Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb:40:61-71.
doi: 10.1016/j.biomaterials.2014.11.011. Epub 2014 Nov 26.

Mesenchymal stromal cells form vascular tubes when placed in fibrin sealant and accelerate wound healing in vivo

Julio J Mendez  1 , Mahboobe Ghaedi  1 , Amogh Sivarapatna  1 , Sashka Dimitrievska  1 , Zhen Shao  2 , Chinedum O Osuji  2 , Derek M Steinbacher  3 , David J Leffell  4 , Laura E Niklason  5
Affiliations

Mesenchymal stromal cells form vascular tubes when placed in fibrin sealant and accelerate wound healing in vivo

Julio J Mendez et al. Biomaterials. 2015 Feb.

Abstract

Non-healing, chronic wounds are a growing public health problem and may stem from insufficient angiogenesis in affected sites. Here, we have developed a fibrin formulation that allows adipose-derived mesenchymal stromal cells (ADSCs) to form tubular structures in vitro. The tubular structures express markers of endothelium, including CD31 and VE-Cadherin, as well as the pericyte marker NG2. The ability for the MSCs to form tubular structures within the fibrin gels was directly dependent on the stoichiometric ratios of thrombin and fibrinogen and the resulting gel concentration, as well as on the presence of bFGF. Fibrin gel formulations that varied in stiffness were tested. ADSCs that are embedded in a stiff fibrin formulation express VE-cadherin and CD31 as shown by PCR, FACS and immunostaining. Confocal imaging analysis demonstrated that tubular structures formed, containing visible lumens, in the stiff fibrin gels in vitro. There was also a difference in the amounts of bFGF secreted by ADSCs grown in the stiffer gels as compared to softer gels. Additionally, hAT-MSCs gave rise to perfusable vessels that were VE-cadherin positive after subcutaneous injection into mice, whereas the softer fibrin formulation containing ADSCs did not. The application of ADSCs delivered in the stiff fibrin gels allowed for the wounds to heal more quickly, as assessed by wound size, amount of granulation tissue and collagen content. Interestingly, following 5 days of healing, the ADSCs remained within the fibrin gel and did not integrate into the granulation tissue of healing wounds in vivo. These data show that ADSCs are able to form tubular structures within fibrin gels, and may also contribute to faster wound healing, as compared with no treatment or to wounds treated with fibrin gels devoid of ADSCs.

Keywords: Adipose derived stromal cells (ADSCs); Endothelial cells; Fibrin; Mesenchymal stromal cells (MSCs); Stiffness; Wound healing.

PubMed Disclaimer

Conflict of interest statement

Conflict of Interest:

L.E.N. has a financial interest in Humacyte, Inc, a regenerative medicine company. Humacyte did not fund these studies, and Humacyte did not affect the design, interpretation, or reporting of any of the experiments herein.

Figures

Figure 1
Figure 1
ADSCs form tube structures within the stiffer fibrin gels (Composition 1) and express endothelial cell markers as assessed by FACS, immunohistochemistry, and qRT-PCR. (A,D) At day 7 of culture, ADSC-derived, NG2 +, cellular tube structures (red) are present in fibrin gel cultures at composed of stiff fibrin formula (A) and not in the less stiff formula (D). (B, E) The stiff fibrin conditions also allow for expression of CD144 in up to 49% of the population over isotype control by FACS (B), whereas only 5% of the population in the softer formula is CD144+ (E). GFP-ADSCs also form hollow tube/luminal structures in Composition 1 fibrin gels at Day 7 (C). A comparison with cells cultured on plastic tissue culture flasks (F) reveals that there is no CD144 population present in this condition. (G,H) PCR data indicates increased EC marker presence in stiff compared to soft fibrin formulation. C1-D7, C1-D14 = fibrin Composition 1 Day 7 and 14, respectively; C2-D7, C1-D14 = fibrin Composition 2 day 7 and 14 respectively. (I, J) Immunostaining for CD31 in fibrin conditions C1 and C2, with an arrow pointing to the lumen. Quantification of PCNA+ cells (K) and caspase + cells (L) in fibrin conditions C1 and C2. Scale bar = 50 μm, nuclei are counterstained with DAPI (blue). N=3 technical replicates for each condition
Figure 2
Figure 2
Rheological and SEM analysis of fibrin gel compositions reveal stiffness and structural differences. (A) Elasticity (G′ full circles) and loss modulus (G″ open circles) of Composition 1 fibrin gels plotted in black, and Composition 2 fibrin gels plotted in red as a function of time. The storage modulus G was 3000 Pa for the Composition 1 gels, and 1650 Pa for Composition 2 gels. The table shows the stoichmetric ratios of fibrinogen, thrombin and aprotinin in Composition 1 and 2 (B–E) SEM micrographs show Composition 1 fibrin gels have fibrils with a higher rugosity, scale bar = 1mm (B) when compared with the fibrils of Composition 1, scale bar = 2mm (C). Additionally, the Composition 1 fibrin gels (D) have a greater porosity than the Composition 2 gels (E), scale bars = 10μm
Figure 3
Figure 3
The presence of bFGF in the culture medium is associated with the increased ability of ADSCs to form tubular structures. (A) bFGF ELISA indicates that there is higher bFGF concentration in the conditioned medium in which ADSCs were grown in Composition 1 gels when compared with Composition 2 gels, at days 7 and 21. N=3 (samples run as duplicates). *p<0.05, bars represent SEM. (B–D) ADSCs grown in Composition 2 fibrin gels in which the medium was spiked with bFGF (C–D), contained cells that formed branching structures (chevrons in C), and also contained cells that formed luminal structures (arrows in D) when compared to the non-bFGF spiked condition (B). Scale bar = 20 μm
Figure 4
Figure 4
ADSCs anastomose with the host vasculature and express endothelial markers. Composition 1 fibrin gels (A–F) or Composition 2 fibrin gels (G-1) containing DiI-labeled ADSCs (red) were subcutaneously implanted in mice and left in place for 7 days. Prior to the sacrifice of the animal, the animal received FITC-dextrans (green) via a tail vein injection. The ADSCs contained within Composition 1 fibrin gels formed clear, multi-branching structures (A–F) that were ADSC derived (DiI+, A, D), and contained FITC-dextrans within the lumens of these structures (C, E). Additionally, these structures were positive for the endothelial cell marker, CD31 (magenta) (B). (G–I) In contrast, very few ADSCs remained in the Composition 2 fibrin gels that were implanted and there was no evidence of FITC-dextran perfused, DiI+ vessels. Scale bar = 50 μm for all images. Nuclei are counterstained with DAPI (blue) in (C,I)
Figure 5
Figure 5
Collagen deposition (shown via Masson’s trichrome stain in blue above) by ADSCs delivered in a stiff fibrin composition (A) Symmetrical 6 mm full-thickness wounds were created on the backs of C57/B6 mice followed by no treatment (B and green arrow in A), administration of stiff formulation fibrin only containing no cells (C, and black arrow in A), or stiff fibrin formulation with 0.5 × 106 ADSCs (D). Significant granulation tissue and collagen deposition at day 5 of wound healing.
Figure 6
Figure 6
ADSCs remain in the periphery of the wound and within the fibrin gels (A) ADSCs were pre-labeled with DiI and embedded within Composition 1 gels and placed topically over full-thickness mouse wounds; ADSCs remain in the periphery of the wound, and remain encapsulated within the fibrin itself. (B–C) Non-labeled ADSCs were placed topically over full thickness wounds in Composition 1 fibrin gels; CD31 (B) expression and SMA expression (C) is evident when compared to either fibrin only (D) or with the wound only, no treatment condition (E), which are negative for both markers. All secondary antibodies are shown in red. The white bar and white arrows in each of the panels identifies the apex of the healing wound. The white arrows point to the location of the fibrin sealant. (F, G). There are a significantly higher number of vessels per field of view in C1 gels as compared to C2 (H). C1 or C2 gels, respectively, delivered onto full-thickness rodent wounds. In the fibrin-only condition (I), there is not a significant gross histological distinction from the wound-only (K), whereas with C1 gels (J), there is more granulation tissue and cellularity compared to C2 gels (L). Nuclei are counterstained with DAPI (blue). Scale bar = 50 μm
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Haubner F, Ohmann E, Pohl F, Strutz J, Gassner HG. Wound healing after radiation therapy: Review of the literature. Radiation Oncology. 2012;7(1) - PMC - PubMed
    1. Singer AJ, Clark RAF. Cutaneous Wound Healing. New England Journal of Medicine. 1999;341(10):738–46. - PubMed
    1. Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, et al. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng. 2007;13(6):1299–312. - PubMed
    1. Shaw TJ, Martin P. Wound repair at a glance. Journal of Cell Science. 2009;122(18):3209–13. - PMC - PubMed
    1. Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, et al. Contribution of Bone Marrow–Derived Cells to Skin: Collagen Deposition and Wound Repair. Stem Cells. 2004;22(5):812–22. - PMC - PubMed

Publication types

MeSH terms