Therapeutic Potential of Vasculogenesis and Osteogenesis Promoted by Peripheral Blood CD34-Positive Cells for Functional Bone Healing
2006; Elsevier BV; Volume: 169; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.060064
ISSN1525-2191
AutoresTomoyuki Matsumoto, Atsuhiko Kawamoto, Ryosuke Kuroda, Masakazu Ishikawa, Yutaka Mifune, Hiroto Iwasaki, Masahiko Miwa, Miki Horii, Saeko Hayashi, Akira Oyamada, Hiromi Nishimura, Satoshi Murasawa, Minoru Doita, Masahiro Kurosaka, Takayuki Asahara,
Tópico(s)Bone fractures and treatments
ResumoFailures in fracture healing are mainly caused by a lack of vascularization. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to differentiate into osteoblasts in vitro; however, the therapeutic potential of CD34+ cells for fracture healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that functional fracture healing is supported by vasculogenesis and osteogenesis via regenerative plasticity of CD34+ cells. Peripheral blood CD34+ cells, isolated from total mononuclear cells of adult human volunteers, showed gene expression of osteocalcin in 4 of 20 freshly isolated cells by single cell reverse transcriptase-polymerase chain reaction analysis. Phosphate-buffered saline, mononuclear cells, or CD34+ cells were intravenously transplanted after producing nonhealing femoral fractures in nude rats. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining at the peri-fracture site demonstrated molecular and histological expression of human-specific markers for endothelial cells and osteoblasts at week 2. Functional bone healing assessed by biomechanical as well as radiological and histological examinations was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data suggest circulating human CD34+ cells have therapeutic potential to promote an environment conducive to neovascularization and osteogenesis in damaged skeletal tissue, allowing the complete healing of fractures. Failures in fracture healing are mainly caused by a lack of vascularization. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to differentiate into osteoblasts in vitro; however, the therapeutic potential of CD34+ cells for fracture healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that functional fracture healing is supported by vasculogenesis and osteogenesis via regenerative plasticity of CD34+ cells. Peripheral blood CD34+ cells, isolated from total mononuclear cells of adult human volunteers, showed gene expression of osteocalcin in 4 of 20 freshly isolated cells by single cell reverse transcriptase-polymerase chain reaction analysis. Phosphate-buffered saline, mononuclear cells, or CD34+ cells were intravenously transplanted after producing nonhealing femoral fractures in nude rats. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining at the peri-fracture site demonstrated molecular and histological expression of human-specific markers for endothelial cells and osteoblasts at week 2. Functional bone healing assessed by biomechanical as well as radiological and histological examinations was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data suggest circulating human CD34+ cells have therapeutic potential to promote an environment conducive to neovascularization and osteogenesis in damaged skeletal tissue, allowing the complete healing of fractures. Whereas embryonic stem cells in the blastocyst stage have the ability to generate any differentiated cells in the body, most adult stem cells have limited potential for postnatal tissue/organ regeneration. Among phenotypically characterized adult stem/progenitor cells,1Slack JM Stem cells in epithelial tissues.Science. 2000; 287: 1431-1433Crossref PubMed Scopus (326) Google Scholar, 2Blau HM Brazelton TR Weimann JM The evolving concept of a stem cell: entity or function?.Cell. 2001; 105: 829-841Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 3Korbling M Estrov Z Adult stem cells for tissue repair—a new therapeutic concept?.N Engl J Med. 2003; 349: 570-582Crossref PubMed Scopus (670) Google Scholar the hematopoietic system has traditionally been considered as an organized, hierarchic system with multipotent, self-renewing stem cells at the top, lineage-committed progenitor cells in the middle, and lineage-restricted precursor cells, which give rise to terminally differentiated cells, at the bottom.4Weissman IL Stem cells: units of development, units of regeneration, and units in evolution.Cell. 2000; 100: 157-168Abstract Full Text Full Text PDF PubMed Scopus (1499) Google Scholar Recently, adult human peripheral blood CD34+ cells have been reported to contain intensive endothelial progenitor cells (EPCs) as well as hematopoietic stem cells (HSCs).5Asahara T Murohara T Sullivan A Silver M van der Zee R Li T Witzenbichler B Schatteman G Isner JM Isolation of putative progenitor endothelial cells for angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7794) Google Scholar Tissue ischemia and cytokines mobilize EPCs from bone marrow (BM) into peripheral blood, and mobilized EPCs specifically home to sites of nascent neovascularization and differentiate into mature endothelial cells (ECs) (vasculogenesis).6Asahara T Masuda H Takahashi T Kalka C Pastore C Silver M Kearne M Magner M Isner JM Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.Circ Res. 1999; 85: 221-228Crossref PubMed Scopus (2946) Google Scholar, 7Takahashi T Kalka C Masuda H Chen D Silver M Kearney M Magner M Isner JM Asahara T Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med. 1999; 5: 434-438Crossref PubMed Scopus (47) Google Scholar In the case of the immunodeficient rat model of acute myocardial infarction, transplanted human CD34+ cells or ex vivo expanded EPCs incorporate into the site of the myocardial neovascularization, differentiate into mature ECs, augment capillary density, inhibit myocardial fibrosis and apoptosis, and preserve the left ventricular function.8Kawamoto A Tkebuchava T Yamaguchi J Nishimura H Yoon YS Milliken C Uchida S Masuo O Iwaguro H Ma H Hanley A Silver M Kearney M Losordo DW Isner JM Asahara T Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia.Circulation. 2003; 107: 461-468Crossref PubMed Scopus (580) Google Scholar, 9Kocher AA Schuster MD Szabolcs MJ Takuma S Burkhoff D Wang J Homma S Edwards NM Itescu S Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function.Nat Med. 2001; 7: 430-436Crossref PubMed Scopus (2364) Google Scholar, 10Kawamoto A Gwon HC Iwaguro H Yamaguchi JI Uchida S Masuda H Silver M Ma H Kearney M Isner JM Asahara T Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia.Circulation. 2001; 103: 634-637Crossref PubMed Scopus (1122) Google Scholar In addition, intravenously transplanted CD34+ cells efficiently incorporate into ischemic tissue.9Kocher AA Schuster MD Szabolcs MJ Takuma S Burkhoff D Wang J Homma S Edwards NM Itescu S Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function.Nat Med. 2001; 7: 430-436Crossref PubMed Scopus (2364) Google Scholar, 11Taguchi A Soma T Tanaka H Kanda T Nishimura H Yoshikawa H Tsukamoto Y Iso H Fujimori Y Stern DM Naritomi H Matsuyama T Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model.J Clin Invest. 2004; 114: 330-338Crossref PubMed Scopus (712) Google Scholar In recent years, in an attempt to meet clinical demands, interest has turned to bone formation as an alternative category of regenerative medicine. It is anticipated that by optimizing the process of fracture repair, a biological approach results in the restoration of normal structure and function in the injured skeletal tissue. Although most fractures typically heal with callus formation that bridges the fracture gap, a significant proportion (5 to 10%) of fractures fail to heal and result in delayed union or persistent nonunion.12Rodriguez-Merchan EC Forriol F Nonunion: general principles and experimental data.Clin Orthop Relat Res. 2004; : 4-12Crossref PubMed Scopus (205) Google Scholar, 13Marsh D Concepts of fracture union, delayed union, and nonunion.Clin Orthop Relat Res. 1998; 355: S22-S30Crossref PubMed Scopus (260) Google Scholar Inappropriate neoangiogenesis is considered to be a crucial factor in failed bone formation and remodeling.14Colnot CI Helms JA A molecular analysis of matrix remodeling and angiogenesis during long bone development.Mech Dev. 2001; 100: 245-250Crossref PubMed Scopus (134) Google Scholar, 15Gerstenfeld LC Cullinane DM Barnes GL Graves DT Einhorn TA Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation.J Cell Biochem. 2003; 88: 873-884Crossref PubMed Scopus (969) Google Scholar Notably, appropriate vascularization is emerging as a prerequisite for bone development and regeneration, and indeed there appears to be a developmental reciprocity between ECs and osteoblasts (OBs).16Karsenty G Wagner EF Reaching a genetic and molecular understanding of skeletal development.Dev Cell. 2002; 2: 389-406Abstract Full Text Full Text PDF PubMed Scopus (1214) Google Scholar Under such recognition, human CD34+ cells, which are capable of generating ECs in an appropriate environment,6Asahara T Masuda H Takahashi T Kalka C Pastore C Silver M Kearne M Magner M Isner JM Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.Circ Res. 1999; 85: 221-228Crossref PubMed Scopus (2946) Google Scholar, 7Takahashi T Kalka C Masuda H Chen D Silver M Kearney M Magner M Isner JM Asahara T Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med. 1999; 5: 434-438Crossref PubMed Scopus (47) Google Scholar have also been reported to differentiate into OBs in vitro.17Long MW Williams JL Mann KG Expression of human bone-related proteins in the hematopoietic microenvironment.J Clin Invest. 1990; 86: 1387-1395Crossref PubMed Scopus (102) Google Scholar, 18Chen JL Hunt P McElvain M Black T Kaufman S Choi ES Osteoblast precursor cells are found in CD34+ cells from human bone marrow.Stem Cells. 1997; 15: 368-377Crossref PubMed Scopus (101) Google Scholar, 19Tondreau T Meuleman N Delforge A Dejeneffe M Leroy R Massy M Mortier C Bron D Lagneaux L Mesenchymal stem cells derive from CD133 positive cells in mobilized peripheral blood and cord blood: proliferation, Oct-4 expression and plasticity.Stem Cells. 2005; 23: 1105-1112Crossref PubMed Scopus (392) Google Scholar In addition, a recent report demonstrated that CD34+ osteoblastic cells line the cavities of the cartilage in the fracture site in a rabbit tibial osteotomy model.20Ford JL Robinson DE Scammell BE Endochondral ossification in fracture callus during long bone repair: the localisation of ‘cavity-lining cells’ within the cartilage.J Orthop Res. 2004; 22: 368-375Crossref PubMed Scopus (28) Google Scholar These observations provoked the hypothesis that human peripheral blood CD34+ cells play a key role in fracture healing via vasculogenesis and osteogenesis. Therefore, we first confirmed that mouse ScaI+ lineage marker− (Lin−) cells, quite similar to human CD34+ cells,7Takahashi T Kalka C Masuda H Chen D Silver M Kearney M Magner M Isner JM Asahara T Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med. 1999; 5: 434-438Crossref PubMed Scopus (47) Google Scholar, 21Otani A Kinder K Ewalt K Otero FJ Schimmel P Friedlander M Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis.Nat Med. 2002; 8: 1004-1010Crossref PubMed Scopus (294) Google Scholar, 22Rafii S Lyden D Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.Nat Med. 2003; 9: 702-712Crossref PubMed Scopus (1462) Google Scholar were mobilized to peripheral blood in the natural course of the fracture healing process. Next, we investigated whether transplantation of circulating human CD34+ cells contributed to both vasculogenesis and osteogenesis for functional bone healing after fracture in an immunodeficient rat model. In the present series of studies, we demonstrate that mouse ScaI+Lin− cells are mobilized into the peripheral blood in the natural course of fracture healing and that human peripheral blood CD34+ cells, containing osteo/endothelial progenitor cells already expressing osteocalcin (OC), which were recruited to the fracture site after systemic delivery, develop a favorable environment for fracture healing by enhancing vasculogenesis and osteogenesis and finally lead to functional recovery from fracture. The present findings have important clinical implications for cell-based therapy that will enhance bone repair after fracture. To confirm the kinetics of ScaI+Lin− cells in the natural course of fracture healing, we detected ScaI+Lin− cells at prefracture and 1, 4, 7, and 14 days after fracture by fluorescence-activated cell sorting (FACS) analysis (n = 3 in each). Peripheral blood cells were aspirated from the hearts of 10-week-old fractured mice 1, 4, 7, and 14 days after fracture and from those of unfractured mice and mixed with phosphate-buffered saline (PBS) containing 5% fetal calf serum (n = 3 in each). MNCs were obtained by a Histopaque-1083 (Sigma Co., St. Louis, MO) density gradient centrifugation at 400 × g for 20 minutes. The light-density MNCs were collected, washed twice with Dulbecco's PBS supplemented with 2 mmol/L ethylenediaminetetraacetic acid, and counted manually. Separation of Lin− cells was performed to deplete mature hematopoietic cells7Takahashi T Kalka C Masuda H Chen D Silver M Kearney M Magner M Isner JM Asahara T Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med. 1999; 5: 434-438Crossref PubMed Scopus (47) Google Scholar, 21Otani A Kinder K Ewalt K Otero FJ Schimmel P Friedlander M Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis.Nat Med. 2002; 8: 1004-1010Crossref PubMed Scopus (294) Google Scholar such as T cells, B cells, natural killer (NK) cells, monocytes/macrophages, granulocytes, and erythrocytes by labeling MNCs with a Lin− separation kit (BD Pharmingen, San Diego, CA), containing biotin-conjugated Mac1, B220, CD3e, Ter119, Ly6G, and CD45R antibodies followed by streptavidin-conjugated magnetic beads and BD IMagnet separation. Then, Lin− MNCs were counted, and the number of ScaI+Lin− cells was calculated from the rate of ScaI+ cells in Lin− MNCs by FACS analysis and the number of Lin− MNCs. Human peripheral blood total MNCs were obtained from healthy male volunteers age 31.7 ± 1.2 years (n = 3). CD34+ cells were isolated from the MNCs by the AutoMACS system (Miltenyi Biotec, Auburn, CA) using anti-CD34 microbeads (Miltenyi Biotec). The CD34+ cell fraction had a purity of >97%, as determined by FACS analysis using a CD34-specific monoclonal antibody (Becton Dickinson, San Jose, CA). Institutional review board approval for the collection of peripheral blood MNCs from healthy human volunteers and informed consent regarding the experimental use of the cells from the volunteers were obtained. Regular flow cytometric profiles were analyzed with a FACS Calibur analyzer and CELLQuest software (Becton Dickinson Immunocytometry Systems, Mountain View, CA). The instrument was aligned and calibrated daily using a four-color mixture of CaliBRITE beads (BD Biosciences) with FACSComp software (BD Bioscience). Dead cells were excluded from the plots' beads on propidium iodide (PI) staining (Sigma Co.). Human CD34+ cells or mouse Lin− cells were washed twice with Hanks' balanced salt solution containing 3.0% fetal calf serum, incubated with 10 μl of FcR blocking reagent to increase the specificity of monoclonal antibodies (Miltenyi Biotec) for 20 minutes at 4°C, and incubated with the monoclonal antibodies for 30 minutes at 4°C. The stained cells were washed three times with PBS containing 3.0% fetal calf serum, resuspended in 0.5 ml of Hanks' balanced salt solution/3% fetal calf serum/propidium iodide, and analyzed by FACScan Caliber flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Cells (1 × 106) were processed through the cytometer, and 3 × 104 cells per sample were analyzed for human CD34+ cell or mouse Lin− cell fraction. The following monoclonal anti-human antibodies were used to characterize the CD34+ cell population: CD34-APC (BD Pharmingen), CD34-FITC (BD Pharmingen), CD45-FITC (BD Pharmingen), CD133-APC (BD Pharmingen), c-Kit-FITC (Nichirei), CD31-FITC (BD Pharmingen), CD105 (BD Pharmingen), VE cadherin (VE-cad)-FITC (BD Pharmingen), KDR-PE (BD Pharmingen), Tie2-PE (BD Pharmingen), IgG1-FITC isotype controls (BD Pharmingen), IgG1-APC isotype controls (BD Pharmingen), and propidium iodide (Sigma Co.). The following monoclonal anti-mouse antibodies were used to characterize the Lin− MNCs: cKit-APC (BD Pharmingen), ScaI-FITC (BD Pharmingen), IgG2a-PE isotype controls (BD Pharmingen), IgG2a-FITC isotype controls (BD Pharmingen), and propidium iodide (Sigma Co.). Female athymic nude rats (F344/N Jcl rnu/rnu; CLEA Japan, Inc.), age 8 to 12 weeks and weighing 150 to 170 g, were used in this study. The rats were fed a standard maintenance diet and provided with water ad libitum. The institutional animal care and use committees of Riken Center for Developmental Biology approved all animal procedures including human cell transplantation. All surgical procedures were performed under anesthesia and normal sterile conditions. Anesthesia was performed with ketamine hydrochloride (60 mg/kg) and xylazine hydrochloride (10 mg/kg) administered intraperitoneally. A lateral parapatellar knee incision on the right limb was made to expose the distal femoral condyle. An animal model of femoral fracture was applied using a modification of the method described by Bonnarens and Einhorn.23Bonnarens F Einhorn TA Production of a standard closed fracture in laboratory animal bone.J Orthop Res. 1984; 2: 97-101Crossref PubMed Scopus (630) Google Scholar To avoid significant displacement of the fracture by obtaining the well-aligned stability of the fracture site, a 1.2-mm-diameter K-wire was inserted from the trochlear groove into the femoral canal in a retrograde manner using a motor-driven drill. The wire was advanced until its proximal end was positioned stably in the greater trochanter, and the distal end was cut close to the articular surface of the knee. A thin saw cut at a depth of a 3 mm was applied mid-shaft after minimal lateral exposure to weaken the bone and to avoid complex fractures. A transverse femoral shaft fracture was then produced in the right femur of each rat using a C-shaped instrument applying three-point bending. After this procedure, each rat received additional surgery to produce a nonunion in the fractured shaft according to the method of Kokubu and colleagues.24Kokubu T Hak DJ Hazelwood SJ Reddi AH Development of an atrophic nonunion model and comparison to a closed healing fracture in rat femur.J Orthop Res. 2003; 21: 503-510Crossref PubMed Scopus (118) Google Scholar The periosteum was cauterized (OPTEMP, variable low temperature cautery; Alcon Manufacturing, Ltd., Fort Worth, TX) circumferentially at a distance of 2 mm on each side of the fracture. The wound was then irrigated with 10 ml of sterile saline, and the muscle and skin were closed in layers with 5-0 nylon sutures. Postoperative pain was managed by administration of subcutaneous injection of buprenorphine hydrochloride after surgery. Unprotected weight bearing was allowed immediately after operation. The left unfractured femur served as a control. Thirty minutes after the production of the fracture, rats received an intravenous transplantation of 1 × 105 CD34+ cells or 1 × 105 total MNCs resuspended with 100 μl of PBS or the same volume of PBS without cells through their tail vein (n = 15 in each group). Three rats were randomly selected from each group and sacrificed for the histological study after radiological evaluation of fracture healing at each time point: weeks 2, 4, and 8. The six remaining rats in each group were sacrificed at week 8 for biomechanical testing as described below. If the fracture was not a stable transverse fracture or if any evidence of deep infection was seen, the animals were excluded from the study and replaced with additional animals. Thus, eight rats with comminuted fractures and six rats with infection identified by radiographs were replaced during the experiment. To target human CD34+ cells or MNCs after intravenous infusion and confirm their recruitment into fracture site, Qtracker 655 cell labeling kit (Quantum Dot Corp.) was applied for the human cells before transplantation in three additional rats in each group according to the manufacturer's instructions. In brief, Qtracker cell labeling kits deliver fluorescent quantum dot (qdot) nanocrystals into the cytoplasm of live cells using a custom targeting peptide and the long-term stability and brightness of qdots make them ideal candidates for live cell targeting and imaging.25Michalet X Pinaud FF Bentolila LA Tsay JM Doose S Li JJ Sundaresan G Wu AM Gambhir SS Weiss S Quantum dots for live cells, in vivo imaging, and diagnostics.Science. 2005; 307: 538-544Crossref PubMed Scopus (7143) Google Scholar, 26Akerman ME Chan WC Laakkonen P Bhatia SN Ruoslahti E Nanocrystal targeting in vivo.Proc Natl Acad Sci USA. 2002; 99: 12617-12621Crossref PubMed Scopus (1357) Google Scholar The 1 × 105 CD34+ cells or MNCs were incubated for 60 minutes with the Qtracker labeling solution (1 μl of Qtracker regent A and B) and 0.2 ml of Dulbecco's modified Eagle's medium in eight-well Lab-Tek chambered coverglass system. The cells were washed twice with Dulbecco's modified Eagle's medium and cell labeling was confirmed under fluorescence microscopy. The labeled cells were intravenously transplanted into each animal 30 minutes after fracture. All animals were sacrificed at day 7 for targeting cell analysis with Qtracker. Total RNA was obtained from the human CD34+ cells immediately after isolation and from the rat tissues in peri-fracture site at day 14 using Trizol (Life Technologies) according to the manufacturer's instructions. The first-strand cDNA was synthesized using the RNA LA PCR kit version 1.1 (Takara), amplified by Taq DNA polymerase (Advantage-GC cDNA PCR kit; Clontech, Palo Alto, CA; and AmpliTaq Gold DNA polymerase; Applied Biosystems, Foster City, CA). PCR was performed using a PCR thermocycler (MJ Research PTC-225). Human CD31 (hCD31), human VE-cadherin (hVE-cad), human osteocalcin (hOC), human collagen1A1 (hCol1A1), human vascular endothelial growth factor (hVEGF), human fibroblast growth factor 2 (hFGF2), and human hepatocyte growth factor (hHGF) were amplified by TaqDNA polymerase (Advantage-GC cDNA PCR kit; Clontech) in the following conditions: 35 cycles of 30-second initial denaturation at 94°C, annealing at 56°C for 1 minute, and 30 seconds of extension at 72°C according to the manufacturer's instructions. Subsequently, PCR products were visualized in 1.5% ethidium bromide-stained agarose gels. Human umbilical vein endothelial cells and hOBs (NHOst cells; Cambrex Bio Science, Walkersville, MD) were used for the positive control for human-specific endothelial and bone-related genes. To avoid interspecies cross-reactivity of the primer pairs between human and rat genes, we designed the following human-specific primers using Oligo software (Takara). None of the primer pairs showed any cross-reactivity to rat genes (data not shown). hCD31 primer sequence (363 bp): sense 5′-ATCGATCAGTGGAACTTTGCCTATT-3′; anti-sense 5′-GTGGCATTTGAGATTTGATAGA-3′; hVE-cad primer sequence (461 bp): sense 5′-ACGCCTCTGTCATGTACCAAATCCT-3′; anti-sense 5′-GGCCTCGACGATGAAGCTGTATT-3′; hOC primer sequence (417 bp): sense 5′-AAGCAAGTAGCGCCAATCT-3′; anti-sense 5′-GGAAGTAGGGTGCCATAACAC-3′; hCol1A1 primer sequence (502 bp): sense 5′-CCTGGCCCCATTGGTAATGTT-3′; anti-sense 5′-CCCCCTCACGTCCAGATTCAC-3′; hVEGF primer sequence (186 bp): sense 5′-CAACATCACCATGCAGATTATGC-3′; anti-sense 5′-CCACAGGGACGGGATTTCTTG-3′; hFGF2 primer sequence (282 bp): sense 5′-AGCACAGTAACACTATCCTGCA-3′; anti-sense 5′-AAACGGAAACGCTCACCATAA-3′; hHGF primer sequence (282 bp): sense 5′-ACGAACACAGCTATCGGGGTA-3′; anti-sense 5′-CATCAAAGCCCTTGTCGGGAT-3′; hGAPDH primer sequence (596 bp): sense 5′-CTGATGCCCCCATGTTCGTC-3′; anti-sense 5′-CACCCTGTTGCTGTAGCCAAATTCG-3′; and rGAPDH primer sequence (320 bp): sense 5′-GTGCCAGCCTCGTCTCATAGA-3′; anti-sense 5′-CGCCAGTAGACTCCACGACAT-3′. Single-CD34+ cells derived from peripheral blood using the MACS system were put individually into PCR tubes with 4.5 μl of lysis buffer containing 50 mmol/L Tris-HCl, 75 mmol/L KCl, 5 U of SuperRNaseIN (Ambion), 7.5 U of PrimeRNase inhibitor (Eppendorf), 0.5% Nonidet P-40, 1 mmol/L dithiothreitol, 50 μmol/L dNTP, and 15 nmol/L MO-dT30 primer (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGTACTT-(dT)30-3′). Tubes were then incubated at 65°C for 2 minutes and cooled to 45°C for 2 minutes. Reverse transcription was then performed by the addition of 100 U of SuperScript III (Invitrogen). After incubation at 45°C for 15 minutes, the reaction was terminated by heating at 65°C for 10 minutes. Next, 1.5 μl of RNA digestion mixture, 2 U of RNase H (Invitrogen), and 25 mmol/L MgCl2 was added into each tube, and RNA digestion was performed by incubating at 37°C for 15 minutes and then inactivating at 65°C for 10 minutes. After RNA digestion, 6.5 μl of reaction mixture [5× terminal transferase buffer (Roche), 3 mmol/L CoCl2, 1.5 mmol/L dATP, and 15 U of TdT (Promega)] was added, poly(A) tailing was performed by incubating at 37°C for 15 minutes, and inactivation was performed at 65°C for 10 minutes. cDNA amplification was performed using ExTaq polymerase (Takara Biochemicals, Japan). In brief, poly(A)-tailed cDNA (13 μl) was split 4 μl each into two tubes containing 16 μl of primary PCR reaction solution containing ExTaq PCR buffer (Takara Biochemicals), 2 mmol/L dNTP, 10 μmol/L MO-dT30 primer, and 1 U of ExTaq polymerase (Takara Biochemicals). PCR was performed with one cycle of 1 minute at 94°C, 2 minutes at 50°C, and 2 minutes at 72°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 2 minutes at 72°C. After combining split tubes into one tube, 2 μl of first-amplified cDNA was added to 18 μl of second PCR mixture, ExTaq PCR buffer (Takara Biochemicals), 2 mmol/L dNTP, 2 μmol/L MO-dT30 primer, and 1 U of ExTaq polymerase (Takara Biochemicals), and second PCR was performed for 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 2 minutes at 72°C. Finally, amplified cDNA purified with Qiagen PCR purification kit according to the manufacturer's procedure, and then PCR analysis for specific gene expression was performed using each purified cDNA. hCD34 primer sequence (91 bp): sense 5′-TGCCTCTTCTGTGGGTGACC-3′; anti-sense 5′-TCCAACCGTCATTGAAACCAG-3′; hOC primer sequence (96 bp): sense 5′-GCTCAATCCGGACTGTGACG-3′; anti-sense 5′-CAGAGCGACACCCTAGACCG-3′; hGAPDH primer sequence (95 bp): sense 5′-GCATTGCCCTCAACGACC-3′; anti-sense 5′-CATGTGGGCCATGAGGTCC-3′. Rats were euthanized with an overdose of ketamine and xylazine. Bilateral femurs were harvested and quickly embedded in optimal cutting temperature compound (Miles Scientific, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at −80°C for histochemical and immunohistochemical staining as described below. Rat femurs in optimal cutting temperature blocks were sectioned, and 6-μm serial sections were mounted on silane-coated glass slides and air-dried for 1 hour before being fixed with 4.0% paraformaldehyde at 4°C for 5 minutes and stained immediately. Histochemical staining (n = 3 in each group) for isolectin B4 (Vector Laboratories, Burlingame, CA) as a rat EC marker or OC (Santa Cruz Biotechnology, Santa Cruz, CA) as a rat OB marker was visualized with diaminobenzidine (Vector Laboratories), and capillary or OB density was morphometrically evaluated as the average value in five randomly selected fields of soft tissue in the peri-fracture site (Figure 3M, zones a and b). To address the location of chondrocytes in the fractured sections, toluidine blue was used for counter staining. Capillaries were recognized as tubular structures positive for isolectin B4. OBs were recognized as lining or floating cells positive for OC on new bone surface. All morphometric studies were performed by two examiners blind to treatment. Laser Doppler perfusion imaging (LDPI) (n = 3 in each group) (Moor Instrument, Wilmington, DE)27Linden M Sirsjo A Lindbom L Nilsson G Gidlof A Laser-Doppler perfusion imaging of microvascular blood flow in rabbit tenuissimus muscle.Am J Physiol. 1995; 269: H1496-H1500PubMed Google Scholar, 28Wardell K Jakobsson A Nilsson GE Laser Doppler perfusion imaging by dynamic light scattering.IEEE T Bio-Med Eng. 1993; 40: 309-316Crossref PubMed Scopus (417) Google Scholar was used to measure serial blood flow in both legs throughout the course of 2 weeks after fracture. The ratio of fractured/intact (contralateral) blood flow was calculated to evaluate the serial blood flow recovery after fracture. The measurement was done under anesthesia with the animals supine and both limbs fully extended. To detect transplanted human cells in the rat fracture site, immunohistochemistry (n = 3) was performed with the following human-specific antibodies: human leukocyte antigen (HLA)-ABC (BD Pharmingen) to
Referência(s)