Injectable, porous, biohybrid hydrogels incorporating decellularized tissue components for soft tissue applications

Abstract

Biodegradable injectable hydrogels have been extensively studied and evaluated in various medical applications such as for bulking agents, drug delivery reservoirs, temporary barriers, adhesives, and cell delivery matrices. Where injectable hydrogels are intended to facilitate a healing response, it may be desirable to encourage rapid cellular infiltration into the hydrogel volume from the tissue surrounding the injection site. In this study, we developed a platform technique to rapidly form pores in a thermally responsive injectable hydrogel, poly(NIPAAm-co-VP-co-MAPLA) by using mannitol particles as porogens. In a rat hindlimb muscle injection model, hydrogels incorporating porosity had significantly accelerated cellular infiltration. To influence the inflammatory response to the injected hydrogel, enzymatically digested urinary bladder matrix (UBM) was mixed with the solubilized hydrogel. The presence of UBM was associated with greater polarization of the recruited macrophage population to the M2 phenotype, indicating a more constructive foreign body response. The hybrid hydrogel positively affected the wound healing outcomes of defects in rabbit adipose tissue with negligible inflammation and fibrosis, whereas scar formation and chronic inflammation were observed with autotransplantation and in saline injected groups. These results demonstrate the value of combining the effects of promoting cell infiltration and mediating the foreign body response for improved biomaterials options soft tissue defect filling applications.

Statement of significance: Our objective was to develop a fabrication process to create porous injectable hydrogels incorporating decellularized tissue digest material. This new hydrogel material was expected to exhibit faster cellular infiltration and a greater extent of pro-M2 macrophage polarization compared to control groups not incorporating each of the functional components. Poly(NIPAAm-co-VP-co-MAPLA) was chosen as the representative thermoresponsive hydrogel, and mannitol particles and digested urinary bladder matrix (UBM) were selected as the porogen and the bioactive decellularized material components respectively. In rat hindlimb intramuscular injection models, this new hydrogel material induced more rapid cellular infiltration and a greater extent of M2 macrophage polarization compared to control groups not incorporating all of the functional components. The hybrid hydrogel positively affected the wound healing outcomes of defects in rabbit adipose tissue with negligible inflammation and fibrosis, whereas scar formation and chronic inflammation were observed with autotransplantation and in saline injected groups. The methodology of this report provides a straightforward and convenient mechanism to promote cell infiltration and mediate foreign body response in injectable hydrogels for soft tissue applications. We believe that the readership of Acta Biomaterialia will find the work of interest both for its specific results and general translatability of the findings.

Keywords: Cell infiltration; Decellularized extracellular matrix; Foreign body response; Hydrogel; Porous biomaterial; Soft tissue repair.

Figure 1

Hydrogel cross-sections. NP: nonporous hydrogel, NPE: nonporous hydrogel without the UBM digest component, PME: porous hydrogel with the UBM digest component.

Figure 2

(a) Compressive modulus of hydrogels. (b) Hydrogel degradation profile of hydrogels. * Significant difference, p < 0.05.

Figure 3

Chemotactic migration of macrophage induced by release products from hydrogels.

Figure 4

In situ pore formation in hydrogels after intramuscular injections. M: muscle, G: hydrogel, Arrows: boundary of pores.

Figure 5

Cell infiltration into injected hydrogels. M: muscle, G: hydrogel, Arrows: foreign body capsule. (a) DAPI staining of rat hindlimb muscle 3 days after hydrogel injection, scale bar = 500 μm. (b) Trichrome staining of rat hindlimb muscle 3 days after hydrogel injection. (c) DAPI staining of rat hindlimb muscle 21 days after hydrogel injection. (d) Trichrome staining of rat hindlimb muscle 21 days after hydrogel injection. (e,f) Relative cell density (muscle = 100%) in injected hydrogel and foreign body capsule 3 days and 21 days after injection.

Figure 5

Cell infiltration into injected hydrogels. M: muscle, G: hydrogel, Arrows: foreign body capsule. (a) DAPI staining of rat hindlimb muscle 3 days after hydrogel injection, scale bar = 500 μm. (b) Trichrome staining of rat hindlimb muscle 3 days after hydrogel injection. (c) DAPI staining of rat hindlimb muscle 21 days after hydrogel injection. (d) Trichrome staining of rat hindlimb muscle 21 days after hydrogel injection. (e,f) Relative cell density (muscle = 100%) in injected hydrogel and foreign body capsule 3 days and 21 days after injection.

Figure 6

Macrophage polarization 3 days after hydrogel injection. M: muscle, G: hydrogel, Arrows: foreign body capsule. (a) CD86 (green) and CD68 (red) staining. (b) CD206 (green) and CD68 (red) staining. (c) CD86 (green)/CD68 (red) and (d) CD206 (green)/CD68 (red) staining at the hydrogel/muscle interface. (e, f) Percentage of CD86⁺ and CD206⁺ cells in CD68⁺ population, respectively. (g) Ratio between CD86⁺ and CD206⁺ cells in CD68⁺ population. * Significant difference, p < 0.05.

Figure 6

Macrophage polarization 3 days after hydrogel injection. M: muscle, G: hydrogel, Arrows: foreign body capsule. (a) CD86 (green) and CD68 (red) staining. (b) CD206 (green) and CD68 (red) staining. (c) CD86 (green)/CD68 (red) and (d) CD206 (green)/CD68 (red) staining at the hydrogel/muscle interface. (e, f) Percentage of CD86⁺ and CD206⁺ cells in CD68⁺ population, respectively. (g) Ratio between CD86⁺ and CD206⁺ cells in CD68⁺ population. * Significant difference, p < 0.05.

Figure 7

Macrophage polarization in hydrogel injection sites after 21 days. M: muscle, G: hydrogel, Arrows: foreign body capsule. (a) CD86 (green) and CD68 (red) staining. (b) CD206 (green) and CD68 (red) staining. (c) CD86 (green)/CD68 (red) and (d) CD206 (green)/CD68 (red) staining at the hydrogel/muscle interface. (e, f) Percentage of CD86⁺ and CD206⁺ cells in CD68⁺ population, respectively. (g) Ratio between CD86⁺ and CD206⁺ cells in CD68⁺ population. * Higher than PM group, # Higher than NP and PM groups, p < 0.05.

Figure 7

Macrophage polarization in hydrogel injection sites after 21 days. M: muscle, G: hydrogel, Arrows: foreign body capsule. (a) CD86 (green) and CD68 (red) staining. (b) CD206 (green) and CD68 (red) staining. (c) CD86 (green)/CD68 (red) and (d) CD206 (green)/CD68 (red) staining at the hydrogel/muscle interface. (e, f) Percentage of CD86⁺ and CD206⁺ cells in CD68⁺ population, respectively. (g) Ratio between CD86⁺ and CD206⁺ cells in CD68⁺ population. * Higher than PM group, # Higher than NP and PM groups, p < 0.05.

Figure 8

Morphology of rabbit adipose tissue 2 weeks after treatment. H: healthy tissue, T: transplant. (a) Trichrome staining. (b) Oil Red staining.

Figure 9

Gross appearance of rabbit adipose tissue 8 weeks after treatment. Arrows: suture markers.

Figure 10

Morphology of rabbit adipose tissue 8 weeks after treatment. H: healthy tissue. (a) Trichrome staining. (b) Oil Red staining. (c) Isolectin (red)/BIOPSY (green)/DAPI staining.

Figure 11

Morphology analysis by adipocyte size and crown-like structure number. (a) Frequency distribution of adipocyte size. (b) Adipocyte size comparison among treatments. (c) Ratio between the quantity of crown-like structure and adipocytes. * Significant difference, p < 0.05.

Scheme 1

(a) In situ formation of pores immediately subsequent to hydrogel injection. (b) Faster cell infiltration and pro-M2 macrophage polarization in the porous injectable hydrogel.