Acellular porcine placental membranes as a novel biomaterial for tissue repair applications

Abstract

Biological dressings derived from the extracellular matrix (ECM) of human placental tissues have proven effective in treating complex skin wounds and other anatomical sites, offering potential for new therapeutic applications. However, the use of human tissues is limited by ethical and biosafety concerns, restricting large-scale production. To address this, biomaterials from placentas of livestock animals offer a cost-effective, accessible alternative without harming animal welfare. Given pigs' large-scale production, short gestation periods, and abundant material availability, this study aimed to produce, characterize, and validate acellular biomembranes derived from decellularized porcine allantochorion for tissue repair. Placental fragments from Duroc sows were decellularized using a protocol involving immersion and orbital shaking in 0.1% SDS and 0.5% Triton X-100, followed by low-frequency ultrasonication. Accelularity was confirmed by total genomic DNA quantification and H&E and DAPI staining for nuclear visualization. Membrane structure and composition were analyzed using histological, immunohistochemical methods, and scanning electron microscopy. Spectroscopic analyses detected physicochemical changes in placental ECM, and biomechanical testing assessed membrane strength and stiffness. Biological functionality was validated through in vitro cell viability and adhesion assays with canine endothelial progenitor cells and L929 murine fibroblasts. In vivo biocompatibility was tested by subcutaneously implanting the biomaterial in rats for histopathological evaluation. Results showed efficient decellularization, with preserved ECM structure. The scaffolds were cytocompatible, supporting cell adhesion and high viability. In vivo testing revealed no immune rejection, confirming biocompatibility and biodegradability. In conclusion, acellular porcine placental biomembranes have the necessary characteristics to be explored as scaffolds for tissue engineering and novel repair therapies.

Keywords: biocompatibility; biomaterial; decellularization; placenta; porcine.

FIGURE 1

Experimental design of the study, highlighting the production and characterization of placental acellular scaffolds. Created with BioRender.com.

FIGURE 2

Production and decellularization efficiency evaluation of porcine decellularized placental biomembranes (n = 5 per group). Photodocumentation of a dissected porcine epitheliochorial placenta, highlighting the chorioallantoic membrane (CAM) (A). CAM macroscopic aspects after each step of the decellularization process, showing the samples gradual whitening (B). Total genomic DNA quantification of placental tissues before and after the decellularization (C). Hematoxylin and Eosin and DAPI staining showing the nuclei absence compared to the native tissue (D–K). **p < 0.05 compared to the native group.

FIGURE 3

Structural evaluation of extracellular matrix components of native and decellularized porcine placental membranes (n = 5 per group). Alcian Blue staining for total glycosaminoglycans (GAGs) content (A–D) and total GAGs semiquantitative analysis (E). Fuchsin-resorcin staining for elastic fibers content (F–I) and elastic fibers semiquantitative analysis (J). Masson’s trichrome staining for total collagen content (K–N) and total collagen content semiquantitative analysis (O).

FIGURE 4

Collagen fibers distribution pattern evaluation of native and decellularized porcine placental membranes (n = 5 per group). Non-polarized and polarized Picrosirius Red staining to differentiate thick collagen fibers stained in reddish and yellowish tones from thin collagen fibers stained in greenish tones in native (A-A ³ ) and decellularized (B-B ³ ) samples. Semiquantitative analysis of thick and thin collagen fibers distribution (C,D). Ultrastructural analysis by scanning electronic microscopy of native (E–L) and decellularized porcine placental membranes (M–T). villi (chorionic villi), bv (blood vessel), tc (trophoblastic cell).

FIGURE 5

Immunohistochemical analysis of key extracellular matrix proteins of native and decellularized porcine placental membrane samples (n = 5 per group). Immunostaining of elastin (A–D), fibronectin (F–I) and laminin (K–N). Semiquantitative comparative analysis of elastin (E), fibronectin (J) and laminin (O) content between native and decellularized placental samples.

FIGURE 6

FTIR-ATR average spectrum of native and decellularized porcine placental membranes (n = 5 per group) with the respective standard deviation (SD) highlighted in gray (A). Integrated band areas associated to triple helical structure of collagens (ratio between amide III and 1,450 cm^-1), elastin content, proteoglycan content and collagen (Amide I and II) of native and decellularized placental samples (B). Raman spectroscopy average spectrum of native and decellularized porcine placental membrane samples (n = 5 per group) with the respective standard deviation (SD) highlighted in gray (C). Score plot of principal component analysis (PCA) for native and decellularized samples (D). PC1 loading plot highlighting spectral differences between groups (E). Integrated band areas associated to the content of Amide I, Amide III, phenylalanine, proline/hydroxyproline, glycosaminoglycans, and elastin (F). ****p < 0.0001 compared to the native tissue.

FIGURE 7

Biomechanical performance evaluation of native and decellularized porcine placental membranes (n = 5 per group). Representative images of native (A) decellularized (B) test specimens used in the assay. Photodocumentation of the biomechanical assay, demonstrating the positioning of the native and decellularized test specimens in the machine, respectively (C,E) and the rupture moment used to measure the mechanical parameters (D,F). Maximum Pulling Force (G), Maximum Elongation (H) and Stiffness (I) of native and decellularized placental samples. Swelling assay performed with decellularized placental membranes, demonstrating the swelling ratio in each evaluated period (J).

FIGURE 8

In vitro cytocompatibility evaluation of porcine placental decellularized membranes (n = 5 per cell type). Scanning electronic microscopy photodocumentation of YS cells (A–F) and L929 fibroblast cells (H–M) on the placental scaffolds ECM fibers, demonstrating their anchoring and adhesion. Viability assay of YS and L929 cells cultured on placental scaffolds (dCAM) (G,N). Absorbance data were converted and expressed in viability percentage. YS vs. dCAM + YS and L929 vs. dCAM + L929.

FIGURE 9

Histopathological assessment of decellularized placental membranes implanted in the subcutaneous tissue of immunocompetent rats for biocompatibility evaluation (n = 5 per group). Hematoxylin and Eosin staining of the implanted region on day 7 (B), 14 (D) and 30 (F) and their respective Sham control groups (A,C,E). Bar graphs represent semiquantitative scores (mean ± SD) for inflammatory infiltrate (G), capsule thickness (H), and collagen deposition (I), comparing Scaffold and Sham groups at each timepoint. Epi (epidermis), SP (subcutaneous pocked), PC (panniculus carnosus), Adip (adipose tissue). Black arrows indicate the scaffolds borders with cell infiltration. **p < 0.05 compared to the Sham group.

FIGURE 10

Histological evaluation of collagen deposition and biomaterial integrity after subcutaneous implantation of decellularized placental membranes in immunocompetent rats (n = 5 per group). Masson’s trichrome staining of the implanted region on day 7 (C), 14 (G) and 30 (K) and their respective Sham control groups (A,E,I). Black arrows indicate the scaffolds position in the tissue. Picrosirius Red staining of the implanted region on day 7 (D), 14 (H) and 30 (L) and their respective Sham control groups (B,F,J). White arrows indicate the scaffolds position in the tissue.