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.

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.

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.

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.

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.

Macro- and microporous polycaprolactone/duck's feet collagen scaffold fabricated by combining facile phase separation and particulate leaching techniques to enhance osteogenesis for bone tissue engineering.

Herein, a facile macro- and microporous polycaprolactone/duck's feet collagen scaffold (PCL/DC) was fabricated and characterized to confirm its applicability in bone tissue engineering. A biomimetic scaffold for bone tissue engineering and regeneration for bone defects is an important element. PCL is a widely applied biomaterial for bone tissue engineering due to its biocompatibility and biodegradability. However, the high hydrophobicity and low cell attachment site properties of PCL lead to an insufficient microenvironment in designing a scaffold. Collagen is a nature-derived biomaterial that is widely used in tissue engineering and has excellent biocompatibility, mechanical properties, and cell attachment moieties. Among the resources from which collagen can be obtained, DC contains a high amount of collagen type I (COL1), is biocompatible, and is cost-effective. In this study, the scaffolds were fabricated by blending DC with PCL in various ratios and applied non-solvent-induced phase separation (NIPS) and thermal-induced phase separation (TIPS) (N-TIPS), solvent casting and particulate leaching (SCPL), and gas foaming method to fabricate macro- and microporous structure. The characterization of the fabricated scaffolds was carried out by morphological analysis, bioactivity test, physicochemical analysis, and mechanical test. In vitro study was carried out by viability test, morphology observation, and gene expression. The results showed that the incorporation of DC enhances the physicochemical and mechanical properties of the scaffolds. Also, a large amount of bone mimetic apatite was formed according to the DC content in the bioactivity test. The in vitro study showed that the PCL/DC scaffold is biocompatible and the existence of apatite and DC formed a favorable microenvironment for cell proliferation and differentiation. Overall, the novel porous PCL/DC scaffold can be a promising biomaterial model for bone tissue engineering and regeneration.

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.

Decellularized liver ECM-based 3D scaffolds: Compositional, physical, chemical, rheological, thermal, mechanical, and in vitro biological evaluations.

The extracellular matrix (ECM) is involved in many critical cellular interactions through its biological macromolecules. In this study, a macroporous 3D scaffold originating from decellularized bovine liver ECM (dL-ECM), with defined compositional, physical, chemical, rheological, thermal, mechanical, and in vitro biological properties was developed. First, protocols were determined that effectively remove cells and DNA while ECM retains biological macromolecules collagen, elastin, sGAGs in tissue. Rheological analysis revealed the elastic properties of pepsin-digested dL-ECM. Then, dL-ECM hydrogel was neutralized, molded, formed into macroporous (~100-200 µm) scaffolds in aqueous medium at 37 °C, and lyophilized. The scaffolds had water retention ability, and were mechanically stable for at least 14 days in the culture medium. The findings also showed that increasing the dL-ECM concentration from 10 mg/mL to 20 mg/mL resulted in a significant increase in the mechanical strength of the scaffolds. The hemolysis test revealed high in vitro hemocompatibility of the dL-ECM scaffolds. Studies investigating the viability and proliferation status of human adipose stem cells seeded over a 2-week culture period have demonstrated the suitability of dL-ECM scaffolds as a cell substrate. Prospective studies may reveal the extent to which 3D dL-ECM sponges have the potential to create a biomimetic environment for cells.

Preliminary assessment of an injectable extracellular matrix from decellularized bovine myocardial tissue.

The goal of this study was to develop an injectable form of decellularized bovine myocardial tissue matrix which could retain high levels of functional ECM molecules, and could gel at physiological temperature. Dissected ventricular tissue was processed by a detergent-based protocol, lyophilized, enzymatically-digested, and neutralized to form the injectable myocardial matrix (IMM). Histochemical analysis, DNA quantification, and agarose gel electrophoresis demonstrated the efficiency of the applied protocol. Chemical, thermal, morphological, and rheological characterization; protein and sulfated glycosaminoglycan (sGAG) content analysis were performed, in vitro biological properties were evaluated. An in vivo histocompatibility and biodegradability study was performed. Histochemistry revealed complete removal of myocardial cells. DNA content analysis revealed a significant decrease (87%) in the nuclear material, while protein and sGAG contents were highly preserved following decellularization. Soluble IMM was capable of turning into gel form at ∼37 °C, indicating selfassembling property. In vitro findings showed the biomaterial was noncytotoxic, nonhemolytic, and supported the attachment and proliferation of mesenchymal stem cells. In vivo study demonstrated IMM was well-tolerated by rats receiving subcutaneous injection. This work demonstrates that the IMM from decellularized bovine myocardial tissue has the potential for use as a feasible regenerative biomaterial in prospective tissue engineering and regenerative medicine studies.

Magneto-sensitive decellularized bone matrix with or without low frequency-pulsed electromagnetic field exposure for the healing of a critical-size bone defect.

Bioactive ECM-based materials mimic the complex composition and structure of natural tissues. Decellularized cancellous bone matrix (DBM) has potential for guiding new bone formation and accelerating the regeneration process. On the other hand, low frequency-pulsed electromagnetic field (LF-PEMF) has been shown to enhance the regeneration capacity of bone defects. The present study sought to explore the feasibility of using DBM and DBM/MNP, and LF-PEMF for treating critical-size bone defects. Firstly, decellularization protocol was optimized to obtain a bioactive DBM, then MNPs were incorporated. Later, the physical, chemical and biological properties of DBM and DBM/MNP were assessed in vitro. MNPs homogeneously distributed into the DBM were not found to be toxic to human osteoblast cultures. Finally, an in vivo study was carried out with DBM and DBM/MNP composites in a bilateral critical-size rat cranial defect model (n = 48) with or without LF-PEMF exposure for 45 and 90 days. The histomorphometric and radiographic evaluations revealed that, while the collagen (positive control) and Sham (negative control) groups showed high incidence of fibrous connective tissue together with low level of osteogenic activity, both the DBM and DBM/MNP-grafted groups significantly promoted new bone tissue formation and angiogenesis, by the appropriate use of LF-PEMF for 90 days.

Biomimetic 3D-Bone Tissue Model.

Various approaches have been evaluated for developing three-dimensional (3D) scaffolds for modeling or engineering of the bone tissue. However, most of such attempts have come up short in mimicking the natural bone tissue extracellular matrix (ECM) microenvironment, especially its natural bioactive content. Here we describe the methodology for the preparation of a natural ECM-based multichannel construct as a biomimetic 3D bone tissue model. We elucidate the construction of the composite scaffold incorporating decellularized small intestinal submucosa ECM, synthetic hydroxyapatite and poly(ε-caprolactone), and the mechanical stimulation of the cell-seeded construct under bioreactor culture.

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.

Magnetic and electrically conductive silica-coated iron oxide/polyaniline nanocomposites for biomedical applications.

This work describes the development of novel dual-stimuli-responsive nanocomposites based on silica-coated iron oxide/polyaniline (Si-MNPs/PANI) for biomedical applications. Si-MNPs/PANI nanocomposites were developed via chemical oxidative polymerization of aniline in the presence of Si-MNPs (25 and 50 wt%). Si-MNPs/PANI were obtained both in nanotubular (SPNTs) and granular (SGTs) forms by altering the synthesis parameters such as acid concentration and mixing process. The effects of nanocomposite morphology were evaluated by investigating their chemical, physical and biological properties. Material characterization was comparatively carried out via SEM, TEM, FTIR, XRD, TGA, room temperature VSM, and electrical resistivity measurements. Biological properties were evaluated by indirect in vitro cytotoxicity and in vitro hemocompatibility analyses according to ISO standards. Results indicated that Si-MNPs/PANI nanocomposites exhibited both magnetically and electrically-responsive properties. Magnetization values of Si-MNPs/PANI nanocomposites increased with increasing Si-MNPs content. However, electrical conductivity was inversely proportional to Si-MNPs content. In addition, SGTs represented remarkably higher electrical conductivity (1.1 S/cm) than SPNTs (4.8 × 10-2 S/cm), but lower saturation magnetization (21 emu/g) compared to SPNTs (27 emu/g). Furthermore, in vitro cytocompatibility and hemocompatibility of the SGTs and SPNTs varied in a dose-dependent manner, suggesting their use in certain doses for biomedical applications. In conclusion, the developed Si-MNPs/PANI, with magnetic sensitivity and electrical conductivity have potential as nanocomposites for utilization in biomedical applications, e.g. biosensing, controlled-drug delivery, bioelectronic systems, tissue engineering and regenerative medicine as active compound. Besides, the selection of the appropriate synthesis protocol allows Si-MNPs/PANI nanocomposites to exhibit superior properties according to the targeted application area.

Therapeutic Applications of Stem Cells and Extracellular Vesicles in Emergency Care: Futuristic Perspectives.

Regenerative medicine (RM) is an interdisciplinary field that aims to repair, replace or regenerate damaged or missing tissue or organs to function as close as possible to its physiological architecture and functions. Stem cells, which are undifferentiated cells retaining self-renewal potential, excessive proliferation and differentiation capacity into offspring or daughter cells that form different lineage cells of an organism, are considered as an important part of the RM approaches. They have been widely investigated in preclinical and clinical studies for therapeutic purposes. Extracellular vesicles (EVs) are the vital mediators that regulate the therapeutic effects of stem cells. Besides, they carry various types of cargo between cells which make them a significant contributor of intercellular communication. Given their role in physiological and pathological conditions in living cells, EVs are considered as a new therapeutic alternative solution for a variety of diseases in which there is a high unmet clinical need. This review aims to summarize and identify therapeutic potential of stem cells and EVs in diseases requiring acute emergency care such as trauma, heart diseases, stroke, acute respiratory distress syndrome and burn injury. Diseases that affect militaries or societies including acute radiation syndrome, sepsis and viral pandemics such as novel coronavirus disease 2019 are also discussed. Additionally, featuring and problematic issues that hamper clinical translation of stem cells and EVs are debated in a comparative manner with a futuristic perspective. Graphical Abstract.

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.

Mesenchymal Stem Cells for Coronavirus (COVID-19)-Induced Pneumonia: Revisiting the Paracrine Hypothesis with New Hopes?

Mesenchymal stem cells (MSCs) bear a promising potential for regenerative medicine therapies and they repair damaged tissue through secretion of immune modulatory and anti-inflammatory molecules acting in a paracrine fashion. Coronavirus disease 2019 (COVID-19) has spread all over the world with high morbidity and mortality rates and there is no specific treatment for this infection. A recent study published in the journal reports that MSC infusion is safe and effective in patients suffering from COVID-19 induced pneumonia. In the light of this study and previous reports, we make additional comments about possible therapeutic effects of MSCs in COVID-19 infection.

Bioethical issues in genome editing by CRISPR-Cas9 technology.

Genome editing technologies have led to fundamental changes in genetic science. Among them, CRISPR-Cas9 technology particularly stands out due to its advantages such as easy handling, high accuracy, and low cost. It has made a quick introduction in fields related to humans, animals, and the environment, while raising difficult questions, applications, concerns, and bioethical issues to be discussed. Most concerns stem from the use of CRISPR-Cas9 to genetically alter human germline cells and embryos (called germline genome editing). Germline genome editing leads to serial bioethical issues, such as the occurrence of undesirable changes in the genome, from whom and how informed consent is obtained, and the breeding of the human species (eugenics). However, the bioethical issues that CRISPR-Cas9 technology could cause in the environment, agriculture and livestock should also not be forgotten. In order for CRISPR-Cas9 to be used safely in all areas and to solve potential issues, worldwide legislation should be prepared, taking into account the opinions of both life and social scientists, policy makers, and all other stakeholders of the sectors, and CRISPR-Cas9 applications should be implemented according to such legislations. However, these controls should not restrict scientific freedom. Here, various applications of CRISPR-Cas9 technology, especially in medicine and agriculture, are described and ethical issues related to genome editing using CRISPR-Cas9 technology are discussed. The social and bioethical concerns in relation to human beings, other organisms, and the environment are addressed.