Hybrid Graphene-Gold Nanoparticle-based Nucleic Acid Conjugates for Cancer-Specific Multimodal Imaging and Combined Therapeutics.

Nanoparticle-based nucleic acid conjugates (NP-NACs) hold great promise for theragnostic (diagnostic and therapeutic) applications. However, several limitations have hindered the realization of their full potential in the clinical treatment of cancer and other diseases. In diagnosis, NP-NACs, combined with conventional optical sensing systems, have been applied for cancer detection in vitro, but low signal-to-noise ratios limit their broad in vivo applications. Meanwhile, the efficiency of NP-NAC-mediated cancer therapies has been limited through the adaptation of alternative pro-survival pathways in cancer cells. The recent emergence of personalized and precision medicine has outlined the importance of both accurate diagnosis and efficient therapeutics in a single platform. As such, we report the controlled assembly of hybrid graphene oxide/gold nanoparticle-based cancer-specific NACs (Au@GO NP-NACs) for multimodal imaging and combined therapeutics. Our developed Au@GO NP-NACs shows excellent surface-enhanced Raman scattering (SERS)-mediated live-cell cancer detection and multimodal synergistic cancer therapy through the use of photothermal, genetic, and chemotherapeutic strategies. Synergistic and selective killing of cancer cells were then demonstrated by using in vitro microfluidic models and nine different cancer cell lines by further incorporating near-infrared photothermal hyperthermia, a Topoisomerase II anti-cancer drug, and cancer targeting peptides. Moreover, with distinctive advantages of the Au@GO NP-NACs for cancer theragnostics, we further demonstrated precision cancer treatment through the detection of cancer cells in vivo using SERS followed by efficient ablation of the tumor. Therefore, our Au@GO NP-NACs could pave a new road for the advanced theragnostics of cancer as well as many other diseases.

Neoadjuvant vs Adjuvant Chemoimmunotherapy for Stage II-IIIB Non-Small Cell Lung Cancer.

BACKGROUND: In patients with resectable non-small cell lung cancer (NSCLC), recent trials demonstrate survival benefit of chemoimmunotherapy over chemotherapy alone in both the neoadjuvant and adjuvant settings. To date, there is no direct comparison between neoadjuvant and adjuvant protocols. We compared neoadjuvant vs adjuvant chemoimmunotherapy for resectable stage II-IIIB NSCLC. METHODS: We queried the National Cancer Database for patients who had undergone an operation for stage II-IIIB NSCLC and who had received neoadjuvant or adjuvant chemoimmunotherapy between 2015 and 2020. We used inverse probability weighting to adjust for confounding variables and used Kaplan-Meier survival curves and Cox regression to explore the relationship between treatment groups and overall survival (OS) at 3 years postoperatively. RESULTS: The inverse probability-weighted cohort represented 2119 weighted patient cases (neoadjuvant, 1034; adjuvant, 1085). Kaplan-Meier analysis demonstrated a significant OS benefit for neoadjuvant chemoimmunotherapy compared with adjuvant chemoimmunotherapy in the weighted cohort (3-year OS: 77% [95% CI, 71%-83%] vs 68% [95% CI, 64%-72%]; P = .035). On adjusted Cox regression, neoadjuvant chemoimmunotherapy was associated with a significant OS benefit (hazard ratio, 0.70; 95% CI, 0.50-0.96; P = .027). Among patients for whom pathologic stage data were available, 25% of patients receiving neoadjuvant chemoimmunotherapy had a pathologic complete response, with an additional 32.5% being downstaged. CONCLUSIONS: Neoadjuvant chemoimmunotherapy confers a significant OS benefit over adjuvant chemoimmunotherapy for patients with resectable stage II-IIIB NSCLC. Although randomized trials are needed to confirm our findings, strong consideration should be given to administering neoadjuvant chemoimmunotherapy to patients who are predetermined to receive systemic treatment.

Identifying Molecular Pathophysiology and Potential Therapeutic Options in Iatrogenic Tracheal Stenosis.

INTRODUCTION: While most patients with iatrogenic tracheal stenosis (ITS) respond to endoscopic ablative procedures, approximately 15% experience a recalcitrant, recurring disease course that is resistant to conventional management. We aimed to explore genetic profiles of patients with recalcitrant ITS to understand underlying pathophysiology and identify novel therapeutic options. METHODS: We collected 11 samples of granulation tissue from patients with ITS and performed RNA sequencing. We identified the top 10 most highly up- and down-regulated genes and cellular processes that these genes corresponded to. For the most highly dysregulated genes, we identified potential therapeutic options that favorably regulate their expression. RESULTS: The dysregulations in gene expression corresponded to hyperkeratinization (upregulation of genes involved in keratin production and keratinocyte differentiation) and cellular proliferation (downregulation of cell cycle regulating and pro-apoptotic genes). Genes involved in retinoic acid (RA) metabolism and signaling were dysregulated in a pattern suggesting local cellular RA deficiency. Consequently, RA also emerged as the most promising potential therapeutic option for ITS, as it favorably regulated seven of the ten most highly dysregulated genes. CONCLUSION: This is the first study to characterize the role of hyperkeratinization and dysregulations in RA metabolism and signaling in the disease pathophysiology. Given the ability of RA to favorably regulate key genes involved in ITS, future studies must explore its efficacy as a potential therapeutic option for patients with recalcitrant ITS.

Development of a Nanohybrid Peptide Hydrogel for Enhanced Intervertebral Disc Repair and Regeneration.

Effective therapeutic approaches to overcome the heterogeneous pro-inflammatory and inhibitory extracellular matrix (ECM) microenvironment are urgently needed to achieve robust structural and functional repair of severely wounded fibrocartilaginous tissues. Herein we developed a dynamic and multifunctional nanohybrid peptide hydrogel (NHPH) through hierarchical self-assembly of peptide amphiphile modified with biodegradable two-dimensional nanomaterials with enzyme-like functions. NHPH is not only injectable, biocompatible, and biodegradable but also therapeutic by catalyzing the scavenging of pro-inflammatory reactive oxygen species and promoting ECM remodeling. In addition, our NHPH method facilitated the structural and functional recovery of the intervertebral disc (IVD) after severe injuries by delivering pro-regenerative cytokines in a sustained manner, effectively suppressing immune responses and eventually restoring the regenerative microenvironment of the ECM. In parallel, the NHPH-enhanced nucleus pulposus cell differentiation and pain reduction in a rat nucleotomy model further validated the therapeutic potential of NHPH. Collectively, our advanced nanoscaffold technology will provide an alternative approach for the effective treatment of IVD degeneration as well as other fibrocartilaginous tissue injuries.

Injectable bioorthogonal hydrogel (BIOGEL) accelerates tissue regeneration in degenerated intervertebral discs.

Intervertebral disc (IVD) degeneration is a leading cause of back pain and precursor to more severe conditions, including disc herniation and spinal stenosis. While traditional growth factor therapies (e.g., TGFß) are effective at transiently reversing degenerated disc by stimulation of matrix synthesis, it is increasingly accepted that bioscaffolds are required for sustained, complete IVD regeneration. Current scaffolds (e.g., metal/polymer composites, non-mammalian biopolymers) can be improved in one or more IVD regeneration demands: biodegradability, noninvasive injection, recapitulated healthy IVD biomechanics, predictable crosslinking, and matrix repair induction. To meet these demands, tetrazine-norbornene bioorthogonal ligation was combined with gelatin to create an injectable bioorthogonal hydrogel (BIOGEL). The liquid hydrogel precursors remain free-flowing across a wide range of temperatures and crosslink into a robust hydrogel after 5-10 min, allowing a human operator to easily inject the therapeutic constructs into degenerated IVD. Moreover, BIOGEL encapsulation of TGFß potentiated histological repair (e.g., tissue architecture and matrix synthesis) and functional recovery (e.g., high water retention by promoting the matrix synthesis and reduced pain) in an in vivo rat IVD degeneration/nucleotomy model. This BIOGEL procedure readily integrates into existing nucleotomy procedures, indicating that clinical adoption should proceed with minimal difficulty. Since bioorthogonal crosslinking is essentially non-reactive towards biomolecules, our developed material platform can be extended to other payloads and degenerative injuries.

Effective Modulation of Inflammation and Oxidative Stress for Enhanced Regeneration of Intervertebral Discs Using 3D Porous Hybrid Protein Nanoscaffold.

Degeneration of fibrocartilaginous tissues is often associated with complex pro-inflammatory factors. These include reactive oxygen species (ROS), cell-free nucleic acids (cf-NAs), and epigenetic changes in immune cells. To effectively control this complex inflammatory signaling, it developed an all-in-one nanoscaffold-based 3D porous hybrid protein (3D-PHP) self-therapeutic strategy for treating intervertebral disc (IVD) degeneration. The 3D-PHP nanoscaffold is synthesized by introducing a novel nanomaterial-templated protein assembly (NTPA) strategy. 3D-PHP nanoscaffolds that avoid covalent modification of proteins demonstrate inflammatory stimuli-responsive drug release, disc-mimetic stiffness, and excellent biodegradability. Enzyme-like 2D nanosheets incorporated into nanoscaffolds further enabled robust scavenging of ROS and cf-NAs, reducing inflammation and enhancing the survival of disc cells under inflammatory stress in vitro. Implantation of 3D-PHP nanoscaffolds loaded with bromodomain extraterminal inhibitor (BETi) into a rat nucleotomy disc injury model effectively suppressed inflammation in vivo, thus promoting restoration of the extracellular matrix (ECM). The resulting regeneration of disc tissue facilitated long-term pain reduction. Therefore, self-therapeutic and epigenetic modulator-encapsulated hybrid protein nanoscaffold shows great promise as a novel approach to restore dysregulated inflammatory signaling and treat degenerative fibrocartilaginous diseases, including disc injuries, providing hope and relief to patients worldwide.

Advanced Drug Delivery Modulation via Hybrid Nanofibers Enhances Stem Cell Differentiation.

Seamlessly integrating soluble factors onto biomedical scaffolds with a precisely manufactured topography for efficient cell control remains elusive since many scaffold fabrication techniques degrade payloads. Surface adsorption of payloads onto synthesized nanoscaffolds retains bioactivity by removing exposure to harsh processing conditions at the expense of inefficient drug loading and uncontrolled release. Herein, we present a nanomaterial composite scaffold paradigm to improve physicochemical surface adsorption pharmacokinetics. As a proof of concept, we integrated graphene oxide (GO) and manganese dioxide (MnO2) nanosheets onto nanofibers to increase loading capacity and tune drug release. Non-degradable GO enhances payload retention, while biodegradable MnO2 enables cell-responsive drug release. To demonstrate the utility of this hybrid nanomaterial scaffold paradigm for tissue engineering, we adsorbed payloads ranging from small molecules to proteins onto the scaffold to induce myogenesis and osteogenesis for multiple stem cell lines. Scaffolds with adsorbed payloads enabled more efficient differentiation than media supplementation using equivalent quantities of differentiation factors. We attribute this increased efficacy to a reverse uptake mechanism whereby payloads are localized around seeded cells, increasing delivery efficiency for guiding differentiation. Additionally, we demonstrate spatial control over cells since differentiation factors are delivered locally through the scaffold. When co-culturing scaffolds with and without adsorbed payloads, only cells seeded on payload-adsorbed scaffolds underwent differentiation. With this modular technology being capable of enhancing multiple differentiation fates for specific cell lines, this technology provides a promising alternative for current tissue engineering scaffolds.

Hybrid SMART spheroids to enhance stem cell therapy for CNS injuries.

Although stem cell therapy holds enormous potential for treating debilitating injuries and diseases in the central nervous system, low survival and inefficient differentiation have restricted its clinical applications. Recently, 3D cell culture methods, such as stem cell­based spheroids and organoids, have demonstrated advantages by incorporating tissue-mimetic 3D cell-cell interactions. However, a lack of drug and nutrient diffusion, insufficient cell-matrix interactions, and tedious fabrication procedures have compromised their therapeutic effects in vivo. To address these issues, we developed a biodegradable nanomaterial-templated 3D cell assembly method that enables the formation of hybrid stem cell spheroids with deep drug delivery capabilities and homogeneous incorporation of 3D cell-matrix interactions. Hence, high survival rates, controlled differentiation, and functional recovery were demonstrated in a spinal cord injury animal model. Overall, our hybrid stem cell spheroids represent a substantial development of material-facilitated 3D cell culture systems and can pave the way for stem cell­based treatment of CNS injuries.

Functional nanoarrays for investigating stem cell fate and function.

Stem cells show excellent potential in the field of tissue engineering and regenerative medicine based on their excellent capability to not only self-renew but also differentiate into a specialized cell type of interest. However, the lack of a non-destructive monitoring system makes it challenging to identify and characterize differentiated cells before their transplantation without compromising cell viability. Thus, the development of a non-destructive monitoring method for analyzing cell function is highly desired and can significantly benefit stem cell-based therapies. Recently, nanomaterial-based scaffolds (e.g., nanoarrays) have made possible considerable advances in controlling the differentiation of stem cells and characterization of the differentiation status sensitively in real time. This review provides a selective overview of the recent progress in the synthesis methods of nanoarrays and their applications in controlling stem cell fate and monitoring live cell functions electrochemically. We believe that the topics discussed in this review can provide brief and concise guidelines for the development of novel nanoarrays and promote the interest in live cell study applications. A method which can not only control but also monitor stem cell fate and function will be a promising technology that can accelerate stem cell therapies.

4D-Printed Transformable Tube Array for High-Throughput 3D Cell Culture and Histology.

3D cell cultures are rapidly emerging as a promising tool to model various human physiologies and pathologies by closely recapitulating key characteristics and functions of in vivo microenvironment. While high-throughput 3D culture is readily available using multi-well plates, assessing the internal microstructure of 3D cell cultures still remains extremely slow because of the manual, laborious, and time-consuming histological procedures. Here, a 4D-printed transformable tube array (TTA) using a shape-memory polymer that enables massively parallel histological analysis of 3D cultures is presented. The interconnected TTA can be programmed to be expanded by 3.6 times of its printed dimension to match the size of a multi-well plate, with the ability to restore its original dimension for transferring all cultures to a histology cassette in order. Being compatible with microtome sectioning, the TTA allows for parallel histology processing for the entire samples cultured in a multi-well plate. The test result with human neural progenitor cell spheroids suggests a remarkable reduction in histology processing time by an order of magnitude. High-throughput analysis of 3D cultures enabled by this TTA has great potential to further accelerate innovations in various 3D culture applications such as high-throughput/content screening, drug discovery, disease modeling, and personalized medicine.

Post-sulfonation of cellulose nanofibrils with a one-step reaction to improve dispersibility.

Cellulose nanofibrils (CNF) were sulfonated and the dispersion quality was compared to unfunctionalized and 2,2,6,6-tetramethylpiperdine-1-oxyl radical (TEMPO) post-oxidation treatment of existing CNF (mechanically fibrillated pulp). A post-sulfonation treatment on existing CNF in chlorosulfonic acid and dimethylformamide (DMF) resulted in sulfonated CNF that retained a fibril-like morphology. There was a small decrease in the cellulose crystallinity index for the sulfonated CNF, but this was much lower than the reported regioselective oxidative bisulfite pretreatment method used to make sulfonated CNF. The current approach was extremely quick, and 5min of reaction time was sufficient to result in significant improvements in dispersibility compared to unfunctionalized CNF. The sulfonated CNF and TEMPO oxidized CNF had better dispersibility compared to the unfunctionalized CNF when dispersed in DMF and water, and in many cases the sulfonated CNF had better dispersibility than the TEMPO CNF. It was found that when CNF was dispersed in DMF the TEMPO CNF formed carboxyl dimethylammonium groups, while the sulfonated CNF formed formate groups.

Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding.

The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.

Kienböck's disease: an approach to treatment.

Since its description in 1910, Kienböck's disease has continued to be a difficult problem for clinicians as well as patients. An incomplete understanding of the etiology as well as the natural history of the disease has led to an assortment of surgical treatment options. The authors present a review of Kienböck's disease and the theories behind different surgical interventions, as well as their current approach to treatment of patients with Kienböck's disease.

Individually Dispersed Wood-Based Cellulose Nanocrystals.

Good dispersion of cellulose nanocrystals (CNCs) in the polymer matrix is one of the key factors to obtaining good properties in the resulting nanocomposites. However, the preparation of individually dispersed CNCs in solvents or in polymer matrices has been a challenge. In this study, individually dispersed wood-based CNCs have been successfully prepared in solvents, including dimethylformamide (DMF), H2O, and a mixture of H2O/DMF, by sonication of moisture-containing CNCs. The CNCs dispersions were characterized by dynamic light scattering (DLS). It is found that CNCs containing above about 3.8 wt % moisture is critical for achieving individually dispersed CNC in solvents. Hydrodynamic radius (Rh) of CNCs is smaller in H2O/DMF co-solvent mixture than that in pure DMF or in pure H2O under same sonication treatment conditions. Experimental results have been corroborated using molecular simulation study.

Gene therapy for bone regeneration.

Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone has the unique ability to regenerate and continuously remodel itself throughout life. However, clinical situations arise when bone is unable to heal itself, as with segmental bone loss, fracture non-union, and failed spinal fusion. This leads to significant morbidity and mortality. Current attempts at improved bone healing have been met with limited success, fueling the development of improved techniques. Gene therapy in many ways represents an ideal approach for augmenting bone regeneration. Gene therapy allows specific gene products to be delivered to a precise anatomic location. In addition, the level of transgene expression as well as the duration of expression can be regulated with current techniques. For bone regeneration, the gene of interest should be delivered to the fracture site, expressed at appropriate levels, and then deactivated once the fracture has healed. Delivery of biological factors, mostly bone morphogenetic proteins (BMPs), has yielded promising results both in animal and clinical studies. There has also been tremendous work on discovering new growth factors and exploring previously defined ones. Finally, significant advances are being made in the delivery systems of the genes, ranging from viral and non-viral vectors to tissue engineering scaffolds. Despite some public hesitation to gene therapy, its use has great potential to expand our ability to treat a variety of human bone and musculoskeletal disorders. It is conceivable that in the near future gene therapy can be utilized to induce bone formation in virtually any region of the body in a minimally invasive manner. As bone biology and gene therapy research progresses, the goal of successful human gene transfer for augmentation of bone regeneration draws nearer.

The future of flexor tendon surgery.

Clinical outcomes following flexor tendon repair have made significant improvements in the last 50 years. In that time standard treatment has evolved from secondary grafting to primary repair with postoperative rehabilitation protocols. Unfortunately, excellent results are not yet attained universally following treatment. Improving understanding of tendon healing at the cellular, molecular, and genetic levels will likely enable surgeons to modulate the normal repair process. We now look toward biologic augmentation of flexor tendon repairs to address the problems of increasing tensile strength while reducing adhesion formation following injury and operative repair.

Characterization of chondrocyte scaffold carriers for cell-based gene therapy in articular cartilage repair.

Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in three-dimensional scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and open-cell polylactic acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At 4 weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs.

Characterization of adenovirus-mediated gene transfer in rabbit flexor tendons.

PURPOSE: Adenoviral vector-based gene therapy is a promising technique for the delivery of growth factors to tendons. The objective of this study was to determine whether rabbit flexor tendons could be transduced effectively by adenoviral vectors and whether the introduction of adenoviral vectors would cause a notable local inflammatory response. METHODS: Recombinant adenoviruses expressing green fluorescent protein (AdGFP) or BMP-13 (AdBMP-13) were constructed and 3 different viral titers (1 x 10(7), 1 x 10(8), and 1 x 10(9)) were tested in this study. The second through fifth tendons of the forepaws and hindpaws of a New Zealand white rabbit were identified surgically and injected with different viral titers of adenoviruses. The fifth tendon was used as a control. The tendons were harvested 12 days after surgery. The retrieved tendons were sectioned to measure transgene expression, as well as for histologic evaluation. RESULTS: At all tested viral titers an efficient dose-dependent transgene expression was detected in all samples at 12 days after injection. At the highest dose the injection sites were notable for lymphocytic infiltration, suggesting that injected adenoviral vectors can illicit some local inflammatory response. Lymphocytic infiltration was much less apparent, however, in the tendons injected with lower titers of adenoviral vectors. There was no evidence of a massive inflammatory response and/or cell death. CONCLUSIONS: Our findings show that adenovirus-based gene therapy is an efficient means of gene delivery to rabbit flexor tendons. Transduction efficiency of transgenes was dose dependent across the tested titers, although adenovirus-induced inflammation was notable only at the highest titer. This indicates that efficient gene transfer without notable local inflammatory response may be achieved by using the lower titers. Although adenovirus-induced inflammation can be minimized by using lower viral titers, its impact on adhesion formation in the long term remains unknown.

Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells.

Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was beta-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells.