Current Stem Cell Research & Therapy, 2012, 7, 00-00
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Endothelial Progenitor Cells (EPCs) and Mesenchymal Stem Cells (MSCs) in Bone Healing Nikolaos C. Keramaris1, Sarandos Kaptanis3, Helen Lucy Moss4, Mattia Loppini5, Spyridon Pneumaticos1 and Nicola Maffulli*,2 1
University of Athens, Medical School, 3rd Department of Orthopaedics, KAT General Hospital, Nikis 2 Kifissia 14561, Athens, Greece; 2Queen Mary University of London, Barts and The London School of Medicine and Dentistry, William Harvey Research Institute, Centre for Sports and Exercise Medicine, Mile End Hospital, 275 Bancroft Road, 3 London E1 4DG, England, UK; Croydon University Hospital, 530 London Road, Croydon, CR7 7YE, England,
UK; 4 Airedale General Hospital, Skipton Road, Steeton, Keighley, West Yorkshire, BD20 6TD, England, UK; 5
University Campus Biomedico of Rome, Department of Orthopaedic and Trauma Surgery, Via Alvaro del Portillo 21,00128 Rome Italy Abstract: Fracture healing is a complex physiological process. Local vascularity at the site of the fracture has been established as one of the most important factors influencing the healing process, and lack of vascularity has been implicated in atrophic non unions. Existing research has primarily involved utilising Mesenchymal Stem Cells (MSCs) to augment bone healing but there remains much scope to explore the role of stem cells in the vascularisation process. Endothelial Progenitor Cells (EPCs) and other Endothelial Cellular populations (ECs) could constitute a valid alternative to MSCs. This systematic review is examining the importance of co-implantation of MSCs and EPCs/ECs for bone healing. A literature search was performed using the Cochrane Library, OVID Medline, OVID EMBASE and Google Scholar, searching for combinations of the terms ‘EPCs’, ‘Endothelial progenitor cells’, ‘angiogenesis’, ‘fracture’, ‘bone’ and ‘healing’. Finally 18 articles that fulfilled our criteria were included in this review. ECs could be of value for the treatment of critical size bone defects as they are known to be capable of forming ectopic, vascularised bone. The co-implantation of ECs with MSCs is more intriguing when we take into account the vast array of complex reciprocal interactions between ECs and MSCs.
Keywords: Angiogenesis, bone and bones, EPCs, endothelial cells, fracture healing, mesenchymal stem cells. INTRODUCTION Bone healing is a remarkable regenerative process. Healing of a fracture can be completed without scarring, but some fractures fail to heal adequately. In the United States, 5-10% of the 5.6 million fractures occurring every year undergo a delay in healing or can fail to heal completely. The vast majority (80%) of these nonunions are atrophic [1]. A major cause of atrophic nonunion is damage to the vascular system and dysfunctional regeneration of the vasculature at the site of the fracture, frequently combined with altered biomechanics and instability [2]. The rate of nonunion escalates from 5-10% in the general population to 46% in patients with severe compromise of blood flow due to extensive vascular injury. The disruption of one of the three arteries of the leg may lead to a threefold increase of delayed or failed union [3].
*Address correspondence to this author at the Centre for Sports and Exercise Medicine, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Mile End Hospital, 275 Bancroft Road, London E1 4DG, England, UK; Email:
[email protected]
1574-888X/12 $58.00+.00
Therefore, a challenge in bone regeneration is its vascularization, because, if adequate perfusion cannot be established quickly, central necrosis of the newly produced bone tissue will occur [4]. Since diffusion of oxygen in the active tissue is limited to about 150 μm from the capillary lumen (the mean of intercapillary distance (ICD) is 304 ± 30 μm), vascularization becomes the cornerstone of the healing process in larger volume tissue-engineered constructs [5]. Previous attempts in engineered angiogenesis have focused on the delivery of angiogenic growth factors, transplantation of proangiogenic cells or fabrication of blood vessel analogs [6-9]. In several studies, angiogenesis in scaffolds has been induced by a number of angiogenic cytokines such as vascular endothelial growth factors (VEGF), platelet derived growth factors (PDGF), and basic fibroblast growth factor (bFGF) [10-17]. Despite promising results, there are some concerns over the cost of multiple cytokines and delivery, potential toxicity, and suboptimal endothelial migration in large tissue grafts. Progenitor cells able to initiate neovascularization, known as endothelial precursor cells (EPCs), were initially identified by Isner and Asahara in 1997 [18]. They are bone © 2012 Bentham Science Publishers
2 Current Stem Cell Research & Therapy, 2012, Vol. 7, No. 4
marrow (BM) cells with properties similar to those of embryonal angioblasts [19]. These progenitors constitute a small population of cells which can proliferate, migrate, and differentiate into the cells that line the lumen of blood vessels. EPCs can also be isolated from peripheral blood and spleen [20]. Prior to the discovery of this cell type, new vessel formation was believed to occur only by the proliferation of existing endothelial cells (ECs). These findings have produced a true paradigm shift in vasculogenesis, which is now considered to happen not only during embryogenesis, but also during adult life [21]. Adult EPCs can be isolated from bone marrow aspirate or peripheral blood. The classical isolation methods include the use of adherence culture of total mononuclear cells (for non adherent cells) or the use of magnetic microbeads coated with antibodies against surface markers like CD133, CD34 or CD31 for mononuclear cell sorting. After isolation, the cells are cultured on fibronectin-coated plates in mediums with specific growth factors, e.g. vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), which facilitate the growth of endothelial-like cells. Isolated bone marrow-derived EPCs represent more immature cells, expressing the early hematopoietic marker CD133 [22]. In general, early EPCs in bone marrow or immediately after their migration into the systemic circulation are positive for CD133/CD34/VEGFR-2, whereas circulating EPCs are positive for CD34/VEGFR-2/CD31/VE-cadherin and obviously lose CD133 and begin to express von Willebrand factor [19]. Bone marrow also contains stem cells for tissues that can roughly be defined as mesenchymatic. In the experiment by Friedenstein and colleagues in 1976 [23], following in vitro culture expansion, clonal cultures derived from BM could be introduced into diffusion chambers in experimental models where the formation of bone, cartilage and stromal elements was observed. The frequency of these marrow CFU-Fs was extremely low, ranging from 1/10,000 to 1/100,000 BM mononuclear cells (MNCs) [24]. This is considerably lower than the frequency of CD34+ haematopoietic progenitor/stem cells (HSCs) that comprise about 1% of the MNC fraction [25]. The progenitor cell lineages giving rise to endothelial and osteoblastic cells (EPCs/ECs and MSCs respectively) have recently been thought to overlap. In vitro, CD34+ and CD133+ cells were not only hematopoietic and vasculogenic, but they were also capable of differentiating into osteoblasts [26], while in vivo, a non-adherent side population of BM cells containing primitive cells was capable of producing both hematopoietic and osteogenic lineages [27]. CD34+ cells are committed to not only endothelial cells but also mural perivascular cells (i.e., pericytes and smooth muscle cells) [28, 29]. The co-implantation of MSCs and Endothelial Cells (EPCs/ECs) could provide an answer to the difficult problem of adequate vascularization for bone healing. This systematic
Keramaris et al.
review intends to clarify the importance of co-implantation of those two cellular populations for bone regeneration strategies. MATERIALS AND METHODS A systematic review of the literature was undertaken. Two authors (NK, SK) searched Medline (1946 to 2012 February Week 1, via OvidSP), EMBASE (1980 to 2012 Week 05, via OvidSP), and the Cochrane Library (on February 2012). The aim was to identify all studies reporting outcomes on bone regeneration with the use of Endothelial Progenitor Cells (EPCs) or other Endothelial Cell (ECs) populations, with or without the co-implantation of Mesenchymal Stem Cells (MSCs). Keywords used were “bone and bones” and “fracture healing”, combined with “endothelial cells”, “mesenchymal stem cell transplantation”, “mesenchymal stem cells”, mapped to subheadings and used as keywords. No systematic reviews or meta-analyses were identified. Sensitive search strategies yielded a total of 1899 abstracts. All journals were considered and all article abstracts reviewed for consideration. Relevant articles were chosen to retrieve and review the full text by consensus of the two authors. Citations of articles retrieved were further crossreferenced. Non-English language articles were excluded. We evaluated all studies included in the review according to an adaptation of the Coleman et al. [30] Methodology Score (Table 1). All studies not describing endpoints relevant to bone regeneration (ectopic or orthotopic, but in vivo) were excluded from the review. Articles scoring zero in over 3 domains in the abstract were not retrieved. Each study was scored for each of the criteria to give a maximum total of 100 points by two authors (NK, SK). Inter-observer variability was measured using the percentage of agreement and the kappa statistic (k) [31]. Statistical analyses were performed with Stata Version 11 [32]. RESULTS Eighteen articles that met our inclusion criteria were incorporated in this review. Independent assessment of these by two authors (NK, SK) using the modified Coleman Methodology Score demonstrated 88.89% agreement by the authors, and Cohen’s kappa was calculated at 0.875 (95% confidence interval: 0.721 to 1.030, p60
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