- Research article
- Open Access
Proliferating and differentiating effects of three different growth factors on pluripotent mesenchymal cells and osteoblast like cells
Journal of Orthopaedic Surgery and Research volume 2, Article number: 27 (2007)
Growth factors are in clinical use to stimulate bone growth and regeneration. BMP-2 is used in long bone and spinal surgery, PDGFbb for the treatment of periodontal defects and children with growth hormone receptor deficiency are treated with IGF-I.
Aim of the present study was the comparative analysis of the effect of these growth factors released from a local drug delivery system on cells of the osteogenic lineage at differing differentiation stages.
The experiments with the mesenchymal cell line C2C12 revealed a proliferating effect of all three growth factors and a differentiating effect of BMP-2 with a dramatic increase in alkaline phosphatase activity. None of the growth factors stimulated cell migration.
Human osteoblast like cells showed similar results with an increase in proliferation after stimulation with IGF-I or PDGFbb. The enzymatic activity of alkaline phosphatase was enhanced only in the cells stimulated with BMP-2. This group showed also more mineralized matrix compared to the other groups.
In conclusion, the growth factors IGF-I and PDGFbb delivered with a local drug delivery system stimulated cell proliferation, whereas BMP-2 showed a dramatic effect on differentiation on osteoblast precursor cells and osteoblast like cells.
Today BMPs are used in spine and orthopaedic surgery, the platelet derived growth factor (PDGFbb) for periodontal treatment  and insulin growth factor-I (IGF-I) to treat children with growth hormone insensitivity syndrome or IGF-I deficiency [2, 3]. These three growth factors belong to different families and initiate their signaling from the cell surface by different receptors and intracellular pathways. The IGF and PDGF signals are transduced via tyrosine kinases [4, 5] and the BMP signal via serine/threonine kinase [6, 7]. Several in vitro and pre clinical in vivo studies have been performed to demonstrate the effect of the growth factors on different cell types and bone [8–13].
For the clinical use of growth factors the delivery system is important . Once a growth factor reaches the site of action, it must remain at the site in an appropriate concentration and long enough for the pharmacological effect. The half life of growth factors in vivo is very short and they are metabolized within a few hours . For the use in bone regeneration, however, an action over a longer time period is necessary. For a therapeutical success these requirements must be met and therefore an adequate carrier must be used for drug delivery. A drug delivery system based on poly(D,L-lactide) (PDLLA) was developed for local release . In previous studies the release profile of the growth factors incorporated in the PDLLA coating by eluting in PBS or cell culture medium was investigated. The incorporated growth factors were released with an initial peak with in the first 2 to 3 days. The peak release is followed by a slow sustained release [16, 17]. Storage of the coated implants over 14 month had no effect on the activity of the incorporated growth factors on osteoblast like cells . The PDLLA serves as a coating for orthopedic implants with incorporated pharmacological agents. Using this application system, aim of the study was the comparison of BMP-2, IGF-I and PDGFbb in their effect on different cell types. Primary human osteoblast like cells were used to investigate the effect of the growth factors on bone forming cells. The used cell line C2C12 differentiates rapidly into myoblasts after reaching confluence . This cell line has also the potential to differentiate to adipocytes  or osteoblast like cells  and therefore serve as a model for pluripotent mesenchymal cells. The potential of these cells to differentiate into the osteoblastic linage is used to test the osteoinductivity of bone grafting materials . Using the C2C12 cells the osteoinductivity and migratory effect of the growth factors was analyzed.
Osteoblast like cells were isolated from tibia plateau after reconstructive surgery with the permission of the local authorities. An informed consent was obtained from all donors. For isolation of the cells, the trabecular bone was minced into little pieces followed by overnight digestion with collagenase Type II according to established protocols  1 × 105 osteoblasts were cultivated in 12 well plates in DMEM/F-12-media with 10% heat inactivated FCS at 37°C and 5% CO2. After cultivation of the cells for 3 days under identical conditions, the implants were added to the culture in a non-contact manner using a tissue culture inserts (0.4 μm pore size, Nunc, Germany). The cells were cultured for further 15 days. One third of the medium was changed every day to ensure only gentle changes in the medium composition and growth factor concentration.
Three parallel test series were performed with pooled cells from different donors. Each test series was done in triplicate.
The mice myoblast cell line C2C12 (ACC 565) was obtained from DSMZ, Braunschweig, Germany. 5 × 104 cells were cultivated in 24 well plates in DMEM with 10% heat inactivated FCS at 37°C and 5% CO2. After a 5 h adherence period medium was changed to DMEM with only 1% heat inactivated FCS to reduce the proliferation activity. The implants were placed into the culture wells and the cells were cultured for three days. The test were conducted in triplicate and repeated two times.
The growth factors were applied to the cell culture from a local drug delivery system. The drug delivery system is based on a Poly(D,L-lactide)-coating (Boehringer, Ingelheim, Germany) on Titanium Kirschner-wires (1.0 mm diameter, Synthes USA) and described in more detail elsewhere .
Three different recombinant human growth factors were used for the experiments:
IGF-I (R&D-Systems, Wiesbaden, Germany), BMP-2 (Osteogenetics, Würzburg, Germany) and PDGFbb (Biomimetics, Franklin, USA).
According to previous experiments [11, 12] the growth factors were incorporated in 5% (w/w) in the PDLLA coating. The amount of growth factor added to the cell cultures was 15 μg/ml (osteoblast) and 10 μg/ml (C2C12).
The difference in the applied growth factor amount (15 μg/ml or 10 μg/ml) is due to the different cell culture approaches (24 well plates or 12 well plates) used and the fact that the factors were applied from coated titanium k-wires. For control served k-wires coated with the carrier PDLLA.
Cell vitality and proliferation was achieved via a non invasive/toxic cell activity assay (alamarBlue, Assay, Biozol, Eching, Germany). For the assay, 10% alamarBlue was added to the cells and incubated for 3 h at 37°C. The absorbance was measured in triplicate spectrophotometrical with a micro plate reader at two wavelengths: 570 and 600 nm in accordance to the instruction of the manufacturer.
The catalytic activity of the alkaline phosphatase (AP) was determined using para-nitrophenyl phosphate (p-NPP, Sigma, Germany) as a substrate of the enzyme. After rinsing the cells the freshly prepared AP-buffer was added and incubated for 30 min at 37°C. The absorbance was read out in triplicate on a micro plate reader by 405 nm wavelength.
The Osteocalcin concentration was quantified with an ELISA (Metra Osteocalcin EIA kit, Quidel, San Diego, CA). De Novo synthesis of collagen type-I was quantified to detect carboxyterminal propeptide of type I collagen (CICP, Quidel, San Diego, CA), a decomposition product of collagen type-I. Supernatant from the cell cultures was used and both ELISA were performed in accordance to the instructions of the manufacturer.
Matrix mineralization was evaluated by the use of Von Kossa stain. Cells were rinsed and fixed with cool methanol for 10 min. After rinsing with water cells were incubated for 30 min at room temperature (RT) with 3% silver nitrate and for 2 min in formaldehyde. Surplus silver nitrate was removed by incubation in 5% sodium thiosulfate for 5 min at RT.
Migration assay was performed by using a well established assay . Briefly, the growth factor coated wires were placed to the lower chamber of polycarbonate-membrane-inserts (8 μm pore size, Nunc, Germany) and covered with DMEM with 0.1% BSA. After 24 h 5 × 104 pluripotent mesenchymal cells (C2C12) were added to the upper chamber and cultured under standard conditions. For positive control, 10% FCS was added to the lower chamber. After 5 h of incubation, the membrane was removed and the cells on the surface carefully abscised. Cells migrated into the membrane were fixed with 4% w/v paraformaldehyd (PFA, Sigma, Germany), stained with 4',6-Diamidino-2-phenylindole (DAPI, Sigma, Germany) and counted under the microscope.
In order to compare the data of the independent test serials, the results of the experimental groups were normalized to the results of the PDLLA-group (control).
Statistical differences were assessed using an ANOVA and Dunnett Post Hoc test was employed for multiple comparison tests at a level of 95% (Software SPSS12.0).
Osteoblast like cells
The results of the growth factor groups and the different assays were normalized to the PDLLA group which was set 100%. This method was chosen for accounting for differences between the serials. All cell cultures were started with a comparable cell number (day 0, Fig. 1a). Over the experimental period of 10 days a significant increase in cell number was detectable in the PDGFbb and IGF-I group compared to the PDLLA group (Fig. 1a). No effect of BMP-2 on cell proliferation was detectable. The enzymatic activity of alkaline phosphatase, however, was significantly higher in the BMP-2 treated osteoblast like cells at days 10 and 15 (Fig. 1b). The two other growth factors had no influence on the AP-activity. The von Kossa stain for mineralized extra cellular matrix after 15 days revealed a clear stimulating effect of BMP-2 on the mineralization (Fig. 2a–d). No effect on collagen-1 and osteocalcin synthesis was observed after treatment with growth factors (data not shown).
C2C12 cell line
All three growth factors, PDGFbb, IGF-I and BMP-2, stimulated significantly the cell proliferation in the myoblast cell line (Fig. 3a). The effect of BMP-2, however, was less pronounced. The alkaline phosphatase activity was only significantly increased after stimulation with BMP-2 compared to the PDLLA group (Fig. 3b). This is also clearly visible in the alkaline phosphatase stain in Figure 2e–h.
In the standard cell culture wells, the pluripotent mesenchymal cell line showed an accumulation around IGF-I coated k-wire (Fig. 2g). This effect was not seen for PDGFbb or BMP-2.
The migration assays (Boyden Chamber) revealed a significant migratory effect of the positive control (FCS) on the cells. None of the growth factors, however, showed an effect on the migratory activity of the pluripotent mesenchymal cell line (Fig. 3c).
The biological stimulation of bone regeneration is a growing field. Several growth factors necessary for bone development, maintenance, and regeneration have been identified. This study aims to compare the effectiveness of three growth factors approved for clinical use released from a local drug delivery system: PDGFbb, IGF-I and BMP-2. Two different cell types were used to investigate the effect of the different growth factors. In both cell types, primary human osteoblast like cells and a murine pluripotent mesenchymal cell line, BMP-2 induced cell differentiation, whereas IGF-I and PDGFbb stimulated cell proliferation. None of the investigated growth factors induced migration in the Boydan chamber assay.
The pluripotent myoblast cell line is a well established system for testing osteoinductivity by using the reversible potential of the cells to differentiate into osteoblastic phenotype after stimulation with osteoinductive factors . The observed effect of BMP-2 on pluripotent mesenchymal cell line is in accordance with previous studies showing the osteoinductivity of this growth factor [23, 24]. The performed Boyden Chamber experiment showed no migratory effect of the used growth factors on the C2C12 cells and this is in accordance with a study by Allen at al. . The proliferating effect was the strongest in the myoblast culture treated with IGF-I followed by PDGFbb and then BMP-2.
The mitogenic effect of the growth factors IGF-I and PDGFbb released from the implant coating on osteoblast like cells and pluripotent mesenchymal cells is also in accordance with previous studies [8, 25, 26]. The proliferating effect of the growth factors seem to be differentiation depending, because BMP-2 stimulated proliferation only in the pluripotent cell line, whereas no effect was shown on the osteoblast like cells.
The results concerning the effect of both factors (PDGFbb and IGF-I) on osteoblast differentiation are controversy. Some studies demonstrated an enhanced collagen and osteocalcin synthesis [8, 27, 28], other studies, however, found no effect [25, 29]. The present study showed also no effect on the activity of alkaline phosphatase, the collagen-1 synthesis and the osteocalcin level in the medium. The stimulating effect of BMP-2 on alkaline phosphatase activity of osteoblast like cells has been reported earlier [30, 31]. The stimulating effect of BMP-2 on osteocalcin expression as described by Spinella-Jaegle et al. was not seen in the present study . This might be due to the different cells used in the experiments. Spinella-Jaegle performed the experiments with the murine preosteoblastic cell line MC3T3 and in the present study primary human osteoblast like cells were used.
In vivo studies on bone healing revealed an expression of the three analyzed growth factors at different healing phases. Cho and coworkers used a mouse fracture model and found BMP-2 expression only at the first day after fracture indicating the role in the very early healing phase . The quantification of IGF-I during rat fracture healing on the protein level revealed no increase in the early phase in comparison to the unfractured tibia. In the phase corresponding to the chondrogenesis and intramembraneaus ossification (days 10, and 15) a significant increase of IGF-I was detectable . The immunohistochemical detection of PDGF during mice fracture healing showed that PDGF is expressed by several cell types during almost the entire healing period .
The different phases of fracture healing are characterized by the presents of different cell types [36, 37]. In addition, the receptors on the cells also vary depending on the differentiation stage of the cell [38, 39]. These data point out that the three investigated factors are important during different healing phases. The controlled temporal regulation of growth factor action is necessary because of the interaction of the different factors. Less information on the interaction of factors is available, but the study by Cirri et al. demonstrated the inhibition of PDGF induced cell proliferation after application of insulin . The simultaneous application of IGF-I, TGF-β1 and PDGF to osteoblast like cells enhanced the in vitro bone formation synergistically . Therefore, for optimal stimulation of bone repair the controlled and local delivery of factors and factor combinations is mandatory [14, 41, 42]. In the present study the growth factors were delivered by using a local drug delivery system. Further studies are now necessary to identify the most potent stimulating factors and the timing of delivery. Based on the implant coating for local drug delivery we will develop a sequential drug release system for the temporally optimized delivery of stimulating factors.
In conclusion, the growth factors IGF-I and PDGFbb delivered with a local drug delivery system stimulated cell proliferation, whereas BMP-2 showed a dramatic effect on differentiation in osteoblast precursor cells and osteoblast like cells.
Nevins M, Giannobile WV, McGuire MK, Kao RT, Mellonig JT, Hinrichs JE, McAllister BS, Murphy KS, McClain PK, Nevins ML, Paquette DW, Han TJ, Reddy MS, Lavin PT, Genco RJ, Lynch SE: Platelet-Derived Growth Factor Stimulates Bone Fill and Rate of Attachment Level Gain: Results of a Large Multicenter Randomized Controlled Trial. J Periodontol. 2005, 76: 2205-2215. 10.1902/jop.2005.76.12.2205.
Wilton P: Treatment with recombinant human insulin-like growth factor I of children with growth hormone receptor deficiency (Laron syndrome). Acta Paediatr Suppl. 1992, 383: 137-142.
Clark RG: Recombinant insulin-like growth factor-1 as a therapy for IGF-1 deficiency in renal failure. Pediatr Nephrol. 2005, 20: 290-294. 10.1007/s00467-004-1714-7.
Schmid C: Insulin-like growth factors. Cell Biology International. 1995, 19: 445-458. 10.1006/cbir.1995.1088.
Bornfeldt KE, Raines EW, Graves LM, Skinner MP, Krebs EG, Ross R: Platelet-derived growth factor. Distinct signal transduction pathways associated with migration versus proliferation. Ann N Y Acad Sci. 1995, 766: 416-430. 10.1111/j.1749-6632.1995.tb26691.x.
Canalis E, Economides AN, Gazzerro E: Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev. 2003, 24: 218-235. 10.1210/er.2002-0023.
Wan M, Cao X: BMP signaling in skeletal development. Biochem Biophys Res Commun. 2005, 328: 651-657. 10.1016/j.bbrc.2004.11.067.
Canalis E: Effect of insulinlike growth factor I on DNA and protein synthesis in cultured rat calvaria. J Clin Invest. 1980, 66: 709-719.
Husmann I, Soulet L, Gautron J, Martelly I, Barritault D: Growth factors in skeletal muscle regeneration. Cytokine Growth Factor Rev. 1996, 7: 249-258. 10.1016/S1359-6101(96)00029-9.
Lind M: Growth factor stimulation of bone healing. Effects on osteoblasts, osteomies, and implants fixation. Acta Orthop Scand Suppl. 1998, 283: 2-37.
Schmidmaier G, Wildemann B, Lübberstedt M, Haas NP, Raschke M: IGF-I and TGF-beta 1 incorporated in a poly(D,L-lactide) implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture. J Biomed Mater Res, Applied Biomat. 2003, 65b: 157-162. 10.1002/jbm.b.10513.
Wildemann B, Kadow-Romacker A, Lubberstedt M, Raschke M, Haas N, Schmidmaier G: Differences in the fusion and resorption activity of human osteoclasts after stimulation with different growth factors released from a polylactide carrier. Calcif Tissu Int. 2005, 76: 50-55. 10.1007/s00223-004-0040-1.
Cesari C, Gatto MR, Malucelli F, Raspanti M, Zucchelli G, Checchi L: Periodontal growth factors and tissue carriers: Biocompatibility and mitogenic efficacy in vitro. J Biomed Mater Res B Appl Biomater. 2006, 76: 15-25.
Luginbuehl V, Meinel L, Merkle HP, Gander B: Localized delivery of growth factors for bone repair. European Journal of Pharmaceutics and Biopharmaceutics. 2004, 58: 197-208. 10.1016/j.ejpb.2004.03.004.
Zapf J, Hauri C, Waldvogel M, Froesch ER: Acute metabolic effects and half-lives of intravenously administered insulinlike growth factors I and II in normal and hypophysectomized rats. J Clin Invest. 1986, 77: 1768-1775.
Schmidmaier G, Wildemann B, Stemberger A, Haas NP, Raschke M: Biodegradable poly(D,L-lactide) coating of implants for continuous release of growth factors. J Biomed Mater Res, Applied Biomat. 2001, 58: 449-455. 10.1002/jbm.1040.
Wildemann B, Lubberstedt M, Haas NP, Raschke M, Schmidmaier G: IGF-I and TGF-beta 1 incorporated in a poly(d,l-lactide) implant coating maintain their activity over long-term storage-cell culture studies on primary human osteoblast-like cells. Biomaterials. 2004, 25: 3639-3644. 10.1016/j.biomaterials.2003.10.058.
Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T: Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol. 1994, 127: 1755-1766. 10.1083/jcb.127.6.1755.
Shin CS, Lecanda F, Sheikh S, Weitzmann L, Cheng SL, Civitelli R: Relative abundance of different cadherins defines differentiation of mesenchymal precursors into osteogenic, myogenic, or adipogenic pathways. J Cell Biochem. 2000, 78: 566-577. 10.1002/1097-4644(20000915)78:4<566::AID-JCB6>3.0.CO;2-K.
Han B, Tang B, Nimni ME: Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix. J Orthop Res. 2003, 21: 648-654. 10.1016/S0736-0266(03)00005-6.
Robey PG, Termine JD: Human bone cells in vitro. Calcif Tissue Int. 1985, 37: 453-460. 10.1007/BF02557826.
Allen DL, Teitelbaum DH, Kurachi K: Growth factor stimulation of matrix metalloproteinase expression and myoblast migration and invasion in vitro. Am J Physiol Cell Physiol. 2003, 284: C805-C815.
Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, Kahn AJ, Suda T, Yoshiki S: Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol. 1991, 113: 681-687. 10.1083/jcb.113.3.681.
Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY, He TC: Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am. 2003, 85-A: 1544-1552.
Hock JM, Canalis E: Platelet-derived growth factor enhances bone cell replication, but not differentiated function of osteoblasts. Endocrinology. 1994, 134: 1423-1428. 10.1210/en.134.3.1423.
Cirri P, Taddei ML, Chiarugi P, Buricchi F, Caselli A, Paoli P, Giannoni E, Camici G, Manao G, Raugei G, Ramponi G: Insulin inhibits platelet-derived growth factor-induced cell proliferation. Mol Biol Cell. 2005, 16: 73-83. 10.1091/mbc.E04-01-0011.
Canalis E, Lian JB: Effects of bone associated growth factors on DNA, collagen and osteocalcin synthesis in cultured fetal rat calvariae. Bone. 1988, 9: 243-246. 10.1016/8756-3282(88)90037-3.
McCarthy T, Centrella M, Canalis E: Regulatory effects of insulin-like growth factors I and II on bone collagen synthesis in rat calvarial cultures. Endocrinology. 1989, 124: 301-309.
Davis J, Tucci M, Franklin L, Russell G, Benghuzzi H: The effects of growth factors on the production of osteopontin and osteocalcin. Biomed Sci Instrum. 2006, 42: 31-36.
Gonnerman KN, Brown LS, Chu TM: Effects of growth factors on cell migration and alkaline phosphatase release. Biomed Sci Instrum. 2006, 42: 60-65.
Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S: BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res. 2003, 18: 1842-1853. 10.1359/jbmr.2003.18.10.1842.
Spinella-Jaegle S, Roman-Roman S, Faucheu C, Dunn FW, Kawai S, Gallea S, Stiot V, Blanchet AM, Courtois B, Baron R, Rawadi G: Opposite effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on osteoblast differentiation. Bone. 2001, 29: 323-330. 10.1016/S8756-3282(01)00580-4.
Cho TJ, Gerstenfeld LC, Einhorn TA: Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res. 2002, 17: 513-520. 10.1359/jbmr.2002.17.3.513.
Wildemann B, Schmidmaier G, Brenner N, Huning M, Stange R, Haas NP, Raschke M: Quantification, Localization, and Expression of IGF-I and TGF-beta1 During Growth Factor-Stimulated Fracture Healing. Calcif Tissue Int. 2004, 74: 388-397. 10.1007/s00223-003-0117-2.
Fujii H, Kitazawa R, Maeda S, Mizuno K, Kitazawa S: Expression of platelet-derived growth factor proteins and their receptor alpha and beta mRNAs during fracture healing in the normal mouse. Histochem Cell Biol. 1999, 112: 131-138. 10.1007/s004180050399.
Dimitriou R, Tsiridis E, Giannoudis PV: Current concepts of molecular aspects of bone healing. Injury. 2005, 36: 1392-1404. 10.1016/j.injury.2005.07.019.
Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA: Fracture healing as a post-natal developmental process: Molecular, spatial, and temporal aspects of its regulation. J Cell Biochem. 2003, 88: 873-884. 10.1002/jcb.10435.
Centrella M, Casinghino S, Kim J, Pham T, Rosen V, Wozney J, McCarthy TL: Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype. Mol Cell Biol. 1995, 15: 3273-3281.
Kim KK, Ji C, Chang W, Wells RG, Gundberg CM, McCarthy TL, Centrella M: Repetitive exposure to TGF-beta suppresses TGF-beta type I receptor expression by differentiated osteoblasts. Gene. 2006, 379: 175-184. 10.1016/j.gene.2006.05.005.
Pfeilschifter J, Oechsner M, Naumann A, Gronwald R, Minne H, Ziegler R: Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between Insulin-like growth factor I, platelet-derived growth factor and transforming growth factor beta. Endocrinology. 1990, 127: 69-75.
Varkey M, Gittens SA, Uludag H: Growth factor delivery for bone tissue repair: an update. Expert Opin Drug Deliv. 2004, 1: 19-36. 10.1517/17425247.1.1.19.
Simpson AH, Mills L, Noble B: The role of growth factors and related agents in accelerating fracture healing. J Bone Joint Surg Br. 2006, 88: 701-705. 10.1302/0301-620X.88B6.17524.
We thank BioMimetic Therapeutics, Inc., USA, for providing the PDGFbb.
The authors gratefully acknowledge the support of the German Research Foundation (KFO 102/2-1, TP 11).
The author(s) declare that they have no competing interests.
BW conceived, supervised, coordinated the study, performed the statistical analysis and wrote the manuscript. NB carried out the experiments with the C2C12 cells ML carried out the experiments with the osteoblast like cells. TV and GS participated in the study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
About this article
Cite this article
Wildemann, B., Burkhardt, N., Luebberstedt, M. et al. Proliferating and differentiating effects of three different growth factors on pluripotent mesenchymal cells and osteoblast like cells. J Orthop Surg Res 2, 27 (2007). https://doi.org/10.1186/1749-799X-2-27
- C2C12 Cell
- Primary Human Osteoblast
- Myoblast Cell Line
- Osteoblast Precursor Cell
- Osteocalcin Synthesis