Abstract
In this study, the osteoinductive and cell-binding properties of three different resorbable polymers were evaluated by human mesenchymal stem cells (MSCs). MSCs were isolated, expanded, and cultivated onto resorbable D,D,L,L-polylactide (PLLA), collagen I/III, and polygalactin-910/polydioxanone (PGPD) scaffolds in vitro. To evaluate the influence of dexamethasone, ascorbic acid, and β-glycerolphosphate (DAG) on osteoblast differentiation, MSCs were incubated in a DAG-enriched medium. After a 28-day period in vitro, the cellular loaded polymers were digested enzymatically by papain and HCl. The Ca2+ content of the biomembranes was evaluated by an o-kresolphthalein-complexon reaction via photometer. A PicoGreen® assay was performed for dsDNA quantification. Significant differences between the number of adherent MSCs were documented (collagen > PLLA > PGPD). Compared to the initial number of adherent cells, all biomaterials induced a significant decrease in cellular adherence after 28 days in vitro. The presence of DAG-enriched culture medium stimulated the cellular proliferation for PLLA and slightly for PGPD, whereas cell proliferation was inhibited when MSCs were cultivated onto collagen I/III. In comparison with the control groups, all biomaterials (PLLA, PGPD, and collagen I/III) showed a significant increase in local Ca2+ accumulation under DAG stimulation after 28 days in vitro. Furthermore, collagen I/III and PLLA scaffolds showed osteoinductive properties without DAG stimulation. These results were verified by immunocytochemical stainings against osteoblast-typical markers (osteopontin and alkaline phosphatase) and completed by calcified matrix detection (von Kossa staining). MSCs were identified by CD105 and CD13 antigen expression. Corresponding to an absence of CD34, CD45, and collagen II expression, we found no chondrogenic or hematopoietic cell differentiation. The results indicate significant differences for the proliferation, differentiation, adherence, and Ca2+ accumulation between the tested polymers in a MSC culture.
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References
Al-Salihi, K. A., and A. R. Samsudin. Bone marrow mesenchymal stem cells differentiation and proliferation on the surface of coral implant. Med. J. Malaysia 59 (Suppl. B):45–46, 2004.
Alpar, B., G. Leyhausen, H. Gunay, and W. Geurtsen. Compatibility of resorbable and nonresorbable guided tissue regeneration membranes in cultures of primary human periodontal ligament fibroblasts and human osteoblast-like cells. Clin. Oral. Investig. 4(4):219–225, 2000.
Ambrose, C. G., and T. O. Claton. Bioabsorbable implants: Review of clinical experience in orthopaedic surgery. Ann. Biomed. Eng. 32(1):171–177, 2004.
Anselme, K. Osteoblast adhesion on biomaterials. Biomaterials 21:667–681, 2000.
Atmani, H., D. Chappard, and M. F. Basle. Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: Effects of dexamethasone and calcitriol. J. Cell Biochem. 89(2):364–372, 2003.
Barbolt, T. A., M. Odin, M. Leger, L. Kangas, J. Hoiste, and S. H. Liu. Biocompatibility evaluation of duramater substitutes in an animal model. Neurol. Res. 23(8):813–820, 2001.
Bielby, R. C., A. R. Boccaccini, J. M. Polak, and L. D. Buttery. In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells. Tissue Eng. 10(9–10):1518–1525, 2004.
Blaheta, R. A., B. Kronenberger, D. Woitaschek, S. Weber, M. Scholz, H. Schuldes, A. Encke, and B. H. Markus. Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity. J. Immunol. Methods 211(1–2):159–169, 1998.
Blaker, J. J., J. E. Gough, V. Maquet, I. Notingher, and A. R. Boccaccini. In vitro evaluation of novel bioactive composites based on bioglass-filled polylactide foams for bone tissue engineering scaffolds. J. Biomed. Mater. Res. 67A(4):1401–1411, 2003.
Blum, B., J. Moseley, L. Miller, K. Richelsoph, and W. Haggard. Measurement of bone morphogenetic proteins and other growth factors in demineralized bone matrix. Orthopedics 27(1 Suppl.):s161–s165, 2004.
Boden, S. D. Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute. Spine 27(16 Suppl. 1):S26–S31, 2002.
Borden, M., M. Attawia, and C. T. Laurencin. The sintered microsphere matrix for bone tissue engineering: In vitro osteoconductivity studies. J. Biomed. Mater. Res. 61:421–429, 2002.
Burridge, A., L. Molony, and L. Kelly. Adhesion plaques: Sites of transmembrane interaction between the extracellular matrix and the actin cytoskeleton. J. Cell Sci. 8:211–229, 1987.
Cartmell, S., K. Huynh, A. Lin, S. Nagaraja, and R. Guldberg. Quantitative microcomputed tomography analysis of mineralization within three-dimensional scaffolds in vitro. J. Biomed. Mater. Res. 69A: 97–104, 2004.
Claes, L., and A. Ignatius. Development of new, biodegradable implants. Chirurg 73(10):990–996, 2002.
Cornet, F., O. Broux, K. Anselme, P. Hardouin, and J. Jeanfils. Effect of dexamethasone on moesin gene expression in rabbit bone marrow stromal cells. Mol. Cell Biochem. 265(1–2):79–83, 2004.
Corsi, K., F. Chellat, L. Yahia, and J. C. Fernandes. Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles. Biomaterials 24(7):1255–1264, 2003.
Datta, N., H. L. Holtorf, V. I. Sikavitsas, J. A. Jansen, and A. G. Mikos. Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells. Biomaterials 26(9):971–977, 2005.
Declercq, H. A., R. M. Verbeeck, L. I. De Ridder, E. H. Schacht, and M. J. Cornelissen. Calcification as an indicator of osteoinductive capacity of biomaterials in osteoblastic cell cultures. Biomaterials 26(24):4964–4674, 2005.
Giavaresi, G., M. Tschon, V. Borsari, J. H. Daly, J. J. Liggat, M. Fini, V. Bonazzi, A. Nicolini, A. Carpi, M. Morra, C. Cassinelli, and R. Giardino. New polymers for drug delivery systems in orthopaedics: in vivo biocompatibility evaluation. Biomed. Pharmacother. 58(8):411–417, 2004.
Gindler, E. M., and J. D. King. Rapid colorimetric determination of calcium in biologic fluids with methylthymol blue. Am. J. Clin. Pathol. 58(4):376–382, 1972.
Glowacki, J. A review of osteoinductive testing methods and sterilization processes for demineralized bone. Cell Tissue Bank 6(1):3–12, 2005.
Gröger, A., S. Kläring, H. A. Merten, J. Holste, C. Kaps, and M. Sittinger. Tissue engineering of bone for mandibular augmentation in immunocompetent minipigs: Preliminary study. Scand. J. Plast. Reconstr. Surg. Hand Surg. 39:129–133, 2003.
Gugala, Z., and S. Gogolewski. Protein adsorption, attachment, growth and activity of primary rat osteoblasts on polylactide membranes with defined surface characteristics. Biomaterials 25(12):2341–2351, 2004.
Gunatillake, P. A., and R. Adhikari. Biodegradable synthetic polymers for tissue engineering. Eur. Cell Mater. 5:1–16, 2003.
Habibovic, P., H. Yuan, C. M. van der Valk, G. Meijer, C. A. van Blitterswijk, and K. de Groot. 3D microenvironment as essential element for osteoinduction by biomaterials. Biomaterials 26(17):3565–3575, 2005.
Han, B., B. Tang, and M. E. Nimni. Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix. J. Orthop. Res. 21(4):648–654, 2003.
Henderson, I. J., B. Tuy, D. Connell, B. Oakes, and W. H. Hettwer. Prospective clinical study of autologous chondrocyte implantation and correlation with MRI at three and 12 months. J. Bone Joint Surg. Br. 2003;85(7):1060–1066.
Hillmann, G., A. Steinkamp-Zucht, W. Geurtsen, G. Gross, and A. Hoffmann. Culture of primary human gingival fibroblasts on biodegradable membranes. Biomaterials 23(6):1461–1469, 2002.
Hu, Y., S. R. Winn, I. Krajbich, and J. O. Hollinger. Porous polymer scaffolds surface-modified with arginine-glycine-aspartic acid enhance bone cell attachment and differentiation in vitro. J. Biomed. Mater. Res. 64A:583–590, 2003.
Igarashi, M., N. Kamiya, M. Hasegawa, T. Kasuya, T. Takahashi, and M. Takagi. Inductive effects of dexamethasone on the gene expression of Cbfa1, Osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts. J. Mol. Histol. 35(1):3–10, 2004.
Ignatius, A. A., and L. E. Claes. In vitro biocompatibility of bioresorbable polymers: Poly(L, DL-lacide) and poly(L-lactide-co-glycolide). Biomaterials 17:831–839, 1996.
Iu, M. F., H. Kaji, H. Sowa, J. Naito, T. Sugimoto, and K. Chihara. Dexamethasone suppresses Smad3 pathway in osteoblastic cells. J. Endocrinol. 185(1):131–138, 2005.
Jäger, M., and A. Wilke. Comprehensive Biocompatibility Testing of a New PMMA Bone Cement versus conventional PMMA Cement in vitro. J. Biomat. Sci. Polym. Ed. 14(11):1283–1298, 2003.
Jäger, M., A. Wild, S. Lensing-Höhn, and R. Krauspe. Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells. Biomed. Tech. (Berl.). 48(9):241–244, 2003.
Jäger, M., M. Sager, A. Knipper, Ö. Degistirici, J. Fischer, G. Kögler, P. Wernet, and R. Krauspe. in vivo and in vitro bone regeneration from cord blood derived mesenchymal stem cells. Orthopäde 33(12):1361–1372, 2004.
Jäger, M., A. Schultheis, B. Westhoff, and R. Krauspe. Osteogenic progenitor cell potency after high-dose chemotherapy (COSS-96). Anticancer Res. 25(2A):947–954, 2005.
Knowles, J. C. Development of a natural degradable polymer for orthopaedic use. J. Med. Eng. Technol. 17(4):129–137, 1993.
Kudelska-Mazur, D., M. Lewandowska-Szumiel, M. Mazur, and J Komender. Osteogenic cell contact with biomaterials influences phenotype expression. Cell Tissue Bank 6(1):55–64, 2005.
Leclerc, N., T. Noh, A. Khokhar, E. Smith, and B. Frenkel. Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20-binding enhancer. Arthritis Rheum. 52(3):929–939, 2005.
Li, X., L. Jin, Q. Cui, G. J. Wang, and G. Balian. Steroid effects on osteogenesis through mesenchymal cell gene expression. Osteoporos. Int. 16(1):101–108, 2005.
Liu, G., Y. Y. Hu, J. N. Zhao, S. J. Wu, Z. Xiong, and R. Lu. Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells. Chin. J. Traumatol. 7(6):358–362, 2004.
Livingston, T. L., S. Gordon, M. Archambault, S. Kadiyala, K. McIntosh, A. Smith, and S. J. Peter. Mesenchymal stem cells combined with biphasic calcium phosphate ceramics promote bone regeneration. J. Mater. Sci. Mater. Med. 14(3):211–218, 2003.
McGowan, K. B., M. S. Kurtis, L. M. Lottman, D. Watson, and R. L. Sah. Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258. Osteoarthritis Cartilage 10(7):580–587, 2002.
Moreira, P. L., Y. H. An, A. R. Santos Jr., and S. C. Genari. In vitro analysis of anionic collagen scaffolds for bone repair. J. Biomed. Mater. Res. 71B(2):229–237, 2004.
Nakamura, O., and A. I. Caplan. Noncollagenous matrix protein-enhanced mineral deposition in osteoblast-like cell culture. J. Bone Miner. Metab. 12:17–25, 1994.
Nguyen, C. A., E. Allemann, G. Schwach, E. Doelker, and R. Gurny. Cell interaction studies of PLA-MePEG nanoparticles. Int. J. Pharmacol. 254:69–72, 2003.
Ogston, N., A. J. Harrison, H. F. Cheung, B. A. Ashton, and G. Hampson. Dexamethasone and retinoic acid differentially regulate growth and differentiation in an immortalised human clonal bone marrow stromal cell line with osteoblastic characteristics. Steroids 67(11):895–906, 2002.
Ohnaka, K., M. Tanabe, H. Kawate, H. Nawata, and R. Takayanagi. Glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts. Biochem. Biophys. Res. Commun. 329(1):177–181, 2005.
Otto, T. E., J. K. Nulend, P. Patka, E. H. Burger, and H. J. Haarman. Effect of (poly)-L-lactic acid on the proliferation and differentiation of primary bone cells in vitro. J. Biomed. Mater. Res. 32(4):513–518, 1996.
Perka, C., O. Schultz, R. S. Spitzer, K. Lindenhayn, G. R. Burmester, and M. Sittinger. Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits. Biomaterials 21:1145–1153, 2000.
Pittenger, M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simoneti, S. Craig, and D. R. Marshak. Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147, 1999.
Porter, R. M., W. R. Huckle, and A. S. Goldstein. Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells. J. Cell Biochem. 90(1):13–22, 2003.
Rezania, A., and K. E. Healy. Integrin subunits responsible for adhesion of human osteoblast-like cells to biomimetic peptide surfaces. J. Orthop. Res. 17:615–623, 1999.
Rocha, L. B., G. Goissis, and M. A. Rossi. Biocompatibility of anionic collagen matrix as scaffold for bone healing. Biomaterials 23(2):449–456, 2002.
Rodriguez, J. P., M. Gonzalez, S. Rios, and V. Cambiazo. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J. Cell Biochem. 93(4):721–731, 2004.
Ronco, P., M. Antoine, B. Baudouin, M. Geniteau-Legendre, B. Lelongt, F. Chatelet, P. Verroust, and A. Vandewalle. Polarized membrane expression of brush-border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone. J. Cell Physiol. 145(2):222–237, 1990.
Rothamel, D., F. Schwarz, A. Sculean, M. Herten, W. Scherbaum, and J. Becker. Biocompatibility of various collagen membranes in cultures of human PDL fibroblasts and human osteoblast-like cells. Clin. Oral Implants Res. 15(4):443–449, 2004.
Shikinami, Y., M. Okuno. Bioresorbable devices made of forged composites of hydroxyapatite (HA) particles and poly-l-lactide (PLLA): Part I. Basic characteristics. Biomaterials 20:859–877, 1999.
Shur, I., R. Socher, and D. Benayahu. Dexamethasone regulation of cFos mRNA in osteoprogenitors. J. Cell Physiol. 202(1):240–245, 2005.
Singer, V. L., L. J. Jones, S. T. Yue, and R. P. Haugland. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal. Biochem. 249(2):228–238, 1997.
Soost, F., S. Koch, C. Stoll, H. Amthauer, C. Grosse-Siestrup, and P. Zorn. Validation of bone conversion in osteoconductive and osteoinductive bone substitutes. Cell Tissue Bank 2(2):77–86, 2001.
Sorrell, J. M., L. Brinon, M. A. Baber, and A. I. Caplan. Cytokines and glucocorticoids differentially regulate APN/CD13 and DPPIV/CD26 enzyme activities in cultured human dermal fibroblasts. Arch. Dermatol. Res. 295(4):160–168, 2003.
Stemberg, F., and A. Wilke. Evaluation of bioresorbable polymers of lactic acid in a culture of human bone marrow cells. J. Biomater. Sci. Polym. Ed. 12(2):171–184, 2001.
Takata, T., H. L. Wang, and M. Miyauchi. Migration of osteoblastic cells on various guided bone regeneration membranes. Clin. Oral Implants Res. 12(4):332–338, 2001.
Tanaka, N., K. Hirose, H. Sakahashi, T. Ishima, and S. Ishii. Usefulness of bioabsorbable thread pins after resection arthroplasty for rheumatoid forefoot reconstruction. Foot Ankle Int. 25(7):496–502, 2004.
Tangada, S. D., R. D. Peterson, and J. D. Funkhouser. Regulation of expression of aminopeptidase N in fetal rat lung by dexamethasone and epidermal growth factor. Biochim. Biophys. Acta 1268(2):191–199, 1995.
Uchimura, E., H. Machida, N. Kotobuki, T. Tihara, S. Kitamusa, M. Ikeuchi, M. Hirose, J. Miyake, and H. Ohgushi. In-situ visualization and quantification of mineralization of cultured osteogenetic cells. Calc. Tissue Int. 73(6):575–583, 2003.
Uzan, B., M. C. de Vernejoul, and M. Cressent. RAMPs and CRLR expressions in osteoblastic cells after dexamethasone treatment. Biochem. Biophys. Res. Commun. 3;321(4):802–808, 2004.
Vinogradov, A. E. Genome size and GC-percent in vertebrates as determined by flow cytometry: The triangular relationship. Cytometry 31(2):100–109, 1998.
Walsh, S., G. R. Jordan, C. Jefferiss, K. Stewart, and J. N. Beresford. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: Relevance to glucocorticoid-induced osteoporosis. Rheumatology (Oxford) 40(1):74–83, 2001.
Wang, H. L., M. Miyauchi, and T. Takata. Initial attachment of osteoblasts to various guided bone regeneration membranes: An in vitro study. J. Peridont. Res. 37:340–355, 2002.
Wild, A., M. Jäger, S. Lensing-Hoehn, A. Werner, and R. Krauspe. Growth behaviour of human mononuclear cells derived from bone marrow and cord blood on a collagen carrier for osteogenic regeneration. Biomed. Tech. (Berl.) 49(9):227–232, 2004.
Yang, L., T. Tao, X. Wang, N. Du, W. Chen, S. Tao, Z. Wang, and L. Wu. Effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro. Chin. Med. J. (Engl.) 116(9):1357–1360, 2003.
Yang, X. B., R. S. Bhatnagar, S. Li, and R. O. Oreffo. Biomimetic collagen scaffolds for human bone cell growth and differentiation. Tissue Eng. 10(7–8):1148–1159, 2004.
Young, H. E., T. A. Steele, R. A. Bray, K. Detmer, L. W. Blake, P. W. Lucas, and A. C. Black Jr. Human pluripotent and progenitor cells display cell surface cluster differentiation markers CD10, CD13, CD56, and MHC class-I. Proc. Soc. Exp. Biol. Med. 221(1):63–71, 1999.
Zhang, J. Y., B. A. Doll, E. J. Beckman, and J. O. Hollinger. Three-dimensional biocompatible ascorbic acid-containing scaffold for bone tissue engineering. Tissue Eng. 9(6):1143–1157, 2003.
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Jäger, M., Feser, T., Denck, H. et al. Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Cultured onto Three Different Polymers In Vitro. Ann Biomed Eng 33, 1319–1332 (2005). https://doi.org/10.1007/s10439-005-5889-2
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DOI: https://doi.org/10.1007/s10439-005-5889-2