Review
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Apr 26, 2015; 7(3): 630-640
Published online Apr 26, 2015. doi: 10.4252/wjsc.v7.i3.630
Recent advances in bone regeneration using adult stem cells
Hadar Zigdon-Giladi, Utai Rudich, Gal Michaeli Geller, Ayelet Evron
Hadar Zigdon-Giladi, Ayelet Evron, Department of Periodontology, School of Graduate Dentistry, Rambam Health Care Campus, Haifa 31096, Israel
Hadar Zigdon-Giladi, Ayelet Evron, Research Institute for Bone Repair, Rambam Health Care Campus, Haifa 31096, Israel
Hadar Zigdon-Giladi, Gal Michaeli Geller, the Rappaport Family Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel
Utai Rudich, Orthopedic Department, Rambam Health Care Campus, Haifa 31096, Israel
Ayelet Evron, Evolutionary Developmental Biology, Faculty of Natural Sciences, Haifa University, Haifa 31096, Israel
Author contributions: Zigdon-Giladi H, Rudich U, Michaeli Geller G and Evron A designed and wrote this review paper.
Conflict-of-interest: The authors declare that they have no conflict of interest.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Hadar Zigdon-Giladi, DMD, PhD, Research Institute for Bone Repair, Rambam Health Care Campus, P.O. Box 9602, Haifa 31096, Israel. hadar@tx.technion.ac.il
Telephone: +972-4-8543606 Fax: +972-4-8542467
Received: November 3, 2014
Peer-review started: November 4, 2014
First decision: November 27, 2014
Revised: December 30, 2014
Accepted: January 18, 2015
Article in press: January 20, 2015
Published online: April 26, 2015

Abstract

Bone is a highly vascularized tissue reliant on the close spatial and temporal association between blood vessels and bone cells. Therefore, cells that participate in vasculogenesis and osteogenesis play a pivotal role in bone formation during prenatal and postnatal periods. Nevertheless, spontaneous healing of bone fracture is occasionally impaired due to insufficient blood and cellular supply to the site of injury. In these cases, bone regeneration process is interrupted, which might result in delayed union or even nonunion of the fracture. Nonunion fracture is difficult to treat and have a high financial impact. In the last decade, numerous technological advancements in bone tissue engineering and cell-therapy opened new horizon in the field of bone regeneration. This review starts with presentation of the biological processes involved in bone development, bone remodeling, fracture healing process and the microenvironment at bone healing sites. Then, we discuss the rationale for using adult stem cells and listed the characteristics of the available cells for bone regeneration. The mechanism of action and epigenetic regulations for osteogenic differentiation are also described. Finally, we review the literature for translational and clinical trials that investigated the use of adult stem cells (mesenchymal stem cells, endothelial progenitor cells and CD34+ blood progenitors) for bone regeneration.

Key Words: Bone tissue, Mesenchymal stem cells, Tissue engineering, Osteogenesis, Angiogenesis, Endothelial progenitor cells

Core tip: Mesenchymal stem cells and blood derived progenitor cells have the potential to open new horizon for the treatment of bone defects. Numerous preclinical and clinical studies demonstrated angiogenic and osteogenic potential of these cells. Additional research is required in order to improve isolation and expansion techniques, as well as long term studies to follow the safety of this cellular approach.


Citation: Zigdon-Giladi H, Rudich U, Michaeli Geller G, Evron A. Recent advances in bone regeneration using adult stem cells. World J Stem Cells 2015; 7(3): 630-640
INTRODUCTION

Incidence of bone fractures that require hospitalization is estimated to be 1 million in the United States alone. About 5%-10% of these fractures present delayed healing or nonunion and represent a major treatment challenge in orthopedic reconstructive surgery[1]. Current gold standard surgical technique for reconstruction of large bony defects involves autologous bone blocks harvested from the fibula, iliac crest, scapula, and radius[2-5]. These procedures require extended hospitalization and a secondary donor site with associated morbidity and complications. In addition to their surgical complications, these techniques are unpredictable and associated with significant graft resorption[6,7]. As an alternative to autologous bone block, recent developments in bone tissue engineering, gene and stem cell therapy are currently examined. A tissue engineering approach offers several potential benefits, including the lack of donor site morbidity, decrease in technical sensitivity of the repair, and most importantly, the ability to closely mimic the in vivo microenvironment in an attempt to recapitulate normal bone development[8].

BONE COMPOSITION

The bone comprises of inorganic and organic components. Two thirds of the bone volume consists of inorganic mineral salts: calcium, phosphate, carbonate, citrate, hydroxyl and ions (magnesium, sodium and fluoride) in the form of hydroxyapatite crystals[9]. The organic component is composed of 99% collagen type-1[10] as well as several growth factors osteocalcin, osteonectin, phosphoproteins, proteoglycans and bone morphogenic proteins (BMPs)[11].

In addition, the bone consists of cellular components: pre-osteoblasts, osteoblasts, osteocytes and osteoclasts. Osteoblasts originate from pluripotent mesenchymal cells, characterized as mononucleated, cuboidal cells located at bone margins. Their life span is between 3 d (in young rabbits) and 8 wk (in humans). Osteoblasts produce the inorganic bone matrix (un-mineralized osteoid) and supervise the mineralization process by secretion of alkaline phosphatase. At the end of the process several osteoblasts remain entrapped in the matrix that they have secreted, and develop into osteocytes[12]. These cells are considered mature osteoblasts. Only 10%-20% of the osteoblasts develop into osteocytes. Osteocytes are smaller than osteoblasts, have a larger nucleus to cytoplasm ratio and have a large number of processes which provide intercellular communication. Osteocytes are thought to be the cells responsible for bone regeneration, thus their presence is crucial[13,14]. The process of bone formation is followed by bone resorption. This process is carried out by osteoclasts that secrete enzymes and reduce the pH at the resorption site. Osteoclasts are large, multinucleated, polarized cells with a life span of 15-20 d. These cells originate from hematopoietic precursors of monocytes in the bone marrow, thus comprise phagocytic properties[15].

EMBRYONIC BONE DEVELOPMENT

The development of bone tissue is controlled by tightly regulated programs of cell proliferation, differentiation, survival, and organization. The bone tissue derives from mesenchymal condensations in which osteochondral progenitors give rise to either chondrocytes to form the cartilage or osteoblasts to form the bone[16]. Within the mesenchymal condensations, cells differentiate into chondrocytes or osteoblasts, and form cartilage and bone under the control of various transcription factors. Differentiation of mesenchymal cells into chondrocytes leads to the production of cartilage structures of the future bones of all the skeleton, except for the craniofacial skeleton and the clavicle. Through the process of endochondral ossification, the cartilage structures are subsequently replaced by bone and bone marrow. In the craniofacial skeleton and the clavicle, differentiation of mesenchymal cells into osteoblasts produces intramembranous bones directly from the mesenchymal condensations through the process of intramembraneous ossification[17]. It is well known that bone mineralization during endochondral and intramembranous ossification is tightly coupled with angiogenesis[18].

BONE REMODELING

Bone remodeling occurs throughout life. During this process small amounts of bone are resorbed by the activity of osteoclasts, followed by the recruitment of osteoblast precursors that differentiate and replace the amount of the removed bone[19]. The remodeling process takes place at anatomically distinct sites, termed basic multicellular units, that are dispersed throughout the skeleton[20]. An exact coupling between bone resorption and bone replacement is essential for successful bone remodeling. Thus, bone resorption by osteoclasts has to be matched with the generation of osteoblasts from precursor cells in order to replace the resorbed bone[21]. In the coupling between bone resorption and bone formation several factors are involved, such as growth factors released from the matrix, secreted and membrane-bound factors of osteoclasts, signals from osteocytes, signals from immune cells and signaling taking place within the osteoblast lineage[22].

FRACTURE HEALING AND TREATMENT OF NONUNION

The biologic process of fracture healing is complex and impacted by various factors including the recruitment of stem/progenitor cells from the bone marrow to the fracture site. The healing occurs via rapid bone regeneration followed by osteoclast-mediated remodeling, resulting in new bone with structural integrity and a geometrical configuration similar to that prior to the injury[2,23]. In most cases, healing is uneventful, but in some cases delayed or impaired healing occurs. The absence of proper blood supply, improper stability, soft tissue damage and different host features such as age, osteoporosis and smoking have been found to contribute to failure in achieving complete restoration. Nonunion fracture is a dramatic example of poor healing and is defined as non-consolidation at the fracture site within 6 mo[4,5,24]. Autologous bone graft is the gold standard treatment for nonunion fractures. Bone block may be harvested from the iliac crest, fibula or calvaria and transplanted at the recipient site. Bone block stimulates bone regeneration by serving as a scaffold for new bone growth (osteoconduction); moreover it contains cells [osteogenesis, e.g., mesenchymal stem cells (MSCs) and osteoblasts] and growth factors (osteoinduction). However, this technique has several disadvantage such as donor-site morbidity, pain and prolonged hospitalization[2-5].

MICROENVIRONMENT AT OSSEOUS TISSUE HEALING SITE

Healing of small bone defects and bone fractures involves complex and well coordinated interactions between cells, cytokines, and a mechanically stable environment with adequate blood supply[25] to generate new bone instead of a fibrous scar[26]. This complex dynamic process begins with secretion of pro-inflammatory cytokines from aggregated platelets, such as interleukin-1 (IL-1), IL-6 or tumor necrosis factor-α, which have chemotactic activity towards endothelial cells, fibroblasts, lymphocytes and monocytes-macrophages[27]. Transforming growth factor β1 (TGFβ1) plays a key role in this process due to its potent chemotactic and proliferative effect on mesenchymal stem cells, therefore modulate the recruitment of bone cells to the healing site[28]. In addition, TGFβ1 induces the production of extracellular bone matrix proteins such as collagen, osteopontin, and alkaline phosphatase from MSCs and osteoblasts[29]. BMPs and vascular endothelial growth factor (VEGF) are additional growth factors that induce osteogenesis and angiogenesis[30,31]. BMPs belong to the TGF-β superfamily and induce the formation of both bone and cartilage by stimulating the cellular events of mesenchymal progenitor cells. VEGF induces angiogenesis that is crucial for delivering oxygen and progenitor cells to the site[26].

RATIONALE FOR USING ADULT STEM/PROGENITOR CELLS FOR BONE REGENERATION

In the process of bone development and bone healing MSCs and adequate vasculature play a central role. Therefore, cell-based therapy strategies to enhance bone regeneration utilize MSCs and endothelial progenitor cells (EPCs).

MSCs contribute to the maintenance of various tissues, especially bone, in adults[32]. In 2006, the International Society for Cellular Therapy proposed that cells with the following characteristics should be considered as MSCs: (1) cells adherent to plastic in culture; (2) presence of CD105, CD73 and CD90 but absence of CD34, CD45, CD14 or CD11b, CD79α or CD19, and HLA-DR molecules; and (3) cells with the capacity to differentiate into osteoblasts, chondrocytes and adipocytes[33]. MSCs can be isolated from the bone marrow, placenta, umbilical cord blood, adipose tissue, muscle[34], brain[35], kidney, heart and many others[36]. While these cells share common characteristics, they are distinctive, and reflect aspects of their tissue of origin[37]. In animal models, transplantation of MSCs formed ectopic bone and improved healing of bone defects[38,39]. In clinical trials, MSCs regenerate bone in non-union long bone defects[40] in children with osteogenesis imperfecta[41] and in maxillofacial surgeries[42,43]. However, several difficulties concerning the use of MSCs still exist: (1) their aspiration involves an invasive procedure and morbidity[44]; (2) age-dependent decline exists in their proliferation and osteogenic differentiation potentials[45]; and (3) inadequate vascularization to MSC composite graft that causes malfunction and death of the transplanted cells[46].

In 2009, a novel study presented a successful use of blood derived endothelial progenitor cells to heal critical size bone defects[47]. Circulating EPCs were discovered in 1997 by Asahara et al[48] and Murohara et al[49]. EPCs participate in neovascularization[50], angiogenesis, vascular repair, blood-flow recovery after tissue ischemia, distraction osteogenesis[51,52], fracture healing[53], bone regeneration[54], and have osteogenic potential[55]. EPCs are mainly located in bone marrow and mobilize into the circulation under the guidance of signals such as granulocyte colony-stimulating factor (G-CSF), VEGF, basic fibroblast growth factor, placental growth factor, erythropoietin, or stromal-derived factor-1 (SDF-1) in order to home to ischemic sites[56,57]. EPCs can be isolated from peripheral blood, cord blood or bone marrow[58] and classically described as cells expressing a combination of an endothelial marker (VEGF receptor-2) and hematopoietic progenitor markers (CD34/CD133). However, this definition is under debate, since none of these markers are fully specific[59,60].

In vitro culture of mononuclear cell fraction in specific conditions consistently produces two distinct EPC subtypes: early EPCs and outgrowth endothelial cells[61] (that are also known as endothelial colony-forming cells[62] or late EPCs because of their late appearance in culture). Early EPCs live < 7 d in culture and have a spindle shape morphology and limited proliferation potential. These cells are CD133, CD31, CD45, VEGFR-2 and CD14 positive[63]. Late outgrowth EPCs emerge 2-3 wk after seeding from the nonmyeloid cell population (CD14 and CD45 negative), present a cobblestone morphology, have a high proliferation potential, express CD34, CD144, and VEGF-R2, but are negative for CD133, CD45 and CD14[63].

Both populations are capable of inducing neovascularization, but with a different mode of action. Whereas early EPCs have limited capacity for population doubling and induce only transient angiogenesis, late outgrowth EPCs can expand to more than 100 population doublings[64]. Early EPCs exert an angiogenic effect mainly by paracrine effect (secretion of proangiogenic growth factors), whereas late outgrowth cells were thought to produce the effect by direct engraftment and may differentiate to osteoblasts in a proper environment[55].

G-CSF-mobilized CD34 positive cells

The use of G-CSF was suggested as a favorable method to harvest abundant CD34+ cells from adult peripheral blood[65]. This alternative approach to isolate EPC was introduced in order to avoid EPC culture and to overcome the scarcity of these cells in the circulation (0.0001% of total mononuclear cells in peripheral blood of human adults)[66]. These cells are also known as endothelial and hematopoietic progenitor cell-rich population and present phenotypic characterization that includes endothelial and hematopoetic markers: positive for CD133, CD31, c-Kit, and CD45 but negative for kinase insert domain protein receptor and CD14. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of these cell revealed weak expression of CD31 and osteocalcin[65]. In vitro and in vivo experiments using these enriched CD34+ cells demonstrated angiogenic and osteogenic properties similar to expanded EPCs[67]. Peripheral blood and cord blood CD34+ cells were used to enhance tissue neovascularization in several preclinical models: stroke induction in immuno-compromised mice, wound healing in diabetic mice, peripheral nerve injuries and spinal cord injuries[68-71]. Based on the belief that improved neovascularization may be an attractive strategy for bone healing, several studies were conducted to investigate the efficacy of circulating CD34+ cells for bone fracture healing and found promising results[67,72,73].

MECHANISMS OF ACTION IN BONE REGENERATION

The idea of stem cell therapy relies on two possible mechanisms of action: (1) Direct pathway - this process starts with homing of the transplanted cells to ischemic or damaged site. This is followed by integration of the cells into the site and differentiation into cells specific to the tissue[74]; and (2) Indirect, paracrine pathway-transplanted cells secrete peptides (growth factors, interleukins, etc.) that modify the environment and recruit resident cells (MSCs and EPCs) to the regenerating site[75].

Homing of transplanted cells into ischemic or injured site allows systemic application of the cells. This includes the recruitment of the cells along a gradient of chemokines and their receptors. The CXCR4-CXCL12 and CX3CR1-CX3CL1 systems are reported to be involved in MSC migration, and among them, SDF-1 alpha and CXCR4 (the SDF-1 receptor) are strong mediators of MSCs and EPCs[76-80].

Following integration into the damaged site, the fate of the cells will be determined by environmental cues and allow osteogenic differentiation of the cells. The osteoblastic differentiation of MSCs was demonstrated in vitro by culturing the cells in the presence of ascorbic acid, inorganic phosphate (beta glycerol phosphate), and dexamethasone. In vivo studies have suggested that transplantation of adult stem cells derived from one tissue are able to engraft into developmentally unrelated tissues and, in certain cases, contribute to their regeneration[81]. Nevertheless, since in vivo transdifferentiation of the cells is more difficult to prove, it was suggested that the supposed differentiation was actually a result of fusion between donor and host cells[82,83]. The second mechanism by which MSCs contribute to bone formation is through indirect paracrine effect. MSCs secrete trophic agents and pro-angiogenic factors [angiopoietin (Ang)-1, -2, Ang-like-1, -2, -3, -4, VEGF, and fibroblast growth factor-2] that recruit resident MSCs to the site of injury and promote angiogenesis that is indispensable for tissue reconstruction[84].

There is only little data regarding the role of EPCs in bone healing. Early outgrowth EPCs exert an angiogenic effect mainly by product secretion, whereas late outgrowth cells were thought to produce the effect by direct engraftment. Direct incorporation of transplanted EPCs into intima layer of blood vessels was demonstrated in several in vivo models[48,54,85]. The indirect role of late EPCs to sustain MSC survival and function was demonstrated in an ectopic subcutaneous model[46]. According to this study, EPC-derived paracrine factors via platelet-derived growth factor BB/platelet-derived growth factor receptor-β signaling regulate MSC engraftment[46]. The mechanism of action of CD34+ cells was suggested by Kuroda et al[86] and includes differentiation of the cells into osteoblasts or endothelial cells and engraftment in the fracture healing site. To confirm this hypothesis, transplanted human cells were labeled in the fracture site using human specific antibodies (immunohistochemestry). Furthermore, human specific endothelial and osteoblastic markers were identified in the fracture site using RT-PCR. Additional mechanism of CD34+ cells in bone healing can be attributed to their paracrine effect, especially VEGF secretion. Recruitment of resident rat endothelial cells and osteoblasts to the fracture site was demonstrated following human CD34+ cell transplantation. Moreover, injection of VEGF antagonist reduced the vasculogenic and osteogenic effect that was observed following transplantation of CD34+ cells into fracture healing sites[86].

EPIGENETIC REGULATION IN BONE REGENERATION

Epigenetic mechanisms play essential roles in stem cell maintenance and differentiation during bone regeneration processes[87,88]. During the differentiation of stem cells into osteogenic cells specific genes are up-regulated, while others are repressed. Epigenetic regulation is a crucial mechanism involved in the control of the complex, dynamic gene expression pattern during stem cell differentiation. Epigenetic modifications lead to functional relevant alterations in the genome without any changes in the nucleotide sequence[89]. Well known examples for epigenetic modifications are DNA methylation and histone modifications[90]. During DNA methylation, through the enzymatic activity of DNA methyl-transferases (Dnmts), a methyl group is covalently bound to the carbon 5 of the cytosine in the CpG sites of the genome[91]. Methylation of cytosine results in the down-regulation of gene expression[90]. In contrast, the absence of DNA methylation is crucial for gene expression[92]. During bone regeneration DNA methylation is essential. As such it has been documented that CpG methylation of the osteocalcin promoter significantly decreases during in vitro osteoblast differentiation of MSCs and thus leads to the up-regulation of osteocalcin expression[87]. In addition, hyper-methylation of the LIN28 promoter (a gene responsible for the maintenance of stemness) reduces LIN28 expression during osteoblast differentiation and hence facilitates osteogenesis[93].

Gene transcription is also regulated by histone modifications[90]. Histones are positively charged proteins that tightly bind to the negatively charged phosphate-sugar backbone of the double stranded DNA. Histone binding to the DNA modulates the accessibility of the DNA for transcription factors[94]. The most investigated histone modifications are acetylation and methylation. Acetylation, mediated by histone acetylases, of the histon’s lysin residue reduces the positive charge of the histone and thus decreases histone binding to the DNA. This in turn increases DNA accessibility for transcription factors and hence leads to increased gene expression. On the other hand, histone deacetylases remove acetyl from histones and therefore enhance histone binding to the DNA. This leads to a closed chromatin structure, reduced DNA accessibility for transcription factors and thus decreased gene expression[95]. Histone acetylation plays an important role during bone regeneration. It has been shown that during osteoblast differentiation both the promoter and the coding region of osteocalcin contain high levels of acetylated H4 and H3 histones, pointing to increased transcriptional accessibility to these sites. Furthermore, it has been demonstrated that down-regulation of histone deacetylase 1 is an important process for osteogenesis[96].

Histone methylation often occurs on lysine and arginine residues. Histone methylation is tightly controlled by the balanced activity between methyl transferases and demethylases[95]. Histone methylation is involved in osteogenic differentiation of MSCs. It has been demonstrated that the activation of CDK1 promotes osteogenic differentiation of MSCs through phosphorylation of EZH2, the catalytic subunit of PRC2, that in turn catalyzes the methylation of histone H3 on lysine 27 (H3K27)[97].

The above mentioned examples are only a few describing the complex involvement of epigenetic regulation in bone regeneration. The importance for the understanding of epigenetics in osteogenic differentiation of stem cells gains growing attendance among scientists coping with the development of new technologies for bone regeneration.

TRANSLATIONAL STUDIES

Cell-based pioneer studies in the field of bone regeneration used non-expanded bone marrow derived MSCs. Autologous bone marrow MSCs were used to treat patients with genetic bone metabolism diseases[41] as well as nonunion fractures[40]. However, due to the limited number of MSCs in the bone marrow (0.01% of bone marrow cell population) the results following non-expanded MSC transplantation are unpredictable[98]. Few years later, technological developments in MSC culture allowed expansion and characterization of these cells. The osteogenic potential of expanded, purified and identified MSCs was extensively studied, with conflicting results[42,99]. These inconsistent results can be related to several factors that influence MSC function: donor related differences (species, age, systemic disorders), insufficient vasculature to the grafted area that caused malfunction and apoptosis of the cells[46] and inadequate osteoblastic differentiation of the transplanted cells in situ. To overcome these disadvantages, attempts were made to improve osteogenic differentiation of MSCs by genetic modification[100] and to enhance vascularization of the graft[101,102]. MSCs that were engineered to express an osteogenic growth factor such as BMP2 and implanted in vivo were shown to differentiate and contribute to the process of bone formation[103-106]. According to their results MSC-BMP2 induced new bone formation in orthotopic (bone defect) and ectopic (intramuscular) models. Histological analysis revealed that MSCs-BMP2 differentiated into osteoblasts and became integrated in newly formed bone alongside recipient cells that also had differentiated into osteoblasts. In contrast, MSCs that were not genetically transformed survived in the damaged tissue as undifferentiated or fibroblastic cells[98]. Since early vascularization of the graft is crucial for successful bone regeneration, Kumar et al[101] examined the vasculogenic and osteogenic potential of MSC expressing BMP2 and VEGF (alone or in combination). In the dual therapy group, improved vasculogenesis preceded increased bone healing, suggesting that increased vascularity during the bone-healing process allows continuous flow of all the necessary nutrients and cells needed for bone regeneration. Another approach to improve vascularization of MSC-composite graft was presented by Koob et al[102] who used co-culture of human umbilical vein endothelial cells (HUVECs) and MSCs. HUVECs and MSCs were seeded alone or in combination into xenograft scaffolds and implanted into calvarial critical-sized defects. Neo-vessel formation was considerably higher in the co-implantation group, suggesting that implanted MSCs supported HUVEC-triggered neovascularization. In addition, implanted MSCs effectively supported bone formation in calvarial defects. However, the human HUVEC-derived neo-vasculature did not improve MSC-triggered bone regeneration in this orthotopic critical-sized defect model[102].

With the novel discovery of circulating EPCs[48], and the detection of cells expressing endothelial markers in newly regenerating bone tissue[107], a new idea to treat bone defects using EPCs was considered. Transplantation of autologous late outgrowth EPCs into critical size gap in sheep tibiae revealed full bridging at 3 mo in 6 out of 7 EPC-transplanted defects while non or minimal new bone formation was observed radiographically in 8 sham-operated defects[47]. These authors also suggested that the effect of EPC is not limited to vasculogenesis. When sheep EPCs were sub-cultured under osteogenic conditions, they changed their morphology and formed nodular aggregates (1-2 mm diameter) following 1-2 wk incubation and stained positively for markers of osteoblastic differentiation[55]. These results were confirmed by other research groups that showed promising results with late EPCs[108,109]. However, none of them utilized expanded human late EPCs. This could be related to difficulties in human late EPC culturing that demands further improvement. The negligible concentration of circulating EPCs in humans is probably the main obstacle in this process; however, their concentration increases following trauma, bone fractures, malignancy, tissue ischemia, physical activity and GMCSF administration[110]. Contrary to the difficulties arising with human late EPC culture, early human EPCs (that have only limited proliferation capacity) are more accessible, therefore can alternatively be used. Seebach et al[54] compared healing of critical size defect in athymic rats following transplantation of human early EPCs/bone marrow MSCs/and coculture of EPCs + MSCs. Early vascularization was significantly improved in the EPC + MSC group or EPC group. Bone formation increased significantly when treated with co-culture of MSCs + EPCs and to a lesser extent in the MSC group. Surprisingly, transplantation of early EPCs that increased early vascularization failed to enhance bone formation. Therefore, it is suggested that there is a synergistic effect and that the initial stage of neovascularization by EPCs is considered to be crucial for complete bone regeneration by MSCs in the late phase[54].

EARLY CLINICAL FINDINGS - BONE REGENERATION USING AUTOLOGOUS STEM/PROGENITOR CELLS

One of the leading research groups in the field of bone regeneration recently published a randomized controlled clinical trial in which human autologous cells were transplanted into bone defects with promising results. Bone repair cells were produced from bone marrow aspirates, isolated, cultured and characterized. Flow cytometry demonstrated that these cells were enriched for mesenchymal and vascular phenotypes. Cell suspension was absorbed onto a gelatin sponge that was transplanted into the extraction site and covered by a bio-absorbable collagen barrier membrane. Six weeks later, a biopsy of the regenerated site revealed highly vascularized, mineralized bone tissue formation[111]. Promising results were also reported following transplantation of human autologous bone marrow derived MSCs to augment cleft palate and maxillary sinus[112,113]. Orthopedic use of bone marrow MSCs to treat long bone fractures was also recently reported[114]. Autologous osteoblasts, isolated from bone marrow and cultured in osteogenic conditioned medium, were transplanted into the fracture two weeks post internal fixation. They showed statistically significant acceleration of fracture healing in the experimental group that was treated with cells compared with the nontransplanted group. Importantly, none of these studies reported on significant inflammatory reaction or serious adverse effects.

From a clinical point of view peripheral blood progenitor cells are more accessible compared with bone marrow derived MSCs. Therefore attempts are made to isolate, culture and transplant blood derived progenitor cells into bone defects. The first clinical case of tibial nonunion treated with peripheral blood progenitors (G-CSF-mobilized CD34+ cells) was published by Kuroda et al[115]. In this case report, delayed healing and nonunion were observed following primary operation that included transplantation of autologous iliac bone block. Nine months later, mobilized CD34+ cells were transplanted into the site and resulted in bone union without any symptoms including pain and gait disturbance[115]. Encouraged by these results, the same research group began a phase I/IIa clinical trial: Autologous local transplantation of G-CSF-mobilized peripheral blood CD34+ cells for patients with tibial or femoral nonunion[110]. Five days following G-CSF injection, each patient’s cells were magnet sorted to separate the CD34+ cells. Treatment included autologous transplantation of G-CSF-mobilized peripheral blood CD34+ cells suspended in atherocollagen gel. Radiological fracture healing at 12 wk was achieved in five of seven (71.4%) patients, which was greater than the threshold (18.1%) predefined by the historical outcome of standard care.

SAFTEY OF ADULT STEM CELL TRANSPLANTATION

Clinical trials that assessed the safety of MSC transplantation for the treatment of graft-vs-host disease, ischemic heart disease, spinal cord injury, and systemic lupus erythematosus, did not find any significant adverse effect[116-120]. Nevertheless, studies that investigated the role of MSCs in tumor-genesis found that MSCs have the ability to interact with tumor cells, support tumor angiogenesis and tumor development by providing the matrix required for new vessels and tumor cell scaffolding[121,122]. Therefore, the potential of MSCs to facilitate growth of preexisting occult tumors raised concerns[123,124]. In vitro transdiffrentiation of MSCs was also reported but may be related to contamination with cancer cells[125,126].

EPCs initiate vasculogenesis, as such, these cells play a critical role in pathological vasculature in tumor growth[121] and metastasis[127]. Recent clinical studies demonstrated an increase in the amount of circulating EPCs in patients suffering from malignant tumors and in cases of invasive or recurrence of the disease[128,129]. These observations underline the need for careful phenotypic, functional, and genetic characterization of EPCs/MSCs before cell administration or therapy. Also, the question of whether patients should be treated with autologous, or donor-derived progenitor-cells remains to be addressed.

PROSPECTIVE

There is a significant clinical need to develop innovative approaches for the treatment of large bone defects, nonunion fractures and alveolar bone atrophy. In recent years, many efforts are being made to regenerate bone using stem cells and progenitor cells. While animal studies and limited human studies have shown efficacy of bone marrow derived MSCs and circulating EPCs, the mechanisms by which these cells improve bone regeneration are still unclear. Numerous factors restrict the clinical use of stem cells: immune rejection of cells that are not of autologous origin; limited amount of available cells for transplantation that requires in vitro cell expansion with xenograft materials; donor related differences (e.g., age and systemic conditions) that affect cell function and the absence of potency test that can predict the in vivo function of cells before transplantation. Moreover, safety and regulation of these procedures have to be performed before clinical trials. To overcome these drawbacks, new technologies that will allow increasing the amount of cells available for transplantation and standardization of growth conditions need to be developed. In addition, understanding the mechanisms of action will enable a breakthrough in bone regeneration induced by cells.

Footnotes

P- Reviewer: Huang YH, Song GB, Yao CL S- Editor: Tian YL L- Editor: Wang TQ E- Editor: Lu YJ

References
1.  Einhorn TA. Enhancement of fracture-healing. J Bone Joint Surg Am. 1995;77:940-956.  [PubMed]  [DOI]
2.  Balogh ZJ, Reumann MK, Gruen RL, Mayer-Kuckuk P, Schuetz MA, Harris IA, Gabbe BJ, Bhandari M. Advances and future directions for management of trauma patients with musculoskeletal injuries. Lancet. 2012;380:1109-1119.  [PubMed]  [DOI]
3.  Colnot C, Romero DM, Huang S, Helms JA. Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects. Clin Orthop Relat Res. 2005;69-78.  [PubMed]  [DOI]
4.  Frölke JP, Patka P. Definition and classification of fracture non-unions. Injury. 2007;38 Suppl 2:S19-S22.  [PubMed]  [DOI]
5.  Tzioupis C, Giannoudis PV. Prevalence of long-bone non-unions. Injury. 2007;38 Suppl 2:S3-S9.  [PubMed]  [DOI]
6.  Lekholm U, Wannfors K, Isaksson S, Adielsson B. Oral implants in combination with bone grafts. A 3-year retrospective multicenter study using the Brånemark implant system. Int J Oral Maxillofac Surg. 1999;28:181-187.  [PubMed]  [DOI]
7.  Iizuka T, Smolka W, Hallermann W, Mericske-Stern R. Extensive augmentation of the alveolar ridge using autogenous calvarial split bone grafts for dental rehabilitation. Clin Oral Implants Res. 2004;15:607-615.  [PubMed]  [DOI]
8.  Mistry AS, Mikos AG. Tissue engineering strategies for bone regeneration. Adv Biochem Eng Biotechnol. 2005;94:1-22.  [PubMed]  [DOI]
9.  Glimcher MJ. The nature of the mineral component of bone and the mechanism of calcification. Instr Course Lect. 1987;36:49-69.  [PubMed]  [DOI]
10.  Muhlemann HR, Zander HA, Halberg F. Mitotic activity in the periodontal tissues of the rat molar. J Dent Res. 1954;33:459-467.  [PubMed]  [DOI]
11.  Buckwalter JA, Cooper RR. Bone structure and function. Instr Course Lect. 1987;36:27-48.  [PubMed]  [DOI]
12.  Thomson BM, Saklatvala J, Chambers TJ. Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts. J Exp Med. 1986;164:104-112.  [PubMed]  [DOI]
13.  Lanyon LE. Osteocytes, strain detection, bone modeling and remodeling. Calcif Tissue Int. 1993;53 Suppl 1:S102-S106; discussion S106-S107.  [PubMed]  [DOI]
14.  Einhorn TA, Majeska RJ, Rush EB, Levine PM, Horowitz MC. The expression of cytokine activity by fracture callus. J Bone Miner Res. 1995;10:1272-1281.  [PubMed]  [DOI]
15.  Roodman GD. Advances in bone biology: the osteoclast. Endocr Rev. 1996;17:308-332.  [PubMed]  [DOI]
16.  Yang Y. Skeletal morphogenesis during embryonic development. Crit Rev Eukaryot Gene Expr. 2009;19:197-218.  [PubMed]  [DOI]
17.  Mundlos S, Olsen BR. Heritable diseases of the skeleton. Part I: Molecular insights into skeletal development-transcription factors and signaling pathways. FASEB J. 1997;11:125-132.  [PubMed]  [DOI]
18.  Percival CJ, Richtsmeier JT. Angiogenesis and intramembranous osteogenesis. Dev Dyn. 2013;242:909-922.  [PubMed]  [DOI]
19.  Frost HM. Tetracycline-based histological analysis of bone remodeling. Calcif Tissue Res. 1969;3:211-237.  [PubMed]  [DOI]
20.  Hattner R, Epker BN, Frost HM. Suggested sequential mode of control of changes in cell behaviour in adult bone remodelling. Nature. 1965;206:489-490.  [PubMed]  [DOI]
21.  Parfitt AM. The coupling of bone formation to bone resorption: a critical analysis of the concept and of its relevance to the pathogenesis of osteoporosis. Metab Bone Dis Relat Res. 1982;4:1-6.  [PubMed]  [DOI]
22.  Pennisi A, Ling W, Li X, Khan S, Wang Y, Barlogie B, Shaughnessy JD, Yaccoby S. Consequences of daily administered parathyroid hormone on myeloma growth, bone disease, and molecular profiling of whole myelomatous bone. PLoS One. 2010;5:e15233.  [PubMed]  [DOI]
23.  Kwong FN, Harris MB. Recent developments in the biology of fracture repair. J Am Acad Orthop Surg. 2008;16:619-625.  [PubMed]  [DOI]
24.  Copuroglu C, Calori GM, Giannoudis PV. Fracture non-union: who is at risk? Injury. 2013;44:1379-1382.  [PubMed]  [DOI]
25.  Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the diamond concept. Injury. 2007;38 Suppl 4:S3-S6.  [PubMed]  [DOI]
26.  Gómez-Barrena E, Rosset P, Lozano D, Stanovici J, Ermthaller C, Gerbhard F. Bone fracture healing: Cell therapy in delayed unions and nonunions. Bone. 2015;70C:93-101.  [PubMed]  [DOI]
27.  Kon T, Cho TJ, Aizawa T, Yamazaki M, Nooh N, Graves D, Gerstenfeld LC, Einhorn TA. Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res. 2001;16:1004-1014.  [PubMed]  [DOI]
28.  Vo TN, Kasper FK, Mikos AG. Strategies for controlled delivery of growth factors and cells for bone regeneration. Adv Drug Deliv Rev. 2012;64:1292-1309.  [PubMed]  [DOI]
29.  Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res. 1998;S7-21.  [PubMed]  [DOI]
30.  Champagne CM, Takebe J, Offenbacher S, Cooper LF. Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2. Bone. 2002;30:26-31.  [PubMed]  [DOI]
31.  Lehmann W, Edgar CM, Wang K, Cho TJ, Barnes GL, Kakar S, Graves DT, Rueger JM, Gerstenfeld LC, Einhorn TA. Tumor necrosis factor alpha (TNF-alpha) coordinately regulates the expression of specific matrix metalloproteinases (MMPS) and angiogenic factors during fracture healing. Bone. 2005;36:300-310.  [PubMed]  [DOI]
32.  Fridenshteĭn AIa, Piatetskiĭ-Shapiro II, Petrakova KV. [Osteogenesis in transplants of bone marrow cells]. Arkh Anat Gistol Embriol. 1969;56:3-11.  [PubMed]  [DOI]
33.  Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315-317.  [PubMed]  [DOI]
34.  Deschaseaux F, Pontikoglou C, Sensébé L. Bone regeneration: the stem/progenitor cells point of view. J Cell Mol Med. 2010;14:103-115.  [PubMed]  [DOI]
35.  Paul G, Özen I, Christophersen NS, Reinbothe T, Bengzon J, Visse E, Jansson K, Dannaeus K, Henriques-Oliveira C, Roybon L. The adult human brain harbors multipotent perivascular mesenchymal stem cells. PLoS One. 2012;7:e35577.  [PubMed]  [DOI]
36.  Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang L. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 2008;3:301-313.  [PubMed]  [DOI]
37.  Hennig T, Lorenz H, Thiel A, Goetzke K, Dickhut A, Geiger F, Richter W. Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6. J Cell Physiol. 2007;211:682-691.  [PubMed]  [DOI]
38.  Petite H, Viateau V, Bensaïd W, Meunier A, de Pollak C, Bourguignon M, Oudina K, Sedel L, Guillemin G. Tissue-engineered bone regeneration. Nat Biotechnol. 2000;18:959-963.  [PubMed]  [DOI]
39.  Bruder SP, Kraus KH, Goldberg VM, Kadiyala S. The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am. 1998;80:985-996.  [PubMed]  [DOI]
40.  Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med. 2001;344:385-386.  [PubMed]  [DOI]
41.  Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, Muul L, Hofmann T. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci USA. 2002;99:8932-8937.  [PubMed]  [DOI]
42.  Steinhardt Y, Aslan H, Regev E, Zilberman Y, Kallai I, Gazit D, Gazit Z. Maxillofacial-derived stem cells regenerate critical mandibular bone defect. Tissue Eng Part A. 2008;14:1763-1773.  [PubMed]  [DOI]
43.  Jafarian M, Eslaminejad MB, Khojasteh A, Mashhadi Abbas F, Dehghan MM, Hassanizadeh R, Houshmand B. Marrow-derived mesenchymal stem cells-directed bone regeneration in the dog mandible: a comparison between biphasic calcium phosphate and natural bone mineral. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;105:e14-e24.  [PubMed]  [DOI]
44.  Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Exp Hematol. 2000;28:707-715.  [PubMed]  [DOI]
45.  Zhou S, Greenberger JS, Epperly MW, Goff JP, Adler C, Leboff MS, Glowacki J. Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts. Aging Cell. 2008;7:335-343.  [PubMed]  [DOI]
46.  Lin RZ, Moreno-Luna R, Li D, Jaminet SC, Greene AK, Melero-Martin JM. Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling. Proc Natl Acad Sci USA. 2014;111:10137-10142.  [PubMed]  [DOI]
47.  Rozen N, Bick T, Bajayo A, Shamian B, Schrift-Tzadok M, Gabet Y, Yayon A, Bab I, Soudry M, Lewinson D. Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects. Bone. 2009;45:918-924.  [PubMed]  [DOI]
48.  Asahara 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;275:964-967.  [PubMed]  [DOI]
49.  Murohara T, Ikeda H, Duan J, Shintani S, Sasaki Ki, Eguchi H, Onitsuka I, Matsui K, Imaizumi T. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000;105:1527-1536.  [PubMed]  [DOI]
50.  Takahashi 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;5:434-438.  [PubMed]  [DOI]
51.  Lee DY, Cho TJ, Lee HR, Park MS, Yoo WJ, Chung CY, Choi IH. Distraction osteogenesis induces endothelial progenitor cell mobilization without inflammatory response in man. Bone. 2010;46:673-679.  [PubMed]  [DOI]
52.  Cetrulo CL, Knox KR, Brown DJ, Ashinoff RL, Dobryansky M, Ceradini DJ, Capla JM, Chang EI, Bhatt KA, McCarthy JG. Stem cells and distraction osteogenesis: endothelial progenitor cells home to the ischemic generate in activation and consolidation. Plast Reconstr Surg. 2005;116:1053-1064; discussion 1064-1065.  [PubMed]  [DOI]
53.  Lee DY, Cho TJ, Kim JA, Lee HR, Yoo WJ, Chung CY, Choi IH. Mobilization of endothelial progenitor cells in fracture healing and distraction osteogenesis. Bone. 2008;42:932-941.  [PubMed]  [DOI]
54.  Seebach C, Henrich D, Kähling C, Wilhelm K, Tami AE, Alini M, Marzi I. Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance early vascularization and bone healing in a critical-sized bone defect in rats. Tissue Eng Part A. 2010;16:1961-1970.  [PubMed]  [DOI]
55.  Bick T, Rozen N, Dreyfuss E, Soudry M, Lewinson D. Osteogenic differentiation of circulating endothelial progenitor cells. J of Bone and Min Res. 2006;22:S143.  [PubMed]  [DOI]
56.  Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, Zeiher AM, Dimmeler S. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med. 2003;9:1370-1376.  [PubMed]  [DOI]
57.  De Falco E, Porcelli D, Torella AR, Straino S, Iachininoto MG, Orlandi A, Truffa S, Biglioli P, Napolitano M, Capogrossi MC. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells. Blood. 2004;104:3472-3482.  [PubMed]  [DOI]
58.  Medina RJ, O’Neill CL, Sweeney M, Guduric-Fuchs J, Gardiner TA, Simpson DA, Stitt AW. Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities. BMC Medical Genomics. 2010;3:18.  [PubMed]  [DOI]
59.  Timmermans F, Plum J, Yöder MC, Ingram DA, Vandekerckhove B, Case J. Endothelial progenitor cells: identity defined? J Cell Mol Med. 2009;13:87-102.  [PubMed]  [DOI]
60.  Barber CL, Iruela-Arispe ML. The ever-elusive endothelial progenitor cell: identities, functions and clinical implications. Pediatr Res. 2006;59:26R-32R.  [PubMed]  [DOI]
61.  Hur J, Yoon CH, Kim HS, Choi JH, Kang HJ, Hwang KK, Oh BH, Lee MM, Park YB. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol. 2004;24:288-293.  [PubMed]  [DOI]
62.  Yoder MC, Mead LE, Prater D, Krier TR, Mroueh KN, Li F, Krasich R, Temm CJ, Prchal JT, Ingram DA. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood. 2007;109:1801-1809.  [PubMed]  [DOI]
63.  Fuchs S, Dohle E, Kolbe M, Kirkpatrick CJ. Outgrowth endothelial cells: sources, characteristics and potential applications in tissue engineering and regenerative medicine. Adv Biochem Eng Biotechnol. 2010;123:201-217.  [PubMed]  [DOI]
64.  Goligorsky MS, Salven P. Concise review: endothelial stem and progenitor cells and their habitats. Stem Cells Transl Med. 2013;2:499-504.  [PubMed]  [DOI]
65.  Mifune Y, Matsumoto T, Kawamoto A, Kuroda R, Shoji T, Iwasaki H, Kwon SM, Miwa M, Kurosaka M, Asahara T. Local delivery of granulocyte colony stimulating factor-mobilized CD34-positive progenitor cells using bioscaffold for modality of unhealing bone fracture. Stem Cells. 2008;26:1395-1405.  [PubMed]  [DOI]
66.  Ingram DA, Caplice NM, Yoder MC. Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells. Blood. 2005;106:1525-1531.  [PubMed]  [DOI]
67.  Matsumoto T, Kawamoto A, Kuroda R, Ishikawa M, Mifune Y, Iwasaki H, Miwa M, Horii M, Hayashi S, Oyamada A. Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing. Am J Pathol. 2006;169:1440-1457.  [PubMed]  [DOI]
68.  Taguchi A, Soma T, Tanaka H, Kanda T, Nishimura H, Yoshikawa H, Tsukamoto Y, Iso H, Fujimori Y, Stern DM. Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest. 2004;114:330-338.  [PubMed]  [DOI]
69.  Sivan-Loukianova E, Awad OA, Stepanovic V, Bickenbach J, Schatteman GC. CD34+ blood cells accelerate vascularization and healing of diabetic mouse skin wounds. J Vasc Res. 2003;40:368-377.  [PubMed]  [DOI]
70.  Kijima Y, Ishikawa M, Sunagawa T, Nakanishi K, Kamei N, Yamada K, Tanaka N, Kawamata S, Asahara T, Ochi M. Regeneration of peripheral nerve after transplantation of CD133+ cells derived from human peripheral blood. J Neurosurg. 2009;110:758-767.  [PubMed]  [DOI]
71.  Kamei N, Kwon SM, Alev C, Nakanishi K, Yamada K, Masuda H, Ishikawa M, Kawamoto A, Ochi M, Asahara T. Ex-vivo expanded human blood-derived CD133+ cells promote repair of injured spinal cord. J Neurol Sci. 2013;328:41-50.  [PubMed]  [DOI]
72.  Ishida K, Matsumoto T, Sasaki K, Mifune Y, Tei K, Kubo S, Matsushita T, Takayama K, Akisue T, Tabata Y. Bone regeneration properties of granulocyte colony-stimulating factor via neovascularization and osteogenesis. Tissue Eng Part A. 2010;16:3271-3284.  [PubMed]  [DOI]
73.  Fukui T, Matsumoto T, Mifune Y, Shoji T, Kuroda T, Kawakami Y, Kawamoto A, Ii M, Kawamata S, Kurosaka M. Local transplantation of granulocyte colony-stimulating factor-mobilized human peripheral blood mononuclear cells for unhealing bone fractures. Cell Transplant. 2012;21:707-721.  [PubMed]  [DOI]
74.  Kørbling M, Estrov Z. Adult stem cells for tissue repair - a new therapeutic concept? N Engl J Med. 2003;349:570-582.  [PubMed]  [DOI]
75.  Tolar J, Le Blanc K, Keating A, Blazar BR. Concise review: hitting the right spot with mesenchymal stromal cells. Stem Cells. 2010;28:1446-1455.  [PubMed]  [DOI]
76.  Kawakami Y, Ii M, Matsumoto T, Kuroda R, Kuroda T, Kwon SM, Kawamoto A, Akimaru H, Mifune Y, Shoji T. SDF-1/CXCR4 Axis in Tie2-Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing. J Bone Miner Res. 2015;30:95-105.  [PubMed]  [DOI]
77.  Marquez-Curtis LA, Janowska-Wieczorek A. Enhancing the migration ability of mesenchymal stromal cells by targeting the SDF-1/CXCR4 axis. Biomed Res Int. 2013;2013:561098.  [PubMed]  [DOI]
78.  Molyneaux KA, Zinszner H, Kunwar PS, Schaible K, Stebler J, Sunshine MJ, O’Brien W, Raz E, Littman D, Wylie C. The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival. Development. 2003;130:4279-4286.  [PubMed]  [DOI]
79.  Liu H, Xue W, Ge G, Luo X, Li Y, Xiang H, Ding X, Tian P, Tian X. Hypoxic preconditioning advances CXCR4 and CXCR7 expression by activating HIF-1α in MSCs. Biochem Biophys Res Commun. 2010;401:509-515.  [PubMed]  [DOI]
80.  Yu J, Li M, Qu Z, Yan D, Li D, Ruan Q. SDF-1/CXCR4-mediated migration of transplanted bone marrow stromal cells toward areas of heart myocardial infarction through activation of PI3K/Akt. J Cardiovasc Pharmacol. 2010;55:496-505.  [PubMed]  [DOI]
81.  Blau HM, Brazelton TR, Weimann JM. The evolving concept of a stem cell: entity or function? Cell. 2001;105:829-841.  [PubMed]  [DOI]
82.  Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature. 2003;425:968-973.  [PubMed]  [DOI]
83.  Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature. 2002;416:542-545.  [PubMed]  [DOI]
84.  Phinney DG. Biochemical heterogeneity of mesenchymal stem cell populations: clues to their therapeutic efficacy. Cell Cycle. 2007;6:2884-2889.  [PubMed]  [DOI]
85.  Hu Y, Davison F, Zhang Z, Xu Q. Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells. Circulation. 2003;108:3122-3127.  [PubMed]  [DOI]
86.  Kuroda R, Matsumoto T, Kawakami Y, Fukui T, Mifune Y, Kurosaka M. Clinical impact of circulating CD34-positive cells on bone regeneration and healing. Tissue Eng Part B Rev. 2014;20:190-199.  [PubMed]  [DOI]
87.  Villagra A, Gutiérrez J, Paredes R, Sierra J, Puchi M, Imschenetzky M, Wijnen Av Av, Lian J, Stein G, Stein J. Reduced CpG methylation is associated with transcriptional activation of the bone-specific rat osteocalcin gene in osteoblasts. J Cell Biochem. 2002;85:112-122.  [PubMed]  [DOI]
88.  Arnsdorf EJ, Tummala P, Castillo AB, Zhang F, Jacobs CR. The epigenetic mechanism of mechanically induced osteogenic differentiation. J Biomech. 2010;43:2881-2886.  [PubMed]  [DOI]
89.  Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003;33 Suppl:245-254.  [PubMed]  [DOI]
90.  Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet. 2002;3:662-673.  [PubMed]  [DOI]
91.  Bird AP. CpG-rich islands and the function of DNA methylation. Nature. 1986;321:209-213.  [PubMed]  [DOI]
92.  Bird AP. Functions for DNA methylation in vertebrates. Cold Spring Harb Symp Quant Biol. 1993;58:281-285.  [PubMed]  [DOI]
93.  Dansranjavin T, Krehl S, Mueller T, Mueller LP, Schmoll HJ, Dammann RH. The role of promoter CpG methylation in the epigenetic control of stem cell related genes during differentiation. Cell Cycle. 2009;8:916-924.  [PubMed]  [DOI]
94.  Zheng C, Hayes JJ. Structures and interactions of the core histone tail domains. Biopolymers. 2003;68:539-546.  [PubMed]  [DOI]
95.  Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693-705.  [PubMed]  [DOI]
96.  Lee HW, Suh JH, Kim AY, Lee YS, Park SY, Kim JB. Histone deacetylase 1-mediated histone modification regulates osteoblast differentiation. Mol Endocrinol. 2006;20:2432-2443.  [PubMed]  [DOI]
97.  Wei Y, Chen YH, Li LY, Lang J, Yeh SP, Shi B, Yang CC, Yang JY, Lin CY, Lai CC. CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells. Nat Cell Biol. 2011;13:87-94.  [PubMed]  [DOI]
98.  Caplan AI. New era of cell-based orthopedic therapies. Tissue Eng Part B Rev. 2009;15:195-200.  [PubMed]  [DOI]
99.  Ben-David D, Kizhner TA, Kohler T, Müller R, Livne E, Srouji S. Cell-scaffold transplant of hydrogel seeded with rat bone marrow progenitors for bone regeneration. J Craniomaxillofac Surg. 2011;39:364-371.  [PubMed]  [DOI]
100.  Hasharoni A, Zilberman Y, Turgeman G, Helm GA, Liebergall M, Gazit D. Murine spinal fusion induced by engineered mesenchymal stem cells that conditionally express bone morphogenetic protein-2. J Neurosurg Spine. 2005;3:47-52.  [PubMed]  [DOI]
101.  Kumar S, Wan C, Ramaswamy G, Clemens TL, Ponnazhagan S. Mesenchymal stem cells expressing osteogenic and angiogenic factors synergistically enhance bone formation in a mouse model of segmental bone defect. Mol Ther. 2010;18:1026-1034.  [PubMed]  [DOI]
102.  Koob S, Torio-Padron N, Stark GB, Hannig C, Stankovic Z, Finkenzeller G. Bone formation and neovascularization mediated by mesenchymal stem cells and endothelial cells in critical-sized calvarial defects. Tissue Eng Part A. 2011;17:311-321.  [PubMed]  [DOI]
103.  Gazit D, Turgeman G, Kelley P, Wang E, Jalenak M, Zilberman Y, Moutsatsos I. Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy. J Gene Med. 1999;1:121-133.  [PubMed]  [DOI]
104.  Turgeman G, Pittman DD, Müller R, Kurkalli BG, Zhou S, Pelled G, Peyser A, Zilberman Y, Moutsatsos IK, Gazit D. Engineered human mesenchymal stem cells: a novel platform for skeletal cell mediated gene therapy. J Gene Med. 2001;3:240-251.  [PubMed]  [DOI]
105.  Yue B, Lu B, Dai KR, Zhang XL, Yu CF, Lou JR, Tang TT. BMP2 gene therapy on the repair of bone defects of aged rats. Calcif Tissue Int. 2005;77:395-403.  [PubMed]  [DOI]
106.  Zachos T, Diggs A, Weisbrode S, Bartlett J, Bertone A. Mesenchymal stem cell-mediated gene delivery of bone morphogenetic protein-2 in an articular fracture model. Mol Ther. 2007;15:1543-1550.  [PubMed]  [DOI]
107.  Lewinson D, Maor G, Rozen N, Rabinovich I, Stahl S, Rachmiel A. Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system. Histochem Cell Biol. 2001;116:381-388.  [PubMed]  [DOI]
108.  Li R, Atesok K, Nauth A, Wright D, Qamirani E, Whyne CM, Schemitsch EH. Endothelial progenitor cells for fracture healing: a microcomputed tomography and biomechanical analysis. J Orthop Trauma. 2011;25:467-471.  [PubMed]  [DOI]
109.  Amini AR, Laurencin CT, Nukavarapu SP. Differential analysis of peripheral blood- and bone marrow-derived endothelial progenitor cells for enhanced vascularization in bone tissue engineering. J Orthop Res. 2012;30:1507-1515.  [PubMed]  [DOI]
110.  Kuroda R, Matsumoto T, Niikura T, Kawakami Y, Fukui T, Lee SY, Mifune Y, Kawamata S, Fukushima M, Asahara T. Local transplantation of granulocyte colony stimulating factor-mobilized CD34+ cells for patients with femoral and tibial nonunion: pilot clinical trial. Stem Cells Transl Med. 2014;3:128-134.  [PubMed]  [DOI]
111.  Kaigler D, Pagni G, Park CH, Braun TM, Holman LA, Yi E, Tarle SA, Bartel RL, Giannobile WV. Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial. Cell Transplant. 2013;22:767-777.  [PubMed]  [DOI]
112.  Behnia H, Khojasteh A, Soleimani M, Tehranchi A, Khoshzaban A, Keshel SH, Atashi R. Secondary repair of alveolar clefts using human mesenchymal stem cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;108:e1-e6.  [PubMed]  [DOI]
113.  Park JB. Use of cell-based approaches in maxillary sinus augmentation procedures. J Craniofac Surg. 2010;21:557-560.  [PubMed]  [DOI]
114.  Kim SJ, Shin YW, Yang KH, Kim SB, Yoo MJ, Han SK, Im SA, Won YD, Sung YB, Jeon TS. A multi-center, randomized, clinical study to compare the effect and safety of autologous cultured osteoblast(Ossron) injection to treat fractures. BMC Musculoskelet Disord. 2009;10:20.  [PubMed]  [DOI]
115.  Kuroda R, Matsumoto T, Miwa M, Kawamoto A, Mifune Y, Fukui T, Kawakami Y, Niikura T, Lee SY, Oe K. Local transplantation of G-CSF-mobilized CD34(+) cells in a patient with tibial nonunion: a case report. Cell Transplant. 2011;20:1491-1496.  [PubMed]  [DOI]
116.  Battiwalla M, Hematti P. Mesenchymal stem cells in hematopoietic stem cell transplantation. Cytotherapy. 2009;11:503-515.  [PubMed]  [DOI]
117.  Lucchini G, Introna M, Dander E, Rovelli A, Balduzzi A, Bonanomi S, Salvade` A, Capelli C, Belotti D, Gaipa . Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft versus host disease in a pediatric population. Biol Blood Marrow Transplant. 2010;16:1293-1301.  [PubMed]  [DOI]
118.  Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient’s bedside: an update on clinical trials with mesenchymal stem cells. J Cell Physiol. 2007;211:27-35.  [PubMed]  [DOI]
119.  Pal R, Venkataramana NK, Bansal A, Balaraju S, Jan M, Chandra R, Dixit A, Rauthan A, Murgod U, Totey S. Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/paraplegia: A pilot clinical study. Cytotherapy. 2009;11:897-911.  [PubMed]  [DOI]
120.  Sun L, Akiyama K, Zhang H, Yamaza T, Hou Y, Zhao S, Xu T, Le A, Shi S. Mesenchymal stem cell transplantation reverses multi organ dysfunction in systemic lupus erythematosus mice and humans. Stem Cells. 2009;27:1421-1432.  [PubMed]  [DOI]
121.  Ahn GO, Brown JM. Role of endothelial progenitors and other bone marrow-derived cells in the development of the tumor vasculature. Angiogenesis. 2009;12:159-164.  [PubMed]  [DOI]
122.  Zhu W, Xu W, Jiang R, Qian H, Chen M, Hu J, Cao W, Han C, Chen Y. Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo. Exp Mol Pathol. 2006;80:267-274.  [PubMed]  [DOI]
123.  Feng B, Chen L. Review of mesenchymal stem cells and tumors: executioner or coconspirator? Cancer Biother Radiopharm. 2009;24:717-721.  [PubMed]  [DOI]
124.  Motaln H, Schichor C, Lah TT. Human mesenchymal stem cells and their use in cell-based therapies. Cancer. 2010;116:2519-2530.  [PubMed]  [DOI]
125.  Garcia S, Bernad A, Martin MC, Cigudosa JC, Garcia- Castro J, de la Fuente R. Pitfalls in spontaneous in vitro transformation of human mesenchymal stem cells. Exp Cell Res. 2010;316:1648-1650.  [PubMed]  [DOI]
126.  Torsvik A, Rosland GV, Svendsen A, Molven A, Immervoll H, McCormack E, Lonning PE, Primon M, Sobala E, Tonn JC. Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: Putting the research field on track - letter. Cancer Res. 2010;70:6393-6396.  [PubMed]  [DOI]
127.  Gao D, Nolan DJ, Mellick AS, Bambino K, McDonnell K, Mittal V. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science. 2008;319:195-198.  [PubMed]  [DOI]
128.  Naik RP, Jin D, Chuang E, Gold EG, Tousimis EA, Moore AL, Christos PJ, de Dalmas T, Donovan D, Rafii S. Circulating endothelial progenitor cells correlate to stage in patients with invasive breast cancer. Breast Cancer Res Treat. 2008;107:133-138.  [PubMed]  [DOI]
129.  Igreja C, Courinha M, Cachaço AS, Pereira T, Cabeçadas J, da Silva MG, Dias S. Characterization and clinical relevance of circulating and biopsy-derived endothelial progenitor cells in lymphoma patients. Haematologica. 2007;92:469-477.  [PubMed]  [DOI]