Acellular scaffold-based approach for in situ genetic engineering of host T-cells in solid tumor immunotherapy.

BACKGROUND: Targeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies. However, its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity. Localized delivery of tumor-specific T-cells using biomaterials has shown promise, however, procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints. METHODS: Polyethylene glycol (PEG)-based three-dimensional (3D) scaffolds were developed and conjugated with positively charged poly-L-lysine (PLL) using carbamide chemistry for efficient loading of lentiviruses (LVs) carrying tumor antigen-specific T-cell receptors (TCRs). The physical and biological properties of the scaffold were extensively characterized. Further, the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin (B16-OVA) tumor model to evaluate the anti-tumor response and the presence of transduced T-cells. RESULTS: Our findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site. In B16-OVA melanoma tumor-bearing mice, the scaffolds efficiently transduce host T-cells with OVA-specific TCRs. These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs, resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines. Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds. CONCLUSION: Our scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors. This approach offers a viable alternative to in vitro manipulation of T-cells, circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells. It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells, thereby, overcoming a major hurdle.

Tetrahydrobiopterin from cyanide-degrading bacterium Bacillus pumilus strain SVD06 induces apoptosis in human lung adenocarcinoma cell (A549).

Tetrahydrobiopterin (BH4) is an essential biological cofactor and a derivative of pterin which is considered potent anticancer agents. In continuation of our previous study on the identification of BH4 from cyanide-degrading Bacillus pumilus, the present study focuses on evaluating the anticancer properties of BH4 on A549, a human lung adenocarcinoma. Anticancer activity analysis shows that BH4 inhibited A549 cell growth after 24 h of incubation with 0.02 mg/mL. In acridine orange/ethidium bromide staining, BH4-treated A549 cells showed apoptotic morphology. BH4 treatment caused cell cycle arrest at G0/G1 phase compared to control cells. BH4 augmented p53 expression in alveolar cancer cells by downregulating MDM2 levels. There was downregulation of casp-3 and upregulation of iNOS gene in BH4-treated A549 cells. Further, docking studies indicated that BH4 had significant interactions with the above proteins affirming the apoptosis mechanism. Thus, BH4 could be considered a potential anticancer drug.

Bilayered skin substitute incorporating rutin nanoparticles for antioxidant, anti-inflammatory, and anti-fibrotic effect.

Hypertrophic scarring in large burns and delayed healing in chronic wounds are consequences of prolonged and aggravated inflammation, sustained infiltration of immune cells, free radical generation, and abundance of inflammatory mediators. Therefore, it is imperative to curb hyperinflammation to expedite wound healing. In this study, rutin nanoparticles (RNPs) were synthesized without an encapsulant and incorporated into eggshell membrane powder-crosslinked gelatin-chitosan cryogels to impart antioxidant and anti-inflammatory properties for treating hyperinflammation. The resultant nanoparticles were found to be 17.53 ± 4.03 nm in size and were stable at room temperature for a month with no visible sedimentation. RNPs were found to be non-cytotoxic and exhibited anti-inflammatory (by increasing IL-10 levels) and antioxidant properties (by controlling the generation of reactive oxygen species and enhancing catalase production in human macrophages). Additionally, RNPs were found to reduce α-SMA expression in fibroblasts, thereby demonstrating their anti-scarring effect. In vivo studies with a bilayered skin substitute constituting an RNP-incorporated cryogel proved that it is biocompatible, does not induce renal toxicity, aids wound healing, and induces better re-epithelialization than the control groups at the initial stages. Thus, RNP-incorporated cryogels containing bilayered skin substitutes are an advanced and novel alternative to commercial dermo-epidermal substitutes that lack anti-inflammatory or anti-scarring properties.

Two-photon polymerization based reusable master template to fabricate polymer microneedles for drug delivery.

Microneedle patches have been widely used in a minimally invasive manner for various drug delivery applications. However, for developing these microneedle patches, master molds are required, which are generally made of metal and are very expensive. Two-photon polymerization (2PP) technique can be used for fabricating microneedles more precisely and at a much lower cost. This study reports a novel strategy for developing microneedle master templates using the 2PP method. The main advantage of this technique is that there is no requirement for post-processing after laser writing, and that for the fabrication of polydimethylsiloxane (PDMS) molds, harsh chemical treatments such as silanization are not required. This is a one-step process for manufacturing of microneedle templates which allows easy replication of negative PDMS molds. This is done by adding resin to the master-template and annealing at a specific temperature, thereby making the PDMS peel-off easy and allowing re-use of the master template multiple times. Using this PDMS mold, two types of polyvinyl alcohol (PVA)-rhodamine (RD) microneedle patches were developed, namely, dissolving (D-PVA) and hydrogel (H-PVA) patches and were characterized using suitable techniques. This technique is affordable, efficient and does not require post-processing for development of microneedle templates required for drug delivery applications.•Two photon polymerization can be used for cost-effective fabrication of polymer microneedles for transdermal drug delivery.•Post-processing or surface-modification procedures are not required for these master templates.•Using a simple annealing step, the master template becomes reusable and robust for peeling off polymers like PDMS.

Novel combination of bioactive agents in bilayered dermal patches provides superior wound healing.

In present study, multifunctional bilayered dermal patches with antibacterial, antioxidant and anti-inflammatory properties were developed using solvent casting or electrospinning methods and compared for performance. Top layer was made up of polycaprolactone (PCL) and chitosan (CS) while bottom layer was made of polyvinyl alcohol (PVA) with curcumin nanoparticles and soluble eggshell membrane protein (SESM) as the wound healing agents. Curcumin nanoparticles showed reduction in the production of reactive oxygen species (ROS) and inflammatory cytokines and markers in mice T cells or human macrophages, confirming their antioxidant and anti-inflammatory properties while SESM improved migration of human adult dermal fibroblasts, suggesting its contribution to wound healing. The dermal patches were hemocompatible and antibacterial and also provided adequate absorption of wound exudates, support and components required for recruitment of cells and deposition of extracellular matrix to enable superior wound healing than its commercial counterpart in a full thickness excision wound model in rats.

A bilayered skin substitute developed using an eggshell membrane crosslinked gelatin-chitosan cryogel.

Commercially available allografts and xenografts pose problems such as high cost, risk of infection transmission and immune rejection of grafts. Thus, bioengineered skin substitutes fabricated from natural biomaterials or synthetic polymers are currently the focus of skin tissue engineering. In this study, eggshell membrane (ESM) powder was used to crosslink a gelatin-chitosan cryogel thereby replacing glutaraldehyde, a known cytotoxic chemical crosslinker. The resultant ESM-crosslinked macroporous cryogel with a pore size ranging between 10 and 350 µm has improved flexibility, biodegradability and biocompatibility compared to a glutaraldehyde-crosslinked cryogel. For healing of large and deep wounds, bilayered scaffolds which exhibit key aspects of skin physiology are being explored. Hence, we fabricated a bilayered substitute by coupling the ESM-crosslinked cryogel (dermal equivalent) to a non-porous, physically-crosslinked gelatin-chitosan film (epidermal equivalent). The epidermal layer provides the requisite barrier properties while the dermal layer facilitates cell attachment and migration for optimal wound healing. Further, chitosan confers antibacterial properties to the cryogel with almost 50% reduction in bacterial viability. Animal studies confirm that the developed bilayered skin substitute is non-allergic, aids wound healing by improving re-epithelialization within 14 days and supports the formation of skin appendages. This system presents a new and alternative treatment option for burn and chronic wounds.

Dual antimicrobial and anticancer activity of a novel synthetic α-helical antimicrobial peptide.

Antimicrobial peptides (AMPs) are increasingly sought-after and researched antimicrobial agents due to its desired pharmacological properties and the continuous diminishing efficacy of antibiotics. In addition to this line of research, the aim of the present study is to determine the antimicrobial and anticancer activity of a de novo designed α-helical peptide. Circular dichroism showed 100% helical nature of the peptide in 10 mM SDS. Notably, the peptide exerted significant antimicrobial activity against the reference and antibiotic-resistant clinical isolates belonging to Pseudomonas sp. at a MIC and MBC of 2 and 8 µM, respectively. The progressive disruption and disturbance of cell membrane in the overall topography was observed in the scanning electron microscopy (SEM) micrographs of Pseudomonas aeruginosa ATCC 27853 treated with the peptide as compared to untreated control. The results of time-kill kinetics showed complete lysis at 3x MIC after 50 min of incubation of the microbe with the peptide. Moreover, the peptide did not lyse human RBCs even at the highest concentration of the peptide (10 mM) and retained its activity upon treatment at 0.5 mg/ml trypsin. Cancer cell lines, viz. A549 and MCF-7 were also found to be sensitive to peptide activity showing 50% reduction in survivability at 4 and 2 µM, respectively; however, L929 cells were unaffected. Drastic membrane permeability and necrotic mode of lysis of peptide-treated-A549 cells were affirmed by propidium iodide and live/dead cell staining. The results showed that the designed peptide could be an efficient drug molecule for clinical studies subjected to successful experiments on animal models.

Symbiotic culture of nanocellulose pellicle: A potential matrix for 3D bioprinting.

Nanocellulose pellicle is produced as a byproduct during the symbiotic culture of bacteria and yeast in kombucha. It shows good mechanical strength, biocompatibility and hydrophilicity. However, it has limited application in tissue engineering due to its low processability. In this work, bacterial cellulose-based sustainable kombucha (KBC) sheet has been produced and it was acid-treated to partially hydrolyse. This controlled process improves its extrusion and shape formation ability. The physical, functional and biological properties were studied to assess its potential as a 3D printed scaffold. Two different cell lines (Human dermal fibroblast cells and mouse osteoblast cells) were used to study the cytocompatibility. Both the cell types showed good attachment, growth and proliferation on the pure and treated KBC. They attained almost full confluence within 3 days. This study indicates that the controlled partial hydrolysis of KBC can make it suitable for 3D printing retaining its mechanical strength and cytocompatibility. This sustainable microbial biopolymer shows the possibility to be used as a bioink for 3D bioprinting.

Three-dimensional directional nerve guide conduits fabricated by dopamine-functionalized conductive carbon nanofibre-based nanocomposite ink printing.

A potential issue in current nerve guides is that they do not transmit electrical nerve impulses between the distal and proximal end of an injured nerve, i.e. a synapse. Conductivity is a desirable property of an ideal nerve guide that is being considered for peripheral nerve regeneration. Most conductive polymers reported for the fabrication of tissue engineering scaffolds, such as polypyrrole and polyaniline, are non-biodegradable and possess weak mechanical properties, and thus cannot be fabricated into 3D structures. Herein, we have designed a new nanocomposite material composed of dopamine, carbon nanofibers (CNF) and polycaprolactone (PCL) for the fabrication of nerve conduits, which facilitates the growth and migration of neurons toward the targeted end of an injured nerve. This support and navigation of the scaffold leads to better sensory and motor function. The results showed that the mechanical properties of the printed PCL increased by 30% in comparison with the pure PCL film, which is comparable with human nerves. The in vitro cell study of human glioma cells showed that the printed lines provided support for neural cell attachment, migration and differentiation toward the targeted end. In contrast, in the absence of printed lines in the scaffold, the cells attach and grow in random directions, forming a flower shape (cell cluster) on the surface of PCL. Thus, the proposed scaffold is a promising candidate for nerve guide application based on its signal transmission and navigating neurons in a correct pathway towards the targeted end.

Correction: Three-dimensional directional nerve guide conduits fabricated by dopamine-functionalized conductive carbon nanofibre-based nanocomposite ink printing.

[This corrects the article DOI: 10.1039/D0RA06556K.].

Effect of nanocomposite coating and biomolecule functionalization on silk fibroin based conducting 3D braided scaffolds for peripheral nerve tissue engineering.

In this work, the effects of carbon nanofiber (CNF) dispersed poly-ε-caprolactone (PCL) nanocomposite coatings and biomolecules functionalization on silk fibroin based conducting braided nerve conduits were studied for enhancing Neuro 2a cellular activities. A unique combination of biomolecules (UCM) and varying concentrations of CNF (5, 7.5, 10% w/w) were dispersed in 10% (w/v) PCL solution for coating on degummed silk threads. The coated silk threads were braided to develop the scaffold structure. As the concentration of CNF increased in the coating, the electrical impedance decreased up to 400 Ω indicating better conductivity. The tensile and dynamic mechanical property analysis showed better mechanical properties in CNF coated samples. In vitro cytocompatibility analysis proved the non-toxicity of the developed braided conduits. Cell attachment, growth and proliferation were significantly enhanced on the biomolecule functionalized nanocomposite coated silk braided structure, exhibiting their potential for peripheral nerve regeneration and recovery.

Human Knee Meniscus Regeneration Strategies: a Review on Recent Advances.

PURPOSE OF REVIEW: Lack of vascularity in the human knee meniscus often leads to surgical removal (total or partial meniscectomy) in the case of severe meniscal damage. However, complete recovery is in question after such removal as the meniscus plays an important role in knee stability. Thus, meniscus tissue regeneration strategies are of intense research interest in recent years. RECENT FINDINGS: The structural complexity and inhomogeneity of the meniscus have been addressed with processing technologies for precisely controlled three dimensional (3D) complex porous scaffold architectures, the use of biomolecules and nanomaterials. The regeneration and replacement of the total meniscus have been studied by the orthopedic and scientific communities via successful pre-clinical trials towards mimicking the biomechanical properties of the human knee meniscus. Researchers have attempted different regeneration strategies which contribute to in vitro regeneration and are capable of repairing meniscal tears to some extent. This review discusses the present state of the art of these meniscus tissue engineering aspects.

Synergistic effect of electrical conductivity and biomolecules on human meniscal cell attachment, growth, and proliferation in poly-ε-caprolactone nanocomposite scaffolds.

Poly-ε-caprolactone (PCL)-based nanocomposite scaffolds with different concentrations of carbon nanofillers (carbon nanofibers (CNFs), nanographite, and exfoliated graphite) have been studied to investigate the effect of electrical conductivity and biomolecule supplementation for enhanced human meniscal cell attachment, growth, and proliferation. The incorporation of carbon nanofillers was found to improve the mechanical and electrical properties. CNF-based nanocomposite scaffolds showed the highest electrical conductivity with significant improvements in mechanical properties (more than 50% tensile strength increase than PCL with 10% (w/w) CNF). All nanocomposite scaffolds were subjected to cytotoxicity studies using primary meniscus cells. The nanocomposite scaffolds showing higher cell viability were selected and tested for meniscal cell attachment and proliferation assays such as total deoxyribonucleic acid content, extracellular matrix secretion, nuclear staining, and cell attachment studies using a scanning electron microscope. When an optimized combination of biomolecules is supplemented in the cell culture medium, a synergistic effect of the electrical conductivity and biomolecule combination is observed, especially in the case of highly conducting CNF (7.5% and 10% (w/w))-based nanocomposite scaffolds. Our findings suggest that electrically conductive scaffolds with optimized biomolecules in cell culture medium can potentially be used for successful human meniscal tissue engineering applications.

An in vitro 3D model using collagen coated gelatin nanofibers for studying breast cancer metastasis.

The study of breast cancer metastasis is limited due to poor knowledge of molecular progression of breast tumor and varied heterogeneity. For a better understanding of tumor metastasis, a reliable 3D in vitro model bridging the gap between 2D cultures and in vivo animal model studies is essential. Our study is focused on two key points: (i) designing a 3D microenvironment for studying metastasis and (ii) simulating the metastasis milieu by inducing epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). An electrospun gelatin nanofiber matrix (EGNF) was fabricated using electrospinning and further dip coated with different concentrations of collagen to obtain surface complexity and mechanical properties, similar to connective tissues. Nanofiber matrices were physically characterized by Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and field-emission scanning electron microscopy (FESEM). The FTIR, AFM, and FESEM results indicated the crosslinking and confirmed the presence of pores in the nanofiber matrices. Comparative studies on biocompatibility, cell attachment, and the proliferation of MCF-7 cells on EGNF and collagen coated gelatin nanofibrous matrix (CCGM) revealed higher cellular attachment and proliferation in CCGM. CCGM with human metastatic breast cancer cell line (MCF-7) was taken to study breast cancer metastasis using estrogen (induces EMT) and progesterone (induces MET) hormones for 24 h. Quantitative real-time PCR was used for quantifying the expression of metastasis related genes, and fluorescence microscopy for verifying the invasion of cells to the matrices. The expression of E-cadherin and matrix metalloproteinase 2 (MMP 2) confirmed the occurrence of EMT and MET. Live cell imaging and cellular attachment showed significant increase of cellular invasion in crosslinked 0.15% CCGM that serves as a suitable non-toxic, biocompatible, and affordable scaffold for studying breast cancer metastasis. Our findings suggested that CCGM can be used as a tissue-like 3D model for studying breast cancer metastatic events in vitro.

Silk-PVA Hybrid Nanofibrous Scaffolds for Enhanced Primary Human Meniscal Cell Proliferation.

In this study, silk fibroin nanofibrous scaffolds were developed to investigate the attachment and proliferation of primary human meniscal cells. Silk fibroin (SF)-polyvinyl alcohol (PVA) blended electrospun nanofibrous scaffolds with different blend ratios (2:1, 3:1, and 4:1) were prepared. Morphology of the scaffolds was characterized using atomic force microscopy (AFM). The hybrid nanofibrous mats were crosslinked using 25 % (v/v) glutaraldehyde vapor. In degradation study, the crosslinked nanofiber showed slow degradation of 20 % on weight after 35 days of incubation in simulated body fluid (SBF). The scaffolds were characterized with suitable techniques for its functional groups, porosity, and swelling ratio. Among the nanofibers, 3:1 SF:PVA blend showed uniform morphology and fiber diameter. The blended scaffolds had fluid uptake and swelling ratio of 80 % and 458 ± 21 %, respectively. Primary meniscal cells isolated from surgical debris after meniscectomy were subcultured and seeded onto these hybrid nanofibrous scaffolds. Meniscal cell attachment studies confirmed that 3:1 SF:PVA nanofibrous scaffolds supported better cell attachment and growth. The DNA and collagen content increased significantly with 3:1 SF:PVA. These results clearly indicate that a blend of SF:PVA at 3:1 ratio is suitable for meniscus cell proliferation when compared to pure SF-PVA nanofibers.

A combination of biomolecules enhances expression of E-cadherin and peroxisome proliferator-activated receptor gene leading to increased cell proliferation in primary human meniscal cells: an in vitro study.

The present study investigates the impact of biomolecules (biotin, glucose, chondroitin sulphate, proline) as supplement, (individual and in combination) on primary human meniscus cell proliferation. Primary human meniscus cells isolated from patients undergoing meniscectomy were maintained in Dulbecco's Modified Eagle's Medium (DMEM). The isolated cells were treated with above mentioned biomolecules as individual (0-100 µg/ml) and in combinations, as a supplement to DMEM. Based on the individual biomolecule study, a unique combination of biomolecules (UCM) was finalized using one way ANOVA analysis. With the addition of UCM as supplement to DMEM, meniscal cells reached 100 % confluency within 4 days in 60 mm culture plate; whereas the cells in medium devoid of UCM, required 36 days for reaching confluency. The impact of UCM on cell viability, doubling time, histology, gene expression, biomarkers expression, extra cellular matrix synthesis, meniscus cell proliferation with respect to passages and donor's age were investigated. The gene expression studies for E-cadherin and peroxisome proliferator-activated receptor (PPAR∆) using RT-qPCR and immunohistochemical analysis for Ki67, CD34 and Vimentin confirmed that UCM has significant impact on cell proliferation. The extracellular collagen and glycosaminoglycan secretion in cells supplemented with UCM were found to increase by 31 and 37 fold respectively, when compared to control on the 4th day. The cell doubling time was reduced significantly when supplemented with UCM. The addition of UCM showed positive influence on different passages and age groups. Hence, this optimized UCM can be used as an effective supplement for meniscal tissue engineering.

Impact of silk fibroin-based scaffold structures on human osteoblast MG63 cell attachment and proliferation.

The present study was carried out to investigate the impact of various types of silk fibroin (SF) scaffolds on human osteoblast-like cell (MG63) attachment and proliferation. SF was isolated from Bombyx mori silk worm cocoons after degumming. Protein concentration in the degummed SF solution was estimated using Bradford method. Aqueous SF solution was used to fabricate three different types of scaffolds, viz, electrospun nanofiber mat, sponge, and porous film. The structures of the prepared scaffolds were characterized using optical microscopy and field emission scanning electron microscopy. The changes in the secondary structure of the proteins and the thermal behavior of the scaffolds were determined by Fourier transform infrared spectroscopy and thermo-gravimetric analysis, respectively. The biodegradation rate of scaffolds was determined by incubating the scaffolds in simulated body fluid for 4 weeks. MG63 cells were seeded on the scaffolds and their attachment and proliferation onto the scaffolds were studied. The MTT assay was carried out to deduce the toxicity of the developed scaffolds. All the scaffolds were found to be biocompatible. The amount of collagen produced by the osteoblast-like cells growing on different scaffolds was estimated.