Structural characterization of seaweed cellulose scaffolds
In our previous study, we developed cellulose scaffolds from green macroalgae species Ulva sp. and Cladophora sp. as potential connective support scaffolds alternatives. Decellularization effectively removed cellular content while preserving structural integrity, as confirmed by SEM, DNA analysis, histology imaging (Fig. 1A-G), and fluorescence imaging, verified cellulose as the primary component. Scaffolds structural analysis revealed distinct morphologies (Fig. 1B,E): Ulva sp. displayed a porous structure with 20.2 ± 4 µm pore size, while Cladophora sp. exhibited a fibrous structure with fibers ranging from 5 µm to over 80 µm, overlaid with microfibrils measuring 55-400 nm (Fig. 1H).
Cell growth analysis demonstrated high viability for up to 40 days in culture, highlighting the distinct impact of each scaffold's structure on cell behavior and proliferation rates. The porous Ulva sp. scaffold supported rapid, multidirectional fibroblast proliferation, reaching saturation by week 3, whereas Cladophora sp. fibrous scaffold promoted elongated cell growth along fiber axes with consistent, linear proliferation over time (Fig. 1I). In vitro Biocompatibility assessments using alamarBlue assays confirmed scaffold non-toxicity, demonstrating their suitability for long-term cell culture.
These findings emphasize the direct correlation between scaffold structural properties and cell behavior, survival, and proliferation, underscoring the potential of macroalgae-derived cellulose scaffolds for biomedical applications such as tissue engineering and wound healing.
Following decellularization, sterilized seaweed cellulose implants (Ø = 8 mm) from porous Ulva sp. (Fig. 2A) and filamentous Cladophora sp. (Fig. 2B) were evaluated for in vivo biocompatibility as alternative implants in a wound-healing model using male Sprague-Dawley rats (250 g, n = 24, 8 weeks old). Animals were divided into test groups (with implants) and controls (without implants) over an 8-week period. Subcutaneous implantations of Ulva sp. and Cladophora sp. SC scaffolds were performed in two upper dorsal incisions (1 cm each) (Fig. 2D,E), with biocompatibility assessed at weeks 1, 4, and 8 post implantation, for tissue with and without the subcutaneous implants (Fig. 2F). Dissected tissue sites (1-2 cm) (Fig. 2H,I) were collected at each time point for histopathological analysis, revealing minimal inflammation, structural stability, and intact shapes with minimal alteration throughout the study.
Both Ulva sp. and Cladophora sp. implants retained consistent dimensions and their original shape from the initial implantation (diameter: Ø = 8 mm; thickness: 2 ± 0.06 mm and 1.7 ± 0.05 mm, respectively) through week 8 of the experiment (Fig. 7D,E). Throughout the experiment, including surgical procedures and handling, the animals exhibited no signs of discomfort or behavioral changes, maintained consistent weight gain, and tolerated the implants well. Although three animals died early from causes unrelated to the test items, as confirmed by the study pathologist, their exclusion, along with the removal of one additional animal, left 20 rats for statistical analysis, establishing the 8-week experiment timeline.
Excision sites with and without SC implants were processed for histopathological evaluations. Assessments were based on adversity criteria, as well as semiquantitative scoring conducted by a board-certified pathologist, for Ulva sp. and Cladophora sp. scaffolds. Global view imaging (Figs. 3, 4, top row) and representative view imaging of the implants' center (Figs. 3, 4, bottom row), highlight their performance as biomaterial implants. Evaluations include inflammation reactions (Hematoxylin & Eosin staining) Fig. 3, collagen depositions (Masson's Trichome staining), Fig. 4, and vascularization developments (anti-CD31 antibody staining) Fig. 5, concluded with cell and tissue semiquantitative scoring Fig. 6.
Additionally, morphological characteristics and the distinct impact of the SC scaffolds structural characteristic on the healing process were studied Fig. 7. Significant differences were observed in the healing processes for both Ulva sp. and Cladophora sp. SC implants. The histological responses of the two SC implants are shown in Figs. 3, 4, 5 and 6, indicating an ongoing healing process during this study. Notably, some SC implant samples exhibited a bent shape due to handling during the formalin fixation, causing deformation before histological staining.
Hematoxylin and eosin (H&E) staining (Fig. 3) revealed distinct patterns of inflammation and cell infiltration at the implantation sites of porous Ulva sp. and fibrous Cladophora sp. scaffolds, compared to control tissues without implants. Both scaffolds elicited inflammatory reactions at week 1, which diminished or resolved entirely by week 8, indicative of an active healing process. However, their responses differed significantly. Ulva sp. implant (Fig. 3A) showed a milder initial inflammatory response at week 1, characterized by polymorphonuclear-hemorrhage interspersed throughout the expanded necrotic implant site. The reduction of polymorphonuclear cells (PMNNs), notably neutrophils, was gradual over the study period, with smaller necrotic cavities persisting among the implant fragments until week 8, suggesting a slower inflammation resolution. In contrast, Cladophora sp. implant (Fig. 3B) exhibited a more pronounced initial inflammatory reaction at week 1, characterized by PMNN clusters at the implant' expanded necrotic center. However, by week 4, the necrotic center diminished, PMNNs had markedly reduced, and none were observed by week 8, indicating faster inflammation resolution and a more accelerated healing process. Control tissues without implants (Fig. 3C) displayed minimal inflammatory reactions at week 1, followed by complete healing in subsequent weeks, highlighting the differences in healing dynamics between the two implants and compared to tissues without implants.
Additionally, foreign body reactions (FBR), notably macrophages (MCs) and foreign body giant cells (FBGCs), were observed at both implant sites at week 1, albeit with distinct differences. The Ulva sp. implant exhibited a gradual increase in FBGC presence by week 4, predominantly scattered throughout necrotic cavities surrounding implant fragments, followed by steady reduction over the 8-week study period. Conversely, the Cladophora sp. implant elicited an earlier resolution of FBR, with transformation of MCs into FBGCs predominantly at the implant periphery. By week 4, FBR at the implant' center significantly diminished, leaving moderate FBGCs activity persisting at the periphery, which further decreased by week 8. This suggests faster integration and recovery process in the Cladophora sp. implant compared with the more prolonged and scattered FBR response in the Ulva sp. implant. Control tissues without implants showed minimal FBR at week 1 and 4, and none by week 8, confirming an earlier and complete healing response in the absence of implants. These observations align with Masson's Trichrome staining (Fig. 4), which demonstrated the gradual replacement of inflammation and FBR with connective tissue in both scaffolds.
Masson's Trichrome staining (Fig. 4) revealed collagen deposition and fibrosis within and around the implant sites, supporting new tissue formation. Both Ulva sp. and Cladophora sp. scaffolds exhibited fibrotic reactions starting at week 4, gradually replacing inflammation and FBR, with scaffold-specific fibrosis-fibroplasia patterns. Ulva sp. implant (Fig. 4A) demonstrated non-homogeneous fibrosis scattered across the implant site, steadily replacing necrotic-FBR areas with mature connective tissue septa among implant fragments. Collagen infiltration and replacement was slower but gradual and more pronounced. However, some necrotic-FBR collections persisted through week 8, indicating ongoing healing. In contrast, Cladophora sp. implant (Fig. 4B) elicited denser and more homogeneous fibrosis, replacing the necrotic center by week 4 and progressively expanding towards the periphery by week 8, with collagen deposition engulfing individual fibers, suggesting more advanced tissue integration. Fibroplasia capsules were observed surrounding both SC implants throughout the 8-week study, reflecting FBR responses and healing progression. Control tissues without implants (Fig. 4C) displayed early fibrosis by week 1 and fully mature connective tissue regeneration by weeks 4 and 8, highlighting faster and more complete healing in the absence of implants. These observations underscore the structural influence of the two SC scaffolds on fibrosis development and healing dynamics.
Anti-CD31/PECAM-1 immunohistochemistry (Fig. 5) revealed endothelial cells (stained brown) within Ulva sp. and Cladophora sp. implant sites at week 8, confirming vascularization and angiogenesis. This was supported by Masson's Trichrome staining imaging (Fig. 4), which showed neovascularization alongside fibroplasia and fibrosis, progressively replacing inflammation and FBR through the study. At week 1, early-stage neovascularization was observed primarily at the periphery of both implant sites. By week 4, blood vessels began forming in the fibrosis implants' center, with distinctdifferences between scaffolds. Ulva sp. implants exhibited displayed a continuous decrease in vascularization at the periphery while extending into the connective tissue septa among necrotic-FBR collections, reflecting slower and ongoing healing. In contrast, Cladophora sp. implants showed neovascularization at both the FBR periphery and fibrosis center by week 4, with vascularization shifting predominantly to the fibrosis center by week 8, forming between the implant' fibers, indicating advanced healing and tissue integration. Control tissues without implants demonstrated early vascularization throughout the scar tissue at week 1, which decreased by week 4 as connective tissue matured. By week 8, vascularization was absent, indicating correlation with the inflammation decrees and the complete healing process in the absence of implants. Overall, Cladophora sp. implants demonstrated significantly greater neovascularization than Ulva sp. at week 8, highlighting their superior capacity to support angiogenesis and integration during healing.
The histopathological semi-quantitative scoring was conducted according to ISO 10993-6:2016 standards, following the reference criteria outlined in Tables E.1 - E.3 of the guideline, to assess inflammation, foreign body reaction (FBR), fibroplasia/fibrosis, and neovascularization at Ulva sp. and Cladophora sp. implantation sites (Table 1, Fig. 6). Tissue cross-sections were evaluated for cell types and tissue responses, using a five-point grading scale (0-4) based on histological changes observed via Hematoxylin & Eosin (H&E), Masson's Trichrome, and anti-CD31 staining over the 8-week study period. Scoring was performed by a board-certified pathologist (Table 1), with representative plots illustrating trends for MCs (yellow) and FBGCs (orange) (Fig. 6B), PMNNs (purple) and necrosis (green) (Fig. 6C), and collagen deposition (blue) and blood cells/vessels (gray) (Fig. 6D). The Ulva sp. implant was represented by a dashed line, Cladophora sp. by a solid line, and control tissues without implants by a dotted line.
Both scaffolds triggered inflammatory responses at week 1, composed primarily of polymorphonuclear neutrophils (PMNN). The Ulva sp. implants exhibited a mild (2) inflammatory response that steadily declined to minimal (1) by week 4 and resolved entirely (0) by week 8. In contrast, Cladophora sp. scaffolds elicited a more pronounced moderate (3) inflammation at week 1, with a steeper resolution, reducing to none (0) by week 4. Control tissues displayed minimal (1) inflammation at week 1, resolving completely by week 4.
Ulva sp. scaffolds exhibited upward trend of MCs and FBGC's by week 4, followed by a downward trend by week 8. FBR-associated MCs increased from minimal (1) FBR at week 1 to mild (2) FBR by week 4, then decreased to minimal (1) FBR by week 8. This transition coincided with FBGC formation, with minimal (1) FBR/FBGCs present at weeks 4 and 8. Whereas Cladophora sp. scaffolds showed a decreasing trend in MCs and an increasing trend in FBGCs. FBR was moderate (3) at week 1, with MCs concentrated at the implant core, decreasing to mild (2) FBR/MCs at weeks 4 and 8, confined to the periphery. FBGC formation progressed outward, increasing from minimal (1) FBR/FBGCs at week 1 to mild (2) at week 4, and reaching moderate (3) FBR/FBGCs by week 8, only at the periphery. Control tissues demonstrated minimal (1) FBR at week 1 and 4, with no FBR (0) by week 8.
Collagen deposition and connective tissue formation varied between scaffolds. The Ulva sp. scaffolds showed a gradual increase in fibrosis, starting as mild (2) at weeks 1 and 4, progressing to moderate (3) by week 8. Fibrosis was scattered among implant fragments, indicating steady but slower tissue remodeling. Cladophora sp. scaffolds exhibited earlier and more homogenous fibrosis, scoring mild (2) at week 1 and moderate (3) by week 4, with complete replacement of necrotic centers and denser fibrosis by week 8. Control tissues exhibited advanced fibrosis early, scoring moderate (3) at week 1 and severe (4) by weeks 4 and 8.
Neovascularization followed different trends for each scaffold. The Ulva sp. implants demonstrated an initial increase in vascularization at weeks 1 and 4 (mild, 2), followed by a decline to minimal (1) by week 8, reflecting slower angiogenesis. In contrast, Cladophora sp. scaffolds maintained steady vascularization, scoring mild (2) throughout the study, with enhanced vessel formation in fibrotic regions. Control tissues exhibited declining vascularization, scoring mild (2) at week 1 and minimal (1) by week 4, with no vascularization (0) by week 8.
FBR associated with the presence of foreign debris remnants, including hair shafts and cotton fibers from surgical residues, was observed at both SC implant sites and surrounding tissues. as well as at the animal tissue without implant. Ulva sp. scaffolds showed mild (2) response at weeks 1 and 4, decreased to minimal (1) by week 8. Whereas Cladophora sp. scaffolds exhibited moderate (3) foreign debris-associated FBR at week 1, decreasing to mild (2) and week 4 following an increase at week 8. Control tissue without implant showed minor (1) FBR linked to surgical debris, which resolved by week 8. Local scant FBR was assessed based on MCs and FBGCs presence surrounding residual material, which did not undergo degradation, scattered at the implant sites.
Overall, both scaffolds demonstrated progressive healing, with inflammation and FBR transitioning to fibrosis and vascularization by week 8. Cladophora sp. scaffolds exhibited faster integration and more advanced tissue remodeling, while Ulva sp. scaffolds supported gradual and steady healing. Control tissues without implants showed earlier and complete healing by week 8, with minimal tissue reactions. Both scaffolds were biocompatible, non-toxic, and effective in promoting tissue regeneration, highlighting their potential as connective support scaffolds alternatives.