Dysfunctional stem and progenitor cells impair fracture healing with age

Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells(MSCs) are directed to replace the bone tissue, while endothelial progenitor cells(EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species,and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics.Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly.     

Human primary endothelial cells stimulate human osteoprogenitor cell differentiation.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly endothelial cells that may be pivotal members of a complex interactive communication network in bone. While cell cooperation was previously established between Human OsteoProgenitor cells (HOP) and Human Umbilical Vein Endothelial Cells (HUVEC) the aim of our study was to investigate if this interaction is specific to Human Endothelial cell types (ECs) from different sources. Osteoblastic cell differentiation analysis performed using different co-culture models with direct contact revealed that Alkaline Phosphatase (Al-P) activity was only increased by the direct contact of HOP with human primary vascular endothelial cell types including endothelial precursor cells (EPCs) isolated from blood cord, endothelial cells from Human Saphen Vein (HSV) while a transformed cell line, the Human Bone Marrow Endothelial Cell Line (HBMEC) did not modify osteoblastic differentiation of HOP. Because connexin 43, a specific gap junction protein, seemed to be involved in HUVEC/HOP cell cooperation, expression by RT-PCR and immunocytochemistry of this gap junctional protein was investigated in EPCs, HSV and HBMEC. Both endothelial cells are positive to this protein and the disruption of gap junction communication using 18alpha-glycyrrhetinic acid treatment decreased the positive effect of these endothelial co-cultures on HOP differentiation as was previously demonstrated for HUVEC and HOP co-cultures. These data seem to indicate that this cross talk between HOP and ECs, through gap junction communication constitutes an additional concept in cell differentiation control.     

间充质干细胞分化为内皮细胞的意义和细胞学基础

间充质干细胞(mesenchymalstemcells,MSCs)主要存在于骨髓中,是多潜能干细胞,在脐血、外周血、脂肪、皮肤等多种组织中也相继分离出MSCs。MSCs具有独特的免疫特性,在异种异体环境内长期存在,使其临床应用前景更为广泛。目前,MSCs的分离培养、诱导分化及鉴定体系已趋成熟,理论上可分化为所有中胚层来源的细胞,内皮细胞来源于中胚层,因此MSCs具有分化为内皮细胞的可能性。本文对MSCs内皮分化意义和细胞学基础及其新近的研究进展作一综述。     

Endothelial progenitor cells for regeneration

Endothelial progenitor cells (EPCs) have been recently isolated from peripheral blood and bone marrow (BM), and shown to be incorporated into sites of physiological and pathological neovascularization in vivo. In contrast to differentiated endothelial cells (ECs), transplantation of EPCs successfully enhanced vascular development by in situ differentiation and proliferation within ischemic organs. Based on such a novel concept of closed up function on EPCs in postnatal neovascularization, the beneficial property of EPC is attractive for cell therapy as well as cell-mediated gene therapy applications targeting regeneration of ischemic tissue.     

内皮祖细胞在炎症损伤修复中的作用和机制

内皮祖细胞（endothelial progenitor cells,EPCs）是出生后,可以在机体内分化为成熟内皮细胞的一种前体细胞,主要来源于骨髓。多种伴有血管内皮细胞损伤的疾病都可引起外周血EPCs数量变化。有研究显示EPCs参与炎性损伤修复,并且外周血EPCs数量与血管内皮损伤程度和疾病预后存在一定的相关关系。EPCs。通过动员、迁移、归巢和分化等步骤修复内皮。炎症反应中受损组织释放的基质细胞衍生因子、血管内皮生长因子可与EPCs相应的受体结合,通过内皮型一氧化氮合酶、基质金属蛋白酶9等途径调节内皮修复过程,这是EPCs分化为内皮细胞过程的主要调控机制。此外,EPCs还可通过旁分泌机制促进相邻的内皮细胞增殖分化。目前,EPCs在炎症领域仅用于内皮炎性损伤和疾病预后评估,但是EPCs在心血管疾病和组织工程领域应用研究的成功,为EPCs在炎症反应的诊断和治疗提供了新的思路。     

The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.     

Dynamic compression promotes proliferation and neovascular networks of endothelial progenitor cells in demineralized bone matrix scaffold seed

Neovascularization is required for bone formation and successful fracture healing. In the process of neovascularization, endothelial progenitor cells (EPCs) play an important role and finish vascular repair through reendothelialization to promote successful fracture healing. In this study, we found that dynamic compression can promote the proliferation and capillary-like tube formation of EPCs in the demineralized bone matrix (DBM) scaffold seed. EPCs isolated from the bone marrow of rats have been cultured in DBM scaffolds before dynamic compression and then seeded in the DBM scaffolds under dynamic conditions. The cells/scaffold constructs were subjected to cyclic compression with 5% strain and at 1 Hz for 4 h/day for 7 consecutive days. By using MTT and real-time PCR, we found that dynamic compression can significantly induce the proliferation of EPCs in three-dimensional culture with an even distribution of cells onto DBM scaffolds. Both in vitro and in vivo, the tube formation assays in the scaffolds showed that the loaded EPCs formed significant tube-like structures. These findings suggest that dynamic compression promoted the vasculogenic activities of EPCs seeded in the scaffolds, which would benefit large bone defect tissue engineering.     

The involvement of endothelial progenitor cells in tumor angiogenesis

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood CD34, VEGFR-2, or AC 133 (CD133) antigen-positive cells, which may home to site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis and growth of certain tumors. This review article will summarize the literature data concerning this new role played by EPCs in tumor angiogenesis.     

BMP2 Genetically Engineered MSCs and EPCs Promote Vascularized Bone Regeneration in Rat Critical-Sized Calvarial Bone Defects

Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.     

Effects of Ca2+-activated potassium and inward rectifier potassium channel on the differentiation of endothelial progenitor cells from human peripheral blood

Endothelial progenitor cells (EPCs) are bone marrow-derived cells that have the propensity to differentiate into mature endothelial cells (ECs). The transplantation of EPCs has been shown to enhance in vivo postnatal neo-vasculogenesis, as well as repair infarcted myocardium. Via the whole-cell patch clamp technique, numerous types of ion channels have been detected in EPCs, including the inward rectifier potassium channel (IKir), Ca²⁺-activated potassium channel (IKCa), and volume-sensitive chloride channel, but their influence on the differentiation of EPCs has yet to be characterized. The present study was designed to investigate: (1) which ion channels have the most significant impact on the differentiation of EPCs; (2) what role ion channels play in the functional development of EPCs; (3) the mRNA and protein expression levels of related ion channel subunits in EPCs. In our study, EPCs were obtained from the peripheral blood of healthy adults and cultured with endothelial growth factors. When EPCs differentiate into mature ECs, they lose expression of the stem cell/progenitor marker CD133, as analyzed by flow cytometry (0.44 ± 0.20 %). However, treatment with the potassium channel inhibitor, tetraethylammonium (TEA) results in an increase in CD133⁺ cells (25.50 ± 7.55 %). In a functional experiment, we observed a reduction in the capacity of TEA treated ECs (differentiated from EPCs) to form capillary tubes when seeded in Matrigel. At the mRNA and protein levels, we revealed several K⁺ subtypes, including KCNN4 for IKCa, KCNNMA1 for BKCa and Kir3.4 for IKir. These results demonstrate for the first time that potassium channels play a significant role in the differentiation of EPCs. Moreover, inhibition of potassium channels may depress the differentiation of EPCs and the significant potassium channel subunits in EPCs appear to be IKCa, BKCa and Kir3.4.     

Effects of estrogen on diverse stem cells and relevant intracellular mechanisms

Sexual dimorphism definitely exists in the pathogenesis of a variety of cardiovascular,neurodegenerative and bone metabolism disorders.Estrogen affects the healing of ischemic myocardium partially through paracrine growth hormone production by bone marrow mesenchymal stem cells(MSCs) and facilitation on mobilization of endothelial progenitors cells(EPCs) to the ischemic myocardium.Estrogen can also inhibit the proliferation of the cardiac fibroblasts.Therefore,estrogen effectively enhances the neovasculariz...     

细胞因子对内皮祖细胞功能影响的研究进展

内皮祖细胞(EPCs)是一种具有较强增殖能力的前体细胞,血管损伤或者缺血会刺激骨髓EPCs动员,迁移、归巢于相应的靶位,然后分化为内皮细胞(ECs),从而参与血管修复和血管新生。因此,EPCs的成功发现为缺血性和血管损伤性疾病的治疗提供了新策略。但是EPCs存在动员率低、靶向性较差和功能不全等问题。大量研究显示细胞因子对EPCs的动员、归巢、增殖和分化等均起着重要的调节作用,同时,通过调控细胞因子能改善EPCs的功能活性,因此选择合适的细胞因子来提高EPCs功能变得非常重要。现总结了近年来细胞因子对EPCs功能影响的研究进展,并提出有待解决的问题和作一定的展望。     

SDF-1 secreted by mesenchymal stem cells promotes the migration of endothelial progenitor cells via CXCR4/PI3K/AKT pathway

Cell-based therapeutics bring great hope in areas of unmet medical needs. Mesenchymal stem cells (MSCs) have been suggested to facilitate neovascularization mainly by paracrine action. Endothelial progenitor cells (EPCs) can migrate to ischemic sites and participate in angiogenesis. The combination cell therapy that includes MSCs and EPCs has a favorable effect on ischemic limbs. However, the mechanism of combination cell therapy remains unclear. Herein, we investigate whether stromal cell-derived factor (SDF)-1 secreted by MSCs contributes to EPC migration to ischemic sites via CXCR4/Phosphoinositide 3-Kinases (PI3K)/protein kinase B (termed as AKT) signaling pathway. First, by a “dual-administration” approach, intramuscular MSC injections were supplemented with intravenous Qdot® 525 labeled-EPC injections in the mouse model of hind limb ischemia. Then, the mechanism of MSC effect on EPC migration was detected by the transwell system, tube-like structure formation assays, western blot assays in vitro. Results showed that the combination delivery of MSCs and EPCs enhanced the incorporation of EPCs into the vasculature and increased the capillary density in mouse ischemic hind limb. The numbers of CXCR4-positive EPCs increased after incubation with MSC-conditioned medium (CM). MSCs contributed to EPC migration and tube-like structure formation, both of which were suppressed by AMD3100 and wortmannin. Phospho-AKT induced by MSC-CM was attenuated when EPCs were pretreated with AMD3100 and wortmannin. In conclusion, we confirmed that MSCs contributes to EPC migration, which is mediated via CXCR4/PI3K/AKT signaling pathway.

     

Insights into the mechanism of vascular endothelial cells on bone biology

In the skeletal system, blood vessels not only function as a conduit system for transporting gases, nutrients, metabolic waste, or cells but also provide multifunctional signal molecules regulating bone development, regeneration, and remodeling. Endothelial cells (ECs) in bone tissues, unlike in other organ tissues, are in direct contact with the pericytes of blood vessels, resulting in a closer connection with peripheral connective tissues. Close-contact ECs contribute to osteogenesis and osteoclastogenesis by secreting various cytokines in the paracrine or juxtacrine pathways. An increasing number of studies have revealed that extracellular vesicles (EVs) derived from ECs can directly regulate maturation process of osteoblasts and osteoclasts. The different pathways focus on targets at different distances, forming the basis of the intimate spatial and temporal link between bone tissue and blood vessels. Here, we provide a systematic review to elaborate on the function of ECs in bone biology and its underlying mechanisms based on three aspects: paracrine, EVs, and juxtacrine. This review proposes the possibility of a therapeutic strategy targeting blood vessels, as an adjuvant treatment for bone disorders.     

Angiotensin II promotes NO production, inhibits apoptosis and enhances adhesion potential of bone marrow-derived endothelial progenitor cells

Endothelial progenitor cells (EPCs) participate in the processes of postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. The level of EPCs present has been found to be directly associated with the outcome of cardiovascular diseases, and could be regulated by stimulatory or inhibitory factors. Given the close relationship between angiotensin II (AngII) and the cardiovascular system, we investigated the effect of AngII on the activities of bone marrow (BM)-derived EPCs. Cells were isolated from BM of rats by density gradient centrifugation. Administration of AngII significantly promoted nitric oxide (NO) release, inhibited EPC apoptosis and enhanced EPC adhesion potential. All of these AngII-mediated effects on EPCs were attenuated by pretreatment with valsartan or L-NAME. Moreover, both LY294002 and wortmannin abolished the anti-apoptotic effect of AngII. Western blot analyses indicated that endothelial NO synthase (eNOS) protein and phosphorylated Akt increased with the treatment of AngII in EPCs. Thus, AngII improved several activities of EPCs through AngII type 1 receptor (AT1R), which may represent a possible mechanism linking AngII and AT1R with angiogenesis. Additionally, AngII-induced NO synthesis through eNOS in EPCs regulates apoptosis and adhesion, and the PI3-kinase/Akt pathway has an essential role in AngII-induced antiapoptosis signaling.     

Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage

The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.     

Endothelial Progenitor Cells Promote Directional Three-Dimensional Endothelial Network Formation by Secreting Vascular Endothelial Growth Factor

Endothelial progenitor cell (EPC) transplantation induces the formation of new blood-vessel networks to supply nutrients and oxygen, and is feasible for the treatment of ischemia and cardiovascular diseases. However, the role of EPCs as a source of proangiogenic cytokines and consequent generators of an extracellular growth factor microenvironment in three-dimensional (3D) microvessel formation is not fully understood. We focused on the contribution of EPCs as a source of proangiogenic cytokines on 3D microvessel formation using an in vitro 3D network model. To create a 3D network model, EPCs isolated from rat bone marrow were sandwiched with double layers of collagen gel. Endothelial cells (ECs) were then cultured on top of the upper collagen gel layer. Quantitative analyses of EC network formation revealed that the length, number, and depth of the EC networks were significantly enhanced in a 3D model with ECs and EPCs compared to an EC monoculture. In addition, conditioned medium (CM) from the 3D model with ECs and EPCs promoted network formation compared to CM from an EC monoculture. We also confirmed that EPCs secreted vascular endothelial growth factor (VEGF). However, networks cultured with the CM were shallow and did not penetrate the collagen gel in great depth. Therefore, we conclude that EPCs contribute to 3D network formation at least through indirect incorporation by generating a local VEGF gradient. These results suggest that the location of EPCs is important for controlling directional 3D network formation in the field of tissue engineering.     

Dual role of parathyroid hormone in endothelial progenitor cells and marrow stromal mesenchymal stem cells

Hematopoietic stem cells derive regulatory information also from parathyroid hormone (PTH). To explore the possibility that PTH may have a role in regulation of other stem cells residing in bone marrow, such as mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) we assessed the effect of this hormone on the in vitro behavior of MSCs and EPCs. We evidenced that MSCs were much more responsive to PTH than EPCs. PTH increased the proliferation rate of MSCs with a diminution of senescence and apoptosis. Taken together, our results may suggest a protective effect of PTH on MSCs that reduces stress phenomena and preserve genome integrity. At the opposite, PTH did not modify the fate of EPCs in culture. J. Cell. Physiol. 222: 474–480, 2010. © 2009 Wiley‐Liss, Inc.     

Differentiation of Rabbit Bone Mesenchymal Stem Cells into Endothelial Cells In Vitro and Promotion of Defective Bone Regeneration In Vivo

Tissue engineering strategies often fail to regenerate bones because of inadequate vascularization, especially in the reconstruction of large segmental bone defects. Large volumes of vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesis are difficult to obtain. In this study, we simulated bone healing by co-culturing differentiated ECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolic acid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairing rabbit mandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells. After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs. Immunohistochemical staining analysis with CD34 showed that the induced cells had the characteristics of ECs and MSC. The induced ECs were co-cultured in vitro, and the induction of MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR) staining experiments were performed, and the Coomassie brilliant blue total protein and ALP activity were measured. The MSCs proliferated and differentiated into osteoblast-like cells through direct contact between the derived ECs and MSCs. The co-cultured cells were seeded on PLGA scaffold to repair 1 cm mandible defects in the rabbit. The effectiveness of the repairs was assessed through soft X-ray and histological analyses. The main findings indicated that MSCs survived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. The results demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis and promoted new bone formation. This study may serve as a basis for the use of in vitro co-culturing techniques as an improvisation to bone tissue engineering for the repair of large bone defects.