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Abstract Background Despite their ability to regenerate as well as autografts, the use of nerve allografts is limited by the need for immunosuppression and the risk of disease transmission. Further, decellularized allografts lacking Schwann cells limit axonal regeneration in long nerve defects. This study evaluated sciatic nerve regeneration in rats implanted with cold- or cryopreserved allografts, and examined the effects of FK506, an immunosuppressant that targets calcineurin function, on motor recovery. Methods Sixty-five male Lewis rats were divided into five groups of 13, each with a 10-mm sciatic nerve gap. Group I received an autograft, whereas Groups II and III received allografts pretreated with cryopreservation and cold preservation, respectively. Groups IV and V were also implanted with cryo- and cold-preserved allografts, but were treated with a low dose of FK506. Motor regeneration was assessed at 20 weeks by the measurement of ankle contracture, compound muscle action potential, maximal isometric tetanic force, wet muscle weight of the tibialis anterior, peroneal nerve histomorphometry, and immunohistochemistry of the reconstructed sciatic nerve. Results Similar motor recovery was observed between the autografts and both types of allografts. The groups treated with FK506 showed improved recovery, particularly in terms of ankle angle and tibialis anterior muscle weight. Histomorphometry revealed a superior myelinated fiber area and nerve ratio in the cold-preserved allograft group, while Group II displayed a less well-organized morphology. Conclusion This study demonstrates that cold- or cryopreserved nerve allografts represent effective alternatives to autografts for peripheral nerve reconstruction, with low-dose FK506 enhancing motor recovery without necessitating immunosuppression. Level of Evidence I Basic Science Level I.

Recent cumulative findings signify the adaptive immunity of materials as a key agenda in tissue healing that can improve regenerative events and outcomes. Modulating immune responses, mainly the recruitment and functions of T and B cells and their further interplay with innate immune cells (e.g., dendritic cells, macrophages) can be orchestrated by materials. For instance, decellularized matrices have been shown to promote muscle healing by inducing T helper 2 (Th2) cell immunity, while synthetic biopolymers exhibit differential effects on B cell responses and fibrosis compared decellularized matrices. We discuss the recent findings on how implantable materials instruct the adaptive immune events and the subsequent tissue healing process. In particular, we dissect the materials’ physicochemical properties (shape, size, topology, degradation, rigidity, and matrix dynamic mechanics) to demonstrate the relations of these parameters with the adaptive immune responses in vitro and the underlying biological mechanisms. Furthermore, we present evidence of recent in vivo phenomena, including tissue healing, cancer progression, and fibrosis, wherein biomaterials potentially shape adaptive immune cell functions and in vivo outcomes. Our discussion will help understand the materials-regulated immunology events more deeply, and offer the design rationale of materials with tunable matrix properties for accelerated tissue repair and regeneration.

Abstract Phosphate bioactive glass has been studied for its advanced biodegradability and active ion release capability. Our previous research found that phosphate glass containing (P2O5)–(Na2O)–(TiO2)–(CaO)–(SrO) or (ZnO) showed good biocompatibility with MG63 and hMSCs. This study further investigated the application of 5 mol% zinc oxide or 17.5 mol% strontium oxide in titanium-doped phosphate glass for bone tissue engineering. Ti-Ca-Na-Phosphate glasses, incorporating 5% zinc oxide or 17.5% strontium oxide, were made with melting quenching technology. The pre-osteoblast cell line MC3T3-E1 was cultured for indirect contact tests with graded diluted phosphate glass extractions and for direct contact tests by seeding cells on glass disks. The cell viability and cytotoxicity were analysed in vitro over 7 days. In vivo studies utilized the tibial defect model with or without glass implants. The micro-CT analysis was performed after surgery and then at 2, 6, and 12 weeks. Extractions from both zinc and strontium phosphate glasses showed no negative impact on MC3T3-E1 cell viability. Notably, non-diluted Zn-Ti-Ca-Na-phosphate glass extracts significantly increased cell viability by 116.8% (P

Abstract Oral cancer stands as a prevalent maligancy worldwide; however, its therapeutic potential is limited by undesired effects and complications. As a medicinal edible fungus, Chaga mushroom (Inonotus obliquus) exhibits anticancer effects across diverse cancers. Yet, the precise mechanisms underlying its efficacy remain unclear. We explored the detailed mechanisms underlying the anticancer action of Chaga mushroom extract in oral cancer cells (HSC-4). Following treatment with Chaga mushroom extracts, we analyzed cell viability, proliferation capacity, glycolysis, mitochondrial respiration, and apoptosis. Our findings revealed that the extract reduced cell viability and proliferation of HSC-4 cells while arresting their cell cycle via suppression of STAT3 activity. Regarding energy metabolism, Chaga mushroom extract inhibited glycolysis and mitochondrial membrane potential in HSC-4 cells, thereby triggering autophagy-mediated apoptotic cell death through activation of the p38 MAPK and NF-κB signaling pathways. Our results indicate that Chaga mushroom extract impedes oral cancer cell progression, by inhibiting cell cycle and proliferation, suppressing cancer cell energy metabolism, and promoting autophagy-mediated apoptotic cell death. These findings suggest that this extract is a promising supplementary medicine for the treatment of patients with oral cancer.

Abstract Epithelial‐stromal interplay through chemomechanical cues from cells and matrix propels cancer progression. Elevated tissue stiffness in potentially malignant tissues suggests a link between matrix stiffness and enhanced tumor growth. In this study, employing chronic oral/esophageal injury and cancer models, it is demonstrated that epithelial–stromal interplay through matrix stiffness and Hedgehog (Hh) signaling is key in compounding cancer development. Epithelial cells actively interact with fibroblasts, exchanging mechanoresponsive signals during the precancerous stage. Specifically, epithelial cells release Sonic Hh, activating fibroblasts to produce matrix proteins and remodeling enzymes, resulting in tissue stiffening. Subsequently, basal epithelial cells adjacent to the stiffened tissue become proliferative and undergo epithelial‐to‐mesenchymal transition, acquiring migratory and invasive properties, thereby promoting invasive tumor growth. Notably, transcriptomic programs of oncogenic GLI2, mechano‐activated by actin cytoskeletal tension, govern this process, elucidating the crucial role of non‐canonical GLI2 activation in orchestrating the proliferation and mesenchymal transition of epithelial cells. Furthermore, pharmacological intervention targeting tissue stiffening proves highly effective in slowing cancer progression. These findings underscore the impact of epithelial‐stromal interplay through chemo‐mechanical (Hh‐stiffness) signaling in cancer development, and suggest that targeting tissue stiffness holds promise as a strategy to disrupt chemo‐mechanical feedback, enabling effective cancer treatment.

Abstract Electrical conductivity is a pivotal biophysical factor for neural interfaces, though optimal values remain controversial due to challenges isolating this cue. To address this issue, conductive substrates made of carbon nanotubes and graphene oxide nanoribbons, exhibiting a spectrum of conductivities from 0.02 to 3.2 S m−1, while controlling other surface properties is designed. The focus is to ascertain whether varying conductivity in isolation has any discernable impact on neural lineage specification. Remarkably, neural‐tissue‐like low conductivity (0.02–0.1 S m−1) prompted neural stem/progenitor cells to exhibit a greater propensity toward neuronal lineage specification (neurons and oligodendrocytes, not astrocytes) compared to high supraphysiological conductivity (3.2 S m−1). High conductivity instigated the apoptotic process, characterized by increased apoptotic fraction and decreased neurogenic morphological features, primarily due to calcium overload. Conversely, cells exposed to physiological conductivity displayed epigenetic changes, specifically increased chromatin openness with H3acetylation (H3ac) and neurogenic‐transcription‐factor activation, along with a more balanced intracellular calcium response. The pharmacological inhibition of H3ac further supported the idea that such epigenetic changes might play a key role in driving neuronal specification in response to neural‐tissue‐like, not supraphysiological, conductive cues. These findings underscore the necessity of optimal conductivity when designing neural interfaces and scaffolds to stimulate neuronal differentiation and facilitate the repair process.

Highlights Nanozyme-based approaches to produce therapeutic hydrogels. Enzymatic mechanisms and multifunctional roles of nanozyme-engineered hydrogels for skin therapy. Therapeutic actions of nanozyme-engineered hydrogels in inflamed skin tissues. Mechanical and immunological aspects of skin therapy guided by nanozyme-engineered hydrogels. Promising directions and challenges of nanozyme-inspired hydrogel platforms.

Abstract Understanding the impact of long‐term opioid exposure on the embryonic brain is critical due to the surging number of pregnant mothers with opioid dependency. However, this has been limited by human brain inaccessibility and cross‐species differences in animal models. Here, a human midbrain model is established that uses hiPSC‐derived midbrain organoids to assess cell‐type‐specific responses to acute and chronic fentanyl treatment and fentanyl withdrawal. Single‐cell mRNA sequencing of 25,510 cells from organoids in different treatment groups reveals that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters synaptic activity and neuron projection. In contrast, acute fentanyl treatment increases dopamine release but does not significantly alter gene expression related to cell lineage development. These results provide the first examination of the effects of opioid exposure on human midbrain development at the single‐cell level.

Periodontal ligament (PDL) cells play a crucial role in maintaining periodontal integrity and function by providing cell sources for ligament regeneration. While biophysical stimulation is known to regulate cell behaviors and functions, its impact on epigenetics of PDL cells has not yet been elucidated. Here, we aimed to investigate the cytoskeletal changes, epigenetic modifications, and lineage commitment of PDL cells following the application of stretch stimuli to PDL. PDL cells were subjected to stretching (0.1 Hz, 10 %). Subsequently, changes in focal adhesion, tubulin, and histone modification were observed. The survival ability in inflammatory conditions was also evaluated. Furthermore, using a rat hypo-occlusion model, we verified whether these phenomena are observed in vivo. Stretched PDL cells showed maximal histone 3 acetylation (H3Ace) at 2 h, aligning perpendicularly to the stretch direction. RNA sequencing revealed stretching altered gene sets related to mechanotransduction, histone modification, reactive oxygen species (ROS) metabolism, and differentiation. We further found that anchorage, cell elongation, and actin/microtubule acetylation were highly upregulated with mechanosensitive chromatin remodelers such as H3Ace and histone H3 trimethyl lysine 9 (H3K9me3) adopting euchromatin status. Inhibitor studies showed mechanotransduction-mediated chromatin modification alters PDL cells behaviors. Stretched PDL cells displayed enhanced survival against bacterial toxin (C12-HSL) or ROS (H2O2) attack. Furthermore, cyclic stretch priming enhanced the osteoclast and osteoblast differentiation potential of PDL cells, as evidenced by upregulation of lineage-specific genes. In vivo, PDL cells from normally loaded teeth displayed an elongated morphology and higher levels of H3Ace compared to PDL cells with hypo-occlusion, where mechanical stimulus is removed. Overall, these data strongly link external physical forces to subsequent mechanotransduction and epigenetic changes, impacting gene expression and multiple cellular behaviors, providing important implications in cell biology and tissue regeneration.

Regeneration of pathological wounds, such as diabetic ulcers, poses a significant challenge in clinical settings, despite the widespread use of drugs. To overcome clinical side effects and complications, drug-free therapeutics need to be developed to promote angiogenesis while overcoming inflammation to restore regenerative events. This study presents a novel bioactive nanozyme based on cobalt-doped nanoglass (namely, CoNZ), which exhibits high enzymatic/catalytic activity while releasing therapeutic ions. Cobalt oxide “Co3O4” tiny crystallites produced in situ through a chemical reaction with H2O2 within CoNZ nanoparticles play a crucial role in scavenging ROS. Results showed that CoNZ-treatment to full-thickness skin wounds in mice significantly accelerated the healing process, promoting neovascularization, matrix deposition, and epithelial lining while reducing pro-inflammatory signs. Notably, CoNZ was highly effective in treating pathological wounds (streptozotocin-induced diabetic wounds). Rapid scavenging of ROS by CoNZ and down-regulation of pro-inflammatory markers while up-regulating tissue healing signs with proliferative cells and activated angiogenic factors contributed to the observed healing events. In vitro experiments involving CoNZ-cultures with macrophages and endothelial cells exposed to high glucose and ROS-generating conditions further confirmed the effectiveness of CoNZ. CoNZ-promoted angiogenesis was attributed to the release of cobalt ions, as evidenced by the comparable effects of CoNZ-extracted ionic medium in enhancing endothelial migration and tubule formation via activated HIF-1α. Finally, we compared the in vivo efficacy of CoNZ with the clinically-available drug deferoxamine. Results demonstrated that CoNZ was as effective as the drug in closing the diabetic wound, indicating the potential of CoNZ as a novel drug-free therapeutic approach.

Abstract Pathological dermal scars such as keloids present significant clinical challenges lacking effective treatment options. Given the distinctive feature of highly stiffened scar tissues, deciphering how matrix mechanics regulate pathological progression can inform new therapeutic strategies. Here, it is shown that pathological dermal scar keloid fibroblasts display unique metamorphoses to stiffened matrix. Compared to normal fibroblasts, keloid fibroblasts show high sensitivity to stiffness rather than biochemical stimulation, activating cytoskeletal‐to‐nuclear mechanosensing molecules. Notably, keloid fibroblasts on stiff matrices exhibit nuclear softening, concomitant with reduced lamin A/C expression, and disrupted anchoring of lamina‐associated chromatin. This nuclear softening, combined with weak adhesion and high contractility, facilitates the invasive migration of keloid fibroblasts through confining matrices. Inhibiting lamin A/C‐driven nuclear softening, via lamin A/C overexpression or actin disruption, mitigates such invasiveness of keloid fibroblasts. These findings highlight the significance of the nuclear mechanics of keloid fibroblasts in scar pathogenesis and propose lamin A/C as a potential therapeutic target for managing pathological scars.

Abstract Cell migration interacting with continuously changing microenvironment, is one of the most essential cellular functions, participating in embryonic development, wound repair, immune response, and cancer metastasis. The migration process is finely tuned by integrin‐mediated binding to ligand molecules. Although numerous biochemical pathways orchestrating cell adhesion and motility are identified, how subcellular forces between the cell and extracellular matrix regulate intracellular signaling for cell migration remains unclear. Here, it is showed that a molecular binding force across integrin subunits determines directional migration by regulating tension‐dependent focal contact formation and focal adhesion kinase phosphorylation. Molecular binding strength between integrin αvβ3 and fibronectin is precisely manipulated by developing molecular tension probes that control the mechanical tolerance applied to cell‐substrate interfaces. This data reveals that integrin‐mediated molecular binding force reduction suppresses cell spreading and focal adhesion formation, attenuating the focal adhesion kinase (FAK) phosphorylation that regulates the persistence of cell migration. These results further demonstrate that manipulating subcellular binding forces at the molecular level can recapitulate differential cell migration in response to changes of substrate rigidity that determines the physical condition of extracellular microenvironment. Novel insights is provided into the subcellular mechanics behind global mechanical adaptation of the cell to surrounding tissue environments featuring distinct biophysical signatures.

The dura mater, as the crucial outermost protective layer of the meninges, plays a vital role in safeguarding the underlying brain tissue. Neurosurgeons face significant challenges in dealing with trauma or large defects in the dura mater, as they must address the potential complications, such as wound infections, pseudomeningocele formation, cerebrospinal fluid leakage, and cerebral herniation. Therefore, the development of dural substitutes for repairing or reconstructing the damaged dura mater holds clinical significance. In this review we highlight the progress in the development of dural substitutes, encompassing autologous, allogeneic, and xenogeneic replacements, as well as the polymeric-based dural substitutes fabricated through various scaffolding techniques. In particular, we explore the development of composite materials that exhibit improved physical and biological properties for advanced dural substitutes. Furthermore, we address the challenges and prospects associated with developing clinically relevant alternatives to the dura mater.

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H2O2. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

Abstract Advancing the technologies for cellular reprogramming with high efficiency has significant impact on regenerative therapy, disease modeling, and drug discovery. Biophysical cues can tune the cell fate, yet the precise role of external physical forces during reprogramming remains elusive. Here the authors show that temporal cyclic‐stretching of fibroblasts significantly enhances the efficiency of induced pluripotent stem cell (iPSC) production. Generated iPSCs are proven to express pluripotency markers and exhibit in vivo functionality. Bulk RNA‐sequencing reveales that cyclic‐stretching enhances biological characteristics required for pluripotency acquisition, including increased cell division and mesenchymal‐epithelial transition. Of note, cyclic‐stretching activates key mechanosensitive molecules (integrins, perinuclear actins, nesprin‐2, and YAP), across the cytoskeletal‐to‐nuclear space. Furthermore, stretch‐mediated cytoskeletal‐nuclear mechano‐coupling leads to altered epigenetic modifications, mainly downregulation in H3K9 methylation, and its global gene occupancy change, as revealed by genome‐wide ChIP‐sequencing and pharmacological inhibition tests. Single cell RNA‐sequencing further identifies subcluster of mechano‐responsive iPSCs and key epigenetic modifier in stretched cells. Collectively, cyclic‐stretching activates iPSC reprogramming through mechanotransduction process and epigenetic changes accompanied by altered occupancy of mechanosensitive genes. This study highlights the strong link between external physical forces with subsequent mechanotransduction process and the epigenetic changes with expression of related genes in cellular reprogramming, holding substantial implications in the field of cell biology, tissue engineering, and regenerative medicine.

We propose an ex vivo T cell expansion system that mimics natural antigen-presenting cells (APCs) for adoptive cell therapy (ACT). Microfiber scaffolds coated with dendritic cell (DC) membrane replicate physicochemical properties of dendritic cells specific for T cell activation such as rapid recognition by T cells, long duration of T cell tethering, and DC-specific co-stimulatory cues. The DC membrane-coated scaffold is first surface-immobilized with T cell stimulatory ligands, anti-CD3 (αCD3) and anti-CD28 (αCD28) antibodies, followed by adsorption of releasable interleukin-2 (IL-2). The scaffolds present both surface and soluble cues to T cells ex vivo in the same way that these cues are presented by natural APCs in vivo. We demonstrate that the DC-mimicking scaffold promotes greater polyclonal expansion of primary human T cells as compared to αCD3/αCD28-functionalized Dynabead. More importantly, major histocompatibility complex molecules derived from the DC membrane of the scaffold allow antigen-specific T cell expansion with target cell-specific killing ability. In addition, most of the expanded T cells (∼97%) can be harvested from the scaffold by density gradient centrifugation. Overall, the DC-mimicking scaffold offers a scalable, modular, and customizable platform for rapid expansion of highly functional T cells for ACT.

Rigidity (or stiffness) of materials and extracellular matrix has proven to be one of the most significant extracellular physicochemical cues that can control diverse cell behaviors, such as contractility, motility, and spreading, and the resultant pathophysiological phenomena. Many 2D materials engineered with tunable rigidity have enabled researchers to elucidate the roles of matrix biophysical cues in diverse cellular events, including migration, lineage specification, and mechanical memory. Moreover, the recent findings accumulated under 3D environments with viscoelastic and remodeling properties pointed to the importance of dynamically changing rigidity in cell fate control, tissue repair, and disease progression. Thus, here we aim to highlight the works related with material/matrix-rigidity-mediated cell and tissue behaviors, with a brief outlook into the studies on the effects of material/matrix rigidity on cell behaviors in 2D systems, further discussion of the events and considerations in tissue-mimicking 3D conditions, and then examination of the in vivo findings that concern material/matrix rigidity. The current discussion will help understand the material/matrix-rigidity-mediated biological phenomena and further leverage the concepts to find therapeutic targets and to design implantable materials for the treatment of damaged and diseased tissues.

Introduction: Osteogenic and angiogenic properties of synthetic bone grafts play a crucial role in the restoration of bone defects. Angiogenesis is recognised for its support in bone regeneration, particularly in larger defects. The objective of this study is to evaluate the new bone formation and neovascularisation of a 3D-printed isosorbide-based novel CSMA-2 polymer in biomimetic gyroid structures. Methods: The gyroid scaffolds were fabricated by 3D printing CSMA-2 polymers with different hydroxyapatite (HA) filler concentrations using the digital light processing (DLP) method. A small animal subcutaneous model and a rat calvaria critical-size defect model were performed to analyse tissue compatibility, angiogenesis, and new bone formation. Results: The in vivo results showed good biocompatibility of the 3D-printed gyroid scaffolds with no visible prolonged inflammatory reaction. Blood vessels were found to infiltrate the pores from day 7 of the implantation. New bone formation was confirmed with positive MT staining and BMP-2 expression, particularly on scaffolds with 10% HA. Bone volume was significantly higher in the CSMA-2 10HA group compared to the sham control group. Discussion and Conclusions: The results of the subcutaneous model demonstrated a favourable tissue response, including angiogenesis and fibrous tissue, indicative of the early wound healing process. The results from the critical-size defect model showcased new bone formation, as confirmed by micro-CT imaging and immunohistochemistry. The combination of CSMA-2 as the 3D printing material and the gyroid as the 3D structure was found to support essential events in bone healing, specifically angiogenesis and osteogenesis.

Abstract At a time of unpredictable challenges for health, one trend is certain: there is an exceedingly high demand for functional implants, particularly bone grafts. This has encouraged the emergence of bone tissue engineering substitutes as an alternative method to conventional bone grafts. However, the current approaches in the field face several limitations that have prevented the ultimate translation into clinical settings. As a result, many attempts have been made to fabricate synthetic bone implants that can offer suitable biological and mechanical properties. Light curable methacrylate-based polymers have ideal properties for bone repair. These materials are also suitable for 3D printing which can be applicable for restoration of both function and aesthetics. The main objective of this research was to investigate the role of calcium phosphate (CaP) incorporation in a mechanically stable, biologically functional and 3D printable polymer for the reconstruction of complex craniofacial defects. The experimental work initially involved the synthesis of (((((((((((3R,3aR,6S,6aR)- hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(ethane-2,1- 48 diyl))bis(oxy))bis(carbonyl))bis(azanediyl))bis(3,3,5-trimethylcyclohexane-5,1- 49 diyl))bis(azanediyl))bis(carbonyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) referred to as CSMA and fabrication of composite discs via a Digital Light Printing (DLP) method. The flow behaviour of the polymer as a function of CaP addition, surface remineralisation potential, in vitro cell culture, using MC3T3 and Adipose-Derived Mesenchymal Stem Cells (ADSCs) and ex ovo angiogenic response was assessed. Finally, in vivo studies were carried out to investigate neo-bone formation at 4- and 8-weeks post-implantation. Quantitative micro-CT and histological evaluation did not show a higher rate of bone formation in CaP filled CSMA composites compared to CSMA itself. Therefore, such polymeric systems hold promising features by allowing more flexibility in designing a 3D printed scaffold targeted at the reconstruction of maxillofacial defects.

Abstract Background Astrocyte is a key regulator of neuronal activity and excitatory/inhibitory balance via gliotransmission. Recently, gliotransmission has been identified as a novel target for neurological diseases. However, using the properties of nanomaterials to modulate gliotransmission has not been uncovered. Results We prepared non-invasive CNT platforms for cells with different nanotopography and properties such as hydrophilicity and conductivity. Using CNT platforms, we investigated the effect of CNT on astrocyte functions participating in synaptic transmission by releasing gliotransmitters. Astrocytes on CNT platforms showed improved cell adhesion and proliferation with upregulated integrin and GFAP expression. In addition, intracellular GABA and glutamate in astrocytes were augmented on CNT platforms. We also demonstrated that gliotransmitters in brain slices were increased by ex vivo incubation with CNT. Additionally, intracellular resting Ca2+ level, which is important for gliotransmission, was also increased via TRPV1 on CNT platforms. Conclusion CNT can improve astrocyte function including adhesion, proliferation and gliotransmission by increasing resting Ca2+ level. Therefore, our study suggests that CNT would be utilized as a new therapeutic platform for central nervous system diseases by modulating gliotransmission. Graphical Abstract

Three-dimensional (3D) bioprinting is a promising and rapidly evolving technology in the field of additive manufacturing. It enables the fabrication of living cellular constructs with complex architectures that are suitable for various biomedical applications, such as tissue engineering, disease modeling, drug screening, and precision regenerative medicine. The ultimate goal of bioprinting is to produce stable, anatomically-shaped, human-scale functional organs or tissue substitutes that can be implanted. Although various bioprinting techniques have emerged to develop customized tissue-engineering substitutes over the past decade, several challenges remain in fabricating volumetric tissue constructs with complex shapes and sizes and translating the printed products into clinical practice. Thus, it is crucial to develop a successful strategy for translating research outputs into clinical practice to address the current organ and tissue crises and improve patients’ quality of life. This review article discusses the challenges of the existing bioprinting processes in preparing clinically relevant tissue substitutes. It further reviews various strategies and technical feasibility to overcome the challenges that limit the fabrication of volumetric biological constructs and their translational implications. Additionally, the article highlights exciting technological advances in the 3D bioprinting of anatomically shaped tissue substitutes and suggests future research and development directions. This review aims to provide readers with insight into the state-of-the-art 3D bioprinting techniques as powerful tools in engineering functional tissues and organs.

Bone defects in patients entail the microenvironment that needs to boost the functions of stem cells (e.g., proliferation, migration, and differentiation) while alleviating severe inflammation induced by high oxidative stress. Biomaterials can help to shift the microenvironment by regulating these multiple events. Here we report multifunctional composite hydrogels composed of photo-responsive Gelatin Methacryloyl (GelMA) and dendrimer (G3)-functionalized nanoceria (G3@nCe). Incorporation of G3@nCe into GelMA could enhance the mechanical properties of hydrogels and their enzymatic ability to clear reactive oxygen species (ROS). The G3@nCe/GelMA hydrogels supported the focal adhesion of mesenchymal stem cells (MSCs) and further increased their proliferation and migration ability (vs. pristine GelMA and nCe/GelMA). Moreover, the osteogenic differentiation of MSCs was significantly stimulated upon the G3@nCe/GelMA hydrogels. Importantly, the capacity of G3@nCe/GelMA hydrogels to scavenge extracellular ROS enabled MSCs to survive against H2O2-induced high oxidative stress. Transcriptome analysis by RNA sequencing identified the genes upregulated and the signalling pathways activated by G3@nCe/GelMA that are associated with cell growth, migration, osteogenesis, and ROS-metabolic process. When implanted subcutaneously, the hydrogels exhibited excellent tissue integration with a sign of material degradation while the inflammatory response was minimal. Furthermore, G3@nCe/GelMA hydrogels demonstrated effective bone regeneration capacity in a rat critical-sized bone defect model, possibly due to an orchestrated capacity of enhancing cell proliferation, motility and osteogenesis while alleviating oxidative stress.

Polymeric hydrogels are fascinating platforms as 3D scaffolds for tissue repair and delivery systems of therapeutic molecules and cells. Among others, methacrylated gelatin (GelMA) has become a representative hydrogel formulation, finding various biomedical applications. Recent efforts on GelMA-based hydrogels have been devoted to combining them with bioactive and functional nanomaterials, aiming to provide enhanced physicochemical and biological properties to GelMA. The benefits of this approach are multiple: i) reinforcing mechanical properties, ii) modulating viscoelastic property to allow 3D printability of bio-inks, iii) rendering electrical/magnetic property to produce electro-/magneto-active hydrogels for the repair of specific tissues (e.g., muscle, nerve), iv) providing stimuli-responsiveness to actively deliver therapeutic molecules, and v) endowing therapeutic capacity in tissue repair process (e.g., antioxidant effects). The nanomaterial-combined GelMA systems have shown significantly enhanced and extraordinary behaviors in various tissues (bone, skin, cardiac, and nerve) that are rarely observable with GelMA. Here we systematically review these recent efforts in nanomaterials-combined GelMA hydrogels that are considered as next-generation multifunctional platforms for tissue therapeutics. The approaches used in GelMA can also apply to other existing polymeric hydrogel systems.

Bioactive phosphate glasses incorporated with Zn2+ (predicted to be useful for bone regeneration applications) were synthesized. Characterization of the bioglasses were done using XRD, FTIR, SEM, and EDS techniques, both prior to and after soaking in simulated body fluid (SBF) solution. The bioactive performance of samples was evaluated by incubated in SBF for different days by means of the pH measuring and comparing the results of XRD and SEM studies. The obtained results show the formation of hydroxyapatite (HAP) layer on the surface of as-developed bioactive glasses. The degradation of the samples evaluated by weight loss and pH of the remnant solution measured as functions of ZnO concentration and immersion time revealed the enhanced formation of HAP layer on the sample surface. The results further indicated the rate of degradation can be monitored by tailoring the concentration of ZnO and immersion time. The studies were also extended to in vitro cytocompatibility and cell proliferation measurements on rMSC using CCK-8 for incubation periods of 24 h and 72 h. These studies have revealed the considerable levels of rMSC proliferation and improved viability within the limits of nontoxic levels. The measurements of antibacterial activity of ZnO-doped phosphate bioactive glasses against E. coli and S. aureus, carried out for 24 h and 48 h have confirmed their potential antibacterial activity. Finally, quantitative analysis of these results confirmed an enhanced in vitro biological and antibacterial activity of the samples due to the ZnO doping and subsequent controlled release of Zn2+ ions in the glass matrix.

Abstract Novel polycaprolactone‐based polyurethane (PCL‐PU) copolymers with hyperelasticity, shape‐memory, and ultra‐cell‐adhesion properties are reported as clinically applicable tissue‐regenerative biomaterials. New isosorbide derivatives (propoxylated or ethoxylated ones) were developed to improve mechanical properties by enhanced reactivity in copolymer synthesis compared to the original isosorbide. Optimized PCL‐PU with propoxylated isosorbide exhibited notable mechanical performance (50 MPa tensile strength and 1150% elongation with hyperelasticity under cyclic load). The shape‐memory effect was also revealed in different forms (film, thread, and 3D scaffold) with 40%–80% recovery in tension or compression mode after plastic deformation. The ultra‐cell‐adhesive property was proven in various cell types which were reasoned to involve the heat shock protein‐mediated integrin (α5 and αV) activation, as analyzed by RNA sequencing and inhibition tests. After the tissue regenerative potential (muscle and bone) was confirmed by the myogenic and osteogenic responses in vitro, biodegradability, compatible in vivo tissue response, and healing capacity were investigated with in vivo shape‐memorable behavior. The currently exploited PCL‐PU, with its multifunctional (hyperelastic, shape‐memorable, ultra‐cell‐adhesive, and degradable) nature and biocompatibility, is considered a potential tissue‐regenerative biomaterial, especially for minimally invasive surgery that requires small incisions to approach large defects with excellent regeneration capacity.

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to further improve the spatiotemporal operability of CRISPR/Cas9.

Therapeutic options are quite limited in clinics for the successful repair of infected/degenerated tissues. Although the prevalent treatment is the complete removal of the whole infected tissue, this leads to a loss of tissue function and serious complications. Herein the dental pulp infection, as one of the most common dental problems, was selected as a clinically relevant case to regenerate using a multifunctional nanotherapeutic approach. For this, a mesoporous bioactive glass nano-delivery system incorporating silicate, calcium, and copper as well as loading epidermal growth factor (EGF) was designed to provide antibacterial/pro-angiogenic and osteo/odontogenic multiple therapeutic effects. Amine-functionalized Cu-doped bioactive glass nanospheres (Cu-BGn) were prepared to be 50–60 nm in size, mesoporous, positive-charged and bone-bioactive. The Cu-BGn could release bioactive ions (copper, calcium and silicate ions) with therapeutically-effective doses. The Cu-BGn treatment to human umbilical vein endothelial cells (HUVEC) led to significant enhancement of the migration, tubule formation and expression of angiogenic gene (e.g. vascular endothelial growth factor, VEGF). Furthermore, the EGF-loaded Cu-BGn (EGF@Cu-BGn) showed pro-angiogenic effects with antibacterial activity against E. faecalis, a pathogen commonly involved in the pulp infection. Of note, under the co-culture condition of HUVEC with E. faecalis, the secretion of VEGF was up-regulated. In addition, the osteo/odontogenic stimulation of the EGF@Cu-BGn was evidenced with human dental pulp stem cells. The local administration of the EGF@Cu-BGn in a rat molar tooth defect infected with E. faecalis revealed significant in vivo regenerative capacity, highlighting the nanotherapeutic uses of the multifunctional nanoparticles for regenerating infected/damaged hard tissues.

Dysfunction in the olfactory system of a person can have adverse effects on their health and quality of life. It can even increase mortality among individuals. Olfactory dysfunction is related to many factors, including post-viral upper respiratory infection, head trauma, and neurodegenerative disorders. Although some clinical therapies such as steroids and olfactory training are already available, their effectiveness is limited and controversial. Recent research in the field of therapeutic nanoparticles and stem cells has shown the regeneration of dysfunctional olfactory systems. Thus, we are motivated to highlight these regenerative approaches. For this, we first introduce the anatomical characteristics of the olfactory pathway, then detail various pathological factors related to olfactory dysfunctions and current treatments, and then finally discuss the recent regenerative endeavors, with particular focus on nanoparticle-based drug delivery systems and stem cells. This review offers insights into the development of future therapeutic approaches to restore and regenerate dysfunctional olfactory systems.

In recent years, nanoparticles have been highly investigated in the laboratory. However, only a few laboratory discoveries have been translated into clinical practice. These findings in the laboratory are limited by trial-and-error methods to determine the optimum formulation for successful drug delivery. A new paradigm is required to ease the translation of lab discoveries to clinical practice. Due to their previous success in antiviral activity, it is vital to accelerate the discovery of novel drugs to treat and manage viruses. Machine learning is a subfield of artificial intelligence and consists of computer algorithms which are improved through experience. It can generate predictions from data inputs via an algorithm which includes a method built from inputs and outputs. Combining nanotherapeutics and well-established machine-learning algorithms can simplify antiviral-drug development systems by automating the analysis. Other relationships in bio-pharmaceutical networks would eventually aid in reaching a complex goal very easily. From previous laboratory experiments, data can be extracted and input into machine learning algorithms to generate predictions. In this study, poly (lactic-co-glycolic acid) (PLGA) nanoparticles were investigated in antiviral drug delivery. Data was extracted from research articles on nanoparticle size, polydispersity index, drug loading capacity and encapsulation efficiency. The Gaussian Process, a form of machine learning algorithm, could be applied to this data to generate graphs with predictions of the datasets. The Gaussian Process is a probabilistic machine learning model which defines a prior over function. The mean and variance of the data can be calculated via matrix multiplications, leading to the formation of prediction graphs—the graphs generated in this study which could be used for the discovery of novel antiviral drugs. The drug load and encapsulation efficiency of a nanoparticle with a specific size can be predicted using these graphs. This could eliminate the trial-and-error discovery method and save laboratory time and ease efficiency.

In recent years, freeform three-dimensional (3D) printing has led to significant advances in the fabrication of artificial tissues with vascularized structures. This technique utilizes a supporting matrix that holds the extruded printing ink and ensures shape maintenance of the printed 3D constructs within the prescribed spatial precision. Since the printing nozzle can be translated omnidirectionally within the supporting matrix, freeform 3D printing is potentially applicable for the fabrication of complex 3D objects, incorporating curved, and irregular shaped vascular networks. To optimize freeform 3D printing quality and performance, the rheological properties of the printing ink and supporting matrix, and the material matching between them are of paramount importance. In this review, we shall compare conventional 3D printing and freeform 3D printing technologies for the fabrication of vascular constructs, and critically discuss their working principles and their advantages and disadvantages. We also provide the detailed material information of emerging printing inks and supporting matrices in recent freeform 3D printing studies. The accompanying challenges are further discussed, aiming to guide freeform 3D printing by the effective design and selection of the most appropriate materials/processes for the development of full-scale functional vascularized artificial tissues.

As one of the most transplanted tissues of the human body, bone has varying architectures, depending on its anatomical location. Therefore, bone defects ideally require bone substitutes with a similar structure and adequate strength comparable to native bones. Light-based three-dimensional (3D) printing methods allow the fabrication of biomimetic scaffolds with high resolution and mechanical properties that exceed the result of commonly used extrusion-based printing. Digital light processing (DLP) is known for its faster and more accurate printing than other 3D printing approaches. However, the development of biocompatible resins for light-based 3D printing is not as rapid as that of bio-inks for extrusion-based printing. In this study, we developed CSMA-2, a photopolymer based on Isosorbide, a renewable sugar derivative monomer. The CSMA-2 showed suitable rheological properties for DLP printing. Gyroid scaffolds with high resolution were successfully printed. The 3D-printed scaffolds also had a compressive modulus within the range of a human cancellous bone modulus. Human adipose-derived stem cells remained viable for up to 21 days of incubation on the scaffolds. A calcium deposition from the cells was also found on the scaffolds. The stem cells expressed osteogenic markers such as RUNX2, OCN, and OPN. These results indicated that the scaffolds supported the osteogenic differentiation of the progenitor cells. In summary, CSMA-2 is a promising material for 3D printing techniques with high resolution that allow the fabrication of complex biomimetic scaffolds for bone regeneration.

Current therapeutic treatments for the repair and/or replacement of damaged skin following disease or traumatic injury is severely limited. The chicken eggshell membrane (ESM) is a unique material: its innate physical and mechanical characteristics offer optimal barrier properties and, as a naturally derived extract, it demonstrates inherent biocompatibility/biodegradability. To further enhance its therapeutic and clinical potential, the ESM can be modified with the thermo-responsive polymer, poly(N-isopropylacrylAmide) (PNIPAAm) as well as the incorporation of (drug-loaded) silver nanoparticles (AgNP); essentially, by a simple change in temperature, the release and delivery of the NP can be targeted and controlled. In this study, ESM samples were isolated using a decellularization protocol, and the physical and mechanical characteristics were profiled using SEM, FT-IR, DSC and DMA. PNIPAAm was successfully grafted to the ESM via amidation reactions and confirmed using FT-IR, which demonstrated the distinctive peaks associated with Amide A (3275 cm−1), Amide B (2970 cm−1), Amide I (1630 cm−1), Amide II (1535 cm−1), CH2, CH3 groups, and Amide III (1250 cm−1) peaks. Confirmation of the incorporation of AgNP onto the stratified membrane was confirmed visually with SEM, qualitatively using FT-IR and also via changes in absorbance at 380 nm using UV-Vis spectrophotometry during a controlled release study for 72 h. The biocompatibility and cytotoxicity of the novel constructs were assessed using human dermal fibroblast (HDFa) and mouse dermal fibroblast (L929) cells and standard cell culture assays. Metabolic activity assessment (i.e., MTS assay), LDH-release profiles and Live/Dead staining demonstrated good attachment and spreading to the samples, and high cell viability following 3 days of culture. Interestingly, longer-term viability (>5 days), the ESM-PNIPAAm and ESM-PNIPAAm (AgNP) samples showed a greater and sustained cell viability profile. In summary, the modified and enhanced ESM constructs were successfully prepared and characterized in terms of their physical and mechanical profiles. AgNP were successfully loaded into the construct and demonstrated a desirable release profile dependent on temperature modulation. Fibroblasts cultured on the extracted ESM samples and ESM-PNIPAAm demonstrated high biocompatibility in terms of high cell attachment, spreading, viability and proliferation rates. As such, this work summarizes the development of an enhanced ESM-based construct which may be exploited as a clinical/therapeutic wound dressing as well as a possible application as a novel biomaterial scaffold for drug development.

Regenerative endodontic treatment based on tissue engineering has recently gained interest in contemporary restorative dentistry. However, low survival rates and poor potential differentiation of stem cells could undermine the success rate of pulp regenerative therapy. Human gingival fibroblast-conditioned medium (hGF-CM) has been considered a potential therapy for tissue regeneration due to its stability in maintaining multiple factors essential for tissue regeneration compared to live cell transplantation. This study aimed to investigate the potency of hGF-CM on stem cells from human dental pulp (DPSC) in pulp regeneration. A series of experiments confirmed that hGF-CM contributes to a significant increase in proliferation, migration capability, and cell viability of DPSC after H2O2 exposure. Moreover, it has been proved to facilitate the odontogenic differentiation of DPSC via qRT-PCR, ALP (alkaline phosphatase), and ARS (Alizarin Red S) staining. It has been discovered that such highly upregulated odontogenesis is related to certain types of ECM proteins (collagen and laminin) from hGF-CM via proteomics. In addition, it is found that the ERK pathway is a key mechanism via inhibition assay based on RNA-seq result. These findings demonstrate that hGF-CM could be beneficial biomolecules for pulp regeneration.

Extracellular vesicles (EVs), including exosomes, carry the genetic packages of RNA, DNA, and proteins and are heavily involved in cell-cell communications and intracellular signalings. Therefore, EVs are spotlighted as therapeutic mediators for the treatment of injured and dysfunctional tissues as well as biomarkers for the detection of disease status and progress. Several key issues in EVs, including payload content and bioactivity, targeting and bio-imaging ability, and mass-production, need to be improved to enable effective therapeutics and clinical translation. For this, significant efforts have been made recently, including genetic modification, biomolecular and chemical treatment, application of physical/mechanical cues, and 3D cultures. Here we communicate those recent technological advances made mainly in the biogenesis process of EVs or at post-collection stages, which ultimately aimed to improve the therapeutic efficacy in tissue healing and disease curing and the possibility of clinical translation. This communication will help tissue engineers and biomaterial scientists design and produce EVs optimally for tissue regenerative therapeutics.

Current gold standard to treat soft tissue injuries caused by trauma and pathological condition are autografts and off the shelf fillers, but they have inherent weaknesses like donor site morbidity, immuno-compatibility and graft failure. To overcome these limitations, tissue-engineered polymers are seeded with stem cells to improve the potential to restore tissue function. However, their interaction with native tissue is poorly understood so far. To study these interactions and improve outcomes, we have fabricated scaffolds from natural polymers (collagen, fibrin and elastin) by custom-designed processes and their material properties such as surface morphology, swelling, wettability and chemical cross-linking ability were characterised. By using 3D scaffolds, we comprehensive assessed survival, proliferation and phenotype of adipose-derived stem cells in vitro. In vivo, scaffolds were seeded with adipose-derived stem cells and implanted in a rodent model, with X-ray microtomography, histology and immunohistochemistry as read-outs. Collagen-based materials showed higher cell adhesion and proliferation in vitro as well as higher adipogenic properties in vivo. In contrast, fibrin demonstrated poor cellular and adipogenesis properties but higher angiogenesis. Elastin formed the most porous scaffold, with cells displaying a non-aggregated morphology in vitro while in vivo elastin was the most degraded scaffold. These findings of how polymers present in the natural polymers mimicking ECM and seeded with stem cells affect adipogenesis in vitro and in vivo can open avenues to design 3D grafts for soft tissue repair.

Ulceration of the oral mucosa is common, can arise at any age and as a consequence of the pain lessens enjoyment and quality of life. Current treatment options often involve the use of topical corticosteroids with poor drug delivery systems and inadequate contact time. In order to achieve local controlled delivery to the lesion with optimal adhesion, we utilized a simple polydopamine chemistry technique inspired by mussels to replicate their adhesive functionality. This was coupled with production of a group of naturally produced polymers, known as polyhydroxyalkanoates (PHA) as the delivery system. Initial work focused on the synthesis of PHA using Pseudomonas mendocina CH50; once synthesized and extracted from the bacteria, the PHAs were solvent processed into films. Polydopamine coating was subsequently achieved by immersing the solvent cast film in a polymerized dopamine solution. Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy confirmed functionalization of the PHA films via the presence of amine groups. Further characterization of the samples was carried out via surface energy measurements and Scanning Electron Microscopy (SEM) micrographs for surface topography. An adhesion test via reverse compression testing directly assessed adhesive properties and revealed an increase in polydopamine coated samples. To further identify the effect of surface coating, LIVE/DEAD imaging and Alamar Blue metabolic activity evaluated attachment and proliferation of fibroblasts on the biofilm surfaces, with higher cell growth in favor of the coated samples. Finally, in vivo biocompatibility was investigated in a rat model where the polydopamine coated PHA showed less inflammatory response over time compared to uncoated samples with sign of neovascularization. In conclusion, this simple mussel inspired polydopamine chemistry introduces a step change in bio-surface functionalization and holds great promise for the treatment of oral conditions.

Using microspherical scaffolds as building blocks to repair bone defects of specific size and shape has been proposed as a tissue engineering strategy. Here, phosphate glass (PG) microcarriers doped with 5 mol % TiO 2 and either 0 mol % CoO (CoO 0%) or 2 mol % CoO (CoO 2%) were investigated for their ability to support osteogenic and vascular responses of human mesenchymal stem cells (hMSCs). Together with standard culture techniques, cell-material interactions were studied using a novel perfusion microfluidic bioreactor that enabled cell culture on microspheres, along with automated processing and screening of culture variables. While titanium doping was found to support hMSCs expansion and differentiation, as well as endothelial cell-derived vessel formation, additional doping with cobalt did not improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled screening of culture parameters for cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing.

Drivers of RNA interference are potent for manipulating gene and protein levels, which enable the restoration of dysregulated mRNA expression that is commonly associated with injuries and diseases. This review summarizes the potential of targeting neuroglial cells, using RNA interference, to treat nerve injuries sustained in the central nervous system. In addition, the various methods of delivering these RNA interference effectors will be discussed.

Cancer stem cells have been shown to be important in tumorigenesis processes, such as tumor growth, metastasis, and recurrence. As such, many three-dimensional models have been developed to establish an ex vivo microenvironment that cancer stem cells experience under in vivo conditions. Cancer stem cells propagating in three-dimensional culture systems show physiologically related signaling pathway profiles, gene expression, cell–matrix and cell–cell interactions, and drug resistance that reflect at least some of the tumor properties seen in vivo . Herein, we discussed the presently available Cancer stem cell three-dimensional culture models that use biomaterials and engineering tools and the biological implications of these models compared to the conventional ones.

Brain diseases including neurodegenerative disorders and tumours are among the most serious health problems, degrading the quality of life and causing massive economic cost. Nanoparticles that load and deliver drugs and genes have been intensively studied for the treatment of brain diseases, and have demonstrated some biological effects in various animal models. Among other efforts taken in the nanoparticle development, targeting of blood brain barrier, specific cell type or local intra-/extra-cellular space is an important strategy to enhance the therapeutic efficacy of the nanoparticle delivery systems. This review underlies the targeting issue in the nanoparticle development for the treatment of brain diseases, taking key exemplar studies carried out in various in vivo models.

Endometriosis is a common benign gynecologic tumor, and it can destroy a patient's life. Surgery and hormone therapy are established therapies for endometriosis. However, there are many cases of recurrent endometriosis after conventional therapies. This report presents a case of a patient who has repetitive recurrence of endometriosis after laparoscopic excision and hormone therapy. A 32-years-old female patient first had laparoscopic surgery to remove endometriosis in 2011. The disease recurred two more times after the first excision. Hormone therapy by dienogest and two more laparoscopic surgeries were done in 2012 and 2014. With acupuncture, moxibustion, fumigation therapy, and herbal medicine therapy, endometriosis didn’t recur during treatment and observation period of 34 months. Menstrual pain numeric rating scales (NRS) decreased from 2 to none. Shortened menstrual cycle (24 days) after second surgery became longer (26.63 ± 2.28 days) after traditional Korean medicine therapy. This case presents the therapeutic potential of TKM for inhibitory effect on the recurrent endometriosis after laparoscopic excision and hormone therapy. Keywords: Boheotang, Dienogest, Endometriosis, Laparoscopic surgery, traditional Korean medicine

Reactive oxygen species (ROS) are continuously produced by skeletal muscle during contractile activity and even at rest. However, the ROS generated from excessive exercise or traumatic damage may produce more ROS than can be neutralized by an antioxidant capacity, which can be harmful to muscle function. In particular, selenium is a known antioxidant that regulates physiological functions such as cell differentiation and anti-inflammatory function. In this study, we developed nano-sized antioxidative biomaterials using selenium to investigate the protective and differentiation effects against C2C12 myoblasts in an H2O2-induced oxidative stress environment. The selenium nanoparticles (SeNPs) were produced with a size of 35.6 ± 4.3 nm and showed antioxidant effects according to the 3,3′,5,5′-tetramethylbenzidine assay. Then, SeNPs were treated to C2C12 cells with or without H2O2. Our results showed that SeNPs reduced C2C12 apoptosis and intracellular ROS levels. Additionally, SeNPs effectively up-regulated in the presence of H2O2, MyoD, MyoG, α-actinin, and myosin heavy chain, which are well known to increase during myoblast differentiation as assayed by qRT-PCR, immunocytochemistry-staining, western blotting. These results demonstrate that SeNPs can accelerate differentiation with its protective effects from the ROS environment and can be applied to the treatment of skeletal muscle in a cellular redox environment.

Iris loczyi is a perennial rhizomatous herb distributed in Central Asia. We examined genomic architecture of the complete chloroplast genome in I. loczyi by assembling the Illumina MiSeq reads using de novo strategy. The chloroplast genome is 150,940 bp in length harboring 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. It exhibits typical quadripartite structure comprising LSC (80,907 bp), SSC (17,853 bp), and a pair of IRs (26,090 bp). Phylogenetic analysis of 20 chloroplast genomes from Asparagales revealed that Iridaceae is a monophyletic group and the I. loczyi is clustered together with the congener, I. sanguinea.

[This corrects the article DOI: 10.1371/journal.pone.0149967.].

For skeletal muscle engineering, scaffolds that can stimulate myogenic differentiation of cells while possessing suitable mechanical properties (e.g. flexibility) are required. In particular, the elastic property of scaffolds is of importance which helps to resist and support the dynamic conditions of muscle tissue environment. Here, we developed highly flexible nanocomposite nanofibrous scaffolds made of polycarbonate diol and isosorbide-based polyurethane and hydrophilic nano-graphene oxide added at concentrations up to 8%. The nano-graphene oxide incorporation increased the hydrophilicity, elasticity, and stress relaxation capacity of the polyurethane-derived nanofibrous scaffolds. When cultured with C2C12 cells, the polyurethane–nano-graphene oxide nanofibers enhanced the initial adhesion and spreading of cells and further the proliferation. Furthermore, the polyurethane–nano-graphene oxide scaffolds significantly up-regulated the myogenic mRNA levels and myosin heavy chain expression. Of note, the cells on the flexible polyurethane–nano-graphene oxide nanofibrous scaffolds could be mechanically stretched to experience dynamic tensional force. Under the dynamic force condition, the cells expressed significantly higher myogenic differentiation markers at both gene and protein levels and exhibited more aligned myotubular formation. The currently developed polyurethane–nano-graphene oxide nanofibrous scaffolds, due to their nanofibrous morphology and high mechanical flexibility, along with the stimulating capacity for myogenic differentiation, are considered to be a potential matrix for future skeletal muscle engineering.

Nanoparticle-based therapeutics delivery holds great promise for the treatment of intractable diseases. The high loading of drug molecules and their precise delivery to target sites are needed to gain optimal therapeutic functions of the nanoparticle delivery system. In this communication, we highlight, among other properties of nanoparticles (e.g. size, shape, surface chemistry, and degradation), the nanoscale topography, which has recently been shown to be an important parameter, ultimately determining drug loading, cell penetration, and body clearance. This nanotopographical aspect is considered to offer a new effective strategy to the development of nanoparticles for drug and gene delivery with enhanced therapeutic outcome.

Tissue engineering is a promising approach for bone regeneration; yet challenges remain that limit successful translation to patients. It is necessary to understand how real-world manufacturing processes will affect the constituent cells and biomaterials that are needed to create engineered bone. Bioactive phosphate glasses processed into microspheres are an attractive platform for expanding bone-forming cells and also for driving their osteogenic differentiation and maturation. The aim of this study was to assess whether Ti-doped phosphate glass microspheres could support osteoblastic cell responses in dynamic cell culture environments. Dynamic culture conditions were achieved using microwell studies under orbital agitation. Dimensionless parameters such as the Froude number were used to inform the choice of agitation speeds, and the impact on cell proliferation and microunit formation was quantified. We found that phosphate glass microspheres doped with titanium dioxide at both 5 and 7 mol% provided a suitable biomaterial platform for effective culture of MG63 osteoblastic cells and was not cytotoxic. Dynamic culture conditions supported expansion of MG63 cells and both 150 and 300 rpm orbital shake resulted in higher cell yield than static cultures at the end of the culture (day 13). The Froude number analysis provided insight into how the microunit size could be manipulated to enable an appropriate agitation speed to be used, while ensuring buoyancy of the microunits. These small-scale experiments and analyses provide understanding of the impact of fluid flow on cell expansion that will have increasing importance when scaling up to process technologies that can deliver clinical quantities of cell-microsphere units. Such knowledge will enable future engineering of living bone-like material using processing systems such as bioreactors that use mixing and agitation for nutrient transfer, therefore introducing cells to dynamic culture conditions.

In recent years, multidrug-resistant (MDR) bacteria have increased rapidly, representing a major threat to human health. This problem has created an urgent need to identify alternatives for the treatment of MDR bacteria. The aim of this study was to identify the antibacterial activity of selenium nanoparticles (SeNPs) and selenium nanowires (SeNWs) against MDR bacteria and assess the potential synergistic effects when combined with a conventional antibiotic (linezolid). SeNPs and SeNWs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), zeta potential, and UV-visible analysis. The antibacterial effects of SeNPs and SeNWs were confirmed by the macro-dilution minimum inhibitory concentration (MIC) test. SeNPs showed MIC values against methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant enterococci (VRE) at concentrations of 20, 80, 320, and >320 μg/mL, respectively. On the other hand, SeNWs showed a MIC value of >320 μg/mL against all tested bacteria. Therefore, MSSA, MRSA, and VRSA were selected for the bacteria to be tested, and SeNPs were selected as the antimicrobial agent for the following experiments. In the time-kill assay, SeNPs at a concentration of 4X MIC (80 and 320 μg/mL) showed bactericidal effects against MSSA and MRSA, respectively. At a concentration of 2X MIC (40 and 160 μg/mL), SeNPs showed bacteriostatic effects against MSSA and bactericidal effects against MRSA, respectively. In the synergy test, SeNPs showed a synergistic effect with linezolid (LZD) through protein degradation against MSSA and MRSA. In conclusion, these results suggest that SeNPs can be candidates for antibacterial substitutes and supplements against MDR bacteria for topical use, such as dressings. However, for use in clinical situations, additional experiments such as toxicity and synergistic mechanism tests of SeNPs are needed.

Genistein (4,5,7-trihydroxyisoflavone) is abundant in various dietary vegetables, especially soybeans, and is known to have not only an estrogenic effect but also an antiadipogenic effect. Atorvastatin (dihydroxy monocarboxylic acid) is a statin used to prevent heart disease. Although genistein and atorvastatin have been reported to possess antiadipogenic effects, their combined effects are still unclear. The aim of the current study was to explore whether the combination of genistein and atorvastatin at low concentrations significantly suppresses adipogenesis in a murine preadipocyte cell line (3T3-L1) compared to treatment with genistein or atorvastatin alone. Our results showed that cotreatment with 50 µM genistein and 50 nM atorvastatin significantly suppressed preadipocyte differentiation, whereas when each compound was used alone, there was no inhibitory effect. Additionally, cotreatment with genistein and atorvastatin significantly downregulated adipogenic marker proteins, including mitogen-activated protein kinases (MAPKs), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), glucocorticoid receptor (GR), and CCAAT/enhancer-binding protein β (C/EBPβ). This is the first evidence of the combined antiadipogenic effects of genistein and atorvastatin. Although additional experiments are required, combinational treatment with genistein and atorvastatin may be an alternative treatment for menopause-associated lipid metabolic disorders and obesity.