- Email: [email protected]
Fate, origin and roles of cells within free bone grafts
J Orthop Sci DOI 10.1007/s00776-014-0673-5
ORIGINAL ARTICLE
Fate, origin and roles of cells within free bone grafts Koichi Yano · Hiroyuki Yasuda · Kunio Takaoka · Masafumi Takahashi · Hiroaki Nakamura · Yuuki Imai · Shigeyuki Wakitani
Received: 5 July 2014 / Accepted: 1 October 2014 © The Japanese Orthopaedic Association 2014
Abstract Background The efficacy of autologous bone grafting in repairing nonunion fractures, large bone defects and spinal instability is widely accepted. However, the cellular and molecular mechanisms underlying new bone formation in bone grafting have yet to be fully elucidated. The purpose of this study was to clarify the fate, origin and the contribution of the cells within the grafted bone. Methods This study was designed to investigate the role and fate of cells contained in the grafted bone and their contribution to new bone formation in the graft in an animal model. Middiaphyseal cylindrical bone samples obtained from green fluorescent protein (GFP) transgenic and wild-type rats were transplanted into the back muscle
K. Yano · H. Yasuda · K. Takaoka · H. Nakamura · Y. Imai · S. Wakitani Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1‑4‑3 Asahimachi, Abeno‑ku, Osaka, Osaka 545‑8585, Japan M. Takahashi Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, 3311‑1 Yakushiji, Shimotsuke, Tochigi 329‑0498, Japan Y. Imai (*) Division of Integrative Pathophysiology, Proteo‑Science Center, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791‑0295, Japan e-mail: y‑[email protected]‑u.ac.jp S. Wakitani (*) Department of Artificial Joint and Biomaterials, Hiroshima University Graduate School of Biomedical Sciences, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima 739‑8553, Japan e-mail: [email protected]
of wild-type and GFP rats, respectively. The transplanted bones were evaluated by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Results Immunohistochemical analyses showed that all the cells in the newly formed bone originated from the grafted bone, and osteoblasts were gradually replaced by host cells. Conversely, osteoclasts were immediately replaced by host cells 2 weeks after the bone graft. In addition, expression of bone morphogenetic protein (Bmp)-4, Bmp receptors and Noggin in the grafted bone was significantly upregulated before new bone formation occurred, indicating that the grafted cells might contribute to the recruitment of mesenchymal cells into the graft bed. Conclusion This study revealed the possible molecular mechanisms of the contribution of cells contained in grafted bone to facilitate new bone formation.
Introduction Autologous bone grafting is a standard modality to promote local osteogenesis in the treatment of nonunion fractures, spinal instability or bone defects caused by high-energy trauma or bone tumor resection. The clinical outcome of the autograft has been acceptable because it has an excellent capacity to promote local bone formation in spite of limitations that include additional surgery to procure the bone graft, donor site morbidities and limited graft mass [1]. Recently developed molecular biology techniques enable identification of cells by marking them with β-galactosidase (β-gal) or green fluorescent protein (GFP), or by detecting the Y-chromosome using a sex-mismatched model [2–4]. However, the osteogenic mechanisms driven by the autograft at the cellular or molecular level have not been fully elucidated. For instance, the fate or role of the cells
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included in the graft has not been clarified, and there has been controvery about whether the grafted cells actually survive and propagate to form new bone. If the cells do not survive, what cellular reactions work to generate new bone in the bone graft? Among the various possible molecular mechanisms underlying new bone formation and fracture healing, the contribution of bone morphogenetic proteins (BMPs) has been studied [5–7]. Based on this background information, this study was designed to address the issues regarding the fate of the cells in the bone graft, the origin of cells contributing to graft-induced new bone formation and the possible contribution of the BMP signaling system at the bone graft location.
demineralized with 10 % ethylenediamine tetraacetic acid (EDTA, pH 7.4) for 6 weeks at 4 °C. Specimens were dehydrated through a graded ethanol series, then embedded in paraffin, sagittally sectioned with 4 μm thickness at the middle of the graft and stained routinely with hematoxylin and eosin (H&E). Bone histomorphometry
Materials and methods
Twenty areas, each of 400 × 250 μm, within the newly formed bone located in both intramedullary ends in each of ten samples from every time point at the same magnification were randomly selected. Analyses were performed using OsteoMeasure (OsteoMetrics, Atlanta, GA, USA) [11].
Animals
Immunohistochemistry (IHC) and TRAP staining
Lewis wild-type rats were purchased from Japan SLC (Shizuoka, Japan). GFP transgenic Lewis strain rats were kindly provided by PhoenixBio Co. (Tochigi, Japan) [8]. Eighty wild-type and 80 GFP transgenic male rats at 6 weeks of age were used in this series of experiments. All experiments were performed in strict accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals at Osaka City University.
After deparaffinization, IHC was performed with the usual protocols using rabbit anti-GFP antibody (1:200, Molecular Probes, Eugene, OR) and mouse monoclonal anti-human underdecarboxylated osteocalcin (OCN) antibody (1:200, Takara Bio Inc., Shiga, Japan) for mature osteoblasts. Subsequently, sections were stained with FITC-conjugated swine anti-rabbit IgG secondary antibody (DAKO, Glostrup, Denmark) and Alexa Fluor 594 goat anti-mouse IgG secondary antibody (Molecular Probes) for 2 h and then counterstained with DAPI. All pictures were taken with a Leica TCS-SP5 confocal laser microscope (Leica Microsystems, Tokyo, Japan). For osteoclasts, TRAP staining was carried out using a staining kit (Cell Garage, Tokyo, Japan) according to the manufacturer’s protocol. The ratio was quantitatively calculated in 20 middle-power visual fields randomly selected from both edges of the internal graft.
Surgical procedure Animals were anesthetized with an intraperitoneal injection of ketamine (30 mg/kg) and xylazine (10 mg/kg). Both diaphyses of the femurs of wild-type rats were surgically harvested under sterile conditions. Soft tissue, including the periosteum, was completely removed from the femurs with a scalpel. Two middiaphyseal cylindrical bone grafts of 5 mm length were cut out with a band saw and transplanted immediately into the bilateral back muscle pouches (one per pouch) of an isogenic GFP transgenic rat (wild-type to GFP series) and vice versa (GFP to wild-type series). Grafts that had been devitalized of cells using liquid nitrogen as previously reported [9, 10] were implanted as controls in the same manner as in the experimental rats. Schedule for harvesting grafts Host rats were euthanized at days 1, 4, 7, 14, 21, 32 and 42 after grafting by an overdose of anesthesia, and the grafts were harvested for histology and RNA extraction.
Real‑time reverse transcriptase‑polymerase chain reaction (RT‑PCR) Total RNA was prepared from homogenized transplanted grafts using ISOGEN. Reverse transcription was performed using Superscript II reverse transcriptase (Invitrogen). Real-time RT-PCR was performed according to the manufacturer’s instructions. TaqMan probes for Bmp-4, Bmpr1a, Bmpr2, Noggin and Gapdh were purchased from Applied Biosystems (Foster City, CA, USA). Normalization to Gapdh was performed as described previously [12]. Experiments were performed on three separate test occasions (n = 4 for each).
Histological sections
Statistical analysis
To prepare histological sections, the harvested bone grafts were fixed with 4 % paraformaldehyde (PFA) and
Data are expressed as the mean ± standard deviation (SD). Statistical differences between two groups were analyzed
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Cell fates in grafted bone
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Fig. 1 a H&E staining of representative grafts in the control (top) and experimental (middle and bottom) groups at days 4, 7, 14, 21, 32 and 42 after grafting. Bottom panels indicate higher magnification of each boxed area of middle panels. Scale bar indicates 1 mm. b Bone
morphometrical analysis in the experimental group. NBV/TV (new bone volume/total tissue volume, %). N. Ob (number of osteoblasts/ mm2). N. Oc (number of osteoclasts/mm2). Data are presented as the mean ± SD
using a two-tailed Student’s t test. Values of p < 0.05 were considered significant.
Results
throughout the 42-day experiment (Fig. 1a). These results indicated that cell-containing grafted bone, but not devitalized bone, could induce new bone formation around the graft, and it was remodeled by bone formation and resorption.
Process of new bone formation and resorption on the graft
Origin of osteoblasts and osteoclasts in newly formed bone
All rats survived without fatal complications. No rejection or foreign body reactions were observed histologically. Figure 1a shows representative sections with H&E staining after grafting. In the experimental group, newly formed bone was found along the endosteum at the edge of the internal graft at day 7 after grafting. New bone formation spread to the center of the graft at day 14 and was increased until day 21, followed by becoming gradually smaller at day 32 and 42 with pseudomarrow containing adipocytes. Temporal changes of the number of osteoblasts were similar to the pattern of new bone area (Fig. 1b). Only a few osteoclasts were found at day 7, but the number increased from day 14 (Fig. 1b). Temporal changes of the number of osteoclasts also resembled the time course of new bone formation and the number of osteoblasts. Conversely, while soft tissues penetrated into the edges of the graft in the control group, no new bone formation was found
To assess the origin of the osteoblasts, sections were analyzed using immunohistochemistry with anti-OCN to label mature osteoblasts and anti-GFP to determine cellular origin. In the experimental group, almost all of the OCN-positive cells (osteoblasts) in the new bone at day 7 originated from cells in the grafted bone when grafting from GFP transgenic to wild-type rats (Fig. 2a, b) and from wild-type to GFP transgenic rats (Fig. 2c, d). After day 7, the population of cells derived from the grafted bone was gradually decreased and completely replaced by host cells at day 42 (Fig. 2). In addition, to examine the origin of the osteoclasts, TRAP-positive cells (osteoclasts) were analyzed with or without the GFP signal. In the experimental group, almost all of the osteoclasts in the new bone originated from the grafted bone at day 7 (Fig. 3). In contrast to boneforming osteoblasts, osteoclasts differentiated from host cells took over from cells derived from the grafted bone at day 14 (Fig. 3).
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K. Yano et al. Fig. 2 Immunohistochemistry of osteocalcin (OCN) and GFP for isografts between GFP transgenic rats and wild-type rats in the experimental group and the rate of contribution of cells in the graft and cells from the host. a Immunofluorescence staining of anti-OCN (red), GFP (green) and DAPI (blue) of a graft from GFP transgenic to wild-type rats from day 7 to day 42. b Quantification of GFP signal in OCN-positive cells in a. c The same as a, but for grafts from wild-type to GFP transgenic rats. d Quantification of the GFP signal in OCN-positive cells in c. Scale bar indicates 40 μm. Data are presented as the mean ± SD
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Time sequential gene expression related to osteogenesis in the bone graft To study temporal gene expression in the grafted bone and newly formed bone, real-time RT-PCR was used. In the experimental group, the expression of Bmp-4 and Noggin mRNA was increased from days 7 to 32 and was higher than in the control, but expression in the control group was not changed (Fig. 4). The expression of Bmpr1a and Bmpr2 mRNA was increased at an earlier time point (day 4) than
13
Bmp-4 and Noggin. In addition, Bmpr1a and Bmpr2 mRNA was increased in the control group as well as the experimental group (Fig. 4).
Discussion Concerning the origin of osteoblasts for new bone formation in autografted bone, cells in the graft mainly contribute to the early phase [4, 13]. However, it has not been clear
Cell fates in grafted bone
a GFP
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Fig. 3 TRAP staining and immunohistochemistry of GFP in isografts between GFP transgenic rats and wild-type rats in the experimental group and the rate of contribution of cells in the graft and cells from the host. a Immunohistochemistry of grafts from GFP transgenic to wild-type rats harvested at day 7 and 14. b Quantification of the GFP signal in TRAP-positive cells in a. c Immunohistochemistry of grafts from wild-type to GFP transgenic rats harvested at day 7 and 14. d Quantification of GFP signal in TRAP-positive cells in c. Scale bar indicates 40 μm. Data are presented as the mean ± SD
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whether cells in the graft express genes related to bone formation [i.e., BMPs, BMP receptors (BMPRs), Noggin], how the cells in the host contribute to local bone formation or where the osteoclasts originated. It was reported that bone grafts without bone marrow treated with freezing or freeze drying do not have bone formation, but grafts with
bone marrow induce new bone formation. However, it is not known how vital cells act as bone inducers and if they interact with host cells [9]. Our experiment model, isografts at a heterotopic site, clarified the contribution of the cells originating from the graft and the host to bone formation and remodeling after
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Bmp-4
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Relative mRNA expression
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Fig. 4 Temporal changes of mRNA expression of Bmp-4, Bmpr1a, Bmpr2 and Noggin in the experimental and control groups by realtime RT-PCR. Asterisk indicates p < 0.05 representing statistically
significant differences between the experimental and control groups. Data are presented as the mean ± SD
grafting. This study focused on osteoblasts, which form new bone, and osteoclasts, which resorb bone. Our study clearly showed bone metabolism at days 7 and 14 when new bone was formed mainly by cells derived from the graft. Cells from the host appeared from day 14 and cells from the host replaced those from the graft, accounting for about 60–80 %. At day 42, in the late phase, almost all of the osteoblasts lining the new bone originated from the host. Our results were equivalent to previous studies that showed cells from the graft contributed to new bone formation in the early phases of bone grafting. However, previous studies carried out the analysis in only one way, namely focusing on the contribution of cells in the graft, and did not show the contribution of cells in the host [4, 13]. In addition, our study showed that osteoclasts in the early phase originated from the graft and almost all of the osteoclasts from day 14 onward were from the host. This phenomenon may be explained as follows: osteoclasts were induced by osteoblasts forming new bone via RANK (receptor activator of nuclear factor kappa-B)-RANK ligand signaling. In the early phase, they were induced from hematopoietic stem cells or cells of monocyte lineage in the graft, but from day 14 they were mainly induced from those in the blood flow of the host [14]. The knowledge that
the survival period of osteoclasts was only 14 days supports this theory [15]. The fact that no new bone formation was observed throughout the experimental period in the control group treated with liquid nitrogen demonstrated that new ectopic bone in the early phase could not be formed without cells in the graft and cells from the host were not subsequently mobilized to undergo osteogenesis. These results are supported by the clinical findings that the union rate is low in allograft treatments, and they are osteoconductive rather than osteoinductive [16]. The possibility that BMP-4 expressed by cells in the graft aids mobilization of host cells to undergo osteogenesis (osteoinductive action) was suggested because the expression of Bmp-4 mRNA was not detected in the control group, but was detected in cells derived from the graft at day 7. The expression of Bmp-4 mRNA was gradually replaced by host cells. The expression of Bmp-4 mRNA may act on mesenchymal stem cells, circulating osteoblastic progenitor cells, muscular cells or fibroblastic cells from the host as an osteoinductive effect [17, 18]. Nakamura et al. [6] showed that implantation of collagen-containing BMP-2 under the back muscle in mouse induced BMP receptors and noggin mRNA expression in undifferentiated mesenchymal
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Cell fates in grafted bone
cells and regenerating muscle fibers around the implant. The cellular and vascular conditions around the graft are important since radiation has a significant adverse effect on bone healing [19, 20]. In the control group, BMP receptors were expressed in spite of transplanting bone without viable cells. This expression of BMP receptors may be induced by hepatocyte growth factor (HGF) [7]. The hematoma and inflammation by the grafting procedure may introduce cascades of cellular events and secretion of cytokines such as interleukin (IL)-1, IL-6, transforming growth factor (TGF)-β, fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF) to the graft, similar to fracture healing [21, 22]. In summary, our study showed for the first time the local mechanism of osteogenesis in bone grafts: cells originating from the graft mainly formed new bone in the early phase of grafting and expression of Bmp-4 by cells originating in the graft may mobilize cells from the host to promote osteogenesis. This study provides new clues to the development of a reasonable and effective method of bone grafting. Acknowledgments We thank Dr. Amu Kawaguchi and Ms Kanako Hata for their technical assistance and PhoenixBio Co. (Tochigi, Japan) for providing GFP-transgenic rats. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 21591954 to KT). Conflict of interest All authors declare that they have no conflicts of interest.
References 1. Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA. Complications of iliac crest bone graft harvesting. Clin Orthop Relat Res. 1996;329:300–9. 2. Kubota K, Soeda J, Misawa R, Mihara M, Miwa S, Ise H, Takahashi M, Miyagawa S. Bone marrow-derived cells fuse with hepatic oval cells but are not involved in hepatic tumorigenesis in the choline-deficient ethionine-supplemented diet rat model. Carcinogenesis. 2008;29(2):448–54. 3. Colnot C, Huang S, Helms J. Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice. Biochem Biophys Res Commun. 2006;350(3):557–61. 4. Gould SE, Rhee JM, Tay B-B, Otsuka NY, Bradford DS. Cellular contribution of bone graft to fusion. J Orthop Res. 2000;18(6):920–7. 5. Yoshimura Y, Nomura S, Kawasaki S, Tsutsumimoto T, Shimizu T, Takaoka K. Colocalization of noggin and bone morphogenetic protein-4 during fracture healing. J Bone Miner Res. 2001;16(5):876–84.
6. Nakamura Y, Wakitani S, Nakayama J, Wakabayashi S, Horiuchi H, Takaoka K. Temporal and spatial expression profiles of BMP receptors and noggin during BMP-2-induced ectopic bone formation. J Bone Miner Res. 2003;18(10):1854–62. 7. Imai Y, Terai H, Nomura-Furuwatari C, Mizuno S, Matsumoto K, Nakamura T, Takaoka K. Hepatocyte growth factor contributes to fracture repair by upregulating the expression of BMP receptors. J Bone Miner Res. 2005;20(10):1723–30. 8. Inoue H, Ohsawa I, Murakami T, Kimura A, Hakamata Y, Sato Y, Kaneko T, Takahashi M, Okada T, Ozawa K, Francis J, Leone P, Kobayashi E. Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun. 2005;329(1):288–95. 9. Burwell RG. Studies in the transplantation of bone. 8. Treated composite homograft-autografts of cancellous bone: an analysis of inductive mechanisms in bone transplantation. J Bone Joint Surg Br. 1966;48(3):532–66. 10. Elves MW. Newer knowledge of the immunology of bone and cartilage. Clin Orthop Relat Res. 1976;120:232–59. 11. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1987;2(6):595–610. 12. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45. 13. Zhang X, Xie C, Lin AS, Ito H, Awad H, Lieberman JR, Rubery PT, Schwarz EM, O’Keefe RJ, Guldberg RE. Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering. J Bone Miner Res. 2005;20(12):2124–37. 14. Nakashima T, Takayanagi H. Osteoclasts and the immune system. J Bone Miner Metab. 2009;27(5):519–29. 15. Marx RE. Bone and bone graft healing. Oral Maxillofac Surg Clin North Am. 2007;19(4):455–66 v. 16. Enneking WF, Campanacci DA. Retrieved human allo grafts: a clinicopathological study. J Bone Joint Surg Am. 2001;83-A(7):971–86. 17. Liu R, Birke O, Morse A, Peacock L, Mikulec K, Little DG, Schindeler A. Myogenic progenitors contribute to open but not closed fracture repair. BMC Musculoskelet Disord. 2011;12:288. 18. Otsuru S, Tamai K, Yamazaki T, Yoshikawa H, Kaneda Y. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice. Biochem Biophys Res Commun. 2007;354(2):453–8. 19. Bouchard JA, Koka A, Bensusan JS, Stevenson S, Emery SE. Effects of irradiation on posterior spinal fusions. A rabbit model. Spine (Phila Pa 1976). 1994;19(16):1836–41. 20. Ames CP, Smith JS, Preul MC, Crawford NR, Kim GE, Nottmeier E, Chamberlain R, Speiser B, Sonntag VK, Dickman CA. Effect of recombinant human bone morphogenetic protein-2 in an experimental model of spinal fusion in a radiated area. Spine (Phila Pa 1976). 2005;30(23):2585–92. 21. Bolander ME. Regulation of fracture repair by growth factors. Proc Soc Exp Biol Med. 1992;200(2):165–70. 22. Einhorn TA, Majeska RJ, Rush EB, Levine PM, Horowitz MC. The expression of cytokine activity by fracture callus. J Bone Miner Res. 1995;10(8):1272–81.
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ORIGINAL ARTICLE
Fate, origin and roles of cells within free bone grafts Koichi Yano · Hiroyuki Yasuda · Kunio Takaoka · Masafumi Takahashi · Hiroaki Nakamura · Yuuki Imai · Shigeyuki Wakitani
Received: 5 July 2014 / Accepted: 1 October 2014 © The Japanese Orthopaedic Association 2014
Abstract Background The efficacy of autologous bone grafting in repairing nonunion fractures, large bone defects and spinal instability is widely accepted. However, the cellular and molecular mechanisms underlying new bone formation in bone grafting have yet to be fully elucidated. The purpose of this study was to clarify the fate, origin and the contribution of the cells within the grafted bone. Methods This study was designed to investigate the role and fate of cells contained in the grafted bone and their contribution to new bone formation in the graft in an animal model. Middiaphyseal cylindrical bone samples obtained from green fluorescent protein (GFP) transgenic and wild-type rats were transplanted into the back muscle
K. Yano · H. Yasuda · K. Takaoka · H. Nakamura · Y. Imai · S. Wakitani Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1‑4‑3 Asahimachi, Abeno‑ku, Osaka, Osaka 545‑8585, Japan M. Takahashi Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, 3311‑1 Yakushiji, Shimotsuke, Tochigi 329‑0498, Japan Y. Imai (*) Division of Integrative Pathophysiology, Proteo‑Science Center, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791‑0295, Japan e-mail: y‑[email protected]‑u.ac.jp S. Wakitani (*) Department of Artificial Joint and Biomaterials, Hiroshima University Graduate School of Biomedical Sciences, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima 739‑8553, Japan e-mail: [email protected]
of wild-type and GFP rats, respectively. The transplanted bones were evaluated by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Results Immunohistochemical analyses showed that all the cells in the newly formed bone originated from the grafted bone, and osteoblasts were gradually replaced by host cells. Conversely, osteoclasts were immediately replaced by host cells 2 weeks after the bone graft. In addition, expression of bone morphogenetic protein (Bmp)-4, Bmp receptors and Noggin in the grafted bone was significantly upregulated before new bone formation occurred, indicating that the grafted cells might contribute to the recruitment of mesenchymal cells into the graft bed. Conclusion This study revealed the possible molecular mechanisms of the contribution of cells contained in grafted bone to facilitate new bone formation.
Introduction Autologous bone grafting is a standard modality to promote local osteogenesis in the treatment of nonunion fractures, spinal instability or bone defects caused by high-energy trauma or bone tumor resection. The clinical outcome of the autograft has been acceptable because it has an excellent capacity to promote local bone formation in spite of limitations that include additional surgery to procure the bone graft, donor site morbidities and limited graft mass [1]. Recently developed molecular biology techniques enable identification of cells by marking them with β-galactosidase (β-gal) or green fluorescent protein (GFP), or by detecting the Y-chromosome using a sex-mismatched model [2–4]. However, the osteogenic mechanisms driven by the autograft at the cellular or molecular level have not been fully elucidated. For instance, the fate or role of the cells
13
K. Yano et al.
included in the graft has not been clarified, and there has been controvery about whether the grafted cells actually survive and propagate to form new bone. If the cells do not survive, what cellular reactions work to generate new bone in the bone graft? Among the various possible molecular mechanisms underlying new bone formation and fracture healing, the contribution of bone morphogenetic proteins (BMPs) has been studied [5–7]. Based on this background information, this study was designed to address the issues regarding the fate of the cells in the bone graft, the origin of cells contributing to graft-induced new bone formation and the possible contribution of the BMP signaling system at the bone graft location.
demineralized with 10 % ethylenediamine tetraacetic acid (EDTA, pH 7.4) for 6 weeks at 4 °C. Specimens were dehydrated through a graded ethanol series, then embedded in paraffin, sagittally sectioned with 4 μm thickness at the middle of the graft and stained routinely with hematoxylin and eosin (H&E). Bone histomorphometry
Materials and methods
Twenty areas, each of 400 × 250 μm, within the newly formed bone located in both intramedullary ends in each of ten samples from every time point at the same magnification were randomly selected. Analyses were performed using OsteoMeasure (OsteoMetrics, Atlanta, GA, USA) [11].
Animals
Immunohistochemistry (IHC) and TRAP staining
Lewis wild-type rats were purchased from Japan SLC (Shizuoka, Japan). GFP transgenic Lewis strain rats were kindly provided by PhoenixBio Co. (Tochigi, Japan) [8]. Eighty wild-type and 80 GFP transgenic male rats at 6 weeks of age were used in this series of experiments. All experiments were performed in strict accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals at Osaka City University.
After deparaffinization, IHC was performed with the usual protocols using rabbit anti-GFP antibody (1:200, Molecular Probes, Eugene, OR) and mouse monoclonal anti-human underdecarboxylated osteocalcin (OCN) antibody (1:200, Takara Bio Inc., Shiga, Japan) for mature osteoblasts. Subsequently, sections were stained with FITC-conjugated swine anti-rabbit IgG secondary antibody (DAKO, Glostrup, Denmark) and Alexa Fluor 594 goat anti-mouse IgG secondary antibody (Molecular Probes) for 2 h and then counterstained with DAPI. All pictures were taken with a Leica TCS-SP5 confocal laser microscope (Leica Microsystems, Tokyo, Japan). For osteoclasts, TRAP staining was carried out using a staining kit (Cell Garage, Tokyo, Japan) according to the manufacturer’s protocol. The ratio was quantitatively calculated in 20 middle-power visual fields randomly selected from both edges of the internal graft.
Surgical procedure Animals were anesthetized with an intraperitoneal injection of ketamine (30 mg/kg) and xylazine (10 mg/kg). Both diaphyses of the femurs of wild-type rats were surgically harvested under sterile conditions. Soft tissue, including the periosteum, was completely removed from the femurs with a scalpel. Two middiaphyseal cylindrical bone grafts of 5 mm length were cut out with a band saw and transplanted immediately into the bilateral back muscle pouches (one per pouch) of an isogenic GFP transgenic rat (wild-type to GFP series) and vice versa (GFP to wild-type series). Grafts that had been devitalized of cells using liquid nitrogen as previously reported [9, 10] were implanted as controls in the same manner as in the experimental rats. Schedule for harvesting grafts Host rats were euthanized at days 1, 4, 7, 14, 21, 32 and 42 after grafting by an overdose of anesthesia, and the grafts were harvested for histology and RNA extraction.
Real‑time reverse transcriptase‑polymerase chain reaction (RT‑PCR) Total RNA was prepared from homogenized transplanted grafts using ISOGEN. Reverse transcription was performed using Superscript II reverse transcriptase (Invitrogen). Real-time RT-PCR was performed according to the manufacturer’s instructions. TaqMan probes for Bmp-4, Bmpr1a, Bmpr2, Noggin and Gapdh were purchased from Applied Biosystems (Foster City, CA, USA). Normalization to Gapdh was performed as described previously [12]. Experiments were performed on three separate test occasions (n = 4 for each).
Histological sections
Statistical analysis
To prepare histological sections, the harvested bone grafts were fixed with 4 % paraformaldehyde (PFA) and
Data are expressed as the mean ± standard deviation (SD). Statistical differences between two groups were analyzed
13
Cell fates in grafted bone
D4
a
D7
D14
D21
D32
D42
Control group
40
240
20
0
1
4
7
14
21
32
Days after grafting (D)
42
90
N. Oc (/mm 2)
60
N. Ob (/mm 2 )
b NBV/TV (%)
Experimental group
160
80
0
1
4
7
14
21
32
Days after grafting (D)
42
60
30
0
1
4
7
14
21
32
42
Days after grafting (D)
Fig. 1 a H&E staining of representative grafts in the control (top) and experimental (middle and bottom) groups at days 4, 7, 14, 21, 32 and 42 after grafting. Bottom panels indicate higher magnification of each boxed area of middle panels. Scale bar indicates 1 mm. b Bone
morphometrical analysis in the experimental group. NBV/TV (new bone volume/total tissue volume, %). N. Ob (number of osteoblasts/ mm2). N. Oc (number of osteoclasts/mm2). Data are presented as the mean ± SD
using a two-tailed Student’s t test. Values of p < 0.05 were considered significant.
Results
throughout the 42-day experiment (Fig. 1a). These results indicated that cell-containing grafted bone, but not devitalized bone, could induce new bone formation around the graft, and it was remodeled by bone formation and resorption.
Process of new bone formation and resorption on the graft
Origin of osteoblasts and osteoclasts in newly formed bone
All rats survived without fatal complications. No rejection or foreign body reactions were observed histologically. Figure 1a shows representative sections with H&E staining after grafting. In the experimental group, newly formed bone was found along the endosteum at the edge of the internal graft at day 7 after grafting. New bone formation spread to the center of the graft at day 14 and was increased until day 21, followed by becoming gradually smaller at day 32 and 42 with pseudomarrow containing adipocytes. Temporal changes of the number of osteoblasts were similar to the pattern of new bone area (Fig. 1b). Only a few osteoclasts were found at day 7, but the number increased from day 14 (Fig. 1b). Temporal changes of the number of osteoclasts also resembled the time course of new bone formation and the number of osteoblasts. Conversely, while soft tissues penetrated into the edges of the graft in the control group, no new bone formation was found
To assess the origin of the osteoblasts, sections were analyzed using immunohistochemistry with anti-OCN to label mature osteoblasts and anti-GFP to determine cellular origin. In the experimental group, almost all of the OCN-positive cells (osteoblasts) in the new bone at day 7 originated from cells in the grafted bone when grafting from GFP transgenic to wild-type rats (Fig. 2a, b) and from wild-type to GFP transgenic rats (Fig. 2c, d). After day 7, the population of cells derived from the grafted bone was gradually decreased and completely replaced by host cells at day 42 (Fig. 2). In addition, to examine the origin of the osteoclasts, TRAP-positive cells (osteoclasts) were analyzed with or without the GFP signal. In the experimental group, almost all of the osteoclasts in the new bone originated from the grafted bone at day 7 (Fig. 3). In contrast to boneforming osteoblasts, osteoclasts differentiated from host cells took over from cells derived from the grafted bone at day 14 (Fig. 3).
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K. Yano et al. Fig. 2 Immunohistochemistry of osteocalcin (OCN) and GFP for isografts between GFP transgenic rats and wild-type rats in the experimental group and the rate of contribution of cells in the graft and cells from the host. a Immunofluorescence staining of anti-OCN (red), GFP (green) and DAPI (blue) of a graft from GFP transgenic to wild-type rats from day 7 to day 42. b Quantification of GFP signal in OCN-positive cells in a. c The same as a, but for grafts from wild-type to GFP transgenic rats. d Quantification of the GFP signal in OCN-positive cells in c. Scale bar indicates 40 μm. Data are presented as the mean ± SD
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Time sequential gene expression related to osteogenesis in the bone graft To study temporal gene expression in the grafted bone and newly formed bone, real-time RT-PCR was used. In the experimental group, the expression of Bmp-4 and Noggin mRNA was increased from days 7 to 32 and was higher than in the control, but expression in the control group was not changed (Fig. 4). The expression of Bmpr1a and Bmpr2 mRNA was increased at an earlier time point (day 4) than
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Bmp-4 and Noggin. In addition, Bmpr1a and Bmpr2 mRNA was increased in the control group as well as the experimental group (Fig. 4).
Discussion Concerning the origin of osteoblasts for new bone formation in autografted bone, cells in the graft mainly contribute to the early phase [4, 13]. However, it has not been clear
Cell fates in grafted bone
a GFP
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Fig. 3 TRAP staining and immunohistochemistry of GFP in isografts between GFP transgenic rats and wild-type rats in the experimental group and the rate of contribution of cells in the graft and cells from the host. a Immunohistochemistry of grafts from GFP transgenic to wild-type rats harvested at day 7 and 14. b Quantification of the GFP signal in TRAP-positive cells in a. c Immunohistochemistry of grafts from wild-type to GFP transgenic rats harvested at day 7 and 14. d Quantification of GFP signal in TRAP-positive cells in c. Scale bar indicates 40 μm. Data are presented as the mean ± SD
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whether cells in the graft express genes related to bone formation [i.e., BMPs, BMP receptors (BMPRs), Noggin], how the cells in the host contribute to local bone formation or where the osteoclasts originated. It was reported that bone grafts without bone marrow treated with freezing or freeze drying do not have bone formation, but grafts with
bone marrow induce new bone formation. However, it is not known how vital cells act as bone inducers and if they interact with host cells [9]. Our experiment model, isografts at a heterotopic site, clarified the contribution of the cells originating from the graft and the host to bone formation and remodeling after
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K. Yano et al.
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Fig. 4 Temporal changes of mRNA expression of Bmp-4, Bmpr1a, Bmpr2 and Noggin in the experimental and control groups by realtime RT-PCR. Asterisk indicates p < 0.05 representing statistically
significant differences between the experimental and control groups. Data are presented as the mean ± SD
grafting. This study focused on osteoblasts, which form new bone, and osteoclasts, which resorb bone. Our study clearly showed bone metabolism at days 7 and 14 when new bone was formed mainly by cells derived from the graft. Cells from the host appeared from day 14 and cells from the host replaced those from the graft, accounting for about 60–80 %. At day 42, in the late phase, almost all of the osteoblasts lining the new bone originated from the host. Our results were equivalent to previous studies that showed cells from the graft contributed to new bone formation in the early phases of bone grafting. However, previous studies carried out the analysis in only one way, namely focusing on the contribution of cells in the graft, and did not show the contribution of cells in the host [4, 13]. In addition, our study showed that osteoclasts in the early phase originated from the graft and almost all of the osteoclasts from day 14 onward were from the host. This phenomenon may be explained as follows: osteoclasts were induced by osteoblasts forming new bone via RANK (receptor activator of nuclear factor kappa-B)-RANK ligand signaling. In the early phase, they were induced from hematopoietic stem cells or cells of monocyte lineage in the graft, but from day 14 they were mainly induced from those in the blood flow of the host [14]. The knowledge that
the survival period of osteoclasts was only 14 days supports this theory [15]. The fact that no new bone formation was observed throughout the experimental period in the control group treated with liquid nitrogen demonstrated that new ectopic bone in the early phase could not be formed without cells in the graft and cells from the host were not subsequently mobilized to undergo osteogenesis. These results are supported by the clinical findings that the union rate is low in allograft treatments, and they are osteoconductive rather than osteoinductive [16]. The possibility that BMP-4 expressed by cells in the graft aids mobilization of host cells to undergo osteogenesis (osteoinductive action) was suggested because the expression of Bmp-4 mRNA was not detected in the control group, but was detected in cells derived from the graft at day 7. The expression of Bmp-4 mRNA was gradually replaced by host cells. The expression of Bmp-4 mRNA may act on mesenchymal stem cells, circulating osteoblastic progenitor cells, muscular cells or fibroblastic cells from the host as an osteoinductive effect [17, 18]. Nakamura et al. [6] showed that implantation of collagen-containing BMP-2 under the back muscle in mouse induced BMP receptors and noggin mRNA expression in undifferentiated mesenchymal
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Cell fates in grafted bone
cells and regenerating muscle fibers around the implant. The cellular and vascular conditions around the graft are important since radiation has a significant adverse effect on bone healing [19, 20]. In the control group, BMP receptors were expressed in spite of transplanting bone without viable cells. This expression of BMP receptors may be induced by hepatocyte growth factor (HGF) [7]. The hematoma and inflammation by the grafting procedure may introduce cascades of cellular events and secretion of cytokines such as interleukin (IL)-1, IL-6, transforming growth factor (TGF)-β, fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF) to the graft, similar to fracture healing [21, 22]. In summary, our study showed for the first time the local mechanism of osteogenesis in bone grafts: cells originating from the graft mainly formed new bone in the early phase of grafting and expression of Bmp-4 by cells originating in the graft may mobilize cells from the host to promote osteogenesis. This study provides new clues to the development of a reasonable and effective method of bone grafting. Acknowledgments We thank Dr. Amu Kawaguchi and Ms Kanako Hata for their technical assistance and PhoenixBio Co. (Tochigi, Japan) for providing GFP-transgenic rats. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 21591954 to KT). Conflict of interest All authors declare that they have no conflicts of interest.
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