Biocompatible adipose extracellular matrix and reduced graphene oxide nanocomposite for tissue engineering applications

Abstract

Despite the immense need for effective treatment of spinal cord injury (SCI), no successful repair strategy has yet been clinically implemented. Multifunctional biomaterials, based on porcine adipose tissue-derived extracellular matrix (adECM) and reduced graphene oxide (rGO), were recently shown to stimulate in vitro neural stem cell growth and differentiation. Nevertheless, their functional performance in clinically more relevant in vivo conditions remains largely unknown. Before clinical application of these adECM-rGO nanocomposites can be considered, a rigorous assessment of the cytotoxicity and biocompatibility of these biomaterials is required. For instance, xenogeneic adECM scaffolds could still harbour potential immunogenicity following decellularization. In addition, the toxicity of rGO has been studied before, yet often in experimental settings that do not bear relevance to regenerative medicine. Therefore, the present study aimed to assess both the in vitro as well as in vivo safety of adECM and adECM-rGO scaffolds. First, pulmonary, renal and hepato-cytotoxicity as well as macrophage polarization studies showed that scaffolds were benign invitro. Then, a laminectomy was performed at the 10th thoracic vertebra, and scaffolds were implanted directly contacting the spinal cord. For a total duration of 6 weeks, animal welfare was not negatively affected. Histological analysis demonstrated the degradation of adECM scaffolds and subsequent tissue remodeling. Graphene-based scaffolds showed a very limited fibrous encapsulation, while rGO sheets were engulfed by foreign body giant cells. Furthermore, all scaffolds were infiltrated by macrophages, which were largely polarized towards a pro-regenerative phenotype. Lastly, organ-specific histopathology and biochemical analysis of blood did not reveal any adverse effects. In summary, both adECM and adECM-rGO implants were biocompatible upon laminectomy while establishing a pro-regenerative microenvironment, which justifies further research on their therapeutic potential for treatment of SCI.

Keywords: Cytotoxicity; Extracellular matrix; Foreign body response; Graphene; Macrophages; Nanocomposite.

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Fig. 1

Synthesis and characterization of adECM and adECM-rGO scaffolds. A) Characterization of GO (left) and rGO (right) via X-ray photoelectron spectroscopy; B) Morphological characterization of the adECM and adECM-rGO solid foams, including reconstructed foams top view from μCT projections (scale bar: 2 mm) with representative graphs of the percentage pore volume depending on pore ranges based on μCT morphometric data; C) scanning electron microscopy of the foams cross section (scale bar: 200 μm); D) Compressive Young's modulus; E) PBS uptake at equilibrium.

Fig. 2

Cytotoxicity and immune response following direct contact with adECM and adECM-rGO scaffolds. A) Confocal microscopy images confirming cell morphology of lung A549, liver HepG2, and kidney HK2 cells from left to right, respectively. Nuclei are labeled in blue, while filamentous actin (F-actin) is stained green. Scalebar represents either 20 μm (left), or 5 μm (right), which applies to all cell lines. B) CCK-8 assay results following 1 and 7 days of culture in direct contact with adECM and adECM-rGO scaffolds for lung A549, liver HepG2, and kidney HK2 cells, from left to right, respectively. Positive control corresponds to culture media and cells, while the negative control corresponds to 10% DMSO. Following direct contact culture of RAW-264.7 macrophages with adECM and adECM-rGO scaffolds, intracellular content of ROS, measured by flow cytometry, and secretion of TNFα and IL-6, measured by ELISA, is presented in (C), (D), and (E), respectively. Confocal microscopy images depict the morphology of RAW-264.7 macrophages (F, G), cultured on the surface of adECM (left) and adECM-rGO (right) scaffolds. Nuclei are labeled in blue, CD80 (F) is labeled in red and CD206 (G) is labeled in green. Scalebar represents 20 μm. Flow cytometry results are presented of CD80 (H) and CD206 (I) expression by RAW-264.7 macrophages, cultured for 24h and 48h on adECM and adECM-rGO scaffolds. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Macroscopic and histological evaluation of the implantation site. Panel A shows pictures that were taken during surgery, before and after implantation for the control (left), adECM (middle), and adECM-rGO (right) treatment groups. Note that for each group, dotted line squares represent the site of laminectomy, in which the exposed spinal cord can be observed. Scalebar represents 0.5 cm. Axial tissue sections, stained with H&E, are presented for the control (B), adECM implantation (C), and adECM-rGO implantation groups (D). These transversal overview images depict the spinal cord (indicated by *), which is surrounded by the vertebra (**). Dorsal to the spinal cord, the site of laminectomy and implantation (***) can be distinguished. Inserts are included to present a more detailed view of cells infiltrating the injury site, represented by dotted line squares. Regarding the adECM-rGO group, yellow arrows show foreign body giant cells (FBGCs) that engulf rGO sheets. Scalebar represents either 1000 μm or 50 μm (inserts). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Immunohistochemical assessment of macrophage polarization. Shown are immunohistochemical stainings of the implantation sites of the control (A), adECM (B) and adECM-rGO (C) treatment groups. For each treatment condition, cells positive for M1-marker CD86 are stained blue, while cells positive for M2-marker CD163 are stained brown. Higher magnification images (inserts) are represented by dotted line squares. For clarification, blue and yellow arrows represent M1-and M2-positive macrophages, respectively. Regarding adECM-rGO, it is noteworthy that foreign body giant cells (FBGCs) seem to express CD86. Scalebar represents either 50 μm or 10 μm (inserts). Quantification of M2-positive macrophage density in cells permm² (n = 6 for each treatment group) is presented in panel D. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Goldner-modified Masson's Trichrome staining of implantation area. To visualize fibrosis and fibrous encapsulation, a Masson-Goldner staining was performed for all treatment groups, including the control (A, with higher magnification image in B), adECM (C, with higher magnification image in D), and adECM-rGO (E, with higher magnification image in F). Higher magnification images are represented by dotted line squares. Collagenous fibrous tissue is stained green, nuclei are stained red. Concerning the higher magnification images, yellow arrows specifically depict fibrosis (D) or fibrotic encapsulation (F). Scalebar represents 500 μm (A-C-E) or 50 μm (B-D-F). Panel G shows the quantification results of fibrosis (in μm) regarding the adECM and adECM-rGO treatment groups. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Biochemical plasma analysis and macroscopic assessment of relevant organs. For each timepoint, 1 (left) and 6 weeks (right) after implantation, plasma levels of total protein (TP; A), cholesterol (CHOL; B), triglycerides (TG; C), glucose (Glc; D), albumin (ALB; E), alkaline phosphatase (ALP; F), alanine aminotransferase (ALAT; G), aspartate aminotransferase (ASAT; H), gamma-glutamyltransferase (GGT; I), creatine kinase-myocardial band (CK-MB; J), creatinine (CREAT; K) and urea (UREA; L) are presented for the control, adECM and adECM-rGO groups. The spinal column, lungs, liver, kidneys, spleen and brain were harvested and pictures are presented in panel M (scalebar represents 1 cm). Organs weights at time of sacrifice are presented in panel N.

Fig. 7

Histological staining (H&E) of relevant organs. To check for potential organ inflammation, structural damage, or accumulation of material components, relevant organs from the control (left), adECM (middle) and adECM-rGO (right) treatment groups. Presented are H&E stainings of lung, liver, kidney, spleen, and brain tissue (from top to bottom). Scalebar represents 50 μm.