Research Article | Open Access
Kentaro Uchida, Gen Inoue, Osamu Matsushita, Kyosuke Horikawa, Hiroyuki Sekiguchi, Wataru Saito, Shotaro Takano, Hisako Fujimaki, Masayuki Miyagi, Masashi Takaso, "Basic Fibroblast Growth Factor-Anchored Multilayered Mesenchymal Cell Sheets Accelerate Periosteal Bone Formation", BioMed Research International, vol. 2017, Article ID 4371460, 8 pages, 2017. https://doi.org/10.1155/2017/4371460
Basic Fibroblast Growth Factor-Anchored Multilayered Mesenchymal Cell Sheets Accelerate Periosteal Bone Formation
Cell-based regenerative therapy has the potential to repair bone injuries or large defects that are recalcitrant to conventional treatment methods, including drugs and surgery. Here, we developed a multilayered cell-based bone formation system using cells coated with fibronectin-gelatin (FN-G) nanofilms. The multilayered mesenchymal cells (MLMCs) were formed after two days of culture and were shown to express higher levels of BMP-2 and VEGF compared to monolayer cultures of MCs. The MLMCs were used as a graft material in combination with a fusion protein consisting of basic fibroblast growth factor (bFGF), polycystic kidney disease (PKD) domain, and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. In femur sites grafted with the MLMCs, significantly higher levels of callus volume and bone mineral content were observed compared to the sham controls. The callus volume and bone mineral content were further increased in femur sites grafted with bFGF-PKD-CBD/MLMCs. Taken together, these results suggest that bFGF-PKD-CBD/MLMCs, which can be simply and rapidly generated in vitro, have the potential to promote bone repair when grafted into large defect sites.
Cell-based regenerative therapy has the potential to repair injured or defect-containing bone that is resistant to conventional medical treatments, including growth-stimulating drugs and surgeries. Mesenchymal stem cells (MSCs) are an attractive autologous source of somatic stem cells for cell-based bone regenerative therapy, as they proliferate actively in vitro and differentiate into bone cells [1–5]. Several types of layered cell technologies, such as cell sheets constructed in temperature-responsive culture dishes [6–8], magnetic liposomes [9, 10], and cell-containing gel layers , have been applied towards the treatment of injured tissues. Although these approaches accelerate tissue healing, the cells may contain intracellular magnetic particles, which may have adverse effects.
To overcome the limitations of cell-layering techniques developed to date, a simple and rapid tissue engineering approach for generating multilayered cells was developed using fibronectin-gelatin (FN-G) nanofilms . This cell-accumulation technique allowed for mouse fibroblast cells to form approximately eight layers in vitro after a 24 h incubation. MSCs secrete trophic factors and accelerate wound healing compared to fibroblasts . Due to these promising results, this method may also be applicable for forming multiple layers of MSCs for use in bone grafting. However, although the cell-accumulation technique has been evaluated in vitro with murine fibroblasts, the potential of this system to promote bone repair in vivo has not been investigated with MSCs.
The exogenous application of growth factors, particularly basic fibroblast growth factor (bFGF), has been shown to promote tissue regeneration when performing bone grafting [14–17]. bFGF is a potent mitogen for MSCs and promotes angiogenesis [18, 19], bone formation [20–24], and nerve regeneration [25, 26]. We previously demonstrated that the subcutaneous injection of a recombinant protein consisting of the polycystic kidney disease (PKD) and Clostridium histolyticum collagenase collagen-binding domains (CBD) fused to basic fibroblast growth factor (bFGF; bFGF-PKD-CBD) had greater skin fibroblast growth-promoting effects in nude mice than native bFGF . More recently, bFGF-PKD-CBD was shown to enhance bone formation at lower concentrations than bFGF alone when loaded onto implantable collagen sheets [28, 29], suggesting that the treatment combination of bFGF-PKD-CBD and a multilayered cell construct consisting of MSCs may promote ectopic bone formation at defect sites.
Here, we constructed multilayered mesenchymal cell (MLMCs) sheet anchored to collagen-binding bFGF using a novel cell tissue engineering technique. The properties and bone formation capacity of this material were evaluated both in vitro and in vivo using a rat femur model.
2. Materials and Methods
2.1. Isolation of Rat Mesenchymal Cells
A specific pathogen-free colony of Sprague-Dawley rats was housed in a semibarrier system with a controlled environment (temperature, °C; humidity, 55% ± 10%; and lighting, 12 h light/dark cycle) at Nippon Charles River Laboratories (Kanagawa, Japan) and were fed a diet of standard rodent chow (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan). The periosteum of distal femurs harvested from 10-week-old male rats, as previously described [28, 30], was used for the isolation of nucleated periosteal cells, which were then plated at 1 × 104 cells/cm2 in 6-well culture plates containing α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells (passage 0 [P0]) were incubated at 37°C in 5% CO2 for 7 days and expression of the mesenchymal cell markers CD29, CD54, and CD90 was confirmed by flow cytometry using antibodies against CD45 (fluorescein isothiocyanate, FITC), CD29 (phycoerythrin, PE), CD54 (PE), and CD90 (peridinin chlorophyll protein, PERCP; Biolegend, San Diego, CA, USA) . All animal procedures were performed in accordance with the approval of the animal ethics committee of Kitasato University.
2.2. Preparation of bFGF and CB-bFGF
2.3. Preparation of Multilayered Mesenchymal Cell Constructs
For the construction of MLMCs, FN-G nanofilms were first prepared on single-cell surfaces using a layer-by-layer assembly approach with a multilayered cell culture kit (Cell Feuille, Sumitomo Bakelite Co., Ltd., Tokyo, Japan). Briefly, 5 × 106 cells/ml of P2 rat mesenchymal cells collected by centrifugation at 300 after trypsinization, were alternatively incubated with 0.2 mg/mL FN (Mw 4.6 × 105) and G (Mw 1.0 × 105) in 50 mM Tris-HCl (pH 7.4) for 1 min at room temperature with mixing at 30 rpm using a Microtube Rotator (TAITEC Co., Saitama, Japan). After each treatment, the cells were washed with 50 mM Tris-HCl (pH 7.4) using centrifugation at 200 for 1 min to remove unadsorbed polymers. After five cycles of the immersion steps, the FN-G nanofilms were coated onto the cell surfaces. A total of 2 × 106 cells coated in the FN-G nanofilm were seeded into a cell culture insert coated with a FN and were further incubated in α-MEM containing 10% FBS at 37°C. Noncoated cells were used as controls. After two days of incubation, the histology and mRNA expression profiles of layered cells were evaluated by cryosection and real-time PCR, respectively, as described below.
2.4. Histology of MLMCs
After two days of incubation, noncoated and FN-G nanofilm-coated MLMCs were fixed in 4% paraformaldehyde at 4°C for 48 h and were then immersed in a carboxymethyl cellulose (CMC) gel, transferred to hexane, and completely frozen using solid CO2. The frozen samples were cut into 6 μm thick sections using a CM 3050S IV cryomicrotome (Leica Instruments, Germany). After drying, the sections were stained with hematoxylin and eosin (HE) using standard procedures (Kureha Special Laboratory Co., Tokyo, Japan).
2.5. Real-Time PCR
Total RNA was extracted from monolayer and multilayered mesenchymal cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and was used as template for first-strand cDNA synthesis using SuperScript III RT (Invitrogen). The PCR reaction mixture consisted of 2 μL cDNA, specific primer set (0.2 μM final concentration), 12.5 μL SYBR Premix Ex Taq (Takara, Kyoto, Japan), and an appropriate amount of ultrapure sterile, deionized water to give a final volume of 25 μL. The sequences of the PCR primer pairs used in this study are listed in Table 1. Quantitative PCR was performed using a Real-Time PCR Detection System (CFX-96; Bio-Rad, CA, USA) and the following PCR cycle parameters: denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. mRNA expression of the target genes was normalized to the levels of GAPDH mRNA ().
2.6. Bone Formation Ability of Multilayered Mesenchymal Cell Construct In Vivo
To investigate the bone formation ability of MLMCs in vivo, FN-G nanofilm-coated MLMCs were seeded onto culture inserts, which contain an atelocollagen membrane on the anterior surface (Koken Co., Ltd., Japan). After two days of incubation at 37°C in 5% CO2, the collagen membranes with MLMCs were cut out from the bottom of the culture insert and were further incubated with or without 0.58 nmoles CB-bFGF solutions at 4°C for 30 min. To prepare rat femurs for receiving the MLMC grafts, leg hair was shaved and a skin incision was then made in the center of the thigh to expose the right femur of the hind leg. MLMCs were then grafted onto the periosteum of the anterior surface of the exposed femur. Sham operated rats without grafting were used as controls. After surgery, the rats were allowed to use their hind legs without restriction. To assess new bone formation, 8 rats in each group were sacrificed with excess CO2 gas 7 days after performing the MLMCs grafting.
2.7. Quantification of New Bone Volume and Bone Mineral Content
Seven days after performing the MLMCs grafts, rats were sacrificed, and the femurs and surrounding muscle were excised. The femurs were placed in 4% paraformaldehyde, stored at 4°C for 48 h, and then immersed in PBS. Micro-CT images of the graft regions were captured using a Microfocus X-ray CT system (inspeXio SMX-90CT; Shimadzu Co., Ltd., Tokyo, Japan) and the following settings: acceleration voltage, 90 kV; current, 110 mA; voxel size, 20 μm/pixel; and matrix size, 1024 × 1024. Images of the entire femur were first obtained, and 10 mm regions of interest (500 slices) were defined at the midfemur. New bone volume and bone mineral content were measured using three-dimensional (3D) image analysis software (Tri-3D-Bon; Ratoc System Engineering Co., Ltd., Tokyo, Japan) as previously described [28, 31].
3.1. Preparation of MLMC Constructs
Isolated periosteal cells were positive for the mesenchymal stem cell markers CD54 and CD29 and CD54 and CD90 and were negative for the hematopoietic cell marker CD45 (Figure 1(a)). After two days of culture, the non-FG-N-coated cells formed a single layer on the cell culture plate inserts. In contrast, the FG-N-coated periosteal mesenchymal cells clearly formed a layer with an average thickness of eight cells (Figure 1(b)).
3.2. Gene Profile of MLMCs
Mesenchymal stem cells secrete trophic factors such as VEGF, TGF-b, BMP-2, and bFGF, which contribute to musculoskeletal regeneration in cell therapies [32–37]. Therefore, to assess the potential contribution of MLMC grafts to bone regeneration, we examined trophic factor gene expression in MLMCs. The expression levels of BMP-2 and VEGF in the MLMCs were significantly higher than those in monolayers of mesenchymal cells (Figure 2). In contrast, no differences in the expression of bFGF or TGβ1 were detected between the multilayered and monolayer mesenchymal cells.
3.3. In Vivo Periosteal Bone Formation by MLMCs and CB-bFGF-Anchored MLMCs
Periosteal bone formation in rat femurs subjected to a sham operation or grafted with MLMCs either alone or in combination with CB-bFGF (CB-bFGF/MLMCs) was evaluated by micro-CT image analysis one week after grafting. In femurs grafted with MLMCs and CB-bFGF/MLMCs, higher levels of bone formation and bone mineral content were observed compared to the femurs of sham operated rats (Figures 3, 4(a), and 4(b)). Notably, the new bone volume and bone mineral content were significantly higher in femurs grafted with CB-bFGF/MLMCs compared to the femurs grafted with MLMCs alone (Figures 3, 4(a), and 4(b)).
To develop a novel and effective method for cell-based regenerative therapy, rat mesenchymal cells were coated with FN/G to promote the rapid formation of cell layers. The resulting MLMCs had higher expression levels of BMP-2 and VEGF compared to monolayer-cultured mesenchymal cells. When grafted into rat femur sites, the MLMCs promoted significantly higher callus formation and the resulting bone had a higher mineral content compared to the bone of the sham controls. Further increases in bone formation and mineral content were observed in femur sites grafted with MLMCs combined with the recombinant protein CB-bFGF. Taken together, the results of the present study suggest that the CB-bFGF/MLMC construct, which can be simply and rapidly generated in vitro, may have the potential to promote bone formation in a clinical setting.
Cell sheet technologies have been successfully developed and used for bone regeneration in several animal models [38, 39]. For example, the transplantation of monolayer-cultured mesenchymal stem cell sheet enhances bone formation in a rat nonunion model . Similarly, mesenchymal stem cell sheets constructed using magnetite nanoparticles stimulated ossification in rat crania . To overcome inherent problems like the fact that nonbiocompatible materials, such as magnetic particles, remain intracellular, here, multilayers of periosteal mesenchymal cells were constructed using an FN-G nanofilm-based technique. We confirmed that the MLMCs were formed after only two days of culture and further demonstrated that transplantation of the MLMCs into rat femurs promoted bone formation. These results suggest that the coating of MSCs with FN-G nanofilms promoted multicell layer formation without leaving residual nonbiocompatible materials and that this tissue engineering technique may be useful for bone regenerative therapy.
Therapeutic cells such as MSCs, embryonic stem cells and endothelial cells secrete numerous trophic factors that contribute to tissue repair [34–37, 40–42]. For example, MSC-conditioned medium accelerates osteogenesis in a distraction osteogenesis mouse model , and endothelial cell-produced BMP-2 promotes the osteogenic differentiation of MSCs. Further, the secretion of VEGF by MSCs on a hydroxyapatite/poly(lactide-co-glycolide) scaffold was speculated to contribute to osteogenesis when the cellular construct was transplanted into the dorsum of nude rats . In addition, the production of VEGF and the observed osteogenic effects were shown to be dependent on the composition of the scaffold. Here, the expression levels of BMP-2 and VEGF were increased in MLMCs compared to monolayer mesenchymal cells. Consistent with this finding, MLMCs stimulated periosteal bone formation in vivo.
To enhance osteogenic activity, the transplantation of MSCs combined with growth factors, particularly bFGF, has been investigated [16, 43]. bFGF has anabolic effects in the process of bone regeneration and also stimulates MSCs proliferation [20–23]. MSCs have been used as vectors for bFGF gene transfer and local drug delivery to promote bone regeneration following distraction osteogenesis in a rabbit model . However, a major concern of gene therapy is the ability to control transgene expression, as the overexpression of growth factors may lead to adverse side effects . We previously established a sustained release system consisting of collagen materials coated with a recombinant bFGF containing a PKD and CBD derived from C. histolyticum ColH and demonstrated that this system accelerates periosteal bone formation and growth in bone fracture and defect models [28–30, 45]. Here, the CB-bFGF/MLMC constructs stimulated periosteal bone formation in a rat femur to a significantly higher extent compared to MLMCs alone, suggesting that the CB-bFGF/MLMC constructs are promising materials for accelerating bone formation in clinical settings.
bFGF and BMP-2 promote mesenchymal proliferation and osteogenic differentiation during bone healing, respectively . Previous studies also reported that the combination of bFGF and BMP2 synergistically promote bone formation [46–48]. For example, the combination of BMP-2 and bFGF enhances osteoblastic differentiation of cultured rat bone marrow mesenchymal stromal cells in vitro and synergistically promotes ectopic bone formation in vivo . In addition, coadministration of bFGF and BMP-2 promotes cranial  and mandibular defects . These findings suggest that the bone forming ability of CB-bFGF/MLMC constructs may result from the actions of bFGF- and MLMC-secreted BMP-2 to stimulate proliferation and promote differentiation, respectively.
Several limitations of the present study warrant mention. First, we did not evaluate whether the CB-bFGF/MLMC construct has the potential to promote bone repair when grafted into large defect sites. Second, whether MSCs generated using this system differentiate into bone in the transplanted site in vivo remains to be determined. Cell tracing should be performed to evaluate the fate of MSCs. Finally, we used rat mesenchymal cells. Further investigation using human mesenchymal cells is needed to examine the potential of CB-bFGF/MLMC constructs in clinical settings.
In conclusion, we developed a novel and simple system for promoting bone formation by combining MLMCs and a bFGF-PKD-CBD fusion protein. Compared to MLMCs alone, the MLMCs/bFGF-PKD-CBD construct significantly increased callus volume and bone mineral content at the fracture sites within two weeks of grafting. These findings suggest that the MLMC/bFGF-PKD-CBD composite is a promising and relatively simple method for promoting bone formation in a clinical setting.
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
All authors provided intellectual contributions to the manuscript and read the final version. Kentaro Uchida, Osamu Matsushita, and Masashi Takaso were responsible for experimental design; Kentaro Uchida, Gen Inoue, Kyosuke Horikawa, Hiroyuki Sekiguchi, Wataru Saito, Shotaro Takano, Hisako Fujimaki, and Masayuki Miyagi were responsible for acquisition of data; Kentaro Uchida, Hiroyuki Sekiguchi, and Masashi Takaso were responsible for analysis and interpretation of data; Kentaro Uchida and Masashi Takaso were responsible for drafting of the manuscript.
This investigation was supported in part by JSPS KAKENHI Grants nos. 25670659, 15K20015, 2681209, and 26460527, the Uehara Memorial Foundation, Kitasato University Research Grant for Young Researchers, Research Grant for Young Physicians and Health Professionals from SRL Inc., and research grants from the Parents’ Association of Kitasato University School of Medicine.
- A. I. Caplan, “Mesenchymal stem cells,” Journal of Orthopaedic Research, vol. 9, no. 5, pp. 641–650, 1991.
- Y. Mabuchi and Y. Matsuzaki, “Prospective isolation of resident adult human mesenchymal stem cell population from multiple organs,” International Journal of Hematology, vol. 103, no. 2, pp. 138–144, 2016.
- Y. Mabuchi, S. Morikawa, S. Harada et al., “LNGFR(+), THY-1(+), VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells,” Stem Cell Reports, vol. 1, no. 2, pp. 152–165, 2013.
- Y. Matsuzaki, Y. Mabuchi, and H. Okano, “Leptin receptor makes its mark on MSCs,” Cell Stem Cell, vol. 15, no. 2, pp. 112–114, 2014.
- S. Morikawa, Y. Mabuchi, Y. Kubota et al., “Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow,” Journal of Experimental Medicine, vol. 206, no. 11, pp. 2483–2496, 2009.
- N. Asakawa, T. Shimizu, Y. Tsuda et al., “Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering,” Biomaterials, vol. 31, no. 14, pp. 3903–3909, 2010.
- A. Kushida, M. Yamato, C. Konno, A. Kikuchi, Y. Sakurai, and T. Okano, “Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces,” Journal of Biomedical Materials Research, vol. 45, no. 4, pp. 355–362, 1999.
- T. Sasagawa, T. Shimizu, S. Sekiya et al., “Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology,” Biomaterials, vol. 31, no. 7, pp. 1646–1654, 2010.
- A. Ito, Y. Takizawa, H. Honda et al., “Tissue engineering using magnetite nanoparticles and magnetic force: Heterotypic layers of cocultured hepatocytes and endothelial cells,” Tissue Engineering, vol. 10, no. 5-6, pp. 833–840, 2004.
- Y. Yamamoto, A. Ito, H. Fujita, E. Nagamori, Y. Kawabe, and M. Kamihira, “Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique,” Tissue Engineering - Part A, vol. 17, no. 1-2, pp. 107–114, 2011.
- L. Grossin, D. Cortial, B. Saulnier et al., “Step-by-step build-up of biologically active cell-containing stratified films aimed at tissue engineering,” Advanced Materials, vol. 21, no. 6, pp. 650–655, 2009.
- A. Nishiguchi, H. Yoshida, M. Matsusaki, and M. Akashi, “Rapid construction of three-dimensional multilayered tissues with endothelial tube networks by the cell-accumulation technique,” Advanced Materials, vol. 23, no. 31, pp. 3506–3510, 2011.
- L. Chen, E. E. Tredget, P. Y. G. Wu, Y. Wu, and Y. Wu, “Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing,” PLoS ONE, vol. 3, no. 4, Article ID e1886, 2008.
- S. H. Bhang, Y. E. Lee, S.-W. Cho et al., “Basic fibroblast growth factor promotes bone marrow stromal cell transplantation-mediated neural regeneration in traumatic brain injury,” Biochemical and Biophysical Research Communications, vol. 359, no. 1, pp. 40–45, 2007.
- M. Ikeda, T. Uemura, K. Takamatsu et al., “Acceleration of peripheral nerve regeneration using nerve conduits in combination with induced pluripotent stem cell technology and a basic fibroblast growth factor drug delivery system,” Journal of Biomedical Materials Research A, vol. 102, no. 5, pp. 1370–1378, 2014.
- X. Jiang, S. Zou, B. Ye, S. Zhu, Y. Liu, and J. Hu, “bFGF-Modified BMMSCs enhance bone regeneration following distraction osteogenesis in rabbits,” Bone, vol. 46, no. 4, pp. 1156–1161, 2010.
- C. Yang, Y. Liu, C. Li, and B. Zhang, “Repair of mandibular defects by bone marrow stromal cells expressing the basic fibroblast growth factor transgene combined with multi-pore mineralized Bio-Oss,” Molecular Medicine Reports, vol. 7, no. 1, pp. 99–104, 2013.
- R. Baffour, J. Berman, J. L. Garb, S. W. Rhee, J. Kaufman, and P. Friedmann, “Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: Dose-response effect of basic fibroblast growth factor,” Journal of Vascular Surgery, vol. 16, no. 2, pp. 181–191, 1992.
- J. Stark, R. Baffour, J. L. Garb et al., “Basic fibroblast growth factor stimulates angiogenesis in the hindlimb of hyperglycemic rats,” Journal of Surgical Research, vol. 79, no. 1, pp. 8–12, 1998.
- H. Kawaguchi, T. Kurokawa, K. Hanada et al., “Stimulation of fracture repair by recombinant human basic fibroblast growth factor in normal and streptozotocin-diabetic rats,” Endocrinology, vol. 135, no. 2, pp. 774–781, 1994.
- T. Nakamura, K. Hanada, M. Tamura et al., “Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats,” Endocrinology, vol. 136, no. 3, pp. 1276–1284, 1995.
- H. Schliephake, F. W. Neukam, A. Löhr, and D. Hutmacher, “The use of basic fibroblast growth factor (bFGF) for enhancement of bone ingrowth into pyrolized bovine bone,” International Journal of Oral and Maxillofacial Surgery, vol. 24, no. 2, pp. 181–186, 1995.
- Y. Tabata, K. Yamada, S. Miyamoto et al., “Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels,” Biomaterials, vol. 19, no. 7-9, pp. 807–815, 1998.
- K. Yamada, Y. Tabata, K. Yamamoto et al., “Potential efficacy of basic fibroblast growth factor incorporated in biodegradable hydrogels for skull bone regeneration,” Journal of Neurosurgery, vol. 86, no. 5, pp. 871–875, 1997.
- H. Fujimaki, K. Uchida, G. Inoue et al., “Oriented collagen tubes combined with basic fibroblast growth factor promote peripheral nerve regeneration in a 15 mm sciatic nerve defect rat model,” Journal of Biomedical Materials Research Part A, vol. 105, no. 1, pp. 8–14, 2017.
- S. Wang, Q. Cai, J. Hou et al., “Acceleration effect of basic fibroblast growth factor on the regeneration of peripheral nerve through a 15-mm gap,” Journal of Biomedical Materials Research, vol. 66A, no. 3, pp. 522–531, 2003.
- N. Nishi, O. Matsushita, K. Yuube, H. Miyanaka, A. Okabe, and F. Wada, “Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 12, pp. 7018–7023, 1998.
- K. Uchida, O. Matsushita, K. Naruse et al., “Acceleration of periosteal bone formation by human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase,” Journal of Biomedical Materials Research - Part A, vol. 102, no. 6, pp. 1737–1743, 2014.
- K. Uchida, O. Matsushita, N. Nishi, G. Inoue, K. Horikawa, and M. Takaso, “Enhancement of periosteal bone formation by basic fibroblast-derived growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase,” Journal of Tissue Engineering and Regenerative Medicine, 2015.
- W. Saito, K. Uchida, M. Ueno et al., “Acceleration of bone formation during fracture healing by injectable collagen powder and human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase,” Journal of Biomedical Materials Research - Part A, vol. 102, no. 9, pp. 3049–3055, 2014.
- H. Sekiguchi, K. Uchida, G. Inoue et al., “Acceleration of bone formation during fracture healing by poly(pro-hyp-gly)10 and basic fibroblast growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase,” Journal of Biomedical Materials Research - Part A, vol. 104, no. 6, pp. 1372–1378, 2016.
- F. Bortolotti, L. Ukovich, V. Razban et al., “In vivo therapeutic potential of mesenchymal stromal cells depends on the source and the isolation procedure,” Stem Cell Reports, vol. 4, no. 3, pp. 332–339, 2015.
- K. Uchida, K. Urabe, K. Naruse, M. Ujihira, K. Mabuchi, and M. Itoman, “Comparison of the cytokine-induced migratory response between primary and subcultured populations of rat mesenchymal bone marrow cells,” Journal of Orthopaedic Science, vol. 12, no. 5, pp. 484–492, 2007.
- C. Gallina, V. Turinetto, and C. Giachino, “A new paradigm in cardiac regeneration: the mesenchymal stem cell secretome,” Stem Cells International, vol. 2015, Article ID 765846, 10 pages, 2015.
- J. He, D. C. Genetos, and J. K. Leach, “Osteogenesis and trophic factor secretion are influenced by the composition of hydroxyapatite/poly(lactide-co-glycolide) composite scaffolds,” Tissue Engineering - Part A, vol. 16, no. 1, pp. 127–137, 2010.
- M. W. Maijenburg, C. E. van der Schoot, and C. Voermans, “Mesenchymal stromal cell migration: possibilities to improve cellular therapy,” Stem Cells and Development, vol. 21, no. 1, pp. 19–29, 2012.
- D. Zisa, A. Shabbir, G. Suzuki, and T. Lee, “Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair,” Biochemical and Biophysical Research Communications, vol. 390, no. 3, pp. 834–838, 2009.
- A. Nakamura, M. Akahane, H. Shigematsu et al., “Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model,” Bone, vol. 46, no. 2, pp. 418–424, 2010.
- K. Shimizu, A. Ito, T. Yoshida, Y. Yamada, M. Ueda, and H. Honda, “Bone tissue engineering with human mesenchymal stem cell sheets constructed using magnetite nanoparticles and magnetic force,” Journal of Biomedical Materials Research Part B: Applied Biomaterials, vol. 82, no. 2, pp. 471–480, 2007.
- Y. Ando, K. Matsubara, J. Ishikawa et al., “Stem cell-conditioned medium accelerates distraction osteogenesis through multiple regenerative mechanisms,” Bone, vol. 61, pp. 82–90, 2014.
- D. Kaigler, P. H. Krebsbach, P. J. Polverini, and D. J. Mooney, “Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells,” Tissue Engineering, vol. 9, no. 1, pp. 95–103, 2003.
- D. Kaigler, P. H. Krebsbach, E. R. West, K. Horger, Y.-C. Huang, and D. J. Mooney, “Endothelial cell modulation of bone marrow stromal cell osteogenic potential,” FASEB Journal, vol. 19, no. 6, pp. 665–667, 2005.
- Y. Zhang, W. Fan, L. Nothdurft et al., “In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for bone morphogenetic protein-7 gene delivery,” Tissue Engineering - Part C: Methods, vol. 17, no. 8, pp. 789–797, 2011.
- T. N. Vo, F. K. Kasper, and A. G. Mikos, “Strategies for controlled delivery of growth factors and cells for bone regeneration,” Advanced Drug Delivery Reviews, vol. 64, no. 12, pp. 1292–1309, 2012.
- M. Ueno, K. Uchida, W. Saito et al., “Acceleration of bone union after structural bone grafts with a collagen-binding basic fibroblast growth factor anchored-collagen sheet for critical-size bone defects,” Biomedical Materials (Bristol), vol. 9, no. 3, Article ID 035014, 2014.
- L. Wang, Y. Huang, K. Pan, X. Jiang, and C. Liu, “Osteogenic responses to different concentrations/ratios of BMP-2 and bFGF in bone formation,” Annals of Biomedical Engineering, vol. 38, no. 1, pp. 77–87, 2010.
- S. Akita, M. Fukui, H. Nakagawa, T. Fujii, and K. Akino, “Cranial bone defect healing is accelerated by mesenchymal stem cells induced by coadministration of bone morphogenetic protein-2 and basic fibroblast growth factor,” Wound Repair and Regeneration, vol. 12, no. 2, pp. 252–259, 2004.
- J. Su, H. Xu, J. Sun, X. Gong, and H. Zhao, “Dual delivery of BMP-2 and bFGF from a new nano-composite scaffold, loaded with vascular stents for large-size mandibular defect regeneration,” International Journal of Molecular Sciences, vol. 14, no. 6, pp. 12714–12728, 2013.
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