiou KA, Kyriacos A, Niederauer GG, Agrawal GM. Agrawal, Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers. Biomaterials. 1996; 17: 93-102.
[12]. Stankevich KS, Gudima A, Filimonov VD, Kluter H, Mamontova EM, Tverdokhlebov SI, Kzhyshkowska J. Surface modification of biomaterials based on high-molecular polylactic acid and their effect on inflammatory reactions of primary human monocyte-derived macrophages: perspective for personalized therapy. Material Science Engineering C. 2015; 51: 117-26.
[13]. Xu W, Shen R, Yan Y, Gao J. Preparation and characterization of electrospun alginate/PLA nanofibers as tissue engineering material by emulsion electrospinning. Journal of the Mechanical Behavior of Biomedical Materials 2017; 65: 428-438.
[14]. Guo Y, Wang D, Song Q, Wu T, Zhuang X, Bao Y, Kong M, Qi Y, Tan S, Zhang Z, Zhang Z. Erythrocyte Membrane-Enveloped Polymeric Nanoparticles as Nanovaccine for Induction of Antitumor Immunity against Melanoma. ACS Nano. 2015; 28: 6918-33.
[15]. Murariu M, Dubois P. PLA composites: From production to properties. Advanced Drug Delivery Reviews. 2016; 13: pii: S0169-409X(16)30102-8.
[16]. Serra T, Planell JA, Navarro M. High-resolution PLA-based composite scaffolds via 3-D printing technology. Acta Biomaterials. 2013; 9: 5521-30.
[17]. Serra T, Mateos-Timoneda MA, Planell JA, Navarro M. 3D printed PLA-based scaffolds: a versatile tool in regenerative medicine. Organogenesis. 2013; 9:239-44.
[18]. Yalçınozan M, Mammadov E. Comparison of Custom-Made 3D Printed Bio-Degradable Plates and Titanium Anatomical Plates at Fracture Treatment. Cyprus J Med Sci. 2021; 6(4): 285-289.
[19]. Chu CR, Monosov, AZ, Amiel, D. (1995) In situ assessment of cell viability within biodegradable polylactic acid polymer matrices. Biomaterial; 16: 1381-4.
[20]. Islam A, Mammadov E, Kendirci R, Aytac E, Cetiner S, Vatansever HS.. In vitro cultivation, characterization and osteogenic differentiation of stem cells from human exfoliated deciduous teeth on 3D printed polylactic acid scaffolds. Iran Red Crescent Med J. 2017; 19 (8).
[21]. Kurt FO, Vatansever HS. Potential clinical use of differentiated cells from embryonic or Mesenchymal stem cells in orthopeadic problems. Curr Stem Cell Res Ther. 2016; 11: 522-529.
[22]. Tuğlu I, Ozdal-Kurt F, Koca H, Saraç A, Barut T. The contribution of differentiated bone marrow stromal stem cell-loaded biomaterial to treatment in critical size defect model in rats. Kafkas Üniversitesi Veteriner Fakultesi Dergisi. 2010; 16: 783-792.
[23]. Goh BS, Che Omar SN, Ubaidah MA, Saim L, Sulaiman S, Chua KH. Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique. Acta Otolaryngol. 2017; 137: 432-441.
[24]. Costantini M, Idaszek J, Szoke K, Jaroszewicz J, Dentini M, Barbetta A, Brinchmann JE, Swieszkowski W. 3D bioprinting of BM-MSCs-loaded ECM biomimetic hydrogels for in vitro neocartilage formation. Biofabrication. 2016; 8: 035002.
[25]. Fujihara Y, Takato T, Hoshi K. Immunological response to tissue-engineered cartilage derived from auricular chondrocytes and a PLLA scaffold in transgenic mice. Biomaterials. 2010; 31: 1227-34.
[26]. Behonick DJ, Xing Lieu S, Buckley JM, Lotz JC, Marcucio RS, Werb Z, Miclau T, Colnot C. Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration. PLoS One. 2007; 2: e1150.
[27]. Caplan AI. Why are MSCs therapeutic? New data: new insight. The Journal of.Pathology. 2009; 217: 318-24.
[28]. Kinoshita T, Gohara R, Koga T, Sueyasu Y, Terasaki H, Rikimaru T, Aizawa H. Visualization of pulmonary arteriovenous malformation by threedimensional computed tomography: a case report. The Kurume Medical Journal. 2003; 50: 161-3.
[29]. Fukushima K, Chang YH, Kimura Y. Enhanced stereocomplex formation of poly(L-lactic acid) and poly(D-lactic acid) in the presence of stereoblock poly(lactic acid). Macromolecular Biosciences. 2007; 7: 829-35.
[30]. Walton M, Cotton NJ. Long-term in vivo degradation of poly-L-lactide (PLLA) in bone. Journal of. Biomaterials. Applications. 2007; 21: 395-411.