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Development of Stepwise Osteogenesis-mimicking Matrices for the Regulation of Mesenchymal Stem Cell Functions

2009; Elsevier BV; Volume: 284; Issue: 45 Linguagem: Inglês

10.1074/jbc.m109.054676

1083-351X

Takashi Hoshiba, Naoki Kawazoe, Tetsuya Tateishi, Guoping Chen,

Bone and Dental Protein Studies

An extracellular microenvironment, including an extracellular matrix (ECM), is an important factor in regulating stem cell differentiation. During tissue development, the ECM is dynamically remodeled to regulate stem cell functions. Here, we developed matrices mimicking ECM remodeling during the osteogenesis of mesenchymal stem cells (MSCs). The matrices were prepared from cultured MSCs controlled at different stages of osteogenesis and referred to as "stepwise osteogenesis-mimicking matrices." The matrices supported the adhesion and proliferation of MSCs and showed different effects on the osteogenesis of MSCs. On the matrices mimicking the early stage of osteogenesis (early stage matrices), the osteogenesis occurred more rapidly than did that on the matrices mimicking undifferentiated stem cells (stem cell matrices) and the late stage of osteogenesis (late stage matrices). RUNX2 was similarly expressed when MSCs were cultured on both the early stage and late stage matrices but decreased on the stem cell matrices. PPARG expression in the MSCs cultured on the late stage matrices was higher than for those cultured on the stem cell and early stage matrices. This increase of PPARG expression was caused by the suppression of the amount of β-catenin and downstream signal transduction. These results demonstrate that the osteogenesis-mimicking matrices had different effects on the osteogenesis of MSCs, and the early stage matrices provided a favorable microenvironment for the osteogenesis. An extracellular microenvironment, including an extracellular matrix (ECM), is an important factor in regulating stem cell differentiation. During tissue development, the ECM is dynamically remodeled to regulate stem cell functions. Here, we developed matrices mimicking ECM remodeling during the osteogenesis of mesenchymal stem cells (MSCs). The matrices were prepared from cultured MSCs controlled at different stages of osteogenesis and referred to as "stepwise osteogenesis-mimicking matrices." The matrices supported the adhesion and proliferation of MSCs and showed different effects on the osteogenesis of MSCs. On the matrices mimicking the early stage of osteogenesis (early stage matrices), the osteogenesis occurred more rapidly than did that on the matrices mimicking undifferentiated stem cells (stem cell matrices) and the late stage of osteogenesis (late stage matrices). RUNX2 was similarly expressed when MSCs were cultured on both the early stage and late stage matrices but decreased on the stem cell matrices. PPARG expression in the MSCs cultured on the late stage matrices was higher than for those cultured on the stem cell and early stage matrices. This increase of PPARG expression was caused by the suppression of the amount of β-catenin and downstream signal transduction. These results demonstrate that the osteogenesis-mimicking matrices had different effects on the osteogenesis of MSCs, and the early stage matrices provided a favorable microenvironment for the osteogenesis. IntroductionStem cells pass through stepwise maturational stages for differentiation into somatic cells. During the stepwise differentiation of stem cells into somatic cells, the expression pattern of transcription factors changes, depending on their maturational stages (1D'Amour K.A. Bang A.G. Eliazer S. Kelly O.G. Agulnick A.D. Smart N.G. Moorman M.A. Kroon E. Carpenter M.K. Baetge E.E. Nat. Biotechnol. 2006; 24: 1392-1401Crossref PubMed Scopus (1510) Google Scholar). The expression of the transcription factors is regulated by the extracellular environment through the modulation of intracellular signaling. To alter the expression of the transcription factors, the extracellular microenvironment surrounding the stem cells changes through the differentiation processes. In particular, the extracellular matrix (ECM) 2The abbreviations used are: ECMextracellular matrixMSCmesenchymal stem cellDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumTCPStissue culture polystyrenePBSphosphate-buffered salineBSAbovine serum albuminCBBCoomassie Brilliant Blue. is dynamically remodeled to activate the intracellular signaling to regulate the differentiation of stem cells into somatic cells (2Daley W.P. Peters S.B. Larsen M. J. Cell Sci. 2008; 121: 255-264Crossref PubMed Scopus (735) Google Scholar, 3Page-McCaw A. Ewald A.J. Werb Z. Nat. Rev. Mol. Cell. Biol. 2007; 8: 221-233Crossref PubMed Scopus (2147) Google Scholar).Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, adipocytes, and osteoblasts (4Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (17775) Google Scholar). When osteoblasts originate from MSCs, the stepwise maturational stages are passed. At the early stage of MSC osteogenesis, the cells express RUNX2 (also known as CBFA1), an osteogenic transcription factor, and the genes controlled by RUNX2, such as ALP (liver/bone/kidney alkaline phosphatase) and SPP1 (osteopontin) (5Malaval L. Modrowski D. Gupta A.K. Aubin J.E. J. Cell. Physiol. 1994; 158: 555-572Crossref PubMed Scopus (322) Google Scholar, 6Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3605) Google Scholar). However, at the early stage of osteogenesis, the cells cannot deposit calcium to form mineralized bone. In order to deposit calcium, the cells must enter the late stage of osteogenesis and express SP7 (also known as osterix), whose expression is regulated by RUNX2 (7Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2733) Google Scholar, 8Nishio Y. Dong Y. Paris M. O'Keefe R.J. Schwarz E.M. Drissi H. Gene. 2006; 372: 62-70Crossref PubMed Scopus (272) Google Scholar), and IBSP (bone sialoprotein 2), whose expression is controlled by Sp7 (7Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2733) Google Scholar). During the osteogenesis processes of MSCs in vivo, the ECM surrounding the cells is dynamically remodeled (9Nakamura M. Sone S. Takahashi I. Mizoguchi I. Echigo S. Sasano Y. J. Histochem. Cytochem. 2005; 53: 1553-1562Crossref PubMed Scopus (60) Google Scholar, 10Sasano Y. Li H.C. Zhu J.X Imanaka-Yoshida K. Mizoguchi I. Kagayama M. Histochem. J. 2000; 32: 591-598Crossref PubMed Scopus (33) Google Scholar, 11Kamiya N. Shigemasa K. Takagi M. J. Oral Sci. 2001; 43: 179-188Crossref PubMed Scopus (30) Google Scholar). Fibronectin and versican are observed in the region of mesenchymal condensation (early stage of osteogenesis) and then disappear in mature mineralized bone (9Nakamura M. Sone S. Takahashi I. Mizoguchi I. Echigo S. Sasano Y. J. Histochem. Cytochem. 2005; 53: 1553-1562Crossref PubMed Scopus (60) Google Scholar, 10Sasano Y. Li H.C. Zhu J.X Imanaka-Yoshida K. Mizoguchi I. Kagayama M. Histochem. J. 2000; 32: 591-598Crossref PubMed Scopus (33) Google Scholar). Decorin is present in unmineralized bone matrix but disappears in mineralized bone (11Kamiya N. Shigemasa K. Takagi M. J. Oral Sci. 2001; 43: 179-188Crossref PubMed Scopus (30) Google Scholar). Biglycan is strongly detected in bone marrow surrounding MSCs but not in unmineralized and mineralized bone matrices (11Kamiya N. Shigemasa K. Takagi M. J. Oral Sci. 2001; 43: 179-188Crossref PubMed Scopus (30) Google Scholar). Similar to in vivo ECM remodeling, the ECM gene expression pattern also changes during the osteogenesis of MSCs in vitro (12Pham Q.P. Kasper F.K. Scott Baggett L. Raphael R.M. Jansen J.A. Mikos A.G. Biomaterials. 2008; 29: 2729-2739Crossref PubMed Scopus (134) Google Scholar). The effects of these ECM proteins on osteogenesis have been studied in vivo and in vitro using gene-deficient animals and cells, gene-overexpressed cells, and surfaces coated with isolated ECM proteins (13Xu T. Bianco P. Fisher L.W. Longenecker G. Smith E. Goldstein S. Bonadio J. Boskey A. Heegaard A.M. Sommer B. Satomura K. Dominguez P. Zhao C. Kulkarni A.B. Robey P.G. Young M.F. Nat. Genet. 1998; 20: 78-82Crossref PubMed Scopus (386) Google Scholar, 14Chen X.D. Shi S. Xu T. Robey P.G. Young M.F. J. Bone Miner. Res. 2002; 17: 331-340Crossref PubMed Scopus (130) Google Scholar, 15Gutierrez J. Osses N. Brandan E. J. Cell. Physiol. 2006; 206: 58-67Crossref PubMed Scopus (37) Google Scholar, 16Riquelme C. Larrain J. Schonherr E. Henriquez J.P. Kresse H. Brandan E. J. Biol. Chem. 2001; 276: 3589-3596Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Moursi A.M. Damsky C.H. Lull J. Zimmerman D. Doty S.B. Aota S. Globus R.K. J. Cell Sci. 1996; 109: 1369-1380Crossref PubMed Google Scholar). However, ECM is formed from complex components to regulate proper cell functioning in vivo (18Adachi E. Hopkinson I. Hayashi T. Int. Rev. Cytol. 1997; 173: 73-156Crossref PubMed Google Scholar). It is expected that the matrices that formed from components similar to in vivo components will give us more precise and detailed insights into the role of ECM remodeling in the osteogenesis of MSCs.There are many reports of the development of acellular matrices from tissues using various decellularization treatments (19Gilbert T.W. Sellaro T.L. Badylak S.F. Biomaterials. 2006; 27: 3675-3683Crossref PubMed Scopus (1689) Google Scholar, 20Ott H.C. Matthiesen T.S. Goh S.K. Black L.D. Kren S.M. Netoff T.I. Taylor D.A. Nat. Med. 2008; 14: 213-221Crossref PubMed Scopus (2047) Google Scholar). In acellular matrices, it is difficult to identify and isolate the matrices at the different maturational stages of stem cells. In contrast, the cells cultured in vitro can secrete ECM proteins and deposit them beneath the cells. Similar to the acellular matrices from the tissue, these deposited ECM proteins can be used as matrices after decellularization (21Datta N. Holtorf H.L. Sikavitsas V.I. Jansen J.A. Mikos A.G. Biomaterials. 2005; 26: 971-977Crossref PubMed Scopus (248) Google Scholar, 22Datta N. Pham Q.P. Sharma U. Sikavitsas V.I. Jansen J.A. Mikos A.G. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2488-2493Crossref PubMed Scopus (346) Google Scholar, 23Chen X.D. Dusevich V. Feng J.Q. Manolagas S.C. Jilka R.L. J. Bone Miner. Res. 2007; 22: 1943-1956Crossref PubMed Scopus (282) Google Scholar). Mikos and co-workers (21Datta N. Holtorf H.L. Sikavitsas V.I. Jansen J.A. Mikos A.G. Biomaterials. 2005; 26: 971-977Crossref PubMed Scopus (248) Google Scholar, 22Datta N. Pham Q.P. Sharma U. Sikavitsas V.I. Jansen J.A. Mikos A.G. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2488-2493Crossref PubMed Scopus (346) Google Scholar) made a matrix from cultured MSC-derived osteoblasts in vitro. This matrix enhanced the osteogenesis of MSCs compared with a Ti surface (21Datta N. Holtorf H.L. Sikavitsas V.I. Jansen J.A. Mikos A.G. Biomaterials. 2005; 26: 971-977Crossref PubMed Scopus (248) Google Scholar). Chen et al. (23Chen X.D. Dusevich V. Feng J.Q. Manolagas S.C. Jilka R.L. J. Bone Miner. Res. 2007; 22: 1943-1956Crossref PubMed Scopus (282) Google Scholar) reported an undifferentiated MSC-derived matrix in vitro and also reported that this matrix suppressed the spontaneous differentiation of MSCs during culture. Therefore, it seems that the matrices formed by cultured cells can serve as an alternative to the acellular matrices of tissue.In this study, we developed matrices mimicking in vivo ECM remodeling during the osteogenesis of human MSCs. To make the matrices mimicking in vivo ECM remodeling during the osteogenesis of MSCs, we prepared the matrices from cultured MSCs controlled at different stages of osteogenesis and referred to them as "stepwise osteogenesis-mimicking matrices." In addition, the effects of stepwise osteogenesis-mimicking matrices on MSC functions, such as proliferation and osteogenesis, were also evaluated. IntroductionStem cells pass through stepwise maturational stages for differentiation into somatic cells. During the stepwise differentiation of stem cells into somatic cells, the expression pattern of transcription factors changes, depending on their maturational stages (1D'Amour K.A. Bang A.G. Eliazer S. Kelly O.G. Agulnick A.D. Smart N.G. Moorman M.A. Kroon E. Carpenter M.K. Baetge E.E. Nat. Biotechnol. 2006; 24: 1392-1401Crossref PubMed Scopus (1510) Google Scholar). The expression of the transcription factors is regulated by the extracellular environment through the modulation of intracellular signaling. To alter the expression of the transcription factors, the extracellular microenvironment surrounding the stem cells changes through the differentiation processes. In particular, the extracellular matrix (ECM) 2The abbreviations used are: ECMextracellular matrixMSCmesenchymal stem cellDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumTCPStissue culture polystyrenePBSphosphate-buffered salineBSAbovine serum albuminCBBCoomassie Brilliant Blue. is dynamically remodeled to activate the intracellular signaling to regulate the differentiation of stem cells into somatic cells (2Daley W.P. Peters S.B. Larsen M. J. Cell Sci. 2008; 121: 255-264Crossref PubMed Scopus (735) Google Scholar, 3Page-McCaw A. Ewald A.J. Werb Z. Nat. Rev. Mol. Cell. Biol. 2007; 8: 221-233Crossref PubMed Scopus (2147) Google Scholar).Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, adipocytes, and osteoblasts (4Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (17775) Google Scholar). When osteoblasts originate from MSCs, the stepwise maturational stages are passed. At the early stage of MSC osteogenesis, the cells express RUNX2 (also known as CBFA1), an osteogenic transcription factor, and the genes controlled by RUNX2, such as ALP (liver/bone/kidney alkaline phosphatase) and SPP1 (osteopontin) (5Malaval L. Modrowski D. Gupta A.K. Aubin J.E. J. Cell. Physiol. 1994; 158: 555-572Crossref PubMed Scopus (322) Google Scholar, 6Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3605) Google Scholar). However, at the early stage of osteogenesis, the cells cannot deposit calcium to form mineralized bone. In order to deposit calcium, the cells must enter the late stage of osteogenesis and express SP7 (also known as osterix), whose expression is regulated by RUNX2 (7Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2733) Google Scholar, 8Nishio Y. Dong Y. Paris M. O'Keefe R.J. Schwarz E.M. Drissi H. Gene. 2006; 372: 62-70Crossref PubMed Scopus (272) Google Scholar), and IBSP (bone sialoprotein 2), whose expression is controlled by Sp7 (7Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2733) Google Scholar). During the osteogenesis processes of MSCs in vivo, the ECM surrounding the cells is dynamically remodeled (9Nakamura M. Sone S. Takahashi I. Mizoguchi I. Echigo S. Sasano Y. J. Histochem. Cytochem. 2005; 53: 1553-1562Crossref PubMed Scopus (60) Google Scholar, 10Sasano Y. Li H.C. Zhu J.X Imanaka-Yoshida K. Mizoguchi I. Kagayama M. Histochem. J. 2000; 32: 591-598Crossref PubMed Scopus (33) Google Scholar, 11Kamiya N. Shigemasa K. Takagi M. J. Oral Sci. 2001; 43: 179-188Crossref PubMed Scopus (30) Google Scholar). Fibronectin and versican are observed in the region of mesenchymal condensation (early stage of osteogenesis) and then disappear in mature mineralized bone (9Nakamura M. Sone S. Takahashi I. Mizoguchi I. Echigo S. Sasano Y. J. Histochem. Cytochem. 2005; 53: 1553-1562Crossref PubMed Scopus (60) Google Scholar, 10Sasano Y. Li H.C. Zhu J.X Imanaka-Yoshida K. Mizoguchi I. Kagayama M. Histochem. J. 2000; 32: 591-598Crossref PubMed Scopus (33) Google Scholar). Decorin is present in unmineralized bone matrix but disappears in mineralized bone (11Kamiya N. Shigemasa K. Takagi M. J. Oral Sci. 2001; 43: 179-188Crossref PubMed Scopus (30) Google Scholar). Biglycan is strongly detected in bone marrow surrounding MSCs but not in unmineralized and mineralized bone matrices (11Kamiya N. Shigemasa K. Takagi M. J. Oral Sci. 2001; 43: 179-188Crossref PubMed Scopus (30) Google Scholar). Similar to in vivo ECM remodeling, the ECM gene expression pattern also changes during the osteogenesis of MSCs in vitro (12Pham Q.P. Kasper F.K. Scott Baggett L. Raphael R.M. Jansen J.A. Mikos A.G. Biomaterials. 2008; 29: 2729-2739Crossref PubMed Scopus (134) Google Scholar). The effects of these ECM proteins on osteogenesis have been studied in vivo and in vitro using gene-deficient animals and cells, gene-overexpressed cells, and surfaces coated with isolated ECM proteins (13Xu T. Bianco P. Fisher L.W. Longenecker G. Smith E. Goldstein S. Bonadio J. Boskey A. Heegaard A.M. Sommer B. Satomura K. Dominguez P. Zhao C. Kulkarni A.B. Robey P.G. Young M.F. Nat. Genet. 1998; 20: 78-82Crossref PubMed Scopus (386) Google Scholar, 14Chen X.D. Shi S. Xu T. Robey P.G. Young M.F. J. Bone Miner. Res. 2002; 17: 331-340Crossref PubMed Scopus (130) Google Scholar, 15Gutierrez J. Osses N. Brandan E. J. Cell. Physiol. 2006; 206: 58-67Crossref PubMed Scopus (37) Google Scholar, 16Riquelme C. Larrain J. Schonherr E. Henriquez J.P. Kresse H. Brandan E. J. Biol. Chem. 2001; 276: 3589-3596Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Moursi A.M. Damsky C.H. Lull J. Zimmerman D. Doty S.B. Aota S. Globus R.K. J. Cell Sci. 1996; 109: 1369-1380Crossref PubMed Google Scholar). However, ECM is formed from complex components to regulate proper cell functioning in vivo (18Adachi E. Hopkinson I. Hayashi T. Int. Rev. Cytol. 1997; 173: 73-156Crossref PubMed Google Scholar). It is expected that the matrices that formed from components similar to in vivo components will give us more precise and detailed insights into the role of ECM remodeling in the osteogenesis of MSCs.There are many reports of the development of acellular matrices from tissues using various decellularization treatments (19Gilbert T.W. Sellaro T.L. Badylak S.F. Biomaterials. 2006; 27: 3675-3683Crossref PubMed Scopus (1689) Google Scholar, 20Ott H.C. Matthiesen T.S. Goh S.K. Black L.D. Kren S.M. Netoff T.I. Taylor D.A. Nat. Med. 2008; 14: 213-221Crossref PubMed Scopus (2047) Google Scholar). In acellular matrices, it is difficult to identify and isolate the matrices at the different maturational stages of stem cells. In contrast, the cells cultured in vitro can secrete ECM proteins and deposit them beneath the cells. Similar to the acellular matrices from the tissue, these deposited ECM proteins can be used as matrices after decellularization (21Datta N. Holtorf H.L. Sikavitsas V.I. Jansen J.A. Mikos A.G. Biomaterials. 2005; 26: 971-977Crossref PubMed Scopus (248) Google Scholar, 22Datta N. Pham Q.P. Sharma U. Sikavitsas V.I. Jansen J.A. Mikos A.G. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2488-2493Crossref PubMed Scopus (346) Google Scholar, 23Chen X.D. Dusevich V. Feng J.Q. Manolagas S.C. Jilka R.L. J. Bone Miner. Res. 2007; 22: 1943-1956Crossref PubMed Scopus (282) Google Scholar). Mikos and co-workers (21Datta N. Holtorf H.L. Sikavitsas V.I. Jansen J.A. Mikos A.G. Biomaterials. 2005; 26: 971-977Crossref PubMed Scopus (248) Google Scholar, 22Datta N. Pham Q.P. Sharma U. Sikavitsas V.I. Jansen J.A. Mikos A.G. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2488-2493Crossref PubMed Scopus (346) Google Scholar) made a matrix from cultured MSC-derived osteoblasts in vitro. This matrix enhanced the osteogenesis of MSCs compared with a Ti surface (21Datta N. Holtorf H.L. Sikavitsas V.I. Jansen J.A. Mikos A.G. Biomaterials. 2005; 26: 971-977Crossref PubMed Scopus (248) Google Scholar). Chen et al. (23Chen X.D. Dusevich V. Feng J.Q. Manolagas S.C. Jilka R.L. J. Bone Miner. Res. 2007; 22: 1943-1956Crossref PubMed Scopus (282) Google Scholar) reported an undifferentiated MSC-derived matrix in vitro and also reported that this matrix suppressed the spontaneous differentiation of MSCs during culture. Therefore, it seems that the matrices formed by cultured cells can serve as an alternative to the acellular matrices of tissue.In this study, we developed matrices mimicking in vivo ECM remodeling during the osteogenesis of human MSCs. To make the matrices mimicking in vivo ECM remodeling during the osteogenesis of MSCs, we prepared the matrices from cultured MSCs controlled at different stages of osteogenesis and referred to them as "stepwise osteogenesis-mimicking matrices." In addition, the effects of stepwise osteogenesis-mimicking matrices on MSC functions, such as proliferation and osteogenesis, were also evaluated.