Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials

ABSTRACTThere is intense interest in developing novel biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Decellularization of existing tissues have formed the basis of one major approach to producing 3D scaffolds for such purposes. In this study, we utilize the native hypanthium tissue of apples and a simple preparation methodology to create implantable cellulose scaffolds. To examine biocompatibility, scaffolds were subcutaneously implanted in wild-type, immunocompetent mice (males and females; 6-9 weeks old). Following the implantation, the scaffolds were resected at 1, 4 and 8 weeks and processed for histological analysis (H&amp;E, Masson’s Trichrome, anti-CD31 and anti-CD45 antibodies). Histological analysis revealed a characteristic foreign body response to the scaffold 1 week post-implantation. However, the immune response was observed to gradually disappear by 8 weeks post-implantation. By 8 weeks, there was no immune response in the surrounding dermis tissue and active fibroblast migration within the cellulose scaffold was observed. This was concomitant with the deposition of a new collagen extracellular matrix. Furthermore, active blood vessel formation within the scaffold was observed throughout the period of study indicating the pro-angiogenic properties of the native scaffolds. Finally, while the scaffolds retain much of their original shape they do undergo a slow deformation over the 8-week length of the study. Taken together, our results demonstrate that native cellulose scaffolds are biocompatible and exhibit promising potential as a surgical biomaterial.</jats:p

Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials.

There is intense interest in developing novel biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Decellularization of existing tissues have formed the basis of one major approach to producing 3D scaffolds for such purposes. In this study, we utilize the native hypanthium tissue of apples and a simple preparation methodology to create implantable cellulose scaffolds. To examine biocompatibility, scaffolds were subcutaneously implanted in wild-type, immunocompetent mice (males and females; 6-9 weeks old). Following the implantation, the scaffolds were resected at 1, 4 and 8 weeks and processed for histological analysis (H&E, Masson's Trichrome, anti-CD31 and anti-CD45 antibodies). Histological analysis revealed a characteristic foreign body response to the scaffold 1 week post-implantation. However, the immune response was observed to gradually disappear by 8 weeks post-implantation. By 8 weeks, there was no immune response in the surrounding dermis tissue and active fibroblast migration within the cellulose scaffold was observed. This was concomitant with the deposition of a new collagen extracellular matrix. Furthermore, active blood vessel formation within the scaffold was observed throughout the period of study indicating the pro-angiogenic properties of the native scaffolds. Finally, while the scaffolds retain much of their original shape they do undergo a slow deformation over the 8-week length of the study. Taken together, our results demonstrate that native cellulose scaffolds are biocompatible and exhibit promising potential as a surgical biomaterial

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).</p

Cellulose scaffold preparation.

<p>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 (<b>C</b>). 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.</p

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.</p

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.</p

Vascularization and Angiogenesis.

<p>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.</p

Extracellular matrix deposition.

<p>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.</p

The physical interaction of myoblasts with the microenvironment during remodeling of the cytoarchitecture.

Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ~13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality