Efficacy and Cytocompatibility of a Pressure Garment—Silicone Composite Dressing Material for Scar Healing

3.1. Vitro Cell Viability by MTT Assay and Analysis All prepared samples (PGF-1000-6, PGF-2000-6, PGF-1000-50, and PGF-2000-50) were selected based on optimal mechanical properties determined by the Chamis model application [18]. The MTT method was employed to evaluate the impact of varying Biopor®AB printing layers on L929 cell viability. Adhering to the 10993-5 standard of cytotoxicity, a <30% reduction in cell viability induced by PGF-Biopor®AB would be categorised as non-cytotoxic. PDMS possesses superior flexibility, film-forming ability, biocompatibility, and resistance to chemical degradation, and the use of medical grade silicone Biopor®AB guarantees biocompatibility and is free from allergens for continuous skin application [19]. As illustrated in Figure 4A, with a recorded <30% reduction in cell viability for both 1 day and 3 days, all samples were confirmed non-cytotoxic, ensuring their safety for patient use in continuous 3-day applications. When examining cell viability (%) over time (cultured for 1 day and 3 days), all samples exhibited a trend of increasing cell viability (Figure 4A). In the case of thin-layer samples (PGF-1000-6 and PGF-2000-6), they initially displayed lower cell viability on day 1 but ultimately reached nearly 100% cell viability by the end of 3 days. This robust cell viability, comparable to commercial SGS (Cica-care©), suggests vigorous cellular activities for repair and regeneration, representing an optimal outcome for scar healing. In contrast, thick-layer samples (PGF-1000-50 and PGF-2000-50) also started with lower cell viability on day 1 but concluded with lower cell viability than commercial SGS (Cica-care©) by the end of 3 days, still achieving over 70% cell viability. To quantify and visually represent the rate of cell proliferation on different PGF-Biopor®AB samples and Cica-care©, microscopic images were captured at the end of day 3. Intriguingly, images depicting both high and low cell densities coexisted within the range of above 70% cell viability (Figure 4B). Among samples exhibiting >30% reduction in cell viability, both PGF-1000-6 and PGF-2000-50 demonstrated nearly identical extents of high cell density, similar to the Cica-care© control (designated as “high cell density”). Microscopic images revealed that cell spreading with an elongated shape indicated robust cellular alignment and migration, signifying the potential for cellular growth and proliferation prohibition in the context of repair and regeneration. The “high cell density” samples, with close to 100% cell viability and comparable cell density to the commercial SGS control (Cica-care©), suggest an effective outcome in scar healing performance. Conversely, PGF-2000-6 and PGF-1000-50 displayed a lower rate of cell density (designated as “low cell density” samples). Furthermore, cells in these samples appeared more circular in microscopic images, indicating lower cellular activities. The coexistence of both high and low cell density in samples with >30% reduction in cell viability indicates that the percentage of cell viability is not directly proportional to the extent of cell density. Given the absence of literature data for scar therapeutics’ performance reference concerning high and low cell density samples, utilizing both types of samples in patient trials becomes necessary. Scar healing is a complex physiological process involving cell viability performance in various aspects such as alignment, movement, migration, and proliferation [20]. Studies have shown that substrate characteristics, including materials, topography, and stiffness, can influence cell behaviour [21]. According to Wu et al.’s study on PDMS surface topography, micro- and nanoscale morphological features in pattern spacing and depth guide cell alignment and migration [21]. Curtis et al. have highlighted the impact of cell tension at topography and topography-induced strain on cellular movement, shape, orientation, and polarity of movement [22]. The guidance of endothelial cells in angiogenesis and regenerating tissues is influenced by both physical guidance (topographic and mechanical) and chemical guidance (adhesive) [22]. Per Ross Harison, topographic reactions are biocompatibility-related [23], with cells exhibiting contact guidance and topographic guidance in regions of extreme curvature [22]. Scar healing of PGF-Biopor®AB addresses inflammation, fibrosis, and angiogenesis. Cross-linking silicone Biopor®AB with hydrophobic SiO₂ provides sufficient chain mobility, stretchability, and low surface energy that improves the bonding between PGF and Biopor®AB [23]. Hydrophobicity and high adhesion of Biopor®AB surface reorganize patterns of screen gauge to build cellular-accessible Biopor®AB microchannels and modify the PGF-Biopor®AB surface structural features [24]. The anisotropic characteristics and Biopor®AB microchannels construct a favorable microenvironment for scar healing therapeutics (Figure 5a,b). Thus, the PGF-Biopor®AB structure promotes cell movement, adhesion, and alignment through microscale spaces, facilitating cell growth and proliferation exhibition, ultimately contributing to tissue repair and regeneration for scar therapeutics (Figure 5c). The development of surface anisotropic characteristics in PGF-Biopor®AB encompasses considerations of viscoelastic properties, roughness, and rigidity. These topological features play a crucial role in influencing cell-surface interactions, thereby impacting cellular physical guidance. Figure 6A depicts the construction of surface structure with screen-marked microchannels. Within this structure (Figure 6B), a concave > convex edge with approximately 200 µm was observed. In the PGF-inlaid “through-the-thickness” structure (Figure 6C), continuous filaments of fibre diameter 50 µm each were observed (Figure 6D). In the PGF-inlaid “through-the-thickness” structure, step and spirality of wrapping yarn built micro features of diameter 30 µm each (Figure 6E). According to literature data, The microscale fibre structure influences cell alignment, cell orientation and cell contact inhibition [22,23,25]. Fibres with a radius of 50 to 230 µm provide sites for cell alignment, cells can move on such concave > convex edges and align on the inside of a bend [22]. Cell orientation and contact inhibition occur on fibres with a radius of 15–20 µm, and cells spread and orient themselves at <50 µm radii of curvature [22]. According to Jiang et al., bending a PDMS slab can create surface waves that induce mechanical stress for contact guidance, and cells can elongate and spread parallel to the direction of surface waves of >8 µm [25]. Referencing such microstructural data, the observed PGF and Biopor®AB-coated micro-dimensions in PGF-Biopor®AB structure fit cellular activities of cell alignment, cell movement, and cell adhesion. Similar to the structural design of a 3D hybrid scaffold, the controllable fibre micro-dimension from PGF and the increased surface-to-volume ratio from screen-printed microchannels can ameliorate cell adhesion, proliferation, and differentiation [26]. Good adhesion from the pressure-screen-printed matrix–fibre interface guarantees good stress transfer from the soft matrix to the stiff fibre, a proper stress transfer of tissue-tension-off-loading mechanical performance for scar therapeutics [27,28]. In addition to the surface anisotropic characteristics, the inherent 100% oxygen permeability from Biopor®AB further enhances the availability of oxygen species, favouring cell adhesion [13]. When cells respond to mechanical anisotropy, the adhesive-viscoelastic surface of PGF-Biopor®AB is sufficiently isotropic to be deformable, enabling cells to exert significant traction, hindering their ability to move, and spread. This viscoelasticity is mechanically analogous to cells grown on spheres with sufficiently small radii of curvature, causing cells to be trapped by PGF-Biopor®AB isotropic curvature and resulting in approximately 30% inhibition of cell viability. This also clarifies why, despite small topographical differences between samples (refer to Table 1 and Figure 4), there is a notable discrepancy in cell density, leading to the classification of samples as high- or low-density. Under pressure-induced compression with warp-insertion shifting, Biopor®AB viscoelasticity allows the bending of such micro features, and the creation of surface waves as that of the PDMS slab induces mechanical stress for contact guidance. Essentially, the construction of micro features and PDMS surface anisotropic characteristics in PGF-Biopor®AB constructs a breathable dressing with surface hydrophobicity [9]. This unique surface hydrophobicity with breathability supports robust cellular activities and barrier properties, facilitating cell growth and proliferation exhibition, thereby fulfilling the optimization goals for multi-scar-healing efficacy. The major cause of hypertrophic scar formation is the excessive proliferation of fibroblasts that causes excess collagen deposition. Drawing insights from surface characteristic studies in scar healing literature, it is suggested that fibroblasts grow on the surface of anisotropic structured patches, promoting cell growth while inhibiting cellular proliferation without a negative effect on cell viability [2]. In the PGF-Biopor®AB scar-healing microenvironment, cell viability (%) serves as an indicator of cellular behaviors related to alignment, movement, adhesion, and proliferation. The anisotropic characteristics of PGF-Biopor®AB and microscale surface anisotropic features should encourage cell growth while same time inhibiting cellular proliferation. The inhibiting excess proliferation of fibroblasts indicates reduced collagen production and cell growth with cell proliferation reduction (without obvious toxicity) reflects potential values of scar inhibition. Therefore, >70% cell viability and the extent of cell density should correlate with corresponding scar healing performance in a patient study. Since cell density images from cell culture can only be qualitatively categorized as high or low and are not quantifiable for analysis, the subsequent in-depth study on surface characteristics and cell viability analysis will focus solely on cell viability (%). By examining the impact of viscoelastic properties, roughness, and rigidity on cell viability (%), the aim is to establish a reference guideline for PDMS surface characteristics in the context of scar therapeutics. 3.2. Trial Patient Study with Non-Healed Wound and Scarring Tissues In addressing non-healed wounds, many multifunctional biodegradable biomaterials have been developed and used in wound care to achieve the functions of inhibiting bacterial growth, promoting angiogenesis, and accelerating healing [2,29,30]. However, the use of such biodegradable biomaterials and silicone products in non-healed scarring wounds is still not suggested by plastic surgeons for problems of ulcers and abnormal hypertrophies. Stretching the polymer inverse opal patterns in an ellipsoidal porous patch signified the function and possibility of anisotropic cell-inducing ability for scar inhibition [30]. To achieve earlier tissue regeneration and overcome the formation of scars during wound healing, PGF-Biopor®AB equips a microchannel structure with high WVTR and the operation of low-pressure incorporation with viscoelasticity helps in avoiding the formation of hypertrophic scars. In the non-healed wound study, a practical trial involving a patient with a degree-2 non-healed scarring wound was conducted over 7 days (Figure 7a,b). Cutting PGF-Biopor®AB and Omnifix© in a circular shape ensured longitudinal, lowest, and consistent pressure application (<10 mmHg) to avoid pressure-induced skin ulcer and hypertrophy. After pressure release by viscoelasticity of Biopor®AB, the scar became flattened with no scabs on day 3. Figure 7bi–biv shows the progress of hypertrophy reduction, with no hypertrophy after 3 days of PGF-Biopor®AB application. 10 days post-injured wounds are in the healing stage of proliferation (Figure 8), a condition of high demands of WVTR from 1900–2109 g/m²/Day [31]. As insufficient WVTR would result in recurring inflammation and delayed healing, open wound healing was administrated with silvex® and aloe upon no hypertrophy. The open wound with aloe ensured sufficient WVTR and the use of silvex® ensured no recurrence of inflammation, both addressing proper/scarless wound healing. PGF-Biopor®AB application got rid of the hypertrophy in 3 days; sufficient WVTR at the healing wound for angiogenesis and silvex® and aloe prevented the recurrence of inflammation. No non-healed scarring wounds, no scabs, and no progressing hypertrophy were found within 7 days, which were evident in an earlier-scarless healed wound (Figure 7bv). No surface hypertrophy and no scabs within 7 days indicate that PGF-Biopor®AB significantly inhibited the formation of scars and helped reduce the wound-healing time. Larger Biopor®AB surface area (from fillers in Biopor®AB) and active delivery characteristics of 3D channel structures are conducive to increasing adhesion sites for cell growth and infiltration, favouring the early healing of non-healed scarring wounds [32]. In accelerated wound repair, the aligned microstructures should provide direct guidance for cellular growth orientation and extension [26]. Further, proper longitudinal pressure incorporation with sufficient ventilation performs the functions of inhibiting excess fibroblast proliferation and reducing collagen production. As a result, enhanced migration and proliferation of surrounding fibroblasts are conducive to early wound closure and a healed wound without scar in the early stage of wound healing. However, due to complications of non-healed scarring wounds, the use of PGF-Biopor®AB on large areas of non-healed scarring wounds should be under the supervision of plastic surgeons. A practical trial involving a patient with degree-2 and degree-3 scarring tissues was conducted over one month, utilizing Vancouver Scar Scale parameters (Figure 9). Significant improvements in surface irregularity were noted, particularly along the scar edges, indicating a reduction in irregularities. Scar thickness investigation revealed a decrease in hypertrophy, with a notable thinning of the superficial scar. In terms of pliability, the scar structure exhibited increased smoothness and enhanced elasticity, transitioning from thicker to finer elastic fibres. Pigmentation also showed a slight reduction in colour. Compared to literature data, typical efficacy for SGS involves wearing it over the scar for 12–24 h per day for at least 2–3 months, while PGT typically shows efficacy over 6 months [34,35]. Thus, the over 70% cell viability echoing one-month efficacy in the PGF-Biopor®AB application confirmed optimization of efficacy through PGF-SGS dual therapy, mechanotherapy, and active-fluid transport. The improvement in surface irregularities, scar thickness reduction, and enhanced overall scar elasticity in shorter treatment time indicate a synergistic effect of multi-therapeutic efficacy. The sustained dynamic hydration performance observed in WVTR, and water uptake capability in previous research further support active-fluid transport, supporting overall scar improvement [13]. In the suggested assisted transdermal delivery system (ATDDS) for PGF-Biopor®AB application, the drug layer containing Aveeno® functions as an effective occlusive-diffusion-penetration source for water hydration. The continuous pressure-driven self-pumping activates warp insertion shifting, facilitating dynamic and sustainable diffusional transport of Aveeno® with water (fluid) in the cellular accessible 3-D channel structure. At a cellular level, the asymmetric “spiral-through-the-thickness” structure with screen-mark microchannels aids effective directional transport, and current >70% cell viability evidenced their accessibility to positive cellular activities [13]. For studying the influence of relative surface roughness (MIU, MMD and SMD) and the influence of stiffness on cell viability, the related parameters were analysed (Table 1). Both the MIU_warp and MIU_weft enhancements supported cell viability (%) increase and a range of MIU from 0.6-1 favoured >70% cell viability. A range of MMD above 0.01 and a range of SMD_warp from 0.2 to 1.6 and SMD_weft from 0.2 to 6 favoured >70% cell viability. Referencing roughness configurations of smaller cells in nanoscale 10–102 nm (take vein endothelial cells as an example), the above PGF-Biopor®AB _SMD is equivalent to around 176–4000 times larger surfaces. Here, the high PGF-Biopor®AB _SMD means the availability of more cell adsorption sites and the provision of sufficient space for cellular activities [36]. Here, high PGF-Biopor®AB _MIU/MMD/SMD is equivalent to the provision of large adsorption sites for bioactive molecules, and the geometrically rougher surfaces enable a higher ability of cell adhesion, growth, and differentiation [36]. The rougher surface with high MIU, MMD and SMD provides a larger solid−liquid interface area, the results of over 70% cell viability indicated high cell growth and differentiation potential in a favourable scar-healing microenvironment [37]. Over 70% cell viability in all selected samples supported not only the scar-healing efficacy of overall scar improvement but also cellularly assessed the functionality of fluid transport microchannels in the 3D channel structure. Referring to the seven elements of the Arzt heptahedron for skin hydration, the mechanical behaviour of both a-keratin and b-keratin of the skin relies heavily on the degree of hydration [35,37,38,39]. Increasing hydration reduces scar tissue stiffness and modulus as the keratin matrix absorbs moisture [36,37,38,39]. Thus, adequate hydration strives to restore collagen tensile strength and elongation in scar tissue for earlier scar maturation [38]. The surface anisotropic characteristics and microscale scar-healing microenvironment all favour cell alignment, adhesion, movement, and proliferation. Here, active-fluid transport strived for adequate hydration, and dual PGF-SGS therapy and mechanotherapy enabled the reorganization of collagen and elastic fibre networks, fulfilling the scar-healing role of scar structure recovery. Concluding from both cell viability and trial patient study results, using a sample of over 70% cell viability, with either low cell density (PGF-1000-50) or high cell density (PGF-1000-6) achieved the same one-month efficacy. This one-month efficacy of both PGF-1000-6 and PGF-1000-50 implies that >70% cell viability can be a sufficient scar healing therapeutic guideline for PDMS composite. Over 70% cell viability is sufficient to achieve scar therapeutics, regardless of low or high cell density. The one-month efficacy results align with the findings of the cell viability study, supporting the above active silicone occlusive healing theory with multi-therapeutics from compression-silicone dual therapy, mechanotherapy, and active-fluid transport. 3.3. Possible PGF-Biopor®AB Scar Healing Mechanism Figure 10 illustrates the Ichikawa diagram elucidating scar-healing functions and associated mechanisms. Optimizing scar healing efficacy involves three key functions: (ⅰ) PGF-SGS dual therapy, (ⅱ) Mechanotherapy incorporating tension-shielding and pressure redistribution, and (ⅲ) Active moisture management. In PGF-SGS dual therapy, SGS therapy is conducted through Biopor®AB-skin contact with Biopor®AB exposure at the outer surface of PGF-Biopor®AB. Medical silicone Biopor®AB performs SGS therapy with inherent characteristics of oxygen permeability and occlusive hydration [13]. The pressure-driven “warp insertions” mobility design accomplishes compression therapy via “warp insertions” shifting to achieve PGF-SGS dual therapy. Collagen fibrils in scarring tissue are arranged in non-parallel arrays and not in alignment with the lines of tension but are sensitive to the local stress profile. The organization of collagen fibrils is also highly reconfigurable, collagen tissues show a nonlinear stress response (stress-stiffening): collagenous tissues stiffen as they are strained (by an external force) [40]. Biopor®AB is composed of at least two polymer networks in PDMS and PVMS. When PDMS/PVMS combined with PGF-textile reinforcement from screen printing, elastic gradient tunability for tensile and shear (from PDMS/PVMS) exhibited high strength and diverse shape transformation ability (Figure 11). Elastic gradient tunability allowed a change from isotropic to anisotropic within the Biopor®AB screen-mark microchannels. PGF-Biopor®AB would become softened when stretched to allow pressure and tension reduction, which fits well with the stress-stiffening properties of collagen, causing a reduction in collagen deposition for less scarring. The Biopor®AB silicone characteristics of oxygen permeability and occlusive hydration also allow water vapor penetration, oxygen penetration, and stratum corneum hydration, demonstrating scar repair function. The oxygen permeability in Biopor®AB provides an oxygen environment that supports reactive oxygen species for the sustained release of pro-inflammatory cytokines. The coexistence of hydrophobicity and permeability, the building of micro hierarchy structure for liquid collection and directional movement, on the other hand, resists bacteria invasion and reduces infection, stopping continuous infections and excess inflammation [9,13,41,42,43]. The Biopor®AB-nylon characteristics in the “warp insertions” mobility design offered higher water-holding capacity, enhanced transdermal water loss and normalized the barrier function. This result of >70% cell viability demonstrates excellent biocompatibility, high cell viability, and proliferation capacity, the one-month scar-healing efficacy and an early scarless healed wound signified the appropriateness in the magnitude of occlusive hydration. The results of cell viability and patient outcomes reflected stimulation of cell growth and proliferation inhibition—elastic gradient tunability reduced collagen deposition while occlusive hydration reduced hypertrophy by keratinocyte hydration, both restoring the homeostasis of the barrier function and correcting the defective permeability. Optimizing scar healing efficacy involves three primary mechanisms (Figure 10): (ⅰ) Integrating and synchronizing PGF and SGS therapies to expedite the scar-healing process, (ⅱ) Reducing tension through viscoelasticity and redistributing pressure via tension-shielding, and (ⅲ) Enhancing permeability through active water uptake for effective moisture management. Addressing the water non-permeability of SGS and mitigating pressure points from PGF, PGF-Biopor®AB harmonizes the benefits of both therapies and eliminates such problems, realizing compression-silicone dual therapy in the first place. Mechanotherapy involves leveraging viscoelastic properties at the structural level, and pressure-driven PGF-Biopor®AB enhances the surface area of Biopor®AB and facilitates warp insertions shifting for tension-shielding and pressure-redistribution [43]. Subsequently, applying viscoelasticity and surface roughness contributes to mechanotherapy, aiding in pressure and tension reduction. Notably, the surface roughness created by the Biopor®AB screen-mark microchannels promotes tension shielding and pressure redistribution. The “warp insertions” shifting, propelled by pressure, generates internal pressure redistribution, collectively supporting mechanotherapy. For active moisture management, the screen-mark microchannels form cellular-accessible pathways for transport, and the “warp insertions” mobility design serves as pressure-driven “pressure operators” for efficient nylon absorbency, enabling water absorption with retention for effective moisture control. Validation through human skin WVTR and 13% water uptake confirmed that the PGF-Biopor®AB microchannel structure was an effective permeability barrier, normalising the barrier function [13]. The microchannels, shaped by screen-mark structures, facilitated the diffusion of water and nutrients from applied creams and oils onto scarred tissues, ensuring both ample moisture for the function of transdermal drug delivery and the construction of an occlusive microenvironment conducive to skin repair and regeneration. These three mechanisms collectively support the core functions of scar healing, resulting in one-month scar-healing efficacy and 7-day scarless-healed wound. The microchannels serve as effective pathways for micro and nanocarriers, contributing to inflammation inhibition and enhanced angiogenesis against fibrosis in the various stages of scar healing. The ability of water-retaining in microchannel structure provides a moist microenvironment crucial for maintaining a balance between collagen progress and lysis, thereby promoting rapid scar inhibition. The PGF-Biopor®AB system excels in water absorption with retention, ensuring effective moisture control.