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Induction of osteogenic markers in differentially treated cultures of embryonic stem cells
© Handschel et al; licensee BioMed Central Ltd. 2008
- Received: 30 July 2007
- Accepted: 10 June 2008
- Published: 10 June 2008
Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation.
Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and β-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR.
ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17.
Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.
- Embryonic Stem Cell
- Osteogenic Differentiation
- Leukemia Inhibitory Factor
- Bone Tissue Engineering
Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton . The reconstruction of damaged or lost bone is a clinical challenge in modern reconstructive surgery. The repair of bone defects still poses a significant problem for many clinicians. In the early decades of bone reconstruction surgeons used artificial tissue substitutes containing metals, ceramics, and polymers to maintain skeletal function . These artificial materials have facilitated surgeons to restore the form and – to some extent – the function of defective bones. Nevertheless, these artificial materials have specific disadvantages, and thus encouraged surgeons to develop alternative approaches including cell-based devices. Transplantation of autografts is a frequently used treatment strategy in routine clinical practice and has gained the "gold standard" in bone reconstructive surgery, despite donor site morbidity and donor shortage .
Modern cell-based bone reconstruction techniques may offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Generally, the combination of scaffolds, bioactive factors, and living cells provides a surgically implantable product for use in tissue regeneration and functional restoration [4, 5]. Numerous attempts were undertaken with various success to restore bone defects by various biomaterials alone [6–10] or in combination with bioactive cytokines such as bone morphogenetic protein (BMP)-7, BMP-2 or BMP-2-mutants [11, 12]. Cell-based strategies in bone tissue engineering use different cell sources including autologous cells as well as allogenic and xenogenic cells [13–16]. There are some reports that use totipotential embryonic stem cells in tissue engineering of bone [17, 18].
Embryonic stem cells (ESCs) are routinely derived from the inner cell mass of blastocysts and represent pluripotential embryonic precursor cells that give rise to all cell types in the developing organism. ESCs have historically been maintained in co-culture with mitotically inactive fibroblasts [19–21]. This co-culture system is unnecessary if the medium is supplemented with leukemia inhibitory factor (LIF) [22, 23]. In the absence of LIF embryonic stem cells will differentiate into a morphologically mixed cell population expressing features of endoderm and mesoderm lineages . By definition ESCs have the potential to differentiate into osteogenic cells under selective culture conditions. Specifically, it has been shown by various investigators that ESCs can differentiate into osteogenic cells under selective culture conditions [17, 18, 25]. However, it is unclear which medium is most suitable to initiate osteogenic differentiation. BMP-2 and a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) are good candidates [19, 25]. Thus, we examined the time-dependent expression of the osteoblastic markers osteopontin , collagen I , alkaline phosphatase , and osteocalcin  in ESC cells.
Culture of ESCs with biomaterials
Feeder-independent murine ESCs were derived from the inner cell mass of blastocysts extracted from C57BL/6 mice. The ESCs were kindly provided by K. Pfeffer (Institute for Microbiology, Heinrich-Heine-University, Germany). The cells were tested to be positive for the stem cell marker Pouf1 (alias Oct4) and Foxd3  (data not shown). A total number of 1.5 × 106 cells per petri dish (10 cm in diameter) were cultured in Dulbecco's Eagle medium (DMEM). The medium was supplemented with 5 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol and 15% fetal calf serum (FCS). The ESCs were divided into four groups and cultured for 25 days as follows: group I; control, supplemented with LIF to prevent differentiation, group II; no additional supplement, group III; supplemented with BMP-2 (10 ng/ml), and group IV; supplemented with DAG (dexamethasone (0.1 μM), ascorbic acid (50 μM) and β-glycerophosphate (10 mM).
To detect mineralization in the differently treated cell cultures, the cells were washed two times with PBS (phosphate-buffered saline) before fixation with 3% glutardialdehyde in PBS for 30 minutes. The cells were washed with distilled water and incubated in 5% silver nitrate (Sigma Aldrich) for 1 hour. The cells were washed again with distilled water. A solution of 5% sodium carbonate and 10% formaldehyde was added for 2 minutes before the cells were washed again and fixed with 1% sodium thiosulfate. Calcium-phosphate deposits stained black [31, 32].
Quantitative real time PCR
Quantitative real time PCR was employed to assess the influence of the biomaterials on gene expression. Total RNA was isolated from specimens using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For cDNA synthesis 800 ng total RNA was used as a template for Superscript II (Invitrogen, Paisley, UK) and OligodT-Primers (Peqlab, Erlangen, Germany) in a total volume of 20 μl. Amplification was performed with 1 μl of cDNA and the following specific primer pairs (MWG-Biotech AG, Ebersberg, Germany): CD34; 5'-CACAGAACTTCCCAGCAAACTC-3' and 5'-CATGTTGTCTTGCTGAATGGCC-3', osteopontin; 5'-CCCGGTGAAAGTGACTGATT-3' and 5'-TTCTTCAGAGGACACAGCATTC-3', osteocalcin; 5'-GCCCTGAGTCTGACAAAGGTA-3' and 5'-GGTGATGGCCAAGACTAAGG-3', collagen type I; 5'-AAGGGGTCTTCCTGGTGAAT-3' and 5'-GGGGTACCACGTTCTCCTC-3', alkaline phosphatases; 5'-AAGGCTTCTTCTTGCTGGTG-3' and 5'-GCCTTACCCTCATGATGTCC-3', and GAPDH; 5'-CAATGAATACGGCTACAGCAAC-3' and 5'-AGGGAGATGCTCAGTGTTGG-3'. For quantitative real time PCR the iCycler Thermal Cycler Base (Bio-Rad Laboratories GmbH, München, Germany) and qPCR MasterMix, No Rox, #RT-QP2X-03NR (Eurogentec, Köln, Germany) was used. The increase in reaction products during PCR was monitored by measuring the increase in fluorescence intensity caused by the binding of SYBR green to double-stranded DNA that accumulated during PCR cycles. Reaction mixtures were set up as suggested by the manufacturer. Threshold cycle values of target genes were standardized against GAPDH expression and normalized to the expression in the control culture (group I). All real time experiments in this study have been performed with regard to the publication of Pfaffl . We have applied the mathematical model given there to eliminate deviations due to sample preparation. In order to apply this model it is necessary to choose a reference gene (e.g. GAPDH) for calculating relative expression levels. The quantitative real time PCR was performed in samples obtained at day 5, 9, 11, 13, 15, 17, 19, 21, 23, and 25 of culture, respectively. Following PCR agarose-gel electrophoresis was performed using β-actin as a reference.
Currently, there are many efforts to establish cell-based strategies in bone tissue engineering. ESCs are one of many different cell populations, which are being tested for their feasibility for these treatment options. The purpose of this investigation was to determine which supplements in culture medium are most suitable to initiate osteogenic differentiation in ESC cultures. In addition, we investigated the kinetics of gene expression during in vitro differentiation.
The results of our microscopical analysis revealed that ESCs cultured in the presence of DAG show by far the highest extent of mineralisation as determined by the occurrence of calcium-phosphate-containing crystals. With respect to extracellular matrix maturation and mineral deposition as crucial steps in the osteogenic cascade , DAG seems to be the most promising supplement for inducing osteogenic differentiation in ESCs. In accordance with our microscopical results, a strong increase of collagen I expression was observed at day 11 in the DAG-treated cells. Stimulation with BMP-2 also increased collagen synthesis. Expression of osteocalcin mRNA followed a different pattern and appeared as a double-peaked curve, when ESCs were supplemented with osteogenic agents (DAG or BMP-2). However, the peak induction of osteocalcin mRNA in the BMP-2-treated cells was lower and delayed as compared to DAG-exposed cells. Taken together, these results support the use of DAG as a potent agent for inducing in vitro differentiation of ESCs into osteoblast-like cells.
There are only few reports addressing osteogenic differentiation of ESCs published in the literature so far [18, 25, 34, 35]. In agreement with these results we describe here that mineralisation is microscopically evident as early as two weeks of culture. Buttery and co-workers also used DAG as a culture supplement and found that mineralisation was detectable when dexamethasone was added only at day 14 or later . By following this protocol the differentiation process was delayed as compared to the findings in our ESC cultures. While Buttery used only microscopical methods for studying osteogenic differentiation, zur Nieden and colleagues performed also gene expression analyses for osteogenic markers . With respect to the time-course of gene expression with an early increase of collagen I and a later increase of osteocalcin transcripts, their data are comparable to our findings as shown above. Unlike to the findings of zur Nieden and colleagues, an early peak of osteocalcin expression and a minor increase of osteopontin were found in the presented study. The differences could be explained by different concentrations of supplements used for cell differentiation. Zur Nieden et al. used 1,25-OH vitamin D3 instead of dexamethasone. According to Zhang et al. vitamin D3 increases osteopontin expression in osteoblasts and inhibits expression of osteocalcin . Chaudhry and co-workers replaced dexamethasone with retinoid acid, which was found to be an inductor of mineralization in three-dimensional scaffolds . Notably, alkaline phosphatase was constitutively expressed at high levels in undifferentiated cells . In this experimental setting the mineralisation process was delayed and was detectable only after day 21. Treatment with DAG appeared to be equal or even superior to BMP-2 stimulation regarding the induction of osteogenic differentiation in ESCs. Other authors have used BMP-2 in combination with osteogenic supplements for this purpose [18, 38].
An advantage of using ESCs instead of tissue-derived progenitor cells is that ESCs are immortal and could potentially provide an unlimited supply of differentiated osteoblast and osteoprogenitor cells for transplantation. In contrast to embryonic cells, the proliferative, self-renewal and differentiation capacity of cells derived from adult tissues generally decreases with age [39, 40]. One major challenge pointing to the use of ESCs lies in overcoming immunological rejection from the transplant recipient. Interestingly, Burt and colleagues performed ESC transplantation in major histocompatibility complex (MHC)-mismatched mice without clinical or histological evidence of graft-versus-host disease (GVHD) . In addition, recent data indicate that ESCs may allow for a low-risk induction of tolerance not requiring any immunosuppression .
In conclusion, ESCs differentiate into osteoblast-like cells in vitro when stimulated with DAG and showed a time-dependent induction of osteogenic markers. Thus, stimulation with these agents is suitable to generate a promising cell population used for bone tissue engineering.
We are very grateful to Prof. Dr. K. Pfeffer, Institut für Mikrobiologie an der Heinrich-Heine-Universität Düsseldorf, who provided the murine ESCs.
- Malara P, Malara B, Drugacz J: Characteristics of maxillofacial injuries resulting from road traffic accidents - a 5 year review of the case records from Department of Maxillofacial Surgery in Katowice, Poland. Head Face Med. 2006, 2: 27-10.1186/1746-160X-2-27.View ArticlePubMedPubMed CentralGoogle Scholar
- Binderman I, Fin N: Bone substitutesorganic, inorganic, and polymeric: Cell material interactions. CRC Handbook of Bioactive Ceramics. Edited by: Yamamuro T, Hench L, Wilson J. 1990, Boca Raton, Florida , CRC Press, 45-51.Google Scholar
- Damien CJ, Parsons JR: Bone graft and bone graft substitutes: a review of current technology and applications. J Appl Biomater. 1991, 2 (3): 187-208. 10.1002/jab.770020307.View ArticlePubMedGoogle Scholar
- Tuan RS, Boland G, Tuli R: Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther. 2003, 5 (1): 32-45. 10.1186/ar614.View ArticlePubMedGoogle Scholar
- Vacanti CA, Vacanti JP: Bone and cartilage reconstruction with tissue engineering approaches. Otolaryngol Clin North Am. 1994, 27 (1): 263-276.PubMedGoogle Scholar
- Cohen SR, Holmes RE, Meltzer HS, Levy ML, Beckett MZ: Craniofacial reconstruction with a fast resorbing polymer: a 6- to 12-month clinical follow-up review. Neurosurg Focus. 2004, 16 (3): E12-10.3171/foc.2004.16.3.13.View ArticlePubMedGoogle Scholar
- Handschel J, Wiesmann HP, Stratmann U, Kleinheinz J, Meyer U, Joos U: TCP is hardly resorbed and not osteoconductive in a non-loading calvarial model. Biomaterials. 2002, 23 (7): 1689-1695. 10.1016/S0142-9612(01)00296-4.View ArticlePubMedGoogle Scholar
- Holmes RE, Cohen SR, Cornwall GB, Thomas KA, Kleinhenz KK, Beckett MZ: MacroPore resorbable devices in craniofacial surgery. Clin Plast Surg. 2004, 31 (3): 393-406, v. 10.1016/j.cps.2004.03.003.View ArticlePubMedGoogle Scholar
- Thaller SR, Hoyt J, Borjeson K, Dart A, Tesluk H: Reconstruction of calvarial defects with anorganic bovine bone mineral (Bio-Oss) in a rabbit model. J Craniofac Surg. 1993, 4 (2): 79-84.View ArticlePubMedGoogle Scholar
- Velich N, Nemeth Z, Hrabak K, Suba Z, Szabo G: Repair of bony defect with combination biomaterials. J Craniofac Surg. 2004, 15 (1): 11-15. 10.1097/00001665-200401000-00006.View ArticlePubMedGoogle Scholar
- Kubler NR, Wurzler KK, Reuther JF, Sieber E, Kirchner T, Sebald W: [Effect of different factors on the bone forming properties of recombinant BMPs]. Mund Kiefer Gesichtschir. 2000, 4 Suppl 2: S465-9. 10.1007/PL00012693.View ArticlePubMedGoogle Scholar
- Terheyden H, Knak C, Jepsen S, Palmie S, Rueger DR: Mandibular reconstruction with a prefabricated vascularized bone graft using recombinant human osteogenic protein-1: an experimental study in miniature pigs. Part I: Prefabrication. Int J Oral Maxillofac Surg. 2001, 30 (5): 373-379. 10.1054/ijom.2001.0032.View ArticlePubMedGoogle Scholar
- Meyer U, Wiesmann HP: Tissue engineering: a challenge of today's medicine. Head Face Med. 2005, 1: 2-10.1186/1746-160X-1-2.View ArticlePubMedPubMed CentralGoogle Scholar
- Puricelli E, Ulbrich LM, Ponzoni D, Filho JJ: Histological analysis of the effects of a static magnetic field on bone healing process in rat femurs. Head Face Med. 2006, 2: 43-10.1186/1746-160X-2-43.View ArticlePubMedPubMed CentralGoogle Scholar
- Handschel J, Wiesmann HP, Depprich R, Kubler NR, Meyer U: Cell-based bone reconstruction therapies--cell sources. Int J Oral Maxillofac Implants. 2006, 21 (6): 890-898.PubMedGoogle Scholar
- Yamaguchi M, Hirayama F, Murahashi H, Azuma H, Sato N, Miyazaki H, Fukazawa K, Sawada K, Koike T, Kuwabara M, Ikeda H, Ikebuchi K: Ex vivo expansion of human UC blood primitive hematopoietic progenitors and transplantable stem cells using human primary BM stromal cells and human AB serum. Cytotherapy. 2002, 4 (2): 109-118. 10.1080/146532402317381811.View ArticlePubMedGoogle Scholar
- Heng BC, Cao T, Stanton LW, Robson P, Olsen B: Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro. J Bone Miner Res. 2004, 19 (9): 1379-1394. 10.1359/JBMR.040714.View ArticlePubMedGoogle Scholar
- zur Nieden NI, Kempka G, Rancourt DE, Ahr HJ: Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenetic protein-2: effect of cofactors on differentiating lineages. BMC Dev Biol. 2005, 5 (1): 1-10.1186/1471-213X-5-1.View ArticlePubMedPubMed CentralGoogle Scholar
- Bielby RC, Boccaccini AR, Polak JM, Buttery LD: In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells. Tissue Eng. 2004, 10 (9-10): 1518-1525. 10.1089/ten.2004.10.1518.View ArticlePubMedGoogle Scholar
- Evans MJ, Kaufman MH: Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981, 292 (5819): 154-156. 10.1038/292154a0.View ArticlePubMedGoogle Scholar
- Burt RK, Verda L, Kim DA, Oyama Y, Luo K, Link C: Embryonic stem cells as an alternate marrow donor source: engraftment without graft-versus-host disease. J Exp Med. 2004, 199 (7): 895-904. 10.1084/jem.20031916.View ArticlePubMedPubMed CentralGoogle Scholar
- Chambers I: The molecular basis of pluripotency in mouse embryonic stem cells. Cloning Stem Cells. 2004, 6 (4): 386-391. 10.1089/clo.2004.6.386.View ArticlePubMedGoogle Scholar
- Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D: Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988, 336 (6200): 688-690. 10.1038/336688a0.View ArticlePubMedGoogle Scholar
- Niwa H, Miyazaki J, Smith AG: Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000, 24 (4): 372-376. 10.1038/74199.View ArticlePubMedGoogle Scholar
- Chaudhry GR, Yao D, Smith A, Hussain A: Osteogenic Cells Derived From Embryonic Stem Cells Produced Bone Nodules in Three-Dimensional Scaffolds. J Biomed Biotechnol. 2004, 2004 (4): 203-210. 10.1155/S111072430431003X.View ArticlePubMedPubMed CentralGoogle Scholar
- McKee MD, Nanci A: Osteopontin: an interfacial extracellular matrix protein in mineralized tissues. Connect Tissue Res. 1996, 35 (1-4): 197-205. 10.3109/03008209609029192.View ArticlePubMedGoogle Scholar
- Bilezikian JP, Raisz LG, Rodan GA: Principles of Bone Biology. 1996, San Diego , Academic Press, Inc.Google Scholar
- Zernik J, Twarog K, Upholt WB: Regulation of alkaline phosphatase and alpha 2(I) procollagen synthesis during early intramembranous bone formation in the rat mandible. Differentiation. 1990, 44 (3): 207-215. 10.1111/j.1432-0436.1990.tb00619.x.View ArticlePubMedGoogle Scholar
- Aubin JE, Liu F: The osteoblast lineage. Principles of Bone Biology. Edited by: Bilezikian JP, Raisz LG, Rodan GA. 1996, San Diego , Academic Press, 51-67.Google Scholar
- Baharvand H, Ashtiani SK, Taee A, Massumi M, Valojerdi MR, Yazdi PE, Moradi SZ, Farrokhi A: Generation of new human embryonic stem cell lines with diploid and triploid karyotypes. Dev Growth Differ. 2006, 48 (2): 117-128. 10.1111/j.1440-169X.2006.00851.x.View ArticlePubMedGoogle Scholar
- Koch TG, Heerkens T, Thomsen PD, Betts DH: Isolation of mesenchymal stem cells from equine umbilical cord blood. BMC Biotechnol. 2007, 7: 26-10.1186/1472-6750-7-26.View ArticlePubMedPubMed CentralGoogle Scholar
- Mizobuchi M, Ogata H, Hatamura I, Koiwa F, Saji F, Shiizaki K, Negi S, Kinugasa E, Ooshima A, Koshikawa S, Akizawa T: Up-regulation of Cbfa1 and Pit-1 in calcified artery of uraemic rats with severe hyperphosphataemia and secondary hyperparathyroidism. Nephrol Dial Transplant. 2006, 21 (4): 911-916. 10.1093/ndt/gfk008.View ArticlePubMedGoogle Scholar
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29 (9): e45-10.1093/nar/29.9.e45.View ArticlePubMedPubMed CentralGoogle Scholar
- zur Nieden NI, Kempka G, Ahr HJ: In vitro differentiation of embryonic stem cells into mineralized osteoblasts. Differentiation. 2003, 71 (1): 18-27. 10.1046/j.1432-0436.2003.700602.x.View ArticlePubMedGoogle Scholar
- Buttery LD, Bourne S, Xynos JD, Wood H, Hughes FJ, Hughes SP, Episkopou V, Polak JM: Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng. 2001, 7 (1): 89-99. 10.1089/107632700300003323.View ArticlePubMedGoogle Scholar
- Zhang R, Ducy P, Karsenty G: 1,25-dihydroxyvitamin D3 inhibits Osteocalcin expression in mouse through an indirect mechanism. J Biol Chem. 1997, 272 (1): 110-116. 10.1074/jbc.272.1.110.View ArticlePubMedGoogle Scholar
- Phillips BW, Belmonte N, Vernochet C, Ailhaud G, Dani C: Compactin enhances osteogenesis in murine embryonic stem cells. Biochem Biophys Res Commun. 2001, 284 (2): 478-484. 10.1006/bbrc.2001.4987.View ArticlePubMedGoogle Scholar
- Yamashita A, Takada T, Narita J, Yamamoto G, Torii R: Osteoblastic differentiation of monkey embryonic stem cells in vitro. Cloning Stem Cells. 2005, 7 (4): 232-237. 10.1089/clo.2005.7.232.View ArticlePubMedGoogle Scholar
- D'Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA: Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res. 1999, 14 (7): 1115-1122. 10.1359/jbmr.19126.96.36.1995.View ArticlePubMedGoogle Scholar
- Quarto R, Thomas D, Liang CT: Bone progenitor cell deficits and the age-associated decline in bone repair capacity. Calcif Tissue Int. 1995, 56 (2): 123-129. 10.1007/BF00296343.View ArticlePubMedGoogle Scholar
- Zavazava N: Embryonic stem cells and potency to induce transplantation tolerance. Expert Opin Biol Ther. 2003, 3 (1): 5-13. 10.1517/147125188.8.131.52.View ArticlePubMedGoogle Scholar
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