The Emerging Role of Decellularized Plant-Based Scaffolds as a New Biomaterial.

The decellularization of plant-based biomaterials to generate tissue-engineered substitutes or in vitro cellular models has significantly increased in recent years. These vegetal tissues can be sourced from plant leaves and stems or fruits and vegetables, making them a low-cost, accessible, and sustainable resource from which to generate three-dimensional scaffolds. Each construct is distinct, representing a wide range of architectural and mechanical properties as well as innate vasculature networks. Based on the rapid rise in interest, this review aims to detail the current state of the art and presents the future challenges and perspectives of these unique biomaterials. First, we consider the different existing decellularization techniques, including chemical, detergent-free, enzymatic, and supercritical fluid approaches that are used to generate such scaffolds and examine how these protocols can be selected based on plant cellularity. We next examine strategies for cell seeding onto the plant-derived constructs and the importance of the different functionalization methods used to assist in cell adhesion and promote cell viability. Finally, we discuss how their structural features, such as inherent vasculature, porosity, morphology, and mechanical properties (i.e., stiffness, elasticity, etc.) position plant-based scaffolds as a unique biomaterial and drive their use for specific downstream applications. The main challenges in the field are presented throughout the discussion, and future directions are proposed to help improve the development and use of vegetal constructs in biomedical research.

Identification of early gene expression changes in primary cultured neurons treated with topoisomerase I poisons.

Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1-CPT, actinomycin D (ActD) and ß-lapachone (ß-Lap)-on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify differentially regulated transcripts in MNs in response to a brief exposure to CPT. Pathway analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. Immediate-early genes like Fos, Egr-1 and Gadd45b were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cell types treated with CPT; Egr-1, Gadd45b and Dyrk3 transcript levels, however, increased in CPT-treated MNs and CNs but decreased in CPT-treated fibroblasts. These transcripts may represent new targets for the development of therapeutic agents that mitigate the off-target effects of chemotherapy on the nervous system.

Precancerous Lesions of the Head and Neck Region and Their Stromal Aberrations: Piecemeal Data.

Head and neck squamous cell carcinomas (HNSCCs) develop through a series of precancerous stages from a pool of potentially malignant disorders (PMDs). Although we understand the genetic changes that lead to HNSCC, our understanding of the role of the stroma in the progression from precancer to cancer is limited. The stroma is the primary battleground between the forces that prevent and promote cancer growth. Targeting the stroma has yielded promising cancer therapies. However, the stroma at the precancerous stage of HNSCCs is poorly defined, and we may miss opportunities for chemopreventive interventions. PMDs already exhibit many features of the HNSCC stroma, such as inflammation, neovascularization, and immune suppression. Still, they do not induce cancer-associated fibroblasts or destroy the basal lamina, the stroma's initial structure. Our review aims to summarize the current understanding of the transition from precancer to cancer stroma and how this knowledge can reveal opportunities and limitations for diagnostic, prognostic, and therapeutic decisions to benefit patients. We will discuss what may be needed to fulfill the promise of the precancerous stroma as a target to prevent progression to cancer.

Evaluation of the orally bioavailable 4-phenylbutyrate-tethered trichostatin A analogue AR42 in models of spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a leading genetic cause for infant death in the world and results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of SMN protein and small molecules that can increase SMN expression are of considerable interest as potential therapeutics. Previous studies have shown that both 4-phenylbutyrate (4PBA) and trichostatin A (TSA) increase SMN expression in dermal fibroblasts derived from SMA patients. AR42 is a 4PBA-tethered TSA derivative that is a very potent histone deacetylase inhibitor. SMA patient fibroblasts were treated with either AR42, AR19 (a related analogue), 4PBA, TSA or vehicle for 5 days and then immunostained for SMN localization. AR42 as well as 4PBA and TSA increased the number of SMN-positive nuclear gems in a dose-dependent manner while AR19 did not show marked changes in gem numbers. While gem number was increased in AR42-treated SMA fibroblasts, there were no significant changes in FL-SMN mRNA or SMN protein. The neuroprotective effect of this compound was then assessed in SMNΔ7 SMA (SMN2+/+;SMNΔ7+/+;mSmn-/-) mice. Oral administration of AR42 prior to disease onset increased the average lifespan of SMNΔ7 SMA mice by ~ 27% (20.1 ± 1.6 days for AR42-treated mice vs. 15.8 ± 0.4 days for vehicle-treated mice). AR42 treatment also improved motor function in these mice. AR42 treatment inhibited histone deacetylase (HDAC) activity in treated spinal cord although it did not affect SMN protein expression in these mice. AKT and GSK3ß phosphorylation were both significantly increased in SMNΔ7 SMA mouse spinal cords. In conclusion, presymptomatic administration of the HDAC inhibitor AR42 ameliorates the disease phenotype in SMNΔ7 SMA mice in a SMN-independent manner possibly by increasing AKT neuroprotective signaling.

Decellularized Spinach Biomaterials Support Physiologically Relevant Mechanical Cyclic Strain and Prompt a Stretch-Induced Cellular Response.

Recently, decellularized plant biomaterials have been explored for their use as tissue engineered substitutes. Herein, we expanded upon the investigation of the mechanical properties of these materials to explore their elasticity as many anatomical areas of the body require biomechanical dynamism. We first constructed a device to secure the scaffold and induce a strain within the physiological range of the normal human adult lung during breathing (12-20 movements/min; 10-20% elongation). Results showed that decellularized spinach leaves can support cyclic strain for 24 h and displayed heterogeneous local strain values (7.76-15.88%) as well as a Poisson's ratio (0.12) similar to that of mammalian lungs (10.67-19.67%; 0.01), as opposed to an incompressible homogeneous standard polymer (such as PDMS (10.85-12.71%; 0.4)). Imaging and mechanical testing showed that the vegetal scaffold exhibited strain hardening but maintained its structural architecture and water retention capacity, suggesting an unaltered porosity. Interestingly, we also showed that cells seeded on the scaffold can also sense the mechanical strain as demonstrated by a nuclear reorientation perpendicular to strain direction (63.3° compared to 41.2° for nonstretched cells), a nuclear location of YAP and increased expression of YAP target genes, a high cytoplasmic calcium level, and an elevated expression level of collagen genes (COL1A1, COL3A1, COL4A1, and COL6A) with an increased collagen secretion at the protein level. Taken together, these data demonstrated that decellularized plant leaf tissues have an inherent elastic property similar to that found in the mammalian system to which cells can sense and respond.

Evaluation of supercritical CO2 sterilization efficacy for sanitizing personal protective equipment from the coronavirus SARS-CoV-2.

The purpose of this research is to evaluate the supercritical carbon dioxide (scCO2) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks, and N95 respirators. Preliminarily, Bacillus atrophaeus were inoculated into different environments (dry, hydrated, and saliva) to imitate coughing and sneezing and serve as a "worst-case" regarding challenged PPE. The inactivation of the microbes by scCO2 sterilization with NovaKill or H2O2 sterilant was investigated as a function of exposure times ranging from 5 to 90 min with a goal of elucidating possible mechanisms. Also, human coronavirus SARS-CoV-2 and HCoV-NL63 were inoculated on the respirator material, and viral activity was determined post-treatment. Moreover, we investigated the reprocessing ability of scCO2-based decontamination using wettability testing and surface mapping. Different inactivation mechanisms have been identified in scCO2 sanitization, such as membrane damage, germination defect, and dipicolinic acid leaks. Moreover, the viral sanitization results showed a complete inactivation of both coronavirus HCoV-NL63 and SARS-CoV-2. We did not observe changes in PPE morphology, topographical structure, or material integrity, and in accordance with the WHO recommendation, maintained wettability post-processing. These experiments establish a foundational understanding of critical elements for the decontamination and reuse of PPE in any setting and provide a direction for future research in the field.

Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds.

The use of plant-based biomaterials for tissue engineering has recently generated interest as plant decellularization produces biocompatible scaffolds which can be repopulated with human cells. The predominant approach for vegetal decellularization remains serial chemical processing. However, this technique is time-consuming and requires harsh compounds which damage the resulting scaffolds. The current study presents an alternative solution using supercritical carbon dioxide (scCO2). Protocols testing various solvents were assessed and results found that scCO2 in combination with 2% peracetic acid decellularized plant material in less than 4 h, while preserving plant microarchitecture and branching vascular network. The biophysical and biochemical cues of the scCO2 decellularized spinach leaf scaffolds were then compared to chemically generated scaffolds. Data showed that the scaffolds had a similar Young's modulus, suggesting identical stiffness, and revealed that they contained the same elements, yet displayed disparate biochemical signatures as assessed by Fourier-transform infrared spectroscopy (FTIR). Finally, human fibroblast cells seeded on the spinach leaf surface were attached and alive after 14 days, demonstrating the biocompatibility of the scCO2 decellularized scaffolds. Thus, scCO2 was found to be an efficient method for plant material decellularization, scaffold structure preservation and recellularization with human cells, while performed in less time (36 h) than the standard chemical approach (170 h).

Plant-Based Scaffolds Modify Cellular Response to Drug and Radiation Exposure Compared to Standard Cell Culture Models.

Plant-based scaffolds present many advantages over a variety of biomaterials. Recent studies explored their potential to be repopulated with human cells and thus highlight a growing interest for their use in tissue engineering or for biomedical applications. However, it is still unclear if these in vitro plant-based scaffolds can modify cell phenotype or affect cellular response to external stimuli. Here, we report the characterization of the mechano-regulation of melanoma SK-MEL-28 and prostate PC3 cells seeded on decellularized spinach leaves scaffolds, compared to cells deposited on standard rigid cell culture substrate, as well as their response to drug and radiation treatment. The results showed that YAP/TAZ signaling was downregulated, cellular morphology altered and proliferation rate decreased when cells were cultured on leaf scaffold. Interestingly, cell culture on vegetal scaffold also affected cellular response to external stress. Thus, SK-MEL-28 cells phenotype is modified leading to a decrease in MITF activity and drug resistance, while PC3 cells showed altered gene expression and radiation response. These findings shed lights on the decellularization of vegetal materials to provide substrates that can be repopulated with human cells to better reproduce a soft tissue microenvironment. However, these complex scaffolds mediate changes in cell behavior and in order to exploit the capability of matching physical properties of the various plant scaffolds to diverse physiological functionalities of cells and human tissue constructs, additional studies are required to better characterize physical and biochemical cell-substrate interactions.

Establishing a reference dataset for the authentication of spinal muscular atrophy cell lines using STR profiling and digital PCR.

Fibroblasts and lymphoblastoid cell lines (LCLs) derived from individuals with spinal muscular atrophy (SMA) have been and continue to be essential for translational SMA research. Authentication of cell lines helps ensure reproducibility and rigor in biomedical research. This quality control measure identifies mislabeling or cross-contamination of cell lines and prevents misinterpretation of data. Unfortunately, authentication of SMA cell lines used in various studies has not been possible because of a lack of a reference. In this study, we provide said reference so that SMA cell lines can be subsequently authenticated. We use short tandem repeat (STR) profiling and digital PCR (dPCR), which quantifies SMN1 and SMN2 copy numbers, to generate molecular identity codes for fibroblasts and LCLs that are commonly used in SMA research. Using these molecular identity codes, we clarify the familial relationships within a set of fibroblasts commonly used in SMA research. This study presents the first cell line reference set for the SMA research community and demonstrates its usefulness for re-identification and authentication of lines commonly used as in vitro models for future studies.

Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs-glyceryl tributyrate (BA3G) and VX563-on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3ß, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling.

The effect of the DcpS inhibitor D156844 on the protective action of follistatin in mice with spinal muscular atrophy.

Spinal muscular atrophy (SMA), a leading genetic cause of pediatric death in the world, is an early-onset disease affecting the motor neurons in the anterior horn of the spinal cord. This degeneration of motor neurons leads to loss of muscle function. At the molecular level, SMA results from the loss of or mutation in the survival motor neuron 1 (SMN1) gene. The number of copies of the nearly duplicated gene SMN2 modulates the disease severity in humans as well as in transgenic mouse models for SMA. Most preclinical therapeutic trials focus on identifying ways to increase SMN2 expression and to alter its splicing. Other therapeutic strategies have investigated compounds which protect affected motor neurons and their target muscles in an SMN-independent manner. In the present study, the effect of a combination regimen of the SMN2 inducer D156844 and the protectant follistatin on the disease progression and survival was measured in the SMNΔ7 SMA mouse model. The D156844/follistatin combination treatment improved the survival of, delayed the end stage of disease in and ameliorated the growth rate of SMNΔ7 SMA mice better than follistatin treatment alone. The D156844/follistatin combination treatment, however, did not provide additional benefit over D156844 alone with respect to survival and disease end stage even though it provided some additional therapeutic benefit over D156844 alone with respect to motor phenotype.

SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR.

Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.

Transcriptome profiling of spinal muscular atrophy motor neurons derived from mouse embryonic stem cells.

Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn-/-) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs.