In recent years, with the increasing research on bone tissue engineering technology for the treatment of bone defects, the research on tissue-engineered bone seed cells, especially vascular endothelial progenitor cells, has also been intensified. In this paper, we review the research progress on the origin of vascular endothelial progenitor cells, their biological intermediate properties, mechanisms of improving vascular neogenesis, their role in bone repair, treatment and applications.
Bone repair is a complex pathophysiological activity involving multiple cells and extracellular matrix, and regulated by multiple growth factors and hormones. In recent years, bone tissue engineering technology has become the focus of research on bone defect repair, and most of its research has focused on the selection and culture of seed cells. Endothelial progenitor cells (EPCs) were first reported by Asahara et al. in 1997. This discovery updated the traditional theory of postnatal angiogenesis and vascular injury repair and provided a new idea for the treatment of ischemic diseases.
1. Origin of EPCs
It is now widely believed that EPCs and hematopoietic stem cells originate from a common stem cell, heman-gioblast. Although the definition and origin of EPCs are still controversial, most studies suggest that EPCs mainly originate from umbilical vein blood, adult peripheral blood, bone marrow, and the EPCs in peripheral blood originate from bone marrow, while the EPCs in umbilical blood originate from the fetal liver. Under normal conditions, the number of EPCs is very small, about 2-3/mL in peripheral blood and about 3.5 times higher in umbilical cord blood. Under the culture conditions containing vascular endothelia growth factor (VEGF) and fibroblast growth factor (FGF), which are suitable for the growth of EPCs, they can proliferate and expand in large numbers. Single nucleated cells obtained from umbilical cord blood, peripheral blood, bone marrow and other specimens, or single nucleated cells selected for CD34 and CD133 positivity, cultured in vitro on substrates coated with fibronectin (FN) can form EPCs, which are monolayers with a striated morphology. CD34, CD133, VEGFR-2+ and other surface marker positive, and can proliferate and differentiate into vascular endothelial cells as precursor cells after in vitro culture. It is not only involved in embryonic angiogenesis, but also exists in umbilical cord blood, peripheral blood and bone marrow, which can have a strong pro-angiogenic effect in the postnatal angiogenesis process and form neovascular EPCs in an angiogenic manner.
2, Biological properties of EPCs
2.1 Surface markers of EPCs
There is no specific surface marker for the identification of EPCs, and most scholars believe that CD34+ cells are the common progenitor cells of hematopoietic stem cells and endothelial progenitor cells.Reyes et al [3] showed that CD34+, vascular endothelial calmodulin-, CD133+ and Flk1+ multi-potent adult progenitor cells (MAPC) in bone marrow progenitor cell (MAPC) is the source of EPCs. It can differentiate into CD34+, CD133+, VEGFR-2+ and Flk1+ adult progenitor cells under the action of VEGF, FGF and IGF-Ⅰ, and can continue to differentiate into mature vascular endothelial cells, which is an important source of endothelial cells.
Initial studies defined EPCs as cells that express both the hematopoietic stem cell surface marker CD34 and the endothelial cell surface marker vascular endothelial growth factor receptor-2 (VEGFR-2). Subsequently, Peichev et al. found that CD133 antigen was present only in vascular endothelial precursor cells and that mature endothelial cells did not express CD133; therefore, they referred to cells expressing CD34+, VEGFR-2+, and CD133+ as functional vascular endothelial progenitor cells. However, some authors have proposed a different view, and Harraz et al. found that CD34- cells gradually differentiated into endothelial cells in conditioned cultures (cultures in which CD34+ cells had been cultured) during their experiments, and therefore concluded that CD34+ cells secreted certain unknown factors to stimulate CD34- cells to differentiate into endothelial cells.
Rehman et al. reported that bone marrow MSCs and CD34-CD14+ monocytes can also form functional vascular endothelial cells in vitro after induction by VEGF and other factors. Induced differentiation of endothelial cells from mononuclear macrophages has also been reported.
2.2 Mobilization of EPCs
Mobilization of progenitor cells in the bone marrow is dictated by the local environment. Activation of e.g. elastase, histone G and matrix protease family (MMPs), by removing the binding of adhesions on stromal cells, acts with hematopoietic stem cell integrins to mobilize cytokines that impede the interaction between stem cells and stromal cells, which ultimately allows stem cells to leave the bone marrow by transendothelial migration.
Physiologically, it is generally believed that ischemia can be a signal to induce mobilization of bone marrow EPCs. Thus, ischemia can upregulate VEGF and release it into the circulation and induce the mobilization of bone marrow precursor cells through MMP-9-dependent induction. In hematology, other factors have been identified including mobilization of bone marrow stem cells, such as harvesting of hematopoietic stem cells from peripheral blood for bone marrow transplantation. In addition, erythropoietin (EPO) can stimulate erythroid cell proliferation and maturation, and in mice and humans can also increase the number of endothelial progenitor cells in peripheral blood. The relationship between serum EPO levels and CD34+ or CD133+ stem cells in the bone marrow of patients with ischemic heart disease supports an important role of endogenous EPO levels as a physiological indicator of the mobilization of EPCs.
However, it is unclear which mobilization factor is most important for increasing EPCs. In animal experiments, VEGF165 rapidly mobilized hematopoietic stem cells and circulating endothelial progenitor cells, whereas angiopoietin-1 (ANG-1) induced a delayed, weaker mobilization of endothelial and hematopoietic progenitor cells. In addition, the first evidence for pharmacological modulation of EPCs levels was provided by studies with the atheroprotective drug MG-CoA reductase inhibitors (statins).
It was shown that statins increased the number and functional activity of EPCs in mice and patients with stable coronary artery disease in vitro. Statins can increase the number of stem cells in the bone marrow, increase the number of EPCs and improve the function of EPCs including proliferation of EPCs, mobilization of EPCs and prevention of senescence and apoptosis of EPCs.
2.3 Differentiation of EPCs
Koyanagi et al. found that calcineurin E and N were expressed at the EPCs-cardiomyocytes contact surface in the co-culture system, and blocking calcineurin E inhibited the transdifferentiation of EPCs, indicating the effect of intercellular interactions on the transdifferentiation of EPCs. Similarly, the notion of transdifferentiation into cardiomyocytes has recently been challenged by Sales et al. who found that EPCs induced by TGF-ß1 in vitro for 10-15 d could change from the initial endothelial phenotype (CD31+/vWF+/αSMA-) to the mesenchymal phenotype (CD31+/α-SMA+) while secreting laminin, fibronectin, and collagen type I and III. However, further studies are needed to confirm the finding that EPCs are transdifferentiated to form MSCs and not from MSC precursor cells that may be mixed with EPCs or through cell fusion mechanisms. The EPCs expressed higher smooth muscle cell markers than mature endothelial cells and were induced by PDGF-BB to differentiate into smooth muscle cells of different phenotypes (contractile or synthetic), suggesting that they may also differentiate into vascular smooth muscle cells.
All of the above studies suggest that the fate of EPCs is not necessarily linear, but may follow other pathways of differentiation under certain conditions, meaning that the “EPCs state” shared by different cells is dynamic and unstable, and may change to other stem (progenitor) progenitor cell states under the influence of external environmental factors, and differentiate into The state of EPCs is dynamic and unstable.
3, Role of EPCs in bone repair
3.1 Mechanism of EPCs in improving vascular neogenesis
Although the role of EPCs in revascularization has been demonstrated, the question now is how EPCs promote revascularization. In the absence of tissue injury, the role of progenitor cells is weak, but in ischemic tissues, genetically labeled bone marrow-derived cells can co-express marker proteins of EC with a wide variation in their effects (ranging from 0% to 90%). Similarly, what is the role of bone marrow-derived cells in post-stroke brain tissue? Reports in the literature vary considerably.
In two studies, one study resulted in a mean of 34% of bone marrow-derived positive cells expressing endothelial markers; while the other study failed to detect cells expressing endothelial markers, with a large number (50%) being detected mainly in a tumor angiogenesis model. Some studies detected only bone marrow-derived cells near blood vessels, but did not express endothelial marker proteins. One possible explanation is that the ischemic model (e.g., the degree of injury or ischemia) significantly affects the role of these cells. Mild ischemia may hardly induce the mobilization of endothelial progenitor cells in the bone marrow, and only a small number of bone marrow progenitor cells are induced to act. The effect of cells after transplantation may also vary depending on the subpopulation of cells (e.g., pure hematopoietic stem cells versus bone marrow cells). Indeed, intravenous infusion of purified bone marrow mononuclear cells or expanded endothelial progenitor cells can produce better results than internal mobilization of bone marrow transplanted cells.
Tie-2-positive bone marrow-derived cells can block tumor angiogenesis by activating suicide genes, and although these cells integrate into the tumor vasculature, they can be detected in the vicinity of the vessels. Thus, EPCs can act similarly to monocytes or macrophages in that they can increase angiogenesis by providing cytokines and growth factors. It has been shown that cultured EPCs of different origins can express growth factors such as VEGF, HGF and IGF-1. Adherent monocytes can be cultured under similar conditions and release VEGF, HGF and G-CSF, but cannot express endothelial marker proteins; EPCs can be incorporated into neovascular structures, suggesting that they are involved in endothelial marker protein expression in vivo; whereas macrophages, which can also release growth factors but cannot be incorporated into vascular structures, can only induce a small increase in post-ischemic tissue neovascularization. A small increase in post-ischemic tissue neovascularization was observed.
The above studies did not confirm the ability of EPCs to participate in the formation of vascular-like structures in vivo, but they could improve the status of vascular neovascularization.
3.2 Role of EPCs in bone repair
EPCs may be involved in neovascularization and endothelial cell renewal through mechanisms such as integration, fusion, and paracrine secretion, and act as seed cells in tissue engineering to promote in vivo vascularization of tissue-engineered bone. The 3 basic processes after bone transplantation are graft vascularization, bone regeneration and bone end fusion, of which vascularization is the key link, its role is throughout the whole graft repair process, and plays a decisive role in the mode and effect of bone regeneration and fusion. After the in vitro construction of tissue-engineered bone, especially the large volume of tissue-engineered bone is implanted in the body, an adequate blood supply must be rapidly established to bring osteoblast precursor cells, related factors, nutrients and other cells involved in bone repair to the local microenvironment, and to carry away metabolic waste and necrosis and decomposition products to provide nutrition for the survival and development of seed cells, and to maintain a physiological environment conducive to this process as a whole. metabolic microenvironment that is conducive to this physiological process.
The basic method of bone tissue engineering is to inoculate seed cells with resorbable biomaterials to form cell-scaffold complexes for implantation into the body. As the scaffold material degrades, the seed cells continue to proliferate and differentiate, secrete matrix and release cytokines, thereby accelerating the repair of bone defects. Currently, there are many studies to promote in vivo vascularization of tissue-engineered bone, and the common ones include: wrapped vascular bundle method, muscle encapsulation pre-constructed with vascular tip method, vascular tip fascia wrapping method, composite vascular endothelial progenitor cells or vascular endothelial cells, and 3D structural modification of scaffold materials and gene transfection and slow/controlled release techniques of pro-angiogenic bioactive factors that facilitate vascularization. Granulocyte colony-stimulating factor mobilization has been found to be effective in increasing the number of circulating EPCs and improving the endothelialization of artificial implant materials. Studies have also shown that EPCs can be used as seed cells in tissue engineering to promote in vivo vascularization of tissue-engineered bone and promote bone repair.
3.3 Treatment and application of EPCs in bone repair
Tissue engineering is simply the in vitro culture of functionally relevant cells grown on natural or synthetic scaffolds to obtain new functional tissues and organs, but most tissues and organs require a microvascular network to supply nutrients and carry away metabolites, which is essential and one of the difficulties in research.
Schmidt et al. isolated and cultured endothelial progenitor cells from human umbilical cord blood and inoculated them together with human vascular smooth muscle cells onto a scaffold and found that they could form capillary-like structures by polyglycolic acid polylactic acid copolymer. Schultheiss et al. constructed tissue-engineered bladders by inoculating smooth muscle cells and bladder epithelial cells after decellularization of porcine small intestine segments that retained vascular structures. In an important area of tissue engineering research, bone tissue engineering research has yielded exciting results in several areas and has had initial clinical applications, and is considered one of the most promising and viable areas of tissue engineering.
These experimental studies suggest that the use of vascular endothelial progenitor cells to construct tissue-engineered bone is promising and has great potential for the treatment of long-segment bone defects.
4.Conclusion and prospect
Vascular endothelial progenitor cells have a very promising application in the field of tissue engineering, but the research has just started and there are still many problems to be solved, such as surface markers and optimized culture system. It is believed that future research will lead to breakthroughs in their vascular phenotype, role in tissue remodeling, and biological properties, and that applied research can make better use of endothelial progenitor cells to improve therapeutic efficiency and reduce adverse effects. As a member of tissue engineering seed cells, EPCs have shown great potential with the emergence of autologous cell therapy and gene modification concepts, and the clinical application of EPCs in the field of regenerative medicine will certainly be more developed.