Abstract
Skin/Tissue Bioprinting
Handheld Skin Printer: In Situ Formation of Planar Biomaterials and Tissues
Acute and complex full-thickness wounds are especially prey to pathogens and dehydration. The preferred treatment for such full-thickness wounds is split-thickness autografting. This involves an acellular scaffold to assist skin reconstitution before distributing healthy skin grafts throughout large wounds. Unfortunately, large portions of the wound remain uncovered due to the lack of material. Often, allografts are used that do not yield desirable outcomes. To supplement such efforts, biological and synthetic skin substitutes have been used to improve wound healing. Minimally modified (chemical crosslinking, freeze drying) collagen is currently the most widely used. Covering with split-thickness autografts reduces wound contraction and promotes migration of neighboring cells to reconstitute the dermal layer in 2 to 3 wk. Other options include tissue-engineered approaches, including cultured epithelial autografts and cell spraying.
Despite these options, a number of barriers hinder widespread usage, such as long processing times, high cost, and the lack of patient customizability. Additive manufacturing methods (e.g., 3D bioprinting) have also been of great interest, but many protein-based biomaterials (collagen I, fibrin, etc.) have poor “printability” properties (low viscosity, long gelation times >1 min). Existing attempts at 3D bioprinting are also disadvantaged by the requirement for laser scanning of wounds that necessitate bulky setups with reduced spatial control. The authors propose a compact, extrusion-based method to bioprint biomaterials and skin tissue that circumvents the existing issues of bioprinting. This biopinter facilitates in situ bioprinting through a handheld device. This contains compartments for cells (suspended in a hydrogel precursor) and a crosslinking solution that aids gelation. These can deposit dermal layers between 0.1 and 0.6 mm thick. The (fully loaded) device is <0.8 kg, can be operated by hand, and deposits bioink with actively driven rollers on compliant surfaces of skin. Syringe pump modules deliver bioink and crosslinker solution at adjustable rates.
Compared to manually pipetted agarose and crosslinker, an approximately 300-µm uniform layer was obtained from the handheld bioprinter. In contrast, manually pipetted hydrogels were dome shaped. Working parameters such as fluid viscosity, flow rate, and deposition thickness/height were adjusted to deposit a wide variety of biomaterials (hence different mechanical properties). These were practically demonstrated using different biomaterials and biomaterial combinations: fibrin-hyaluronic acid (HA)/collagen I, fibrin-HA, collagen I–alginate, and alginate, which spanned a 5-fold change in Young’s modulus. Composite gels were further deposited at different thicknesses before imaging using fluorescent labels (gross morphology) and scanning electron microscopes (microstructural features). Independent control of microfluidic cartridges provided exquisite control over gelation conditions. These were further applied to printing an array of striped, spotted, arrays/undulated sheets, and so on.
A bioink containing hyaluronic acid, fibrinogen, and type I collagen was used for depositing dermal fibroblasts and epithelial keratinocytes through the handheld printer in vitro. Cells were found to be highly viable, with significant cell proliferation (after day 3) observed. Fluorescently stained cells also showed suitable morphology and cell adhesion. Stripes of bilayers containing fibroblasts and keratinocytes were efficiently printed. The skin bioprinter was further used on mouse and pig models. These produced channels that were highly reproducible, as assessed by green fluorescence (impregnated microparticles). In a pig model (20 mm × 40 mm) excision wound, a deposited fibrin-HA sheet improved the wound, showing improved reepithelialization after 20 days postwounding. Thus, this handheld platform was able to print both natural and synthetic biomaterials, including payloads containing cellular material. It is a promising approach for difficult to heal pathological wounds. (Hakimi, N.; et al. Lab Chip.
In Situ Bioprinting of Autologous Skin Cells Accelerates Wound Healing of Extensive Excisional Full-Thickness Wounds
Chronic wounds (diabetic, venous, pressure ulcers, and burn wounds) cost $25 billion to treat in the United States alone. Early wound management through excision and coverage is vital to prevent the wounds from worsening with time, leading to tissue damage and long-term pathological scarring. Such steps increase patient survival (after extensive burn trauma), which promotes closure and prompt wound protection. Delayed and/or underperforming treatments give rise to extensive scarring that leads to disfigurement and loss of range of motion. While split-thickness autografts are regarded as the “gold standard” for treating severe wounds, there is often a limit in availability. Allografts are also an option but risk immune rejection.
Dermal substitutes have been developed to improve wound healing, but these are costly and have dissatisfactory cosmetic outcomes. Tissue-engineered methods have given rise to complex and more lifelike features showing promise for chronic wound management. However, customization and adequate wound cover are bottlenecks in their effective utilization. While cell therapy is also promising, they fail to generate complex skin structures required for successful skin restoration. Bioprinting with inkjet printers, on the other hand, facilitates the deposition of biological materials and cells with high viability onto predefined locations. Exploiting such technology, the authors integrate imaging technology to capture wound topography for in situ cell delivery. Both fibroblasts and keratinocytes are deposited to specific locations of the wound, mimicking skin complexity and promoting wound restoration.
A bioprinter was developed according to the following criteria: 1) portability and ease of transportation, 2) ability to identify and measure wound dimensions, 3) deliver multiple cell types for individual wound customization, 4) easily sterilized, 5) ease of operation, and 6) cost-effective. The principle of bioprinting operation involves real-time 3D scanning before forming a wound model. The wound depth (z-axis) is then used to determine the layers that correspond to the dermis and epidermis. A cartridge-based system is used to deposit a fibrinogen and collagen matrix mixture containing cells while separate nozzles deposit thrombin to gelate and form fibrin. This allows the user to define spacing between individually deposited drops. The integrated scanning system facilitates the rapid measurement and customization of a wound into a printed dual fibroblast-keratinocyte cell layer for the customized treatment of large wounds.
To evaluate bioprinted skin in live subjects, full thickness wounds (3cm × 2.5cm) were generated in athymic nude mice before treating with fibrin and collagen (matrix) or printed skin tissue (printed) and compared to untreated wounds. No mortalities, infection, and skin irritation were reported. Among the groups, printed skin demonstrated the fastest closure compared to matrix and untreated wounds. Crucially, printed skin cells accelerated closure by 40% (3 wk compared to 5 wk postsurgery). Histology also revealed the clearest presence of skin epithelium in the bioprinted group. The skin bioprinter was transferred to Yorkshire pigs with 10-cm × 10-cm wounds. In addition to matrix, allogeneic, and untreated groups, an autologous printed cell group was also included. A significant finding was that the autologous group decreased wound size, the degree of contraction, and reepithelialization relative to the other groups. Histology of the autologous group showed good wound healing, including features such as rete peg epithelial projections as well as keratinized stratified squamous epithelium. Allogeneic and matrix-treated wounds exhibited delayed wound healing by contrast. The autologous group had improved dermal architecture with a lower degree of acute inflammation.
Vascular expression was evaluated using CD31 immunostaining, which showed larger, more mature vessels in the dermis for the autologous compared to the other groups. The others had smaller, immature blood vessels by comparison. α–Smooth muscle actin (αSMA) was confined to the periphery of the larger blood vessels (autologous group), whereas it was distributed throughout the dermis of the untreated and matrix-treated groups. This suggested a greater quantity of myofibroblast-mediated hypertrophic scarring 8 wk postwound—a lower quality of wound healing. This bioprinting method was then evaluated against FibriJet, a cell spraying technique. Histological evaluation confirmed improved healing outcomes from bioprinting. In summary, the bioprinting system demonstrates significant promise in accelerating wound healing. Further efforts involve the addition of melanocytes, adipose, and hair follicle cells to improve the extent of restoration for burns, ulcers, and even deep tissue injuries. (Albanna, M.; et al. Sci. Rep.
Rapid Continuous 3D Printing of Customizable Peripheral Nerve Guidance Conduits
Peripheral nervous system (PNS) injuries involve at least half a million procedures in both the United States and Europe combined. The “gold standard” PNS repair strategy involves harvesting a graft with complete nerve grafting involving suturing complete nerves without introducing tension to repair short nerve gaps (
Several fabrication techniques, including electrospinning, microdrilling, and molding, have been used to shape natural (collagen, chitosan, agarose, etc.) and synthetic (silicone, polyglycolide, gelatin methacryloyl) materials. Although an extrusion-based 3D printing approach was recently applied to fabricate NGCs, limitations have emerged, including low printing resolution, compromised structural integrity, and limited printing speed. In response, the authors propose a digital light processing (DLP)–based rapid, continuous 3D printer to generate NGCs with customized shapes and properties to guide peripheral nerve regeneration. The rapid continuous 3D printer uses a 405-nm visible light LED for photopolymerization (no ultraviolet light damage) with a digital micromirror device (DMD) chip to control the process. This process is digitalized and controlled by a computer, resulting in great reproducibility and flexibility to optimize design parameters with vastly reduced turnaround time relative to traditional manufacturing (e.g., molding).
Continuous 3D printing was performed on a range of NGCs from simple (hollow conduit) to complex (an anatomically appropriate-sized conduit for facial nerve repair). These were printed within 10 min, even for complex NGCs. Matching the mechanical properties of the NGC graft is critical to prevent damage to growing nerves as well as to ensure implant viability. Because peripheral nerve tissue elastic modulus ranges between 0.5 and 13 MPa, photo-induced free radical polymerization is required to fine-tune NGC mechanical properties by modulating the extent of crosslinking. To ensure a sufficiently robust and tunable composite material, polyethylene glycol diacrylate (PEGDA) and gelatin methacrylate (GelMA) were combined with a strongly water-soluble photoinitiator, allowing an increase of Young’s modulus from 0.3 to 2 MPa. Increased light exposure intensity further increased it to 4.5 MPa. Not only was the 3D printer able to obtain complex customizable shapes, but it also provided requisite material properties.
The customized NGCs (4 linear microchannels and 2 sleeve design) were then used to guide nerve regeneration in vivo. In transgenic mice (expressing cyan fluorescence under neuron-specific activity), the sciatic nerve was completely resected, which led to nerve retraction, creating a nerve gap of approximately 4 mm. Following 11 wk of implantation, NGC grafts were harvested and regenerating nerves observed through histology to branch into the microchannels (proximal end), merging into 1 nerve at the distal end. Histology also revealed that NGCs facilitated nerve regeneration, and neurons (expressing fluorescent proteins) were found throughout the NGC.
Excitingly, promising results were observed in functional recovery tests. At early time points (2 d), no sensation was observed posttransection. However, the mice were observed to have regained sensation after several weeks (3–10 wk). Therefore, the authors demonstrate 3D-printing technology that creates complex architecture NGCs efficiently. Having suitable features and properties facilitates sciatic recovery in vivo along with corresponding function. This suggests it has clinical potential for peripheral nerve repair. (Zhu, W.; et al. Mater. Today.
Wound Monitoring
Smart Bandage for Monitoring and Treatment of Chronic Wounds
Injuries that reach the dermis depth significantly increase the likelihood of infections, making their management highly critical. Some wounds defy normal healing, where the stages of inflammation, proliferation, and remodeling do not occur as expected. Certain health conditions such as diabetes increase the propensity of pathological/chronic wounds. Up to 90% of ulcers from chronic wounds result in bacterial infections (e.g., Staphylococcus aureus), which leads to limb amputations. Ideally, coverings form a barrier against environmental pathogens and prevent infection by detecting and releasing antibacterial agents in response. Infections frequently occur in chronic wounds, and thus patients seek continuous monitoring capability without removing wound dressings.
Recent technology development has given rise to flexible sensors to monitor parameters that provide critical information (e.g., pH). Whereas normal wounds range between 5.5 and 6.5 pH while healing, infected chronic wounds have pH above 6.5. Such pH readings could send an alert regarding a possible instance of infection. Advanced dressings can even be designed to release antibiotic therapeutics locally (as a self-responding polymer technology) upon detecting particular parameters. Thus, a next-generation dressing with integrated electronics may detect bacterial infections in situ and release antibiotics locally, on demand.
A smart bandage was designed with the following components: sensors (pH, temperature), microheater, thermo-responsive drug-encapsulated biomaterials, and wireless electronics to control drug release. Thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) particles were embedded in alginate hydrogels. Other components in the design also included a flexible heater, microcontroller, transparent medical tape (creating a thin [<3-mm] wearable platform), and low-cost sensing and integrated disposable heaters. The authors then demonstrated that the sensors exhibited a linear response with minimal drift (6 mV) over 12 h. The smart dressing demonstrated an average of 8 °C increase per applied voltage (V). It also exhibited a response time of <5 min as well as dynamic cycling switching between temperatures over a 10-min period.
The drug-release performance was further evaluated. To model drug release, rhodamine B (a fluorescent model drug) was loaded into PNIPAM particles. Increasing the temperature from 25 to 37 °C gave rise to a 4- or 10-fold increase in drug release (alginate and phosphate-buffered saline [PBS], respectively). The smart dressing was further capable of releasing drugs in a dynamic manner using a 30-min on, 30-min off regimen. Increasing heat released cefazolin antibiotics faster, whereas lower temperatures reduced their release rate. Its functionality was then evaluated in a scratch test performed in the presence of bacteria (S. aureus). Scratching a confluent keratinocyte (epidermal cell) monolayer mimics wound healing where cells migrate to close this gap. Since bacterial infections inhibit normal keratinocyte function, this can be used to evaluate the smart dressing performance. Promisingly, the closure of the gap occurred to a similar degree of the positive control (free cefazolin). Measures of cell viability and total cell numbers were consequently the highest compared to the free biomaterial (alginate, PNIPAM) groups. This suggests that the thermo-responsive biomaterial was able to release the antibiotic and restrict the behavior of S. aureus compared to the antibiotic-free cultures.
Further evaluation of the dressing’s function showed inhibition of bacteria, as well as its pH-triggered inhibition properties. The entire package was sufficiently flexible to conform to human skin and secured with transparent medical tape. Thus, the authors demonstrated the development of a closed-loop wireless Bluetooth low-energy dressing for the automated monitoring and treatment of chronic wounds. Further evaluation in animal models can improve its likelihood for biomedical translation. (Mostafalu, P.; et al. Small
Detection of Redox State Evolution during Wound Healing Process Based on a Redox Sensitive Wound Dressing
Wound healing undergoes changes in redox state due to different combinations of reactive oxygen species (ROS), reactive nitrogen species (RNS), antioxidants, and other corresponding enzymes. Wound healing is also a highly complex yet ordered process that requires varying amounts of ROS to trigger cell signaling pathways and antibacterial radicals. Current techniques to probe this process are invasive, potentially interfering with the wound-healing process and even generating local trauma that alters the redox state. Noninvasive methods are further limited in their spatiotemporal acquisition properties. Thus, novel methods are required to probe the spatiotemporal redox state without interfering with the wound-healing process. Surface-enhanced Raman scattering (SERS) imaging offers benefits, including noninvasiveness, short image acquisition times, and high sensitivity. Using infrared excitation allows SERS signal collection from subcutaneous tissue.
A redox-sensitive wound dressing was fabricated by functionalizing anthraquinone derivatives on gold nanoshells before loading them on the surface of a common chitosan dressing. This allowed the collection of SERS spectra in vivo from acute wounds. Scanning electron microscope imaging showed that the gold nanoshells were 150 to 200 nm in diameter, which were distributed evenly onto the chitosan membrane. SERS spectra intensity at 1606 and 1666/cm also demonstrated homogeneity throughout the wound dressing.
SERS probes subcutaneously injected into mice led to a change in potential shortly after the addition of excess glucose, which affected redox potential. Redox potential at the wound edge was highly reduced, while the center of the wound was highly oxidized. Redox potential at the wound edge increased over the next few days while there was a distinct transformation to a reduced state in the center. The authors reason that antioxidants can be recruited from healthy tissues at the wound edge, whereas injured tissue releases ROS that generate an oxidative microenvironment.
To demonstrate the wound-monitoring capabilities through SERS spectra, intraperitoneal doses of lower glucose were injected. This was observed to have a minimal effect on wound healing with similar SERS spectra. When the glucose dosage was increased 10-fold, redox potentials remained significantly below physiological levels throughout, coinciding with poorer wound-healing outcomes. This demonstrates how imaging redox potential through SERS probes may facilitate the noninvasive monitoring of wound status. Thus, the authors propose that the SERS imaging of redox potential in wounds could benefit from evaluating wound-healing mechanisms, as well as tracking therapeutic outcomes in wound complications due to advanced age, diabetes, or immunoimpairment. (Sun, J.; et al. Anal. Chem.
Evaluation of Scar Quality after Treatment of Superficial Burns of the Hands and Face with Dressilk or Biobrane: An Intra-Individual Comparison
Scars occur following traumatic wound/injuries such as burns. Surveys suggest that visible scarring is well correlated with greater distress and stigma. Postburn contractures created by scars also limit limb function and impede daily activities. To date, a number of wound dressings have been proposed to increase wound healing and decrease the degree of scarring. Two candidate dressings are Biobrane, a biocomposite made from a semipermeable silicone membrane mechanically bonded to a flexible knitted nylon fabric with porcine collagen type I. This can be used to treat superficial partial-thickness to mid-dermal burns following tissue debridement and full-thickness burns.
Studies have also shown a return to normal or near-normal quality of life following 2 to 4 wk with respect to pain, range of motion, grip strength, and appearance. While it was the standard of care for superficial burns by the author’s clinic, delivery issues have led to a search for functional and cost-effective alternatives. Dressilk, a silk fibroin dressing, has a high potential for wound healing as it is found to reduce inflammation and improve collagen regeneration. It is also semitransparent (allowing observation), can be sterilized, and is approximately 10× cheaper compared to Biobrane. The 2 dressings were then evaluated for the long-term scarring of the hand and face following the treatment of superficial burns. The visible scars were then evaluated using subjective scores such as the Vancouver Scar Scale (VSS; after 6, 12 mo), Patient Scar Scale (PSAS), and Observer Scar Scale (OSAS; after 6 mo). In the follow-up, treated and untreated areas were found to have no significant differences.
Objective measures such as Mexameter (skin color), Tewameter (assessment of transepidermal water loss), Cutometer (elasticity measurement), and O2C (measurement of cell metabolism) were performed to assess the efficacy of both dressings. Both wound dressings did not result in differences in skin elasticity. Transepidermal water loss readings indicate similar levels of skin softness and hydration. These values were found to be at similar levels to normal skin levels for both dressings. Burn wounds and scars express high trans-epidermal water loss (TEWL) levels, with the normal levels only returning 6 to 13 mo after injury. After 6 mo, Dressilk expressed higher levels compared to Biobrane, although this was not significant after 12 mo. Overall, Dressilk was found to have similar performance in normalizing skin quality compared to Biobrane. Thus, Dressilk is a suitable alternative to treat superficial burns, especially because of its cost-effectiveness. (Schiefer, J. L.; et al. Burns
Bioactive Wound Dressings
Anti-IL-6 Eluting Immunomodulatory Biomaterials Prolong Skin Allograft Survival
Skin autografts represent the gold standard to assist wound closure, which is especially critical in patients with severe burns and chronic wounds. However, these are infeasible for patients with large-sized burns (e.g., covering more than 15% of the body), infected wounds, donor site skin insufficiency, and those with poor tolerability for surgery. One therapeutic option involves using skin allografts (including cryopreserved tissue) to ensure wound closure. Evidence suggests they are more effective than synthetic skin substitutes (e.g., biomaterials, natural materials). Crucially, allografts promote tissue vascularization (in a few days), thereby providing the necessary oxygen and nutrients.
The greatest drawback of allografts is its potential for rejection. While immunosupression is an option, systemic administration is not advisable in burn patients due to their increased exposure to infections. Instead, localized immune delivery strategies are favored. There is growing evidence to suggest that intragraft inflammatory responses instigate alloimmune responses. Interleukin (IL)–6 has been identified as a key inflammatory cytokine that is reported to increase significantly in burn patients. Furthermore, higher levels also coincide with poorer outcomes for burn patients. To address the production of excessive IL6 by dendritic cells, hydrogels were employed for the controlled release of anti-inflammatory molecules. These hydrophilic biopolymers have significant usage in a number of emerging areas of biomedicine and preserve the activity of incorporated compounds. The authors then employed a photo-crosslinkable strategy to generate an immune-privileged microenvironment to suppress inflammatory responses.
Gelatin methacrylate (GelMA) 5% was crosslinked to contain IL6 antibodies and photo-cured to create an adhesive hydrogel. The IL6 antibodies underwent sustained release from the hydrogel over 70 h. To assess its beneficial properties to allograft survival, skin from BALB/c donor mice was transplanted fully onto a mismatched C57BL/6 recipient mouse overlaying a GelMA–anti-IL6 hydrogel. These were compared to control mice that received intravenous injections of anti-IL6, GelMa only (no antibody), and no intervention. Whereas the untreated allografts shrank and became rejected, the GelMA/anti-IL6 ones remained viable even compared to the grafts assisted with systemic injections. Histology showed the GelMA integrating well with the syngeneic skin transplant.
When GelMA hydrogels were implanted, the allografts were found to blacken (signifying necrosis) with significant cell infiltration. Further analysis of immune cell populations revealed that T-cell and monocyte infiltration was suppressed by locally released IL6 antibodies. This led to reduced release of inflammatory cytokines. Further analysis of draining lymphatic nodes showed that GelMA/anti-IL6 hydrogels led to reduced node size and reduced inflammatory T cells. Supporting this evidence, Tregs were also found in higher numbers in the antibody-eluted hydrogels. Inflammation also gives rise to increased fibrosis (matrix accumulation) in lymphatic nodes. Histology analysis also revealed that anti-IL6 hydrogels reduced the amount of inflammation compared to untreated and empty gel controls. Furthermore, locally released IL6 antibodies do not affect systemic immunity. This suggests that the antibody-loaded hydrogels may be suitable to manage burns using allograft without risking opportunistic infections. This study also represents a suitable proof of concept, suggesting that other immunomodulatory agents such as CTLA4-Ig and tacrolimus/sirolimus could be incorporated into the hydrogels to improve the viability of allografts. This will improve treatment outcomes by increasing the tolerance for allografts. (Uehara, M.; et al. Sci. Reps.
Superhydrophobic Hierarchical Fiber/Bead Composite Membranes for Efficient Treatment of Burns
Burns are a significant global public health problem; autologous skin graft remains the “gold standard” for wound repair. However, they encounter issues such as graft morbidity (due to harvesting) as well as limited availability of healthy skin. This shortage has led to a demand for artificial biomaterials as alternatives. Wound healing is a complex, multistaged process (inflammation, proliferation, regeneration) that is complicated by pathogens, dehydration, and trauma caused by surgery or wound maintenance. Thus, wound dressings should ideally possess these attributes:1) prevent pathogen colonization, 2) minimize water loss, and 3) facilitate skin regeneration by serving as biological templates. Among the emerging synthetic biomaterials, electrospinning techniques have proven to be highly versatile, generating porous, high surface area structures to support cell growth and efficient nutrient/metabolite exchange. Coaxial electrospinning has also created multicomponent core/shell fibers for the controlled delivery of drugs and therapeutic agents. A further benefit is the fabrication of superhydrophobic surfaces that prevent microbial adhesion.
The authors create a hierarchical composite fibrous structure comprising a poly(lactic acid):poly(vinyl pyrrolidone) (PLA:PVP)/PLA:poly(ethylene glycol) (PLA:PEG) core/shell fibrous membrane (FM) prepared by coaxial electrospinning. A layer of PLA beads was electrosprayed on the FM, which increased its hydrophobicity and reduced bacterial adhesion. To tune drug release, antimicrobial peptides (AMPs) were loaded into the shell layer, while curcumin was encapsulated into its core. While the earlier-released AMPs could induce prompt bacteria killing and acute inflammation, the subsequent release of anti-inflammatory and antioxidative curcumin would accelerate wound healing. Different compositions were developed having a similar appearance—randomly oriented, intertwined fibrous mats with uniform fiber diameters. Due to its superior tensile strength, 7:3 (PLA:PVP w/w) FMs were used for the remaining experiments.
The FMs were found to exhibit good biocompatibility, with fibroblasts proliferating readily and having an elongated, spindle-like morphology, suggesting favorable cytocompatibility and favorable cell-substrate adhesion. Within the composite, a drug-loading efficiency of 70% and 79% was obtained from the core (AMP) and shell (curcumin), respectively. Characteristically, they gave an initial burst release in the first hours before a more gradual profile subsequently. Favorably, AMPs were released faster (~90% within 3 d) than curcumin—more than 5 d, which suggests it is suited to suppress bacterial infections and manage inflammation. The authors then demonstrated that increasing the volume of electrosprayed volume led to increased PLA bead deposition. This increased the water contact angle, making the FM significantly more hydrophobic. Its value rose
The FMs were also demonstrated to possess good antibacterial properties on both gram-positive and gram-negative bacteria (S. aureus and Escherichia coli, respectively). Bacteria attached at sparse densities and exhibited a shriveled appearance, suggesting poor viability. Following this, various FMs were evaluated on full-thickness burn wounds on rat dorsum. The superhydrophobic drug loaded composite fibrous membrane (DLCFM) demonstrated the greatest extent of wound closure with ~75% by day 7. Increased wound healing led to a thicker epidermis by day 14 compared to control groups. The restored collagen extracellular matrix was also found to be more regular by comparison. High tumor necrosis factor–α and interleukin-1β activity in the controls (compared to DLCFM) also suggested greater inflammatory activity. Such rationally designed dressings that elute drugs at various stages and display antibacterial activity (due to superhydrophobicity) lay the path for novel dressings for effective burn treatments for the future. (Li, W.; et al. Acta Biomater.
Potent Laminin-Inspired Antioxidant Regenerative Dressing Accelerates Wound Healing in Diabetes
Chronic wounds (e.g., foot ulcers) can be a particularly dangerous threat in diabetic patients due to its high occurrence and close association with limb amputations. While recent therapeutic efforts to heal chronic wounds have involved a myriad of drugs, proteins, and tissue-engineered constructs, substantial regulatory hurdles have meant that there has been limited adoption. Regranex, a recombinant platelet-derived growth factor gel, comes with increased cancer risk during overuse. These novel approaches also suffer from limited product shelf life, poor understanding of their mechanism(s) of action, and other unexpected systemic side effects.
In response, the authors have developed a biomaterial that does not contain bioactive factors to serve as a restorative wound dressing. This is based on the functional motifs of laminin and that integrin α3β1 activation is necessary for fibroblast and epidermal keratinocyte movement. They hypothesized that laminin-derived peptides (A5G81) can bind to antioxidative thermo-responsive polymers, poly(polyethylene glycol cocitric acid-co-N-isopropylacrylamide) (PPCN), thereby enhancing wound closure. The authors first attempted to identify the specific peptide/amino acid sequences (A5G81) necessary to promote fibroblast (dermis) and keratinocyte (epidermis) attachment. Various lengths of peptides were tethered to glass slips and compared to the full A5G81 and a truncated sequence that was demonstrably inactive.
Full-length A5G81 effected a 2-fold increase in migration distance, whereas negative controls showed few adherent cells and limited migration. A number of cytokine-mediated signaling, cell surface receptor signaling pathways, and keratinocyte development biomarkers were upregulated. Activity associated with cell proliferation was similarly boosted by the peptides. Thereafter, the thermo-responsive gel (A5G81-PPCN) was synthesized to present A5G81 and compared with the gel containing inactive peptides. The final peptide concentration was held equal at 1 mM. The peptide-modified gel transitioned at 24 °C, allowing liquid application before transitioning to become a gel at bodily temperatures. Rinsing with cold saline reverses this transition, facilitating gentle removal and minimizing trauma.
A5G81-PPCN and RGD-PCN (positive control) supported long-term fibroblast culture with adequate cell spreading observed, whereas unmodified PPCN and inactive gels did not display similar cell-matrix interactions. A5G81-PPCN increased the proportion of cells in the DNA synthesis phase, with up to 27% observed on day 10. Engagement with cell integrins α3 and α6 were necessary for cell proliferation. In splinted wounds generated on diabetic mice, A5G81-PPCN, RGD-PCN, inactive, and empty gels were topically applied. A5G81-PPCN achieved 2.25× more effective closure compared to other groups (day 10). Seventy-nine percent wound closure was observed at day 15 and full closure observed by day 22 (significantly shorter than other groups). Histology was then used to assess wound quality. A5G81-PPCN promoted the thickest granulation tissue and minimized the epithelial gap. Keratin 10, a biomarker of tissue differentiation, was also strongly expressed, as was integrin α3 (cell adhesion) and minimal expression of F4/80, a biomarker for macrophages.
The thermo-responsive A5G81-PPCN gel was then benchmarked against Promogran Prisma wound dressing (Systagenix, San Antonio, TX). The A5G81-PPCN gel outperformed in terms of wound closure as well as in conditions filled with fluid. Using an approach that involved promoting cell migration, adhesion (using laminin-derived peptides), and antioxidative biomaterials, the authors demonstrated a novel thermo-responsive gel to enhance wound closure. Further efforts will involve implementation in larger animal studies. (Zhu, Y.; et al. Proc Natl Acad Sci U S A.
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.