Amorphous-Crystalline Calcium Phosphate Coating Promotes In Vitro Growth of Tumor-Derived Jurkat T Cells Activated by Anti-CD2/CD3/CD28 Antibodies.

A modern trend in traumatology, orthopedics, and implantology is the development of materials and coatings with an amorphous-crystalline structure that exhibits excellent biocopatibility. The structure and physico-chemical and biological properties of calcium phosphate (CaP) coatings deposited on Ti plates using the micro-arc oxidation (MAO) method under different voltages (200, 250, and 300 V) were studied. Amorphous, nanocrystalline, and microcrystalline statesof CaHPO4 and ß-Ca2P2O7 were observed in the coatings using TEM and XRD. The increase in MAO voltage resulted in augmentation of the surface roughness Ra from 2.5 to 6.5 µm, mass from 10 to 25 mg, thickness from 50 to 105 µm, and Ca/P ratio from 0.3 to 0.6. The electrical potential (EP) of the CaP coatings changed from -456 to -535 mV, while the zeta potential (ZP) decreased from -53 to -40 mV following an increase in the values of the MAO voltage. Numerous correlations of physical and chemical indices of CaP coatings were estimated. A decrease in the ZP magnitudes of CaP coatings deposited at 200-250 V was strongly associated with elevated hTERT expression in tumor-derived Jurkat T cells preliminarily activated with anti-CD2/CD3/CD28 antibodies and then contacted in vitro with CaP-coated samples for 14 days. In turn, in vitro survival of CD4+ subsets was enhanced, with proinflammatory cytokine secretion of activated Jurkat T cells. Thus, the applied MAO voltage allowed the regulation of the physicochemical properties of amorphous-crystalline CaP-coatings on Ti substrates to a certain extent. This method may be used as a technological mechanism to trigger the behavior of cells through contact with micro-arc CaP coatings. The possible role of negative ZP and Ca2+ as effectors of the biological effects of amorphous-crystalline CaP coatings is discussed. Micro-arc CaP coatings should be carefully tested to determine their suitability for use in patients with chronic lymphoid malignancies.

Zn- or Cu-containing CaP-Based Coatings Formed by Micro-Arc Oxidation on Titanium and Ti-40Nb Alloy: Part II-Wettability and Biological Performance.

This work describes the wettability and biological performance of Zn- and Cu-containing CaP-based coatings prepared by micro-arc oxidation on pure titanium (Ti) and novel Ti-40Nb alloy. Good hydrophilic properties of all the coatings were demonstrated by the low contact angles with liquids, not exceeding 45°. An increase in the applied voltage led to an increase of the coating roughness and porosity, thereby reducing the contact angles to 6° with water and to 17° with glycerol. The free surface energy of 75 ± 3 mJ/m2 for all the coatings were determined. Polar component was calculated as the main component of surface energy, caused by the presence of strong polar PO43- and OH- bonds. In vitro studies showed that low Cu and Zn amounts (~0.4 at.%) in the coatings promoted high motility of human adipose-derived multipotent mesenchymal stromal cells (hAMMSC) on the implant/cell interface and subsequent cell ability to differentiate into osteoblasts. In vivo study demonstrated 100% ectopic bone formation only on the surface of the CaP coating on Ti. The Zn- and Cu-containing CaP coatings on both substrates and the CaP coating on the Ti-40Nb alloy slightly decreased the incidence of ectopic osteogenesis down to 67%. The MAO coatings showed antibacterial efficacy against Staphylococcus aureus and can be arranged as follows: Zn-CaP/Ti > Cu-CaP/TiNb, Zn-CaP/TiNb > Cu-CaP/Ti.

Costimulatory Effect of Rough Calcium Phosphate Coating and Blood Mononuclear Cells on Adipose-Derived Mesenchymal Stem Cells In Vitro as a Model of In Vivo Tissue Repair.

Calcium phosphate (CaP) materials do not always induce ectopic vascularization and bone formation; the reasons remain unclear, and there are active discussions of potential roles for post-implantation hematoma, circulating immune and stem cells, and pericytes, but studies on adipose-derived stem cells (AMSCs) in this context are lacking. The rough (average surface roughness Ra = 2-5 µm) scaffold-like CaP coating deposited on pure titanium plates by the microarc oxidation method was used to investigate its subcutaneous vascularization in CBA/CaLac mice and in vitro effect on cellular and molecular crosstalk between human blood mononuclear cells (hBMNCs) and AMSCs (hAMSCs). Postoperative hematoma development on the CaP surface lasting 1-3 weeks may play a key role in the microvessel elongation and invasion into the CaP relief at the end of the 3rd week of injury and BMNC migration required for enhanced wound healing in mice. Satisfactory osteogenic and chondrogenic differentiation but poor adipogenic differentiation of hAMSCs on the rough CaP surface were detected in vitro by differential cell staining. The fractions of CD73+ (62%), CD90+ (0.24%), and CD105+ (0.41%) BMNCs may be a source of autologous circulating stem/progenitor cells for the subcutis reparation, but allogenic hBMNC participation is mainly related to the effects of CD4+ T cells co-stimulated with CaP coating on the in vitro recruitment of hAMSCs, their secretion of angiogenic and osteomodulatory molecules, and the increase in osteogenic features within the period of in vivo vascularization. Cellular and molecular crosstalk between BMNCs and AMSCs is a model of effective subcutis repair. Rough CaP surface enhanced angio- and osteogenic signaling between cells. We believe that preconditioning and/or co-transplantation of hAMSCs with hBMNCs may broaden their potential in applications related to post-implantation tissue repair and bone bioengineering caused by microarc CaP coating.

Calcium Phosphate Coating Prepared by Microarc Oxidation Affects hTERT Expression, Molecular Presentation, and Cytokine Secretion in Tumor-Derived Jurkat T Cells.

Calcium phosphate (CaP) materials are among the best bone graft substitutes, but their use in the repair of damaged bone in tumor patients is still unclear. The human Jurkat T lymphoblast leukemia-derived cell line (Jurkat T cells) was exposed in vitro to a titanium (Ti) substrate (10 × 10 × 1 mm3) with a bilateral rough (average roughness index (Ra) = 2-5 µm) CaP coating applied via the microarc oxidation (MAO) technique, and the morphofunctional response of the cells was studied. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectroscope (EDX) analyses showed voltage-dependent (150-300 V) growth of structural (Ra index, mass, and thickness) and morphological surface and volume elements, a low Ca/PaT ratio (0.3-0.6), and the appearance of crystalline phases of CaHPO4 (monetite) and ß-Ca2P2O7 (calcium pyrophosphate). Cell and molecular reactions in 2-day and 14-day cultures differed strongly and correlated with the Ra values. There was significant upregulation of hTERT expression (1.7-fold), IL-17 secretion, the presentation of the activation antigens CD25 (by 2.7%) and CD95 (by 5.15%) on CD4+ cells, and 1.5-2-fold increased cell apoptosis and necrosis after two days of culture. Hyperactivation-dependent death of CD4+ cells triggered by the surface roughness of the CaP coating was proposed. Conversely, a 3.2-fold downregulation in hTERT expression increased the percentages of CD4+ cells and their CD95+ subset (by 15.5% and 22.9%, respectively) and inhibited the secretion of 17 of 27 test cytokines/chemokines without a reduction in Jurkat T cell survival after 14 days of coculture. Thereafter, cell hypoergy and the selection of an hTERT-independent viable CD4+ subset of tumor cells were proposed. The possible role of negative zeta potentials and Ca2+ as effectors of CaP roughness was discussed. The continuous (2-14 days) 1.5-6-fold reductions in the secretion of vascular endothelial growth factor (VEGF) by tumor cells correlated with the Ra values of microarc CaP-coated Ti substrates seems to limit surgical stress-induced metastasis of lymphoid malignancies.

Cellular and Molecular Basis of Osteoblastic and Vascular Niches in the Processes of Hematopoiesis and Bone Remodeling (A Short Review of Modern Views).

In evolutionary processes, human bone marrow has formed as an organ depot of various types of cells that arise from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Vital HSC activity is controlled through molecular interactions with the niche microenvironment. The review describes current views on the formation of key molecular and cellular components of the HSC niche, which ensure maintenance of home ostasis in stem cell niches, obtained from studies of their role in regulating the proliferation and differentiation of HSCs, including the physiological, reparative and pathological remodeling of bone tissue. Due to rapid developments in biotechnology, tissue bioengineering, and regenerative medicine, information can be useful for developing biomimetic and bioinspired materials and implants that provide an effective bone/bone marrow recovery process after injuries and, to a greater extent, diseases of various etiologies.

Comparative Analysis of Biological Properties of Large-Scale Expanded Adult Neural Crest-Derived Stem Cells Isolated from Human Hair Follicle and Skin Dermis.

INTRODUCTION: The adult neural crest-derived stem cells (NCSCs) have significant perspectives for use in regenerative medicine. The most attractive sources for adult NCSC isolation are the hair follicles (HF) and skin dermis (SD) because of easy access and minimally invasive biopsy. The aim of this study was to compare the biological properties of HF- and SD-derived NCSCs after their large-scale expansion. METHODS: The conventional explant method was used to obtain HF NCSCs. For the isolation of SD NCSCs, a new combined technique consisting of preplating and subsequent culturing in 3D blood plasma-derived fibrin hydrogel was applied. The studied cells were characterized by flow cytometry, ICC, qPCR, Bio-Plex multiplex assay, and directed multilineage differentiation assays. RESULTS: We have obtained both adult SD and HF NCSCs from each skin sample (n = 5). Adult SD and HF NCSCs were positive for key neural crest markers: SOX10, P75 (CD271), NESTIN, SOX2, and CD349. SD NCSCs showed a higher growth rate during the large-scale expansion compared to HF NCSCs (p < 0.01). Final population of SD NCSCs also contained more clonogenic cells (p < 0.01) and SOX10+, CD271+, CD105+, CD140a+, CD146+, CD349+ cells (p < 0.01). Both HF and SD NCSCs had similar gene expression profiling and produced growth factors, but some quantitative differences were detected. Adult HF and SD NCSCs were able to undergo directed differentiation into neurons, Schwann cells, adipocytes, and osteoblasts. CONCLUSION: The HF and SD are suitable sources for large-scale manufacturing of adult NCSCs with similar biological properties. We demonstrated that the NCSC population from SD was homogenous and displayed significantly higher growth rate than HF NCSCs. Moreover, SD NCSC isolation is cheaper, easier, and minimally time-consuming method.

Concept of Hematopoietic and Stromal Niches for Cell-Based Diagnostics and Regenerative Medicine (a Review).

BACKGROUND: R. Schofield (1978) proposed a hypothesis of hematopoietic stem cells (HSCs) niche (specialized cell microenvironment). An existence of osteoblastic and vascular niches for HSCs has been postulated since 2003. At the same time, the discussion about the existence and functioning of niche for multipotent mesenchymal stromal cells (MMSCs) is just beginning to develop. The design of artificial materials capable of biomimetical reproductionof the cellular and tissue microenvironment based on ideas and main elements borrowed from wildlife is an experimental approach in search of the stem cell niches. RESULTS: Recent attempts to model the microterritories (niches) for HSCs have been undertaken and the behavior of cells in such structures has been investigated. However, the main quantitative factors involved in the original design of stem cell microterritories remain unknown. At the modern stage, the topography, hierarchy, and the size of the niches have to be determined, because the definition of the niches as morphological (structural and functional) units (microterritories), which provides the conditions for vital activity of stem cells, implies finite values of its parameters. The aim of this review was the critical review of key milestones of the niche concept for HSCs and MMSCs as we understood it. CONCLUSION: We speculated our definition of the stem cell niche, proposed and described certain stages (postulation; morphofunctional; topographical; quantitative; bioengineering) of the niche theory development. Prospective directions of the niche designing for cell-based diagnostics and regenerative medicine were noted.

Nanoscale Electrical Potential and Roughness of a Calcium Phosphate Surface Promotes the Osteogenic Phenotype of Stromal Cells.

Mesenchymal stem cells (MSCs) and osteoblasts respond to the surface electrical charge and topography of biomaterials. This work focuses on the connection between the roughness of calcium phosphate (CP) surfaces and their electrical potential (EP) at the micro- and nanoscales and the possible role of these parameters in jointly affecting human MSC osteogenic differentiation and maturation in vitro. A microarc CP coating was deposited on titanium substrates and characterized at the micro- and nanoscale. Human adult adipose-derived MSCs (hAMSCs) or prenatal stromal cells from the human lung (HLPSCs) were cultured on the CP surface to estimate MSC behavior. The roughness, nonuniform charge polarity, and EP of CP microarc coatings on a titanium substrate were shown to affect the osteogenic differentiation and maturation of hAMSCs and HLPSCs in vitro. The surface EP induced by the negative charge increased with increasing surface roughness at the microscale. The surface relief at the nanoscale had an impact on the sign of the EP. Negative electrical charges were mainly located within the micro- and nanosockets of the coating surface, whereas positive charges were detected predominantly at the nanorelief peaks. HLPSCs located in the sockets of the CP surface expressed the osteoblastic markers osteocalcin and alkaline phosphatase. The CP multilevel topography induced charge polarity and an EP and overall promoted the osteoblast phenotype of HLPSCs. The negative sign of the EP and its magnitude at the micro- and nanosockets might be sensitive factors that can trigger osteoblastic differentiation and maturation of human stromal cells.

Water-Based Suspensions of Iron Oxide Nanoparticles with Electrostatic or Steric Stabilization by Chitosan: Fabrication, Characterization and Biocompatibility.

Present day biomedical applications, including magnetic biosensing, demand better understanding of the interactions between living systems and magnetic nanoparticles (MNPs). In this work spherical MNPs of maghemite were obtained by a highly productive laser target evaporation technique. XRD analysis confirmed the inverse spinel structure of the MNPs (space group Fd-3m). The ensemble obeyed a lognormal size distribution with the median value 26.8 nm and dispersion 0.362. Stabilized water-based suspensions were fabricated using electrostatic or steric stabilization by the natural polymer chitosan. The encapsulation of the MNPs by chitosan makes them resistant to the unfavorable factors for colloidal stability typically present in physiological conditions such as pH and high ionic force. Controlled amounts of suspensions were used for in vitro experiments with human blood mononuclear leukocytes (HBMLs) in order to study their morphofunctional response. For sake of comparison the results obtained in the present study were analyzed together with our previous results of the study of similar suspensions with human mesenchymal stem cells. Suspensions with and without chitosan enhanced the secretion of cytokines by a 24-h culture of HBMLs compared to a control without MNPs. At a dose of 2.3, the MTD of chitosan promotes the stimulating effect of MNPs on cells. In the dose range of MNPs 10-1000 MTD, chitosan "inhibits" cellular secretory activity compared to MNPs without chitosan. Both suspensions did not caused cell death by necrosis, hence, the secretion of cytokines is due to the enhancement of the functional activity of HBMLs. Increased accumulation of MNP with chitosan in the cell fraction at 100 MTD for 24 h exposure, may be due to fixation of chitosan on the outer membrane of HBMLs. The discussed results can be used for an addressed design of cell delivery/removal incorporating multiple activities because of cell capability to avoid phagocytosis by immune cells. They are also promising for the field of biosensor development for the detection of magnetic labels.