Influence of cell-penetrating peptides on the activity and stability of virus-based nanoparticles.

Viral nanoparticles represent potential natural versatile platforms for targeted gene and drug delivery. Improving the efficiency of gene transfer mediated by viral vectors could not only enhance their therapeutic potential, but also contribute to understanding the limitations in interactions of nanoparticles with cells and the development of new therapeutic approaches. In this study, four cell-penetrating peptides (CPPs), cationic octaarginine (R8), histidine-rich peptides (LAH4 and KH27K) and fusogenic peptide (FUSO), are investigated for their effect on infection by mouse polyomavirus (MPyV) or on transduction of reporter genes delivered by MPyV or related viral vectors. Peptides noncovalently associated with viral particles enhance gene transfer (with the exception of FUSO). Removal of cellular heparan sulfates by the heparinase does not significantly change the enhancing potential of CPPs. Instead, CPPs influences the physical state of viral particles: R8 slightly destabilizes the intact virus, KH27K induces its aggregation and LAH4 promotes disassembly and aggregation of the particles that massively and rapidly associate with cells. The findings indicate that peptides acting as transduction-enhancing agents of polyomavirus-based nanoparticles modulate their physical state, which can be an important prerequisite for sensitization of cells and determination of the further fate of viral particles inside cells.

Positive impact of dynamic seeding of mesenchymal stem cells on bone-like biodegradable scaffolds with increased content of calcium phosphate nanoparticles.

One of the main aims of bone tissue engineering, regenerative medicine and cell therapy is development of an optimal artificial environment (scaffold) that can trigger a favorable response within the host tissue, it is well colonized by resident cells of organism and ideally, it can be in vitro pre-colonized by cells of interest to intensify the process of tissue regeneration. The aim of this study was to develop an effective tool for regenerative medicine, which combines the optimal bone-like scaffold and colonization technique suitable for cell application. Accordingly, this study includes material (physical, chemical and structural) and in vitro biological evaluation of scaffolds prior to in vivo study. Thus, porosity, permeability or elasticity of two types of bone-like scaffolds differing in the ratio of collagen type I and natural calcium phosphate nanoparticles (bCaP) were determined, then analyzes of scaffold interaction with mesenchymal stem cells (MSCs) were performed. Simultaneously, dynamic seeding using a perfusion bioreactor followed by static cultivation was compared with standard static cultivation for the whole period of cultivation. In summary, cell colonization ability was estimated by determination of cell distribution within the scaffold (number, depth and homogeneity), matrix metalloproteinase activity and gene expression analysis of signaling molecules and differentiation markers. Results showed, the used dynamic colonization technique together with the newly-developed collagen-based scaffold with high content of bCaP to be an effective combined tool for producing bone grafts for bone implantology and regenerative medicine.

Evaluation of collagen/hydroxyapatite electrospun layers loaded with vancomycin, gentamicin and their combination: Comparison of release kinetics, antimicrobial activity and cytocompatibility.

The aim of this study was to develop a biodegradable nanostructured electrospun layer based on collagen (COL), hydroxyapatite nanoparticles (HA), vancomycin hydrochloride (V), gentamicin sulphate (G) and their combination (VG) for the treatment of prosthetic joint infections and the prevention of infection during the joint replacement procedure. COL/HA layers containing different amounts of HA (0, 5 and 15 wt%) were tested for the in vitro release kinetics of antibiotics, antimicrobial activity against MRSA, gentamicin-resistant Staphylococcus epidermidis and Enterococcus faecalis isolates and cytocompatibility using SAOS-2 bone-like cells. The results revealed that the COL/HA layers released high concentrations of vancomycin and gentamicin for 21 days and performed effectively against the tested clinically-relevant bacterial isolates. The presence of HA in the collagen layers was found not to affect the release kinetics of the vancomycin from the layers loaded only with vancomycin or its combination with gentamicin. Conversely, the presence of HA slowed down the release of gentamicin from the COL/HA layers loaded with gentamicin and its combination with vancomycin. The combination of both antibiotics exerted a positive effect on the prolongation of the conversion of vancomycin into its degradation products. All the layers tested with different antibiotics exhibited potential antibacterial activity with respect to both the tested staphylococci isolates and enterococci. The complemental effect of vancomycin was determined against both gentamicin-resistant Staphylococcus epidermidis and Enterococcus faecalis in contrast to the application of gentamicin as a single agent. This combination was also found to be more effective against MRSA than is vancomycin as a single agent. Importantly, this combination of vancomycin and gentamicin in the COL/HA layers exhibited sufficient cytocompatibility to SAOS-2, which was independent of the HA content. Conversely, only gentamicin caused the death of SAOS-2 independently of HA content and only vancomycin stimulated SAOS-2 behaviour with an increased concentration of HA in the COL/HA layers. In conclusion, COL/HA layers with 15 wt% of HA impregnated with vancomycin or with a combination of vancomycin and gentamicin offer a promising treatment approach and the potential to prevent infection during the joint replacement procedures.

Comparison of ground sections, paraffin sections and micro-CT imaging of bone from the epiphysis of the porcine femur for morphometric evaluation.

The aim of this study was to compare data on the volume fraction of bone and the thickness of the cortical compact bone acquired during microcomputed tomography (micro-CT) analysis with data acquired from identical samples using stereological analysis of either decalcified paraffin sections or ground sections. Additionally, we aimed to compare adjacent tissue samples taken from the major trochanter of the porcine femur to map the basic biological variability of trabecular bone. Fifteen pairs of adjacent tissue blocks were removed from the major trochanter of the proximal epiphyses of porcine femurs (female pigs aged 24-39 months, weight=59.16±8.15kg). In each sample, the volume of the cortical compact bone, the volume of the trabecular bone, and the thickness of the cortical compact bone was assessed using micro-CT. Afterwards, half of the samples were decalcified and processed using paraffin histological sections. Another half was processed into ground sections. The volume and thickness of bone was assessed in histological sections using stereological techniques. There were no significant differences in the bone volumes and thicknesses measured by micro-CT and the corresponding values quantified in decalcified sections. Similarly, there were no differences between the results from micro-CT and the analysis of the corresponding ground sections. Histomorphometric studies based on relatively low numbers of undecalcified ground sections or demineralized paraffin sections of bone yield data on bone volume and the thickness of cortical compact bone that is comparable with three-dimensional micro-CT examination. The pilot data on the variability of cortical compact bone and trabecular bone volumes in the porcine major trochanter provided in this study aim for planning experiments in the field of bone healing and implantology.

Dry versus hydrated collagen scaffolds: are dry states representative of hydrated states?

Collagen composite scaffolds have been used for a number of studies in tissue engineering. The hydration of such highly porous and hydrophilic structures may influence mechanical behaviour and porosity due to swelling. The differences in physical properties following hydration would represent a significant limiting factor for the seeding, growth and differentiation of cells in vitro and the overall applicability of such hydrophilic materials in vivo. Scaffolds based on collagen matrix, poly(DL-lactide) nanofibers, calcium phosphate particles and sodium hyaluronate with 8 different material compositions were characterised in the dry and hydrated states using X-ray microcomputed tomography, compression tests, hydraulic permeability measurement, degradation tests and infrared spectrometry. Hydration, simulating the conditions of cell seeding and cultivation up to 48 h and 576 h, was found to exert a minor effect on the morphological parameters and permeability. Conversely, hydration had a major statistically significant effect on the mechanical behaviour of all the tested scaffolds. The elastic modulus and compressive strength of all the scaffolds decreased by ~95%. The quantitative results provided confirm the importance of analysing scaffolds in the hydrated rather than the dry state since the former more precisely simulates the real environment for which such materials are designed.

Hyaluronic acid in complexes with surfactants: The efficient tool for reduction of the cytotoxic effect of surfactants on human cell types.

The cationic surfactants carbethoxypendecinium bromide (Septonex) and cetyltrimethylammonium bromide (CTAB) are known to be harmful for certain cell types (bacteria, fungi, mammal cells, etc.). Colloidal complexes of these surfactants with negatively-charged hyaluronic acid (HyA) were prepared for potential drug and/or universal delivery applications. The complexes were tested for their cytotoxic effect on different human cell types - osteoblasts, keratinocytes and fibroblasts. Both the CTAB-HyA and Septonex-HyA complexes were found to reduce the cytotoxicity induced by surfactants alone concerning all the tested concentrations. Moreover, we suggested the limits of HyA protection provided by the surfactant-HyA complexes, e.g. the importance of the amount of HyA applied. We also determined the specific sensitivity of different cell types to surfactant treatment. Keratinocytes were more sensitive to CTAB, while osteoblasts and fibroblasts were more sensitive to Septonex. Moreover, it was indirectly shown that CTAB combines lethal toxicity with cell metabolism induction, while Septonex predominantly causes lethal toxicity concerning fibroblasts. This comprehensive study of the effect of surfactant-HyA complexes on various human cell types revealed that HyA represents a useful CTAB or Septonex cytotoxic effect modulator at diverse levels. Potential applications for these complexes include drug and/or nucleic acid delivery systems, diagnostic dye carriers and cosmetics production.

Initial cell adhesion of three cell types in the presence and absence of serum proteins.

With the development of a wide range of new biomaterials for the sensing of different cell behaviour, it is important to consider whether the cells tested in vitro are in direct contact with the material or whether cell-biomaterial contact is mediated by an interfacial layer of proteins originating from the culture medium or from the cells themselves. Thus, this study describes the differences between the cell adhesion mediated by proteins originating from foetal bovine serum and without the presence of such proteins 2 h following cell seeding exemplarily with different cell types (an osteoblastic cell line, primary fibroblasts, and mesenchymal stem cells). Three of the examined cell types were found to react differently to differing conditions in terms of cell shape, area, and number. Nevertheless, the expression and localization of the various proteins involved in cell adhesion and signalling (CD44, vinculin, talin, actin, focal adhesion kinase, Rho-GTPases and extracellular signal-regulated kinases 1 and 2) were, in general, similar with respect to all the cell types tested, albeit varying according to the presence or absence of serum. Moreover, no classical focal adhesions were formed during cell adhesion without serum proteins, while different signalling pathways were involved in this process. The study systematically describes and discusses the cell adhesion of three different human cell types to a well-known substrate without the presence of external proteins and it is hoped that this knowledge will be subsequently applied in biomaterial applications in which the presence of external proteins is undesirable (e.g. for biosensing purposes).

The release kinetics, antimicrobial activity and cytocompatibility of differently prepared collagen/hydroxyapatite/vancomycin layers: Microstructure vs. nanostructure.

The aim of this study was to develop an osteo-inductive resorbable layer allowing the controlled elution of antibiotics to be used as a bone/implant bioactive interface particularly in the case of prosthetic joint infections, or as a preventative procedure with respect to primary joint replacement at a potentially infected site. An evaluation was performed of the vancomycin release kinetics, antimicrobial efficiency and cytocompatibility of collagen/hydroxyapatite layers containing vancomycin prepared employing different hydroxyapatite concentrations. Collagen layers with various levels of porosity and structure were prepared using three different methods: by means of the lyophilisation and electrospinning of dispersions with 0, 5 and 15wt% of hydroxyapatite and 10wt% of vancomycin, and by means of the electrospinning of dispersions with 0, 5 and 15wt% of hydroxyapatite followed by impregnation with 10wt% of vancomycin. The maximum concentration of the released active form of vancomycin characterised by means of HPLC was achieved via the vancomycin impregnation of the electrospun layers, whereas the lowest concentration was determined for those layers electrospun directly from a collagen solution containing vancomycin. Agar diffusion testing revealed that the electrospun impregnated layers exhibited the highest level of activity. It was determined that modification using hydroxyapatite exerts no strong effect on vancomycin evolution. All the tested samples exhibited sufficient cytocompatibility with no indication of cytotoxic effects using human osteoblastic cells in direct contact with the layers or in 24-hour infusions thereof. The results herein suggest that nano-structured collagen-hydroxyapatite layers impregnated with vancomycin following cross-linking provide suitable candidates for use as local drug delivery carriers.

The effects of different cross-linking conditions on collagen-based nanocomposite scaffolds-an in vitro evaluation using mesenchymal stem cells.

Nanocomposite scaffolds which aimed to imitate a bone extracellular matrix were prepared for bone surgery applications. The scaffolds consisted of polylactide electrospun nano/sub-micron fibres, a natural collagen matrix supplemented with sodium hyaluronate and natural calcium phosphate nano-particles (bioapatite). The mechanical properties of the scaffolds were improved by means of three different cross-linking agents: N-(3-dimethylamino propyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an ethanol solution (EDC/NHS/EtOH), EDC/NHS in a phosphate buffer saline solution (EDC/NHS/PBS) and genipin. The effect of the various cross-linking conditions on the pore size, structure and mechanical properties of the scaffolds were subsequently studied. In addition, the mass loss, the swelling ratio and the pH of the scaffolds were determined following their immersion in a cell culture medium. Furthermore, the metabolic activity of human mesenchymal stem cells (hMSCs) cultivated in scaffold infusions for 2 and 7 days was assessed. Finally, studies were conducted of cell adhesion, proliferation and penetration into the scaffolds. With regard to the structural stability of the tested scaffolds, it was determined that EDC/NHS/PBS and genipin formed the most effectively cross-linked materials. Moreover, it was discovered that the genipin cross-linked scaffold also provided the best conditions for hMSC cultivation. In addition, the infusions from all the scaffolds were found to be non-cytotoxic. Thus, the genipin and EDC/NHS/PBS cross-linked scaffolds can be considered to be promising biomaterials for further in vivo testing and bone surgery applications.