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First published online April 3, 2008
Stem Cells Vol. 26 No. 6 June 2008, pp. 1395 -1405
doi:10.1634/stemcells.2007-0820; www.StemCells.com
© 2008 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

Local Delivery of Granulocyte Colony Stimulating Factor-Mobilized CD34-Positive Progenitor Cells Using Bioscaffold for Modality of Unhealing Bone Fracture

Yutaka Mifunea,b, Tomoyuki Matsumotoa,b, Atsuhiko Kawamotoa, Ryosuke Kurodab, Taro Shojia,b, Hiroto Iwasakia, Sang-Mo Kwona, Masahiko Miwab, Masahiro Kurosakab, Takayuki Asaharaa,c

aStem Cell Translational Research, Institute of Biomedical Research and Innovation/RIKEN Center for Developmental Biology, Kobe, Japan;
bDepartment of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan;
cDepartment of Regenerative Medicine Science, Tokai University School of Medicine, Kanagawa, Japan

Key Words. CD34 stem cells • CD34 progenitors • CD34 cell dose • Cellular therapy • Cell transplantation • Osteoblast • Endothelial cell • Stem cell transplantation

Correspondence: Correspondence: Takayuki Asahara, M.D., Ph.D., Stem Cell Translational Research, Institute of Biomedical Research and Innovation/RIKEN Center for Developmental Biology, 2-2 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan. Telephone: 81-78-304-5772; Fax: 81-78-304-5263; e-mail: Asa777{at}aol.com; or Tomoyuki Matsumoto, M.D., Ph.D., Stem Cell Translational Research, Institute of Biomedical Research and Innovation/RIKEN Center for Developmental Biology, 2-2 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan. Telephone: 81-78-304-5772; Fax: 81-78-304-5263; e-mail: matsun{at}m4.dion.ne.jp

Received on October 18, 2007; accepted for publication on March 23, 2008.

First published online in STEM CELLS EXPRESS  April 3, 2008.


   ABSTRACT
Top
 Footnotes
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 
We recently reported that i.v. transplantation of adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, contributes to fracture healing through the enhancement of vasculogenesis and osteogenesis. However, the scarcity of CD34+ cells in the adult human is a critical issue for the future clinical application of this method. To overcome this issue, we assessed in vitro and in vivo capacity of granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) human CD34+ cells for vasculogenesis and osteogenesis. First, we confirmed the differentiation capability of GM-PB CD34+ cells into osteoblasts in vitro. Second, local transplantation of GM-PB CD34+ cells on atelocollagen scaffold was performed in nude rats in a model of unhealing fractures. Immunostaining for human leukocyte antigen-ABC of tissue samples 1 week after fracture and cell therapy showed the superior incorporation after local transplantation compared with systemic infusion. Third, the effects of local transplantation of 105 (Hi), 104 (Mid), or 103 (Lo) doses of GM-PB CD34+ cells or phosphate-buffered saline (PBS) on fracture healing were compared. Extrinsic vasculogenic and osteogenic differentiation of GM-PB CD34+ cells, enhancement of the intrinsic angio-osteogenesis by recipient cells, augmentation of blood flow recovery at the fracture sites, and radiological and histological confirmation of fracture healing were observed only in the Hi and Mid groups but not in the Lo and PBS groups. These results strongly suggest that local transplantation of GM-PB CD34+ cells with atelocollagen scaffold is a feasible strategy for therapeutic vasculogenesis and osteogenesis needed for fracture healing.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Footnotes
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 
Adult stem/progenitor cells play important roles in tissue homeostasis and have important implications for regenerative medicine. A number of reports have suggested that stem cells derived from a variety of adult tissues are capable of maintaining, regenerating, and repairing other tissues derived from all three germ layers [1–3].

Endothelial progenitor cells (EPCs), which are involved in vascular development in the embryonic stage [4, 5], were first identified as CD34+ cells in adult peripheral blood (PB) [6]. Adult EPCs, originating mainly from bone marrow (BM), are mobilized into PB and recruited into neovascularization sites. Recruited EPCs contribute to postnatal neovascularization by proliferating, differentiating and migrating [6–8]. The therapeutic application of EPCs has been attempted for neovascularization in animal models of hind limb, myocardial, and cerebral ischemia/infarction [9–17] and wound healing [18].

Recent progress in stem cell biology has brought human EPCs/CD34+ cells to light for other fields of regenerative medicine. Eghbali-Fatourechi et al. [19] first identified circulating osteocalcin (OC)-positive cells in adult human, demonstrating osteogenic gene expression and mineralized nodule formation in vitro and bone regeneration in vivo. Chen et al. [20] identified osteoblast (OB) precursor cells in human BM CD34+ cells. Tondreau et al. [21] reported that granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) CD133+ cells, another EPC-enriched population, can act as mesenchymal stem cells and contribute to osteogenesis in vitro. These findings strongly suggest the therapeutic potential of BM-derived CD34+ cells for osteogenesis, as well as vasculogenesis. We previously reported that human circulating CD34+ cells, systemically transplanted into immunodeficient rats with nonhealing fracture, were recruited into fracture sites, contributed to a favorable environment for fracture healing by enhancing vasculogenesis and osteogenesis, and finally led to functional recovery from fracture [22]. Although our previous animal study demonstrating the efficacy of systemic transplantation of CD34+ cells may encourage the application of cell-based therapy in patients with unhealing fracture, local transplantation, but not systemic infusion, of CD34+ cells may need to be considered for future clinical trials for the following reasons: (a) intravenous infusion is generally known as a less safe method than local administration because of adverse systemic effects, and (b) local transplantation may allow reduction of effective cell dose compared with systemic administration, and this may overcome the critical issue in the clinical application: scarcity of circulating CD34+ cells in adults. Use of granulocyte colony-stimulating factor (G-CSF) is also a favorable method to harvest abundant CD34+ cells from adult PB in clinical situation.

In the current preclinical study, we proved the hypothesis that local transplantation of GM-PB CD34+ cells may contribute to fracture healing through vasculogenesis and osteogenesis at the lower dose compared with the systemic infusion used in our previous study [22]. We also demonstrated the in vitro differentiation potential of human GM-PB CD34+ cells into OBs as a supportive mechanistic study.


   MATERIALS AND METHODS
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 
Preparation of Human Cells
We purchased human GM-PB CD34+ cells obtained from a healthy female (43 years old, African-American) and human BM total mononuclear cells (MNCs) obtained from a healthy female (25 years old, African-American) from Lonza (Valais, Switzerland, http://www.lonza.com/group/en.html).

Flow Cytometry Studies
Regular flow cytometric profiles were analyzed with a FACSCalibur analyzer (BD Biosciences, San Diego, http://www.bdbiosciences.com) and CELLQuest software (Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com) as described previously [22].

Induction of Osteogenic Differentiation of GM-PB CD34+ Cells In Vitro
We seeded 5 x 104 BM-MNCs in six-well plates with {alpha}-minimum essential medium ({alpha}-MEM) (Gibco-BRL, Tokyo, http://www.gibcobrl.com) supplemented with 10% fetal bovine serum (FBS) (Vitromex, Vilshofen, Germany) and 2 mM L-glutamine (Gibco-BRL). After 2 weeks of culture, we collected the medium with floating cells and centrifuged it at 7,500g for 15 minutes at 4°C; we then collected the supernatant as conditioned medium (CM) and stored at –80°C for further use.

To induce mesenchymal stem cells from GM-PB CD34+ cells similarly to the previous method using BM-MNCs [21], we seeded 105 GM-PB CD34+ cells in six-well plates with {alpha}-MEM supplemented with 10% FBS, 2 mM L-glutamine, and 10% CM during the first 7 days of culture. Then, we cultured the cells in {alpha}-MEM supplemented with 10% FBS and 2 mM L-glutamine for the next 2 weeks.

Finally, to induce osteogenic differentiation, 105 cells were plated in six-well plates for 3 weeks under specific osteogenic conditions using {alpha}-MEM supplemented with 10% FBS, 2 mM L-glutamine, 60 µM ascorbic acid (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 10 mM β-glycerophosphate (Sigma-Aldrich), and 0.1 µM dexamethasone (Sigma-Aldrich). Cells (105 cells) were also cultured with {alpha}-MEM supplemented with 10% FBS and 2 mM L-glutamine as a negative control.

Unhealing Fracture Model and Local/Systemic Transplantation of GM-PB CD34+ Cells
Female athymic nude rats (F344/N Jcl rnu/rnu; CLEA Japan, Tokyo, http://www.clea-japan.com) ages 8–12 weeks and weighing 150–170 g were used in this study. Nonhealing fracture was induced in femur with cauterizing periosteum according to the methods of Einhorn [23] and Kokubu et al. [24]. Immediately after the creation of nonhealing fracture, rats received local transplantation of 105 (Hi), 104 (Mid), or 103 (Lo) GM-PB CD34+ cells suspended in 100 µl of phosphate-buffered saline (PBS) using atelocollagen (Koken, Tokyo, http://www.kokenmpc.co.jp/english), which is a bovine-derived bioscaffold, or the same volume of PBS without cells using the same scaffold (n= 15 in the Hi group and n= 12 in all other groups). Atelocollagen was used as a bio-absorbable scaffold retaining the cells in the transplanted site [25, 26]. The left, unfractured femur of each animal served as a control. In addition, three rats received i.v. (systemic) transplantation of 105 GM-PB CD34+ cells suspended in 100 µl of PBS through the tail vein immediately after the creation of the fracture model.

Tissue Harvesting
Three rats receiving local or i.v. transplantation of 105 GM-PB CD34+ cells were sacrificed at week 1 for histological examination. Three rats in each group that received local transplantation were euthanized at weeks 2 and 4. Remaining rats receiving local transplantation were sacrificed at week 8. Bilateral femurs were harvested and quickly embedded as described previously [22].

Morphometric Evaluation of Capillary Density and OB Density
Histochemical staining with isolectin B4 (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) as a rat endothelial cell (EC) marker or immunostaining for OC (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) as a rat and human OB marker was performed. Capillary or OB density was morphometrically evaluated as the average value in five randomly selected fields of soft tissue in the perifracture site. To address the location of chondrocytes in fractured sections, toluidine blue was used for counterstaining. All morphometric studies were performed by two blinded examiners.

Reverse Transcriptase Polymerase Chain Reaction Analysis of RNA Isolated from GM-PB CD34+ Cells and Tissue Samples at the Perifracture Site
Total RNA was obtained from GM-PB CD34+ cells and the rat tissues in perifracture sites at week 2 using Trizol (Life Technologies, Gaithersburg, MD, http://www.lifetech.com) according to the manufacturer's instructions. The synthesization of first-strand cDNA and polymerase chain reaction (PCR) were performed as described previously [22]. Subsequently, PCR products were visualized in 1.5% ethidium bromide-stained agarose gels. Human umbilical vein endothelial cells and human OBs (NHOst cells; Cambrex) were used for positive controls for human-specific endothelial and bone-related genes.

To avoid interspecies cross-reactivity of the primer pairs between human and rat genes, we designed human-specific primers using Oligo software (Takara Bio, Shiga, Japan, http://www.takara-bio.com). Each primer sequence is shown in supplemental online Information 1. No primers showed cross-reactivity to rat genes (data not shown).

Real-Time PCR Analysis to Detect Expression of Cytokines in Recipients Following Human GM-PB CD34+ Cell Transplantation
Total RNA was obtained from the rat tissues at perifracture sites at week 2 as described for reverse transcription (RT)-PCR analysis. After the first-strand cDNA was synthesized, the converted cDNA samples (2 µl) were amplified in triplicate by real-time PCR (ABI Prism 7700, Applied Biosystems, Foster City, CA, http://www3.appliedbiosystems.com/index.htm) in a final volume of 20 µl using SYBR Green Master Mix reagent (Applied Biosystems). Melting curve analysis was performed to ensure that only a single product was amplified using Dissociation Curves software (Applied Biosystems). Specificity of the reactions was confirmed by 2.0% agarose gel electrophoresis. Results were obtained using sequence detection software (ABI Prism 7700) and evaluated using Excel (Microsoft, Redmond, WA, http://www.microsoft.com). Each primer sequence is shown in supplemental online Information 2.

Physiological Assessment of Tissue Perfusion by Laser Doppler Perfusion Imaging
Laser Doppler perfusion imaging (LDPI) (Moor Instruments, Wilmington, DE, http://www.moor.co.uk) [27, 28] was used to measure blood flow in both legs at 0, 1, 2, and 3 weeks postfracture. The ratio of fractured to intact (contralateral) blood flow was calculated to evaluate serial blood flow recovery after fracture.

Immunohistochemical Staining
To detect the transplanted human cells in the rat tissue, immunohistochemistry was performed with following human-specific antibodies: human leukocyte antigen (HLA)-ABC (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) to detect various lineage of human cells, Ulex europaeus lectin type 1 (UEA-1) (Vector Laboratories) for human ECs, human-specific osteocalcin (hOC) (Biomedical Technologies, Inc., Stoughton, MA, http://www.btiinc.com/index.html) for human OBs, and smooth muscle actin (SMA) for both human and rat smooth muscle cells. The secondary antibodies for each immunostaining were as follows: Alexa Fluor 594-conjugated goat anti-mouse IgG1 (Molecular Probes, Tokyo, http://probes.invitrogen.com) for HLA-ABC staining, Cy3-conjugated streptavidin (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for UEA-1 staining, Alexa Fluor 488-conjugated goat anti-mouse IgG2a (Molecular Probes) for SMA, and Cy3-conjugated AffiniPure goat anti-rabbit IgG(H+L) (Jackson Immunoresearch Laboratories) for hOC. 4,6-Diamidino-2-phenylindole solution was applied for 5 minutes for nuclear staining.

Radiographic Assessment of the Fracture Healing
Radiographs of the fractured hind limbs were taken on weeks 0, 2, 4, and 8 following creation of the fracture. Fracture union was identified by the presence of a bridging callus on two cortices. Radiographs of each animal were examined by three blinded observers.

Histological Assessment of the Fracture Healing
Toluidine blue staining was performed to histologically evaluate the process of endochondral ossification at weeks 2, 4, and 8. The degree of fracture healing was evaluated using a five-point scale proposed by Allen et al. [29].

Statistical Analysis
The results were statistically analyzed using a software package (Statview 5.0; Abacus Concepts Inc, Berkeley, CA, http://www.abacus.com/abacus/home). All values were expressed as mean ± SE. Paired t tests were performed for comparison of data before and after treatment, and unpaired t tests were performed for comparison of local and i.v. transplantation. The multiple comparisons among groups were made using a one-way analysis of variance. Post hoc analysis was performed by Fisher's Protected Least Significant Difference test. The comparison of radiological results was performed with a {chi}2 test. A probability value of <.05 was considered to denote statistical significance.


   RESULTS
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 References
 
Phenotypic Characterization of GM-PB CD34+ Cells
GM-PB CD34+ cells from the healthy females used in this study were analyzed by flow cytometry. GM-PB CD34+ cell fraction was mainly positive for CD133, CD31, c-Kit, and CD45 but negative for kinase insert domain protein receptor and CD14 (supplemental online Fig. 1A). RT-PCR analysis of the GM-PB CD34+ cells revealed weak expression of the human-specific gene of CD31 (hCD31) and OC (hOC) but no expression of VE-cadherin (hVE-cad) and collagen1A1 (hCol1A1) (supplemental online Fig. 1B). RT-PCR analysis of the bovine-derived atelocollagen that we used as a scaffold showed no expression of human-specific genes, suggesting no cross-reaction of human-specific primers with bovine mRNAs.

In Vitro Differentiation of Human GM-PB CD34+ Cells into OBs
During the primary culture for mesenchymal stem cell induction, part of the GM-PB CD34+ cells exhibited a fibroblast-like spindle shape (Fig. 1A), proliferating quickly to form colonies (Fig. 1B). Treatment for 3 weeks with specific conditions for osteogenic induction resulted in a morphological transformation of the cells from long and spindle-like into a cuboidal shape (Fig. 1C). In contrast, no transformation was observed in the negative control group (Fig. 1D). As shown in Figure 1E, following osteogenic induction, matrix mineralization (calcium deposition) was clearly demonstrated by alizarin red staining. In contrast, no mineralization was observed in negative control wells (Fig. 1F). As shown in Figure 1G, mRNA of hOC and hCol1A1 was highly expressed in the CD34+ cells after osteogenic induction compared with those not induced. These results indicate that human GM-PB CD34+ cells are capable of differentiating into OBs under specific culture conditions.


Figure 1
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  		Figure 1. Osteogenic differentiation of granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) CD34+ cells in vitro. (A, B): Morphology of the GM-PB CD34+ cells, which were cultured in {alpha}-minimal essential medium ({alpha}-MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 10% conditioned medium (CM) during the first 7 days and in the same medium without CM for the next 1 week, changed into fibroblast-like spindle shape (x200) (A). Then, these spindle-shaped cells proliferated quickly to form colonies (x40) (B). (C, D): After osteogenic induction with {alpha}-MEM supplemented with 10% FBS, 2 mM L-glutamine, 60 µM ascorbic acid, 10 mM β-glycerophosphate, and 0.1 µM dexamethasone, the cell morphology changed from spindle-shaped to a cuboidal shape (arrowheads) (x40) (C). In contrast, no transformation was observed in cultured CD34+ cells with {alpha}-MEM supplemented with 10% FBS and 2 mM L-glutamine only (no osteogenic induction) (x40) (D). (E, F): In wells with osteogenesis-inducing conditions, the matrix mineralization was clearly demonstrated by alizarin red staining, indicating existence of calcium (E). In contrast, no mineralization was observed in noninducing conditions (x40) (F). (G): The mRNA of hOC and hCol1A1 was markedly expressed in osteogenesis-induced cells but not in noninduced cells. Abbreviations: hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; hOC, human-specific osteocalcin.

 
More Efficient Incorporation of Human GM-PB CD34+ Cells at Fracture Site Following Local Transplantation Compared with Systemic Infusion
To test the potential therapeutic superiority of local transplantation over systemic infusion, rats received 105 GM-PB CD34+ cells locally or intravenously and were sacrificed 1 week later to obtain tissue samples at the fracture sites. Immunostaining for HLA-ABC was performed to quantify the number of recruited human cells in the rat granulation area (Fig. 2A, 2B) and newly bone formed area (Fig. 2D, 2E). The double immunostaining for HLA-ABC and SMA demonstrated more abundant recruitment of human cells in the granulation area compared with the inner layer of SMA-positive smooth muscle cells, which were morphologically compatible with endothelial cells in arterioles, in both groups (Fig. 2A, 2B). The number of human cells in the granulation area was significantly higher in the local transplantation group compared with the i.v. infusion group (local, 105.6 ± 14.8; i.v., 38.0 ± 16.6 cells per mm2; p < .05) (Fig. 2C). Similarly, the number of human cells in the newly formed bone area was significantly higher in the local transplantation group than in the i.v. infusion group (local, 96.0 ± 10.5; i.v., 37.9 ± 15.4 cells per mm2; p < .05) (Fig. 2F). These findings strongly indicate that local transplantation is superior to i.v. infusion in terms of efficiency of human GM-PB CD34+ cell incorporation into fracture sites.


Figure 2
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  		Figure 2. Recruitment efficiency of locally or intravenously transplanted human CD34+ cells. Number of human CD34+ cells incorporating into the fracture site 1 week after the cell transplantation was compared between local and i.v. administration groups. (A, B): Representative double immunostaining for HLA-ABC (red) and SMA (green) in granulation areas of local (A) and i.v. (B) transplantation groups. (C): Number of HLA-ABC-positive cells in granulation area. *, p < .05 (n= 3 in each group). (D, E): Representative immunostaining for HLA-ABC in newly formed bone area in local (D) and i.v. (E) transplantation groups. (F): Number of HLA-ABC-positive cells in newly formed bone area. *, p < .05 (n= 3 in each group). Abbreviations: HLA, human leukocyte antigen; SMA, smooth muscle actin.

 
Vasculogenesis and Osteogenesis Induced by Human GM-PB CD34+ Cells
Next, we performed experiments to characterize the transplanted human CD34+ cells incorporating into fracture sites. To histologically validate the phenomenon of human cell-derived vasculogenesis, histochemical staining for UEA-1, a human-specific EC marker, was performed using the tissue samples obtained 2 weeks after local cell transplantation. Differentiated human ECs in the vasculature of the perifracture area were detected as UEA-1-positive cells in the Hi and Mid GM-PB CD34+ cell groups but not in the Lo and PBS groups (Fig. 3A–3F). These findings suggest that GM-PB CD34+ cells have the potential to differentiate into ECs; however, transplantation of more than 104 CD34+ cells may be necessary for significant vasculogenesis. To further verify this phenomenon, RT-PCR analysis of tissue RNA isolated from the perifracture site for human-specific EC markers (hVE-cad and hCD31) was performed (Fig. 3G). The expression ratio of hVE-cad to rat glyceraldehyde-3-phosphate dehydrogenase (rGAPDH) was significantly greater in the Hi group than in all others; the ratio was also higher in the Mid group than in the Lo and PBS groups (Hi, 0.579 ± 0.043; Mid, 0.399 ± 0.023; Lo, 0.197 ± 0.011; PBS, 0.191 ± 0.018; p < .01 for Hi vs. Lo or PBS group; p < .05 for Hi vs. Mid and for Mid vs. Lo or PBS group). The expression ratio of hCD31 to rGAPDH was also greater in the Hi group than in the other groups, and the ratio was higher in the Mid group than in the Lo and PBS groups. The ratio was also significantly higher in the Lo group than in the PBS group (Hi, 0.974 ± 0.064; Mid, 0.563 ± 0.023; Lo, 0.429 ± 0.025; PBS group, 0.159 ± 0.011; p < .01 for Hi vs. PBS group; p < .05 for Hi vs. Mid or Lo, for Mid vs. Lo or PBS, and for Lo vs. PBS group) (Fig. 3H).


Figure 3
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  		Figure 3. Fracture site vasculogenesis and osteogenesis derived from human CD34+ cells. (A–F): Representative double immunostaining for UEA-1 (red) and SMA (green) using tissue samples of the fracture sites at week 2 (original magnification, x200 [A–D], x400 [E, F]). Differentiated human endothelial cells (ECs) identified as UEA-1 positive cells (red) were observed only in animals receiving a high dose (Hi) (A, E) or a middle dose (Mid) (B, F) of CD34+ cells but not in those treated with a low dose (Lo) (C) or PBS (D). (G): Reverse transcriptase (RT) PCR analysis of tissue RNA isolated from the perifracture sites demonstrated the expression of human-specific EC markers (hVE-cad, hCD31) in animals treated with Hi, Mid, and Lo doses of CD34+ cells but not in animals receiving PBS. Cultured human umbilical vein endothelial cells were used for the Posi. For the Nega, no RNA was applied. (H): The ratio of gene expression of hVE-cad to rGAPDH in perifracture site was significantly greater in the Hi group than in other groups, as well as in the Mid group compared with the Lo and PBS groups. The ratio of gene expression of hCD31 to rGAPDH was significantly greater in the Hi group than in the other groups, as well as in the Mid group compared with Lo and PBS groups. **, p < .01; *, p < .05 (n= 4 in each group). (I–L): Representative immunostaining for hOC (green) using samples collected from fracture sites at week 2 (x100). Differentiated human osteoblasts (OBs) identified as hOC-positive cells (green) were observed in animals receiving Hi (I) and Mid (J) doses, but not in the Lo (K) and PBS (L) groups. (M): RT-PCR analysis using RNA isolated from the perifracture sites demonstrated the expression of human-specific bone-related markers (hOC and hCol1A1) in animals treated with Hi, Mid, and Lo doses of CD34+ cells but not in animals receiving PBS. Cultured human OBs were used for the Posi. (N): The ratio of gene expression of hOC to rGAPDH in perifracture sites was significantly greater in the Hi group than in the other groups, and the ratio of gene expression of hCol1A1 to rGAPDH was significantly greater in the Hi group than in the other groups, as well as in the Mid group compared with the Lo and PBS groups. **, p < .01; *, p < .05 (n= 4 in each group). Abbreviations: hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; Hi, 105; hOC, human-specific osteocalcin; hVE-cad, human VE-cadherin; Lo, 103; Mid, 104; Nega, negative control; PBS, phosphate-buffered saline; Posi, positive control; rGAPDH, rat glyceraldehyde-3-phosphate dehydrogenase; SMA, smooth muscle actin; UEA-1, Ulex europaeus lectin type 1.

 
To identify osteogenesis from human CD34+ cells, immunohistochemical staining for human-specific OC, an OB marker, was performed using tissue samples obtained 2 weeks after cell transplantation. Differentiated human OBs derived from high and middle doses of GM-PB CD34+ cells were detected as hOC-positive cells in the perifracture area (Fig. 3I, 3J), whereas hOC-positive cells were not identified in the low-dose group of GM-PB CD34+ cells or the PBS group (Fig. 3K, 3L). These findings suggest that transplanted GM-PB CD34+ cells may have the potential to differentiate into OBs; however, a greater than middle dose of CD34+ cells may be necessary for significant osteogenesis, as suggested in the vasculogenesis assessment. RT-PCR analysis also demonstrated dose-dependent expression of human-specific bone-related markers (hOC and hCol1A1) following human GM-PB CD34+ cell transplantation (Fig. 3M). The expression ratio of hOC to rGAPDH was significantly greater in the Hi group than in the other groups (Hi, 0.591 ± 0.032; Mid, 0.279 ± 0.021; Lo, 0.287 ± 0.014; PBS, 0.288 ± 0.011; p < .05 for Hi vs. Mid, Lo, or PBS group). The expression ratio of hCol1A1 to rGAPDH was also significantly greater in the Hi group than in the other groups, as well as in the Mid group compared with the Lo and PBS groups (Hi, 0.988 ± 0.077; Mid, 0.366 ± 0.030; Lo, 0.227 ± 0.021; PBS, 0.031 ± 0.004, respectively; p < .01 for Hi vs. PBS group; p < .05 for Hi vs. Mid or Lo, for Mid vs. Lo or PBS, and for Lo vs. PBS groups) (Fig. 3N).

These results indicate that human GM-PB CD34+ cells dose-dependently differentiate into both EC and OB lineages in the fracture-induced environment. For efficient vasculogenesis and osteogenesis, local transplantation of at least 104 CD34+ cells may be essential in this animal model.

Enhancement of Intrinsic Angiogenesis and Osteogenesis in Animals Receiving GM-PB CD34+ Cells
Enhanced angiogenesis and osteogenesis by the paracrine effect of the transplanted cells on recipients' cells were confirmed by immunostaining for rat-specific markers. Vascular staining with isolectin B4, a rat-specific marker for EC, using tissue samples collected 2 weeks postfracture, demonstrated enhancement of intrinsic neovascularization around the endochondral ossification area in animals treated with high and middle doses of GM-PB CD34+ cells (Fig. 4A). Capillary density was significantly greater in the Hi group compared with the other groups, as well as in the Mid group compared with the Lo and PBS groups (Hi, 1,136.8 ± 95.5; Mid, 928.7 ± 61.6; Lo, 752.5 ± 49.3; PBS, 616.5 ± 57.4 cells per mm2, respectively; p < .01 for Hi vs. PBS group; p < .05 for Hi vs. Mid or Lo group and for Mid vs. Lo or PBS group) (Fig. 4B).


Figure 4
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  		Figure 4. Enhancement of neovascularization and osteogenesis by recipient cells following granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) CD34+ cell transplantation. (A): Representative vascular staining with isolectin B4 using tissue samples of perifracture sites collected at week 2 in the Hi, Mid, or Lo dose of GM-PB CD34+ cells and the PBS group (x100). (B): Neovascularization assessed by capillary density at week 2 was significantly greater in the Hi group than all other groups, as well as in the Mid group compared with the Lo and PBS groups. **, p < .01; *, p < .05 (n= 4 in each group). (C): Representative osteoblast staining with rOC at week 2 in animals treated with a Hi, Mid, or Lo dose of CD34+ cells or PBS alone (x200). (D): Osteogenesis assessed by osteoblast density at week 2 was significantly greater in the Hi group than other groups, as well as in the Mid group compared with the PBS group. **, p < .01; *, p < .05 (n= 4 in each group). (E, F): Gene expression of intrinsic cytokines for angiogenesis and osteogenesis at week 2. The mRNA expression ratio of rVEGF (C) and rBMP-2 (D) to rGAPDH at the fracture sites was significantly greater in the Hi group than other groups, as well as in the Mid group compared with the Lo and PBS groups. **, p < .01; *, p < .05 (n= 4 in each group). Abbreviations: Hi, 105; Lo, 103; Mid, 104; PBS, phosphate-buffered saline; rOC, rat osteocalcin; rBMP-2, rat bone morphogenic protein 2; rVEGF, rat vascular endothelial growth factor.

 
OB staining with anti-rat OC using tissue samples collected 2 weeks postfracture revealed augmentation of intrinsic osteogenesis in the area of new bone formation in animals treated with high and middle doses of GM-PB CD34+ cells (Fig. 4C). OB density was significantly greater in the Hi group than in the other groups, and density in the Mid group was significantly higher than that in the PBS group (Hi, 641.3 ± 54.3; Mid, 399.6 ± 21.3; Lo, 301.7 ± 12.4; PBS group, 213.6 ± 19.2 cells per mm2, respectively; p < .01 for Hi vs. PBS group; p < .05 for Hi vs. Mid or Lo group and for Mid vs. PBS group) (Fig. 4D).

A possible explanation for the enhancement of intrinsic angiogenesis and osteogenesis following CD34+ cell therapy is the upregulation of angiogenesis- and osteogenesis-related cytokines at the perifracture site. Accordingly, we performed real-time RT-PCR to quantify the expression of rat vascular endothelial growth factor (rVEGF) and rat bone morphogenetic protein 2 (rBMP-2) around the fracture sites. The expression ratio of rVEGF to rGAPDH at week 2 was greater in animals receiving a high dose of GM-PB CD34+ cells compared with other groups, and the ratio in the middle-dose group was higher than that in the low-dose and PBS groups (Hi, 1.131 ± 0.284; Mid, 1.085 ± 0.269; Lo, 1.037 ± 0.215; PBS, 1.035 ± 0.231, respectively; p < .01 for Hi vs. Lo or PBS group; p < .05 for Hi vs. Mid group and for Mid vs. Lo or PBS group) (Fig. 4E). The expression ratio of rBMP-2 to rGAPDH at week 2 was also significantly greater in animals receiving a high dose of GM-PB CD34+ cells compared with other groups, and the ratio in the middle-dose group was higher than that in the low-dose and PBS groups (Hi, 1.028 ± 0.276; Mid, 0.991 ± 0.271; Lo, 0.923 ± 0.216; PBS, 0.907 ± 0.244, respectively; p < .01 for Hi vs. Lo or PBS and for Mid vs. PBS group; p < .05 for Hi vs. Mid group and for Mid vs. Lo group) (Fig. 4F). These results indicate that high and middle doses of human GM-PB CD34+ cells enhance both intrinsic angiogenesis and osteogenesis, at least in part by upregulating rVEGF and rBMP-2 at the fracture sites.

Improvement of Blood Flow in Animals Receiving GM-PB CD34+ Cells After Fracture
To evaluate blood flow recovery at the fracture sites, LDPI was serially examined after fracture. LDPI analysis demonstrated severely low blood flow at the fracture site 1 hour after fracture creation (week 0) and subsequent recovery at weeks 1, 2, and 3 in all groups (Fig. 5A). In all groups, the ratio of fractured to intact (contralateral) blood flow significantly increased by week 1. There was no significant difference in the blood flow ratio of fractured to intact (contralateral) limbs 1 hour after fracture creation among any group, whereas the ratio at week 1 was significantly higher in animals receiving a high-dose of GM-PB CD34+ cells compared with the other groups, as well as in the middle-dose group compared with the low-dose and PBS groups (Hi, 1.587 ± 0.042; Mid, 1.397 ± 0.013; Lo, 1.193 ± 0.054; PBS, 1.190 ± 0.042, respectively; p < .05 for Hi vs. Mid, Lo, or PBS and for Mid vs. Lo or PBS group). At week 2, the blood flow ratio was still significantly higher in the Hi group compared with Lo and PBS groups, as well as in the Mid group compared with PBS group (Hi, 1.515 ± 0.035; Mid, 1.485 ± 0.015; Lo, 1.370 ± 0.040; PBS, 1.350 ± 0.020, respectively; p < .05 for Hi vs. Lo or PBS and for Mid vs. PBS group). At week 3, the flow ratio was similar in all groups (Hi, 1.445 ± 0.045; Mid, 1.485 ± 0.019; Lo, 1.495 ± 0.054; PBS, 1.498 ± 0.055, respectively; p= not significant) (Fig. 5B). These results indicate that local transplantation of human GM-PB CD34+ cells contributes to rapid improvement of tissue perfusion at the fracture site in a dose-dependent manner.


Figure 5
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  		Figure 5. Improvement of blood flow at fracture sites following CD34+ cell transplantation. (A): Representative laser Doppler perfusion imaging (LDPI) at week 0 (1 hour after fracture), 1, 2 and 3 are shown. In these digital color-coded images, maximum perfusion values are indicated in white, medium values in green to yellow, and lowest values in dark blue. The skin blood flow within fracture site (red square) and intact contralateral site (black square) were evaluated as mean flux, and the ratio of the mean flux in the fractured site to that in the contralateral site (mean flux ratio) was calculated. (B): Severe reduction of the blood flow was observed 1 hour after nonhealing fracture was created (week 0) in all groups, whereas the mean flux ratio at week 1 was significantly higher in the Hi group compared with other groups, as well as in the Mid group compared with the Lo and PBS groups. At week 2, the ratio was significantly higher in the Hi group compared with the Lo and PBS groups, as well as in the Mid group compared with the PBS group. *, p < .05. Abbreviations: Hi, 105; Lo, 103; Mid, 104; PBS, phosphate-buffered saline.

 
Morphological Fracture Healing in Animals Receiving GM-PB CD34+ Cell Transplantation
Morphological fracture healing was evaluated by radiographic and histological examinations. Thirty-three percent of animals at week 4 and all animals at week 8 that received a high-dose of CD34+ cells, as well as 11% of animals at week 4 and 50% of animals at week 8 that received a middle dose CD34+ cells, demonstrated fractures that radiographically appeared healed with bridging callus formation. Fracture sites in all animals receiving a low dose of CD34+ cells or PBS showed no bridging callus formation and subsequently displayed nonunions after 8 weeks, which is consistent with the previous reports of the natural course of this animal model [23, 24] (Fig. 6A, 6B).


Figure 6
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  		Figure 6. Radiographical and histological evidence of fracture healing following CD34+ cell transplantation. (A): Representative radiographs of fractured sites at weeks 0, 2, 4, and 8 in each group. (B): The fracture healing ratio in all groups. At week 8, in 100% of animals receiving a Hi dose of CD34+ cells and in 50% of animals receiving a Mid dose of CD34+ cells, the fracture radiographically healed with bridging callus formation. Fracture sites in all animals receiving a Lo dose of CD34+ cells or PBS showed no bridging callus formation and fell into nonunions, an outcome consistent with the previous report showing natural course of this animal model. *, p < .05. (C, D): Histological evaluation with toluidine blue staining demonstrated enhanced endochondral ossification consisting of numerous chondrocytes and newly formed trabecular bone at week 2, bridging callus formation at week 4, and complete union at week 8 in animals receiving Hi and Mid doses of CD34+ cells. In contrast, although a thick callus formation was observed at week 2, the healing process stopped by week 4, and finally the callus was absorbed at week 8 in animals receiving a Lo dose of CD34+ cells or PBS. The degree of fracture healing assessed by the classification of Allen et al. [29] was significantly higher in the Hi group than in the other groups at week 8, as well as the Mid group compared with the Lo and PBS groups. **, p < .01; *, p < .05. Abbreviations: GM-PB, granulocyte colony-stimulating factor-mobilized peripheral blood; Hi, 105; Lo, 103; Mid, 104; PBS, phosphate-buffered saline.

 
Fracture healing was also histologically evaluated with toluidine blue staining. The degree of fracture healing at week 8 was assessed by the classification of Allen et al. [29] and was significantly higher in the Hi group compared with the other groups, as well as the Mid group compared with the Lo and PBS groups (Hi, 3.8 ± 0.13; Mid, 2.1 ± 0.16; Lo, 0.4 ± 0.48; PBS, 0.0 ± 0.00, respectively; p < .01 for Hi vs. Lo or PBS group and Mid vs. PBS group; p < .05 for Hi vs. Mid group and Mid vs. Lo group) (Fig. 6C, 6D). These results indicate that a nonhealing femoral fracture in a rat created by periosteal cauterization may be healed in a dose-dependent manner by local administration of human GM-PB CD34+ cells.


   DISCUSSION
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 References
 
Although most fractures typically heal with callus formation that bridges the fracture gap, a significant proportion (5%–10%) of fractures fail to heal and result in delayed union or persistent nonunion, caused mainly by inappropriate neoangiogenesis [30–33]. The importance of angiogenesis in bone formation/fracture healing has been noted since as early as 1763 [30], and adequate blood supply has been considered to be a key contributor to the osteogenic process [34].

In various fields of regenerative medicine, therapeutic neovascularization induced by EPC transplantation has been preclinically and even clinically tested in ischemic diseases, and promising outcomes have been reported [11–17]. Apart from potential for vasculogenic induction, adult human CD34+ cells, an EPC/hematopoietic stem cell (HSC)-enriched population, are also capable of differentiation into cardiomyocytes in vitro [35] and into cardiomyocytes and smooth muscle cells in vivo [11]. Human circulating CD133+ cells and BM CD34+ cells, both of which are EPC/HSC-enriched fractions, have been reported to differentiate into OBs in vitro [20–21]. These findings suggest that human CD34+ cells obtained from BM or PB may have the potential to differentiate into not only hematopoietic and endothelial lineages but also mesenchymal lineages, including osteogenic cells. On the basis of these studies, we previously investigated and reported the efficacy of i.v. transplantation of human circulating CD34+ cells for morphological and physiological recovery from unhealing fracture [22].

In the present study, we successfully confirmed the in vitro differentiation of human GM-PB CD34+ cells into OBs, which show matrix mineralization and calcium deposition, as well as the expression of hOC and hCol1A1 mRNA. Following our previous study, the efficacy of local transplantation of adult human GM-PB CD34+ cells was also examined in an unhealing fracture model of nude rats. To compare the incorporation efficiency between local and systemic transplantation, the human cells located in fracture sites 1 week after cell administration were detected by immunostaining for HLA-ABC. Quantification of the histological staining revealed 2.7-fold and 2.6-fold more incorporation of GM-PB CD34+ cells in the granulation area and the newly formed bone area following local transplantation with atelocollagen compared with i.v. infusion. Although homing efficiency of CD34+ cells after systemic infusion in this study was similarly impressive to that in previous reports of hind limb ischemia [14], myocardial infarction [13], and fracture [22], the cell recruitment was more efficient following local transplantation compared with systemic infusion. The result suggests that local transplantation may overcome the critical issue of CD34+ cell scarcity in clinical situations. Therefore, the effect of local transplantation of not only Hi, which was the same dose as in the previous study for i.v. infusion [22] but also Mid and Lo GM-PB CD34+ cells was examined in this study. Immunohistochemistry and RT-PCR analysis for human-specific markers revealed that direct vasculogenesis and osteogenesis by transplanted GM-PB CD34+ cells were detected in the Hi and Mid groups but not in the Lo and PBS groups. In regards to the paracrine effects of the transplanted cells on the recipient cells, immunostaining for rat-specific markers indicated significant enhancement of intrinsic angiogenesis and osteogenesis in the Hi and Mid groups but not in the other groups. We hypothesized that various cytokines and growth factors secreted from GM-PB CD34+ cells may exert a paracrine effect for intrinsic angio-osteogenesis. Endogenous vascular endothelial growth factor (VEGF) is one of the most important molecules for endochondral bone formation [36–38], and VEGF is expressed in the same temporal and spatial pattern in the fracture callus as occurs during bone development [39, 40]. VEGF activity is essential for normal angiogenesis and appropriate callus architecture and mineralization in response to bone injury [41–43]. Another growth factor, bone morphogenic protein (BMP), is also a key molecule for the process of fracture healing [44–46]. BMPs are newly synthesized by callus-forming cells near the fracture site, and BMP-2 stimulates angiogenesis via upregulation of VEGF at the fracture sites [47]. In this study, real-time RT-PCR analysis revealed overexpression of rVEGF and rBMP-2 at the perifracture sites of the Hi and Mid groups, which may be one of the mechanisms underlying the intrinsic angio-osteogenesis. Physiological assessment by LDPI showed significant recovery of blood flow at the fracture sites in the Hi and Mid groups but not in the other groups. Finally, radiological healing of the fractures was observed only in the Hi and Mid groups. The frequency of the healing was 100% in the Hi group, 50% in the Mid group, and 0% in the Lo and PBS groups at week 8. This radiological outcome was consistent with histological evaluation of the fracture healing by the classification of Allen et al. [29]. These findings strongly suggest that local transplantation of GM-PB CD34+ cells may have the potential to repair fractures by autocrine and paracrine mechanisms of neovascularization and osteogenesis. Most importantly, the significant efficacy of the local transplantation of GM-PB CD34+ cells is found at doses equal to or more than middle dose, which is lower than the effective dose of i.v. infusion found in the previous study [22]. In contrast, transplantation of the low dose of human CD34+ cells did not significantly contribute to vasculo-osteogenesis for fracture repair. The results regarding dose effects in this preclinical study might provide helpful information for establishing a clinical strategy for this novel modality.

The mechanism of multilineage differentiation potential in human CD34+ cells into ECs and OBs is still being investigated. However, single-cell PCR assessment indicated the expression of OC in 4 of 20 CD34+ cells in our previous study [22], suggesting that direct differentiation of human CD34+ cells into OBs may contribute to the multipotent plasticity even at a low rate. Microenvironmental interaction between vascular and osteoblastic lineage cells through paracrine regulatory factors and direct cellular communications may also be involved in developing CD34+ cells and may be conducive to fracture healing. We speculate that an enhanced vasculogenesis signal may cause the cellular commitment and development of CD34+ cells into osteoblastic cells as a cooperative organogenesis mechanism.

Several research groups have demonstrated the usefulness of local transplantation of total BM cells for fracture healing [48–51]. Hernigou et al. reported that in 88% of patients with noninfected nonunions of the tibia, bone union was achieved by percutaneous grafting of autologous total BM cells accompanied by external fixation or cast immobilization [51]. Compared with transplantation of purified CD34+ cells, total BM cell transplantation does not require a magnetic cell sorting process, indicating that time and cost of the cell preparation can be diminished. However, our group recently reported that intramyocardial transplantation of human GM-PB total MNCs represents a possible risk of severe hemorrhagic myocardial infarction in nude rats through the excessive inflammation induced by abundant infiltration of hematopoietic cells [52]. Thus, additional preclinical/clinical studies would be warranted to compare the feasibility, safety, and efficacy of both strategies for bone repair.

Concerning future clinical application, the biological risk of G-CSF may also become a problem. G-CSF has been used in thousands of clinical cases; however, severe complications, such as spleen rupture, interstitial pneumonitis, and acute coronary syndrome, are rarely reported [53]. This rare but potential risk of G-CSF, as well as the high cost of CD34+ cell isolation, would need to be overcome in the future.


   CONCLUSION
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 References
 
In conclusion, the present findings suggest that local transplantation of GM-PB CD34+ cells could be a promising clinical strategy for enhancing bone repair in patients suffering from unhealing fracture.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
References
 
The authors indicate no potential conflicts of interest.


   FOOTNOTES
 
Author contributions: Y.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; T.M. and A.K.: conception and design, administrative support, manuscript writing; R.K., T.S., H.I., S.-M.K., and M.M.: conception and design, administrative support; M.K. and T.A.: conception and design, financial support, final approval of manuscript.


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Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 

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