Endothelial Cell Behavior and Nitric Oxide Production on a-C:H:SiOx-Coated Ti-6Al-4V Substrate.

This paper focuses on the surface modification of the Ti-6Al-4V alloy substrate via a-C:H:SiOx coating deposition. Research results concern the a-C:H:SiOx coating structure, investigated using transmission electron microscopy and in vitro endothelization to study the coating. Based on the analysis of the atomic radial distribution function, a model is proposed for the atomic short-range order structure of the a-C:H:SiOx coating, and chemical bonds (C-O, C-C, Si-C, Si-O, and Si-Si) are identified. It is shown that the a-C:H:SiOx coating does not possess prolonged cytotoxicity in relation to EA.hy926 endothelial cells. In vitro investigations showed that the adhesion, cell number, and nitric oxide production by EA.hy926 endothelial cells on the a-C:H:SiOx-coated Ti-6Al-4V substrate are significantly lower than those on the uncoated surface. The findings suggest that the a-C:H:SiOx coating can reduce the risk of endothelial cell hyperproliferation on implants and medical devices, including mechanical prosthetic heart valves, endovascular stents, and mechanical circulatory support devices.

Engineered Fibrous NiTi Scaffolds with Cultured Hepatocytes for Liver Regeneration in Rats.

At present, the use of alternative systems to replenish the lost functions of hepatic metabolism and partial replacement of liver organ failure is relevant, due to an increase in the incidence of various liver disorders, insufficiency, and cost of organs for transplantation, as well as the high cost of using the artificial liver systems. The development of low-cost intracorporeal systems for maintaining hepatic metabolism using tissue engineering, as a bridge before liver transplantation or completely replacing liver function, deserves special attention. In vivo applications of intracorporeal fibrous nickel-titanium scaffolds (FNTSs) with cultured hepatocytes are described. Hepatocytes cultured in FNTSs are superior to their injections in terms of liver function, survival time, and recovery in a CCl4-induced cirrhosis rats' model. 232 animals were divided into 5 groups: control, CCl4-induced cirrhosis, CCl4-induced cirrhosis followed by implantation of cell-free FNTSs (sham surgery), CCl4-induced cirrhosis followed by infusion of hepatocytes (2 mL, 107 cells/mL), and CCl4-induced cirrhosis followed by FNTS implantation with hepatocytes. Restoration of hepatocyte function in the FNTS implantation with the hepatocytes group was accompanied by a significant decrease in the level of aspartate aminotransferase (AsAT) in blood serum compared to the cirrhosis group. A significant decrease in the level of AsAT was noted after 15 days in the infused hepatocytes group. However, on the 30th day, the AsAT level increased and was close to the cirrhosis group due to the short-term effect after the introduction of hepatocytes without a scaffold. The changes in alanine aminotransferase (AlAT), alkaline phosphatase (AlP), total and direct bilirubin, serum protein, triacylglycerol, lactate, albumin, and lipoproteins were similar to those in AsAT. The survival time of animals was significantly longer in the FNTS implantation with hepatocytes group. The obtained results showed the scaffolds' ability to support hepatocellular metabolism. The development of hepatocytes in FNTS was studied in vivo using 12 animals using scanning electron microscopy. Hepatocytes demonstrated good adhesion to the scaffold wireframe and survival in allogeneic conditions. Mature tissue, including cellular and fibrous, filled the scaffold space by 98% in 28 days. The study shows the extent to which an implantable "auxiliary liver" compensates for the lack of liver function without replacement in rats.

Morphofunctional reaction of leukocytes and platelets in in vitro contact with a-C:H:SiOx -coated Ti-6Al-4V substrate.

The article deals with the plasma-assisted chemical vapor deposition of 0.3-1.4 µm thick a-C:H:SiOx films in a mixture of argon and polyphenylmethylsiloxane vapor onto the Ti-6Al-4V alloy substrate, which is often used as an implant material. The a-C:H:SiOx film structure is studied by the Fourier-transform infrared and Raman spectroscopies. The pull-off adhesion test assesses the adhesive strength of a-C:H:SiOx films, and the ball-on-disk method is employed to measure their wear rate and friction coefficient. According to these studies, a-C:H:SiOx films are highly adhesive to the Ti-6Al-4V substrate, have low (0.056) friction coefficient and wear rate (9.8 × 10-8  mm3  N-1  m-1 ) in phosphate-buffered saline at 40°C. In vitro studies show neither thrombogenicity nor cytotoxicity of the a-C:H:SiOx film for the human blood mononuclear cells (hBMNCs). The in vitro contact between the hBMNC culture and a-C:H:SiOx films 0.8-1.4 µm thick deposited onto Ti-6Al-4V substrates reduces a 24-hour secretion of pro-inflammatory cytokines and chemokines IL-8, IL-17, TNFα, RANTES, and MCP-1. This reduction is more significant when the film thickness is 1.4 µm and implies its potential anti-inflammatory effect and possible application in cardiovascular surgery. The dependence is suggested for the concentration of anti-inflammatory cytokines and chemokines and the a-C:H:SiOx film thickness, which correlates with the surface wettability and electrostatic potential. The article discusses the possible applications of the anti-inflammatory effect and low thrombogenicity of a-C:H:SiOx films in cardiovascular surgery.

RF Magnetron Sputtering of Substituted Hydroxyapatite for Deposition of Biocoatings.

Functionalization of titanium (Ti)-based alloy implant surfaces by deposition of calcium phosphates (CaP) has been widely recognized. Substituted hydroxyapatites (HA) allow the coating properties to be tailored based on the use of different Ca substitutes. The formation of antibacterial CaP coatings with the incorporation of Zn or Cu by an RF magnetron sputtering is proposed. The influence of RF magnetron targets elemental composition and structure in the case of Zn-HA and Cu-HA, and the influence of substrate's grain size, the substrate's temperature during the deposition, and post-deposition heat treatment (HT) on the resulting coatings are represented. Sintering the targets at 1150 °C resulted in a noticeable structural change with an increase in cell volume and lattice parameters for substituted HA. The deposition rate of Cu-HA and Zn-HA was notably higher compared to stochiometric HA (10.5 and 10) nm/min vs. 9 ± 0.5 nm/min, respectively. At the substrate temperature below 100 °C, all deposited coatings were found to be amorphous with an atomic short-range order corresponding to the {300} plane of crystalline HA. All deposited coatings were found to be hyper-stochiometric with Ca/P ratios varying from 1.9 to 2.5. An increase in the substrate temperature to 200 °C resulted in the formation of equiaxed grain structure on both coarse-grained (CG) and nanostructured (NS) Ti. The use of NS Ti notably increased the scratch resistance of the deposited coatings from18 ± 1 N to 22 ± 2 N. Influence of HT in air or Ar atmosphere is also discussed. Thus, the deposition of Zn- or Cu-containing CaP is a complex process that could be fine-tuned using the obtained research results.

Nitrogen-doped titanium dioxide films fabricated via magnetron sputtering for vascular stent biocompatibility improvement.

Nowadays, vascular stents are commonly used to treat cardiovascular diseases. This article focuses on the influence of nitrogen doping of titanium dioxide thin films, utilized for coating metallic stents to improve their biological properties and biocompatibility. The hereby-investigated titanium oxide thin films are fabricated by magnetron sputtering in a reactive gas atmosphere consisting of argon and oxygen in the first case and argon, nitrogen and oxygen in the second case. Control of the nitrogen and oxygen gas flow rates, and hence their mixing ratios, allows adjustment of the nitrogen-doping level within the titanium dioxide thin films. A correlation of the thin film internal structure on the in vitro behavior of human mesenchymal stem cells derived from adipose tissue is hereby demonstrated. Different nitrogen doping levels affect the surface energy, the wettability, the cell adhesion and thus the cellular proliferation on top of the thin films. The surface colonization of cells on titanium dioxide thin films decreases up to a nitrogen-doping level of âˆ¼ 3.75 at.%, which is associated with a decreasing polar component of the surface energy. For non-doped titanium dioxide thin films, a weak chondrogenesis of adult human adipose-derived mesenchymal stem cells with lower chondrogenic differentiation compared to glass is observed. An increasing nitrogen-doping level leads to linear increase in the chondrogenic differentiation rate, which is comparable to the control value of uncoated glass. Other investigated differentiated cell types do not display this behavior.

UMAOH Calcium Phosphate Coatings Designed for Drug Delivery: Vancomycin, 5-Fluorouracil, Interferon α-2b Case.

Drug delivery systems based on calcium phosphate (CaP) coatings have been recently recognized as beneficial drug delivery systems in complex cases of bone diseases for admission of drugs in the localized area, simultaneously inducing osteoinduction because of the bioavailable Ca and P ions. However, micro-arc oxidation (MAO) deposition of CaP does not allow for the formation of a coating with sufficient interconnected porosity for drug delivery purposes. Here, we report on the method to deposit CaP-based coatings using a new hybrid ultrasound-assisted MAO (UMAOH) method for deposition of coatings for drug delivery that could carry various types of drugs, such as cytostatic, antibacterial, or immunomodulatory compositions. Application of UMAOH resulted in coatings with an Ra roughness equal to 3.5 µm, a thickness of 50-55 µm, and a combination of high values of internal and surface porosity, 39 and 28%, respectively. The coating is represented by the monetite phase that is distributed in the matrix of amorphous CaP. Optimal conditions of coating deposition have been determined and used for drug delivery by impregnation with Vancomycin, 5-Fluorouracil, and Interferon-α-2b. Cytotoxicity and antimicrobial activity of the manufactured drug-carrying coatings have been studied using the three different cell lines and methicillin-resistant S. aureus.

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.

Osteogenic Differentiation Factors of Multipotent Mesenchymal Stromal Cells in the Current Understanding.

BACKGROUND: Molecular genetic mechanisms, signaling pathways, conditions, factors, and markers of the osteogenic differentiation of mesenchymal stem cells (MSCs) are being actively studied and are among the most studied areas in the field of cellular technology. This attention is largely due to the mounting contradictions in the seemingly classical knowledge and the constant updating of results in the analyzed areas. In this regard, we focus on the main classical concepts and some new factors and mechanisms that have a noticeable regulatory effect on the differentiation potential of postnatal MSCs. RESULTS: This review considers the importance of the sources of MSCs for the realization of their differentiation potential, molecular genetic factors and signaling pathways of MSC differentiation, the role of inflammatory cytokines and chemokines in osteogenesis, biomechanical signals, and the effect of conformational changes in the cellular cytoskeleton on MSC differentiation. CONCLUSION: It is concluded that it is necessary to move from studies focused on the effects of local genes to those taking multiple measurements of the gene-regulatory profile and the biomolecules critical for the implementation of numerous, incompletely studied osteogenic factors of endogenous and exogenous origin. Among the cornerstones of future (epi)genetic studies, whether osteomodulatory effects are realized through specific signaling pathways and/or whether cross-signaling with known genes drives the osteogenic differentiation of MSCs remains to be determined.

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.

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.

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.

Zn- or Cu-Containing CaP-Based Coatings Formed by Micro-arc Oxidation on Titanium and Ti-40Nb Alloy: Part I-Microstructure, Composition and Properties.

Zn- and Cu-containing CaP­based coatings, obtained by micro-arc oxidation process, were deposited on substrates made of pure titanium (Ti) and novel Ti-40Nb alloy. The microstructure, phase, and elemental composition, as well as physicochemical and mechanical properties, were examined for unmodified CaP and Zn- or Cu-containing CaP coatings, in relation to the applied voltage that was varied in the range from 200 to 350 V. The unmodified CaP coatings on both types of substrates had mainly an amorphous microstructure with a minimal content of the CaHPO4 phase for all applied voltages. The CaP coatings modified with Zn or Cu had a range from amorphous to nano- and microcrystalline structure that contained micro-sized CaHPO4 and Ca(H2PO4)2·H2O phases, as well as nano­sized ß­Ca2P2O7, CaHPO4, TiO2, and Nb2O5 phases. The crystallinity of the formed coatings increased in the following order: CaP/TiNb < Zn-CaP/TiNb < Cu-CaP/TiNb < CaP/Ti < Zn-CaP/Ti < Cu-CaP/Ti. The increase in the applied voltage led to a linear increase in thickness, roughness, and porosity of all types of coatings, unlike adhesive strength that was inversely proportional to an increase in the applied voltage. The increase in the applied voltage did not affect the Zn or Cu concentration (~0.4 at%), but led to an increase in the Ca/P atomic ratio from 0.3 to 0.7.

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.

Two-stage approach for surgical treatment of tetralogy of Fallot in underweight children: Clinical and morphological outcomes.

BACKGROUND: Two-stage surgery including right ventricular outflow tract (RVOT) stenting with subsequent total surgical repair (TSG) has been suggested as a promising curative option in infants with tetralogy of Fallot (ToF) having comorbidities such as low body weight. However, data on clinical outcomes of such approach and tissue response to RVOT stenting in underweight infants are scarce. METHODS: We recruited 16 underweight (<3 kg; average weight, 2.2 ± 0.4 and 4.7 ± 0.9 kg at the time of RVOT stenting and TSG, respectively) infants (1-3 months of age, average 28.2 ± 4.3 and 100.2 ± 22.3 days at the time of RVOT stenting and TSG, respectively) with ToF and performed RVOT stenting with the subsequent TSG. Excised stents were embedded into epoxy resin and stained by toluidine blue and basic fuchsin. RESULTS: Fifteen infants had a favorable clinical outcome, probably due to the rapid increase in the body weight, blood oxygen saturation, and left ventricular end-diastolic volume to body surface area ratio indicative of improved pulmonary perfusion. Histological analysis revealed an endothelial cell monolayer at the stent surface with notable neovascularization of stented tissues, which could potentially explain the abovementioned clinical and echocardiography improvements. The only death occurred immediately after RVOT stenting and was caused by a massive subdural hematoma, possibly provoked by grade 2 intraventricular hemorrhage 12 days before the stenting. CONCLUSIONS: We confirm RVOT stenting with the subsequent TSG as a safe and efficient surgical approach for the treatment of underweight children with ToF.

Modification of the Ceramic Implant Surfaces from Zirconia by the Magnetron Sputtering of Different Calcium Phosphate Targets: A Comparative Study.

In this study, thin calcium phosphate (Ca-P) coatings were deposited on zirconia substrates by radiofrequency (RF) magnetron sputtering using different calcium phosphate targets (calcium phosphate tribasic (CPT), hydroxyapatite (HA), calcium phosphate monobasic, calcium phosphate dibasic dehydrate (DCPD) and calcium pyrophosphate (CPP) powders). The sputtering of calcium phosphate monobasic and DCPD powders was carried out without an inert gas in the self-sustaining plasma mode. The physico-chemical, mechanical and biological properties of the coatings were investigated. Cell adhesion on the coatings was examined using mesenchymal stem cells (MSCs). The CPT coating exhibited the best cell adherence among all the samples, including the uncoated zirconia substrate. The cells were spread uniformly over the surfaces of all samples.

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.

Detection in vitro and quantitative estimation of artificial microterritories which promote osteogenic differentiation and maturation of stromal stem cells.

Extracellular matrix can regulate multipotent mesenchymal stromal cells (MMSC) differentiation, with potential applications for tissue engineering. A relief of mineralized bone takes essential effect on MMSC fate. Nevertheless, delicate structure and a hierarchy of niches for stromal stem cells and its quantitative parameters are not practically known. Here, we describe the protocol to define the basic approach providing a guiding for in vitro identification of quantitative features of artificial calcium phosphate niches which promotes osteogenic differentiation and maturation of stromal stem cell.