Inorganic nanorods direct neuronal differentiation of bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells and are considered a potential source for tissue and organ repair due to their self-renewal, proliferation, and differentiation abilities. However, in most cases, MSCs are needed to be stimulated with external growth factors to promote their proliferation and differentiation. Over the past decade, it has been demonstrated that nanomaterials could facilitate MSC proliferation and differentiation, and excellent efforts are carried out to investigate their possible modulating pattern and mechanisms for MSC differentiation. Europium hydroxide (EuIII(OH)3) nanorods (EHN) are well-researched for their biomimicking properties and act as a substitute for growth factors that induce cell proliferation, migration, and differentiation. In the current study, the human MSCs were chosen as anin vitromodel for evaluating the role of EHN in modulating the differentiation process of MSCs into neuronal and glial lineages. The characterization of MSCs and differentiated neuronal cells observed by flow cytometry, confocal, and gene marker expression studies supported our hypothesis that the EHNs are pro-angiogenic and pro-neurogenic. Finally, altogether our results suggest that EHNs have the potential to play an essential part in developing novel treatment strategies for neurodegenerative diseases and spinal cord injuries based on the nanomedicine approach.

Modulatory Effects of Biosynthesized Gold Nanoparticles Conjugated with Curcumin and Paclitaxel on Tumorigenesis and Metastatic Pathways-In Vitro and In Vivo Studies.

BACKGROUND: Breast cancer is the most common cancer in women globally, and diagnosing it early and finding potential drug candidates against multi-drug resistant metastatic breast cancers provide the possibilities of better treatment and extending life. METHODS: The current study aimed to evaluate the synergistic anti-metastatic activity of Curcumin (Cur) and Paclitaxel (Pacli) individually, the combination of Curcumin-Paclitaxel (CP), and also in conjugation with gold nanoparticles (AuNP-Curcumin (Au-C), AuNP-Paclitaxel (Au-P), and AuNP-Curcumin-Paclitaxel (Au-CP)) in various in vitro and in vivo models. RESULTS: The results from combination treatments of CP and Au-CP demonstrated excellent synergistic cytotoxic effects in triple-negative breast cancer cell lines (MDA MB 231 and 4T1) in in vitro and in vivo mouse models. Detailed mechanistic studies were performed that reveal that the anti-cancer effects were associated with the downregulation of the expression of VEGF, CYCLIN-D1, and STAT-3 genes and upregulation of the apoptotic Caspase-9 gene. The group of mice that received CP combination therapy (with and without gold nanoparticles) showed a significant reduction in the size of tumor when compared to the Pacli alone treatment and control groups. CONCLUSIONS: Together, the results suggest that the delivery of gold conjugated Au-CP formulations may help in modulating the outcomes of chemotherapy. The present study is well supported with observations from cell-based assays, molecular and histopathological analyses.

The Anti-Inflammatory and Cytoprotective Efficiency of Curvularin, a Fungal Macrolactone against Lipopolysaccharide-Induced Inflammatory Response in Nucleus Pulposus Cells: An In Vitro Study.

STUDY DESIGN: Developing an in vitro model for assessing the anti-inflammatory properties of curvularin. PURPOSE: To evaluate the efficacy of natural fungal macrolactone as a therapeutic drug against lipopolysaccharide (LPS)-induced inflammation in primary human nucleus pulposus cells (NPCs) in vitro. OVERVIEW OF LITERATURE: Lumbar disk disease is a common cause of lower back pain (LBP) and sciatica. It is an established fact that inflammation, rather than mechanical compression on the nerve root, plays a role in the cause of LBP and sciatica. Current treatment options for reducing inflammation are either nonsteroidal anti-inflammatory drugs or steroids, prolonged use of which can potentially lead to adverse effects such as gastrointestinal disturbances and renal and cardiac issues. Hence, there is a need for better antiinflammatory drugs with no or minimal complications for treating inflammation-induced LBP and sciatica. Curvularin (Cur), a fungal macrolactone, is known for its anti-inflammatory activity, but nothing is known about its impact on inflammation due to disk pathologies. METHODS: Primary NPCs were cultured and characterized by flow cytometry and immunocytochemistry using the CD24 antibody and treated with 10 µg/mL LPS for 36 hours and then treated with Cur, betamethasone, and dexamethasone (10 µg/mL) for 48 hours, after which cell cycle analysis, cell viability assay, and gene expression studies (quantitative polymerase chain reaction [PCR] and quantitative real-time-PCR) were conducted. The NPCs treated with Cur downregulated the expression of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1ß, and IL-6); matrix metalloproteinases (MMPs; MMP-2 and MMP-3), ADAMTS; and apoptotic marker (cytochrome c). RESULTS: In our study, Cur-treated cells showed enhanced expression of collagen 9A1 and insulin-like growth factor receptor 1, indicating the recovery of NPCs from inflammatory assault. CONCLUSIONS: Based on observations, the anti-inflammatory properties of Cur render it an excellent drug molecule for treating disk degeneration nonsurgically, by direct injection into spinal disks when treating LBP and sciatica.

Evaluation of Anti-inflammatory and Regenerative Efficiency of Naringin and Naringenin in Degenerated Human Nucleus Pulposus Cells: Biological and Molecular Modeling Studies.

STUDY DESIGN: Development of an in vitro model for assessing the anti-inflammatory efficacies of naringin (Nar) and naringenin (NG). PURPOSE: To evaluate the efficacy of natural flavonoids as therapeutic drugs against anti-inflammatory processes in the nucleus pulposus (NP) cells using in-vitro and in-silico methods. OVERVIEW OF LITERATURE: Intervertebral disc (IVD) disease is a common cause of low back pain. Chronic inflammation and degeneration play a significant role in its etiopathology. Thus, a better understanding of anti-inflammatory agents and their role in IVD degeneration and pro-inflammatory cytokines expression is necessary for pain management and regeneration in IVD. METHODS: We performed primary cell culture of NP cells; immunocytochemistry; gene expression studies of cytokines, metalloproteases, extracellular proteins, and apoptotic markers using quantitative polymerase chain reaction and reverse transcription-polymerase chain reaction (RT-PCR); cytotoxicity assay (MTT); and molecular docking studies using AutoDock 4.2 software (Molecular Graphics Laboratory, La Jolla, CA, USA) to confirm the binding mode of proteins and synthesized complexes. We calculated the mean±standard deviation values and performed analysis of variance and t-test using SPSS ver. 17.0 (SPSS, Inc., Chicago, IL, USA). RESULTS: Molecular docking showed that both Nar and NG bind to the selected genes of interest. Semi-quantitative RT-PCR analysis reveals differential gene expression of collagen (COL)9A1, COL9A2, COL9A3, COL11A2, COMT (catechol-O-methyltransferase), and THBS2 (thrombospondin 2); up regulation of ACAN (aggrecan), COL1A1, COL11A1, interleukin (IL)6, IL10, IL18R1, IL18RAP, metalloprotease (MMP)2, MMP3, MMP9, ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5), IGF1R (insulin-like growth factor type 1 receptor), SPARC (secreted protein acidic and cysteine rich), PARK2 (parkin), VDR (vitamin D receptor), and BCL2 (B-cell lymphoma 2); down regulation of IL1A, CASP3 (caspase 3), and nine genes with predetermined concentrations of Nar and NG. CONCLUSIONS: The present study evaluated the anti-inflammatory and regenerative efficiencies of Nar and NG in degenerated human NP cells. Altered gene expressions of cytokines, metalloproteases, extracellular proteins, apoptotic genes were dose responsive. The molecular docking (in silico) studies showed effective binding of these native ligands (Nar and NG) with genes identified as potent inhibitors of inflammation. Thus, these natural flavonoids could serve as anti-inflammatory agents in the treatment of low back pain and sciatica.

Novel biosynthesized gold nanoparticles as anti-cancer agents against breast cancer: Synthesis, biological evaluation, molecular modelling studies.

Cancer therapeutics development has been a challenge due to their untoward side effects and cytotoxicity. Phytochemical anti-cancer drugs have several advantages over chemical chemotherapeutic drugs as they are less cytotoxic and have a greater pharmacological advantage. However, lack of targeting ability limits the use of phytochemicals at a great extent for a successful therapeutic strategy. Gold nanoparticles (AuNPs) have long been used to load the therapeutic cargo and provided significant advantages over conventional chemo-therapeutics. In this present study, we report the synthesis and testing of various biosynthesized AuNPs (b-AuNPs) using naturally derived phytochemicals (Curcumin: Cur, Turmeric: Tur, Quercetin: Qu and Paclitaxel: Pacli). The synthesized b-AuNPs have been well characterized by different Physicochemical techniques. Cytotoxic potential of these b-AuNPs were evaluated in different breast cancer cells either in an individual or in a combination forms. We have observed the maximum therapeutic activity in a combination of all four types of b-AuNPs (AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli) as compared to their pristine administration. Further, mechanistic studies of these compounds revealed that, combinations of AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli were significantly effective in inhibiting cell proliferation, apoptosis, angiogenesis, colony formation and spheroid formation predicting a synergistic effect when compared to individual treatment against different breast cancer cell lines (MCF-7 and MDA-MB 231). Interestingly the nanoconjugates alone or in combinations did not show cytotoxicity towards human embryonic normal kidney cell line (HEK 293), demonstrating the biocompatibility. Together the results demonstrated the potential anti-cancer properties of b-AuNPs in a combinatorial approach that could be the future of cancer nanomedicine.

Europium Hydroxide Nanorods (EHNs) Ameliorate Isoproterenol-Induced Myocardial Infarction: An in Vitro and in Vivo Investigation.

Cardiovascular diseases (CVDs) are one of the leading causes of global morbidity and mortality. Among these, the ischemic heart disease (IHD) or coronary artery disease (CAD) accounts for the major deaths due to CVDs. Several approaches followed to treat the ischemic heart diseases are limited due to various adverse effects and cost of treatment. Recently, nanotechnology has revolutionized the field of biomedical research by introducing various technologies to improve the health care, using a nanomedicine approach. In this context, our group has well-established the europium hydroxide nanorods (EHNs), which promote the formation of new blood vessels (angiogenesis) through reactive oxygen species (ROS) and nitric oxide (NO)-mediated signaling pathways. Further, these pro-angiogenic nanorods were also reported to exhibit a mild to nontoxic nature toward mammalian cells and mouse models. Henceforth, in the present study, myocardial ischemia (MI) was created in Wistar rats using isoproterenol (ISO), a well-established model for investigating MI. For the first time, the effect of the pro-angiogenic nanorods (EHNs) on the ischemic condition was validated using several assays, which revealed that the ischemia and cardiotoxicity induced by ISO were ameliorated by EHNs in both H9C2 rat cardiomyocytes (in vitro) and Wistar rats (in vivo). Considering the above results, we believe that EHN could be developed as alternative treatment strategies for myocardial ischemia therapy and other ischemic diseases where angiogenesis plays a significant role, in the near future.

Efficiency of dual siRNA-mediated gene therapy for intervertebral disc degeneration (IVDD).

BACKGROUND CONTEXT: One of the common causes of low back pain is intervertebral disc degeneration. The pathophysiology of disc degeneration involves apoptosis of nucleus pulposes cells and degradation of extra cellular matrix (ECM). Caspase 3 plays a central role in apoptosis and the ADAMTS5 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 5) gene plays a critical role in ECM degradation. Hence, we hypothesized that if one can silence these two genes, both apoptosis and ECM degradation can be prevented, thereby preventing the progression and even reverse disc degeneration. PURPOSE: The purpose of this study is to demonstrate the regenerative potential of small interfering RNA (siRNA) designed against Caspase 3 and ADAMTS5 genes in an in vitro and animal model of disc degeneration. STUDY DESIGN: In vitro study followed by in vivo study in a rabbit model. METHODS: In vitro studies were done using the human hepatocellular carcinoma (Hep G2) cell line for validating the efficacy of liposomal siRNA in controlling the expression of genes (Caspase 3 and ADAMTS5). Later, siRNA's validation was done in a rabbit annular punctured model by administering siRNA's individually (Caspase 3 and ADAMTS5) and in combination Caspase3-ADAMTS5) for assessing their synergistic effect in down regulating the gene expression in the degenerative discs. Annular punctured intervertebral discs of the rabbit were injected with siRNA formulations (single and dual) and phosphate buffer saline, one week after initial puncture. Magnetic resonance imaging (MRI) scans were done before and after siRNA treatment (1, 4 and 8 weeks) for assessing the progression of disc degeneration. The histopathology and real time polymerase chain reaction (RT-PCR) studies were done for evaluating their efficacy. We did not receive any funding for conducting the study, and we do not have a conflict of interest with any researchers or scientific groups. RESULTS: The observations made from both in vitro and in vivo studies indicate the beneficial effects of siRNA formulation in down regulating the expression of Caspase 3 and ADAMTS5 genes. The MRI and histopathological evaluation showed that the disc degeneration was progressive in phosphate buffer saline and AT5-siRNA injected discs but the discs that received Caspase 3-siRNA and dual siRNA (Cas3-AT5-siRNA) formulation showed signs of recovery and regeneration 4 and 8 weeks after injection. The efficacy of siRNA designed against Cas3 and AT5 was also assessed in both in vitro and in vivo experiments by using RT-PCR analysis and the results showed downregulation of Caspase 3 gene in Caspase 3-siRNA group, but there was no significant downregulation of ADAMTS5 gene in ADAMTS5-siRNA group (ie, indicated by fold change). Synergistic effect was observed in the group that received dual siRNA (Cas3-AT5 siRNA) formulation. CONCLUSIONS: This experiment suggests that intervention by siRNA treatment significantly reduced the extent of apoptosis in the discs. CLINICAL SIGNIFICANCE: Delivery of siRNA directly into spinal discs has a potential in treating disc degeneration nonsurgically.

Development of Novel Animal Model for Studying Scoliosis Using a Noninvasive Method and Its Validation through Gene-Expression Analysis.

STUDY DESIGN: To induce scoliosis in young female Wistar rats using a noninvasive method and to validate this model. PURPOSE: To induce scoliosis in a rat model noninvasively by bracing and to study the corresponding gene-expression profile in the spine and different organs. OVERVIEW OF LITERATURE: Scoliosis involves abnormal lateral curvature of the spine, the causes of which remain unclear. In the literature, it is suggested that scoliosis is genetically heterogeneous, as there are multiple factors involved directly or indirectly in its pathogenesis. Clinical and experimental studies were conducted to understand the etiology of anatomical alterations in the spine and internal organs, as the findings could help clinicians to establish new treatment approaches. METHODS: Twelve female Wistar rats aged 21 days were chosen for this study. Customized braces and real-time polymerase chain reaction (RT-PCR) primers for rats were designed using Primer 3 software. Radiological analysis (X-rays), histopathological studies, SYBR green, and RT-PCR analysis were performed. RESULTS: The spines of six rats were braced in a deformed position, which resulted in a permanent structural deformity as confirmed by X-ray studies. The remaining rats were used as controls. Quantitative studies of the expression of various genes (osteocalcin, pleiotrophins, matrix metalloproteinase-2 [MMP2] and MMP9, TIMP, interleukins 1 and 6, tumor necrosis factor-α) showed their differential expression and significant upregulation (p<0.05) in different organs of scoliotic rats in comparison to those in control rats. Histopathological findings showed tissue necrosis and fibrosis in the brain, retina, pancreas, kidney, liver, and disc of scoliotic rats. CONCLUSIONS: Bracing is a noninvasive method for inducing scoliosis in an animal model with 100% reliability and with corresponding changes in gene expression. Scoliosis does not just involve a spine deformity, but can be referred to as a systemic disease on the basis of the pathological changes observed in various internal organs.

Enchondroma Protuberans of the Transverse Process of D8 Vertebra Extending to the 7th and 8th Ribs: A Rare Case Report.

BACKGROUND: Enchondroma protuberans (EP) is rare, benign cartilaginous bone tumor arising from the intramedullary cavity of long bones and usually protrudes beyond the cortex with an exophytic growth pattern resembling osteochondroma. This study reports on a rare case of EP arising from the transverse process of the D8 vertebra and extending to the adjacent 7th and 8th ribs and the paraspinal tissues. METHODS: A 45-year-old female patient came in with complaints of upper back pain radiating up to the left costal margin for the past 6 months. There were no parasthesias, and there was no history of any sensory or motor symptoms. On physical examination there was midline and left paraspinal tenderness over the D6 to D8 region. Anteroposterior and lateral X-ray images revealed a well-defined oval ossific mass lesion over the lateral aspect of the D8 vertebra, extending to the 7th and 8th ribs on the left side, and multiple bridging osteophytes were noted. Computed tomography scan showed an ossific mass lesion arising from the D8 transverse process with extension to the adjacent 7th and 8th ribs; its margins were well defined, with no periosteal reaction. Magnetic resonance imaging showed a well-defined expansile mass lesion arising from the transverse process of the D8 vertebra matrix; it was was isointense with adjacent marrow and had no evidence of calcifications or vascular flow voids and no encroachment into the spinal canal. RESULTS: Complete resection of the mass lesion with the adjacent part of the 7th and 8th ribs and with intramedullary curettage was performed and sent for histopathologic examination. Histopathology showed bony trabeculae with normal mucosal elements, and a mild hypercellularity with binucleation. Chondrocytes in the myxoid matrix located in round lacunae were compatible with enchondroma, with no evidence of atypia. The postoperative period was uneventful, and after 12 months there were no signs of recurrence noted in computed tomography scan. CONCLUSIONS: EP is a rare presentation in the dorsal spine; it should be considered in the differential diagnosis of osteochondroma, enchondroma, chondrosarcoma, and periosteal chondroid tumors.

Understanding the molecular biology of intervertebral disc degeneration and potential gene therapy strategies for regeneration: a review.

Intervertebral disc degeneration (IVDD) is a multi-factorial process characterized by phenotypic and genotypic changes, which leads to low back pain and disability. Prolonged imbalance between anabolism and catabolism in discs alters their composition resulting in progressive loss of proteoglycans and hydration leading to IVDD. The current managements for IVDD are only able to relieve the symptoms but do not address the underlying pathology of degeneration. Researchers have tried to find out differences between the aging and degeneration of the disc. Intense attempts are in progress for identifying the various factors responsible for disc degeneration, as well as strategies for regeneration. Recently biological approaches have gained thrust in the field of IVDD. The present review illustrates the current understanding of intervertebral disc degeneration and aims to put forth recent advancements in regeneration strategies involving different biological therapies such as growth factor, cell, and gene therapy. The potentials and consequences of these therapies are also extensively discussed along with citing the most suitable method, that is, the gene therapy in detail. Initially, gene therapy was mediated by viral vectors but recent progress has enabled researchers to opt for non-virus-mediated gene therapy methods, which ensure that there are no risks of mutagenicity and infection in target cells. With constant efforts, non-virus-mediated gene therapy may prove to be an extremely powerful tool in treatment of IVDD in future.