- Email: [email protected]
Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects
Bone 45 (2009) 918–924
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Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects Nimrod Rozen a,b,1, Tova Bick b,1, Alon Bajayo c, Ben Shamian b, Michal Schrift-Tzadok d, Yankel Gabet c, Avner Yayon d,e,2, Itai Bab c,2, Michael Soudry b,2, Dina Lewinson b,⁎ a
Department of Orthopaedic Surgery, Ha'emek Medical Center, Afula 18101, Israel Research Institute for Bone Repair, Orthopaedic Surgery A, Rambam Health Care Campus, 8, Ha'aliya st., Bat-Galim, Haifa 31096, Israel Bone Laboratory, Institute of Dental Sciences, The Hebrew University of Jerusalem, Jerusalem 91120, Israel d ProChon Biotech Ltd., Nes-Ziona 70400, Israel e ProCore BioMed Ltd., Nes-Ziona 70400, Israel b c
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Article history: Received 4 June 2009 Revised 28 July 2009 Accepted 29 July 2009 Available online 6 August 2009 Edited by: T. Einhorn Keywords: Bone regeneration Critical-size gap Endothelial progenitor cells (EPCs) Sheep Micro-computed tomography
a b s t r a c t The angiogenic events that accompany bone regeneration function as a “limiting factor” and are the primary regulatory mechanisms that direct the healing process. The general aim of this study was to test whether blood-derived progenitor cells that have endothelial characteristics (EPC), when applied to a large segmental defect, would promote bone regeneration. We established a critical-sized gap platform in sheep tibiae. Our model system takes advantage of the physiological wound healing process that occurs during the first two weeks following injury, and results in the gap being filled with scar tissue. EPC were expanded ex-vivo and 2 × 107 cells/0.2 ml were implanted into a wedged-shaped canal excavated in the fibrotic scar tissue. Sham treated sheep served as controls. Bone regeneration was followed every two weeks for three months by Xray radiography. At the end of the experimental period, the regenerating segments were subjected to microcomputed tomographic (μCT) analysis. While minimal bone formation was detected in sham-treated sheep, six out of seven autologous EPC-transplanted sheep showed initial mineralization already by 2 weeks and complete bridging by 8–12 weeks post EPC transplantation. Histology of gaps 12 weeks post sham treatment showed mostly fibrotic scar tissue. On the contrary, EPC transplantation led to formation of dense and massive woven bone all throughout the defect. The results of this preclinical study open new therapeutic opportunities for the treatment of large scale bone injuries. © 2009 Elsevier Inc. All rights reserved.
Introduction Healing of large bone defects represents a great challenge to reconstructive surgery. Bone must be regenerated in order to fill in the defect and restore structure and function. Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Therefore angiogenesis plays a pivotal role in successful bone regeneration [1,2]. Present treatments are accompanied with pain and risk of infection, hemorrhage, cosmetic disability, nerve damage and loss of function [3]. Thus, there is a significant need for an alternative strategy for the treatment of severe bone loss and delayed or non-union fracture. An emerging approach to damage repair is tissue engineering which involves treatment with stem cells [4]. Cell-based therapies
⁎ Corresponding author. Fax: +972 48543606. E-mail address: [email protected] (D. Lewinson). 1 Nimrod Rozen and Tova Bick contributed equally to this work. 2 Avner Yayon, Itai Bab and Michael Soudry have equal academic contributions to the project, in spite of the difference in their area of expertise. 8756-3282/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2009.07.085
used until now involved fresh bone marrow or ex-vivo expanded mesenchymal stem cells, usually combined with scaffolds. Although promising clinical results have been achieved [5–7], the necessity of anaesthesia and an invasive manipulation requiring a minimum of two surgeries, is a disadvantage. Therefore, there is a need to isolate stem or progenitor cells from a more accessible source, such as peripheral blood. Endothelial progenitor cells (EPC) that reside in the bone marrow have been identified and isolated also from peripheral blood of adult human and umbilical cord [8,9]. These cells have been shown to participate in postnatal neovascularization, in revascularization of ischaemic hind-limb and in acute myocardial infarction ischaemia [10–12]. Distraction osteogenesis (DO), which is a bone regeneration system, is another example of ischaemic regenerating tissue [2,13]. Moreover, systemically injected EPC were shown to incorporate within the ischaemic regenerate [2,13–16]. Early studies from our group in a sheep DO model described the appearance of cellular colonies of vascular nature, that stain immunopositive for Tie-2 and factor VIII-related antigen, the origin of which was not clear [17,18]. These observations led us to hypothesize that EPC
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Fig. 1. Healing critical size gaps in sheep tibia. 2D μCT image of midsagittal plane showing fixation screw holes (asterisks). Full line marks midline between screw holes. Dotted lines mark proximal and distal borders of reference volume. Left, sham-treated tibia; right, EPC-treated tibia.
contribute to the healing process by increasing vascularization and new bone formation and might be used for enhancement of bone regeneration in large or compromised bone defects. The results obtained in the present study demonstrate that local transplantation of autologous EPC brought about complete bridging and may therefore be used as a therapeutic strategy for critical-sized gap bone fracture repair.
Materials and methods Isolation and expansion of sheep EPC Ten milliliters of peripheral venous blood was removed from sheep jugular vein into heparinized tubes. The mononuclear fraction (MNC) was separated using Lymphoprep™ (Axis-Shield PoC AS, Oslo
Fig. 2. EPC grow exponentially after trypsinization and reseeding. Magnification × 10 (A), × 40 (B). Sheep EPC stain positive for cytoplasmic von-Willebrand factor: positive staining (C) vs. no staining in control (without primary Ab) (D); incorporate Dil-Ac-LDL (stained by the red fluorescent dye, E); stain positive for flk-1 (F); form tubes in Matrigel (G). C–G, Magnification × 10. Scale = 500 μm in A, C, D, F, G and 200 μm in B and E.
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Norway) and suspended in endothelial basal medium-2 (EBM-2; Clonetics, Walkersville, MD, USA), supplemented with EGM-2MV SingleQuot® (containing 5% fetal bovine serum, vascular endothelial growth factor (VEGF), fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1 and ascorbic acid) and plated on fibronectin (Sigma Chemical Co., St. Louis, MO) -coated 10 ml plates. Cells were grown at 37 °C with humidified 95% air/5% CO2. After 4 days of culture, non-adherent cells were discarded by gentle washing with PBS, and fresh medium was applied. Adherent EPC were detected 5– 7 days after seeding, split by brief trypsinization using 0.5% trypsin/ 0.2% EDTA (Biological Industries Ltd., Beit Haemek, Israel) and dispersed. Cells reached confluence after 4–6 days. Characterization of sheep EPC Tube formation in Matrigel For demonstration of capillary tube formation, 250 μl of growth factor-reduced Matrigel (BD Biosciences Discovery Labware, Bedford, MA) was added per well of a 24-well plate and allowed to polymerize at 37 °C for at least 30 min. 5 × 104 adherent cells (suspected to be EPC) were suspended in 300 μl EGM2-MV medium and seeded onto Matrigel [19]. The cells were incubated at 37 °C with humidified 95% air/5% CO2 for 5–24 h. The tube networks were observed with an Olympus inverted microscope (Olympus, CKX41). Ac-LDL incorporation Endothelial cells have the capacity to internalize Ac-LDL. In order to demonstrate whether the cells suspected to be EPC are able to
internalize Ac-LDL, adherent cells were incubated with 10 mg/ml AcLDL coupled with fluorescent 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbocyanide perchlorate (DiI-Ac-LDL) (Molecular Probes Inc., Eugene, Oregon, USA) for 3–24 h [19]. Cells were visualized with an Olympus CXK41 inverted fluorescent microscope. Immunohistochemistry von-Willebrand factor (vWf) and Flk-1 On day 7 of culture, adherent cells were trypsinized, seeded and grown on chamber-slides for 2–3 days. Slides were subjected to immunohistochemistry/fluorescence to detect the expression of vWf or flk-1, respectively. Both proteins are regarded to be specifically expressed by endothelial cells. In brief, following fixation with 4% paraformaldehyde for 10 min at 4 °C and endogenous peroxidase inactivation immunostaining was performed. After 3 washes with PBS, slides were incubated with rabbit anti human vWf diluted 1:50 (DAKO, Glostrup, Denmark) or with mouse anti Flk-1 (VEGFR2) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA. USA) for 1 h, RT. Negative control slides were incubated without the Ab. For visualization of positive vWf, after 3 washes with PBS, slides were incubated for 10 min with HRP polymer conjugate (SuperPicTure™ Polymer Detection Kit — Zymed Laboratories, San Francisco, CA, USA) followed by 5 min incubation with AEC (RED) substrate (SuperPicTure™ Polymer Detection Kit — Zymed Laboratories, San Francisco, CA, USA). To visualize positive FLK-1, slides were incubated for 1 h in the dark with Rhodamine Red-X Affinity pure goat anti mouse IgG (H + L) RRX diluted 1:50 (Jackson Immunoresearch, Laboratories, Inc. West Grove,
Fig. 3. Representative X-ray radiographic follow-ups of sham and EPC-transplanted sheep tibiae. No significant new bone formation is observed in sham-transplanted bones (left side). EPC-transplanted bones showed a gradual filling of the defect until full defect bridging at 3 months (right side).
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PA, USA). Cells were visualized with an Olympus CXK41 inverted fluorescent microscope. Surgical procedures All surgical procedures and animal handling were approved by the Institutional Animal Care Committee of The Technion. Critical-sized gap surgery Fifteen, 2 years old female sheep (~ 70 kg), were operated in this study. Sheep were anaesthesized by 0.1 mg/kg bw Xylzine and 10 mg/ kg bw Ketamin iv and induction was achieved by 4–6 mg/kg bw Propofol iv. Maintenance was by 1.5–2.0% isofluran intubation + positive pressure ventilation of 100% oxygen, 15 breaths/min. Continuous infusion of Fentanyl, (5–10 mg/kg bw) was used as analgesic. To prevent infection, 20 mg/kg bw iv Cephazoline was injected before surgery and SID Ceporex (15 mg/kg bw) was applied for10 days post operation or longer, as necessary. To expose the bone, a longitudinal incision of 10–12 cm was made along the posterior–lateral aspect of the right tibia, a few centimeters above the ankle joint and a few centimeters below the knee joint. A 4.5 stainless steel plate with 10 holes was then adjusted to the morphology of the bone by bending. The plate was fixed to the posterior aspect of the tibia with 4.5 mm trans-cortical screws, 4 proximal screws and 4 distal screws, leaving a central space of 3.5 cm. Then, a cylinder of 3.2 cm was cut out of the tibia, under continuous saline rinse. Afterwards, the wound was closed layer by layer. Post
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surgery, the limb was fixed using a plaster cast for 2 weeks, excluding the ankle and knee joints for free movement. EPC transplantation Two weeks following the first procedure, sheep were anaesthetized as described above. A limited incision just above the gap was performed reaching the regeneration scar tissue that fills the gap. A Vwedge-shaped canal, about 5 mm deep and 3 mm wide was cut out all along the gap. The piece removed was then trimmed-off its narrow edge leaving a topped-tailed tissue. 2 × 107 autologous EPC/0.2 ml saline or 0.2 ml saline (sham-transplanted control) were dropped into the canal, covered with the trimmed tissue and then the wound was closed by layers as described above. Evaluation of the healing process Radiological follow-up X-ray radiographs were taken every 2 weeks until sacrifice after 12 weeks following cell implantation. Upon sacrifice, the plates and screws were removed and tibia specimens approximately 6 cm long, that included the healing gap and adjacent proximal and distal screw holes, were excised and immersed in phosphate-buffered formalin for 48 h and then stored in 70% ethanol. Micro-computed tomography Whole specimens were scanned using a desktop μCT imaging system (μCT 40, Scanco Medical, Bruettisellen, Switzerland). The X-ray tube parameters were set to 70 kV and 90 mA. The integration
Fig. 4. Representative three-dimensional μCT images of frontal aspects and midline longitudinal and horizontal slices from sham and EPC-treated specimen. Note dense appearance of newly formed bone in EPC-treated specimen.
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time was 200 ms. Scans were performed at a 36 μm resolution in all three spatial dimensions. The region of interest used for further analysis (a total of 20 mm along the diaphyseal shaft) extended 10 mm on either side of the midline between the last proximal and first distal screw holes (Fig. 1). Then, two dimensional μCT images were reconstructed in 1024× 1024 pixel matrices using a standard convolution — Back projection procedure with a Shepp and Logan filter. Prior to calculations, a constrained 3D Gaussian filter was used to partly suppress the noise in the volumes (filter width = 0.8; filter support, one voxel width). Threshold was determined using the global thresholding procedure at 23% of the maximal grayscale intensity [20]. The following morphometric parameters were calculated: (i) union diameter (calculated from the total volume measurement); (ii) bone volume (BV). In addition, we determined the material density of the newly formed bone. To this end the attenuation coefficient (cm− 1) of each pixel was calculated and the histogram of bone material concentration was derived for each animal to calculate the mean degree of bone mineralization and mineralization distribution curve. The hydroxyapatite content (mg HA/cm3) was standardized with a manufacturer supplied phantom of 5 different HA densities embedded in soft-tissue equivalent resin. Beam hardening effects were corrected in the reconstruction process with a correction curve adapted to individual scans, as supplied by the manufacturer. The average material density (degree of mineralization) was calculated from the whole bone sample region used for architectural analyses [21].
The μCT analysis showed complete bridging of the tibia gap in all 4 (randomly chosen from the 6 successfully-repaired) EPC-treated sheep. Remarkably, the newly formed bridging bone had a dense trabecular appearance with few small marrow spaces and its external appearance was relatively regular (Figs. 1 and 4). Among the 5 shamtreated sheep 2 displayed discontinuous bridging. The remaining 3 specimens showed minute bridging by narrow strands of the newly formed dense bone (Figs. 1 and 4). These differences were reflected by the quantitative μCT analysis which demonstrated increased mean union diameter in the EPC-treated sheep (Fig. 5A). Notably, the amount of the newly formed bone bridging the gap was ~3.5-fold higher in the EPC — than in the sham-treated sheep (Fig. 5B). Both treatment groups showed similar material densities (Fig. 5C) suggesting that the EPC affect mainly the amount of mineralized matrix formed rather than its quality. Histology of gaps 12 weeks post sham treatment showed mostly fibrotic scar tissue that is rich in collagen fibers and quite abundantly vascularized (Figs. 6A and B). Cartilage undergoing endochondral ossification and some trabecular bone were observed in areas bordering the osteotomized bone (Fig. 6C). On the contrary, EPC transplantation led to formation of dense and massive woven bone with small spaces all throughout the defect (Fig. 6D). In some areas
Histology Central longitudinal midsagittal sections, 3 mm thick, were sawedout of EPC or sham-transplanted 12 weeks regenerating gaps, subjected to decalcification (Calci-Clear Rapid, National Diagnostic, Atlanta, Georgia, USA) and processed for paraffin embedding. Five μm sections were stained with H&E, H&E–alcian blue or Masson trichrome. Statistical analysis Quantitative μCT parameters are expressed as mean ± SEM. After testing for normality and equal variance, differences between sham and EPC-treated sheep were analyzed using t-test (Sigmastat version 2.03, SPSS Inc.). Differences were considered significant at p b 0.05. Results Expansion and characterization of EPC Total sheep MNC were seeded. Immediately after seeding cells appeared round, but after 3 to 5 days, attached cells appeared as elongated and spindle shaped. Following re-plating, they rapidly replicated from several cells to colonies and formed a monolayer of homogenous appearance (Figs. 2A and B). The endothelial characteristics of the cells were verified by their positive immunostaining for vWf (Figs. 2C and D) and fetal liver kinase (flk-1/KDR), also known as vascular endothelial growth factor receptor 2 (VEGFR2), (Fig. 2F), ability to rapidly incorporate acetylated LDL (ac-LDL) (Fig. 2E) and form capillary-like structures in Matrigel-based media (Fig. 2G). Bone regeneration Bone regeneration bridging the whole length of the defect was observed in 6 out of 7 experimental sheep implanted with autologous EPC. Four sham-operated gaps showed no bone regeneration at all, while the other 4 showed minimal degrees of bone regeneration. X-ray radiography follow-up showed that bone regeneration in the defects that were implanted with autologous EPC started already by 2–4 weeks following implantation. It gradually increased in volume and density until complete bridging was observed by 10 to 12 weeks after implantation. Representative x-ray follow-ups are demonstrated in Fig. 3.
Fig. 5. EPC stimulates bridging across critical size gap in sheep tibia. (A) Mean diameter of newly formed bone within reference volume; (B) overall amount of newly formed bone in reference volume; (C) material density (degree of mineralization) of newly formed bone. Data are mean ± SE in 5 sham and 4 EPC specimens. ⁎, p b 0.05.
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Fig. 6. Sham-transplanted gaps (A–C): H&E stained section of a gap 12 weeks post sham transplantation. Scar tissue is rich in collagen fibers and quite abundantly vascularized (A). Masson trichrome stained specifically the collagen fibers and exemplified their orientation (B). H&E–alcian blue stained cartilage undergoing endochondral ossification was observed in areas bordering the osteotomized bone (C). EPC-transplanted gaps (D–F): H&E stained EPC-transplanted gaps, showed dense and massive woven bone with small spaces all throughout the defect (D). Polarized-light illumination exemplified the orientation of the collagen fibers in the woven bone areas (upper part of the picture) and of the more mature lamellar bone (lower part of the picture) (E). Remnants of cartilage or chondroid bone stained with H&E–alcian blue could be seen in limited areas (F). Scale = 1.00 mm in A and D and 2.00 mm in B, C, E and F.
remodeling into more mature lamellar bone could be observed (Fig. 6E). Remnants of cartilage or chondroid bone could be seen in limited areas (Fig. 6F). Discussion The results of the present study indicated that autologous peripheral blood-derived sheep EPC can completely heal a tibia critical-sized defect within 3 months. EPC are thought to derive from bone marrow, mobilize to the peripheral circulation and incorporate into ischaemic and injured bone tissues and then differentiate into mature endothelial cells [2,14–16,22]. Currently, EPC can be identified using a combination of several cell specific markers [23]. The bloodderived isolated and expanded sheep EPC formed tubes in Matrigel, incorporated Ac-LDL and were immunopositive for VEGFR2/Flk-1 and vWf. In the present study we used a reproducible large animal procedure that optimally serves as a good preclinical model for testing for bone regeneration by implants. There are only minor differences in bone composition between sheep and humans [24]. Twenty million EPC were implanted into a longitudinal wedged-shaped canal created in the 2 week old scar tissue that has developed throughout the 3.2 cm gap. In this newly-developed approach we took advantage of the fibrous scar tissue serving as a natural scaffold bypassing the need for any artificial carriers. X-ray follow-up, μCT and histology of control sham-transplanted tibia gap showed no or minimal bone formation. However, already at 2–4 weeks post transplantation, 6 out of 7 EPCtransplanted gaps showed first signs of bone formation that gradually expanded throughout the gap until by 3 months, full bridging was achieved. We speculate that the failure in one sheep was due either to incompetence of its autologous cells for some unknown reason or was related to the status of this specific sheep. Quantitatively, the μCT analysis demonstrated that the transplanted EPC lead to an overall larger bony bridge, as shown by the increased BV. Notably, the actual newly formed bone tissue in the EPC and shamtreated sheep appears similar in terms of geometric and material densities, suggesting a quantitative rather than qualitative effect of the transplanted cells. The mechanism by which EPC regenerated bone is
still unclear. The most obvious explanation would be their contribution to angiogenesis and vasculogenesis, processes that are indispensable for bone formation [1]. However, histological observations of biopsies of 2 week old scar tissue from the gap showed an abundance of vascular elements, thus challenging this assumption (Lewinson et al., unpublished results). Alternatively, we hypothesize that EPC might transform into osteogenic cells in an appropriate microenvironment [25]. Indeed we have shown that EPC, when cultured in osteogenic conditions, formed alizarin-red, von Kossa and osteocalcin positive nodules [26]. Another possibility is that EPC induce recruitment, proliferation and differentiation of skeletal progenitors by a paracrine mechanism [27–30]. Interestingly, it has been shown that BMP-2 and -4 are selectively expressed by late outgrowth EPC [31]. Further experiments are now being conducted in our laboratory in order to clarify the paracrine potential of EPC on mesenchymal stem cells. The extraordinary contribution of EPC for bone healing during the repair process of a large bone defect opens new therapeutic opportunities for future trauma, neoplastic and other compromised patients. Acknowledgments The authors wish to thank the staff of the Animal House for their help with the surgeries and the devoted animal care. Funding sources: Ministry of Industry and Commerce, Israel Government — Nofar no. 3417; Magneton no. 37151. Purchase of the μCT system was supported in part by ISF grant to IB (9007/01). References [1] Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cell Mater 2008;15:100–14. [2] Lee DY, Cho TJ, Kim JA, Lee HR, Yoo WJ, Chung CY, et al. Mobilization of endothelial progenitor cells in fracture healing and distraction osteogenesis. Bone 2008;42: 932–41. [3] Mistry AS, Mikos AG. Tissue engineering strategies for bone regeneration. Adv Biochem Eng Biotechnol 2005;94:1–22. [4] Bancroft GN, Mikos AG. Bone tissue engineering by cell transplantation. In: Ikada Y, Oshima N, editors. Tissue engineering for therapeutic use 5. Elsevier, New York, p. 151.
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[19] Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 2004;104:2752–60. [20] Muller R, Ruegsegger P. Micro-tomographic imaging for the nondestructive evaluation of trabecular bone architecture. Stud Health Technol Inform 1997;40: 61–79. [21] Borah B, Dufresne TE, Ritman EL, Jorgensen SM, Liu S, Chmielewski PA, et al. Longterm risedronate treatment normalizes mineralization and continues to preserve trabecular architecture: sequential triple biopsy studies with micro-computed tomography. Bone 2006;39(2):345–52. [22] Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 2004;95:343–53. [23] Liew A, Barry F, O'Brien T. Endothelial progenitor cells: diagnostic and therapeutic considerations. BioEssays 2006;28:261–70. [24] Pearce AI, Richards RG, Milz S, Schneider E, Pearce SG. Animal models for implant biomaterial research in bone: a review. Eur Cell Mater 2007;13:1–10. [25] Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, Gustafsson E, et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 2007;13:952–61. [26] Bick T, Rozen N, Dreyfuss E, Soudry M, Lewinson D. Osteogenic differentiation of circulating endothelial progenitor cells. J Bone Miner Res 2007;22:S143. [27] Kado M, Lee JK, Hidaka K, Miwa K, Murohara T, Kasai K, et al. Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells. Biochem Biophys Res Commun 2008;377(2): 413–8. [28] Mathieu C, Sii-Felice K, Fouchet P, Etienne O, Haton C, Mabondzo A, et al. Endothelial cell-derived bone morphogenetic proteins control proliferation of neural stem/progenitor cells. Mol Cell Neurosci 2008;38:569–77. [29] Imura T, Tane K, Toyoda N, Fushiki S. Endothelial cell-derived bone morphogenetic proteins regulate glial differentiation of cortical progenitors. Eur J Neurosci 2008;27:1596–606. [30] Shao JS, Aly ZA, Lai CF, Cheng SL, Cai J, Huang E, et al. Vascular Bmp Msx2 Wnt signaling and oxidative stress in arterial calcification. Ann NY Acad Sci 2007;1117: 40–50. [31] Smadja DM, Bièche I, Silvestre JS, Germain S, Cornet A, Laurendeau I, et al. Bone morphogenetic proteins 2 and 4 are selectively expressed by late outgrowth endothelial progenitor cells and promote neoangiogenesis. Arterioscler Thromb Vasc Biol 2008;28(12):2137–43.
Contents lists available at ScienceDirect
Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects Nimrod Rozen a,b,1, Tova Bick b,1, Alon Bajayo c, Ben Shamian b, Michal Schrift-Tzadok d, Yankel Gabet c, Avner Yayon d,e,2, Itai Bab c,2, Michael Soudry b,2, Dina Lewinson b,⁎ a
Department of Orthopaedic Surgery, Ha'emek Medical Center, Afula 18101, Israel Research Institute for Bone Repair, Orthopaedic Surgery A, Rambam Health Care Campus, 8, Ha'aliya st., Bat-Galim, Haifa 31096, Israel Bone Laboratory, Institute of Dental Sciences, The Hebrew University of Jerusalem, Jerusalem 91120, Israel d ProChon Biotech Ltd., Nes-Ziona 70400, Israel e ProCore BioMed Ltd., Nes-Ziona 70400, Israel b c
a r t i c l e
i n f o
Article history: Received 4 June 2009 Revised 28 July 2009 Accepted 29 July 2009 Available online 6 August 2009 Edited by: T. Einhorn Keywords: Bone regeneration Critical-size gap Endothelial progenitor cells (EPCs) Sheep Micro-computed tomography
a b s t r a c t The angiogenic events that accompany bone regeneration function as a “limiting factor” and are the primary regulatory mechanisms that direct the healing process. The general aim of this study was to test whether blood-derived progenitor cells that have endothelial characteristics (EPC), when applied to a large segmental defect, would promote bone regeneration. We established a critical-sized gap platform in sheep tibiae. Our model system takes advantage of the physiological wound healing process that occurs during the first two weeks following injury, and results in the gap being filled with scar tissue. EPC were expanded ex-vivo and 2 × 107 cells/0.2 ml were implanted into a wedged-shaped canal excavated in the fibrotic scar tissue. Sham treated sheep served as controls. Bone regeneration was followed every two weeks for three months by Xray radiography. At the end of the experimental period, the regenerating segments were subjected to microcomputed tomographic (μCT) analysis. While minimal bone formation was detected in sham-treated sheep, six out of seven autologous EPC-transplanted sheep showed initial mineralization already by 2 weeks and complete bridging by 8–12 weeks post EPC transplantation. Histology of gaps 12 weeks post sham treatment showed mostly fibrotic scar tissue. On the contrary, EPC transplantation led to formation of dense and massive woven bone all throughout the defect. The results of this preclinical study open new therapeutic opportunities for the treatment of large scale bone injuries. © 2009 Elsevier Inc. All rights reserved.
Introduction Healing of large bone defects represents a great challenge to reconstructive surgery. Bone must be regenerated in order to fill in the defect and restore structure and function. Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Therefore angiogenesis plays a pivotal role in successful bone regeneration [1,2]. Present treatments are accompanied with pain and risk of infection, hemorrhage, cosmetic disability, nerve damage and loss of function [3]. Thus, there is a significant need for an alternative strategy for the treatment of severe bone loss and delayed or non-union fracture. An emerging approach to damage repair is tissue engineering which involves treatment with stem cells [4]. Cell-based therapies
⁎ Corresponding author. Fax: +972 48543606. E-mail address: [email protected] (D. Lewinson). 1 Nimrod Rozen and Tova Bick contributed equally to this work. 2 Avner Yayon, Itai Bab and Michael Soudry have equal academic contributions to the project, in spite of the difference in their area of expertise. 8756-3282/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2009.07.085
used until now involved fresh bone marrow or ex-vivo expanded mesenchymal stem cells, usually combined with scaffolds. Although promising clinical results have been achieved [5–7], the necessity of anaesthesia and an invasive manipulation requiring a minimum of two surgeries, is a disadvantage. Therefore, there is a need to isolate stem or progenitor cells from a more accessible source, such as peripheral blood. Endothelial progenitor cells (EPC) that reside in the bone marrow have been identified and isolated also from peripheral blood of adult human and umbilical cord [8,9]. These cells have been shown to participate in postnatal neovascularization, in revascularization of ischaemic hind-limb and in acute myocardial infarction ischaemia [10–12]. Distraction osteogenesis (DO), which is a bone regeneration system, is another example of ischaemic regenerating tissue [2,13]. Moreover, systemically injected EPC were shown to incorporate within the ischaemic regenerate [2,13–16]. Early studies from our group in a sheep DO model described the appearance of cellular colonies of vascular nature, that stain immunopositive for Tie-2 and factor VIII-related antigen, the origin of which was not clear [17,18]. These observations led us to hypothesize that EPC
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Fig. 1. Healing critical size gaps in sheep tibia. 2D μCT image of midsagittal plane showing fixation screw holes (asterisks). Full line marks midline between screw holes. Dotted lines mark proximal and distal borders of reference volume. Left, sham-treated tibia; right, EPC-treated tibia.
contribute to the healing process by increasing vascularization and new bone formation and might be used for enhancement of bone regeneration in large or compromised bone defects. The results obtained in the present study demonstrate that local transplantation of autologous EPC brought about complete bridging and may therefore be used as a therapeutic strategy for critical-sized gap bone fracture repair.
Materials and methods Isolation and expansion of sheep EPC Ten milliliters of peripheral venous blood was removed from sheep jugular vein into heparinized tubes. The mononuclear fraction (MNC) was separated using Lymphoprep™ (Axis-Shield PoC AS, Oslo
Fig. 2. EPC grow exponentially after trypsinization and reseeding. Magnification × 10 (A), × 40 (B). Sheep EPC stain positive for cytoplasmic von-Willebrand factor: positive staining (C) vs. no staining in control (without primary Ab) (D); incorporate Dil-Ac-LDL (stained by the red fluorescent dye, E); stain positive for flk-1 (F); form tubes in Matrigel (G). C–G, Magnification × 10. Scale = 500 μm in A, C, D, F, G and 200 μm in B and E.
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Norway) and suspended in endothelial basal medium-2 (EBM-2; Clonetics, Walkersville, MD, USA), supplemented with EGM-2MV SingleQuot® (containing 5% fetal bovine serum, vascular endothelial growth factor (VEGF), fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1 and ascorbic acid) and plated on fibronectin (Sigma Chemical Co., St. Louis, MO) -coated 10 ml plates. Cells were grown at 37 °C with humidified 95% air/5% CO2. After 4 days of culture, non-adherent cells were discarded by gentle washing with PBS, and fresh medium was applied. Adherent EPC were detected 5– 7 days after seeding, split by brief trypsinization using 0.5% trypsin/ 0.2% EDTA (Biological Industries Ltd., Beit Haemek, Israel) and dispersed. Cells reached confluence after 4–6 days. Characterization of sheep EPC Tube formation in Matrigel For demonstration of capillary tube formation, 250 μl of growth factor-reduced Matrigel (BD Biosciences Discovery Labware, Bedford, MA) was added per well of a 24-well plate and allowed to polymerize at 37 °C for at least 30 min. 5 × 104 adherent cells (suspected to be EPC) were suspended in 300 μl EGM2-MV medium and seeded onto Matrigel [19]. The cells were incubated at 37 °C with humidified 95% air/5% CO2 for 5–24 h. The tube networks were observed with an Olympus inverted microscope (Olympus, CKX41). Ac-LDL incorporation Endothelial cells have the capacity to internalize Ac-LDL. In order to demonstrate whether the cells suspected to be EPC are able to
internalize Ac-LDL, adherent cells were incubated with 10 mg/ml AcLDL coupled with fluorescent 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbocyanide perchlorate (DiI-Ac-LDL) (Molecular Probes Inc., Eugene, Oregon, USA) for 3–24 h [19]. Cells were visualized with an Olympus CXK41 inverted fluorescent microscope. Immunohistochemistry von-Willebrand factor (vWf) and Flk-1 On day 7 of culture, adherent cells were trypsinized, seeded and grown on chamber-slides for 2–3 days. Slides were subjected to immunohistochemistry/fluorescence to detect the expression of vWf or flk-1, respectively. Both proteins are regarded to be specifically expressed by endothelial cells. In brief, following fixation with 4% paraformaldehyde for 10 min at 4 °C and endogenous peroxidase inactivation immunostaining was performed. After 3 washes with PBS, slides were incubated with rabbit anti human vWf diluted 1:50 (DAKO, Glostrup, Denmark) or with mouse anti Flk-1 (VEGFR2) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA. USA) for 1 h, RT. Negative control slides were incubated without the Ab. For visualization of positive vWf, after 3 washes with PBS, slides were incubated for 10 min with HRP polymer conjugate (SuperPicTure™ Polymer Detection Kit — Zymed Laboratories, San Francisco, CA, USA) followed by 5 min incubation with AEC (RED) substrate (SuperPicTure™ Polymer Detection Kit — Zymed Laboratories, San Francisco, CA, USA). To visualize positive FLK-1, slides were incubated for 1 h in the dark with Rhodamine Red-X Affinity pure goat anti mouse IgG (H + L) RRX diluted 1:50 (Jackson Immunoresearch, Laboratories, Inc. West Grove,
Fig. 3. Representative X-ray radiographic follow-ups of sham and EPC-transplanted sheep tibiae. No significant new bone formation is observed in sham-transplanted bones (left side). EPC-transplanted bones showed a gradual filling of the defect until full defect bridging at 3 months (right side).
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PA, USA). Cells were visualized with an Olympus CXK41 inverted fluorescent microscope. Surgical procedures All surgical procedures and animal handling were approved by the Institutional Animal Care Committee of The Technion. Critical-sized gap surgery Fifteen, 2 years old female sheep (~ 70 kg), were operated in this study. Sheep were anaesthesized by 0.1 mg/kg bw Xylzine and 10 mg/ kg bw Ketamin iv and induction was achieved by 4–6 mg/kg bw Propofol iv. Maintenance was by 1.5–2.0% isofluran intubation + positive pressure ventilation of 100% oxygen, 15 breaths/min. Continuous infusion of Fentanyl, (5–10 mg/kg bw) was used as analgesic. To prevent infection, 20 mg/kg bw iv Cephazoline was injected before surgery and SID Ceporex (15 mg/kg bw) was applied for10 days post operation or longer, as necessary. To expose the bone, a longitudinal incision of 10–12 cm was made along the posterior–lateral aspect of the right tibia, a few centimeters above the ankle joint and a few centimeters below the knee joint. A 4.5 stainless steel plate with 10 holes was then adjusted to the morphology of the bone by bending. The plate was fixed to the posterior aspect of the tibia with 4.5 mm trans-cortical screws, 4 proximal screws and 4 distal screws, leaving a central space of 3.5 cm. Then, a cylinder of 3.2 cm was cut out of the tibia, under continuous saline rinse. Afterwards, the wound was closed layer by layer. Post
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surgery, the limb was fixed using a plaster cast for 2 weeks, excluding the ankle and knee joints for free movement. EPC transplantation Two weeks following the first procedure, sheep were anaesthetized as described above. A limited incision just above the gap was performed reaching the regeneration scar tissue that fills the gap. A Vwedge-shaped canal, about 5 mm deep and 3 mm wide was cut out all along the gap. The piece removed was then trimmed-off its narrow edge leaving a topped-tailed tissue. 2 × 107 autologous EPC/0.2 ml saline or 0.2 ml saline (sham-transplanted control) were dropped into the canal, covered with the trimmed tissue and then the wound was closed by layers as described above. Evaluation of the healing process Radiological follow-up X-ray radiographs were taken every 2 weeks until sacrifice after 12 weeks following cell implantation. Upon sacrifice, the plates and screws were removed and tibia specimens approximately 6 cm long, that included the healing gap and adjacent proximal and distal screw holes, were excised and immersed in phosphate-buffered formalin for 48 h and then stored in 70% ethanol. Micro-computed tomography Whole specimens were scanned using a desktop μCT imaging system (μCT 40, Scanco Medical, Bruettisellen, Switzerland). The X-ray tube parameters were set to 70 kV and 90 mA. The integration
Fig. 4. Representative three-dimensional μCT images of frontal aspects and midline longitudinal and horizontal slices from sham and EPC-treated specimen. Note dense appearance of newly formed bone in EPC-treated specimen.
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time was 200 ms. Scans were performed at a 36 μm resolution in all three spatial dimensions. The region of interest used for further analysis (a total of 20 mm along the diaphyseal shaft) extended 10 mm on either side of the midline between the last proximal and first distal screw holes (Fig. 1). Then, two dimensional μCT images were reconstructed in 1024× 1024 pixel matrices using a standard convolution — Back projection procedure with a Shepp and Logan filter. Prior to calculations, a constrained 3D Gaussian filter was used to partly suppress the noise in the volumes (filter width = 0.8; filter support, one voxel width). Threshold was determined using the global thresholding procedure at 23% of the maximal grayscale intensity [20]. The following morphometric parameters were calculated: (i) union diameter (calculated from the total volume measurement); (ii) bone volume (BV). In addition, we determined the material density of the newly formed bone. To this end the attenuation coefficient (cm− 1) of each pixel was calculated and the histogram of bone material concentration was derived for each animal to calculate the mean degree of bone mineralization and mineralization distribution curve. The hydroxyapatite content (mg HA/cm3) was standardized with a manufacturer supplied phantom of 5 different HA densities embedded in soft-tissue equivalent resin. Beam hardening effects were corrected in the reconstruction process with a correction curve adapted to individual scans, as supplied by the manufacturer. The average material density (degree of mineralization) was calculated from the whole bone sample region used for architectural analyses [21].
The μCT analysis showed complete bridging of the tibia gap in all 4 (randomly chosen from the 6 successfully-repaired) EPC-treated sheep. Remarkably, the newly formed bridging bone had a dense trabecular appearance with few small marrow spaces and its external appearance was relatively regular (Figs. 1 and 4). Among the 5 shamtreated sheep 2 displayed discontinuous bridging. The remaining 3 specimens showed minute bridging by narrow strands of the newly formed dense bone (Figs. 1 and 4). These differences were reflected by the quantitative μCT analysis which demonstrated increased mean union diameter in the EPC-treated sheep (Fig. 5A). Notably, the amount of the newly formed bone bridging the gap was ~3.5-fold higher in the EPC — than in the sham-treated sheep (Fig. 5B). Both treatment groups showed similar material densities (Fig. 5C) suggesting that the EPC affect mainly the amount of mineralized matrix formed rather than its quality. Histology of gaps 12 weeks post sham treatment showed mostly fibrotic scar tissue that is rich in collagen fibers and quite abundantly vascularized (Figs. 6A and B). Cartilage undergoing endochondral ossification and some trabecular bone were observed in areas bordering the osteotomized bone (Fig. 6C). On the contrary, EPC transplantation led to formation of dense and massive woven bone with small spaces all throughout the defect (Fig. 6D). In some areas
Histology Central longitudinal midsagittal sections, 3 mm thick, were sawedout of EPC or sham-transplanted 12 weeks regenerating gaps, subjected to decalcification (Calci-Clear Rapid, National Diagnostic, Atlanta, Georgia, USA) and processed for paraffin embedding. Five μm sections were stained with H&E, H&E–alcian blue or Masson trichrome. Statistical analysis Quantitative μCT parameters are expressed as mean ± SEM. After testing for normality and equal variance, differences between sham and EPC-treated sheep were analyzed using t-test (Sigmastat version 2.03, SPSS Inc.). Differences were considered significant at p b 0.05. Results Expansion and characterization of EPC Total sheep MNC were seeded. Immediately after seeding cells appeared round, but after 3 to 5 days, attached cells appeared as elongated and spindle shaped. Following re-plating, they rapidly replicated from several cells to colonies and formed a monolayer of homogenous appearance (Figs. 2A and B). The endothelial characteristics of the cells were verified by their positive immunostaining for vWf (Figs. 2C and D) and fetal liver kinase (flk-1/KDR), also known as vascular endothelial growth factor receptor 2 (VEGFR2), (Fig. 2F), ability to rapidly incorporate acetylated LDL (ac-LDL) (Fig. 2E) and form capillary-like structures in Matrigel-based media (Fig. 2G). Bone regeneration Bone regeneration bridging the whole length of the defect was observed in 6 out of 7 experimental sheep implanted with autologous EPC. Four sham-operated gaps showed no bone regeneration at all, while the other 4 showed minimal degrees of bone regeneration. X-ray radiography follow-up showed that bone regeneration in the defects that were implanted with autologous EPC started already by 2–4 weeks following implantation. It gradually increased in volume and density until complete bridging was observed by 10 to 12 weeks after implantation. Representative x-ray follow-ups are demonstrated in Fig. 3.
Fig. 5. EPC stimulates bridging across critical size gap in sheep tibia. (A) Mean diameter of newly formed bone within reference volume; (B) overall amount of newly formed bone in reference volume; (C) material density (degree of mineralization) of newly formed bone. Data are mean ± SE in 5 sham and 4 EPC specimens. ⁎, p b 0.05.
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Fig. 6. Sham-transplanted gaps (A–C): H&E stained section of a gap 12 weeks post sham transplantation. Scar tissue is rich in collagen fibers and quite abundantly vascularized (A). Masson trichrome stained specifically the collagen fibers and exemplified their orientation (B). H&E–alcian blue stained cartilage undergoing endochondral ossification was observed in areas bordering the osteotomized bone (C). EPC-transplanted gaps (D–F): H&E stained EPC-transplanted gaps, showed dense and massive woven bone with small spaces all throughout the defect (D). Polarized-light illumination exemplified the orientation of the collagen fibers in the woven bone areas (upper part of the picture) and of the more mature lamellar bone (lower part of the picture) (E). Remnants of cartilage or chondroid bone stained with H&E–alcian blue could be seen in limited areas (F). Scale = 1.00 mm in A and D and 2.00 mm in B, C, E and F.
remodeling into more mature lamellar bone could be observed (Fig. 6E). Remnants of cartilage or chondroid bone could be seen in limited areas (Fig. 6F). Discussion The results of the present study indicated that autologous peripheral blood-derived sheep EPC can completely heal a tibia critical-sized defect within 3 months. EPC are thought to derive from bone marrow, mobilize to the peripheral circulation and incorporate into ischaemic and injured bone tissues and then differentiate into mature endothelial cells [2,14–16,22]. Currently, EPC can be identified using a combination of several cell specific markers [23]. The bloodderived isolated and expanded sheep EPC formed tubes in Matrigel, incorporated Ac-LDL and were immunopositive for VEGFR2/Flk-1 and vWf. In the present study we used a reproducible large animal procedure that optimally serves as a good preclinical model for testing for bone regeneration by implants. There are only minor differences in bone composition between sheep and humans [24]. Twenty million EPC were implanted into a longitudinal wedged-shaped canal created in the 2 week old scar tissue that has developed throughout the 3.2 cm gap. In this newly-developed approach we took advantage of the fibrous scar tissue serving as a natural scaffold bypassing the need for any artificial carriers. X-ray follow-up, μCT and histology of control sham-transplanted tibia gap showed no or minimal bone formation. However, already at 2–4 weeks post transplantation, 6 out of 7 EPCtransplanted gaps showed first signs of bone formation that gradually expanded throughout the gap until by 3 months, full bridging was achieved. We speculate that the failure in one sheep was due either to incompetence of its autologous cells for some unknown reason or was related to the status of this specific sheep. Quantitatively, the μCT analysis demonstrated that the transplanted EPC lead to an overall larger bony bridge, as shown by the increased BV. Notably, the actual newly formed bone tissue in the EPC and shamtreated sheep appears similar in terms of geometric and material densities, suggesting a quantitative rather than qualitative effect of the transplanted cells. The mechanism by which EPC regenerated bone is
still unclear. The most obvious explanation would be their contribution to angiogenesis and vasculogenesis, processes that are indispensable for bone formation [1]. However, histological observations of biopsies of 2 week old scar tissue from the gap showed an abundance of vascular elements, thus challenging this assumption (Lewinson et al., unpublished results). Alternatively, we hypothesize that EPC might transform into osteogenic cells in an appropriate microenvironment [25]. Indeed we have shown that EPC, when cultured in osteogenic conditions, formed alizarin-red, von Kossa and osteocalcin positive nodules [26]. Another possibility is that EPC induce recruitment, proliferation and differentiation of skeletal progenitors by a paracrine mechanism [27–30]. Interestingly, it has been shown that BMP-2 and -4 are selectively expressed by late outgrowth EPC [31]. Further experiments are now being conducted in our laboratory in order to clarify the paracrine potential of EPC on mesenchymal stem cells. The extraordinary contribution of EPC for bone healing during the repair process of a large bone defect opens new therapeutic opportunities for future trauma, neoplastic and other compromised patients. Acknowledgments The authors wish to thank the staff of the Animal House for their help with the surgeries and the devoted animal care. Funding sources: Ministry of Industry and Commerce, Israel Government — Nofar no. 3417; Magneton no. 37151. Purchase of the μCT system was supported in part by ISF grant to IB (9007/01). References [1] Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cell Mater 2008;15:100–14. [2] Lee DY, Cho TJ, Kim JA, Lee HR, Yoo WJ, Chung CY, et al. Mobilization of endothelial progenitor cells in fracture healing and distraction osteogenesis. Bone 2008;42: 932–41. [3] Mistry AS, Mikos AG. Tissue engineering strategies for bone regeneration. Adv Biochem Eng Biotechnol 2005;94:1–22. [4] Bancroft GN, Mikos AG. Bone tissue engineering by cell transplantation. In: Ikada Y, Oshima N, editors. Tissue engineering for therapeutic use 5. Elsevier, New York, p. 151.
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