Effect of Calcium-Infiltrated Hydroxyapatite Scaffolds on the Hematopoietic Fate of Human Umbilical Vein Endothelial Cells

Abstract

Foamed hydroxyapatite offers a three-dimensional scaffold for the development of bone constructs, mimicking perfectly the in vivo bone structure. In vivo, calcium release at the surface is assumed to provide a locally increased gradient supporting the maintenance of the hematopoietic stem cells niche. We fabricated hydroxyapatite scaffolds with high surface calcium concentration by infiltration, and used human umbilical vein endothelial cells (HUVECs) as a model to study the effects on hematopoietic lineage direction. HUVECs are umbilical vein-derived and thus possess progenitor characteristics, with a prospective potential to give rise to hematopoietic lineages. HUVECs were cultured for long term on three-dimensional porous hydroxyapatite scaffolds, which were either infiltrated biphasic foams or untreated. Controls were cultured in two-dimensional dishes. The release of calcium into culture medium was determined, and cells were analyzed for typical hematopoietic and endothelial gene expressions, surface markers by flow cytometry, and hematopoietic potential using colony-forming unit assays. Our results indicate that the biphasic foams promoted a hematopoietic lineage direction of HUVECs, suggesting an improved in vivo-like scaffold for hematopoietic bone tissue engineering.

Keywords: Calcium; Hemangioblast; Hematopoietic stem cells; Human umbilical vein endothelial cells; Hydroxyapatite.

Fig. 1

SEM images of high porosity scaffolds implemented for HUVEC culture; (A) HA scaffold, (B) infiltrated HA scaffold, (C) EDS result for the Ca-rich position on the infiltrated HA scaffold, and (D) EDS result for the surface area on the infiltrated HA.

Fig. 2

Measurement of Ca²⁺ concentration in cell culture media over 42 days of culture. Data are given as means ± standard deviation from n=3 biological repeats.

Fig. 3

RT-PCR result for CD34, PTPRC (CD45), VWF (vWF) and PECAM1 (CD31) gene expression at (A) day 5, (B) day 15, and (C) day 42. Data are given as means ± standard deviation. Statistical significant differences were (***p<0.001, **p < 0.01, *p < 0.05) as measured with Student’s t-test.

Fig. 4

FACS analyses of (A) CD31⁻CD34⁺ hematopoietic progenitor cells, (B) CD31⁻CD34⁺ endothelial progenitor cells, and, (C) CD31⁺CD34⁻ mature endothelial cells of HUVECs cultured on scaffolds of infiltrated hydroxyapatite (HA), plain HA, and controls without scaffolds after 15 and 42 days. Data are given as means ± standard deviation from n=3 biological repeats, asterisks indicate statistical significant differences (***p<0.001, **p < 0.01, *p < 0.05).

Fig. 5

FACS analyses of Lin⁻CD34⁺CD38⁻ hematopoietic stem cells of HUVECs cultured on scaffolds of infiltrated hydroxyapatite (HA), plain HA, and controls without scaffolds after (A) 15 and (B) 42 days. Data are given as means ± standard deviation from n=3 biological repeats, asterisks indicate statistical significant differences (***p<0.001, **p < 0.01, *p < 0.05).

Fig. 6

FACS analyses of CD45⁺ hematopoietic cells, CD235a⁺ erythrocytes, and Lin (linage marker) positive cells of HUVECs cultured on scaffolds of infiltrated hydroxyapatite (HA), plain HA, and controls without scaffolds after (A) 15 and (B) 42 days. Data are given as means ± standard deviation from n=3 biological repeats, asterisks indicate statistical significant differences (***p<0.001, **p < 0.01, *p < 0.05).

Fig. 7

FACS analyses of (A) CD3⁺, CD4⁺, CD8⁺, and CD19⁺ hematopoietic cells of HUVECs cultured on scaffolds of infiltrated hydroxyapatite (HA), plain HA, and controls without scaffolds after 15 days in culture; and (B) CD31⁻CD34⁺CD133⁺KDR⁺ expression characterizing hemangioblasts within fresh HUVECs and HUVECs cultured on scaffolds of infiltrated hydroxyapatite (HA), plain HA, and controls without scaffolds after 15 days. Data are given as means ± standard deviation from n=3 biological repeats, asterisks indicate statistical significant differences (***p<0.001, **p < 0.01, *p < 0.05).