Effect of Calcium-infiltrated Hydroxyapatite Scaffolds on 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 bi-phasic foams or untreated. Controls were cultured in two-dimensional dishes. The release of calcium into culture medium was determined, and cells were analysed 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.

1. Introduction

Hemangioblasts are the bi-potential angio-hematopoietic stem cells that give rise to both hematopoietic stem cells and endothelial cells [1]. The hypothesis that there is a common angio-hematopoietic progenitor was developed by Sabin more than 50 years ago [2], based on the close association of hematopoietic cells and vascular cell lineages [3,4]. Indeed, primitive erythroblasts and endothelial cells are known to develop from the extra-embryonic mesoderm [5,6]. Hemangioblasts have been isolated from mouse embryonic stem cells and have been shown to be capable of differentiating into both endothelial cells and hematopoietic lineages [7,8]. Cogle et al. [9] reported that human retinal neovascularization originated from human hematopoietic stem cells (HSCs), which also suggests HSCs have functional hemangioblast activity. Grant et al. [10] found that adult HSCs provided functional hemangioblast activity in a mouse model, producing both blood cells and endothelial cells in neovascularization.

Human Umbilical Vein Endothelial Cells (HUVECs), derived from human umbilical veins [11], are a well-known source of primary endothelial cells (ECs) [12]. The isolated human endothelial progenitor cells (EPCs) from HUVECs expressed KDR, CD133 and CD34, as described by Mou et al. [13]. HUVECs are used as a model system of ECs to study the regulation of endothelial cell function, the mechanical response of ECs, the development of atherosclerosis, and angiogenesis [14]. Cells with characteristics of HUVECs can be isolated from immune-privileged fetal tissue [15] and express a high level of fetal immune privilege marker, CD95 (Fas) ligand [16]. Thus, HUVECs might have the capability to differentiate into cells with immune cell character.

We hypothesized that there is a strong relationship between the HSCs environment in vivo and hemangioblast cell fate. If a cell population with potential hemangioblast character, such as HUVECs, is placed in a hematopoietic microenvironment this could induce differentiation to hematopoietic cell lineages. In vivo, HSCs proliferate and reside in a microenvironment in the bone marrow, called the endosteal niche, close to the surface of the trabecular bone [17]. In order to induce hematopoietic differentiation of HUVECs, we implemented three-dimensional hydroxyapatite (HA) scaffolds that simulate the architecture and chemical composition of in vivo bone. A calcium-rich HA scaffold with 90% porosity was used in long-term culture to achieve a similar physical and chemical environment as the HSC endosteal niche. According to Adams et al. [18], there is an extremely high calcium concentration near the endosteal niche, and a high ionic Ca concentration has been shown to affect HSCs fate. They reported that there are fewer HSCs in bone marrow and relative more HSCs in circulation in calcium receptor (CaR) deficient mice model. In consequence, we added a soluble Ca-rich phase into the HA as a second phase to provide soluble Ca²⁺, which can be released at the scaffold’s surface during the culture time. We investigated the effects of the scaffold and the calcium-rich second phase on the hematopoietic fate of HUVECs in long-term culture.

2. Materials and Methods

In this study, we cultured HUVECs on plain HA scaffolds and calcium-rich biphasic HA scaffolds, the latter being processed by infiltration of calcium slats into the partially sintered scaffold followed by sintering to high density. The effects of these scaffolds on the cell fate of HUVECs were compared to negative controls using no scaffolds.

2.1 Scaffold Preparation

The HA scaffold was prepared by a direct foaming process. A suspension was made by mixing deionized water with calcium-deficient HA (Sigma-Aldrich, MO, USA) powder with 30vol% solids. Ammonium polymethacrylate polyelectrolyte dispersant (Darvan C, RT Vanderbilt Co., Norwalk CT) was then added to the de-ionized water (DI water) and the pH adjusted to 5.5 with hydrochloric acid (36.5 – 38.0%) (J.T.Baker, PA, USA). The suspension was subsequently mixed at 2500 RPM for 20 minutes, and a cationic surfactant (benzethonium chloride, Sigma-Aldrich, MO, USA) was added and mixed at 2500 RPM speed for 2 minutes. In the final step, 10% volume of heptane was added to create an emulsion by mixing at high speed for 2 minutes.

The emulsion was poured into a paper mold in an incubator with 60% humidity and kept for 1 hour; subsequently, the humidity was decreased to 40% until the foam had fully dried. The samples were then sintered in air at 1000°C to burn away the mold and partially sinter the HA foam while retaining open porosity in the struts. The calcium-rich biphasic HA foams were processed by infiltrating the partially sintered HA. The foams were infiltrated with a 2 mol/L solution of calcium nitrate for 24 hours in an evacuated container to make sure all the air was removed from the partially sintered foam. The foams were then immersed in ammonia hydroxide solution at pH12–13 for 1 hour to precipitate calcium hydroxide and calcium carbonate within the struts of the foam and on the surface of the struts. After drying, the foam was fired at 900°C and fully sintered to high density at 1300° C to achieve the biphasic HA and calcium oxide foam. Based on our previous work [19], the calcium oxide formed CaCO₃, when exposed to air and this phase was detected by x-ray diffraction of the ground foam. The phase-pure HA foams were processed by the same heat treatments but without the infiltration with the calcium salt.

2.2 Scaffold Characterization

The HA and infiltrated HA scaffolds were coated with palladium using a sputter coater, and examined in a XL30 scanning electron microscope (Philips/FEI Company, Hillsboro, OR). During scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS) was applied on the surface of the scaffolds for Ca/P ratio examination.

2.3 Cell Culture

HUVECs from the human vascular endothelium were obtained from ATCC (American Type Culture Collection) (Manassas, VA) at passage 15. The HUVECs were pre-cultured and passaged for expansion of cell numbers to passage 20. HUVECs were seeded into 24-well plates at a density of 5,000 cells/cm² in 0.5 ml of F12-K medium (ATCC, Manassas, VA), containing 10% Fetal bovine serum (PAA), and 1% antibiotic-antimycotic pre-mix (Invitrogen) (100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL Amphotericin B). Culture conditions included a blank well for the negative control containing no scaffold, plain HA scaffold, and biphasic calcium-infiltrated HA scaffold. Cells were cultured in an incubator at 37°C and in a humidified atmosphere of 95% air and 5% CO₂. The F12-K culture medium was replaced every two to three days. At 0 days, 5 days, 15 days and 42 days, cells were harvested for gene expression analyses, fluorescence-activated cell sorting (FACS), and colony-forming unit (CFU) assay.

2.4 Gene Expression Analysis

Gene expression analyses were performed on pre-seeded HUVECs (day 0 control) and HUVECs harvested from cultures after day 5, 15, and 42 by real-time reverse transcription polymerase chain reaction (RT-PCR). RNA was extracted from the cell lysate using an AllPrep DNA/RNA mini-kit (Qiagen, Valencia, CA). The extracted RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Inventoried TaqMan probes (Applied Biosystems, Foster City, CA) were used to quantify gene expression for β-actin (housekeeping gene used as internal normalizer), CD31, CD144, CD34, CD45 and von Willebrand Factor (vWF) (Life Technologies). The StepOnePlus Real Time PCR-System was used, and data were collected by the StepOne Software version 2.0 (Life Technologies). A negative PCR no-template control included water.

2.5 Flow Cytometry

Fresh HUVECs and HUVECs cultured for 5, 15, and 42 days were analyzed for their surface marker expression profiles by FACS. HUVECs at day 0 were also analyzed as control sample. Cells were rinsed twice with PBS (without calcium and magnesium), and after detaching with EDTA-trypsin (Life Technologies), single cells were re-suspended and were incubated with blocking buffer containing 20% FcR block (Miltenyi, San Diego, CA), 0.5% BSA, and 2mM EDTA (Sigma-Aldrich) in Dulbecco's Phosphate-Buffered Saline (DPBS) without calcium and magnesium (Life Technologies), pH 7.2. Controls included non-stained cells, and cells incubated with corresponding isotype controls (Becton Dickinson, Bedford, MA). For surface markers staining, cells were incubated with fluorochrome-conjugated antibodies, FITC-Linage cocktail (CD3, CD16, CD19, CD20, CD14 and CD56), PerCPCy5-CD34, BV421-CD31, PE-CD235a, APC-H7-CD45, AF647-CD309 (KDR), and AF700-CD38 (all Becton Dickinson). Additional fluorochrome-conjugated antibodies were BV510-CD19, PE-CD34, FITC-CD133, and CD3/CD4/CD8a monoclonal Antibody Cocktail (APC-CD8a, FITC-CD4, PerCP-eFluor 710-CD3) (all Ebioscience (San Diego, CA)).

Compensation beads (Becton Dickinson) were used to compensate for potential spectral fluorochrome overlaps. Cells were analyzed with a FACS Aria II (Becton Dickinson). Raw data were analyzed with FlowJo software version 9.5.2 (Tree Star, Ashland, OR); a forward versus side scatter gate was applied to exclude cell debris and cell doublets.

2.6 Live Cell Sorting

HUVECs that had been cultured for 15 days were sorted for those cells that expressed hematopoietic stem cell markers, defined as lin⁻/CD34⁺/CD38⁻. Cells were rinsed twice with DPBS (without calcium and magnesium), and after detaching with EDTA-trypsin (Life Technologies) or TryplE (Life Technologies), single cells were re-suspended and were incubated with blocking buffer containing 20% FcR block (Miltenyi, San Diego, CA), 0.5% BSA, and 2mM EDTA (Sigma-Aldrich) in Dulbecco's Phosphate-Buffered Saline (DPBS) without calcium and magnesium (Life Technologies), pH 7.2. Controls included non-stained cells. For staining of surface markers, cells were incubated with fluorochrome-conjugated antibodies, FITC-Linage cocktail (CD3, CD16, CD19, CD20, CD14 and CD56) (Becton Dickinson), AF700-CD38 (Becton Dickinson), PE-CD34 (ebioscience) and Sytox Blue Dead Cell Stain (Invitrogen detection technologies, Eugene, OR). The HSC gate-sorted cells were cultured for five days in HSC-specific culture medium (StemSpam SFEM (StemCell Technologies)), containing 1% StemSpan CC100 (StemCell Technologies), and 1% Anti-anti (Invitrogen) antibiotic-antimycotic (100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL Amphotericin B).

2.7 Colony Forming Unit Assay

We used the Colony Forming Unit Assay (CFU) to investigate the potential of HUVECs to form colonies of cells of hematopoietic lineages. Day 0 HUVECs and cultured HUVECs from days 15 and 42 were assayed in ultra-low adherence Petri dishes in a complete MethoCult methylcellulose-based assay according to manufacturer’s instructions (StemCell Technologies) for 14 days. In addition, cultured and subsequently sorted cells expressing HSC markers (as described in section 2.6) were assayed under the same conditions. Colonies were observed by phase microscopy (Zeiss Invertoskop C, Carl Zeiss, Jena, Germany) and counted. Four different types of colonies were identified and their frequencies compared to expected numbers as given by the manufacturer: These included: CFU-E (Colony Forming Unit – Erythrocyte), BFU-E (Burst Forming Unit – Erythrocyte), CFU-GM (Colony Forming Unit – Granulocytes, Macrophage) and CFU-GEMM (Colony Forming Unit – Granulocyte, Erythrocyte, Macrophage, Megakaryocyte).

2.8 Measurement of Medium Calcium Ion Concentrations

The calcium ion concentrations were measured from the medium samples to detect the calcium released from the scaffolds over the culture period. All concentrations were measured using a Cobas 221b blood analyzer (Roche Diagnostics, Indianapolis, IN).

3. Statistical Methods

The data are given as means from three biological repeats ± standard deviation. One-way ANOVA with Turkey’s test was used to analyse statistical significance, with p≤0.05 considered being significant.

4. Results

4.1 Characterization of the HA Scaffolds

In this long-term HUVEC culture, the HA and infiltrated HA scaffolds provided a 3D structure for cell growth and proliferation. As the infiltrated HA scaffolds were produced based on HA scaffolds, there is no observable difference between the architecture of the two kinds of scaffolds. SEM images of the open-porous structure of the scaffolds are shown in (Fig. 1). Each piece of scaffold has high porosity, with large, connected pores. The total porosities in the scaffolds were approximately 90%, with open porosities of approximately 87%, as reported previously [20]. Using EDS (Fig. 1 C), the Ca/P ratio was approximately 1.70 at certain positions, while the stoichiometric Ca/P ratio for HA is 1.67. The Ca/P ratio for the whole surface area was approximately 1.57 (Fig. 1D), which indicates that the scaffold is deficient HA.

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.

4.2 Measurement of Calcium Ion Concentrations

During the 42 days of culture, we monitored the Ca⁺² ion concentration in the culture medium (Fig. 2). Whereas cultures with infiltrated HA had a significantly higher Ca²⁺ concentration than control cultures without scaffold, cultures with non-infiltrated HA demonstrated similar Ca²⁺ concentrations to controls without scaffold. The cultures with infiltrated HA gave a burst of high Ca²⁺ concentration at the first three days, probably due to the rapid dissolution of the calcium-rich (CaCO₃) second phase. While the Ca²⁺ concentration subsequently decreased after three days of culture for the infiltrated samples, the Ca²⁺ release was still maintained above the Ca²⁺ release of the control cultures and the pure HA scaffold over the entire 40 days.

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.

4. 3 Gene Expression Analyses

Gene expressions of HUVECs were analyzed by RT-PCR (Fig. 3 A, B and C) at days 5, 15, and 42 of culture. Results were normalized to freshly isolated day 0 cell samples. We analyzed genes typical for mature endothelium (VWF coding for vWF), endothelial progenitor cells and mature endothelium (PECAM1 coding for surface CD31), hematopoietic stem cells and endothelial progenitors (CD34 coding for surface CD34), and mature hematopoietic cells (PTPRC coding for surface CD45). After five days in culture there was no significant difference between the three conditions in terms of gene expressions for CD34, CD45, and CD31; expression of CD45 was not detected at all. vWF expression was significantly higher in the control condition than in the scaffold conditions. However, this significant difference for vWF expression was absent at days 15 and 42, with the vWF expression increasing for all three conditions over time. After 15 days, CD34 expression of the control was statistical significantly higher than that of cultures with scaffolds. After 42 days, the CD34 expression of the control was still higher than that of cultures with scaffolds, although not statistical significantly. CD45 expression was detectable from day 15 on to day 42, and cultures with scaffolds demonstrated significantly higher expression than the control without scaffold at both time points; at day 15, cultures on infiltrated HA scaffolds showed significantly higher CD45 expression than those on non-infiltrated HA. Throughout culture, CD31 expression was maintained and there was no difference between culture conditions.

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.

4. 4 Flow Cytometry

Based on our data on increased CD45 gene expression in cultures with HA scaffolds, we were specifically interested in investigating the potential hematopoietic lineage differentiation potential of HUVECs. We analyzed the percentages of HUVECs at two time points, day 15 and 42, at which the mature hematopoietic marker CD45 first appeared. The antibodies we used included CD235a for erythrocytes, CD45 for mature hematopoietic cells, a hematopoietic lineage cocktail (lin) for mature lineage cells (including T-lymphocytes, B-lymphocytes, NK-lymphocytes, monocytes, neutrophils, eosinophils, granulocytes, and macrophages), CD34 and CD31 in combination for detection of several hematopoietic/endothelial cell types (including CD31⁻CD34⁺ for hematopoietic progenitor cells, CD31⁺CD34⁺ for endothelial progenitor cells and hemangioblasts, and CD31⁺CD34⁻ for mature endothelial cells), and a combination of lin⁻CD34⁺CD38⁻ for hematopoietic stem cells.

We observed a significant increase of CD31⁻CD34⁺ hematopoietic progenitors (Fig. 4A) when HUVECs were cultured on infiltrated HA, but not for plain HA, after long-term culture of 42 days. In addition, the percentages of CD31⁺CD34⁺ endothelial progenitors (Fig. 4B) were significantly lower in cultures with HA (both infiltrated and plain HA) when compared to controls. Also, the percentages of CD31⁺CD34⁻ endothelial cells (Fig. 4C) were significantly lower in cultures with HA; this was the case for both time points examined (days 15 and 42) for infiltrated HA, but only for day 15 for plain HA.

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).

Percentages of hematopoietic stem cells, defined as lin⁻CD34⁺CD38⁻ cells (Fig. 5), at shorter-term culture of 15 days (Fig. 5A) were initially lower in controls than on HA scaffolds, but increased after 42 days of culture (Fig. 5B), from about 0.67% at day 15 to 4.39% at day 42. In comparison, the percentages of hematopoietic stem cells on both types of scaffolds remained stable throughout culture at days 15 and 42 on HA scaffolds.

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).

We also investigated the expression of mature hematopoietic surface markers, including CD45, CD235a, and various hematopoietic lineages using a lineage (lin) cocktail (Fig. 6). After 15 days of culture (Fig. 6A), the percentages of cells on HA scaffolds expressing CD235a and lin increased compared to controls, a statistical significantly higher percentage of lin⁺ cells could be observed in cells cultured on infiltrated HA scaffold. By 42 days of culture (Fig. 6B), the percentages of cells expressing hematopoietic markers including lin, CD45, and CD235a were significantly higher for cells cultured on the infiltrated HA scaffold than control cells without scaffolds; in addition, CD45 expression was significantly higher on infiltrated scaffolds than on plain scaffolds. Cells cultured on the plain HA also demonstrated significantly higher expression of CD45 and CD235a than controls without scaffolds. We further investigated the expression of characteristic T-cell antigens CD3, CD4, and CD8a, and B-cell antigen CD19 (Fig. 7A). At day 0, HUVECs did not express any T-cell or B-cell markers, which indicated that there were no mature hematopoietic cells present. At day 15, we could detect hematopoietic cells in the HA and infiltrated HA conditions. The percentages of cells expressing mature hematopoietic cell markers (CD3, CD4 and CD19) were significantly higher in the HA condition than in the controls. In the infiltrated HA condition, more cells expressed the T-cell markers CD3 and CD4 than cells in the control condition without scaffolds. Compared to day 0 samples, the percentage of CD31⁻/CD34⁺/CD133⁺/KDR⁺ cells, which characterizes hemangioblasts, sharply decreased from 0.65 to 0.02% in all three conditions at day 15 (Fig. 7 B). Yet, cells cultured on both, HA and the infiltrated HA, had higher percentage of the CD31⁻/CD34⁺/CD133⁺/KDR⁺ cells than the control. There was no significant difference between the HA and the infiltrated HA condition.

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).

4.5 Live Cell-sorting

We also sorted cells from HUVEC cultures that expressed characteristic HSC markers (Lin⁻/CD34⁺/CD38⁻). The percentages of the selected cells were 0.7% in controls, 1.0% in the HA condition, and 1.1% in the infiltrated HA condition.

4.5 Colony Unit Forming Assay

In order to investigate the possible potential of HUVECs having the hematopoietic progenitor feature to form colonies of various hematopoietic lineages, we performed CFU assays (Table 1). HUVECs without culturing were assayed as day 0 and did not show any colony formation. Also, HUVECs cultured for 15 or 42 days without scaffolds (control) did not form any colonies, indicating that HUVECs in conventional culture do not have any hematopoietic colony formation potential. HUVECs that had been cultured for 15 or 42 days on infiltrated and plain HA scaffolds, however, demonstrated the formation of colonies. The presence of CFU-E and CFU-GM in the assay proved the existence of functional hematopoietic progenitors in HA and Infiltrated HA conditions after 15 days and 42 days of culture. In addition, in order to investigate the hematopoietic functional potential of hematopoietic-oriented cells, the CFU assays were also performed with HSC marker-sorted cells that were subjected to a 5-day culture period in HSC medium (Table. 2). Interestingly, no colonies could be found in the control condition (no scaffold), but CFU-E and CFU-GM colonies were detected in both scaffold cultures (non-infiltrated and infiltrated HA scaffolds). The differences between the controls and the HA or the infiltrated HA conditions were significant. However, there was no significant difference between the HA and the infiltrated HA condition.

Table 1.

Colony-forming Unit (CFU) assay of HUVECs. Fresh cells at day 0 and cells that had been cultured for 15 or 42 days in control conditions (no scaffold), on plain hydroxyapatite (HA) scaffold, or infiltrated HA scaffold were cultured for 14 days in methyl cellulose-based hematopoietic assay medium. Colony types were counted microscopically, and data are given as means ± standard deviation from n=3 biological repeats.

Sample	CFU-E	BFU-E	CFU-GM	CFU-GEMM
Day 0	0	0	0	0
Day 15 Control	0	0	0	0
Day 15 HA	0.67±0.67	0	0.67±0.67	0
Day 15 Infiltrated HA	0.67±0.67	0	0.67±0.67	0
Day 42 Control	0	0	0	0
Day 42 HA	0.67±0.67	0	1.00±1.00	0
Day 42 Infiltrated HA	0.67±0.67	0	0.33±0.33	0

Table 2.

Colony-forming Unit (CFU) assay of HUVECs. Cells that had been cultured for 15 in control conditions (no scaffold), on plain hydroxyapatite (HA) scaffold, or infiltrated HA scaffold were selected for their expression of HSC markers Lin⁻/CD34⁺/CD38⁻ and cultured in HSC medium for 5 days. Subsequently, the harvested cells were cultured for 14 days in methyl cellulose-based hematopoietic assay medium. Colony types were counted microscopically, and data are given as means ± standard deviation from n=3 biological repeats.

Sample	CFU-E	BFU-E	CFU-GM	CFU-GEMM
Day 15 Control	0	0	0	0
Day 15 HA	4.33±1.67	0	6.67±0.33	0
Day 15 Infiltrated HA	6.00±2.00	0	8.00±1.00	0

5. Discussion

In our research, we performed long-term culture of HUVECs for six weeks to mimic important aspects of the endosteal niche microenvironment in bone marrow using three-dimensional HA-based calcium phosphate scaffolds. We hypothesized that simulating basic aspects of the endosteal niche, including the three-dimensional architecture and the ability to release calcium ions, could promote hematopoietic differentiation. In long-term culture of HUVECs, cells with characteristics of hematopoietic stem cells (HSCs) appeared, and more cells expressing mature hematopoietic markers were present at the end of the culture. Cells were also tested in terms of their functionality and potential using the CFU assay; the results demonstrated that, at least a fraction of the cells were able to differentiate into erythrocytes and form macrophages.

In HSC in vitro culture, endothelial cells have been shown to be an important component for maintenance of the HSC niche [21], perhaps due to the hemangioblast which has been described as a common precursor for endothelial and hematopoietic cells during development [7,22–26] as well as in the adult [27–29]. Definitive proof of the existence of adult hemangioblasts was provided by discovering adult HSCs that had functional hemangioblast activity during retinal neovascularization [30]. CD34 positive adult hemangioblasts have also been discovered in bone marrow and peripheral blood [31–34]; however, it is not well understood how chemical and physical signals (such as calcium or the three-dimensional environment) can contribute to hematopoietic lineage differentiation of endothelial cells. During early embryonic development, the hemogenic endothelium can give rise to hematopoietic cell types (for review, see [35]). The hemogenic endothelium has been defined as displaying an endothelial phenotype and morphology, and having the capacity to form hematopoietic offspring and endothelial tubules/sheets in culture ([36]; for review, see [37]). The overlap in the expression of hematopoietic and endothelial markers, for vascular endothelial cadherin, CD31, CD34, and CD45, suggests a close developmental relationship between hematopoietic cells and endothelial cells ([38–40], reviewed in [37]).

In our work, we found that HUVECs, which are commonly used as a mature endothelial cell lineage model, obtained HSC functionality after long-term culture. A low percentage of HUVECs with hemangioblast characteristics could be detected as determined by CD31⁻/CD34⁺/CD133⁺/KDR⁺ expression. The significantly higher percentage of cells expressing markers of mature hematopoietic cells (erythrocyte marker CD235a, lineage markers and monocyte marker CD45) in scaffold culture compared to controls indicates that endothelial cells can be stimulated into the hematopoietic lineage by culture on HA scaffolds. Some of the cells also expressed lymphocyte marker CD4. In addition, CFU activity of cells that had been cultured on HA scaffolds, but not those that had been cultured in two-dimensional Petri dishes, suggests that HA scaffolds promote the hematopoietic lineage direction. HUVECS, which were sorted for HSC markers after culture on scaffolds, demonstrated an enhanced hematopoietic potential. HA scaffolds have been applied in tissue engineering [41,42] and stem cell culture [43–46], commonly for bone tissue engineering. For example, osteogenic differentiation of mesenchymal stem cells has been reported, using hydroxyapatite alginate scaffolds [46] or hydroxyapatite nanoparticles [47]. In addition the long-term maintenance of bone marrow-derived HSCs could be achieved in bioreactors with HA scaffolds [48].

Calcium is known to be important for stem cell differentiation and proliferation [49]. In HSCs, different calcium receptors have been described (CaR [18] and GPCRs [50]). These receptors are required to maintain HSCs near the endosteal surface of the bone [51], indicating that HSC maintenance is sensitive to Ca²⁺. We found that calcium-infiltrated HA scaffolds induced HUVECs to hematopoietic fates, suggesting that the local surface calcium ion concentration might play an important role in the hematopoietic differentiation of endothelial cells.

6. Conclusion

In this study, we investigated the effects of three-dimensional, porous, calcium-infiltrated HA scaffolds on HUVECs in long-term culture. In culture on scaffolds, some HUVECs acquired hematopoietic characteristics. HA scaffolds per se increased percentages of cells positive for erythrocyte markers and CFU activity, with calcium infiltration significantly increasing the percentages of CD45 and hematopoietic lineage marker positive cells. These results indicate that high calcium HA scaffolds support the hematopoietic lineage direction of HUVECs, suggesting that calcium-infiltrated hydroxyapatite provides an improved in vivo-like scaffold for hematopoietic bone marrow tissue engineering.