Pharmaceutical Formulations Containing Graphene and 5-Fluorouracil for Light-Emitting Diode-Based Photochemotherapy of Skin Cancer.

Nonmelanoma skin cancer (NMSC) is the most common cancer worldwide, among which 80% is basal cell carcinoma (BCC). Current therapies' low efficacy, side effects, and high recurrence highlight the need for alternative treatments. In this work, a partially reduced nanographene oxide (p-rGOn) developed in our laboratory was used. It has been achieved through a controlled reduction of nanographene oxide via UV-C irradiation that yields small nanometric particles (below 200 nm) that preserve the original water stability while acquiring high light-to-heat conversion efficiency. The latter is explained by a loss of carbon-oxygen single bonds (C-O) and the re-establishment of sp2 carbon bonds. p-rGOn was incorporated into a Carbopol hydrogel together with the anticancer drug 5-fluorouracil (5-FU) to evaluate a possible combined PTT and chemotherapeutic effect. Carbopol/p-rGOn/5-FU hydrogels were considered noncytotoxic toward normal skin cells (HFF-1). However, when A-431 skin cancer cells were exposed to NIR irradiation for 30 min in the presence of Carbopol/p-rGOn/5-FU hydrogels, almost complete eradication was achieved after 72 h, with a 90% reduction in cell number and 80% cell death of the remaining cells after a single treatment. NIR irradiation was performed with a light-emitting diode (LED) system, developed in our laboratory, which allows adjustment of applied light doses to achieve a safe and selective treatment, instead of the standard laser systems that are associated with damages in the healthy tissues in the tumor surroundings. Those are the first graphene-based materials containing pharmaceutical formulations developed for BCC phototherapy.

Fabrication of Polymer/Graphene Biocomposites for Tissue Engineering.

Graphene-based materials (GBM) are considered one of the 21st century's most promising materials, as they are incredibly light, strong, thin and have remarkable electrical and thermal properties. As a result, over the past decade, their combination with a diverse range of synthetic polymers has been explored in tissue engineering (TE) and regenerative medicine (RM). In addition, a wide range of methods for fabricating polymer/GBM scaffolds have been reported. This review provides an overview of the most recent advances in polymer/GBM composite development and fabrication, focusing on methods such as electrospinning and additive manufacturing (AM). As a future outlook, this work stresses the need for more in vivo studies to validate polymer/GBM composite scaffolds for TE applications, and gives insight on their fabrication by state-of-the-art processing technologies.

Graphene Oxide Topical Administration: Skin Permeability Studies.

Nanostructured carriers have been widely used in pharmaceutical formulations for dermatological treatment. They offer targeted drug delivery, sustained release, improved biostability, and low toxicity, usually presenting advantages over conventional formulations. Due to its large surface area, small size and photothermal properties, graphene oxide (GO) has the potential to be used for such applications. Nanographene oxide (GOn) presented average sizes of 197.6 ± 11.8 nm, and a surface charge of -39.4 ± 1.8 mV, being stable in water for over 6 months. 55.5% of the mass of GOn dispersion (at a concentration of 1000 µg mL-1) permeated the skin after 6 h of exposure. GOn dispersions have been shown to absorb near-infrared radiation, reaching temperatures up to 45.7 °C, within mild the photothermal therapy temperature range. Furthermore, GOn in amounts superior to those which could permeate the skin were shown not to affect human skin fibroblasts (HFF-1) morphology or viability, after 24 h of incubation. Due to its large size, no skin permeation was observed for graphite particles in aqueous dispersions stabilized with Pluronic P-123 (Gt-P-123). Altogether, for the first time, Gon's potential as a topic administration agent and for delivery of photothermal therapy has been demonstrated.

High-Yield Production of Nano-Lateral Size Graphene Oxide by High-Power Ultrasonication.

Nanographene oxide (GOn) constitutes a nanomaterial of high value in the biomedical field. However, large scale production of highly stable aqueous dispersions of GOn is yet to be achieved. In this work, we explored high-power ultrasonication as a method to reduce particle size of GO and characterized the impact of the process on the physicochemical properties of the material. GOn was obtained with lateral dimensions of 99 ± 43 nm and surface charge of -39.9 ± 2.2 mV. High-power ultrasonication enabled an improvement of stability features, particularly by resulting in a decrease of the average particle size, as well as zeta potential, in comparison to GO obtained by low-power exfoliation and centrifugation (287 ± 139 nm; -29.7 ± 1.2 mV). Remarkably, GOn aqueous dispersions were stable for up to 6 months of shelf-time, with a global process yield of 74%. This novel method enabled the production of large volumes of highly concentrated (7.5 mg mL-1) GOn aqueous dispersions. Chemical characterization of GOn allowed the identification of characteristic oxygen functional groups, supporting high-power ultrasonication as a fast, efficient, and productive process for reducing GO lateral size, while maintaining the material's chemical features.

In vitro temporal HIF-mediated deposition of osteochondrogenic matrix governed by hypoxia and osteogenic factors synergy.

Musculoskeletal interfaces are naturally hypoxic. An understanding of key interactions occurring between different cell populations and their environment is critical for native tissue recapitulation. Here, an enthesis coculture model (preosteoblasts and tendon cells) was used to understand the influence of hypoxia (5% O2 ) and osteogenic medium (OM) supplementation in cells' phenotype modulation. In single cultures, preosteoblasts were found to undergo osteogenic impairment, while tendon cells underwent a maturation process through extracellular matrix (ECM) rescue. When in co-culture, hypoxia and osteoinduction promoted a temporal chondro/osteogenic pathway activation, as observed by an early deposition of cartilaginous ECM associated with HIF1A stabilization and RUNX2 activation, and later hypertrophic differentiation resulting from HIF2A translocation and SOX9 activation. Moreover, the presence of OM under hypoxia was shown to influence the extracellular ROS/HIF1A interplay. Overall, this study revealed a link between biochemical factors and cell-cell crosstalk, providing a molecular framework for hypoxic control and modulation of cells' fate toward enthesis-like phenotypes.

Near-Infrared Radiation-Based Mild Photohyperthermia Therapy of Non-Melanoma Skin Cancer with PEGylated Reduced Nanographene Oxide.

Using a one-step thermal reduction and non-covalent chemical functionalization process, PEGylated reduced nanographene oxide (rGOn-PEG) was produced from nanographene oxide (GOn) and characterized in terms of particle size, dispersion stability, chemistry, and photothermal properties, in view of its use for photothermal therapy (PTT) of non-melanoma skin cancer. GOn infrared spectrum presented more intense bands assigned to oxygen containing functional groups than observed for rGOn-PEG. GOn C/O ratio decreased more than 50% comparing with rGOn-PEG and nitrogen was present in the latter (N at % = 20.6) due to introduction of PEG-NH2. Thermogravimetric analysis allowed estimating the amount of PEG in rGOn-PEG to be of about 56.1%. Simultaneous reduction and PEGylation increased the lateral dimensions from 287 ± 139 nm to 521 ± 397 nm, as observed by transmission electron microscopy and dynamic light scattering. rGOn-PEG exhibited ≈13-fold higher absorbance in the near-infrared radiation (NIR) region, as compared to unmodified GOn. Low power (150 mW cm-2) NIR irradiation using LEDs resulted in rGOn-PEG heating up to 47 °C, which is within the mild PTT temperature range. PEGylation strongly enhanced the dispersibility of rGOn in physiological media (phosphate buffered saline, fetal bovine serum, and cell culture medium) and also improved the biocompatibility of rGOn-PEG, in comparison to GOn (25-250 µg mL-1). After a single NIR LED irradiation treatment of 30 min, a decrease of ≈38% in A-431 cells viability was observed for rGOn-PEG (250 µg mL-1). Together, our results demonstrate the potential of irradiating rGOn-PEG using lower energy, cheaper, smaller, and safer LEDs, as alternative to high power lasers, for NIR mild hyperthermia therapy of cancer, namely non-melanoma skin cancer.

Platelet-rich Blood Derivatives for Tendon Regeneration.

Tendon injuries constitute a significant healthcare problem with variable clinical outcomes. The complex interplay of tissue homeostasis, degeneration, repair, and regeneration makes the development of successful delivery therapeutic strategies challenging. Platelet-rich hemoderivatives, a source of supra-physiologic concentrations of human therapeutic factors, are a promising application to treat tendon injuries from the perspective of tendon tissue engineering, although the outcomes remain controversial.

A Physiology-Inspired Multifactorial Toolbox in Soft-to-Hard Musculoskeletal Interface Tissue Engineering.

Musculoskeletal diseases are increasing the prevalence of physical disability worldwide. Within the body, musculoskeletal soft and hard tissues integrate through specific multitissue transitions, allowing for body movements. Owing to their unique compositional and structural gradients, injuries challenge the native interfaces and tissue regeneration is unlikely to occur. Tissue engineering strategies are emerging to emulate the physiological environment of soft-to-hard tissue interfaces. Advances in biomaterial design enable control over biophysical parameters, but biomaterials alone are not sufficient to provide adequate support and guide transplanted cells. Therefore, biological, biophysical, and biochemical tools can be integrated into a multifactorial toolbox, steering prospective advances toward engineering clinically relevant soft-to-hard tissue interfaces.

Customizable Composite Fibers for Engineering Skeletal Muscle Models.

Engineering tissue-like scaffolds that can mimic the microstructure, architecture, topology, and mechanical properties of native tissues while offering an excellent environment for cellular growth has remained an unmet need. To address these challenges, multicompartment composite fibers are fabricated. These fibers can be assembled through textile processes to tailor tissue-level mechanical and electrical properties independent of cellular level components. Textile technologies also allow control of the distribution of different cell types and the microstructure of fabricated constructs and the direction of cellular growth within the 3D microenvironment. Here, we engineered composite fibers from biocompatible cores and biologically relevant hydrogel sheaths. The fibers are mechanically robust to being assembled using textile processes and could support adhesion, proliferation, and maturation of cell populations important for the engineering of skeletal muscles. We also demonstrated that the changes in the coating of the multicompartment fibers could potentially enhance myogenesis in vitro.

Mesenchymal Stem Cells Empowering Tendon Regenerative Therapies.

Tendon tissues have limited healing capacity. The incidence of tendon injuries and the unsatisfactory functional outcomes of tendon repair are driving the search for alternative therapeutic approaches envisioning tendon regeneration. Cellular therapies aim at delivering adequate, regeneration-competent cell types to the injured tendon and toward ultimately promoting its reconstruction and recovery of functionality. Mesenchymal stem cells (MSCs) either obtained from tendons or from non-tendon sources, like bone marrow (BM-MSCs) or adipose tissue (ASCs), have been receiving increasing attention over the years toward enhancing tendon healing. Evidences from in vitro and in vivo studies suggest MSCs can contribute to accelerate and improve the quality of tendon healing. Nonetheless, the exact mechanisms underlying these repair events are yet to be fully elucidated. This review provides an overview of the main challenges in the field of cell-based regenerative therapies, discussing the role of MSCs in boosting tendon regeneration, particularly through their capacity to enhance the tenogenic properties of tendon resident cells.

A Textile Platform Using Continuous Aligned and Textured Composite Microfibers to Engineer Tendon-to-Bone Interface Gradient Scaffolds.

Tendon-to-bone interfaces exhibit a hierarchical multitissue transition. To replicate the progression from mineralized to nonmineralized tissue, a novel 3D fibrous scaffold is fabricated with spatial control over mineral distribution and cellular alignment. For this purpose, wet-spun continuous microfibers are produced using polycaprolactone (PCL)/ gelatin and PCL/gelatin/hydroxyapatite nano-to-microparticles (HAp). Higher extrusion rates result in aligned PCL/gelatin microfibers while, in the case of PCL/gelatin/HAp, the presence of minerals leads to a less organized structure. Biological performance using human adipose-derived stem cells (hASCs) demonstrates that topography of PCL/gelatin microfibers can induce cytoskeleton elongation, resembling native tenogenic organization. Matrix mineralization on PCL/gelatin/HAp wet-spun composite microfibers suggest the production of an osteogenic-like matrix, without external addition of osteogenic medium supplementation. As proof of concept, a 3D gradient structure is produced by assembling PCL/gelatin and PCL/gelatin/HAp microfibers, resulting in a fibrous scaffold with a continuous topographical and compositional gradient. Overall, the feasibility of wet-spinning for the generation of continuously aligned and textured microfibers is demonsrated, which can be further assembled into more complex 3D gradient structures to mimic characteristic features of tendon-to-bone interfaces.

Enthesis Tissue Engineering: Biological Requirements Meet at the Interface.

IMPACT STATEMENT: The main goal of this review is to give an overview of cell-based and tissue engineered strategies for tendon-to-bone interface. The essential role of cells in tendon-to-bone interface development, healing, and regeneration, is underpinned by the physiological status of the junction. Therefore, recent studies underlining the effect of oxygen concentration and production of growth factors are reviewed. A critical view is made on the use of two-dimensional versus three-dimensional culture systems and mechanical stimulation. An overview of advances on bioengineered strategies in light of the biological/cellular requirements of enthesis will contribute to innovations in tendon-to-bone engineering and regeneration.

Exploring platelet lysate hydrogel-coated suture threads as biofunctional composite living fibers for cell delivery in tissue repair.

To engineer functional tissue substitutes, it is required a multi-component, multi-scale approach that combines both physical, chemical and biological cues. Fiber-based techniques have been explored in the field of tissue engineering to produce structures recapitulating tissue architecture and mechanical properties. In this work, we engineered biofunctional composite living fibers (CLF) as multi-compartment fibers with a mechanically competent core and a hydrogel layer. For this purpose, commercial silk suture threads were coated with a platelet lysate (PL) hydrogel by first embedding the threads in a thrombin solution and then incubating in PL. The fabrication set-up was optimized and the biological performance was studied by encapsulating human adipose-derived stem cells (hASCs). The developed coating process rendered CLF with a homogenous PL hydrogel layer covering suture threads. Encapsulated hASCs were viable up to 14 d in culture and were able to align at the surface of the core fiber and deposit collagen types I and III. In summary, the study shows that PL-hASCs hydrogel coated suture threads represent a simple multi-compartment and multifunctional system, with PL hydrogel offering biofunctionality to guide the biological activities of encapsulated cells in addition to the replication of tissue-level mechanical support provided by the suture threads.

Metabolic Disease Epidemics: Emerging Challenges in Regenerative Medicine.

The interplay between cell/tissue damage caused by metabolic dysfunction and regenerative potential remains elusive. The tissue engineering and regenerative medicine (TERM) field is now facing a worldwide epidemic of obesity. This Forum article uncovers prospective questions to be addressed in TERM toward the development of effective regenerative therapies adjusted to these new demands.

Cellular Complexity at the Interface: Challenges in Enthesis Tissue Engineering.

The complex heterogeneous cellular environment found in tendon-to-bone interface makes this structure a challenge for interface tissue engineering. Orthopedic surgeons still face some problems associated with the formation of fibrotic tissue or re-tear occurring after surgical re-attachment of tendons to the bony insertion or the application of grafts. Unfortunately, an understanding of the cellular component of enthesis lags far behind of other well-known musculoskeletal interfaces, which blocks the development of new treatment options for the healing and regeneration of this multifaceted junction. In this chapter, the main characteristics of tendon and bone cell populations are introduced, followed by a brief description of the interfacial cellular niche, highlighting molecular mechanisms governing tendon-to-bone attachment and mineralization. Finally, we describe and critically assess some challenges faced concerning the use of cell-based strategies in tendon-to-bone healing and regeneration.

Bi-directional modulation of cellular interactions in an in vitro co-culture model of tendon-to-bone interface.

OBJECTIVES: This work aimed at studying in vitro interactions between human tendon-derived cells (hTDCs) and pre-osteoblasts (pre-OBs) that may trigger a cascade of events involved in enthesis regeneration. MATERIALS AND METHODS: The effect of 5 osteogenic medium (OM) conditions over the modulation of hTDCs and pre-OBs towards the tenogenic and osteogenic phenotypes, respectively, was studied. Three different medium conditions were chosen for subsequently establishing a direct co-culture system in order to study the expression of bone, tendon and interface-related markers. RESULTS: A higher matrix mineralization and ALP activity was observed in co-cultures in the presence of OM. Higher transcription levels of bone- (ALPL, RUNX2, SPP1) and interface-related genes (ACAN, COMP) were found in co-cultures. The expression of aggrecan was influenced by the presence of OM and cell-cell interactions occurring in co-culture. CONCLUSIONS: The present work assessed both the influence of OM on cell phenotype modulation and the importance of co-culture models while promoting cell-cell interactions and the exchange of soluble factors in triggering an interface-like phenotype to potentially modulate enthesis regeneration.

Magnetotherapy: The quest for tendon regeneration.

Tendons are mechanosensitive tissues that connect and transmit the forces generated by muscles to bones by allowing the conversion of mechanical input into biochemical signals. These physical forces perform the fundamental work of preserving tendon homeostasis assuring body movements. However, overloading causes tissue injuries, which leads us to the field of tendon regeneration. Recently published reviews have broadly shown the use of biomaterials and different strategies to attain tendon regeneration. In this review, our focus is the use of magnetic fields as an alternative therapy, which has demonstrated clinical relevance in tendon medicine because of their ability to modulate cell fate. Yet the underlying cellular and molecular mechanisms still need to be elucidated. While providing a brief outlook about specific signalling pathways and intracellular messengers as framework in play by tendon cells, application of magnetic fields as a subcategory of physical forces is explored, opening up a compelling avenue to enhance tendon regeneration. We outline here useful insights on the effects of magnetic fields both at in vitro and in vivo levels, particularly on the expression of tendon genes and inflammatory cytokines, ultimately involved in tendon regeneration. Subsequently, the potential of using magnetically responsive biomaterials in tendon tissue engineering is highlighted and future directions in magnetotherapy are discussed.

Continuous Exposure to Simulated Hypergravity-Induced Changes in Proliferation, Morphology, and Gene Expression of Human Tendon Cells.

Gravity influences physical and biological processes, especially during development and homeostasis of several tissues in the human body. Studies under altered gravity have been receiving great attention toward a better understanding of microgravity-, hypogravity (<1 g)-, or hypergravity (>1 g)-induced alterations. In this work, the influence of simulated hypergravity over human tendon-derived cells (hTDCs) was studied at 5, 10, 15, and 20 g for 4 or 16 h, using a large diameter centrifuge. Main results showed that 16 h of simulated hypergravity limited cell proliferation. Cell area was higher in hTDCs cultured at 5, 10, and 15 g for 16 h, in comparison to 1 g control. Actin filaments were more pronounced in hTDCs cultured at 5 and 10 g for 16 h. Focal adhesion kinase (FAK) was mainly expressed in focal adhesion sites upon hypergravity stimulation, in comparison to perinuclear localization in control cells after 16 h; and FAK number/cell increased with increasing g-levels. A tendency toward an upregulation of tenogenic markers was observed; scleraxis (SCX), tenascin C (TNC), collagen type III (COL3A1), and decorin (DCN) were significantly upregulated in hTDCs cultured at 15 g and COL3A1 and DCN were significantly upregulated in hTDCs cultured at 20 g. Overall, simulated hypergravity affected the behavior of hTDCs, with more pronounced effects in the long-term period (16 h) of stimulation.

The effects of platelet lysate patches on the activity of tendon-derived cells.

Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. STATEMENT OF SIGNIFICANCE: Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards simultaneously reducing post-implantation surgical site infections and promoting tendon regeneration for prospective in vivo applications.

Gravity, Tissue Engineering, and the Missing Link.

The influence of microgravity and hypergravity on living systems has attracted significant attention, but the use of these tools in tissue engineering (TE) remains relatively unexplored. This Forum article highlights an emerging field of research to uncover new potential applications at the interface between altered gravity and TE.