Comprehensive genetic analysis of early host body reactions to the bioactive and bio-inert porous scaffolds

Abstract

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1β and IL-10 are important cytokines in tissue responses to biomaterials because IL-1β promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1β was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations.

Figure 1. FTIR/ATR spectra of PMB- and collagen-coated PE films.

Figure 2. The appearance of scaffolds on day 7 after operation.

All scaffolds were attached on the subcutaneous tissue. Scaffolds with a pore size of 157 µm (A, C, and E) and 32 µm (B, D, and F) were implanted. Angiogenesis (arrows) and fibrous tissue encapsulation were compared among non-coat (A and B), PMB-coated (C and D), and collagen-coated (E and F) scaffolds. All scaffold diameters are 6 mm.

Figure 3. Macroscopic observation of implanted scaffold with non-coat (A), PMB-coated (B), and collagen-coated (C) scaffold which were sliced and stained with HE.

Scaffold mean pore size was 157 µm. Scaffolds were attached to rat dorsal skin and encapsulated with layer of fibrous tissue (arrows). Some scaffold skeletons (S) are detached from sliced sample. Bars  = 1 mm.

Figure 4. Microscopic histological observations by HE staining (A, C, and E) and CD68 immunostaining (B, D, and F) of non-coat (A and B), PMB-coated (C and D), and collagen-coated (E and F) scaffolds at boundary between scaffold and tissue.

Small vessels (arrow heads) and macrophages (arrows) were observed. The mean pore size of the scaffolds was 157 µm. Seven days after operation. Some scaffold skeletons (S) are detached from sliced samples. Bars  = 50 µm.

Figure 5. Global analysis of host body responses to PMB-coated and collagen-coated scaffolds.

All genes having valid expression levels in non-coated, collagen-coated, and PMB-coated scaffolds were plotted.

Figure 6. Selected genes that were expressed differently between collagen-coated and PMB-coated and were related to tissue regeneration and inflammation.

Closed circle, wound healing promotion factors; Open circle; inflammatory factors; Closed square, uncertain about tissue regeneration.