Fig 1.
Cellulose scaffold preparation.
Macroscopic appearance of a freshly cut apple hypanthium tissue (A) and the translucent cellulose scaffold biomaterial post-decellularization and absent of all native apple cells or cell debris (B). H&E staining of cross sectioned decellularized cellulose scaffold (C). The cell walls thickness and the absence of native apple cells following decellularization are shown. The 3D acellular and highly porous cellulose scaffold architecture is clearly revealed by scanning electron microscopy (D). Scale bar: A-B = 2mm, C-D = 100μm.
Fig 2.
Cellulose scaffolds implantation and resection.
The subcutaneous implantations of cellulose scaffolds biomaterial were performed on the dorsal region of a C57BL/10ScSnJ mouse model by small skin incisions (8 mm) (A). Each implant was measured before their implantation for scaffold area comparison (B). Cellulose scaffolds were resected at 1 week (D), 4 weeks (E) and 8 weeks (F) after the surgeries and macroscopic pictures were taken (control skin in C). The changes in cellulose scaffold surface area over time are presented (G). The pre-implantation scaffold had an area of 26.30±1.98mm2. Following the implantation, the area of the scaffold declined to 20.74±1.80mm2 after 1 week, 16.41±2.44mm2 after 4 weeks and 13.82±3.88mm2 after 8 weeks. The surface area of the cellulose scaffold has a significant decrease of about 12mm2 (48%) after 8 weeks implantation (* = P<0.001; n = 12–14).
Fig 3.
Biocompatibility and cell infiltration.
Cross sections of representative cellulose scaffolds stained with H&E and anti-CD45. These global view show the acute moderate-severe anticipated foreign body reaction at 1 week (A), the mild chronic immune and subsequent cleaning processes at 4 weeks (B) and finally, the cellulose scaffold assimilated into the native mouse tissue at 8 weeks (C). Higher magnification regions of interest (D-F), see inset (A-C), allow the observation of all the cell type population within biomaterial assimilation processes. At 1 week, we can observe populations of granulocytes, specifically; polymorphonuclear (PMN) and eosinophils that characterize the acute moderate to severe immune response, a normal reaction to implantation procedures (D). At 4 weeks, a decreased immune response can be observed (mild to low immune response) and the population of cells within the epidermis surrounding scaffolds now contain higher levels of monocytes and lymphocytes characterizing chronic response (E). Finally, at 8 weeks, the immune response has completely resorbed with the epidermis tissue now appearing normal (F). The immune response observed with H&E staining is confirmed using anti-CD45 antibody, a well known markers of leukocytes (G-I). The population of cells within the scaffold are now mainly macrophages, multinucleated cells and active fibroblasts. Scale bars: A-C = 1mm, D-F = 100μm and G-I = 500μm.
Fig 4.
Extracellular matrix deposition.
Cross sections of representative cellulose scaffolds stained with Masson’s Trichrome (A-C). After 1 week post-implantation, the magnification of region of interest in (A), see inset, show the lack of collagen structures inside the collagen scaffold (D, G). As fibroblast cells start to invade the scaffold, collagen deposits inside the cellulose scaffold can be sparsely observed after 4 weeks (E, H). Concomitant with the observation of activated fibroblast (spindle shaped cells) inside the cellulose scaffold, collagen network is clearly visible inside the cavities after 8 weeks (F, I). Scale bars: A-C = 1mm, D-F = 100μm and G-I = 20μm. * = collagen fibers; black arrows = cellulose cell wall; white arrow = fibroblast.
Fig 5.
Vascularization and Angiogenesis.
Macroscopic observations of blood vessels directly in the surrounding tissues around the cellulose scaffold (A). Confirmation of angiogenesis within the cellulose scaffold by the observation of multiple blood vessel cross sections in H&E staining (B) and Masson’s Trichrome staining (C) micrographs. The angiogenesis process was also confirmed with anti-CD31 staining to identify endothelial cells within the cellulose scaffold (D). Scale bars: A = 1mm, B = 50μm and C-D = 20μm. White arrows = blood vessels.