Porous tantalum-composited gelatin nanoparticles hydrogel integrated with mesenchymal stem cell-derived endothelial cells to construct vascularized tissue in vivo

Abstract

The ideal scaffold material of angiogenesis should have mechanical strength and provide appropriate physiological microporous structures to mimic the extracellular matrix environment. In this study, we constructed an integrated three-dimensional scaffold material using porous tantalum (pTa), gelatin nanoparticles (GNPs) hydrogel, and seeded with bone marrow mesenchymal stem cells (BMSCs)-derived endothelial cells (ECs) for vascular tissue engineering. The characteristics and biocompatibility of pTa and GNPs hydrogel were evaluated by mechanical testing, scanning electron microscopy, cell counting kit, and live-cell assay. The BMSCs-derived ECs were identified by flow cytometry and angiogenesis assay. BMSCs-derived ECs were seeded on the pTa-GNPs hydrogel scaffold and implanted subcutaneously in nude mice. Four weeks after the operation, the scaffold material was evaluated by histomorphology. The superior biocompatible ability of pTa-GNPs hydrogel scaffold was observed. Our in vivo results suggested that 28 days after implantation, the formation of the stable capillary-like network in scaffold material could be promoted significantly. The novel, integrated pTa-GNPs hydrogel scaffold is biocompatible with the host, and exhibits biomechanical and angiogenic properties. Moreover, combined with BMSCs-derived ECs, it could construct vascular engineered tissue in vivo. This study may provide a basis for applying pTa in bone regeneration and autologous BMSCs in tissue-engineered vascular grafts.

Keywords: bone marrow mesenchymal stem cell; endothelial cell; gelatin nanoparticles hydrogel; porous tantalum; vascularization.

Figure 1.

Design of pTa sample. (a) The body-centered cubic unit cell. (b) CAD model of disc-shaped pTa. (c) Disc-shaped pTa used in biological experiment (φ 6 mm×H 2 mm).

Figure 2.

Schematic diagram of animal model. (a) Incision of skin tissue on the back. (b) Exposure of subcutaneous tissue. (c) Implanted composite scaffold material in subcutaneous tissue. (d) Incision suture.

Figure 3.

Mechanical properties and surface morphologies of pTa. (a) Compression experiment. (b) Stress–strain curve. (c) Surface morphologies of pTa. (d) Flat morphology and the metallic luster of dark gray were shown. These images were magnified ×30, ×100, and ×200, respectively (scale bar = 100μm).

Figure 4.

Characteristic and morphology of pTa-GNPs hydrogel scaffold. (a) Time dependence of storage (G′) and loss(G″) modulus of GNPs. (b) Frequency dependence of storage (G′) and loss (G″) modulus of GNPs. (c) Viscosity and shear-thinning behavior of GNPs. (d) Representative rheological measurement showing the healing behavior of the hydrogels during a cycle of destructive shearing and recovery. (e) GNPs’ hydrogels have injectability and adaptability to irregular shapes before completely solidified. (f and g) The appearance of pTa and pTa-GNPs hydrogel scaffold. (h and i) The SEM images showed the surface morphology of pTa-GNPs hydrogel scaffold and the representative microstructures were observed. The images were magnified ×30, ×100, and ×300, respectively (scale bar = 100 μm).

Figure 5.

Culture and identification of BMSCs. (a) Light microscopic observation of the third-generation BMSCs (scale bar = 100 μm). (b) Flow cytometry identification of cell phenotype. CD44 and CD90 were expressed, but CD34a and CD45 were scarcely expressed. (c) Alkaline phosphatase was showed after 2 weeks of culture in osteogenic induction medium (scale bar =100 μm). (d) After 3 weeks of culture in osteogenic induction medium, Alizarin Red staining showed calcified nodules (scale bar =100 μm). (e) The oil red O staining showed the intracellular lipid droplets after 3 weeks of culture in adipogenic induction medium (scale bar = 100 μm).

Figure 6.

(a) Light microscope images of endothelial differentiated BMSCs (scale bar = 100 μm). (b and c) basal characterization of ECs CD31 expression in BMSCs and endothelial differentiated BMSCs by immunofluorescence. Blue fluorescence signifies DAPI, green indicates CD31 (scale bar = 100 mm). (d) Flow cytometry analysis showed the conversion of ECs in BMSCs group is 0.3±0.1% (scale bar = 100 mm). (e) In endothelial differentiated BMSCs group, the conversion of ECs is 31.5±1%. (f) BMSCs remained around in tube formation assay (scale bar=100 mm). (g) Endothelial differentiated BMSCs formed visible tube-like structures on matrigel after 12 h (scale bar = 100 mm).

Figure 7.

(a) SEM morphology of BMSCs attached to pTa scaffolds after 3 days of culture (marked by white arrows; scale bar = 20 μm). (b) The CCK-8 assay showed the proliferation of BMSCs after co-culture with pTa for 1, 3, 5, and 7 days. (c) Live assay of GNPs hydrogel. On day 1 of co-culture, a small number of BMSCs adhered to GNPs hydrogel. (d) On Day 3 of co-culture, the BMSCs adhered nonhomogeneous to GNPs hydrogel, and several cell aggregates were formed. (e) The trend of cell proliferation on GNPs hydrogel was evidently on Day 5 of co-culture (scale bar = 200 μm).

Figure 8.

The effect of pTa-GNPs hydrogel scaffold associated with BMSCs and BMSCs-derived ECs on angiogenesis. (a–d) Van Gieson’s staining of histological sections of four groups’ implants at 4 weeks post-operation revealed the presence of numerous blood vessels (a: control; b: BMSCs; c: endothelial differentiated BMSCs; d: endothelial differentiated BMSCs+BMSCs). White arrows marked the newly formed blood vessels. The large image was magnified ×100 with a scale bar = 100μm; the small image was magnified ×200 with a scale bar = 100μm (marked by white arrows). (e) Histomorphometric analysis of the samples after 28 days of implantation. Quantitative results of vascular lumens determined the microvessel density. Bars represent the mean microvessel density of four groups of implants ± SD. Data were analyzed by analysis of variance (ANOVA) followed by post-hoc multiple comparisons using Tukey's test. *P < 0.05.

Scheme 1.

(a) Prepare schematic of GNPs hydrogel. (b) The formation mechanism of GNPs colloidal network.

Scheme 2.

Schematic for constructing vascularized scaffolds in subcutaneous tissue of mouse with pTa-GNPs hydrogel scaffold combined with BMSCs and BMSCs-derived ECs. BMSCs were harvested from mouse tibia and femur. PTa-GNPs hydrogel scaffold were seeded with BMSCs and BMSCs-derived ECs, respectively. Composite material was implanted in subcutaneous tissue of mouse for 4 weeks.