Characterization of different biodegradable scaffolds in tissue engineering

Abstract

The aim of the present study was to compare the characteristics of acellular dermal matrix (ADM), small intestinal submucosa (SIS) and Bio‑Gide scaffolds with acellular vascular matrix (ACVM)‑0.25% human‑like collagen I (HLC‑I) scaffold in tissue engineering blood vessels. The ACVM‑0.25% HLC‑I scaffold was prepared and compared with ADM, SIS and Bio‑Gide scaffolds via hematoxylin and eosin (H&E) staining, Masson staining and scanning electron microscope (SEM) observations. Primary human gingival fibroblasts (HGFs) were cultured and identified. Then, the experiment was established via the seeding of HGFs on different scaffolds for 1, 4 and 7 days. The compatibility of four different scaffolds with HGFs was evaluated by H&E staining, SEM observation and Cell Counting Kit‑8 assay. Then, a series of experiments were conducted to evaluate water absorption capacities, mechanical abilities, the ultra‑microstructure and the cytotoxicity of the four scaffolds. The ACVM‑0.25% HLC‑I scaffold was revealed to exhibit the best cell proliferation and good cell architecture. ADM and Bio‑Gide scaffolds exhibited good mechanical stability but cell proliferation was reduced when compared with the ACVM‑0.25% HLC‑I scaffold. In addition, SIS scaffolds exhibited the worst cell proliferation. The ACVM‑0.25% HLC‑I scaffold exhibited the best water absorption, followed by the SIS and Bio‑Gide scaffolds, and then the ADM scaffold. In conclusion, the ACVM‑0.25% HLC‑I scaffold has good mechanical properties as a tissue engineering scaffold and the present results suggest that it has better biological characterization when compared with other scaffold types.

Figure 1.

Observation of scaffold materials. (A) ACVM-HLC-I, (B) ADM, (C) Bio-Gide and (D) SIS. ACVM-HLC-I scaffold presented as an ivory-white, translucent and non-elastic vascular wall with a folded and damaged lumen. ADM presented with an ivory-white, translucent, honeycomb structure with a rough surface. Bio-Gide was pale yellow-white, with translucent rectangular patches and a smooth surface. SIS presented as a pale ivory, translucent membrane. ACVM-HLC-I, acellular vascular matrix-human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa.

Figure 2.

Culture of HGFs. (A) Day 7 of primary culture of HGFs (inverted microscope; magnification, ×400). (B) Day 15 of second generation of HGFs (inverted microscope; magnification, ×400). The primary culture of HGFs exhibited clear outlines and large, spherical or elliptic nuclei. Second generation cells were spindle-shaped and fibroblast-like. HGF, human gingival fibroblast.

Figure 3.

Immunofluorescent staining of HGFs. (A) Cytoplasm of HGFs with positive staining for vimentin (fluorescein isothiocyanate; magnification, ×200). (B) Cytoplasm of HGFs with positively stained for S100A4 (Rhodamine; magnification, ×200). Vimentin and S100A4 are markers for mesenchymal cells. DAPI is a marker of cell nuclei. HGF, human gingival fibroblast; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4.

MTT assay. The cell adhesion ability of different concentrations of ACVM-HLC-I was evaluated by MTT assay on days 1, 4 and 7. ACVM-HLC-I, acellular vascular matrix-human-like collagen I. *P<0.05 vs. 0.25 mg/ml, ^#P<0.05 vs. day 1, ^&P<0.05 vs. day 4.

Figure 5.

Hematoxylin and eosin staining of the scaffolds (magnification, ×400). (A) ACVM-HLC-I, (B) ADM, (C) Bio-Gide and (D) SIS. ACVM-HLC-I contained red reticular fibers and collagen fibers. ADM exhibited a looser meshwork of collagen fibers, and Bio-Gide presented a looser meshwork of collagen fibers. SIS contained staggered and irregular collagen fibers. ACVM-HLC-I, acellular vascular matrix-human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa.

Figure 6.

Masson staining of the scaffolds (magnification, ×400). (A) ACVM-HLC-I, (B) ADM, (C) Bio-Gide and (D) SIS. Collagen fibers are stained green and muscle fibers are stained red. ACVM-HLC-I collagen fibers were soft and dense; the collagen fibers in each layer of ADM were dyed green, and muscle fibers were dyed red; the ADM collagen fiber structure was thin. The Bio-Gide collagen fibers were very thick and sparse. SIS consisted mostly of green collagen fibers and a small amount of red muscle fibers; the SIS collagen fibers were relatively sparse and hard. ACVM-HLC-I, acellular vascular matrix-human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa.

Figure 7.

Scanning electron microscopy images of the scaffolds (magnification, ×1,000). (A) ACVM, (B) ADM, (C) Bio-Gide, (D) SIS and (E) ACVM-0.25% HLC-I. The surface fibers of the ACVM-0.25% HLC-I scaffold were smaller and slender. ACVM, acellular vascular matrix; ACVM-0.25% HLC-I, AVCM-0.25% human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa.

Figure 8.

Stress-time curves of the scaffolds. (A) ACVM-0.25% HLC-I, (B) ADM, (C) Bio-Gide and (D) SIS. The ADM scaffold exhibited the greatest tensile strength. ACVM-0.25% HLC-I, acellular vascular matrix-0.25% human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa; Rm, maximum pressure strength.

Figure 9.

Stress-strain curves of the scaffolds. (A) ACVM-0.25% HLC-I, (B) ADM, (C) Bio-Gide and (D) SIS. The ADM scaffold exhibited the greatest tensile strength. ACVM-0.25% HLC-I, acellular vascular matrix-0.25% human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa.

Figure 10.

Hematoxylin and eosin staining indicating the histology of the scaffolds seeded with HGFs and cultured for 7 days (magnification, ×400). (A) Cells-ACVM-0.25% HLC-I, (B) Cells-ADM, (C) Cells-Bio-Gide and (D) Cells-SIS. HGFs were abundant and well-distributed in the central part of the ACVM-0.25% HLC-I scaffold. The ADM and Bio-Gide scaffold presented reduced cell proliferation. Almost no cell proliferation was visible on the surface of the SIS scaffold. ACVM-0.25% HLC-I, acellular vascular matrix-0.25% human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa; HGF, human gingival fibroblast.

Figure 11.

Scanning electron microscopy images of the four scaffolds seeded with HGFs and cultured for 7 days (magnification, ×800). (A) Cells-ACVM-0.25% HLC-I, (B) Cells-ADM, (C) Cells-Bio-Gide and (D) Cells-SIS. A large number of HGFs were visible on the surface of the ACVM-0.25% HLC-I scaffold. The SIS scaffold had the smallest number of HGFs on the surface. ACVM-0.25% HLC-I, acellular vascular matrix-0.25% human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa; HGF, human gingival fibroblast.

Figure 12.

Number of HGFs in the scaffolds as determined by Cell Counting Kit-8 assay. A large number of proliferative HGFs was observed in the ACVM-0.25% HLC-I scaffold. There is no significant difference in the number of cells between ADM and Bio-Gide scaffolds. The lowest number of proliferative HGFs was observed in the SIS scaffold. ACVM-0.25% HLC-I, acellular vascular matrix-0.25% human-like collagen I; ADM, acellular dermal matrix; SIS, small intestinal submucosa; HGF, human gingival fibroblast. *P<0.05 vs. ACVM-0.25% HLC-I, ^#P<0.05 vs. 24 h, ^&P<0.05 vs. 48 h.