Table of contents

The aim of this study was to obtain hierarchical scaffolds combining 3D printing and two electrofluidodynamic methods. The multi-layered scaffold is composed by 3D printed struts, electrospun fibers obtained from poly(-caprolactone) and electrosprayed spheres produced from hydrophobically modified chitosan, namely chitosan grafted with linoleic acid (CHLA). Since CHLA has been used for the first time in the electrospraying (electro dynamic spraying, EDS) process, the formation of spheres needed an optimization process. The EDS process was strongly affected by the solvent mixture composition, concentration of acid used for CHLA dissolution and solution flow rate. By using the optimized electrospraying conditions, uniformly distributed spheres have been obtained, decorating struts and nanofibers. Preliminary biological tests with mouse preosteoblasts (MC3T3-E1) were performed to investigate the effect of the hierarchical scaffold on cell seeding efficacy. Results showed that the hierarchical structure enhances cell seeding efficacy, respect to the 3D printed struts alone, preventing that the cells passed through the struts during the seeding. Moreover, the addition of the electrosprayed nanoparticles does not affect the cell seeding efficiency. The versatility of the proposed structure, with the added value of CHLA nanoparticles decoration could be suitable for several applications in tissue engineering, mainly related to drug delivery systems.

Immunotherapy has emerged as a novel cancer treatment over the last decade, however, efficacious responses to mono-immunotherapy have only been achieved in a relatively small portion of patients whereas combinational immunotherapies often lead to concurrent side effects. It has been proved that the tumor microenvironment (TME) is responsible for tumor immune escape and the ultimate treatment failure. Recently, there has been remarkable progress in both the understanding of the TME and the applications of nanotechnological strategies, and reviewing the emerging immune-regulatory nanosystems may provide valuable information for specifically modulating the TME at different immune stages. In this review, we focus on comprehending the recently-proposed T-cell-based tumor classification and identifying the most promising targets for different tumor phenotypes, and then summarizing the nanotechnological strategies to best target corresponding immune-related factors. For future precise personalized immunotherapy, tailor-made TME modulation strategies conducted by well-designed nanosystems to alleviate the suppressive TME and then promote anti-tumor immune responses will significantly benefit the clinical outcomes of cancer patients.

Nanozymes are nanomaterials with enzyme-like characteristics. As a new generation of artificial enzymes, nanozymes have the advantages of low cost, good stability, simple preparation, and easy storage, allowing them to overcome many of the limitations of natural enzymes in enzymatic therapy. Currently, most reported nanozymes exhibit oxidoreductase-like activities and can regulate redox balance in cells. Nanozymes with superoxide dismutase and catalase activity can be used to scavenge reactive oxygen species (ROS) for cell protection, while those with peroxidase and oxidase activity can generate ROS to kill harmful cells, such as tumor cells and bacteria. In this review, we summarize recent progress in nanozyme-based medicine for enzymatic therapy and highlight the opportunities and challenges in this field for future study.

Advanced biomaterials are increasingly used for numerous medical applications from the delivery of cancer-targeted therapeutics to the treatment of cardiovascular diseases. The issues of foreign body reactions induced by biomaterials must be controlled for preventing treatment failure. Therefore, it is important to assess the biocompatibility and cytotoxicity of biomaterials on cell culture systems before proceeding to in vivo studies in animal models and subsequent clinical trials. Direct use of biomaterials on animals create technical challenges and ethical issues and therefore, the use of non-animal models such as stem cell cultures could be useful for determination of their safety. However, failure to recapitulate the complex in vivo microenvironment have largely restricted stem cell cultures for testing the cytotoxicity of biomaterials. Nevertheless, properties of stem cells such as their self-renewal and ability to differentiate into various cell lineages make them an ideal candidate for in vitro screening studies. Furthermore, the application of stem cells in biomaterials screening studies may overcome the challenges associated with the inability to develop a complex heterogeneous tissue using primary cells. Currently, embryonic stem cells, adult stem cells, and induced pluripotent stem cells are being used as in vitro preliminary biomaterials testing models with demonstrated advantages over mature primary cell or cell line based in vitro models. This review discusses the status and future directions of in vitro stem cell-based cultures and their derivatives such as spheroids and organoids for the screening of their safety before their application to animal models and human in translational research.

Despite the positive achievements attained, the treatment of male urethral strictures and hypospadiases still remains a challenge, particularly in cases of severe urethral defects. Complications and the need for additional interventions in such cases are common. Also, shortage of autologous tissue for graft harvesting and significant morbidity in the location of harvesting present problems and often lead to staged treatment. Tissue engineering provides a promising alternative to the current sources of grafts for urethroplasty. Since the first experiments in urethral substitution with tissue engineered grafts, this topic in regenerative medicine has grown remarkably, as many different types of tissue-engineered grafts and approaches in graft design have been suggested and tested in vivo. However, there have been only a few clinical trials of tissue-engineered grafts in urethral substitution, involving hardly more than a hundred patients overall. This indicates that the topic is still in its inception, and the search for the best graft design is continuing. The current review focuses on the state of the art in urethral regeneration with tissue engineering technology. It gives a comprehensive overview of the components of the tissue-engineered graft and an overview of the steps in graft development. Different cell sources, types of scaffolds, assembling approaches, options for vascularization enhancement and preclinical models are considered.

Nanomaterials (NMs) have revolutionized multiple aspects of medicine by enabling novel sensing, diagnostic, and therapeutic approaches. Advancements in processing and fabrication have also allowed significant expansion in the applications of the major classes of NMs based on polymer, metal/metal oxide, carbon, liposome, or multi-scale macro-nano bulk materials. Concomitantly, concerns regarding the nanotoxicity and overall biocompatibility of NMs have been raised. These involve putative negative effects on both patients and those subjected to occupational exposure during manufacturing. In this review, we describe the current state of testing of NMs including those that are in clinical use, in clinical trials, or under development. We also discuss the cellular and molecular interactions that dictate their toxicity and biocompatibility. Specifically, we focus on the reciprocal interactions between NMs and host proteins, lipids, and sugars and how these induce responses in immune and other cell types leading to topical and/or systemic effects.

Osteoarthritis (OA) is a leading cause of chronic disability. It is a progressive disease, involving pathological changes to the entire joint, resulting in joint pain, stiffness, swelling, and loss of mobility. There is currently no disease-modifying pharmaceutical treatment for OA, and the treatments that do exist suffer from significant side effects. An increasing understanding of the molecular pathways involved in OA is leading to many potential drug targets. However, both current and new therapies can benefit from a targeted approach that delivers drugs selectively to joints at therapeutic concentrations, while limiting systemic exposure to the drugs. Delivery systems including hydrogels, liposomes, and various types of particles have been explored for intra-articular drug delivery. This review will describe progress over the past several years in the development of polymer-based particles for OA treatment, as well as their in vitro, in vivo, and clinical evaluation. Systems based on biopolymers such as polysaccharides and polypeptides, as well as synthetic polyesters, poly(ester amide)s, thermoresponsive polymers, poly(vinyl alcohol), amphiphilic polymers, and dendrimers will be described. We will discuss the role of particle size, biodegradability, and mechanical properties in the behavior of the particles in the joint, and the challenges to be addressed in future research.

For decades, collagen has been among the most widely used biomaterials with several biomedical applications. Recently, researchers have shown a keen interest in collagen obtained from marine sources because of its biocompatibility, biodegradability, ease of extractability, safety, low immunogenicity, and low production costs. A wide variety of marine collagen-based scaffolds have been developed for bone tissue engineering, and these scaffolds display excellent biological effects. This review aims to provide an overview of the biological effects of marine collagen in bone engineering, such as promoting osteogenesis and collagen synthesis, inhibiting inflammation, inducing the differentiation of cartilage, and improving bone mineral density. Marine collagen holds great promise as a biomaterial in bone tissue engineering.

Phototherapy including photothermal therapy (PTT) and photodynamic therapy (PDT), as non-invasive therapy approaches, have gained accumulated attention for cancer treatment in past years. PTT and PDT can generate local hyperthermia effects and reactive oxygen species (ROS) respectively, for tumor eradication. To improve the therapeutic performance while minimizing the reverse side effects of phototherapy, extensive efforts have been devoted to developing stimuli-activatable (e.g. pH, redox, ROS, enzyme, etc) nanomaterials for tumor-specific delivery/activation of the phototherapeutics. In this review, we first overviewed the recent advances of the engineered stimuli-responsive nanovectors for the phototherapy of cancer. We particularly summarized the progress of stimuli-activatable nanomaterials-based combinatory therapy strategies for augmenting the performance of phototherapy. We further discuss challenges for the clinical translation of nanomaterials-based phototherapy.

As a promising non-invasive treatment method, phototherapy has attracted extensive attention in the field of combined cancer therapy. Among various optical agents, organic ones have been considered as a promising clinical phototheranostic agent due to its high safety and non-toxic property. In addition, due to the clear structure, facile processability, organic optical agents can be easily endowed with multiple imaging and phototherapeutic functions, significantly simplifying the relatively complex system of imaging-guided combined cancer therapy. This review summarizes the recent research on organic optical agents in imaging-guided combined cancer therapy. The application of organic optical agents in a variety of combined cancer therapeutic modes guided by imaging are introduced respectively, including photodynamic and photothermal combined therapy, phototherapy-combined cancer chemotherapy, and phototherapy-combined cancer immunotherapy. Finally, the concluding remarks and the future prospects are discussed.

Nanocarriers (NCs) for delivery anticancer therapeutics have been under development for decades. Although great progress has been achieved, the clinic translation is still in the infancy. The key challenge lies in the biological barriers which lie between the NCs and the target spots, including blood circulation, tumor penetration, cellular uptake, endo-/lysosomal escape, intracellular therapeutics release and organelle targeting. Each barrier has its own distinctive microenvironment and requires different surface charge. To address this challenge, charge-reversal polymeric NCs have been a hot topic, which are capable of overcoming each delivery barrier, by reversing their charges in response to certain biological stimuli in the tumor microenvironment. In this review, the triggering mechanisms of charge reversal, including pH, enzyme and redox approaches are summarized. Then the corresponding design principles of charge-reversal NCs for each delivery barrier are discussed. More importantly, the limitations and future prospects of charge-reversal NCs in clinical applications are proposed.

With the improvement of living standards, cancer has become a great challenge around the world during last decades, meanwhile, abundant nanomaterials have been developed as drug delivery system (DDS) or cancer theranostic agents (CTAs) with their outstanding properties. However, low multifunctional efficiency and time-consuming synthesis limit their further applications. Nowadays, green chemistry, in particular, the concept of atom economy, has defined new criteria for the simplicity and efficient production of biomaterials for nanomedicine, which not only owns the property of spatio-temporal precision imaging, but also possess the ability to treat cancer. Interestingly, metal-organic framework (MOF) is an excellent example to meet the requirements behind this concept and has great potential for next-generation nanomedicine. In this review, we summarize our recent researches and inspiring progresses in designing DDS and CTA built from MOF, aiming to show the simplicity, control, and versatility, and provide views on the development of MOF-based nanomedicine in the future.

Vitreoretinal surgery is an essential approach to treat proliferative diabetic vitreopathy, retinal detachment, retinal tear, ocular trauma, and macular holes. The removal of the natural vitreous and the replacement with substitutes are critical steps for retina reattachment. Vitreous substitutes including silicone oil (SiO), air, sulfur hexafluoride (SF₆), and perfluoropropane (C₃F₈), have been widely applied in clinical practice. However, these substitutes are reported to cause complications such as emulsification, high intraocular pressure, and lens opacification. Polymeric hydrogels are a kind of material with favorable physical, mechanical properties, and adaptable biocompatibility, thus being highly expected to be ideal vitreous substitutes. Despite years of research, very few polymeric hydrogels can be applied practically in the vitreous cavity. In this review, we focus on the development of polymeric natural-based hydrogels and synthetic hydrogels. Particularly, we pay attention to recent advances in the novel stimuli-response and self-assembly supramolecular hydrogels. Characterized by easy injectability and long residence time, this kind of hydrogel becomes the potentially promising candidates for ideal vitreous substitutes. Finally, we evaluate the current challenges and provide the future directions of vitreous substitutes.

Ferroptosis is a new type of programmed cell death, which is expected to become an important strategy of cancer treatment. Traditional strategies for inducing iron death are small molecule inducers based on biological agents. However, because of their poor water solubility, low cell targeting ability and fast metabolism in vivo, it is difficult for molecular drugs to play the long-acting role of ferroptosis induction. With the further study of ferroptosis and development of nanotechnology, nanomaterials have been proved to be more efficient drugs for inducing ferroptosis than those biological drugs. Therein, iron-based nanomaterials can directly release high concentrations of irons and increase reactive oxygen species levels in cells, which produce a better induction effect for ferroptosis. Whereas, it is challenging to differentiate nanoparticle-induced ferroptosis and traditional inducing strategies, elucidate the detailed mechanisms and further classify the synthetical methods of nanomaterials. For better guidance on the development of anticancer strategies, comprehensive summary of the latest developments of ferroptosis related nanomaterials, especially iron-based nanomaterials are in urgent need. In the paper, we summarized the main mechanisms of ferroptosis, highlighted the latest developments of nanomaterials for ferroptosis, and emphasized the advantages of iron-based nanomaterials for ferroptosis. The future prospect in this field was also discussed, paving the way for the related nanomaterials in the clinical cancer therapy.

Articular cartilage has an avascular structure with a poor ability for self-repair; therefore, many challenges arise in cases of trauma or disease. It is of utmost importance to identify the proper biomaterial for tissue repair that has the capability to direct cell recruitment, proliferation, differentiation, and tissue integration by imitating the natural microenvironment of cells and transmitting an orchestra of intracellular signals. Cartilage extracellular matrix (cECM) is a complex nanostructure composed of divergent proteins and glycosaminoglycans (GAGs), which regulate many functions of resident cells. Numerous studies have shown the remarkable capacity of ECM-derived biomaterials for tissue repair and regeneration. Moreover, given the importance of biodegradability, biocompatibility, 3D structure, porosity, and mechanical stability in the design of suitable scaffolds for cartilage tissue engineering, demineralized bone matrix (DBM) appears to be a promising biomaterial for this purpose, as it possesses the aforementioned characteristics inherently. To the best of the authors' knowledge, no comprehensive review study on the use of DBM in cartilage tissue engineering has previously been published. Since so much work is needed to address DBM limitations such as pore size, cell retention, and so on, we decided to draw the attention of researchers in this field by compiling a list of recent publications. This review discusses the implementation of composite scaffolds of natural or synthetic origin functionalized with cECM or DBM in cartilage tissue engineering. Cutting-edge advances and limitations are also discussed in an attempt to provide guidance to researchers and clinicians.

Nucleic acid-based gene therapy has recently made important progress toward clinical implementation, and holds tremendous promise for the treatment of some life-threatening diseases, such as cancer and inflammation. However, the on-demand delivery of nucleic acid therapeutics in target cells remains highly challenging. The development of delivery systems responsive to specific pathological cues of diseases is expected to offer promising alternatives for overcoming this problem. Among them, the reactive oxygen species (ROS)-responsive delivery systems, which in response to elevated ROS in cancer cells or activated inflammatory cells, can deliver nucleic acid therapeutics on-demand via ROS-induced structural and assembly behavior changes, constitute a promising approach for cancer and anti-inflammation therapies. In this short review, we briefly introduce the ROS-responsive chemical structures, ROS-induced release mechanisms and some representative examples to highlight the current progress in constructing ROS-responsive delivery systems. We aim to provide new insights into the rational design of on-demand gene delivery vectors.

The use of medicinal plants is as ancient as human civilization. The development of phytochemistry and pharmacology facilitates the identification of natural bioactive compounds and their mechanisms of action, including against cancer. The efficacy and the safety of a bioactive compound depend on its optimal delivery to the target site. Most natural bioactive compounds (phenols, flavonoids, tannins, etc) are unable to reach their target sites due to their low water solubility, less cellular absorption, and high molecular weight, leading to their failure into clinical translation. Therefore, many scientific studies are going on to overcome the drawbacks of natural products for clinical applications. Several studies in India, as well as worldwide, have proposed the development of natural products-based nanoformulations to increase their efficacy and safety profile for cancer therapy by improving the delivery of natural bioactive compounds to their target site. Therefore, we are trying to discuss the development of natural products-based nanoformulations in India to improve the efficacy and safety of natural bioactive compounds against cancer.

An efficient long-term intracellular growth factor release system in simulated microgravity for osteogenic differentiation was prepared based on polylactic acid (PLA) and polyhydroxyalkanoate (PHA) nanoparticles (NPs) for loading of bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 7 (BMP7) (defined as sB2-PLA-NPs and sB7-PHA-NPs), respectively, associated with osteogenic differentiation of human adipose derived stem cells (hADSCs). On account of soybean lecithin (SL) as biosurfactants, sB2-PLA-NPs and sB7-PHA-NPs had a high encapsulation efficiency (>80%) of BMPs and uniform small size (<100 nm), and showed a different slow-release to provide BMP2 in early stage and BMP7 in late stages of osteogenic differentiation within 20 d, due to degradation rate of PLA and PHA in cells. After uptake into hADSCs, by comparison with single sB2-PLA-NPs or sB7-PHA-NPs, the Mixture NPs compound of sB2-PLA-NP and sB7-PHA-NP with a mass ratio of 1:1, can well-promote ALP activity, expression of OPN and upregulated related osteo-genes. Directed osteo-differentiation of mixture NPs was similar to result of sustained free-BMP2 and BMP7-supplying (sFree-B2&B7) in simulated microgravity, which demonstrated the reliability and stability of Mixture NPs as a long-term osteogenic differentiation system in space medicine and biology in future.

Critical limb ischemia (CLI) is a severe type of peripheral artery disease (PAD) which occurs due to an inadequate supply of blood to the limb extremities. Patients with CLI often suffer from extreme cramping pain, impaired wound healing, immobility, cardiovascular complications, amputation of the affected limb and even death. The conventional therapy for treating CLI includes surgical revascularization as well as restoration of angiogenesis using growth factor therapy. However, surgical revascularization is only suitable for a small percentage of CLI patients and is associated with a high perioperative mortality rate. The use of growth factors is also limited in terms of their poor therapeutic angiogenic potential, as observed in earlier clinical studies which could be attributed to their poor bio-availability and non-specificity issues. Therefore, to overcome the aforesaid disadvantages of conventional strategies there is an urgent need for the advancement of new alternative therapeutic biomaterials to treat CLI. In the past few decades, various research groups, including ours, have been involved in developing different pro-angiogenic nanomaterials. Among these, zinc oxide nanoflowers (ZONFs), established in our laboratory, are considered one of the more potent nanoparticles for inducing therapeutic angiogenesis. In our earlier studies we showed that ZONFs promote angiogenesis by inducing the formation of reactive oxygen species and nitric oxide (NO) as well as activating Akt/MAPK/eNOS cell signaling pathways in endothelial cells. Recently, we have also reported the therapeutic potential of ZONFs to treat cerebral ischemia through their neuritogenic and neuroprotective properties, exploiting angio-neural cross-talk. Considering the excellent pro-angiogenic properties of ZONFs and the importance of revascularization for the treatment of CLI, in the present study we comprehensively explore the therapeutic potential of ZONFs in a rat hind limb ischemia model (established by ligating the hind limb femoral artery), an animal model that mimics CLI in humans. The behavioral studies, laser Doppler perfusion imaging, histopathology and immunofluorescence as well as estimation of serum NO level showed that the administration of ZONFs could ameliorate ischemia in rats at a faster rate by promoting therapeutic angiogenesis to the ischemic sites. Altogether, the present study offers an alternative nanomedicine approach employing ZONFs for the treatment of PADs.

Single-mode magnetic resonance imaging (MRI) contrast agents (CAs) in clinical settings are easily disturbed by calcification, bleeding, and adipose signals, which result in inaccurate diagnoses. In this study, we developed a highly efficient T₁–T₂ dual-mode MRI CA using an ultra-small gadolinium oxide-decorated magnetic iron oxide nanocrystal (GMIO). The gadolinium element could effectively alter the magnetic properties of the GMIO from soft-ferromagnetism to superparamagnetism. In addition, when the Gd/Fe ratio was 15% (designated as GMIO-2), the GMIO-2 possessed the best superparamagnetism and highest magnetism. Subsequently, T₁ and T₂ values of GMIO-2 were measured through a series of turbo spin-echo images and then multi-spin echo sequence, respectively. Based on this, T₁ and T₂ relaxivities of GMIO-2 were calculated and were the highest (r₁: 1.306 m M⁻¹ s⁻¹ and r₂: 234.5 m M⁻¹ s⁻¹) when compared to other groups. The cytotoxicity of GMIO-2 was negligible under a wide range of dosages, thus exhibiting excellent cell biocompatibility. Moreover, GMIO-2 could quickly diffuse into cells, leading to its effective accumulation. The systemic delivery of GMIO-2 resulted in an excellent T₁–T₂ dual-mode MRI contrast effect in kidneys, which is expected to improve the diagnosis of kidney lesions. Therefore, this work provides a promising candidate for the development of a T₁–T₂ dual-mode MRI CA.

Tissue engineering scaffolds have transformed from passive geometrical supports for cell adhesion, extension and proliferation to active, dynamic systems that can in addition, trigger functional maturation of the cells in response to external stimuli. Such 'smart' scaffolds require the incorporation of active response elements that can respond to internal or external stimuli. One of the key elements that direct the cell fate processes is mechanical stress. Different cells respond to various types and magnitudes of mechanical stresses. The incorporation of a pressure-sensitive element in the tissue engineering scaffold therefore, will aid in tuning the cell response to the desired levels. Boron nitride nanotubes (BNNTs) are analogous to carbon nanotubes and have attracted considerable attention due to their unique amalgamation of chemical inertness, piezoelectric property, biocompatibility and, thermal and mechanical stability. Incorporation of BNNTs in scaffolds confers them with piezoelectric property that can be used to stimulate the cells seeded on them. Biorecognition and solubilization of BNNTs can be engineered through surface functionalization with different biomolecules. Over the years, the importance of BNNT has grown in the realm of healthcare nanotechnology. This review discusses the salient properties of BNNTs, the influence of functionalization on their in vitro and in vivo biocompatibility, and the uniqueness of BNNT-incorporated tissue engineering scaffolds.

In situ forming tissue adhesives based on biopolymers offer advantages over conventional sutures and staples in terms of biocompatibility, biodegradability, ease of application and improved patient compliance and comfort. Here, we describe the evaluation of in situ gelling hydrogel system based on dextran dialdehyde (DDA) obtained by periodate oxidization of dextran and chitosan hydrochloride (CH) as tissue adhesive. The hydrogel was prepared by reacting aldehyde functions in DDA with the amino functions in CH via Schiff's reaction. The gelation reaction was instantaneous and took just 4 s. The DDA-CH hydrogel as tissue adhesive was evaluated on a sheep lung parenchymal injury model and a pig aortic model and was compared with the commercially available tissue sealant, Bioglue®. The DDA-CH glue could completely seal the sheep lung incision site even at inflation with air way pressure of 30 cm of H₂O with no air leak observed in the incision sites (n = 8) in any of the animals. Histological analyses showed mild inflammation after 2 weeks, comparable to Bioglue®. Resorption of test material by giant cells with no adverse effect on lung parenchyma was seen after 3 months. The DDA-CH glue was also very effective in sealing aortic incisions in a pig model (n = 4) with no failures and aneurisms. The endoluminal surface of the sealed incision in all cases showed intact apposition with adequate healing across the incision. No tissue necrosis or inflammation of endothelial surface could be seen grossly. Our studies show that the DDA-CH hydrogel could function as an effective sealant for the prevention of air and blood leaks following lung and vascular surgery.

Bone cancer is a malignant tumor that originates in the bone and destroys the healthy bone tissues. Of the various types of bone tumors, osteosarcoma is the most commonly diagnosed primary bone malignancy. The standard treatment for primary malignant bone tumors comprises surgery, chemotherapy and radiotherapy. Owing to the lack of proven treatments, different forms of alternative therapeutic approaches have been examined in recent decades. Among the new therapeutic methodologies, nanotechnology-based anticancer therapy has paved the way for new targeted strategies for bone cancer treatment and bone regeneration. They include approaches such as the co-delivery of multiple drug cargoes, the enhancement of their biodistribution and transport properties, normalizing accumulation and the optimization of drug release profiles to overcome shortcomings of the existing therapy. This review examines the standard treatments for osteosarcoma, their lacunae, and the evolving therapeutic strategies based on nanocarrier-mediated combinational drug delivery systems, and future perspectives for osteosarcoma therapy.

Cellular metabolites play a crucial role in promoting and regulating cellular activities, but it has been difficult to monitor these cellular metabolites in living cells and in real time. Over the past decades, iterative development and improvements of fluorescent probes have been made, resulting in the effective monitoring of metabolites. In this review, we highlight recent progress in the use of fluorescent probes for tracking some key metabolites, such as adenosine triphosphate, cyclic adenosine monophosphate, cyclic guanosine 5'-monophosphate, Nicotinamide adenine dinucleotide (NADH), reactive oxygen species, sugar, carbon monoxide, and nitric oxide for both whole cell and subcellular imaging.

Magnesium and its alloys have the potential to serve as a revolutionary class of biodegradable materials, specifically in the field of degradable implants for orthopedics. However, the corrosion rate of commercially pure magnesium is high and does not match the rate of regeneration of bone tissues. In this work, magnesium alloys containing zinc and cerium, either alone or in combination, were investigated and compared with commercially-pure magnesium as biomaterials. The microstructure, mechanical properties, corrosion resistance, and response of osteoblasts in vitro were systematically assessed. Results reveal that alloying with Ce results in grain refinement and weakening of texture. The tensile test revealed that the ternary alloy offered the best combination of elastic modulus (41.1 ± 0.5 GPa), tensile strength (234.5 ± 4.5 MPa), and elongation to break (17.1 ± 0.4%). The ternary alloy was also the most resistant to corrosion (current of 0.85 ± 0.05 × 10⁻⁴ A cm⁻²) in simulated body fluid than the other alloys. The response of MC3T3-E1 cells in vitro revealed that the ternary alloy imparts minimal cytotoxicity. Interestingly, the ternary alloy was highly efficient in supporting osteogenic differentiation, as revealed by the expression of alkaline phosphatase and calcium deposition. In summary, the extruded Mg alloy containing both Zn and Ce exhibits a combination of mechanical properties, corrosion resistance, and cell response that is highly attractive for engineering biodegradable orthopedic implants.

Retinal prostheses have been developed to restore vision in blind patients suffering from such diseases as retinitis pigmentosa. In our previous studies, we developed a retinal prosthesis called dye-coupled film by chemical coupling of photoelectric dyes, which absorb light and then generate electrical potential, with a polyethylene film surface. The dye-coupled film is nontoxic, and we recovered the vision of a monkey with macular degeneration. The amount of dye on the dye-coupled film, however, decreased to one-third after five months in the monkey's eye. The photoelectric dye consists of a cation with photoresponsivity and a bromide ion (Br⁻). Therefore, an anion-exchange reaction could be applied to the dye-coupled film to improve its durability. In this study, the anion-exchange reaction was conducted using bis(trifluoromethanesulfonyl)imide ion (TFSI⁻), which has lower nucleophilicity than Br⁻. First, the long-term durability was examined without using animal subjects and in a short period. Subsequently, an elemental analysis was performed to confirm the exchange between Br⁻ and TFSI⁻, and chemical properties, such as photoresponsivity and durability, before and after the anion exchange, were evaluated. It was quantitatively confirmed that the long-term durability of dye-coupled films can be evaluated in an in vitro environment and in a short period of one-thirtieth by utilizing a saline solution at 60 °C, compared with an in vivo environment. In addition, the durability of the dye-coupled film with TFSI⁻ was improved to 270%–320% compared with that of the dye-coupled film with Br⁻.

Biofabrication has been adapted in engineering patient-specific biosynthetic grafts for bone regeneration. Herein, we developed a three-dimensional (3D) high-resolution, room-temperature printing approach to fabricate osteoconductive scaffolds using calcium phosphate cement (CPC). The non-aqueous CPC bioinks were composed of tetracalcium phosphate, dicalcium phosphate anhydrous, and Polyvinyl butyral (PVB) dissolved in either ethanol (EtOH) or tetrahydrofuran (THF). They were printed in an aqueous sodium phosphate bath, which performs as a hardening accelerator for hydroxyapatite formation and as a retainer for 3D microstructure. The PVB solvents, EtOH or THF, affected differently the slurry rheological properties, scaffold microstructure, mechanical properties, and osteoconductivity. Our proposed approach overcomes limitations of conventional fabrication methods, which require high-temperature (>50 °C), low-resolution (>400 μm) printing with an inadequate amount of large ceramic particles (>35 μm). This proof-of-concept study opens venues in engineering high-resolution, implantable, and osteoconductive scaffolds with predetermined properties for bone regeneration.

Photodynamic therapy is a new technology for disease diagnosis and treatment in modern medical clinics. The main advantages of photodynamic therapy are low toxicity and side effects, a wide range of applications, no drug resistance, and no obvious trauma in the treatment process. However, to achieve effective photodynamic therapy, new photosensitizer carriers need to be constructed, which can selectively deliver photosensitizers into tumor tissues. In this work, a photoactivatable antibody–Chlorin e6 conjugate with a dual-function to target tumor tissue and realize cancer photodynamic therapy is constructed. Both in vitro and in vivo experiments indicate that the antibody–Chlorin e6 conjugate has the ability to target tumors rapidly and efficiently, and has the ability to generate reactive oxygen species and kill tumor cells. Overall, this photoactivable antibody–Chlorin e6 conjugate may provide a promising strategy to address the current challenges of cancer photodynamic therapy.

Akermanite (Aker) has been widely used for bone regeneration through regulating osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). Previously, we developed an injectable Aker/sodium alginate (Aker/SA) hydrogel to facilitate bone regeneration. However, the effect of this injectable hydrogel on the in vivo response, particularly the inflammatory response, has not been fully understood. Here, to elucidate the response following the implantable of Aker/SA hydrogel, we investigated the interaction among Aker/SA hydrogel, inflammatory cells and cells involved in bone regeneration (BMSCs). Specifically, we cultured macrophages (RAW 264.7 cell line) with the extract liquid of Aker/SA and assessed their phenotypic changes. Subsequently, BMSCs (2 × 10⁵ cells per 24 well) were cultured with different conditioned media including that of Aker/SA hydrogel-activated macrophages to investigate their effect on cell migration. Finally, Aker/SA hydrogel was injected subcutaneously (1 × 10⁶ cells ml⁻¹) in rat to verify its effect in vivo. The in vitro results indicated that Aker/SA hydrogel activated macrophages towards M2 phenotype and stimulated macrophages to express anti-inflammatory factors. In addition, the conditioned medium collected from Aker-activated macrophages could accelerate the migration of BMSCs in 24 h. Consistent with the in vitro results, when the Aker/SA hydrogel was injected subcutaneously, more M2 macrophages could be observed than when the SA solution was injected after 7 d. Besides, when BMSCs were delivered via subcutaneous injection, more BMSCs were recruited by the Aker/SA hydrogel than the SA solution. All these results suggest that the Aker/SA hydrogel can modulate the immune environment at the implantation site and subsequently recruit BMSCs, which can be one of the mechanisms through which the Aker/SA hydrogel accelerates new bone formation.

Additive manufacturing or three-dimensional (3D) printing technology is increasingly being employed in biochemical as well as clinical applications and more importantly in fabrication of microfluidic devices. However, the microfluidic community mainly relies on photolithography for fabrication of a defined mask, which is both tedious and expensive requiring clean room settings as well as limited to the generation of two-dimensional features. In this work, we 3D printed nanoclay-reinforced Pluronic ink as a sacrificial material, which exhibited shear thinning behavior and superior printability allowing the fabrication of unsupported or overhanging templates of channels with uniform diameter and circular cross-sections. To highlight the potential and effectiveness of the presented approach, we fabricated a human blood vessel-on-a-chip model with curved as well as straight channels. These channels were then lined up with human umbilical vein endothelial cells (HUVECs) and subjected to a dynamic culture for 10 d to explore the effect of shear stress on HUVEC morphology based on the location of HUVECs in the devices. Overall, we presented a highly affordable, practical and useful approach in manufacturing of polydimethylsiloxane-based devices with closed microfluidic channels, which holds great potential for a numerous applications, such as but not limited to organ-on-a-chip, microfluidics, point-of-care devices and drug screening platforms.

Bioceramic morphology plays a crucial role in bone repair and regeneration. It is extensively utilized in bone scaffold synthesis due to its better biological system activity and biocompatibility. Here, ultra-long tricalcium phosphate (UTCP) was synthesized with the assistance of the ultrasonication method. The UTCP was modified as a scaffold by the reinforcement of a methacrylate chitosan (MAC) polymer. The functionality of UTCP, UTCP/MAC, and methotrexate (MTX)-loaded composites was characterized through Fourier transform infrared spectroscopy. The crystalline natures are investigated by x-ray diffraction, and the results show the UTCP crystalline phase is not altered after the reinforcement of the MAC polymer and loading of MTX drugs. The morphological analyses were observed through electron microscopic analysis, and polymer-coated rod structures were observed. The UTCP/MAC composite mechanical stress was increased from 1813 Pa of UTCP to 4272 Pa. MTX loading and release at 79.0% within 3 h and 76.15% at 20 h, respectively, were achieved. The UTCP/MAC and UTCP/MAC/MTX's osteoblast-like (MG-63) cell viability was investigated, and the MTX-loaded UTCP/MAC composite exhibits good viability behavior up to 96.0% in 14 d. The results confirm the higher compatibility of the composite and profitable cell growth. It may be suitable for bone implantation preparation, and it helps in faster regeneration of bone tissue after in vivo and clinical evaluation.

There are limitations in current medications of articular cartilage injuries. Although injectable bioactive hydrogels are promising options, they have decreased biomechanical performance. Researchers should consider many factors when providing solutions to overcome these challenges. In this study, we created an injectable composite hydrogel from chitosan and human acellular cartilage extracellular matrix (ECM) particles. In order to enhance its mechanical properties, we reinforced this hydrogel with microporous microspheres composed of the same materials as the structural building blocks of the scaffold. Articular cartilage from human donors was decellularized by a combination of physical, chemical, and enzymatic methods. The decellularization efficiency was assessed by histological analysis and assessment of DNA content. We characterized the composite constructs in terms of storage modulus, gelation time, biocompatibility, and differentiation potential. The results showed that mechanical behavior increased with an increase in microsphere content. The sample that contained 10% microsphere had an enhanced storage modulus of up to 90 kPa. Biocompatibility and preliminary differentiation investigations revealed that this composite hydrogel might have potential benefits for cartilage tissue engineering.

Periodontitis is a chronic inflammatory disease characterized by loss of attachment and destruction of the periodontium. Decellularized sheet, as an advanced tissue regeneration engineering biomaterial, has been researched and applied in many fields, but its effects on periodontal regeneration remain unclear. In this study, the biological properties of decellularized human periodontal ligament cell (dHPDLC) sheets were evaluated in vitro. Polycaprolactone/gelatin (PCL/GE) nanofibers were fabricated as a carrier to enhance the mechanical strength of the dHPDLC sheet. 15-deoxy-${{{\Delta }}^{12,14}}$-prostaglandin J₂ (15d-PGJ₂) nanoparticles were added for anti-inflammation and regeneration improvement. For in vivo analysis, dHPDLC sheets combined with 15d-PGJ₂ nanoparticles, with or without PCL/GE, were implanted into rat periodontal defects. The periodontal regeneration effects were identified by microcomputed tomography (micro-CT) and histological staining, and immunohistochemistry. The results revealed that DNA content was reduced by 96.6%. The hepatocyte growth factor, vascular endothelial growth factor, and basic fibroblast growth factor were preserved but reduced. The expressions or distribution of collagen I and fibronectin were similar in dHPDLC and nondecellularized cell sheets. The dHPDLC sheets maintained the intact structure of the extracellular matrix. It could be recellularized by allogeneic human periodontal stem ligament cells and retain osteoinductive potential. Newly formed bone, cementum, and PDL were observed in dHPDLC sheets combined with 15d-PGJ₂ groups, with or without PCL/GE nanofibers, for four weeks post-operation in vivo. Bringing together all these points, this new construct of dHPDLC sheets can be a potential candidate for periodontal regeneration in an inflammatory environment of the oral cavity.

In this study, the extraction conditions of Nostoc commune Vauch polysaccharide (NCVP) were optimized by single factor and orthogonal experiments. Then, the NCVP microcapsules (NCVPM) were prepared. After analyzing the microcapsule structural and thermal characteristics, the skin wound healing ability was studied by establishing back trauma rat models. Results showed that the NCVP yield was 10.37% under the following optimum conditions: 210 min extraction time, solid–liquid ratio of 1:50 and extraction temperature of 90 °C. The overall performance of the microcapsule was the best when the concentration of sodium alginate, calcium chloride and chitosan was 2%, 3% and 0.3%, respectively. NCVPM had spherical morphology, typical microcapsule structural characteristics and good thermal stability, and NCVP was dispersed in the microcapsules. NCVPM showed good biocompatibility and biodegradability, which met the requirements for slow-release polymer materials. After 14 days of treatment, the wound healing rate was 92.4%, the cells were arranged neatly and regularly, the cell nucleus became large and elliptical, the cell had a tendency to divide, and the fibers and microvessel were significantly more. By evaluating the mechanism, NCVPM could increase the content of hydroxyproline and glutathione to protect cells from oxidative damage, leading in turn to accelerated wound healing and shorter wound healing times. It could also accelerate cell division, collagen and microvascular production by increasing transcription levels of vascular endothelial growth factor mRNA and miRNA-21.

One of the biggest hindrances in tissue engineering in recent decades has been the complexity of the prevascularized channels of the engineered scaffold, which was still lower than that of human tissues. Another relative difficulty was the lack of precision molding capability, which restricted the clinical applications of the huge engineered scaffold. In this study, a promising approach was proposed to prepare hydrogel scaffold with prevascularized channels by liquid bath printing, in which chitosan/β-sodium glycerophosphate served as the ink hydrogel, and gelation/nanoscale bacterial cellulose acted as the supporting hydrogel. Here, the ink hydrogel was printed by a versatile nozzle and embedded in the supporting hydrogel. The ink hydrogel transformed into liquid effluent at low temperature after the cross-linking of gelatin by microbial transglutaminase (mTG). No residual template was seen on the channel surface after template removal. This preparation had a high degree of freedom in the geometry of the channel, which was demonstrated by making various prevascularized channels including circular, branched, and tree-shaped networks. The molding accuracy of the channel was assessed by studying the roundness of the cross section of the molded hollow channel, and the effect of the mechanical properties by adding bacterial cellulose to the supporting hydrogel was analyzed. Human umbilical vein endothelial cells were injected into the aforementioned channels which formed a confluent and homogeneous distribution on the surface of the channels. Altogether, these results showed that this approach can construct hydrogel scaffolds with complex and accurate molding prevascularized channels, and hs great potential to resolve the urgent vascularization issue of bulk tissue-engineering scaffold.

Acellular matrix is a type of promising biomaterial for wound healing promotion. Although acellular bovine and porcine tissues have proven effective, religious restrictions and risks of disease transmission remain barriers to their clinical use. Acellular fish skin (AFS), given its similarity to human skin structure and without the aforementioned disadvantages, is thus seen as an attractive alternative. This study aims to fabricate AFS from the skin of black carp (Mylopharyngodon piceus), evaluate its physical and mechanical properties and assess its impact on wound healing. The results showed that AFS has a highly porous structure, along with high levels of hydrophilicity, water-absorption property and permeability. Furthermore, physical characterization showed the high tensile strength of AFS in dry and wet states, and high stitch tear resistance, indicating great potential in clinical applications. Cell Counting Kit-8 was used to test the viability of L929 cells when culturing in the extracts of AFS. Compared with the control group, there is no significant difference in optical density value when culturing in the extracts of AFS at days 1, 3 and 7 (*p > 0.05). In vivo wound healing evaluation then highlighted its promotion of angiogenesis and collagen synthesis, its function in anti-inflammation and acceleration in wound healing. Therefore, this study suggests that AFS has potential as a promising alternative to mammal-derived or traditional wound dressing.

In this study, nanofibrous matrices of poly(L-lactic acid)-hydroxyapatite (PLLA-HAp) were successfully fabricated by three-dimensional (3D) electrospinning for use in the treatment of irregular bone damages. Compressibility analysis showed that 3D nanofibrous grafts occupied at least 2-fold more volume than their 2D form and they can easily take shape of the defect zone with irregular geometry. Moreover, the compression moduli of the PLLA and PLLA-HAp grafts were calculated as 8.0 ± 3.0 kPa and 11.8 ± 3.9 kPa, respectively, while the strain values of the same samples at the maximum load of 600 kPa were 164 ± 28% and 130 ± 20%, respectively. Treatment of the grafts with aqueous sodium hydroxide solution increased the surface roughness and thus the alloplastic graft materials (PLLA-HAp/M) protecting the fiber morphology were produced successfully. Then, platelet-rich plasma (PRP) was loaded into the surface modified grafts and activated with 10% calcium chloride. The efficiency of the activation was evaluated with flow cytometry and it was found that after activation the percentages of CD62 (P-selectin) and CD41/61 (glycoprotein IIb/IIIa) proteins increased approximately 4-fold. Surface hydrophilicity and biological activity of the PLLA-HAp grafts were enhanced by fibrin coating after PRP activation. The in vitro cell culture studies which were carried out by using mouse pre-osteoblasts (MC3T3-E1) showed that graft materials supported by PRP increased cellular proliferation and osteogenic differentiation significantly. The in vivo results demonstrated that compared with bare PLLA-HAp/M grafts, the PRP loaded grafts (PRP-PLLA-HAp/M) induced significantly greater bone formation based on computed tomography, histological and immunohistochemical analyses. Our findings suggest that 3D PLLA nanofibrous matrices can be used as a graft material for irregular bone defects especially when combined with PRP as an osteogenic induction agent.

There is a need for effective wound healing through rapid wound closure, reduction of scar formation, and acceleration of angiogenesis. Hydrogel is widely used in tissue engineering, but it is not an ideal solution because of its low vascularization capability and poor mechanical properties. In this study, gelatin methacrylate (GelMA) was tested as a viable option with tunable physical properties. GelMA hydrogel incorporating a vascular endothelial growth factor (VEGF) mimicking peptide was successfully printed using a three-dimensional (3D) bio-printer owing to the shear-thinning properties of hydrogel inks. The 3D structure of the hydrogel patch had high porosity and water absorption properties. Furthermore, the bioactive characterization was confirmed by cell culture with mouse fibroblasts cell lines (NIH 3T3) and human umbilical vein endothelial cells. VEGF peptide, which is slowly released from hydrogel patches, can promote cell viability, proliferation, and tubular structure formation. In addition, a pig skin wound model was used to evaluate the wound-healing efficacy of GelMA-VEGF hydrogel patches; the results suggest that the GelMA-VEGF hydrogel patch can be used for wound dressing.

Wound healing is an urgent problem that impacts quality of life, and the need for biomaterials suitable for the treatment of skin wound healing disease is increasing annually. Innovative biomaterials and treatments for skin abrasions are being relentlessly researched and established in order to improve treatment efficacy. Here, we describe a novel electrospun polymeric nanofibrous scaffold enriched with pharmaceutical bioactive materials extracted from Morinda citrifolia (MC), which demonstrated efficient skin wound healing therapy due to its excellent human skin keratinocyte proliferation and adhesion in in vitro analysis. Surface morphological analysis was used to reveal the nano-architectural structure of the electrospun scaffolds. The fabricated nanofibers displayed good antibacterial efficacy by creating an inhibitory zone for the pathogenic microbes studied. MC supported active healing due to the presence of pharmaceuticals associated with wound healing, as revealed by the results of gas chromatography–mass spectrometry and the prediction of activity spectra for substances (PASS) analysis. Since MC is a multi-potential therapeutic herbal plant, it was found that the linoleic acid, olelic acid, and diethyl phthalate present in the extract supported the wound healing proteins glycogen-synthase-kinase-3-β-protein and Protein Data Bank—1Q5K with binding energies of −4.6, −5.2, and −5.9 kcal mol⁻¹, as established by the results of in silico analysis. Thus, by being hydrophilic in nature, targeting wound proteins, increasing the proliferation and adhesion of keratinocytes and combating pathogens, the nanofibrous scaffolds endowed with MC extract proved to be an effective therapeutic material for skin wound dressing applications.

Lipofilling is a popular technique for soft tissue augmentation, limited by unpredictable graft survival. This study aimed at exploring the effect of hydrogel from acellular porcine adipose tissue (HAPA) on angiogenesis and survival of adipose tissue used for lipofilling. The effect of HAPA on adipose-derived stem cells (ADSCs) proliferation, adipogenic differentiation, and vascular endothelial growth factor (VEGF) secretion were evaluated in hypoxia and normoxia in vitro. For the in vivo study, adipose tissue with phosphate buffered saline, ADSCs, and HAPA (with or without ADSCs) were co-injected subcutaneously into nude mice. HAPA–ADSCs mixture (tissue engineering adipose tissue) was also grafted. Gross observation, volume measurement, and ultrasound observation were assessed. For histological assessment, hematoxylin and eosin, perilipin, cluster of differentiation 31 (CD31), Ki67, and transferase-mediated d-UTP nick end labelling (TUNEL) staining were performed. HAPA improved ADSCs proliferation, VEGF secretion, and adipogenic differentiation under normoxia and hypoxia conditions in vitro study. For the in vivo study, HAPA showed improved volume retention and angiogenesis, and reduced cell apoptosis when compared to ADSCs-assisted lipofilling and pure lipofilling. In conclusion, HAPA could maintain ADSCs viability and improve cell resistant to hypoxia and might be a promising biomaterial to assist lipofilling.

Cytocompatible bioactive surface treatments conferring antibacterial properties to osseointegrated dental implants are highly requested to prevent bacteria-related peri-implantitis. Here we focus on a newly designed family of mesoporous coatings based on zirconia (ZrO₂) microstructure doped with gallium (Ga), exploiting its antibacterial and pro-osseo-integrative properties. The ZrO₂ films were obtained via sol–gel synthesis route using Pluronic F127 as templating agent, while Ga doping was gained by introducing gallium nitrate hydrate. Chemical characterization by means of x-ray photoelectron spectroscopy and glow discharge optical emission spectroscopy confirmed the effective incorporation of Ga. Then, coatings morphological and structural analysis were carried out by transmission electron microscopy and selected area electron diffraction unveiling an effective stabilization of both the mesoporous structure and the tetragonal ZrO₂ phase. Specimens' cytocompatibility was confirmed towards gingival fibroblast and osteoblasts progenitors cultivated directly onto the coatings showing comparable metabolic activity and morphology in respect to controls cultivated on polystyrene. The presence of Ga significantly reduced the metabolic activity of the adhered oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in comparison to untreated bulk zirconia (p < 0.05); on the opposite, Ga ions did not significantly reduce the metabolism of the oral commensal Streptococcus salivarius (p > 0.05) thus suggesting for a selective anti-pathogens activity. Finally, the coatings' ability to preserve cells from bacterial infection was proved in a co-culture method where cells and bacteria were cultivated in the same environment: the presence of Ga determined a significant reduction of the bacteria viability while allowing at the same time for cells proliferation. In conclusion, the here developed coatings not only demonstrated to satisfy the requested antibacterial and cytocompatibility properties, but also being promising candidates for the improvement of implantable devices in the field of implant dentistry.

Several biofabrication methods are being investigated to produce scaffolds that can replicate the structure of the extracellular matrix. Direct-write, near-field electrospinning of polymer solutions and electrowriting of polymer melts are methods which combine fine fiber formation with computer-guided control. Research with such systems has focused primarily on synthetic polymers. To better understand the behavior of biopolymers used for direct-writing, this project investigated changes in fiber morphology, size, and variability caused by varying gelatin and acetic acid concentration, as well as process parameters such as needle gauge and height, stage speed, and interfiber spacing. Increasing gelatin concentration at a constant acetic acid concentration improved fiber morphology from large, planar structures to small, linear fibers with a median of 2.3 µm. Further varying the acetic acid concentration at a constant gelatin concentration did not alter fiber morphology and diameter throughout the range tested. Varying needle gauge and height further improved the median fiber diameter to below 2 µm and variability of the first and third quartiles to within ±1 µm of the median. Additional adjustment of stage speed did not impact the fiber morphology or diameter. Repeatable interfiber spacings down to 250 µm were shown to be capable with the system. In summary, this study illustrates the optimization of processing parameters for direct-writing of gelatin to produce fibers on the scale of collagen fibers. This system is thus capable of replicating the fibrous structure of musculoskeletal tissues with biologically relevant materials which will provide a durable platform for the analysis of single cell-fiber interactions to help better understand the impact scaffold materials and dimensions have on cell behavior.

Bulk metallic glasses (BMGs) are a class of amorphous metals that exhibit high strength, ductility paired with wear and corrosion resistance. These properties suggest that they could serve as an alternative to conventional metallic implants that suffer wear and failure. In the present study, we investigated Platinum (Pt)-BMG biocompatibility in bone applications. Specifically, we investigated osteoclast formation on flat and nanopatterned Pt_57.5Cu_14.7Ni_5.3P_22.5 (atomic percent) as well as titanium (control). Specifically, receptor activator of NF-κB (RANK) ligand-induced murine bone marrow derived mononuclear cell fusion was measured on multiple nanopatterns and was found to be reduced on nanorods (80 and 200 nm in diameter) and was associated with reduced tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase (MMP9) expression. Evaluation of mesenchymal stem cell (MSC) to osteoblast differentiation on nanopatterned Pt-BMG showed significant reduction in comparison to flat, suggesting that further exploration of nanopatterns is required to have simultaneous induction of osteoblasts and inhibition of osteoclasts.In vivo studies were also pursued to evaluate the biocompatibility of Pt-BMG in comparison to titanium. Rods of each material were implanted in the femurs of mice and evaluated by x-ray, mechanical testing, micro-computed tomography (micro-CT), and histological analysis. Overall, Pt-BMG showed similar biocompatibility with titanium suggesting that it has the potential to improve outcomes by further processing at the nanoscale.

We demonstrate a benign and straightforward method to modify the chitosan (CH) by carbamoylation. The free amines on CH are converted into carbamyl functionalities by reacting with potassium cyanate (KCNO). One wt% CH solution, when reacted with KCNO ⩾ 0.1 M, leads to the sol–gel transition of CH through the hydrogen bonding to form carbamoylated chitosan (CCH) hydrogel. Gelation time of CCH decreases with an increase in the KCNO concentration and an interconnected porous network is formed as observed under SEM. Rheological studies show that while one wt% CH solution is a viscous liquid, the CCH hydrogel with 0.5 M KCNO has a storage modulus (G') of 10⁴ Pa. The CCH hydrogel is proved to be non-cytotoxic and promotes the attachment and growth of the small lung cancer model A549, and the neuroblastoma SH-SY5Y cell lines. CCH hydrogel also promotes the differentiation of SH-SY5Y cells into neuronal cells, as supported by immunostaining and thus demonstrating its utility as a versatile scaffold for three-dimensional cell-culture systems.

Commonly recognized mechanisms of the xenogeneic-extracellular matrix-based regenerative medicine include timely degradation, release of bioactive molecules, induced differentiation of stem cells, and well-controlled inflammation. This process is most feasible for stromal tissue reconstruction, yet unsuitable for non-degradable scaffold and prefabricated-shaped tissue regeneration, like odontogenesis. Treated dentin matrix (TDM) has been identified as a bioactive scaffold for dentin regeneration. This study explored xenogeneic porcine TDM (pTDM) for induced odontogenesis. The biological characteristics of pTDM were compared with human TDM (hTDM). To investigate its bioinductive capacities on allogeneic dental follicle cells (DFCs) in the inflammation microenvironment, pTDM populated with human DFCs were co-cultured with human peripheral blood mononuclear cells (hPBMCs), and pTDM populated with rat DFCs were transplanted into rat subcutaneous model. The results showed pTDM possessed similar mineral phases and bioactive molecules with hTDM. hDFCs, under the induction of pTDM and hTDM, expressed similar col-I, osteopontin and alkaline phosphatase (ALP) (all expressed by odontoblasts). Whereas, the expression of col-I, dentin matrix protein-1 (DMP-1) and bone sialoprotein (BSP) were down-regulated when cocultured with hPBMCs. The xenogeneic implants inevitably initiated Th1 inflammation (up-regulated CD8, TNF-α, IL-1β, etc) in vivo. However, the biomineralization of pre-dentin and cementum were still processed, and collagen fibrils, odontoblast-like cells, fibroblasts contributed to odontogenesis. Although partially absorbed at 3 weeks, the implants were positively expressed odontogenesis-related-proteins like col-I and DMP-1. Taken together, xenogeneic TDM conserved ultrastructure and molecules for introducing allogeneic DFCs to odontogenic differentiation, and promoting odontogenesis and biomineralization in vivo. Yet effective immunomodulation methods warrant further explorations.

Data on how the immune system reacts to decellularized scaffolds after implantation is scarce and difficult to interpret due to many heterogeneous parameters such as tissue-type match, decellularization method and treatment application. The engraftment of these scaffolds must prove safe and that they remain inert to the recipient's immune system to enable successful translational approaches and potential future clinical evaluation. Herein, we investigated the immune response after the engraftment of three decellularized scaffold types that previously showed potential to repair a uterine injury in the rat. Protocol (P) 1 and P2 were based on Triton-X100 and generated scaffolds containing 820 ng mg⁻¹ and 33 ng mg⁻¹ donor DNA per scaffold weight, respectively. Scaffolds obtained with a sodium deoxycholate-based protocol (P3) contained 160 ng donor DNA per mg tissue. The total number of infiltrating cells, and the population of CD45⁺ leukocytes, CD4⁺ T-cells, CD8a⁺ cytotoxic T-cells, CD22⁺ B-cells, NCR1⁺ NK-cells, CD68⁺ and CD163⁺ macrophages were quantified on days 5, 15 and 30 after a subcutaneous allogenic (Lewis to Sprague Dawley) transplantation. Gene expression for the pro-inflammatory cytokines INF-γ, IL-1β, IL-2, IL-6 and TNF were also examined. P1 scaffolds triggered an early immune response that may had been negative for tissue regeneration but it was stabilized after 30 d. Conversely, P3 initiated a delayed immune response that appeared negative for scaffold survival. P2 scaffolds were the least immunogenic and remained similar to autologous tissue implants. Hence, an effective decellularization protocol based on a mild detergent was advantageous from an immunological perspective and appears the most promising for future in vivo uterus bioengineering applications.

Currently, valve replacement surgery is the only therapy for the end-stage valvular diseases because of the inability of regeneration for diseased heart valves. Bioprosthetic heart valves (BHVs), which are mainly derived from glutaraldehyde (GA) crosslinked porcine aortic heart valves or bovine pericardium, have been widely used in the last decades. However, it is inevitable that calcification and deterioration may occur within 10–15 years, which are still the main challenges for the BHVs in clinic. In this study, N-Lauroylsarcosine sodium salt (SLS) combined with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were utilized to decellularize and crosslink the heart valves instead of GA treatment. The obtained BHVs exhibited excellent extracellular matrix stability and mechanical properties, which were similar with GA treatment. Moreover, the obtained BHVs exhibited better in vitro biocompatibilities than GA treatment. After subcutaneous implantation for 30 d, the obtained BHVs showed mitigated immune response and reduced calcification compare with GA treatment. Therefore, all the above results indicated that the treatment of SLS-based decellularization combined with EDC/NHS crosslink should be a promising method to fabricate BHVs which can be used in clinic in future.

Biomaterials constructed exclusively of sintered microspheres have great potential in tissue engineering scaffold applications, offering the ability to create shape-specific scaffolds with precise controlled release yet to be matched by traditional additive manufacturing methods. The problem is that these microsphere-based scaffolds are limited in their stiffness for applications such as bone regeneration. Our vision to solve this problem was borne from a hierarchical structure perspective, focusing on the individual unit of the structure: the microsphere itself. In a core–shell approach, we envisioned a stiff core to create a stiff microsphere unit, with a polymeric shell that would enable sintering to the other microsphere units. Therefore, the current study provided a comparison of macroscopic biomaterials built on either polymer microspheres or polymer-coated hard glass microspheres. Identical polycaprolactone (PCL) polymer solutions were used to fabricate microspheres and as a thin coating on soda lime glass microspheres (hard phase). The materials were characterized as loose particles and as scaffolds via scanning electron microscopy, thermogravimetry, differential scanning calorimetry, Raman spectroscopy, mechanical testing, and a live/dead analysis with human umbilical cord-derived Wharton's jelly cells. The elastic modulus of the scaffolds with the thinly coated hard phase was about five times higher with glass microspheres (up to about 25 MPa) than pure polymer microspheres, while retaining the structure, cell adhesion, and chemical properties of the PCL polymer. This proof-of-concept study demonstrated the ability to achieve at least a five-fold increase in macroscopic stiffness via altering the core microsphere units with a core–shell approach.

Polyethylene glycol diacrylate (PEGDA) is an important class of photosensitive polymer with many tissue engineering applications. This study compared PEGDA and polycaprolactone (PCL) nanofiber matrix (NFM) coated PEGDA, referred to as PCL-PEGDA, scaffolds for their application in multiple tissue repair such as articular cartilage, nucleus pulposus of the intervertebral disc (IVD). We examined each scaffold morphology, porosity, swelling ratio, degradation, mechanical strength, and in vitro cytocompatibility properties. A defect was created in Sprague Dawley rat tail IVD by scraping native cartilage tissue and disc space, then implanting the scaffolds in the disc space for 4 weeks to evaluate in vivo efficacy of multi-tissue repair. Maintenance of disc height and creation of a new cell matrix was assessed to evaluate each scaffold's ability to repair the tissue defect. Although both PEGDA and PCL-PEGDA scaffolds showed similar porosity ∼73%, we observed distinct topographical characteristics and a higher effect of degradation on the water-absorbing capacity for PEGDA compared to PCL-PEGDA. Mechanical tests showed higher compressive strength and modulus of PCL-PEGDA compared to PEGDA. In vitro cell studies show that the PCL NFM layer covering PEGDA improved osteoblast cell adhesion, proliferation, and migration into the PEGDA layer. In vivo studies concluded that the PEGDA scaffold alone was not ideal for implantation in rat caudal disc space without PCL nanofiber coating due to low compressive strength and modulus. In vivo results confirm that the PCL-PEGDA scaffold-maintained disc space and created a proteoglycan and collagen-rich new tissue matrix in the defect site after 4 weeks of scaffold implantation. We concluded that our developed PCL-PEGDA has the potential to be used in multi-tissue defect site repair.

Bio-adhesives are essential for wound healing because of their convenience and safety. Although widely used as biomaterials, silk fibroin's (SF's) further application as bio-adhesive is hindered due to its weak stickiness with tissue and slow gelation speed. Here, a dopamine-modified SF-based bio-adhesive is fabricated by using genipin as the chemical cross-linking agent. Furthermore, metal ions have been used to adjust the adhesion property of the bio-adhesive. The experimental results shows that the dopamine-modified SF-based composite holds a better stickiness except slow gelation speed. The doping of Cu²⁺ and Fe³⁺ can accelerate the gelation of the bio-adhesive. Compared with Cu²⁺, Fe³⁺ has a stronger effect on the gelation speed of the bio-adhesive, which is positive correlative to the concentration of Fe³⁺. The adhesive has injectability and degradability. In addition, the SF-based adhesive has good biocompatibility and good improvement for cell migration in vitro. The SF-based bio-adhesive holds potential application in the field of rapid fixation of wounds.

Cardiomyocyte (CM) transplantation is a promising option for regenerating infarcted myocardium. However, poor cell survival and residence rates reduce the efficacy of cell transplantation. Gelatin (GA) hydrogel as a frequently-used cell carrier is a possible approach to increase the survival rate of CMs. In this study, microbial transglutaminase (mTG) and chemical crosslinkers glutaraldehyde, genipin, and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide were employed to prepare GA hydrogels. The mechanical properties and degradation characteristics of these hydrogels were then evaluated. Neonatal rat CMs (NRCMs) were isolated and inoculated on the surface of these hydrogels or encapsulated in mTG-hydrogels. Cellular growth morphology and beating behavior were observed. Cellular viability and immunofluorescence were analyzed. Intracellular Ca²⁺ transient and membrane potential propagation were detected using fluorescence dyes (Fluo-3 and di-4-ANEPPS, respectively). Results showed that the chemical crosslinkers exhibited high cytotoxicity and resulted in high rates of cell death. By contrast, mTG-hydrogels showed excellent cell compatibility. The CMs cultured in mTG-hydrogels for a week expressed CM maturation markers. The NRCMs begun independently beating on the third day of culture, and their beating synchronized after a week of culture. Furthermore, intracellular Ca²⁺ transient events with periodicity were observed. In conclusion, the novel mTG-crosslinked GA hydrogel synthesized herein has good biocompatibility, and it supports CM adhesion, growth, and maturation.

Hydrogels with tunable properties are highly desirable in tissue engineering applications as they can serve as artificial extracellular matrix to control cellular fate processes, including adhesion, migration, differentiation, and other phenotypic changes via matrix induced mechanotransduction. Poly(γ-glutamic acid) (PGA) is an natural anionic polypeptide that has excellent biocompatibility, biodegradability, and water solubility. Moreover, the abundant carboxylic acids on PGA can be readily modified to introduce additional functionality or facilitate chemical crosslinking. PGA and its derivatives have been widely used in tissue engineering applications. However, no prior work has explored orthogonal crosslinking of PGA hydrogels by thiol–norbornene (NB) chemistry. In this study, we report the synthesis and orthogonal crosslinking of PGA-norbornene (PGANB) hydrogels. PGANB was synthesized by standard carbodiimide chemistry and crosslinked into hydrogels via either photopolymerization or enzymatic reaction. Moduli of PGA hydrogels were readily tuned by controlling thiol–NB crosslinking conditions or stoichiometric ratio of functional groups. Orthogonally crosslinked PGA hydrogels were used to evaluate the influence of mechanical cues of hydrogel substrate on the phenotype of naïve human monocytes and M0 macrophages in 3D culture.

A tantalum/tantalum nitride (Ta/TaN) multilayered coating is deposited on plasma-nitridedAZ91 Mg alloy. The top TaN layer undergoes O₂ + Ar plasma etching to improve the antibacterial properties and Mg plasma immersion ion implantation (MgPIII) is performed to enhance the biocompatibility and wound healing capability. A uniform, compact, homogeneous, and columnar nanostructured MgPIII and plasma-etched TaN layer with a cluster size of about 17 nm, surface roughness of 0.28 nm, and needle morphology is observed. Although, plasma etching increases the corrosion current density (i_corr) from 0.02 to 0.19 µA cm⁻² due to larger surface roughness and different potentials between sharp points and smooth points, MgPIII decreases i_corr from 0.19 to 0.02 µA cm⁻² besides a more positive corrosion potential. The amounts of Mg⁺² released to the simulated body fluid (SBF) diminishes from 89.63 ± 0.54 to 60.30 ± 0.47 mg l⁻¹ cm⁻² indicating improved corrosion resistance. Under fever conditions (40 °C), i_corr decreases by 63%, but the open circuit potential does not change due to the constant chemical composition of the surface as well as thicker double layer and less defects, as confirmed by the larger amount of Mg⁺² of 71.49 ± 0.22 mg l⁻¹ cm⁻² leached to the SBF. In the self-healing process which occurs via the reactions between the tantalum intermediate layer and electrolytes and penetrating ions through the defects as well as formation of oxide compounds, creation and propagation of defects are deterred as shown the 24 h destructive polarization test in SBF. The combination of plasma etching and MgPIII enhance not only the bacterial resistance and biocompatibility of the super-hard TaN layer by providing the rougher surface on TaN–P–Mg, but also the nano-mechanical properties and anticorrosion properties. As a result, the hardness increases by 7%, elastic modulus decreases by 19%, and the stiffness increases by 21%.

The development of clinically advanced multifaceted therapeutic materials for osteosarcoma is at the forefront of cancer research. Accordingly, this work presents the design of a multifunctional magnetic nanocomposite composed of maghemite, strontium doped hydroxyapatite and silica nanoparticles prospectively holding indispensable therapeutic features such as magnetic hyperthermia, in vitro biomineralization, sustained drug release and intrinsic radiopacity for the treatment of osteosarcoma. The optimal composition has been identified by sequentially modulating the ratio of precursors of the magnetic nanocomposite synthesized by sol–gel technique. Structural and morphological characterization by x-ray diffraction, fourier transform infrared spectrum, Brunauer–Emmet–Teller and transmission electron microscopy analyses followed by VSM, hyperthermia and micro-CT analyses essentially assisted in the selective configuration of biofunctional properties. Results exemplify that MSHSr1 has a saturation magnetization of 47.4 emu g⁻¹ and attained hyperthermia temperature (42 °C) at a very low exposure time of 4 min. MSHSr1 is further unique with respect to its exceptional x-ray attenuation ability (contrast enhancement 154.5% in digital radiography; CT number 3100 HU), early biomimetic mineralization (in vitro) evident by the formation of spheroidal apatite layer (Ca/P ratio 1.33) harvested from FESEM–EDX analysis and controlled release of Doxorubicin, the clinically used chemotherapeutic drug: 87.7% at 120 h in tumour analogous pH (6.5) when compared to physiological pH (71.3% at 7.4). MTT assay complemented with cytoskeleton (F-actin) staining of human osteosarcoma (HOS) cells affirm biocompatibility of MSHSr1. In vitro biomineralization authenticated by Alizarin red S and von Kossa staining has been further corroborated by semi-quantitative calcium estimation of HOS cells cultured with MSHSr1 for two weeks. The results therefore validate the multifunctionality of MSHSr1, and hence could be proposed as a combinatorial therapeutic nanocomposite for osteosarcoma treatment.

For wound healing applications, a scaffold of biocompatible/porous networks is crucial to support cell proliferation and spreading. Therefore, -polycaprolactone (PCL) nanofibrous scaffolds containing co-dopants of strontium/selenium in hydroxyapatite (HAP) were modified with different contributions of graphene oxide (GO) via the laser ablation technique. The obtained compositions were investigated using XRD, TEM and FESEM. It was evident that fiber diameters were in the range of 0.15–0.30 µm and 0.35–0.83 µm at the lowest and highest concentration of GO respectively, while the maximum height of the roughness progressed to 393 nm. The toughness behavior was promoted from 5.77 ± 0.21 to 9.16 ± 0.29 MJ m⁻³ upon GO from the lowest to the highest contribution, while the maximum strain at break reached 148.1% ± 0.49% at the highest concentration of GO. The cell viability indicated that the fibrous scaffold was biocompatible. The investigation of the HFB4 cell attachments towards the fibrous compositions showed that with the increase of GO, cells tended to grow intensively through the scaffolds. Furthermore, the proliferation of cells was observed to be rooted in the porous structure and spreading on the surface of the scaffold. This progression of cells with an increase in GO content may provide a simple strategy not only to enhance the mechanical properties, but also to manipulate a nanofibrous scaffold with proper behaviors for biomedical applications.

Nowadays, heart disease, especially myocardial infarction, is one of the most astoundingly unfortunate causes of mortality in the world. That is why special attention has been paid toward tissue engineering techniques for curing and regeneration of heart tissue. In this study, poly(N-isopropyl acrylamide) (PNIPAAm), a temperature-sensitive injectable hydrogel, was selected as a minimally invasive scaffold to accommodate, carry, and release of niosomal rosuvastatin to the inflicted area for inducing angiogenesis and thus accelerating the healing process. The characteristics of PNIPAAm were studied by scanning electron microscopy, rheology tests, and Fourier transform infrared spectroscopy. The properties of the niosomal rosuvastatin release system, including particle size distribution, zeta potential, encapsulation efficiency (EE), and drug release, were also studied. The results showed that niosomes (358 nm) had a drug EE of 78% and a loading capacity of 53%. The drug was sustainably released from the system up to about 54% in 5 d. Cellular studies showed no toxicity to the endothelial cell lines, and the niosomal drug with a concentration of 7.5 nM enhanced cell proliferation, and cell migration increased from 72% to 90% compared to the control sample. Therefore, the controlled-release of niosomal rosuvastatin enhanced angiogenesis in a dose-dependent manner. Taken together, these advantages suggest that PNIPAAm-based niosomal hydrogel provides a promising candidate as an angiogentic injectable scaffold for potential cardiac tissue regeneration.

A microfluidic technique is presented for micropatterning protein domains and cell cultures within permanently bonded organs-on-chip devices. This method is based on the use of polydimethylsiloxane layers coupled with the plasma ablation technique for selective protein removal. We show how this technique can be employed to generate a multi-organ in vitro model directly within a microscale platform suitable for pharmacokinetic-based drug screening. We miniaturized a liver model based on micropatterned co-cultures in dual-compartment microfluidic devices. The cytotoxic effect of liver-metabolized Tegafur on colon cancer cell line was assessed using two microfluidic devices where microgrooves and valves systems are used to model drug diffusion between culture compartments. The platforms can reproduce the metabolism of Tegafur in the liver, thus killing colon cancer cells. The proposed plasma-enhanced microfluidic protein patterning method thus successfully combines the ability to generate precise cell micropatterning with the intrinsic advantages of microfluidics in cell biology.

The use of composites such as hydroxyapatite (HA)/TiO₂ in bioapplications has attracted increasing attention in recent years. Herein, for the enhancement wetting ability and biocompatibility, the HA/TiO₂ composite was subjected to different treatments to improve nanoparticle (NP) distribution and surface energy with an aim of mitigating nanotoxicity concerns. The treatments included ultrasonication, high-temperature annealing, and addition of a dispersant and surfactant, sodium dodecylbenzenesulfonate (SDBS). Contact angle measurement tests revealed the effect of SDBS addition on the distribution of TiO₂ NPs on the HA surface: a decrease in the contact angle and, thus, an increase in the wetting ability of the HA/TiO₂ composite were observed. The combination of annealing and SDBS addition treatments allowed for guest TiO₂ particles to be uniformly distributed on the surface of the host HA particles, showing a rapid conversion from a hydrophobic to superhydrophilic property. In vitro investigation suggested that the cell viabilities of annealed HA/TiO₂, SDBS-added HA/TiO₂, and SDBS-added and annealed HA/TiO₂ reached 89.7%, 94.7%, and 95.8%, respectively, while those of HA and untreated HA/TiO₂ were 80.3% and 86.9%, respectively. The modified composites exhibited lower cytotoxicities than the unmodified systems (HA and HA/TiO₂). Furthermore, the cell adhesion behavior of the composites was confirmed through actin-4',6-Diamidino-2-phenylindole (DAPI) staining, which showed negligible changes in the cytoskeleton architecture of the cells. This study confirmed that a modified HA/TiO₂ composite has potential for bioapplications.

In the present study, an injectable bone substitute system which utilized porous bioglass (BG)-derived granules supplemented with hyaluronic acid (Hya), was evaluated. Hya plays ultimate role in wound healing, promoting cell motility. The BG were synthesized by a simple and low sintering temperature process without any foreign phase incorporation. Furthermore, the physical properties in the porous scaffold were optimized to investigate the in vitro and in vivo performance. The porous BG60 scaffolds system showed excellent bioactivity in an in vitro simulated body fluid test in which the ions dissolved from the composite materials influenced apatite growth, countered the acidic pH, and increased material degradation. In an in vitro study with pre-osteoblasts cells (MC3T3-E1), the porous scaffold supported cell adhesion and proliferation. A post-implantation study conducted in femoral defects showed implant degradation and surprisingly fast bone formation just after 2 weeks of implantation. Initial in vivo degradation of Hya promotes releasing ions which regulates the bone forming cells, clues to tissue repair, and regeneration. On the other hand it also prevent the scattering of BG granule after grafting at implant site. The faster dissolution of the porous BG scaffold increased the resorption of the composite material and hence, facilitated bone tissue regeneration. Our findings suggest that the porous BG scaffold could potentially be used as an injectable bone substitute for fast, early bone regeneration applications.

Organ decellularization is one of the promising technologies of regenerative medicine, which allows obtaining cell-free extracellular matrix (ECM), which provide preservation of the composition, architecture, vascular network and biological activity of the ECM. The method of decellularization opens up wide prospects for its practical application not only in the field of creating full-scale bioengineered structures, but also in the manufacture of vessels, microcarriers, hydrogels, and coatings. The main goal of our work was the investigation of structure and biological properties of lyophilized decellularized Wistar rat liver fragments (LDLFs), as well as we assessed the regenerative potential of the obtained ECM. We obtained decellularized liver of a Wistar rat, the vascular network and the main components of the ECM of tissue were preserved. H&E staining of histological sections confirmed the removal of cells. DNA content of ECM is equal to 0.7% of native tissue DNA content. Utilizing scanning probe nanotomogrphy method, we showed sinuous, rough topography and highly nanoporous structure of ECM, which provide high level of mouse 3T3 fibroblast and Hep-G₂ cells biocompatibility. Obtained LDLF had a high regenerative potential, which we studied in an experimental model of a full-thickness rat skin wound healing: we observed the acceleration of wound healing by 2.2 times in comparison with the control.

Periodontitis is a chronic, multifactorial, inflammatory disease characterized by the progressive destruction of the periodontal tissues. Guided tissue regeneration (GTR), involving the use of barrier membranes, is one of the most successful clinical procedures for periodontal therapy. Nevertheless, rapid degradation of the membranes and membrane-related infections are considered two of the major reasons for GTR clinical failure. Recently, integration of non-antibiotic, antimicrobial materials to the membranes has emerged as a novel strategy to face the bacterial infection challenge, without increasing bacterial resistance. In this sense, bismuth subsalicylate (BSS) is a non-antibiotic, metal-based antimicrobial agent effective against different bacterial strains, that has been long safely used in medical treatments. Thus, the aim of the present work was to fabricate fibrillar, non-rapidly bioresorbable, antibacterial GTR membranes composed of polycaprolactone (PCL), gelatin (Gel), and BSS as the antibacterial agent. PCL-G-BSS membranes with three different BSS concentrations (2 wt./v%, 4 wt./v%, and 6 wt./v%) were developed by electrospinning and their morphology, composition, water wettability, mechanical properties, Bi release and degradation rate were characterized. The Cytotoxicity of the membranes was studied in vitro using human osteoblasts (hFOB) and gingival fibroblasts (HGF-1), and their antibacterial activity was tested against Aggregatibacter actinomycetemcomitans, Escherichia coli, Porphyromonas gingivalis and Staphylococcus aureus. The membranes obtained exhibited adequate mechanical properties for clinical application, and appropriate degradation rates for allowing periodontal defects regeneration. The hFOB and HGF-1 cells displayed adequate viability when in contact with the lixiviated products from the membranes, and, in general, displayed antibacterial activity against the four bacteria strains tested. Thus, the PCL-G-BSS membranes showed to be appropriate as potential barrier membranes for periodontal GTR treatments.

The elasticity, topography, and chemical composition of cell culture substrates influence cell behavior. However, the cellular responses to in vivo extracellular matrix (ECM), a hydrogel of proteins (mainly collagen) and polysaccharides, remain unknown as there is no substrate that preserves the key features of native ECM. This study introduces novel collagen hydrogels that can combine elasticity, topography, and composition and reproduce the correlation between collagen concentration (C) and elastic modulus (E) in native ECM. A simple reagent-free method based on radiation-cross-linking altered ECM-derived collagen I and hydrolyzed collagen (gelatin or collagen peptide) solutions into hydrogels with tunable elastic moduli covering a broad range of soft tissues (E = 1–236 kPa) originating from the final collagen density in the hydrogels (C = 0.3%–14%) and precise microtopographies (⩾1 μm). The amino acid composition ratio was almost unchanged by this method, and the obtained collagen hydrogels maintained enzyme-mediated degradability. These collagen hydrogels enabled investigation of the responses of cell lines (fibroblasts, epithelial cells, and myoblasts) and primary cells (rat cardiomyocytes) to soft topographic cues such as those in vivo under the positive correlation between C and E. These cells adhered directly to the collagen hydrogels and chose to stay atop or spontaneously migrate into them depending on E, that is, the density of the collagen network, C. We revealed that the cell morphology and actin cytoskeleton organization conformed to the topographic cues, even when they are as soft as in vivo ECM. The stiffer microgrooves on collagen hydrogels aligned cells more effectively, except HeLa cells that underwent drastic changes in cell morphology. These collagen hydrogels may not only reduce in vivo and in vitro cell behavioral disparity but also facilitate artificial ECM design to control cell function and fate for applications in tissue engineering and regenerative medicine.

In the present study, β-tricalcium phosphate (β-TCP) scaffolds with various amounts of bredigite (Bre) were fabricated by the space holder method. The effect of bredigite content on the structure, mechanical properties, in vitro bioactivity, and cell viability was investigated. The structural assessment of the composite scaffolds presented interconnected pores with diameter of 300–500 μm with around 78%–82% porosity. The results indicated that the compressive strength of the scaffolds with 20% bredigite (1.91 MPa) was improved in comparison with scaffolds with 10% bredigite (0.52 MPa), due to the reduction of the average pore and grain sizes. Also, the results showed that the bioactivity and biodegradability of β-TCP/20Bre were better than that of β-TCP/10Bre. Besides, in this study, the release kinetics of ciprofloxacin (CPFX) loaded β-TCP/Bre composites as well as the ability of scaffolds to function as a sustained release drug carrier was investigated. Drug release pattern of β-TCP/bredigite-5CPFX scaffolds exhibited the rapid burst release of 43% for 3 h along with sustained release (82%) for 32 h which is favorable for bone infection treatment. Antibacterial tests revealed that the antibacterial properties of β-TCP/bredigite scaffolds are strongly related to the CPFX concentration, wherein the scaffold containing 5% CPFX showed the most significant zone of inhibition (33 ± 0.5 mm) against Staphylococcus aureus. The higher specific surface areas of nanostructure β-TCP/bredigite scaffolds containing CPFX lead to an initial rapid release followed by constant drug delivery. MTT assay showed that the cell viability of β-TCP/bredigite scaffold loading with up to 1%–3% CPFX (95 ± 2%), is greater than for scaffolds containing 5% CPFX (84 ± 2%). In Overall, it may suggested that β-TCP/bredigite containing 1%–3% CPFX possesses great cell viability and antibacterial activity and be employed as bactericidal biomaterials and bone infection treatment.

Drug-eluting bioresorbable vascular scaffolds (BVSs) have emerged as a potential breakthrough for the treatment of coronary artery stenosis, providing mechanical support and drug delivery followed by complete resorption. Restenosis and thrombosis remain the primary limitations in clinical use. The study aimed to identify potential markers of restenosis and thrombosis analyzing the vascular wall cell transcriptomic profile modulation triggered by BVS at different values of shear stress (SS). Human coronary artery endothelial cells and smooth muscle cells were cultured under SS (1 and 20 dyne cm⁻²) for 6 h without and with application of BVS and everolimus 600 nM. Cell RNA-Seq and bioinformatics analysis identified modulated genes by direct comparison of SS conditions and Gene Ontology (GO). The results of different experimental conditions and GO analysis highlighted the modulation of specific genes as semaphorin 3E, mesenchyme homeobox 2, bone morphogenetic protein 4, (heme oxygenase 1) and selectin E, with different roles in pathological evolution of disease. Transcriptomic analysis of dynamic vascular cell cultures identifies candidate genes related to pro-restenotic and pro-thrombotic mechanisms in an in-vitro setting of BVS, which are not adequately contrasted by everolimus addition.

Elimination of tumor cells is still a therapeutic challenge for breast cancer (BC) in men and women. Mammospheres serve as valuable in vitro tools for evaluating tumor behavior and sensitivity to anticancer treatments. Graphene nanosheets with unique physicochemical properties have been considered as potential biomedical approaches for drug delivery, bioimaging, and therapy. Graphene oxide (GO) and graphene quantum dots (GQDs) are suitable nanocarriers for hydrophobic and low bioaccessible anti-tumor materials like curcumin. Despite extensive studies on the potential application of graphene nanosheets in medicine, our knowledge of how different cells function and respond to these nanoparticles remains limited. Here, we evaluated cell death in mammospheres from MCF-7 and primary tumor cells in response to curcumin loaded on graphene nanosheets. Mammospheres were exposed to graphene oxide-curcumin (GO-Cur) and graphene quantum dots-curcumin (GQDs-Cur), and the incidence of cell death was evaluated by Hoechst 33342/propidium iodide double staining and flow cytometry. Besides, the expression of miR-21, miR-29a, Bax, and Bcl-2 genes were assessed using RT-qPCR. We observed, GO, and GQDs had no cytotoxic effect on Kerman male breast cancer/71 (KMBC/71) and MCF-7 tumor cells, while curcumin induced death in more than 50% of tumor cells. GO-Cur and GQDs-Cur synergistically enhanced anti-tumor activity of curcumin. Moreover, GQDs-Cur induced cell death in almost all cells of KMBC/71 mammospheres (99%; p < 0.0001). In contrast, GO-Cur induced cell death in only 21% of MCF-7 mammosphere cells (p < 0.0001). Also, the expression pattern of miR-21, miR-29a, and Bax/Bcl-2 ratio in KMBC/71 and MCF-7 mammospheres was different in response to GO-Cur and GQDs-Cur. Although KMBC/71 and MCF-7 tumor cells had similar clinical features and displayed similar responses to curcumin, more investigations are needed to clarify the detailed molecular mechanisms underlying observed differences in response to GO-Cur and GQDs-Cur.

Macrophages play a central role in the host response and the integration of implant materials. The nanostructured TiF_x/TiO_x coating (FOTi) on titanium surfaces has shown multiple properties, including antibacterial properties and bioactivity. However, little is known about the effects of these coatings on the regulation of macrophage activity and the subsequent immunomodulatory effects on osteogenesis. In this study, the behavior of macrophages on the FOTi samples was evaluated, and conditioned medium was collected and used to stimulate MC3T3-E1 cells in vitro. The results showed that the FOTi samples stimulated macrophage elongation and promoted the production of proinflammatory cytokines at 24 h, while induced macrophage polarization to the anti-inflammatory M2 phenotype at 72 h. Furthermore, the immune microenvironment generated by macrophage/ FOTi samples interactions effectively promoted the osteogenic differentiation of MC3T3-E1 cells, as evidenced by improved cell adhesion, enhanced alkaline phosphatase activity and extracellular matrix mineralization, and upregulated osteogenesis-related gene expression. In summary, the FOTi samples mediated macrophage phenotype behaviors and induced beneficial immunomodulatory effects on osteogenesis, which could be a potential strategy for the surface modification of bone biomaterials.

Osteoporosis is detrimental to the health of skeletal structure and significantly increases the risks of bone fracture. Moreover, bone regeneration is adversely impaired by increased osteoclastic activities as a result of osteoporosis. In this study, we developed a novel formulation of injectable bone cement based on calcium phosphate silicate cement (CPSC) and leuprolide acetate (LA). Several combinations of LA-CPSC bone cement were characterized and, it is found that LA could increase the setting time and compressive strength of CPSC in a concentration-dependent manner. Moreover, the in vitro results revealed that LA-CPSC was biocompatible and able to encourage the osteoblast proliferation via the mTOR signalling pathway. Furthermore, the LA-CPSC was implanted in the osteoporotic rats to evaluate its effectiveness to repair bone fractures under the osteoporotic conditions. The biomarker study and micro-CT analyses indicated that LA-CPSC could effectively reduce the osteoclast activities and promote the bone regeneration. In conclusion, our study demonstrated that LA-CPSC injectable bone cement should be a viable solution to repair bone fractures under the osteoporotic conditions.

Bone reconstruction in the oral and maxillofacial region presents particular challenges related to the development of biomaterials with osteoinductive properties and suitable physical characteristics for their surgical use in irregular bony defects. In this work, the preparation and bioactivity of chitosan–gelatin (ChG) hydrogel beads loaded with either bioactive glass nanoparticles (nBG) or mesoporous bioactive glass nanospheres (nMBG) were studied. In vitro testing of the bionanocomposite beads was carried out in simulated body fluid, and through viability and osteogenic differentiation assays using dental pulp stem cells (DPSCs). In vivo bone regenerative properties of the biomaterials were assessed using a rat femoral defect model and compared with a traditional maxillary allograft (Puros®). ChG hydrogel beads containing homogeneously distributed BG nanoparticles promoted rapid bone—like apatite mineralization and induced the osteogenic differentiation of DPSCs in vitro. The bionanocomposite beads loaded with either nBG or nMBG also produced a greater bone tissue formation in vivo as compared to Puros® after 8 weeks of implantation. The osteoinductivity capacity of the bionanocomposite hydrogel beads coupled with their physical properties make them promissory for the reconstruction of irregular and less accessible maxillary bone defects.

Myocardial reperfusion treatment for ischemic infarction may cause lethal injury of cardiomyocytes, which is known as ischemia/reperfusion (I/R) injury. As a kind of prospective biomaterial with superior properties, the application of bioactive glasses (BGs) in myocardial tissue engineering have received great interests. In this study, the cardioprotective effect and relevant mechanism of BG on myocardial reperfusion injury were investigated in vitro. H9c2 cardiomyocytes were pretreated with BG extracts and then cultured in hypoxic environment for 30 min followed by reoxygenation for 1 h. The activity of released lactate dehydrogenase (LDH) and the content of malondialdehyde (MDA) in H9c2 cells were tested by assay kits. Cell viability was analyzed by Live/Dead staining assay and the number of living cells was detected by Cell Counting Kit-8 (CCK-8) assay. The cytoskeletal protein F-actin was stained and observed under inverted fluorescence microscope. Mitochondrial membrane potential (MMP) level, reactive oxygen species (ROS) production and apoptosis ratio were evaluated by fluorescent observation and flow cytometry simultaneously. The gene expressions relevant to apoptosis were detected by quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results showed that BG extracts effectively inhibited hypoxia/reoxygenation (H/R)-induced cell injury by suppressing oxidative stress and mitochondrial permeability transition (MPT) within H9c2 cells. Meanwhile, apoptosis caused by H/R injury was alleviated and three classic apoptotic signaling pathways were proved to be regulated by BG extracts. Further analysis showed that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was up-regulated in H/R-induced H9c2 cells by BG extracts, leading to relieved cellular apoptosis. These results indicated that BG might exert cardioprotective effect in reperfusion injury when applied in myocardial tissue regeneration and repair.

In this study, the silk fibroin/nano-hydroxyapatite/hyaluronic acid (SF/nHAp/HA) composite scaffolds with different HA contents were developed by blending, cross-linking and freeze-drying, and their physicochemical properties and cell biocompatibility in vitro were subsequently studied. It was observed that the molecular conformation of the composite scaffolds was mainly composed of silk I and a small amount of the β-sheets structure. On enhancing the HA content, the pore size of the scaffold decreased, while the porosity, water absorption, swelling ratio and mechanical properties were observed to increase. In particular, the SF/nHAp/HA scaffold with a 5.0 wt% ratio exhibited the highest water absorption and mechanical properties among the developed materials. In addition, the in vitro cytocompatibility analysis showed that the bone marrow mesenchymal stem cells exhibited excellent cell proliferation and osteogenic differentiation ability on the SF/nHAp/5.0 wt%HA scaffolds, as compared with the other scaffolds. It can be concluded that the developed composite scaffolds represent a promising class of materials for the bone tissue repair and regeneration.

The cancer microenvironment influences tumor progression and metastasis and is pivotal to consider when designing in vivo-like cancer models. Current preclinical testing platforms for cancer drug development are mainly limited to 2D cell culture systems that poorly mimic physiological environments and traditional, low throughput animal models. The aim of this work was to produce a tunable testing platform based on 3D printed scaffolds (3DPS) with a simple geometry that, by extracellular components and response of breast cancer reporter cells, mimics patient-derived scaffolds (PDS) of breast cancer. Here, the biocompatible polysaccharide alginate was used as base material to generate scaffolds consisting of a 3D grid containing periostin and hydroxyapatite. Breast cancer cell lines (MCF7 and MDA-MB-231) produced similar phenotypes and gene expression levels of cancer stem cell, epithelial–mesenchymal transition, differentiation and proliferation markers when cultured on 3DPS and PDS, contrasting conventional 2D cultures. Importantly, cells cultured on 3DPS and PDS showed scaffold-specific responses to cytotoxic drugs (doxorubicin and 5-fluorouracil) that were different from 2D cultured cells. In conclusion, the data presented support the use of a tunable alginate-based 3DPS as a tumor model in breast cancer drug discovery.

Cell alignment plays an essential role in cytoskeleton reorganization, extracellular matrix remodeling, and biomechanical properties regulation of tissues such as vascular tissues, cardiac muscles, and tendons. Based on the natural-oriented features of cells in native tissues, various biomimetic scaffolds have been reported with the introduction of well-arranged ultrafine fibers to induce cell alignment. However, it is still a challenge to fabricate scaffolds with suitable mechanical properties, biomimetic microenvironment, and ability to promote cell alignment. In this paper, we propose an integrated 3D printing system to fabricate multi-scale hierarchical scaffolds combined with meso-, micro-, and nano-fibrous filaments, in which the meso-, micro-, and nano-fibers fabricated via fused deposition modeling, melt electrospining writing, and solution electrospining can provide structural support, promote cell alignment, and create a biomimetic microenvironment to facilitate cell function, respectively. The plasma surface modification was performed improve the surface wettability of the scaffolds by measuring the contact angle. The obtained in vitro biological results validate the ability of multi-scale hierarchical scaffolds to enhance cell adhesion and proliferation, and promote cell alignment with the guidance of the aligned microfibers produced via melt electrospining writing in hierarchical scaffolds.

Chitosan (CS) hydrogels have been widely used throughout basic tissue engineering and regenerative medicine research and it is very desirable to develop advanced CS materials with superior mechanical and topographical properties for more extensive applications. Herein, we present the design of a double crosslinking pure CS hydrogel material via the synergic effect of the chemical covalent network, hydrophobic interactions, enhanced intermolecular hydrogen bonding and the formation of the CS crystallite. The resultant pure CS hydrogel possesses increases in strength and toughness by two orders of magnitude (fracture energy ∼7.733 J m⁻²; maximal compression stress ∼10.81 MPa, elastic modulus ∼1.33 MPa). We utilize ¹H NMR and FT-IR to prove the success of chemical modification. The results of Raman spectra and WXRD have proved the existence of physical interaction between CS hydrogels and microcrystals, thus explaining the enhancement mechanism of mechanical strength of CS hydrogel. The live and death results also show that MSCs can grow well on CS hydrogels, and the results of CCK-8 indicate low cytotoxicity of CS hydrogels. This CS hydrogel shows great potential applications in tissue engineering and regenerative medicine.

In this study, we offer new insights into the contrasting effects of electrospun fiber orientation on microglial polarization under normoxia and hypoxia, and establish for the first time, the intrinsically protective roles of electrospun meshes against hypoxia-induced microglial responses. First, resting microglia were cultured under normoxia on poly(caprolactone) fibers possessing two distinctly different fiber orientations. Matrix-guided differences in cell shape/orientation and differentially expressed Rho GTPases (RhoA, Rac1, Cdc42) were well-correlated with the randomly oriented fibers inducing a pro-inflammatory phenotype and the aligned fibers sustaining a resting phenotype. Upon subsequent hypoxia induction, both sets of meshes offered protection from hypoxia-induced damage by promoting a radical phenotypic switch and beneficially altering the M2/M1 ratio to different extents. Compared to 2D hypoxic controls, meshes significantly suppressed the expression of pro-inflammatory markers (IL-6, TNF-α) and induced drastically higher expression of anti-inflammatory (IL-4, IL-10, VEGF-189) and neuroprotective (Nrf-2) markers. Consistent with this M2 polarization, the expression of Rho GTPases was significantly lower in the mesh groups under hypoxia compared to normoxic culture. Moreover, meshes—particularly with aligned fibers—promoted higher cell viability, suppressed caspase 3/8 and LC-3 expression and promoted LAMP-1 and LAMP-2 expression, which suggested the mitigation of apoptotic/autophagic cell death via a lysosomal membrane-stabilization mechanism. Notably, all protective effects under hypoxia were observed in the absence of additional soluble cues. Our results offer promise for leveraging the intrinsic therapeutic potential of electrospun meshes in degenerative diseases where microglial dysfunction, hypoxia and inflammation are implicated.