Diagnostic use of circulating cells and sub-cellular bio-particles.

In the bloodstream or other physiological fluids, "circulating cells and sub-cellular bio-particles" include many microscopic biological elements such as circulating tumor cells (CTCs), cell-free DNA (cfDNA), exosomes, microRNAs, platelets, immune cells, and proteins are the most well-known and investigated. These structures are crucial biomarkers in healthcare and medical research for the early detection of cancer and other disorders, enabling treatment to commence before the onset of clinical symptoms and enhancing the efficacy of treatments. As the size of these biomarkers to be detected decreases and their numbers in body fluids diminishes, the detection materials, ranging from visual inspection to advanced microscopy techniques, begin to become smaller, more sensitive, faster, and more effective, thanks to developing nanotechnology. This review first defines the circulating cells and subcellular bio-particles with their biological, physical, and mechanical properties and second focuses on their diagnostic importance, including their most recent applications as biomarkers, the biosensors that are utilized to detect them, the present obstacles that must be surmounted, and prospective developments in the domain. As technology advances and biomolecular pathways are deepens, diagnostic tests will become more sensitive, specific, and thorough. Finally, integrating recent advances in the diagnostic use of circulating cells and bioparticles into clinical practice is promising for precision medicine and patient outcomes.

Effects of liquid-to-solid ratio and gamma irradiation on the rheology and cytocompatibility of a beta-tricalcium phosphate-based injectable bone substitute.

Injectable bone substitute (IBS) materials are commonly used to fill irregular-shaped bone voids in non-load-bearing areas and can offer greater utility over those which are in prefabricated powder, granule, or block forms. This work investigates the impact of liquid-to-solid ratio (LSR) on the rheology and cytocompatibility of IBSs formulated from bioactive glass particles and ß-tricalcium phosphate (ß-TCP) in glycerol and poly(ethylene glycol) (PEG). IBS formulations of varying LSR were prepared and packed in 3 cc open-bore syringes and sterilized via gamma irradiation (10 kGy, 25 kGy). Gamma-irradiated formulations with high PEG content required the highest (73 N) mechanical force for injection from syringes. Oscillatory viscosity measurements revealed that the viscosity of samples was directly proportional to glycerol content. PEG and glycerol displayed competing effects on the washout resistance and cohesiveness of samples, which were based on total weight loss in media and Ca2+ ion release, respectively. Cell viability in 24-h extracts of 10 kGy gamma-sterilized and 25 kGy gamma-irradiated samples were 22.94% and 56.53%, respectively. The research highlights the complex interplay of IBS components on IBS rheology and, moreover, the cytotoxicity behaviors of beta-tricalcium phosphate-based injectable bone substitutes by in vitro experiments.

The role of decellularized cell derived extracellular matrix in the establishment and culture ofin vitrobreast cancer tumor model.

Decades of research have shown that two-dimensional cell culture studies are insufficient for preclinical cancer diagnosis and treatment, and that cancer cells in three-dimensional (3D) culture systems have better cell-cell and cell-matrix interactions, gene expression, heterogeneity, and structural complexity that more closely resemblein vivotumors. Researchers are still optimizing 3D culturing settings for different cancers. Despite promising tumor spheroid research, tumor cell-only aggregates lack the tumor microenvironment and cannot model tumors. Here, MCF-7 breast cancer cell derived decellularized extracellular matrix (CD-dECMs) were obtained and converted into autologous, biologically active, biocompatible, and non-immunogenic hydrogels to be used as micro-environment in both organoid formation and culture. For the production of organoids, CD-dECM doping concentrations ranging from 0.1 mg ml-1to 1.5 mg ml-1were evaluated, and the lowest concentration was found to be the most effective. For organoid culture, 8 mg ml-1CD-dECM, 4 mg ml-1rat tendon collagen type I (Col I) (4 mg ml-1) and a 1:1 (v/v) mixture of these two were used and the most viable and the biggest organoids were discovered in CD-dECM/Col I (1:1) group. The results show that autologous CD-dECM can replace hydrogels in tumor organoid generation and culture at low and high concentrations, respectively.

Spheroid Engineering in Microfluidic Devices.

Two-dimensional (2D) cell culture techniques are commonly employed to investigate biophysical and biochemical cellular responses. However, these culture methods, having monolayer cells, lack cell-cell and cell-extracellular matrix interactions, mimicking the cell microenvironment and multicellular organization. Three-dimensional (3D) cell culture methods enable equal transportation of nutrients, gas, and growth factors among cells and their microenvironment. Therefore, 3D cultures show similar cell proliferation, apoptosis, and differentiation properties to in vivo. A spheroid is defined as self-assembled 3D cell aggregates, and it closely mimics a cell microenvironment in vitro thanks to cell-cell/matrix interactions, which enables its use in several important applications in medical and clinical research. To fabricate a spheroid, conventional methods such as liquid overlay, hanging drop, and so forth are available. However, these labor-intensive methods result in low-throughput fabrication and uncontrollable spheroid sizes. On the other hand, microfluidic methods enable inexpensive and rapid fabrication of spheroids with high precision. Furthermore, fabricated spheroids can also be cultured in microfluidic devices for controllable cell perfusion, simulation of fluid shear effects, and mimicking of the microenvironment-like in vivo conditions. This review focuses on recent microfluidic spheroid fabrication techniques and also organ-on-a-chip applications of spheroids, which are used in different disease modeling and drug development studies.

Surface channel patterned and endothelialized poly(glycerol sebacate) based elastomers.

Prevascularization of tissue equivalents is critical for fulfilling the need for sufficient vascular organization for nutrient and gas transport. Hence, endothelial cell culture on biomaterials is of great importance for researchers. Numerous alternate strategies have been suggested in this sense, with cell-based methods being the most commonly employed. In this study, poly (glycerol sebacate) (PGS) elastomers with varying crosslinking ratios were synthesized and their surfaces were patterned with channels by using laser ablation technique. In order to determine an ideal material for cell culture studies, the elastomers were subsequently mechanically, chemically, and biologically characterized. Following that, human umbilical vein endothelial cells (HUVECs) were seeded into the channels established on the PGS membranes and cultured under various culture conditions to establish the optimal culture parameters. Lastly, the endothelial cell responses to the synthesized PGS elastomers were evaluated. Remarkable cell proliferation and impressive cellular organizations were noticed on the constructs created as part of the investigation. On the concrete output of this research, arrangements in various geometries can be created by laser ablation method and the effects of various molecules, drugs or agents on endothelial cells can be evaluated. The platforms produced can be employed as an intermediate biomaterial layer containing endothelial cells for vascularization of tissue-engineered structures, particularly in layer-by-layer tissue engineering approaches.

In vitro selection of DNA aptamers against human osteosarcoma.

BACKGROUND: Osteosarcoma is a highly malignant bone tumor, most frequently occurring in the rapid bone growth phase. Effective treatment of this disease is hindered by the lack of specific probes for early diagnosis and the fast cancer widespread. METHODS: To find such probes, the cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) methodology was implemented against the human osteosarcoma MG-63 cell line towards the selection of new specific aptamers. After 10 rounds of selection, the aptamer DNA pool was Sanger sequenced and the sequences were subjected to a bioinformatic analysis that included sequence alignment, phylogenetic relationship, and secondary structure prediction. RESULTS: A DNA aptamer (OS-7.9), with a dissociation constant (Kd) value in the nanomolar range (12.8 ± 0.9 nM), revealed high affinity against the target cells at the physiological temperature. Furthermore, the selected aptamer also recognized lung carcinoma and colon colorectal adenocarcinoma cell lines, which are reported as common metastasis sites of osteosarcoma. CONCLUSIONS: These results suggest that OS-7.9 could recognize a common protein expressed in these cancer cells, possibly becoming a potential molecular probe for early diagnosis and targeted therapies for metastatic disease. Moreover, to the best of our knowledge, this was the first attempt to generate a DNA aptamer (OS-7.9 aptamer) against the MG-63-cell line by cell-SELEX.

Fabrication and characterization of carbon aerogel/poly(glycerol-sebacate) patches for cardiac tissue engineering.

Cardiovascular diseases (CVDs) are responsible for the major number of deaths around the world. Among these is heart failure after myocardial infarction whose latest therapeutic methods are limited to slowing the end-state progression. Numerous strategies have been developed to meet the increased demand for therapies regarding CVDs. This study aimed to establish a novel electrically conductive elastomer-based composite and assess its potential as a cardiac patch for myocardial tissue engineering. The electrically conductive carbon aerogels (CAs) used in this study were derived from waste paper as a cost-effective carbon source and they were combined with the biodegradable poly(glycerol-sebacate) (PGS) elastomer to obtain an electrically conductive cardiac patch material. To the best of our knowledge, this is the first report about the conductive composites obtained by the incorporation of CAs into PGS (CA-PGS). In this context, the incorporation of the CAs into the polymeric matrix significantly improved the elastic modulus (from 0.912 MPa for the pure PGS elastomer to 0.366 MPa for the CA-PGS) and the deformability (from 0.792 MPa for the pure PGS to 0.566 MPa for CA-PGS). Overall, the mechanical properties of the obtained structures were observed similar to the native myocardium. Furthermore, the addition of CAs made the obtained structures electrically conductive with a conductivity value of 65 × 10-3S m-1which falls within the range previously recorded for human myocardium. Thein vitrocytotoxicity assay with L929 murine fibroblast cells revealed that the CA-PGS composite did not have cytotoxic characteristics. On the other hand, the studies conducted with H9C2 rat cardiac myoblasts revealed that final structures were suitable for MTE applications according to the successes in cell adhesion, cell proliferation, and cell behavior.

Multi-layered in vitro 3D-bone model via combination of osteogenic cell sheets with electrospun membrane interlayer.

In this study, it was aimed to present an approach for the development of multi-layered tissue engineering constructs by using cell sheet engineering. Briefly, MC3T3-E1 mouse pre-osteoblast cells were cultured in temperature-responsive plates (Nunc Upcell®) in the presence of osteogenic medium and the resulting cell sheets were laminated with electrospun poly(L-lactic acid) (PLLA) membranes to obtain viable three-dimensional, thick constructs. The constructs prepared without PLLA membranes were used as control. The cell viability and death in the resulting structures were investigated by microscopic and colorimetric methods. The in vitro performance of the structures was discussed comparatively. Alkaline phosphatase (ALP) activity, collagen and sulfated glycosaminoglycan (sGAG) content values were calculated. The presented approach shows potential for engineering applications of complex tissues with at least two or more microenvironments such as osteochondral, corneal or vascular tissues.

Synthesis of Conductive Carbon Aerogels Decorated with ß-Tricalcium Phosphate Nanocrystallites.

There has been substantial interest in research aimed at conductive carbon-based supports since the discovery that the electrical stimulus can have dramatic effect on cell behavior. Among these carbon-aerogels decorated with biocompatible polymers were suggested as future materials for tissue engineering. However, high reaction temperatures required for the synthesis of the aerogels tend to impair the stability of the polymeric networks. Herein, we report a synthetic route towards carbon-aerogel scaffolds decorated with biocompatible ceramic nanoparticles of tricalcium phosphate. The composites can be prepared at temperature as high as 1100 °C without significant effect on the morphology of the composite which is comparable with the original aerogel framework. Although the conductivity of the composites tends to decrease with the increasing ceramic content the measured conductivity values are similar to those previously reported on polymer-functionalized carbon-aerogels. The cell culture study revealed that the developed constructs support cell proliferation and provide good cell attachment suggesting them as potentially good candidates for tissue-engineering applications.

Hybrid Cornea: Cell Laden Hydrogel Incorporated Decellularized Matrix.

The decellularization protocols applied on the corneal stromal constructs in the literature usually fail to provide a corneal matrix with sufficient mechanical and optical properties since they alter the extracellular matrix (ECM) microstructure. In this study, to overcome these limitations, a hybrid cornea stromal construct was engineered by combining gelatin methacrylate (GelMA) and decellularized ECM. Photo-cross-linking of impregnated cell laden GelMA in situ using different UV cross-linking energies (3200, 6210, and 6900 µJ/cm2) and impregnation times (up to 24 h) within a decellularized bovine cornea enhanced light transmission and restored lost mechanical features following the harsh decellularization protocol. The light transmittance value for optimized hybrid constructs (53.6%) was increased nearly 10 fold compared to that of decellularized cornea (5.84%). The compressive modulus was also restored up to 6 fold with calculated values of 5040 and 870 kPa for the hybrid and decellularized samples, respectively. These values were found to be quite close to that of native cornea (48.5%, 9790 kPa). ATR-FTIR analyses were carried out to confirm the final chemical structure. The degradation profiles showed similar decomposition behaviors to that of native cornea. In vitro culture studies showed a high level of cell viability and cell proliferation rate was found remarkable up to the 14th day of the culture period regardless of selected UV energy level. The methodology used in the preparation of the hybrid cornea stromal constructs in this study is a promising approach toward the development of successful corneal transplants.

Preparation of MC3T3-E1 cell sheets through short-term osteogenic medium application.

Cell sheet engineering is an emerging field based on the acquisition of cells together with their extracellular matrix (ECM) and is used not only in vitro but also in regeneration studies of various tissues in the clinic. Within this scope, wide variety of cell types have been investigated in terms of sheet formation and underlying mechanism. MC3T3-E1 is a mouse pre-osteoblast cell line that has greatly attracted researchers' attention for bone tissue engineering (BTE) applications thanks to its high proliferation and differentiation properties. The potential of MC3T3-E1 cells on sheet formation and the effects of culture conditions have not been investigated in detail. This study aims to examine the effects of growth and osteogenic medium on cell sheet formation of MC3T3-E1. As a result of this study; intact, ECM-rich, transferable cell sheets at the beginning of the mineralization phase of the differentiation process were obtained by using osteogenic medium. Hereafter, 3D tissue model can be constructed by stacking MC3T3 cell sheets in vitro. This 3D model can conveniently be used for the development of novel biomaterials and in vitro drug screening applications to reduce the need for animal experiments.

Evaluation of collagen foam, poly(l-lactic acid) nanofiber mesh, and decellularized matrices for corneal regeneration.

Corneal tissue engineering efforts to obtain corneal tissue matrices through various types of materials for the replacement of damaged tissues. In this study, three different corneal constructs were prepared and evaluated in terms of morphological, optical, and biological characteristics. Type-I collagen was used to obtain collagen foam scaffolds through dehydrothermal crosslinking, while poly(l-lactic acid) (PLLA) was used to produce both random and aligned oriented electrospun corneal constructs. Bovine corneas were decellularized as third matrix. Software analyses showed that average pore size of collagen scaffolds was 88.207 ± 29.7 µm, while the average fiber diameter of aligned and random PLLA scaffolds were 0.69 ± 0.03 and 0.65 ± 0.03 µm, respectively. Degradation profiles revealed that collagen foam exhibits high degradation (20% mass loss) while electrospun PLLA scaffolds hold low degradation (9% mass loss) rates at day-28. Transmittance values of the obtained scaffolds were calculated as 92, 80, and 70% for collagen, PLLA, and decellularized cornea constructs, respectively. The evaluation of stromal keratocyte behavior on the constructs revealed that the cells exhibited their own morphology mostly on the aligned PLLA constructs, while they were mostly active on random PLLA electrospun corneal scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2157-2168, 2018.

Bi-layered constructs of poly(glycerol-sebacate)-ß-tricalcium phosphate for bone-soft tissue interface applications.

This study aims to establish a facile protocol for the preparation of a bi-layered poly(glycerol-sebacate) (PGS)/ß-tricalcium phosphate (ß-TCP) construct and to investigate its potential for bone-soft tissue engineering applications. The layered structure was prepared by distributing the ceramic particles within a prepolymer synthesized in a microwave reactor followed by a cross-linking of the final construct in vacuum (<10mbar). The vacuum stage led to the separation of cross-linked elastomer (top) and ceramic (bottom) phases. Results showed that addition of ß-TCP particles to the elastomer matrix after the polymerization led to an increase in compression strength (up to 14±2.3MPa). Tensile strength (σ), Young's modulus (E), and elongation at break (%) values were calculated as 0.29±0.03MPa and 0.21±0.03; 0.38±0.02 and 1.95±0.4; and 240±50% and 24±2% for PGS and PGS/ß-TCP bi-layered constructs, respectively. Morphology was characterized by using Scanning Electron Microscopy (SEM) and micro-computed tomography (µ-CT). Tomography data revealed an open porosity of 35% for the construct, mostly contributed from the ceramic phase since the elastomer side has no pore. Homogeneous ß-TCP distribution within the elastomeric structure was observed. Cell culture studies confirmed biocompatibility with poor elastomer-side and good bone-side cell attachment. In a further study to investigate the osteogenic properties, the construct were loaded with BMP-2 and/or TGF-ß1. The PGS/ß-TCP bi-layered constructs with improved mechanical and biological properties have the potential to be used in bone-soft tissue interface applications where soft tissue penetration is a problem.