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.

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).

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.

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.

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.

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.

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.