Bioactive Membranes for Stem Cell Regulation and Tissue Engineering

Abstract

To address the challenges of skin damage and chronic wound healing, researchers have combined cell biology, materials science, engineering technology, and skin tissue engineering to develop functional skin substitutes that promote regeneration and repair. Bioactive materials play a critical role in this process, providing structural support and regulating cellular behavior and accelerating tissue regeneration through structural changes, modifications, and the release of bioactive factors. This paper aims to design and prepare bioactive materials with microstructures to regulate stem cells through these microstructures and material properties, ultimately achieving tissue repair. Using photolithography technology, we designed and fabricated several PDMS films with curvatures and microstructures of different dimensions, exploring the conditions that promote osteogenic differentiation of pre-osteoblasts. Additionally, to study the effects of nanostructures much smaller than cell sizes on stem cells, we used electrospinning technology to prepare PVDF nanofibrous membranes with different orientations. By adjusting the annealing temperature, we obtained PVDF nanofibrous membranes with optimal piezoelectric properties. Furthermore, by treating with oxygen plasma, we crosslinked the electroactive PVDF membrane with environmentally friendly P34HB membrane to prepare a bilayer wound dressing like skin. To maximize the efficacy of the wound dressing, we enhanced the piezoelectric properties of the PVDF membrane by adding zinc oxide nanoparticles and combined it with a hydrophilic layer loaded with drug to form an asymmetric wound dressing structure. The detailed content is as follows.

In chapter 1, we begin by reviewing the importance of tissue engineering and the significance of bioactive membranes in this field. The main types of wounds, including infected wounds, diabetic wounds, and burn wounds, are detailed, along with their pathological causes and treatment characteristics. Following this, we categorize bioactive materials into two major types based on their raw material properties: natural bioactive materials and synthetic bioactive materials. For natural bioactive materials, we focus on collagen-based, silk fibroin-based, and cellulose-based bioactive materials, detailing their preparation and material properties. For synthetic bioactive materials, we review polyurethane-based, polyhydroxyalkanoates-based, and PVDF-based bioactive materials, including their preparation and material properties. We then summarize the factors to consider when designing bioactive materials (such as morphology, porosity, stem cell regulation functions, etc.) and the main preparation methods (microfabrication, 3D/4D printing, and electrospinning). Finally, we summarize recent research on the application of bioactive materials in tissue engineering and propose potential future directions. In recent years, bioactive materials such as electroactive materials, nanomaterials, and natural polymers have demonstrated superior performance, effectively promoting cell proliferation, stem cell differentiation, angiogenesis, and immune regulation. These advanced technologies and materials offer new design concepts and therapeutic approaches for skin regenerative medicine, with the potential to significantly improve healing outcomes and the quality of life for patients.

In designing bioactive materials, we first considered the impact of structure on cells. The biological and morphological responses of both single cells and multicellular assemblies are typically regulated by external physical stimuli, such as topographical features and dimensions. Microstructures of different sizes exhibit various regulatory effects on cell proliferation, migration, and differentiation. However, the influence of cell-scale curvature on cell differentiation and its underlying mechanisms remains unclear. In chapter 2, we fabricated micron-scale microgrooves and micropores with different curvature radii and systematically analyzed the behavior and function of cells on these structures. Interestingly, we found that microstructures with a cell-scale curvature radius (50 μm) provided the greatest force on cells, thereby improving cell spreading and promoting the osteogenic differentiation of pre-osteoblasts. Additionally, the osteogenic effect of MC3T3-E1 cells was superior on microgrooves compared to other microstructures, which may be attributed to the two-dimensional forces generated by narrow continuous curvatures. This study outlines cell differentiation induced by cell-scale microstructures, providing a foundational basis for designing tissue engineering scaffolds for stem cell differentiation.

After exploring the effects of micron-scale structures on cells, it is also necessary to test the impact of nanoscale structures on stem cells. In addition, smart electroactive materials that can dynamically regulate stem cell fate without external stimuli have garnered increasing attention. We identified an electroactive biomaterial, PVDF, which can promote stem cell differentiation through the piezoelectric effect. However, the complex interactions between cells and materials in a complex microenvironment involve substrate reactions, ion exchange, and changes in membrane potential. The mechanisms by which smart materials affect stem cells are not yet fully understood. In chapter 3, we used electrospinning technology to fabricate fibrous membranes composed of disordered and highly oriented polyvinylidene fluoride (PVDF) nanofibers to compare their effects on stem cells. After heat treatment, the oriented annealed PVDF (AA) and random annealed PVDF (RA) membranes exhibited a high fraction of the β phase. By comparing the osteogenic capacity, calcium activity, and F-actin distribution of bone marrow mesenchymal stem cells (BMSCs) cultured with these PVDF nanofibers, we proposed that stem cells autonomously regulate their differentiation by remodeling the cytoskeleton on the electrospun membranes. The greater adhesion area, active calcium influx, and electrical stimulation supported the greater osteogenic effect of BMSCs on RA compared to AA. This mechanism provides a theoretical basis for the design and preparation of stem cells regulation tissue engineering scaffolds and contributes to further research on cell and microenvironment interactions.

After confirming the significant promotion of stem cells by nanofibrous membranes, we began designing wound dressings to meet the needs for hemostasis and antibacterial effects. Effective wound healing requires an environment that is quick to stop bleeding, free from contamination, antibacterial, and breathable. However, developing an ideal wound dressing that simultaneously possesses all these functions presents a significant challenge. In chapter 4, we designed a skin-mimicking wound dressing that not only replicates the bilayer structure of skin but also offers good breathability and protective functions. This wound dressing consists of a hydrophilic poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) membrane coated with zinc oxide nanoparticles and a hydrophobic polyvinylidene fluoride (PVDF) membrane. Additionally, we used plasma treatment to crosslink the two layers, improving the overall mechanical performance of the wound dressing. The crosslinked fibrous membrane exhibited uniform stress distribution during stretching. Due to its unique structure, the wound dressing demonstrated effective exudate management, antibacterial functionality, and hemostatic performance. The hydrophobic layer directs wound exudate to the hydrophilic layer, while the zinc oxide nanoparticles act as a barrier against external bacteria and release zinc ions to inhibit bacterial growth in the exudate. Moreover, the wound dressing investigated great water vapor transmission rate (WVTR), suitable hemolysis rate, and the high healing rate during in vitro wound healing. This skin-mimicking wound dressing shows great potential as a solution for treating chronic and infected wounds.

In chapter 5, we focus on the systemic issues of wound dressings for treating burn wounds, including non-adhesion wound dressing, antibacterial property, and scarless healing during the whole processes to avoid chronic wound formation. The prepared double-layer Janus dressing for burn wound healing combines different hydrophilic and hydrophobic properties to address varying needs of the wound. The outer hydrophobic layer prevents the invasion of external contaminants and microorganisms, while the inner hydrophilic layer promotes a moist environment conducive to cell migration and tissue regeneration. Additionally, this dressing is loaded with flavonoid drugs, providing sustained drug release to enhance antibacterial, anti-inflammatory, and antioxidant effects, thereby accelerating the healing process. Experimental results indicate that the double-layer Janus dressing significantly reduces infection, shortens healing time, and improves healing quality, offering an efficient and intelligent treatment solution for burn patients.

Overall, based on the studied systems and current advancements, the next generation of bioactive membranes for tissue engineering repair has been envisioned. These bioactive membranes will not only possess exceptional mechanical properties and biocompatibility but will also integrate advanced biomaterials and technologies to achieve intelligent, theranostic, and multi-tissue integrated repair.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Jinlian HU (Supervisor), Tingwu Qin (External Co-Supervisor) & Hiu Wai Raymond LAM (Co-supervisor)