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.

Biomimetic mineralization of platelet lysate/oxidized dextran cryogel as a macroporous 3D composite scaffold for bone repair.

Scaffold development approaches using autologous sources for tissue repair are of great importance in obtaining bio-active/-compatible constructs. Platelet-rich plasma (PRP) containing various growth factors and platelet lysate (PL) derived from PRP are autologous products that have the potential to accelerate the tissue repair response by inducing a transient inflammatory event. Considering the regenerative capacity of PRP and PL, PRP/PL-based scaffolds are thought to hold great promise for tissue engineering as a natural source of autologous growth factors and a provider of mechanical support for cells. Here, a bio-mineralized PRP-based scaffold was developed using oxidized dextran (OD) and evaluated for future application in bone tissue engineering. Prepared PL/OD scaffolds were incubated in simulated body fluid (SBF) for 7, 14 and 21 d periods. Mineralized PL/OD scaffolds were characterized using Fourier transform infrared spectroscopy, x-ray diffraction spectroscopy, scanning electron microscopy (SEM), thermogravimetric analysis, porosity and compression tests. SEM and energy-dispersive x-ray spectroscopy analyses revealed mineral accumulation on the PL/OD scaffold as a result of SBF incubation.In vitrocytotoxicity andin vitrohemolysis tests revealed that the scaffolds were non-toxic and hemocompatible. Additionally, human osteoblasts (hOBs) exhibited good attachment and spreading behavior on the scaffolds and maintained their viability throughout the culture period. The alkaline phosphatase activity assay and calcium release results revealed that PL/OD scaffolds preserved the osteogenic properties of hOBs. Overall, findings suggest that mineralized PL/OD scaffold may be a promising scaffold for bone tissue engineering.

Risk assessment approach for analyzing risk factors to overcome pandemic using interval-valued q-rung orthopair fuzzy decision making method.

The process of developing and implementing sustainable strategies to prevent spread of COVID-19 for society typically requires integrating all social, technological, economic, governmental aspects in a systematic way. Since the clear understanding of risk factors contribute to the success of the strategies applied against COVID-19, a risk assessment procedure is applied in this study to properly evaluate risk factors cause to spread of pandemic as a multi-complex decision problem. Therefore, due to the evaluation of risk factors, which often involves uncertain information, the model is constructed based on interval-valued q-rung orthopair fuzzy-COmplex PRoportional ASsessment (IVq-ROF-COPRAS) method. While the developed framework is efficient to enhance the quality of decisions by implementing more realistic, precise, and effective application procedure under uncertain environment, it has capability to help governments for developing comprehensive strategies and responses. According to the results of the proposed risk analysis model, the top three risk factors are "The Approach that Prioritizes the Economy in Policies", "Insufficient Process Control in Normalization" and "Lack of Epidemic Management Culture in Individuals and Businesses". Lastly, to show applicability and efficiency of the model sensitivity and comparative analysis were conducted at the end of the study.

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.

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.

Determining the location of isolation hospitals for COVID-19 via Delphi-based MCDM method.

The existing contagious epidemic disease, [SARS]-CoV-2, has been one of the biggest public health problems that humankind combatting against since December 2019. Answering the increase in the number of infected patients during the pandemic is one of the biggest challenges for healthcare systems, where resources have already been employed by a significant number of patients. While assigning most of the resources to infected people is an effective way in a short-term planning, its bitter effects on regular healthcare cannot be undervalued. Moreover, within this plan the risk of spreading the disease to other patients and healthcare providers is another risk that should not be underestimated. Therefore, in this study, we proposed the Delphi-based multicriteria decision-making (MCDM) framework for selecting the most appropriate location for an isolation hospital serving only epidemic-based patients with mild to moderate symptoms. The integrated framework consists of Delphi, Best-Worst Method, and interval type-2 fuzzy Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS) methodologies. Nine most effecting criteria are considered in the evaluation of five alternative locations in a real case study conducted at the European side of Istanbul. Ataturk Airport is determined as the best location to set up an isolation hospital based on determined nine evaluation criteria. The effectiveness and robustness of the framework are analyzed through comparative and sensitivity analyses.

Macroporous elastic cryogels based on platelet lysate and oxidized dextran as tissue engineering scaffold: In vitro and in vivo evaluations.

In this study, three-dimensional macroporous cryogels were developed from platelet lysate (PL) and different concentrations of oxidized dextran (OD; 0.5, 1, 2, 4%). Subsequently, PL/OD scaffolds were characterized for potential use in tissue engineering applications. The pore size and morphology of the resulting cryogels were visualized using scanning electron microscopy (SEM). The pore size distributions were determined using mercury intrusion porosimetry (MIP). In vitro hydrolytic degradation, water uptake, mechanical properties and hemocompatibility were investigated. Extraction test was carried out to evaluate potential in vitro toxic effects of the PL/OD. The in vitro adhesion, proliferation, chondrogenic differentiation, and extracellular matrix production of human adipose stem cells (hASCs) on PL/OD cryogels were evaluated. In vivo biodegradation of the cryogels was investigated at the subcutaneous dorsal site of rats. SEM and MIP results indicated that PL/OD had a macroporous pore structure with pore sizes ranging between 10 and 200 µm. The cryogels completely degraded within 90-240 days post-implantation, depending on OD concentration. Histochemical analysis revealed high levels of cell and tissue infiltration into the pores of PL/OD. In vitro cytotoxicity findings indicated that the extracts of PL/OD0.5, PL/OD1 and PL/OD2 showed no cytotoxic effect, whereas that of PL/OD4 exhibited a moderate cytotoxic effect on cell cultures. hASCs seeded on PL/OD2 retained their viability and showed extensive spreading and filopodia formation after 7 days. PL/OD2 also supported the chondrogenesis of hASCs in the presence of chondro-inductive factors. Given all the results, PL/OD could have potential as a scaffold for tissue engineering applications.

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.

A CD34+ Cell Enrichment Protocol of Hematopoietic Stem Cells in a Well-Established Quality Management System.

Allogeneic stem cell transplantation applications have improved tremendously over the past quarter of a century. The use of new immunosuppressive protocols and elimination of T cells by CD34+ cell enrichment or T cell depletion on apheresis products increases the chance of using partially matched or haploidentical grafts. This is without increasing the risk of graft-versus-host disease, which is observed as a major complication of hematopoietic stem cell transplantation. The aim of this protocol is to evaluate the results obtained from 6 different process cycles performed on 6 different days. We used the CliniMACS Plus system located in our Cell and Tissue Manufacturing Center Quality Control Unit which is already calibrated as a class D room and includes a class A microbiological safety cabinet inside. The average purity of the end products was 95.66%, excluding only one end product which was 70%; this was higher than the values in current studies in the field. Superior to the reported studies, the CD3 quantity in each end product was below the dedicated thresholds. BactecTM FX40 blood culture system test results were detected as negative for each end product. Endotoxin testing suggested the absence of endotoxin within the products. The consistent outcomes obtained from these 6 different process cycles confirmed that the CliniMACS® Plus process cycles performed in accordance with our well-defined quality management system procedure is sufficient for the routine application of high-quality and safe CD34+ enrichment processes within our clean room area.

Encapsulation of bone marrow-MSCs in PRP-derived fibrin microbeads and preliminary evaluation in a volumetric muscle loss injury rat model: modular muscle tissue engineering.

Repair of volumetric muscle loss (VML) injuries is a complicated endeavour which necessitates the collaborative use of different regenerative approaches and technologies. Herein is proposed the development of fibrin-based microbeads (FMs) alone or as a bone marrow mesenchymal stem cell (MSC) encapsulation matrix for modular muscle engineering. FMs were generated through the ionotropic gelation of alginate and fibrinogen obtained from the platelet-rich plasma of whole blood, and then removing the alginate by citrate treatment. FMs were first characterized by FT-IR, SEM and water uptake tests. Then, the stability of FMs and the mitochondrial dehydrogenase activity of the MSCs encapsulated in FMs were evaluated under in vitro culture conditions. Eventually, the regenerative capacity of the cell-devoid and MSCs-encapsulated FMs was evaluated in a rat VML injury model involving 8 × 4×4 mm3-size bilateral defects in the biceps femoris muscles. The histochemical, immunohistochemical and semi-quantitative histomorphological scoring results retrieved at 30, 60 and 180 days demonstrated that the cell-devoid FMs supported muscle regeneration to a great extent. Moreover, MSCs-encapsulated FMs were more effective in shortening the regeneration period of the injured tissue of the rat VML, resulting in good myofibre orientation, while the Sham group resulted in incomplete repair with fibrotic scar tissue formations.

Comparative evaluation of nano and bulk tin dioxide cytotoxicity on dermal fibroblasts by real-time impedance-based and conventional methods.

In this study, the possible cellular effects of tin dioxide (SnO2) nanoparticles, together with its bulk form, on mouse dermal fibroblasts (DFs) were revealed using in vitro assays. Particle characterizations were carried out with AFM, Braun-Emmet-Teller, and DLS analyses. The cells were treated with nano and bulk SnO2 at concentrations of 0.1, 1, 10, 50, and 100 µg/mL for 6, 24, and 48 h. At the end of the exposure periods, the morphology, viability, particle uptake, and membrane leakage statuses of the cells were evaluated. Furthermore, real-time monitoring of cell responses was performed by using an impedance-based label-free system. Findings showed that at concentrations of 0.1-10 µg/mL, cells had similar doubling time to that of control cells (20.4 ± 2.6 h), while the doubling time of cells exposed to 100 µg/mL of nano and bulk SnO2 increased slightly (P ˃ 0.05) to 25.1 ± 3.9 h and 26.2 ± 5.9 h, respectively. The results indicated that DFs exhibited a similar toxicity response to nano and bulk SnO2; thus, 50 and 100 µg/mL of nano and bulk SnO2 had mild toxic effects on DFs. In conclusion, this study provides information and insight necessary for the safe use of SnO2 in medical and consumer products.

Osteogenic differentiation of mesenchymal stem cells using hybrid nanofibers with different configurations and dimensionality.

Novel, hybrid fibrinogen/polylactic acid (FBG/PLA) nanofibers with different configuration (random vs aligned) and dimensionality (2-D vs 3-D environment) were used to control the overall behavior and the osteogenic differentiation of human adipose-derived mesenchymal stem cells (ADMSCs). Aligned nanofibers in both the 2-D and 3-D configurations are proved to be favored for osteodifferentiation. Morphologically, we found that on randomly configured nanofibers, the cells developed a stellate-like morphology with multiple projections; however, time-lapse analysis showed significantly diminished cell movements. Conversely, an elongated cell shape with advanced cell spreading and extended actin cytoskeleton accompanied with significantly increased cell mobility were observed when cells attached on aligned nanofibers. Moreover, a clear tendency for higher alkaline phosphatase activity was also found on aligned fibers when ADMSCs were switched to osteogenic induction medium. The strongest accumulation of Alizarin red (AR) and von Kossa stain at 21 days of culture in osteogenic medium were found on 3-D aligned constructs while the rest showed lower and rather undistinguishable activity. Quantitative reverse transcription-polymerase chain reaction analysis for Osteopontin (OSP) and RUNX 2 generally confirmed this trend showing favorable expression of osteogenic genes activity in 3-D environment particularly in aligned configuration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2065-2074, 2017.

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.

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.

Real-time monitoring of mesenchymal stem cell responses to biomaterial surfaces and to a model drug by using quartz crystal microbalance.

In this study, the mesenchymal stem cell (MSC) responses to biomaterial surfaces and to an anti-microtubule drug (vinblastine) were detected by using the quartz crystal microbalance (QCM). Gold electrodes with different coatings were subjected to MSCs under flow conditions; thus, crystal frequency decreased due to the adhesion of MSCs on the crystal. For evaluation of cell-drug interactions, vinblastine was introduced to MSCs attached onto the surfaces. The changes in frequency indicated the binding of drug to cell microtubules. The present study demonstrates the suitability of QCM as an invaluable tool for the real-time monitoring of cell-surface and cell-drug interactions.

In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells.

The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7-8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun-Emmet-Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 µg/mL for 6, 24 and 48h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50-100 µg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.

Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds.

Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.