Evaluation of Human Osteoblasts on NIPS Micro-Patterned PCL Carriers Containing Nanohydroxyapatite and Reduced Graphene Oxide Using PSµM.

The content and surface topology of tissue engineering scaffolds are two important parameters in regulating the cell behavior. In this study, a phase separation micromolding (PSµM) method was implemented to develop micro-groove-imprinted poly(ε-caprolactone) (PCL)-nano hydroxyapatite (nHAp)-reduced graphene oxide (rGO) ternary blend constructs. Physical and chemical characterizations of cell-devoid constructs were performed by FTIR, XRD, TGA, DSC, porosity, swelling, wettability analysis, tensile and compression mechanical tests. The in vitro biological performance of human osteoblasts cultured on micro-patterned blend constructs was evaluated by MTT and alamarBlue viability assays. The findings revealed that nHAp and rGO significantly promote cell viability and proliferation, while the micro-pattern determines the direction of cell migration. Alkaline phosphatase and Ca2+ analyses were carried out to determine the osteogenic properties of cell-laden constructs. This study describes a simple method to generate topologically modified ternary blend PCL/nHAp/rGO constructs using the PSµM method, which contributes to cell proliferation and migration, which is particularly important in regenerative medicine.

Decellularized biological scaffold and stem cells from autologous human adipose tissue for cartilage tissue engineering.

Here, the in vitro engineering of a cartilage-like tissue by using decellularized extracellular matrix scaffold (hECM) seeded with human adipose stem cells (hASCs) which can both be isolated from the human waste adipose tissue is described. Cell-free, highly fibrous and porous hECM was produced using a protocol containing physical (homogenization, centrifugation, molding) and chemical (crosslinking) treatments, characterized by SEM, histochemistry, immunohistochemistry and in vitro cell interaction study. A construct of hECM seeded with hASCs was cultured in chondrogenic medium (with TGF-ß3 and BMP-6) for 42 days. SEM and histology showed that the biological scaffold was highly porous and had a compact structure suitable for handling and subsequent cell culture stages. Cells successfully integrated into the scaffold and had good cellular viability and continuity to proliferate. Constructs showed the formation of cartilage-like tissue with the synthesis of cartilage-specific proteins, Collagen type II and Aggrecan. Dimethylmethylene blue dye binding assay demonstrated that the GAG content of the constructs was in tendency to increase with time confirming chondrogenic differentiation of hASCs. The results support that human waste adipose tissue is an important source for decellularized hECM as well as stem cells, and adipose hECM scaffold provides a suitable environment for chondrogenic differentiation of hASCs.

Functions of Mesenchymal Stem Cells in Cardiac Repair.

Myocardial infarction (MI) and heart failure (HF) are significant contributors of mortality worldwide. Mesenchymal stem cells (MSCs) hold a great potential for cardiac regenerative medicine-based therapies. Their therapeutic potential has been widely investigated in various in-vitro and in-vivo preclinical models. Besides, they have been tested in clinical trials of MI and HF with various outcomes. Differentiation to lineages of cardiac cells, neovascularization, anti-fibrotic, anti-inflammatory, anti-apoptotic and immune modulatory effects are the main drivers of MSC functions during cardiac repair. However, the main mechanisms regulating these functions and cross-talk between cells are not fully known yet. Increasing line of evidence also suggests that secretomes of MSCs and/or their extracellular vesicles play significant roles in a paracrine manner while mediating these functions. This chapter aims to summarize and highlight cardiac repair functions of MSCs during cardiac repair.

Evaluation of the stability of standard reference genes of adipose-derived mesenchymal stem cells during in vitro proliferation and differentiation.

Mesenchymal stem cells (MSCs) from a variety of sources are being used in pre-clinical and clinical studies. The choice of optimal source for treatment of diseases requires quantitative evaluation of self-renewal, proliferation and differentiation potencies of MSCs. For this purpose, quantitative real-time polymerase chain reaction (qRT-PCR) technique is used to determine the expression of genes. qRT-PCR requires the normalization of the gene expression levels by the use of reference genes in order to obtain accurate and reliable results. There is a limited number of studies focused on the selection of reference genes that are appropriate and reliable for MSCs. Thus, no single reference gene has yet been found for use in the in vitro proliferation and differentiation of MSCs. The aim of this study is to investigate the stability of the expression of widely used reference genes during the in vitro proliferation and differentiation of human adipose-derived mesenchymal stem cells (hASCs). For this purpose, 13 reference genes commonly used in MSC studies were selected. As a result, the expression stabilities of EF1α, RPLP0 and RPL13A genes were found to be high and were predicted to be suitable for use as reference genes for normalization in hASC studies. The GAPDH was identified as the gene with the lowest expression stability and evaluated to be an unsuitable reference gene for hASC differentiation studies. This piece of information could be crucial for the selection of appropriate reference genes and accurate measurement of gene expression in hASC studies.

3D Bioprinting of Tissue Models with Customized Bioinks.

The ordered assembly of multicellular structures mimicking native tissues has lately come into prominence for various applications of biomedicine. In this respect, three-dimensional bioprinting (3DP) of cells and other biologics through additive manufacturing techniques has brought the possibility to develop functional in vitro tissue models and perhaps creating de novo transplantable tissues or organs in time. Bioinks, which can be defined as the printable analogues of the extracellular matrix, represent the foremost component of 3DP. In this chapter, we attempt to elaborate the major classes of bioinks which are prevalently being evaluated for the 3DP of a wide range of tissue models.

Autologous protein-based scaffold composed of platelet lysate and aminated hyaluronic acid.

This study describes a protein-based scaffold using platelet rich plasma (PRP), aminated hyaluronic acid (HA-NH2) and Genipin for potential use in regenerative applications as an autologous tissue engineering scaffold. Human PRP was subjected to three freeze-thaw cycles for obtaining platelet lysates (PL). HA-NH2 was synthesized from hyaluronic acid. PL/HA-NH2 scaffolds were fabricated using different concentrations of genipin (0.05, 0.1 and 0.2%) and HA-NH2 (10, 20 and 30 mg/mL). Mechanical, physical, and chemical properties of the scaffolds were comprehensively investigated. The compressive test findings revealed that crosslinking with 0.1 and 0.2% genipin improved the mechanical properties of the scaffolds. SEM evaluations showed that the scaffolds exhibited an interconnected and macroporous structure. Besides, porosimetry analysis indicated a wide distribution of the scaffold pore-size. Rheological findings demonstrated that the G' values were higher than the G″ values, indicating that PL/HA-NH2 scaffolds had typical viscoelastic properties. In vitro biocompatibility studies showed that the scaffolds were both cytocompatible and hemocompatible. Alamar Blue test indicated that human adipose mesenchymal stem cells (hASCs) were able to attach, spread and proliferate on the scaffolds for 21 days-duration. Our findings clearly indicate that PL/HA-NH2 can be a promising autologous candidate scaffold for tissue engineering applications.

Differential gene expression profiling of human adipose stem cells differentiating into smooth muscle-like cells by TGFß1/BMP4.

Regenerative repair of the vascular system is challenging from the perspectives of translational medicine and tissue engineering. There are fundamental hurdles in front of creating bioartificial arteries, which involve recaputilation of the three-layered structure under laboratory settings. Obtaining and maintaining smooth muscle characteristics is an important limitation, as the transdifferentiated cells fail to display mature phenotype. This study aims to shed light on the smooth muscle differentiation of human adipose stem cells (hASCs). To this end, we first acquired hASCs from lipoaspirate samples. Upon characterization, the cells were induced to differentiate into smooth muscle (SM)-like cells using a variety of inducer combinations. Among all, TGFß1/BMP4 combination had the highest differentiation efficiency, based on immunohistochemical analyses. hSM-like cell samples were compared to hASCs and to the positive control, human coronary artery-smooth muscle cells (hCA-SMCs) through gene transcription profiling. Microarray findings revealed the activation of gene groups that function in smooth muscle differentiation, signaling pathways, extracellular modeling and cell proliferation. Our results underline the effectiveness of the growth factors and suggest some potential variables for detecting the SM-like cell characteristics. Evidence in transcriptome level was used to evaluate the TGFß1/BMP4 combination as a previously unexplored effector for the smooth muscle differentiation of adipose stem cells.

Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Human Stem Cell Research.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely utilized method for evaluating the gene expressions in stem cell research. This method enables researchers to obtain fast and precise results, but the accuracy of the data depends on certain factors, such as those associated with biological sample preparation and PCR efficiency. In order to achieve accurate and reliable results, it is of utmost importance to designate the reference genes, the expressions of which are suitable to all kinds of experimental conditions. Hence it is vital to normalize the qRT-PCR data by using the reference genes. In recent years, it has been found that the expression levels of reference genes widely used in stem cell research present a substantial amount of variation and are not necessarily suitable for normalization. This chapter at hand stresses the significance of selecting suitable reference genes from the point view of human stem cell research.

Intrinsically Conductive Polymer Nanocomposites for Cellular Applications.

Intrinsically conductive polymer nanocomposites have a remarkable potential for cellular applications such as biosensors, drug delivery systems, cell culture systems and tissue engineering biomaterials. Intrinsically conductive polymers transmit electrical stimuli between cells, and induce regeneration of electroactive tissues such as muscle, nerve, bone and heart. However, biocompatibility and processability are common issues for intrinsically conductive polymers. Conductive polymer composites are gaining importance for tissue engineering applications due to their excellent mechanical, electrical, optical and chemical functionalities. Here, we summarize the different types of intrinsically conductive polymers containing electroactive nanocomposite systems. Cellular applications of conductive polymer nanocomposites are also discussed focusing mainly on poly(aniline), poly(pyrrole), poly(3,4-ethylene dioxythiophene) and poly(thiophene).

Clinical Applications of Injectable Biomaterials.

Regenerative medicine is an interdisciplinary field that aims to regenerate the lost or diseased tissues through the combinational use of cells, biomolecules and/or biomaterials. Injectable biomaterials have been comprehensively evaluated for use in this field for their prominent properties, such as ease of handling, providing a better integration of the native tissue by filling irregular defects and having controllable chemical and physical properties. This class of biomaterials can be developed from natural or synthetic origin materials, decellularized matrices or from combinations of materials to form composites. Injectable biomaterials enable minimally invasive approach when compared with traditional open surgeries, which can reduce the cost, and speed up the recovery time for the patients. Cells, growth factors and/or bioactive molecules can be effectively delivered to the target tissue using injectable biomaterials, making them desirable for a number of clinical applications. This chapter gives an overview on injectable biomaterials and their clinical applications in soft, hard, and cardiovascular tissue regeneration.

Investigations on clonazepam-loaded polymeric micelle-like nanoparticles for safe drug administration during pregnancy.

Medication during pregnancy is often a necessity for women to treat their acute or chronic diseases. The goal of this study is to evaluate the potential of micelle-like nanoparticles (MNP) for providing safe drug usage in pregnancy and protect both foetus and mother from medication side effects. Clonazepam-loaded MNP were prepared from copolymers [polystyrene-poly(acrylic acid) (PS-PAA), poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) and distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-poly(ethylene glycol) (PEG-DSPE)] with varying monomer ratios and their drug-loading efficiency, drug release ratio, particle size, surface charge and morphology were characterised. The cellular transport and cytotoxicity experiments were conducted on clonazepam and MNP formulations using placenta-choriocarcinoma-BeWo and brain-endothelial-bEnd3 cells. Clonazepam-loaded PEG5000-PLA4500 MNP reduced the drug transport through BeWo cells demonstrating that MNP may lower foetal drug exposure, thus reduce the drug side effects. However, lipofectamine modified MNP improved the transport of clonazepam and found to be promising for brain and in-utero-specific drug treatment.

A comparative study on the in vitro cytotoxic responses of two mammalian cell types to fullerenes, carbon nanotubes and iron oxide nanoparticles.

The present study was designed to evaluate and compare the time- and dose-dependent cellular response of human periodontal ligament fibroblasts (hPDLFs), and mouse dermal fibroblasts (mDFs) to three different types of nanoparticles (NPs); fullerenes (C60), single walled carbon nanotubes (SWCNTs) and iron (II,III) oxide (Fe3O4) nanoparticles via in vitro toxicity methods, and impedance based biosensor system. NPs were characterized according to their morphology, structure, surface area, particle size distribution and zeta potential by using transmission electron microscopy, X-ray diffraction, Brunauer-Emmett-Teller, dynamic light scattering and zeta sizer analyses. The Mössbauer spectroscopy was used in order to magnetically characterize the Fe3O4 NPs. The hPDLFs and mDFs were exposed to different concentrations of the NPs (0.1, 1, 10, 50 and 100 µg/mL) for predetermined time intervals (6, 24 and 48 h) under controlled conditions. Subsequently, NP exposed cells were tested for viability, membrane leakage and generation of intracellular reactive oxygen species. Additional to in vitro cytotoxicity assays, the cellular responses to selected NPs were determined in real time using an impedance based biosensor system. Taken together, information obtained from all experiments suggests that toxicity of the selected NPs is cell type, concentration and time dependent.

Magnetic biocomposite scaffold based on decellularized tendon ECM and MNP-deposited halloysite nanotubes: physicochemical, thermal, rheological, mechanical andin vitrobiological evaluations.

The development of new three-dimensional biomaterials with advanced versatile properties is critical to the success of tissue engineering (TE) applications. Here, (a) bioactive decellularized tendon extracellular matrix (dECM) with a sol-gel transition feature at physiological temperature, (b) halloysite nanotubes (HNT) with known mechanical properties and bioactivity, and (c) magnetic nanoparticles (MNP) with superparamagnetic and osteogenic properties were combined to develop a new scaffold that could be used in prospective bone TE applications. Deposition of MNPs on HNTs resulted in magnetic nanostructures without agglomeration of MNPs. A completely cell-free, collagen- and glycosaminoglycan- rich dECM was obtained and characterized. dECM-based scaffolds incorporated with 1%, 2% and 4% MNP-HNT were analysed for their physical, chemical, andin vitrobiological properties. Fourier-transform infrared spectroscopy, x-ray powder diffractometry and vibrating sample magnetometry analyses confirmed the presence of dECM, HNT and MNP in all scaffold types. The capacity to form apatite layer upon incubation in simulated body fluid revealed that dECM-MNP-HNT is a bioactive material. Combining dECM with MNP-HNT improved the thermal stability and compressive strength of the macroporous scaffolds upto 2% MNP-HNT.In vitrocytotoxicity and hemolysis experiments showed that the scaffolds were essentially biocompatible. Human bone marrow mesenchymal stem cells adhered and proliferated well on the macroporous constructs containing 1% and 2% MNP-HNT; and remained metabolically active for at least 21 din vitro. Collectively, the findings support the idea that magnetic nanocomposite dECM scaffolds containing MNP-HNT could be a potential template for TE applications.

Decellularized Bone Matrix/45S5 Bioactive Glass Biocomposite Hydrogel-Based Constructs with Angiogenic and Osteogenic Properties: Ex Ovo and Ex Vivo Evaluations.

Decellularized extracellular matrix is often used to create an in vivo-like environment that supports cell growth and proliferation, as it reflects the micro/macrostructure and molecular composition of tissues. On the other hand, bioactive glasses (BG) are surface-reactive glass-ceramics that can convert to hydroxyapatite in vivo and promote new bone formation. This study is designed to evaluate the key properties of a novel angiogenic and osteogenic biocomposite graft made of bovine decellularized bone matrix (DBM) hydrogel and 45S5 BG microparticles (10 and 20 wt%) to combine the existing superior properties of both biomaterial classes. Morphological, physicochemical, mechanical, and thermal characterizations of DBM and DBM/BG composite hydrogels are performed. Their in vitro biocompatibility is confirmed by cytotoxicity and hemocompatibility analyses. Ex vivo chick embryo aortic arch and ex ovo chick chorioallantoic membrane (CAM) assays reveal that the present pro-angiogenic property of DBM hydrogels is enhanced by the incorporation of BG. Histochemical stainings (Alcian blue and Alizarin red) and digital image analysis of ossification on hind limbs of embryos used in the CAM model reveal the osteogenic potential of biomaterials. The findings support the notion that the developed DBM/BG composite hydrogel constructs have the potential to be a suitable graft for bone repair.

Regeneration of Volumetric Muscle Loss Using MSCs Encapsulated in PRP-Derived Fibrin Microbeads.

Volumetric muscle loss (VML) is one of the major types of soft tissue injury frequently encountered worldwide. In case of VML, the endogenous regenerative capacity of the skeletal muscle tissue is usually not sufficient for complete healing of the damaged area resulting in permanent functional musculoskeletal impairment. Therefore, the development of new tissue engineering approaches that will enable functional skeletal muscle regeneration by overcoming the limitations of current clinical treatments for VML injuries has become a critical goal. Platelet-rich plasma (PRP) is an inexpensive and relatively effective blood product with a high concentration of platelets containing various growth factors and cytokines involved in wound healing and tissue regeneration. Due to its autologous nature, PRP has been a safe and widely used treatment option for various wound types for many years. Recently, PRP-based biomaterials have emerged as a promising approach to promote muscle tissue regeneration upon injury. This chapter describes the use of PRP-derived fibrin microbeads as a versatile encapsulation matrix for the localized delivery of mesenchymal stem cells and growth factors to treat VML using tissue engineering strategies.

Advanced liposome and polymersome-based drug delivery systems: Considerations for physicochemical properties, targeting strategies and stimuli-sensitive approaches.

Liposomes and polymersomes are colloidal vesicles that are self-assembled from lipids and amphiphilic polymers, respectively. Because of their ability to encapsulate both hydrophilic and hydrophobic therapeutics, they are of great interest in drug delivery research. Today, the applications of liposomes and polymersomes have expanded to a wide variety of complex therapeutic molecules, including nucleic acids, proteins and enzymes. Thanks to their chemical versatility, they can be tailored to different drug delivery applications to achieve maximum therapeutic index. This review article evaluates liposomes and polymersomes from a perspective that takes into account the physical and biological barriers that reduce the efficiency of the drug delivery process. In this context, the design approaches of liposomes and polymersomes are discussed with representative examples in terms of their physicochemical properties (size, shape, charge, mechanical), targeting strategies (passive and active) and response to different stimuli (pH, redox, enzyme, temperature, light, magnetic field, ultrasound). Finally, the challenges limiting the transition from laboratory to practice, recent clinical developments, and future perspectives are addressed.

Advances in Regenerative Medicine and Biomaterials.

The low regenerative potential of the human body hinders proper regeneration of dysfunctional or lost tissues and organs due to trauma, congenital defects, and diseases. Tissue or organ transplantation has hence been a major conventional option for replacing the diseased or dysfunctional body parts of the patients. In fact, a great number of patients on waiting lists would benefit tremendously if tissue and organs could be replaced with biomimetic spare parts on demand. Herein, regenerative medicine and advanced biomaterials strive to reach this distant goal. Tissue engineering aims to create new biological tissue or organ substitutes, and promote regeneration of damaged or diseased tissue and organs. This approach has been jointly evolving with the major advances in biomaterials, stem cells, and additive manufacturing technologies. In particular, three-dimensional (3D) bioprinting utilizes 3D printing to fabricate viable tissue-like structures (perhaps organs in the future) using bioinks composed of special hydrogels, cells, growth factors, and other bioactive contents. A third generation of multifunctional biomaterials could also show opportunities for building biomimetic scaffolds, upon which to regenerate stem cells in vivo. Besides, decellularization technology based on isolation of extracellular matrix of tissue and organs from their inhabiting cells is presented as an alternative to synthetic biomaterials. Today, the gained knowledge of functional microtissue engineering and biointerfaces, along with the remarkable advances in pluripotent stem cell technology, seems to be instrumental for the development of more realistic microphysiological 3D in vitro tissue models, which can be utilized for personalized disease modeling and drug development. This chapter will discuss the recent advances in the field of regenerative medicine and biomaterials, alongside challenges, limitations, and potentials of the current technologies.

Bioactive composite hydrogels as 3D mesenchymal stem cell encapsulation environment for bone tissue engineering: in vitro and in vivo studies.

Although decellularized bone matrix (DBM) has often been used in scaffold form for osteogenic applications, its use as a stem cell encapsulation matrix adaptable to surgical shaping procedures has been neglected. This study aimed to investigate the feasibility of utilizing solubilized DBM and nanohydroxyapatite (nHAp)-incorporated DBM hydrogels as encapsulation matrix for bone marrow-derived MSCs (BM-MSCs). First, DBM and DBM/nHAp hydrogels were assessed by physical, chemical, turbidimetric, thermal, and mechanical methods; then, in vitro cytocompatibility and in vitro hemocompatibility were investigated. An in vivo study was performed to evaluate the osteogenic properties of hydrogels alone or with BM-MSCs encapsulated in them. The findings revealed that hydrogels retained high levels of collagen and glycosaminoglycans after successful decellularization. They were found to be cytocompatible and hemocompatible in vitro, and were able to gel with sufficient mechanical stability at physiological temperature. BM-MSCs survived in culture for at least 2 weeks as metabolically active when encapsulated in both DBM and DBM/nHAp. Preliminary in vivo study showed that DBM-nHAp has higher osteogenicity than DBM. Moreover, BM-MSC encapsulated DMB/nHAp showed predominant bone-like tissue formation at 30 days in the rat ectopic site compared to its cell-free form.

Fabrication and Molecular Modeling of Navette-Shaped Fullerene Nanorods Using Tobacco Mosaic Virus as a Nanotemplate.

To date, metallization studies have been performed with the nanometer-scale template, Tobacco Mosaic Virus (TMV). Here we show that fullerenes as well can be deposited on TMV coat protein in a controlled manner. Two methods were followed for the coating process. First, underivatized fullerene was dispersed in different solvents to bring the underivatized fullerene and wild-type TMV together. Improved depositions were obtained with the fullerene dicarboxylic derivative synthesized via the Bingel method. The form of the coating was analyzed by transmission electron microscopy. Our results demonstrate that the coating efficiency with the carboxy derivative was much better compared to the underivatized fullerene. The goal of coupling a carbon nanoparticle to a biological molecule, the viral coat of TMV, was achieved with the carboxy derivative of fullerene, resulting in the production of navette-shaped nanorods. The interactions between carboxyfullerenes and TMV were investigated through modeling with computational simulations and Gaussian-based density functional theory (DFT) calculations using the Gaussian09 program package. The theoretical calculations supported the experimental findings. This inexpensive and untroublesome method promises new fullerene hybrid nanomaterials in particular shapes and structures.

Development of a multicellular 3D-bioprinted microtissue model of human periodontal ligament-alveolar bone biointerface: Towards a pre-clinical model of periodontal diseases and personalized periodontal tissue engineering.

While periodontal (PD) disease is among principal causes of tooth loss worldwide, regulation of concomitant soft and mineralized PD tissues, and PD pathogenesis have not been completely clarified yet. Besides, relevant pre-clinical models and in vitro platforms have limitations in simulating human physiology. Here, we have harnessed three-dimensional bioprinting (3DBP) technology for developing a multi-cellular microtissue model resembling PD ligament-alveolar bone (PDL-AB) biointerface for the first time. 3DBP parameters were optimized; the physical, chemical, rheological, mechanical, and thermal properties of the constructs were assessed. Constructs containing gelatin methacryloyl (Gel-MA) and hydroxyapatite-magnetic iron oxide nanoparticles showed higher level of compressive strength when compared with that of Gel-MA constructs. Bioprinted self-supporting microtissue was cultured under flow in a microfluidic platform for >10 days without significant loss of shape fidelity. Confocal microscopy analysis indicated that encapsulated cells were homogenously distributed inside the matrix and preserved their viability for >7 days under microfluidic conditions. Immunofluorescence analysis showed the cohesion of stromal cell surface marker-1+ human PDL fibroblasts containing PDL layer with the osteocalcin+ human osteoblasts containing mineralized layer in time, demonstrating some permeability of the printed constructs to cell migration. Preliminary tetracycline interaction study indicated the uptake of model drug by the cells inside the 3D-microtissue. Also, the non-toxic levels of tetracycline were determined for the encapsulated cells. Thus, the effects of tetracyclines on PDL-AB have clinical significance for treating PD diseases. This 3D-bioprinted multi-cellular periodontal/osteoblastic microtissue model has potential as an in vitro platform for studying processes of the human PDL.