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

Abstract

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.

Figure 1

Images of vascularized constructs containing human EPCs and MPCs, explanted after 7 days in vivo. Left column, collagen, center column, fibrin, right column, PuraMatrix. (A) Macro images of vascularized constructs. Bar, 5 mm. (B) H&E stained images of vascularized constructs, showing patent, erythrocyte-filled lumens. Bar, 100 μm. (C) Construct sections antibody-stained for human CD31, showing erythrocyte-filled lumens lined with EPCs. Bar, 100 μm. (D) Construct sections antibody-stained for CD31 (red) and αSMA (green) showing EPC-lined vessels (one example per image marked with *) overlaid with αSMA-expressing perivascular cells. Bar, 50 μm, nuclei blue. (E) Quantification of blood vessel density in three concentrations of collagen and fibrin ECMs, and PuraMatrix ECM containing EPCs and MPCs. Dashed line indicates average value of microvessel density in Matrigel containing EPCs and MPCs. Microvessel density on left axis, dark bars, % vascular area on right axis, light bars. * indicates significance for microvessel density and % vascular area between 6 and 7 mg/mL collagen groups. † indicates significance between PuraMatrix and all concentrations of collagen and fibrin for microvessel density and % vascular area.

Figure 2

Images of vascularized constructs containing only human EPCs explanted after 7 days in vivo. (A) H&E stained images of vascularized constructs, showing patent, erythrocyte-filled lumens. Bar, 100 μm. (B) Construct sections antibody-stained for human CD31, showing erythrocyte-filled lumens lined with EPCs. Bar, 100 μm. (C) Construct sections antibody-stained for CD31 (red) and αSMA (green) showing EPC-lined vessels (one example per image marked with *) overlaid with αSMA-expressing perivascular cells. Bar, 50 μm, nuclei blue. (D) Quantification of blood vessel density in 5 mg/mL collagen, 10 mg/mL fibrin, and PuraMatrix ECMs containing EPCs. Microvessel density on left axis and dark bars, % vascular area on right axis and light bars. * indicates significance of PuraMatrix microvessel density and % vascular area compared to collagen and fibrin.

Figure 3

Images of vascularized constructs containing only human MPCs explanted after 7 days in vivo. (A) H&E stained images of vascularized constructs, showing patent, erythrocyte-filled lumens. Bar, 100 μm. (B) Construct sections antibody-stained for endothelial marker VE-Cadherin, showing erythrocyte-filled, endothelialized lumens. Bar, 100 μm. (C) Construct sections antibody-stained for VE-Cadherin (red) and αSMA (green) showing vessels (one example per image marked with *) overlaid with αSMA-expressing perivascular cells. Bar, 50 μm, nuclei blue. (D) Quantification of blood vessel density in 5 mg/mL collagen, 10 mg/mL fibrin, and PuraMatrix ECMs containing MPCs. Microvessel density on left axis and dark bars, % vascular area on right axis and light bars.

Figure 4

Rheological characterization of collagen, fibrin, and PuraMatrix ECMs. (A) Representative raw data showing storage modulus, G′, from 10-3 strain units to 10-1, for three concentrations of collagen and fibrin ECMs, and Matrigel and PuraMatrix. (B) Average values of storage modulus, G′ (grey bars), and loss modulus, G″ (white bars), for each ECM, average of multiple preparations, average value from 10-3 to 10-2 strain units. (C) Phase shift, δ, for each ECM from 10-3 to 10-1 strain units, average of multiple preparations. Collagen and fibrin data are average of multiple preparations from 5, 6, and 7 mg/mL, and 10, 15, and 20 mg/mL, respectively.