A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged ß cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged ß cells.

Decellularized kidney capsule as a three-dimensional scaffold for tissue regeneration.

Tissue regeneration is thought to have considerable promise with the use of scaffolds designed for tissue engineering. Although polymer-based scaffolds for tissue engineering have been used extensively and developed quickly, their ability to mimic the in-vivo milieu, overcome immunogenicity, and have comparable mechanical or biochemical properties has limited their capability for repair. Fortunately, there is a compelling method to get around these challenges thanks to the development of extracellular matrix (ECM) scaffolds made from decellularized tissues. We used ECM decellularized sheep kidney capsule tissue in our research. Using detergents such as Triton-X100 and sodium dodecyl sulfate (SDS), these scaffolds were decellularized. DNA content, histology, mechanical properties analysis, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), biocompatibility, hemocompatibility and scanning electron microscope (SEM) imaging were measured. The results showed that the three-dimensional (3D) structure of the ECM remained largely intact. The scaffolds mentioned above had several hydrophilic properties. The best biocompatibility and blood compatibility properties were reported in the SDS method of 0.5%. The best decellularization scaffold was introduced with 0.5% SDS. Therefore, it can be proposed as a scaffold that has ECM like natural tissue, for tissue engineering applications.

Resveratrol Nanoformulation Inhibits Invasive Breast Cancer Cell Growth through Autophagy Induction: An In Vitro Study.

OBJECTIVE: The aim of this study was to synthesize chitosan nanoparticles (Cs NPs) for resveratrol (RSV) delivery and assess their effectiveness in inducing autophagy in MDA-MB 231 cells. MATERIALS AND METHODS: In this experimental study, Pure and RSV-loaded Cs NPs (RSV. Cs NPs) were prepared via the ionic gelation method, and their physicochemical properties were characterized using standard techniques, and RSV release was measured in vitro. MDA-MB 231 cells were incubated with RSV, Cs NPs, and RSV. Cs NPs and Half-maximal inhibitory concentration (IC50) values were calculated following the MTT test. Cell viability was assessed by lactate dehydrogenase (LDH) assay, and autophagy was evaluated using the real-time polymerase chain reaction (PCR). RESULTS: NP formation was confirmed with the analysis of FTIR spectra. Pure and RSV. Cs NPs had 36.7 and 94.07 nm sizes with 18.3 and 27 mV zeta potentials, respectively. Above 60% of RSV entrapped within NPs was released in an initial burst manner followed by a gradual release till 72 hours. Cs and RSV. Cs NPs restrained cell proliferation at lower concentrations. RSV. Cs NPs showed the highest anticancer effect and stimulated autophagy, indicated by increased Beclin-1 ATG5, ATG7, LC3A, and P62 expression. CONCLUSION: RSV. Cs NPs show promising effects in inhibiting invasive breast cancer (BC) cells in vitro by inducing autophagy.

Computational and in vitro analyses on synergistic effects of paclitaxel and thymoquinone in suppressing invasive breast cancer cells.

BACKGROUND: In the present experiment, we evaluated the impact of thymoquinone (TQ) and paclitaxel (PTX) treatment on MDA-MB-231 cell line growth inhibition via controlling apoptosis/autophagy. MATERIALS AND RESULTS: MDA-MB-231cells were exposed to PTX (0, 25, 50, 75, and 100 nM), TQ (0, 25, 50, 75, and 100 µM), and combinations for 48 h. After the MTT assessment, dose-response curves and IC50 values were calculated, and the combination synergism was evaluated using the Compusyn software. Following the treatment with PTX, TQ, and combinations at IC50 doses, the expression of apoptosis and autophagy genes was assessed in cells. The GraphPad Prism program was used to analyze the data, and Tukey's test at p < 0.05 was then run. PTX, TQ, and their combinations inhibited MDA-MB-231cell proliferation and viability dose-dependently. TQ reduced the effective concentration (IC50) of PTX in co-treatment groups. PTX and TQ showed antagonistic effects when cell proliferation declined above 70%. Antagonistic effects shifted into additive and synergistic effects upon increasing PTX concentration, indicated by diminished cell proliferation below 70%. PTX-TQ co-treatment significantly enhanced P53 and BAX expression while reducing Bcl-2 expression. Also, their combination increased Beclin-1, ATG-5, and ATG-7 expression in treated cells. CONCLUSION: Effective concentrations of TQ and PTX had synergic effects and inhibited breast cancer cells via prompting apoptosis and autophagy in vitro.

Bifunctional tissue-engineered composite construct for bone regeneration: The role of copper and fibrin.

Bifunctional tissue engineering constructs promoting osteogenesis and angiogenesis are essential for bone regeneration. Metal ion-incorporated scaffolds and fibrin encapsulation attract much attention due to low cost, nontoxicity, and tunable control over ion and growth factor release. Herein, we investigated the effect of Cu.nHA/Cs/Gel scaffold and fibrin encapsulation on osteogenic and angiogenic differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) in vitro and in vivo. Cu-laden scaffolds were synthesized using salt leaching/freeze drying and were characterized using standard techniques. WJMSCs were isolated from the human umbilical cord and characterized. WJMSCs with or without encapsulating in fibrin were seeded onto scaffolds, followed by differentiating into the osteogenic lineage for 7 and 21 days. Osteogenic and angiogenic differentiation were evaluated using real-time polymerase chain reaction, western blot, and Alizarin red staining. Then, scaffolds were implanted into critical-sized calvarial bone defects in rats and histological assessments were performed using hematoxylin/eosin, Masson's trichrome, and CD31 immunohistochemical staining at 4 and 12 weeks. The scaffolds had good physicochemical and biological characteristics suitable for cell attachment and growth. Cu and fibrin increased the expression of ALP, RUNX2, OCN, COLI, VEGF, and HIF1α in differentiated WJMSCs. Implanted scaffolds were also biocompatible and were integrated well with the host tissue. Increased collagen condensation, mineralization, and blood vessel formation were observed in Cu-laden scaffolds. The fibrin-encapsulated groups showed the highest collagen and cell densities, immune cell infiltration, and bone trabeculae. CD31-positive cell population increased with fibrin encapsulation and seeding onto Cu-laden scaffolds. Adding Cu to scaffolds and encapsulating cells in fibrin are promising methods that guide osteogenesis and angiogenesis cellular signaling, leading to better bone regeneration.

The anti-cancer effect of chitosan/resveratrol polymeric nanocomplex against triple-negative breast cancer; an in vitro assessment.

Herein, the authors synthesised chitosan nanoparticles (Cs NPs) as a resveratrol (RSV) carrier and evaluated their efficacy in stimulating apoptosis in MDA-MB 231 cells. Blank (Cs NPs) and RSV- Cs NPs (RSV-Cs NPs) were synthesised via ionic gelation and characterised by using fourier-transform infrared spectroscopy (FTIR), Scanning electron microscope, dynamic light scattering/Zeta potential and RSV release. MDA-MB 231 cells were treated with RSV, Cs NPs and RSV-Cs NPs (24, 48, and 72 h), followed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Cell toxicity was evaluated using lactate dehydrogenase assay, and real-time polymerase chain reaction was performed to explore apoptosis induction. FTIR spectra confirmed the NPs via the formation of cross-linking bonds. Cs and RSV-Cs NPs sizes were about 75 and 198 nm with 14 and 24 mV zeta potentials. The RSV entrapment efficiency was 52.34 ± 0.16%, with an early rapid release followed by a sustained manner. Cs and RSV-Cs NPs inhibited cell proliferation at lower concentrations and IC50 values. RSV-Cs NPs had the most cytotoxic effect and stimulated intrinsic apoptotic pathway, indicated by increased Bcl-2-associated x (BAX), BAX/Bcl-2 ratio, P53 expressions, reduced Bcl-2 and upregulated caspases 3, 8 and 9. RSV-Cs NPs have a great potential to suppress invasive breast cancer cell proliferation by targeting mitochondrial metabolism and inducing the intrinsic apoptotic pathway.

Immunotherapy and immunoengineering for breast cancer; a comprehensive insight into CAR-T cell therapy advancements, challenges and prospects.

BACKGROUND: Breast cancer (BC) is a highly prevalent solid cancer with a high-rise infiltration of immune cells, turning it into a significant candidate for tumor-specific immunotherapies. Chimeric antigen receptor (CAR)-T cells are emerging as immunotherapeutic tools with genetically engineered receptors to efficiently recognize and attack tumor cells that express specific target antigens. Technological advancements in CAR design have provided five generations of CAR-T cells applicable to a wide range of cancer patients while boosting CAR-T cell therapy safety. However, CAR-T cell therapy is ineffective against breast cancer because of the loss of specified antigens, the immunosuppressive nature of the tumor and CAR-T cell-induced toxicities. Next-generation CAR-T cells actively pass through the tumor vascular barriers, persist for extended periods and disrupt the tumor microenvironment (TME) to block immune escape. CONCLUSION: CAR-T cell therapy embodies advanced immunotherapy for BC, but further pre-clinical and clinical assessments are recommended to achieve maximized efficiency and safety.

Fabrication, characterization, and optimization of a novel copper-incorporated chitosan/gelatin-based scaffold for bone tissue engineering applications.

Introduction: Fabricating composite scaffolds with improved physicochemical properties as artificial microenvironments are of great interest in bone tissue engineering. Given advantageous properties of nano-hydroxyapatite/chitosan/gelatin (nHA/Cs/Gel) scaffolds, the present study aimed to synthesize a modified nHA/Cs/Gel biomimetic scaffold with improved features. Methods: Pure and copper (Cu)-substituted nHA was synthesized using the chemical precipitation method under controlled pH and temperature. Pure and Cu-substituted nHA/Cs/Gel scaffolds were fabricated by salt-leaching/freeze-drying method. Physicochemical characteristics of nanoparticles and scaffolds were explored using XRD, FTIR, FE-SEM/EDX, and ICP. Besides, scaffold mechanical strength, degradation, porosity, swelling, biomineralization, and cytocompatibility were assessed. Results: Pure and Cu-substituted nHA were synthesized and characterized with appropriate Cu substitution and improved physical properties. All scaffolds were highly porous (porosity > 98%) and Cu incorporation reduced porosity from 99.555 ± 0.394% to 98.69 ± 0.80% while enlarged the pore size to more than100 µm. Cu-substitution improved the scaffold mechanical strength and the best result was observed in nHA.Cu5%/Cs/Gel scaffolds by the compressive strength 88.869 ± 19.574 MPa. Furthermore, 3% and 5% Cu-substituted nHA enhanced the scaffold structural stability and supported osteoblast spread, adhesion, survival, mineralization, and proliferation. Moreover, long-term and sustainable Cu release from scaffolds was observed within 28 days. Conclusion: Cu-substituted nHA/Cs/Gel scaffolds mimic the porous structure and mechanical strength of cancellous bone, along with prolonged degradation and Cu release, osteoblast attachment, viability, calcium deposition, and proliferation. Taken together, our results indicate the upgraded properties of nHA.Cu5%/Cs/Gel scaffolds for future applications in bone tissue engineering.

Adipose tissue-derived stem cells: a comparative review on isolation, culture, and differentiation methods.

Adipose tissue-derived stem cells (ADSCs) are an available source of mesenchymal stem cells with the appropriate capacity to in vitro survive, propagate, and differentiate into cells from three lineages of ectoderm, mesoderm, and endoderm. The biological features of ADSCs depend on the donor physiology and health status, isolation procedure, culture conditions, and differentiation protocols used. Adipose tissue samples are provided by surgery and lipoaspiration-based methods and subjected to various mechanical and chemical digestion techniques to finally generate a heterogeneous mixture named stromal vascular fraction (SVF). ADSCs are purified through varied cell populations that exist within SVF and cultured under standard conditions to give rise to a highly rich resource of stem cells directly applied in the clinic or differentiated into a wide range of cells. The development and optimization of conventional isolation, expansion, and differentiation methods seem noteworthy to preserve the desirable biological functions of ADSCs in pre-clinical and clinical investigations.

Application of nanoparticles in bone tissue engineering; a review on the molecular mechanisms driving osteogenesis.

The introduction of nanoparticles into bone tissue engineering strategies is beneficial to govern cell fate into osteogenesis and the regeneration of large bone defects. The present study explored the role of nanoparticles to advance osteogenesis with a focus on the cellular and molecular pathways involved. Pubmed, Pubmed Central, Embase, Scopus, and Science Direct databases were explored for those published articles relevant to the involvement of nanoparticles in osteogenic cellular pathways. As multifunctional compounds, nanoparticles contribute to scaffold-free and scaffold-based tissue engineering strategies to progress osteogenesis and bone regeneration. They regulate inflammatory responses and osteo/angio/osteoclastic signaling pathways to generate an osteogenic niche. Besides, nanoparticles interact with biomolecules, enhance their half-life and bioavailability. Nanoparticles are promising candidates to promote osteogenesis. However, the interaction of nanoparticles with the biological milieu is somewhat complicated, and more considerations are recommended on the employment of nanoparticles in clinical applications because of NP-induced toxicities.

Osteosarcoma, personalized medicine, and tissue engineering; an overview of overlapping fields of research.

INTRODUCTION: Osteosarcoma is a common bone malignancy in patients of all ages. Surgical and chemotherapy interventions fail to shrink tumor growth and metastasis. The development of efficient patient-specific therapeutic strategies for osteosarcoma is of great interest in tissue engineering and personalized medicine. The present manuscript aimed to review the advancements in tissue engineering and personalized medicine strategies to overcome osteosarcoma and the relevant biological aspects as well as the current tumor models in vitro and in vivo. RESULTS: Tissue engineering and personalized medicine contribute to gene/cell engineering and cell-based therapies specific to genomic and proteomic profiles of individual patients to improve the current treatment options. Also, tissue engineering scaffolds provide physical support to missing bones, could trap cancer cells and deliver immune cells. Taken together, these strategies suppress tumor growth, angiogenic potential, and the subsequent metastasis as well as elicit desirable immune responses against tumor mass. DISCUSSION: Advanced and high-throughput gene and protein identification technologies have facilitated the recognition of genomic and proteomic profiles of patients to design and develop patient-specific treatments. The pre-clinical studies showed promising outcomes to inhibit tumor growth and invasion but controversial results compared to clinical investigations make the importance of more clinical reports inevitable. The experimental tumor models assist the evolution of effective treatments by understanding the mechanisms of tumor progression. CONCLUSION: Tissue engineering and personalized medicine strategies seem encouraging alternatives to conventional therapies against osteosarcoma. Modeling the tumor microenvironment coupled with pre-clinical results give new intelligence into the translation of strategies into the clinic.

Natural and herbal compounds targeting breast cancer, a review based on cancer stem cells.

Cancer stem cells (CSCs) are known as the major reason for therapy resistance. Recently, natural herbal compounds are suggested to have a significant role in inhibiting the breast cancer stem cells (BCSCs). The aim of this study was to explore the effective natural herbal compounds against BCSCs.This review article was designed based on the BCSCs, mechanisms of therapy resistance and natural herbal compounds effective to inhibit their activity. Therefore, Science direct, PubMed and Scopus databases were explored and related original articles were investigated from 2010 to 2019. BCSCs use different mechanisms including special membrane transporters, anti-apoptotic, pro-survival, and self-renewal- related signaling pathways. Natural herbal compounds could disturb these mechanisms, therefore may inhibit or eradicate the BCSCs. Studies show that a broad range of plants, either as a food or medicine, contain anti-cancer agents that phenolic components and their different derivatives share a large quantity. Natural herbal compounds play a pivotal role in the eradication of BCSCs, through the inhibition of biological activities and induction of apoptosis. Although it is necessary to conduct more clinical investigation.

New Findings on Breast Cancer Stem Cells: A Review.

Since the introduction of the "cancer stem cell" theory, significant developments have been made in the understanding of cancer and the heterogenic structure of tumors. In 2003, with the isolation of cancer stem cells from the first solid tumor, breast cancer, and recognition of the tumorigenicity of these cells, this theory suggested that the main reason for therapy failure might be the presence of cancer stem cells. This review article describes breast cancer stem cell origin, the related cellular and molecular characteristics, signaling pathways, and therapy resistance mechanisms. The databases PubMed, SCOPUS, and Embase were explored, and articles published on these topics between 1992 and 2015 were investigated. It appears that this small subpopulation of cells, with the capacity for self-renewal and a high proliferation rate, originate from normal stem cells, are identified by specific markers such as CD44(+)/CD24(-/low), and enhance a tumor's capacity for metastasis, invasion, and therapy resistance. Cancer stem cell characteristics depend on their interactions with their microenvironment as well as on the inducing factors and elements. Although uncertainties about breast cancer stem cells exist, many of researchers believe that cancer stem cells should be considered as possible therapeutic targets.