Bioinspired and Photo-Clickable Thiol-Ene Bioinks for the Extrusion Bioprinting of Mechanically Tunable 3D Skin Models.

Bioinks play a fundamental role in skin bioprinting, dictating the printing fidelity, cell response, and function of bioprinted 3D constructs. However, the range of bioinks that support skin cells' function and aid in the bioprinting of 3D skin equivalents with tailorable properties and customized shapes is still limited. In this study, we describe a bioinspired design strategy for bioengineering double crosslinked pectin-based bioinks that recapitulate the mechanical properties and the presentation of cell-adhesive ligands and protease-sensitive domains of the dermal extracellular matrix, supporting the bioprinting of bilayer 3D skin models. Methacrylate-modified pectin was used as a base biomaterial enabling hydrogel formation via either chain-growth or step-growth photopolymerization and providing independent control over bioink rheology, as well as the mechanical and biochemical cues of cell environment. By tuning the concentrations of crosslinker and polymer in bioink formulation, dermal constructs were bioprinted with a physiologically relevant range of stiffnesses that resulted in strikingly site-specific differences in the morphology and spreading of dermal fibroblasts. We also demonstrated that the developed thiol-ene photo-clickable bioinks allow for the bioprinting of skin models of varying shapes that support dermis and epidermis reconstruction. Overall, the engineered bioinks expand the range of printable biomaterials for the extrusion bioprinting of 3D cell-laden hydrogels and provide a versatile platform to study the impact of material cues on cell fate, offering potential for in vitro skin modeling.

One-Pot Microfluidics to Engineer Chitosan Nanoparticles Conjugated with Antimicrobial Peptides Using "Photoclick" Chemistry: Validation Using the Gastric Bacterium Helicobacter pylori.

Surface bioconjugation of antimicrobial peptides (AMP) onto nanoparticles (AMP-NP) is a complex, multistep, and time-consuming task. Herein, a microfluidic system for the one-pot production of AMP-NP was developed. Norbornene-modified chitosan was used for NP production (NorChit-NP), and thiolated-AMP was grafted on their surface via thiol-norbornene "photoclick" chemistry over exposure of two parallel UV LEDs. The MSI-78A was the AMP selected due to its high activity against a high priority (level 2) antibiotic-resistant gastric pathogen: Helicobacter pylori (H. pylori). AMP-NP (113 ± 43 nm; zeta potential 14.3 ± 7 mV) were stable in gastric settings without a cross-linker (up to 5 days in pH 1.2) and bactericidal against two highly pathogenic H. pylori strains (1011 NP/mL with 96 µg/mL MSI-78A). Eradication was faster for H. pylori 26695 (30 min) than for H. pylori J99 (24 h), which was explained by the lower minimum bactericidal concentration of soluble MSI-78A for H. pylori 26695 (32 µg/mL) than for H. pylori J99 (128 µg/mL). AMP-NP was bactericidal by inducing H. pylori cell membrane alterations, intracellular reorganization, generation of extracellular vesicles, and leakage of cytoplasmic contents (transmission electron microscopy). Moreover, NP were not cytotoxic against two gastric cell lines (AGS and MKN74, ATCC) at bactericidal concentrations. Overall, the designed microfluidic setup is a greener, simpler, and faster approach than the conventional methods to obtain AMP-NP. This technology can be further explored for the bioconjugation of other thiolated-compounds.

Skin models of cutaneous toxicity, transdermal transport and wound repair.

Skin is widely used as a drug delivery route due to its easy access and the possibility of using relatively painless methods for the administration of bioactive molecules. However, the barrier properties of the skin, along with its multilayer structure, impose severe restrictions on drug transport and bioavailability. Thus, bioengineered models aimed at emulating the skin have been developed not only for optimizing the transdermal transport of different drugs and testing the safety and toxicity of substances but also for understanding the biological processes behind skin wounds. Even though in vivo research is often preferred to study biological processes involving the skin, in vitro and ex vivo strategies have been gaining increasing relevance in recent years. Indeed, there is a noticeably increasing adoption of in vitro and ex vivo methods by internationally accepted guidelines. Furthermore, microfluidic organ-on-a-chip devices are nowadays emerging as valuable tools for functional and behavioural skin emulation. Challenges in miniaturization, automation and reliability still need to be addressed in order to create skin models that can predict skin behaviour in a robust, high-throughput manner, while being compliant with regulatory issues, standards and guidelines. In this review, skin models for transdermal transport, wound repair and cutaneous toxicity will be discussed with a focus on high-throughput strategies. Novel microfluidic strategies driven by advancements in microfabrication technologies will also be revised as a way to improve the efficiency of existing models, both in terms of complexity and throughput.

Metal-Organic Frameworks Applications in Synergistic Cancer Photo-Immunotherapy.

Conventional cancer therapies, such as radiotherapy and chemotherapy, can have long-term side effects. Phototherapy has significant potential as a non-invasive alternative treatment with excellent selectivity. Nevertheless, its applicability is restricted by the availability of effective photosensitizers and photothermal agents, and its low efficacy when it comes to avoiding metastasis and tumor recurrence. Immunotherapy can promote systemic antitumoral immune responses, acting against metastasis and recurrence; however, it lacks the selectivity displayed by phototherapy, sometimes leading to adverse immune events. The use of metal-organic frameworks (MOFs) in the biomedical field has grown significantly in recent years. Due to their distinct properties, including their porous structure, large surface area, and inherent photo-responsive properties, MOFs can be particularly useful in the fields of cancer phototherapy and immunotherapy. MOF nanoplatforms have successfully demonstrated their ability to address several drawbacks associated with cancer phototherapy and immunotherapy, enabling an effective and low-side-effect combinatorial synergistical treatment for cancer. In the coming years, new advancements in MOFs, particularly regarding the development of highly stable multi-function MOF nanocomposites, may revolutionize the field of oncology.

3D Bioprinting: An Enabling Technology to Understand Melanoma.

Melanoma is a potentially fatal cancer with rising incidence over the last 50 years, associated with enhanced sun exposure and ultraviolet radiation. Its incidence is highest in people of European descent and the ageing population. There are multiple clinical and epidemiological variables affecting melanoma incidence and mortality, such as sex, ethnicity, UV exposure, anatomic site, and age. Although survival has improved in recent years due to advances in targeted and immunotherapies, new understanding of melanoma biology and disease progression is vital to improving clinical outcomes. Efforts to develop three-dimensional human skin equivalent models using biofabrication techniques, such as bioprinting, promise to deliver a better understanding of the complexity of melanoma and associated risk factors. These 3D skin models can be used as a platform for patient specific models and testing therapeutics.

An injectable, dual crosslinkable hybrid pectin methacrylate (PECMA)/gelatin methacryloyl (GelMA) hydrogel for skin hemostasis applications.

Biomaterials for effective hemorrhage control are urgently needed in clinics as uncontrolled bleeding is associated with high mortality. Herein, we developed an injectable and in situ photo-crosslinkable hybrid hemostatic hydrogel by combining pectin methacrylate (PECMA) and gelatin methacryloyl (GelMA). This modular material system combines ionic- and photo-crosslinking chemistries to design interpenetrating networks (IPN) exhibiting tunable rheology, highly porous structure, and controllable swelling and mechanical properties. By simply changing the calcium (0-15 mM) and polymer (1.5-7%) content used for the sequential crosslinking of hydrogels via calcium gelation and UV-photopolymerization, it was possible to precisely modulate the injectability, degradation, and swelling ratio. Moreover, it is demonstrated that PECMA/GelMA hydrogels present good cytocompatibility and uniquely synergize the hemostatic properties of calcium ions on PECMA, the amine residues on GelMA, and the highly porous network toward rapid blood absorption and fast coagulation effect. An in vitro porcine skin bleeding model confirmed that the hydrogel could be directly injected into the wound and rapidly photo-crosslinked, circumventing the bleeding and decreasing the coagulation time by 39%. Importantly, the crosslinked hydrogel could be easily removed to prevent secondary wound injury. Overall, this injectable hybrid PECMA/GelMA hydrogel stands as a promising hemostatic material.

Engineering Modular Half-Antibody Conjugated Nanoparticles for Targeting CD44v6-Expressing Cancer Cells.

Gastric cancer (GC) remains a major cause of death worldwide mainly because of the late detection in advanced stage. Recently, we proposed CD44v6 as a relevant marker for early detection of GC, opening new avenues for GC-targeted theranostics. Here, we designed a modular nanoscale system that selectively targets CD44v6-expressing GC cells by the site-oriented conjugation of a new-engineered CD44v6 half-antibody fragment to maleimide-modified polystyrene nanoparticles (PNPs) via an efficient bioorthogonal thiol-Michael addition click chemistry. PNPs with optimal particle size (200 nm) for crossing a developed biomimetic CD44v6-associated GC stromal model were further modified with a heterobifunctional maleimide crosslinker and click conjugated to the novel CD44v6 half-antibody fragment, obtained by chemical reduction of full antibody, without affecting its bioactivity. Collectively, our results confirmed the specific targeting ability of CD44v6-PNPs to CD44v6-expressing cells (1.65-fold higher than controls), highlighting the potential of CD44v6 half-antibody conjugated nanoparticles as promising and clinically relevant tools for the early diagnosis and therapy of GC. Additionally, the rational design of our nanoscale system may be explored for the development of several other nanotechnology-based disease-targeted approaches.

3D Cell Culture Models as Recapitulators of the Tumor Microenvironment for the Screening of Anti-Cancer Drugs.

Today, innovative three-dimensional (3D) cell culture models have been proposed as viable and biomimetic alternatives for initial drug screening, allowing the improvement of the efficiency of drug development. These models are gaining popularity, given their ability to reproduce key aspects of the tumor microenvironment, concerning the 3D tumor architecture as well as the interactions of tumor cells with the extracellular matrix and surrounding non-tumor cells. The development of accurate 3D models may become beneficial to decrease the use of laboratory animals in scientific research, in accordance with the European Union's regulation on the 3R rule (Replacement, Reduction, Refinement). This review focuses on the impact of 3D cell culture models on cancer research, discussing their advantages, limitations, and compatibility with high-throughput screenings and automated systems. An insight is also given on the adequacy of the available readouts for the interpretation of the data obtained from the 3D cell culture models. Importantly, we also emphasize the need for the incorporation of additional and complementary microenvironment elements on the design of 3D cell culture models, towards improved predictive value of drug efficacy.

Tissue-specific engineering: 3D bioprinting in regenerative medicine.

Despite its complexity, the human body is composed of only four basic tissue types, namely epithelial, connective, muscular and nervous tissues. Notably, each tissue is an assemblage of similarly functional cells united in performing a specific function. Instead of mimicking functionality mechanically, three-dimensional (3D) bioprinting based on histological categories is a strategy designed with multiple materials and techniques, which is a versatile technology able to form functional organ structures in line with simplicity. This review aims to provide an overview of tissue-specific 3D bioprinting based on the biological characteristics of four tissue types, including the histological features, biomaterials and corresponding applications. It first briefly introduces the goals of tissue-specific bioprinting and then summarizes the major techniques and identification of particular material development. Moreover, its remarkable regenerative power in replacement therapy and novel outbreak in particular tissues are assembled by epithelial, connective, nerve and muscle tissues. Finally, we discuss challenges and future prospects of tissue-specific based 3D bioprinting in biomedicine, hoping to further inspire the development.

Bioprinting a Multifunctional Bioink to Engineer Clickable 3D Cellular Niches with Tunable Matrix Microenvironmental Cues.

The properties of the surrounding cell environment are major determinants of cell response in 3D. However, the ability to unravel how these cues dictate the biological function in bioprinted constructs is limited by the lack of extracellular matrix (ECM)-mimetic bioinks with fully controllable properties. In this study, a multifunctional bioink that uniquely combines the independent control over the biochemical and biophysical cues that regulate cell fate with the bioorthogonal nature of thiol-norbornene photoclick chemistry is designed for the extrusion bioprinting of bioinspired 3D cellular niches with tunable properties. The bioink rheology is controlled by ionic gelation, being dependent on both the type and content of divalent ions (calcium and barium), while the mechanical and biochemical properties of hydrogels are tailored via a post printing thiol-ene reaction. Bioprinted cell-adhesive and protease-degradable hydrogels modulate cell proliferation and ECM deposition in a matrix-stiffness dependent manner over 14 days of culture regardless of cell spreading, demonstrating the ability to probe the effect of matrix cues on cell response. This bioink can be used as a versatile platform where building blocks can be rationally combined for the bioprinting of functional cell- and tissue-specific constructs with controlled cellular behavior.

Cell-instructive pectin hydrogels crosslinked via thiol-norbornene photo-click chemistry for skin tissue engineering.

Cell-instructive hydrogels are attractive for skin repair and regeneration, serving as interactive matrices to promote cell adhesion, cell-driven remodeling and de novo deposition of extracellular matrix components. This paper describes the synthesis and photocrosslinking of cell-instructive pectin hydrogels using cell-degradable peptide crosslinkers and integrin-specific adhesive ligands. Protease-degradable hydrogels obtained by photoinitiated thiol-norbornene click chemistry are rapidly formed in the presence of dermal fibroblasts, exhibit tunable properties and are capable of modulating the behavior of embedded cells, including the cell spreading, hydrogel contraction and secretion of matrix metalloproteases. Keratinocytes seeded on top of fibroblast-loaded hydrogels are able to adhere and form a compact and dense layer of epidermis, mimicking the architecture of the native skin. Thiol-ene photocrosslinkable pectin hydrogels support the in vitro formation of full-thickness skin and are thus a highly promising platform for skin tissue engineering applications, including wound healing and in vitro testing models. STATEMENT OF SIGNIFICANCE: Photopolymerizable hydrogels are attractive for skin applications due to their unique spatiotemporal control over the hydrogel formation. This study reports the design of a promising photo-clickable pectin hydrogel which biophysical and biochemical properties can be independently tailored to control cell behavior. A fast method for the norbornene-functionalization of pectin was developed and hydrogels fabricated through UV photoinitiated thiol-norbornene chemistry. This one-pot click reaction was performed in the presence of cells using cell-adhesive and matrix metalloproteinase-sensitive peptides, yielding hydrogels that support extensive cell spreading. Keratinocytes seeded on top of the fibroblast-loaded hydrogel formed a compact epidermis with morphological resemblance to human skin. This work presents a new protease-degradable hydrogel that supports in vitro skin formation with potential for skin tissue engineering.

Traditional Therapies for Skin Wound Healing.

Significance: The regeneration of healthy and functional skin remains a huge challenge due to its multilayer structure and the presence of different cell types within the extracellular matrix in an organized way. Despite recent advances in wound care products, traditional therapies based on natural origin compounds, such as plant extracts, honey, and larvae, are interesting alternatives. These therapies offer new possibilities for the treatment of skin diseases, enhancing the access to the healthcare, and allowing overcoming some limitations associated to the modern products and therapies, such as the high costs, the long manufacturing times, and the increase in the bacterial resistance. This article gives a general overview about the recent advances in traditional therapies for skin wound healing, focusing on the therapeutic activity, action mechanisms, and clinical trials of the most commonly used natural compounds. New insights in the combination of traditional products with modern treatments and future challenges in the field are also highlighted. Recent Advances: Natural compounds have been used in skin wound care for many years due to their therapeutic activities, including anti-inflammatory, antimicrobial, and cell-stimulating properties. The clinical efficacy of these compounds has been investigated through in vitro and in vivo trials using both animal models and humans. Besides the important progress regarding the development of novel extraction methods, purification procedures, quality control assessment, and treatment protocols, the exact mechanisms of action, side effects, and safety of these compounds need further research. Critical Issues: The repair of skin lesions is one of the most complex biological processes in humans, occurring throughout an orchestrated cascade of overlapping biochemical and cellular events. To stimulate the regeneration process and prevent the wound to fail the healing, traditional therapies and natural products have been used with promising results. Although these products are in general less expensive than the modern treatments, they can be sensitive to the geographic location and season, and exhibit batch-to-batch variation, which can lead to unexpected allergic reactions, side effects, and contradictory clinical results. Future Directions: The scientific evidence for the use of traditional therapies in wound healing indicates beneficial effects in the treatment of different lesions. However, specific challenges remain unsolved. To extend the efficacy and the usage of natural substances in wound care, multidisciplinary efforts are necessary to prove the safety of these products, investigate their side effects, and develop standard controlled trials. The development of good manufacturing practices and regulatory legislation also assume a pivotal role in order to improve the use of traditional therapies by the clinicians and to promote their integration into the national health system. Current trends move to the development of innovative wound care treatments, combining the use of traditional healing agents and modern products/practices, such as nanofibers containing silver nanoparticles, Aloe vera loaded into alginate hydrogels, propolis into dressing films, and hydrogel sheets containing honey.

Influence of Aloe vera on water absorption and enzymatic in vitro degradation of alginate hydrogel films.

This study investigates the influence of Aloe vera on water absorption and the in vitro degradation rate of Aloe vera-Ca-alginate hydrogel films, for wound healing and drug delivery applications. The influence of A. vera content (5%, 15% and 25%, v/v) on water absorption was evaluated by the incubation of the films into a 0.1 M HCl solution (pH 1.0), acetate buffer (pH 5.5) and simulated body fluid solution (pH 7.4) during 24h. Results show that the water absorption is significantly higher for films containing high A. vera contents (15% and 25%), while no significant differences are observed between the alginate neat film and the film with 5% of A. vera. The in vitro enzymatic degradation tests indicate that an increase in the A. vera content significantly enhances the degradation rate of the films. Control films, incubated in a simulated body fluid solution without enzymes, are resistant to the hydrolytic degradation, exhibiting reduced weight loss and maintaining its structural integrity. Results also show that the water absorption and the in vitro degradation rate of the films can be tailored by changing the A. vera content.

Advanced biofabrication strategies for skin regeneration and repair.

Skin is the largest organ of human body, acting as a barrier with protective, immunologic and sensorial functions. Its permanent exposure to the external environment can result in different kinds of damage with loss of variable volumes of extracellular matrix. For the treatment of skin lesions, several strategies are currently available, such as the application of autografts, allografts, wound dressings and tissue-engineered substitutes. Although proven clinically effective, these strategies are still characterized by key limitations such as patient morbidity, inadequate vascularization, low adherence to the wound bed, the inability to reproduce skin appendages and high manufacturing costs. Advanced strategies based on both bottom-up and top-down approaches offer an effective, permanent and viable alternative to solve the abovementioned drawbacks by combining biomaterials, cells, growth factors and advanced biomanufacturing techniques. This review details recent advances in skin regeneration and repair strategies, and describes their major advantages and limitations. Future prospects for skin regeneration are also outlined.