Type I collagen, fibrin and PuraMatrix matrices provide permissive environments for human endothelial and mesenchymal progenitor cells to form neovascular networks

The field of tissue engineering seeks to create metabolically demanding, functional tissues, which will require blood vessel networks capable of forming rapidly in a variety of extracellular matrix (ECM) environments. We tested whether human endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) could form microvascular networks in type I collagen, fibrin and an engineered peptide hydrogel, PuraMatrix, in 7 days in vivo in immune-deficient mice. These results are compared to those previously published, based on the Matrigel ECM. Perfused blood vessels formed in all three types of ECM within 7 days. Collagen at 5 and 6 mg/ml and 10 mg/ml fibrin supported vessel formation at 30-60 vessels/mm(2), and PuraMatrix enabled vessel formation to 160 vessels/mm(2), significantly greater than collagen or fibrin. Vessels were composed of EPCs with perivascular cells on their abluminal surfaces. EPCs injected alone formed a low density of blood vessels in collagen and PuraMatrix, while MPCs injected alone resulted in sparse vessel networks in all ECMs tested. A rheometer was used to determine whether the ECMs which supported vascularization had bulk physical properties similar to or distinct from Matrigel. Collagen and fibrin were the stiffest matrices to support extensive vascularization, with storage moduli in the range 385-510 Pa, while Matrigel, at 80 Pa, and PuraMatrix, at 5 Pa, were far more compliant. Thus, EPCs and MPCs were capable of vasculogenesis in environments having disparate physical properties, although vascular density was greater in more compliant ECMs. We propose that EPC/MPC-mediated vascularization is a versatile technology which may enable the development of engineered organs.     

Co‐culture of adipose‐derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co‐implantation of adipose‐derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co‐cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell–fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co‐implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary‐like structures by co‐implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary‐like structures near the top of the construct, indicating that perfusion of the cell–fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

Priming of late endothelial progenitor cells with erythropoietin before transplantation requires the CD131 receptor subunit and enhances their angiogenic potential

Summary. Background: Endothelial colony‐forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases. Erythropoietin (EPO) is a cytokine that promotes angiogenesis after ischemic injury. EPO receptors (EPORs) classically include two EPOR subunits, but may also associate with the β‐common chain (CD131) in a newly identified receptor involved in EPO cytoprotective effects. Objective: The aim was to take advantage of the proangiogenic properties of EPO to enhance ECFC graft efficiency. We postulated that priming ECFCs by adding epoietin α in culture medium prior to experiments might increase their angiogenic properties. We also explored the role of the CD131 subunit in EPO priming of ECFCs. Methods and Results: By western blotting on cord blood ECFC lysates, we showed that EPOR and CD131 expression increased significantly after EPO priming. These proteins coimmunoprecipitated and colocalized, suggesting that they are covalently bound in ECFCs. EPO at 5 IU mL^?1 significantly stimulated proliferation, wound healing, migration and tube formation of ECFCs. EPO priming also increased ECFC resistance to H₂O₂‐induced apoptosis and survival in vivo. Similarly, in vivo studies showed that, as compared with non‐primed ECFC injection, 5 IU mL^?1 EPO‐primed ECFCs, injected intravenously 24 h after hindlimb ischemia in athymic nude mice, increased the ischemic/non‐ischemic ratios of hindlimb blood flow and capillary density. These effects were all prevented by CD131 small interfering RNA transfection, and involved the phosphoinositide 3‐kinase–Akt pathway. Conclusion: These results highlight the potential role of EPO‐primed ECFCs for cell‐based therapy in hindlimb ischemia, and underline the critical role of CD131 as an EPO coreceptor.     

Vascularization of prevascularized and non-prevascularized fibrin-based human adipose tissue constructs after implantation in nude mice

Adipose regeneration strategies have been hampered by the inability to supply an adequate vascular supply following implantation. Vascularization in vitro, also called prevascularization, is a promising method that could promote the vascularization of engineered adipose tissue constructs upon implantation. In this study we compared the ability of prevascularized-to-non-prevascularized fibrin-based human adipose tissue to promote vascularization. Human adipose tissue-derived stromal cells (ASCs) and different mixtures (1:1, 1:2 and 1:5) of ASCs with human umbilical vein endothelial cells (HUVECs) were cultured in fibrin at two different densities (1.0 × 10(6) and 10 × 10(6) cells/ml) for 7 days. Histological analysis revealed that prevascular structures formed in 1:5 ASC/HUVEC fibrin-based constructs seeded with a total of 10 × 10(6) cells/ml. These constructs and ASC-only constructs were implanted subcutaneously in athymic mice for 7 days and generated lipid-containing grafts. The numbers and densities of blood vessels within the ASC/HUVEC constructs were similar to those of ASC-only constructs. Furthermore, immunostaining studies demonstrated human-derived vasculature within a few of the ASC/HUVEC and ASC-only constructs. A subset of this human-derived vasculature contained erythrocytes, indicating integration with the host vasculature. In conclusion, our study indicated no difference in the rate of vascularization of prevascularized ASC/HUVEC and non-prevascularized ASC-only fibrin-based constructs, suggesting that prevascularization of these fibrin-based constructs does not promote vascularization. Our results further indicated that not only endothelial cells, but also ASCs may contribute to the formation of vascular lumina upon implantation. This finding is interesting, since it demonstrates the possibility of vascularized adipose tissue engineering from a single cell source.     

Analysis of circulating mesenchymal progenitor cells in arterial and venous blood after fracture

Mesenchymal progenitor cells (MPCs) play a critical role in fracture healing. Increasing evidence suggests that circulating MPCs in peripheral blood are mobilized during fracture healing and may contribute to fracture repair. However, to date, there have been no reports comparing the number of circulating MPCs in arterial blood (AB) with that in venous blood (VB) after fracture. In this study, we investigated the numbers of MPCs in AB, VB and bone marrow (BM) after fracture in rabbits via the colony‐forming unit–fibroblasts (CFU‐Fs) assay, and clarified the time‐course change. After femoral fracture in one side, the number of BM‐MPCs in the contralateral femur increased from day 1 to day 7. Correspondingly, the number of circulating MPCs in AB and VB increased. The number of circulating MPCs in AB was highest at post‐fracture day 4, whereas that in VB was highest at post‐fracture day 1, with significant difference compared to the control. Circulating MPCs in AB and VB after fracture may serve as new cell sources for bone tissue engineering. As the peaks of the number of circulating MPCs in AB and VB after fracture were different, our findings may provide new insights about when to collect circulating MPCs after fracture and from which blood to obtain them. Copyright © 2012 John Wiley & Sons, Ltd.     

Hyperosmolarity and hypoxia induce chondrogenesis of adipose-derived stem cells in a collagen type 2 hydrogel

Apart from soluble growth factors, various other biophysicochemical cues are known to promote chondrogenesis. Under physiological conditions, cartilage in the joint comprises a hyperosmotic and hypoxic environment. Therefore, in this study, we examined the inductive effects of hyperosmotic and/or hypoxic conditions on adipose stem cells (ASCs) and compared them with conventional TGFβ1‐induction. After encapsulation in collagen type II hydrogels and specific induction, ASCs were assessed for viability, proliferation, morphology and chondrogenic differentiation potential. Viability was similar under all conditions, with low proliferative activity. After 4 days, hypoxia and/or hyperosmolarity did not affect round cell morphology, while cells were mainly stretched in the TGFβ1‐induced group. At 21 days, the TGFß1‐treated group had aggregated into a cell nodule. Hyperosmolarity mimicked this aggregation to a lesser extent, whereas cells under hypoxia stretched out after 21 days, with a combined effect in the hypoxic/hyperosmotic group. Both individual and combined hyperosmotic and/or hypoxic conditions significantly upregulated SOX5, SOX9, COMP and Link‐p gene expression compared with the non‐induced group, and to similar levels as the TGFβ1‐induced group. GAG synthesis in both hydrogel and medium was increased under hypoxic conditions, whereas hyperosmolarity decreased GAG formation in the hydrogels, but increased GAG formation in the medium. We conclude that in a joint mimicking the three‐dimensional (3D) micro‐environment, a combination of hyperosmolarity and hypoxia is able to induce chondrogenesis to the same extent as TGFβ1. This might lead to an interesting alternative when considering short‐term triggering in a one‐step surgical procedure for the treatment of cartilaginous defects. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures

To introduce a functional vascular network into tissue‐engineered bone equivalents, human endothelial colony forming cells (ECFCs) and multipotent mesenchymal stromal cells (MSCs) can be cocultured. Here, we studied the impact of donor variation of human bone marrow‐derived MSCs and cord blood‐derived ECFCs on vasculogenesis and osteogenesis using a 3D in vitro coculture model. Further, to make the step towards cocultures consisting of cells derived from a single donor, we tested how induced pluripotent stem cell (iPSC)‐derived human endothelial cells (iECs) performed in coculture models. Cocultures with varying combinations of human donors of MSCs, ECFCs, or iECs were prepared in Matrigel. The constructs were cultured in an osteogenic differentiation medium. Following a 10‐day culture period, the length of the prevascular structures and osteogenic differentiation were evaluated for up to 21 days of culture. The particular combination of MSC and ECFC donors influenced the vasculogenic properties significantly and induced variation in osteogenic potential. In addition, the use of iECs in the cocultures resulted in prevascular structure formation in osteogenically differentiated constructs. Together, these results showed that close attention to the source of primary cells, such as ECFCs and MSCs, is critical to address variability in vasculogenic and osteogenic potential. The 3D coculture model appeared to successfully generate prevascularized constructs and were sufficient in exceeding the ~200 μm diffusion limit. In addition, iPSC‐derived cell lineages may decrease variability by providing a larger and potentially more uniform source of cells for future preclinical and clinical applications.     

Co‐transplantation of Wharton's jelly mesenchymal stem cell‐derived osteoblasts with differentiated endothelial cells does not stimulate blood vessel and osteoid formation in nude mice models

A major challenge in bone tissue engineering is the lack of post‐implantation vascular growth into biomaterials. In the skeletal system, blood vessel growth appears to be coupled to osteogenesis—suggesting the existence of molecular crosstalk between endothelial cells (ECs) and osteoblastic cells. The present study (performed in two murine ectopic models) was designed to determine whether co‐transplantation of human Wharton's jelly mesenchymal stem cell‐derived osteoblasts (WJMSC‐OBs) and human differentiated ECs enhances bone regeneration and stimulates angiogenesis, relative to the seeding of WJMSC‐OBs alone. Human WJMSC‐OBs and human ECs were loaded into a silicate‐substituted calcium phosphate (SiCaP) scaffold and then ectopically implanted at subcutaneous or intramuscular sites in nude mice. At both subcutaneous and intramuscular implantation sites, we observed ectopic bone formation and osteoids composed of host cells when WJMSC‐OBs were seeded into the scaffold. However, the addition of ECs was associated with a lower level of osteogenesis, and we did not observe stimulation of blood vessel ingrowth. in vitro studies demonstrated that WJMSC‐OBs lost their ability to secrete vascular endothelial growth factor and stromal cell‐derived factor 1—including when ECs were present. In these two murine ectopic models, our cell‐matrix environment combination did not seem to be optimal for inducing vascularized bone reconstruction.     

Bone marrow mesenchymal stem cells,platelet‐rich plasma and nanohydroxyapatite–type I collagen beads were integral parts of biomimetic bone substitutes for bone regeneration

Platelet rich plasma (PRP), which includes many growth factors, can activate osteoid production, collagen synthesis and cell proliferation. Nanohydroxyapatite‐type I collagen beads (CIB), which mimetic natural bone components, are not only flexible fillers for bone defect but also encourage osteogenesis. Bone marrow mesenchymal stem cells (BMSCs) are often used as an abundant cell source for tissue engineering. We used a rabbit model to combine PRP, CIB and BMSCs (CIB+PRP+BMSC) into a bone‐like substitute to study its impact on bone regeneration, when compared to defect alone, PRP, CIB+PRP, and PRP+BMSC. CIB+PRP upregulated more alkaline phosphatase (ALP) activity in BMSCs than PRP alone at 4 weeks postoperation. CIB+PRP+BMSC and PRP+BMSC did not differ significantly in DNA content, total collagen content, and ALP activity at 8 weeks. In histological assay, both CIB+PRP+BMSC and PRP+BMSC showed more bone regeneration at 4 and 8 weeks. Higher trabecular bone volume in tissue volume (BV/TV) (31.15±2.67% and 36.93±1.01%), fractal dimension (FD) (2.30±0.18 and 2.65±0.02) and lower trabecular separation (Tb.Sp) (2.30±0.18 and 1.35±0.16) of CIB+PRP+BMSC than of other groups at 4 and 8 weeks, and approach to of bone tissue (BV/TV=24.35±2.13%; FD=2.65±0.06; Tb.Sp=4.19±0.95). CIB+PRP+BMSC significantly enhanced new bone formation at 4 week. Therefore, nanohydroxyapatite‐type I collagen beads combined with PRP and BMSCs produced a bone substitute with efficiently improved bone regeneration that shows promise to repair bone defects. Copyright © 2012 John Wiley & Sons, Ltd.