• Jin Jeon1, Min Suk Lee1 and Hee Seok Yang1,2*

Differentiated osteoblasts derived decellularized extracellular matrix topromote osteogenic differe

2020년 9월 22일 업데이트됨

Abstract

The extracellular matrix (ECM) can directly or indirectly influence on regulation of cell functions such as cell adhesion, migration, proliferation and differentiation. The cell derived ECM (CD ECM) is a useful in vitro model for studying the comprehensive functions of CD-ECM because it maintains a native-like structure and composition. In this study, the CD-ECM is obtained and a test is carried out to determine the effectiveness of several combinations of decellularized methods. These methods were used to regulate the optimal ECM compositions to be induced by osteogenic differentiation using primary isolated osteoblasts.

Background Extracellular matrix (ECM) is specialized architecture composed of extracellular proteins which are known to interact with various cells and influence the regulation of cell behaviors such as cell adhesion, migration, proliferation and differentiation [1–3]. ECM is composed of diverse molecules such as collagen, fibronectin and other proteins that are interlaced with proteoglycans [4]. The composition and structure of ECM can be changed by the phenotype of the resident cells and the function of the tissues or organs. In turn, the ECM can affect the phenotype and behavior of the resident cells [5–7]. Moreover, the ECM can modulate the signal transduction activated by various bioactive molecules, such as growth factors and cytokines [8]. The ECM from tissues or whole organs has been studied as biomaterials which comprises intestinal submucosa, heart valve, blood vessel, skin, nerve, tendon, ligament, urinary bladder, vocal fold, amniotic membrane, heart, liver, and lung in tissue engineering and regenerative medicine [9–16]. Tissue-derived decellularized ECM (TD-ECM) obtained from tissues have properties that preserve the structures of their respective tissues. However, they may have several problems such as tissue scarcity, host responses, and pathogen transfer [17–19].Recently to address these problems, many studies havebeen carried out using ECM derived from culturedcells. Cell-derived ECM (CD-ECM) from culturedcells has several advantages over TD- ECM. In theCD-ECM, it is easy to eliminate pathogen transferand maintain pathogen-free condition. The CD-ECMalso provides the desired geometry and porosity withoutthe limitation of poor cell penetration. Moreover,the CD-ECM can be derived from autologous cells tomake autologous CD-ECM scaffolds [20, 21].The CD-ECM contains specific molecules secreted bythe cells as well as growth serum proteins during proliferation.The composition of CD-ECM molecules canchange according to the differentiation medium composition.Thus, our approach involves the development ofmore osteoinductive culture conditions that impact theability of differentiated CD-ECMs to induce re seededcell functions. Osteogenic differentiated ECM (ODECM)is used to produce collagen type-I, fibronectin,biglycan and decorin. The collagen type-I can not onlyupregulated alkaline phosphatase (ALP) and osteopontin(OPN), but the decorin and biglycan also influence differentiationof osteoblasts [22–26]. In addition to thepreparation of OD-ECM, hydroxyapatite (HA) has beendeposited during osteoblasts maturation. The developmentof OD-ECM involved osteoconductive HA of nativecomponents which induced cellular differentiation.The ALP activity and messenger RNA levels of osteoblastscultured in HA surface were increased at the earlystage of osteogenic differentiation, and osteocalcin expressionwas also increased at the late stage [27, 28].The aim of this study was to investigate the effect of differentcompositions of OD-ECM by different stages ofosteogenesis. We cultured confluent osteoblasts on a tissueculture plate. The osteoblasts were then decellularizedafter treatment of differentiate medium to prepare for thedifferent stages of OD-ECM. They were treated during 2and 4 weeks. We investigated the re-seeded osteoblasteffect on different compositions of ECM as followed: GMECM(normal osteoblast ECM), OD-ECM-2 W (osteogenicdifferentiated ECM during 2 weeks culture), andOD-ECM-4 W (osteogenic differentiated ECM during4 weeks culture). Differentiation and maturation of reseededosteoblasts were determined by analysis of theknown indicators of the osteoblast phenotype, calcification,mineralization, and protein activity under a growthmedium and osteogenic differentiation medium.

Keywords

Cell-derived extracellular matrix, Decellularization, Osteogenic differentiation

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