Cells behave distinctly within sponges and hydrogels due to differences of internal structure

Abstract

Different forms of biomaterials, including microspheres, sponges, hydrogels, and nanofibers, have been broadly used in cartilage regeneration; however, effects of internal structures of the biomaterials on cells and chondrogenesis remain largely unexplored. We hypothesized that different internal structures of sponges and hydrogels led to phenotypic disparity of the cells and may lead to disparate chondrogenesis. In the current study, the chondrocytes in sponges and hydrogels of chitosan were compared with regard to cell distribution, morphology, gene expression, and production of extracellular matrix. The chondrocytes clustered or attached to the materials with spindle morphologies in the sponges, while they distributed evenly with spherical morphologies in the hydrogels. The chondrocytes proliferated faster with elevated gene expression of collagen type I and down-regulated gene expression of aggracan in sponges, when compared with those in the hydrogels. However, there was no significant difference of the expression of collagen type II between these two scaffolds. Excretion of both glycosaminoglycan (GAG) and collagen type II increased with time in vitro, but there was no significant difference between the sponges and the hydrogels. There was no significant difference in secretion of GAG and collagen type II in the two scaffolds, while the levels of collagen type I and collagen type X were much higher in sponges compared with those in hydrogels during an in vivo study. Though the chondrocytes displayed different phenotypes in the sponges and hydrogels, they led to comparable chondrogenesis. An optimized design of the biomaterials could further improve chondrogenesis through enhancing functionalities of the chondrocytes.

FIG. 1.

Environmental scanning electron micrographs. (a) chitosan sponges, (b) chitosan hydrogels.

FIG. 2.

The diffusion of bromophenol blue and release of bovine serum albumin (BSA). (a) The diffusion dynamics in the scaffolds in sponges (left) and hydrogels (right) Top views (a, b, c, d) and side views (e, f, g, h). (b) Quantitative BSA release from the sponges and hydrogels (n=3). Color images available online at www.liebertpub.com/tea

FIG. 3.

Actin staining of the chondrocytes in the sponges (a, c) and hydrogels (b, d). Actin was stained red, while the cell nucleus was blue. Arrow: stretched cell cytoskeletal and cell aggregation; Arrowhead: a round morphology and even distribution. Color images available online at www.liebertpub.com/tea

FIG. 4.

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test for cytotoxicity. Data were normalized as the percentage of Tissue Culture Plate Surface (TCPS). *p<0.05 compared with the latex rubber group (n=4).

FIG. 5.

Proliferation of the chondrocytes in the sponges and hydrogels. *p<0.05 compared with sponges (n=4).

FIG. 6.

The viability of the chondrocytes by fluorescein diacetate (FDA)-PI staining. Confocal microscopy photographs of FDA-PI staining image showed chondrocytes on the surface of sponges at (a) 1 day and (c) 21 days and in hydrogels at 1 day (b) and 21 days (d) after cell seeding. The live cells were dyed by FDA (green), and the dead cells were stained by PI (red). Color images available online at www.liebertpub.com/tea

FIG. 7.

Alcian blue staining and immunohistochemical staining for collagen II in vitro. (left) Alcian blue staining of sponges (a, c) and hydrogels (b, d). (right) Collagen II immunohistochemical staining for sponges (a, c) and hydrogels (b, d). Scale bars: 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 8.

GAG content on day 14 and day 28 after seeding in hydrogels and sponges (*p<0.05, n=3). GAG, glycosaminoglycan.

FIG. 9.

Immunohistochemical staining for collagen type I and collagen type X for in vitro scaffold constructs at day 28. (a) collagen type I staining of the sponges. (b) collagen type I staining of the hydrogels. (c) collagen type X staining of the sponges. (d) collagen type X staining of the hydrogels. Scale bars: 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 10.

The mRNA expression of collagen type I (A), collagen type II (B), and aggrecan (C) in vitro (*p<0.05, and **p<0.01, n=3).

FIG. 11.

Alcian blue staining and immunohistochemical staining for collagen II in vivo. The constructs were cultured for 1 week in vitro before being transplanted into mice subcutaneous cavities for another 2 weeks and 4 weeks. (A) Alcian blue staining of sponges (a, c) and hydrogels (b, d). (B) Collagen II staining for sponges (a, c) and hydrogels (b, d). Scale bars: 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 12.

Immunohistochemical staining for collagen I and collagen X for in vivo scaffold constructs. The constructs were cultured for 1 week in vitro before being transplanted into mice subcutaneous cavities for another 2 weeks and 4 weeks.(A) Collagen I staining for sponges (a, c) and hydrogels (b, d). (B) Collagen X staining for sponges (a, c) and hydrogels (b, d). Scale bars: 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 13.

The schematic illustration of different behaviors of chondrocytes in sponges and hydrogels. Chondrocytes tended to stretch out and form little clusters in sponges (left), while they maintained a round morphology and were distributed evenly within hydrogels (right). Color images available online at www.liebertpub.com/tea