Multiscale characterization of electrospun non-wovens for corneal regeneration: Impact of microstructure on mechanical, optical and biological properties.

The multiscale approach in designing substrates for regenerative medicine endows them with beneficial properties determining their performance in the body. Substrates for corneal regeneration should reveal the proper transparency, mechanical properties and microstructure to maintain the functionality of the regenerated tissue. In our study, series of non-wovens with different fibres orientation (random (R), aligned (A)), topography (shish-kebab (KK), core-shell (CS)) and thickness were fabricated via electrospinning. The samples were assessed for mechanical (static tensile test) and optical properties (spectroscopy UV-Vis). The research evaluated the impact of different microstructures on the viability and morphology of three cell lines (Hs 680, HaCaT and RAW 264.7). The results showed how the fibres arrangement influenced mechanical behaviour of the non-wovens. The randomly oriented fibres were more elongated (up to 50 mm) and had a lower maximum tensile force (up to 0.46 N). In turn, the aligned fibres were characterized by lower elongation (up to 19 mm) and higher force (up to 1.45 N). The conducted transparency tests showed the relation between thickness (of the non-woven and fibres) and morphology of the substrate and light transmission. To simulate the in vivo conditions, prior to the light transmission studies, samples were immersed in water. All the samples exhibited high transparency after immersion in water (>80%). The impact of various morphologies was observed in the in vitro studies. All the samples proved high cells viability. Moreover, the substrate morphology had a significant impact on the orientation and arrangement of the fibroblast cytoskeleton. The aligned fibres were oriented in exactly the same direction. The conducted research proved that, by altering the non-wovens microstructure, the properties can be adjusted so as to induce the desirable cellular reaction. This indicates the high potential of electrospun fibres in terms of modulating the corneal cell behaviour in response to the implanted substrate.

Strategies to Mitigate Biofouling of Nanocomposite Polymer-Based Membranes in Contact with Blood.

An extracorporeal blood purification method called continuous renal replacement therapy uses a porous hollow-fiber polymeric membrane that is exposed to prolonged contact with blood. In that condition, like with any other submerged filtration membrane, the hemofilter loses its properties over time and use resulting in a rapid decline in flux. The most significant reason for this loss is the formation of a biofilm. Protein, blood cells and bacterial cells attach to the membrane surface in complex and fluctuating processes. Anticoagulation allows for longer patency of vascular access and a longer lifespan of the membrane. Other preventive measures include the modification of the membrane itself. In this article, we focused on the role of nanoadditives in the mitigation of biofouling. Nanoparticles such as graphene, carbon nanotubes, and silica effectively change surface properties towards more hydrophilic, affect pore size and distribution, decrease protein adsorption and damage bacteria cells. As a result, membranes modified with nanoparticles show better flow parameters, longer lifespan and increased hemocompatibility.

Correlation between porosity and physicochemical and biological properties of electrospinning PLA/PVA membranes for skin regeneration.

Electrospinning is an increasingly popular technique for obtaining scaffolds for skin regeneration. However, electrospun scaffolds may also have some disadvantages, as the densely packed fibers in the scaffold structure can limit the penetration of skin cells into the inner part of the material. Such a dense arrangement of fibers can cause the cells to treat the 3D material as 2D one, and thus cause them to accumulate only on the upper surface. In this study, bi-polymer scaffolds made of polylactide (PLA) and polyvinyl alcohol (PVA) electrospun in a sequential or a concurrent system were investigated in a different PLA:PVA ratio (2:1 and 1:1). The properties of six types of model materials were investigated and compared i.e.; the initial materials electrospun by the sequential (PLA/PVA, 2PLA/PVA) and the concurrent system (PLA||PVA) and the same materials with removed PVA fibers (PLA/rPVA, 2PLA/rPVA, PLA||rPVA). The fiber models were intended to increase the porosity and coherent structure parameters of the scaffolds. The applied treatment involving the removal of PVA nanofibers increased the size of interfibrous pores formed between the PLA fibers. Ultimately, the porosity of the PLA/PVA scaffolds increased from 78 % to 99 %, and the time of water absorption decreased from 516 to 2 s. The change in wettability was induced by a synergistic effect of decrease in roughness after washing out and the presence of residual PVA fibers. The chemical analysis carried out confirmed the presence of PVA residues on the PLA fibers (FTIR-ATR study). In vitro studies were performed on human keratinocytes (HaKaT) and macrophages (RAW264.7), for which penetration into the inner part of the PLAIIPVA scaffold was observed. The new proposed approach, which allows the removal of PVA fibers from the bicomponent material, allows to obtain a scaffold with increased porosity, and thus better permeability for cells and nutrients.

Functionalized Halloysite Nanotubes as Potential Drug Carriers.

The aim of the work was to examine the possibility of using modified halloysite nanotubes as a gentamicin carrier and to determine the usefulness of the modification in terms of the effect on the amount of the drug attached, its release time, but also on the biocidal properties of the carriers. In order to fully examine the halloysite in terms of the possibility of gentamicin incorporating, a number of modifications of the native halloysite were carried out prior to gentamicin intercalation with the use of sodium alkali, sulfuric and phosphoric acids, curcumin and the process of delamination of nanotubes (expanded halloysite) with ammonium persulfate in sulfuric acid. Gentamicin was added to unmodified and modified halloysite in an amount corresponding to the cation exchange capacity of pure halloysite from the Polish Dunino deposit, which was the reference sample for all modified carriers. The obtained materials were tested to determine the effect of surface modification and their interaction with the introduced antibiotic on the biological activity of the carrier, kinetics of drug release, as well as on the antibacterial activity against Escherichia coli Gram-negative bacteria (reference strain). For all materials, structural changes were examined using infrared spectroscopy (FTIR) and X-ray diffraction (XRD); thermal differential scanning calorimetry with thermogravimetric analysis (DSC/TG) was performed as well. The samples were also observed for morphological changes after modification and drug activation by transmission electron microscopy (TEM). The conducted tests clearly show that all samples of halloysite intercalated with gentamicin showed high antibacterial activity, with the highest antibacterial activity for the sample modified with sodium hydroxide and intercalated with the drug. It was found that the type of halloysite surface modification has a significant effect on the amount of gentamicin intercalated and then released into the surrounding environment but does not significantly affect its ability to further influence drug release over time. The highest amount of drug released among all intercalated samples was recorded for halloysite modified with ammonium persulfate (real loading efficiency above 11%), for which high antibacterial activity was found after surface modification, before drug intercalation. It is also worth noting that intrinsic antibacterial activity was found for non-drug-intercalated materials after surface functionalization with phosphoric acid (V) and ammonium persulfate in the presence of sulfuric acid (V).

Effect of Ionic and Non-Ionic Surfactant on Bovine Serum Albumin Encapsulation and Biological Properties of Emulsion-Electrospun Fibers.

Emulsion electrospinning is a method of modifying a fibers' surface and functional properties by encapsulation of the bioactive molecules. In our studies, bovine serum albumin (BSA) played the role of the modifier, and to protect the protein during the electrospinning process, the W/O (water-in-oil) emulsions were prepared, consisting of polymer and micelles formed from BSA and anionic (sodium dodecyl sulfate-S) or nonionic (Tween 80-T) surfactant. It was found that the micelle size distribution was strongly dependent on the nature and the amount of the surfactant, indicating that a higher concentration of the surfactant results in a higher tendency to form smaller micelles (4-9 µm for S and 8-13 µm for T). The appearance of anionic surfactant micelles reduced the diameter of the fiber (100-700 nm) and the wettability of the nonwoven surface (up to 77°) compared to un-modified PCL polymer fibers (100-900 nm and 130°). The use of a non-ionic surfactant resulted in better loading efficiency of micelles with albumin (about 90%), lower wettability of the nonwoven fabric (about 25°) and the formation of larger fibers (100-1100 nm). X-ray photoelectron spectroscopy (XPS) was used to detect the presence of the protein, and UV-Vis spectrophotometry was used to determine the loading efficiency and the nature of the release. The results showed that the location of the micelles influenced the release profiles of the protein, and the materials modified with micelles with the nonionic surfactant showed no burst release. The release kinetics was characteristic of the zero-order release model compared to anionic surfactants. The selected surfactant concentrations did not adversely affect the biological properties of fibrous substrates, such as high viability and low cytotoxicity of RAW macrophages 264.7.

Assessment of sheep knee joint after ACL replacement with Achilles tendon autograft and PLA-based implant.

In this study, we propose a new approach in the anterior cruciate ligament (ACL) replacement to provide stability and integration with bone tunnel. A polylactide (PLA)-based tubular implant was used to support the graft stabilization in femoral and tibial bones and to stimulate the healing process after (ACL) replacement on a sheep model. The ACL was replaced with an autologous Achilles tendon split graft. The tendon-to-bone healing in the model was analyzed after 6 and 12 weeks. Two groups of animals were compared, i.e. the group with the PLA-based implant used in the ACL replacement and the control group without the implant. The knee joints were mechanically and clinically evaluated, including the histopathology tests, to determine their stability and integrity. The results indicated that the bioresorbable PLA-based tubular implant may facilitate integration of the tendon graft with bone. Remodeling the allograft inside the implant improves the joint mobility from the first week of healing: no pathological changes were observed at the surgery site and in the animals' mobility. After 6 and 12 weeks of healing no significant changes in the mechanical parameters of the knee joint were observed, regarding the joint failure force, knee displacement, angular mobility range and joint stiffness. Relatively small values of the non-destructive tests in the knee displacement, already 6 weeks after surgery, indicated the early stabilization of the knee joint. The studies showed that the failure forces of knee joints after the ACL replacement with the PLA-based implant are lower than those of an intact joint, although their biomechanical features, including strain-at- failure, are similar. The biomechanical parameters of the knee joint were significantly improved due to the selected method of attaching the autograft ends to the femoral and tibial bone surfaces. After 12 weeks the intra-tunnel tendon-bone site with the PLA implant revealed the better tibia-femur joint mechanical stability, linear force-strain function and the decreasing strain-to-failure value, as compared to the control group.

Effects of Montmorillonite and Gentamicin Addition on the Properties of Electrospun Polycaprolactone Fibers.

Electrospinning was used to obtain multifunctional fibrous composite materials with a matrix of poly-ɛ-caprolactone (PCL) and 2 wt.% addition of a nanofiller: montmorillonite (MMT), montmorillonite intercalated with gentamicin sulphate (MMTG) or gentamicin sulphate (G). In the first stage, the aluminosilicate gallery was modified by introducing gentamicin sulfate into it, and the effectiveness of the intercalation process was confirmed on the basis of changes in the clay particle size from 0.5 µm (for MMT) to 0.8 µm (for MMTG), an increase in the interplanar distance d001 from 12.3 Å (for MMT) to 13.9 Å (for MMTG) and altered clay grain morphology. In the second part of the experiment, the electrospinning process was carried out in which the polymer nonwovens with and without the modifier were prepared directly from dichloromethane (DCM) and N,N-dimethylformamide (DMF). The nanocomposite fibrous membranes containing montmorillonite were prepared from the same polymer solution but after homogenization with the modifier (13 wt.%). The degree of dispersion of the modifier was evaluated by average microarray analysis from observed area (EDS), which was also used to determine the intercalation of montmorillonite with gentamicin sulfate. An increase in the size of the fibers was found for the materials with the presence of the modifier, with the largest diameters measured for PCL_MMT (625 nm), and the smaller ones for PCL_MMTG (578 nm) and PCL_G (512 nm). The dispersion of MMT and MMTG in the PCL fibers was also confirmed by indirect studies such as change in mechanical properties of the nonwovens membrane, where the neat PCL nonwoven was used as a reference material. The addition of the modifier reduced the contact angle of PCL nonwovens (from 120° for PCL to 96° for PCL_G and 98° for PCL_MMTG). An approximately 10% increase in tensile strength of the nonwoven fabric with the addition of MMT compared to the neat PCL nonwoven fabric was also observed. The results of microbiological tests showed antibacterial activity of all obtained materials; however, the inhibition zones were the highest for the materials containing gentamicin sulphate, and the release time of the active substance was significantly extended for the materials with the addition of montmorillonite containing the antibiotic. The results clearly show that the electrospinning technique can be effectively used to obtain nanobiocomposite fibers with the addition of nonintercalated and intercalated montmorillonite with improved strength and increased stiffness compared to materials made only of the polymer fibers, provided that a high filler dispersion in the spinning solution is obtained.

Chemical Modification as a Method of Improving Biocompatibility of Carbon Nonwovens.

It was shown that carbon nonwoven fabrics obtained from polyacrylonitrile fibers (PAN) by thermal conversion may be modified on the surface in order to improve their biological compatibility and cellular response, which is particularly important in the regeneration of bone or cartilage tissue. Surface functionalization of carbon nonwovens containing C-C double bonds was carried out using in situ generated diazonium salts derived from aromatic amines containing both electron-acceptor and electron-donor substituents. It was shown that the modification method characteristic for materials containing aromatic structures may be successfully applied to the functionalization of carbon materials. The effectiveness of the surface modification of carbon nonwoven fabrics was confirmed by the FTIR method using an ATR device. The proposed approach allows the incorporation of various functional groups on the nonwovens' surface, which affects the morphology of fibers as well as their physicochemical properties (wettability). The introduction of a carboxyl group on the surface of nonwoven fabrics, in a reaction with 4-aminobenzoic acid, became a starting point for further modifications necessary for the attachment of RGD-type peptides facilitating cell adhesion to the surface of materials. The surface modification reduced the wettability (θ) of the carbon nonwoven by about 50%. The surface free energy (SFE) in the chemically modified and reference nonwovens remained similar, with the surface modification causing an increase in the polar component (ɣp). The modification of the fiber surface was heterogeneous in nature; however, it provided an attractive site of cell-materials interaction by contacting them to the fiber surface, which supports the adhesion process.

Structure and Pathologies of Articular Cartilage.

The aim of the review was to describe a complex microstructure and biomechanical properties of the articular cartilage as well as a current review of its pathologies encountered in veterinary practice. The articular cartilage with its unique features: complex microarchitecture, significant mechanical durability and elasticity, lacking blood, lymphatic vessels, and innervation, seems to stand in contradiction to the laws of biology. It can be involved in a vast majority of diseases, from osteoarthrosis as a result of natural aging process to more complex in nature like osteochondromatosis. The primary role of articular cartilage is to provide the surface for movement in any single joint in the body. Therefore, its diseases lead to physical impairment and deterioration of the quality of life. Treatment of articular cartilage poses a formidable challenge in both modern human and animal medicine.

Effects of Process Parameters on Structure and Properties of Melt-Blown Poly(Lactic Acid) Nonwovens for Skin Regeneration.

Skin regeneration requires a three-dimensional (3D) scaffold for cell adhesion, growth and proliferation. A type of the scaffold offering a 3D structure is a nonwoven material produced via a melt-blown technique. Process parameters of this technique can be adapted to improve the cellular response. Polylactic acid (PLA) was used to produce a nonwoven scaffold by a melt-blown technique. The key process parameters, i.e., the head and air temperature, were changed in the range from 180-270 °C to obtain eight different materials (MB1-MB8). The relationships between the process parameters, morphology, porosity, thermal properties and the cellular response were explored in this study. The mean fiber diameters ranged from 3 to 120 µm. The average material roughness values were between 47 and 160 µm, whereas the pore diameters ranged from 5 to 400 µm. The calorimetry thermograms revealed a correlation between the temperature parameters and crystallization. The response of keratinocytes and macrophages exhibited a higher cell viability on thicker fibers. The cell-scaffold interaction was observed via SEM after 7 days. This result proved that the features of melt-blown nonwoven scaffolds depended on the processing parameters, such as head temperature and air temperature. Thanks to examinations, the most suitable scaffolds for skin tissue regeneration were selected.

How Surface Properties of Silica Nanoparticles Influence Structural, Microstructural and Biological Properties of Polymer Nanocomposites.

The aim of this work was to study effect of the type of silica nanoparticles on the properties of nanocomposites for application in the guided bone regeneration (GBR). Two types of nanometric silica particles with different size, morphology and specific surface area (SSA) i.e., high specific surface silica (hss-SiO2) and low specific surface silica (lss-SiO2), were used as nano-fillers for a resorbable polymer matrix: poly(L-lactide-co-D,L-lactide), called PLDLA. It was shown that higher surface specific area and morphology (including pore size distribution) recorded for hss-SiO2 influences chemical activity of the nanoparticle; in addition, hydroxyl groups appeared on the surface. The nanoparticle with 10 times lower specific surface area (lss-SiO2) characterized lower chemical action. In addition, a lack of hydroxyl groups on the surface obstructed apatite nucleation (reduced zeta potential in comparison to hss-SiO2), where an apatite layer appeared already after 48 h of incubation in the simulated body fluid (SBF), and no significant changes in crystallinity of PLDLA/lss-SiO2 nanocomposite material in comparison to neat PLDLA foil were observed. The presence and type of inorganic particles in the PLDLA matrix influenced various physicochemical properties such as the wettability, and the roughness parameter note for PLDLA/lss-SiO2 increased. The results of biological investigation show that the bioactive nanocomposites with hss-SiO2 may stimulate osteoblast and fibroblast cells'proliferation and secretion of collagen type I. Additionally, both nanocomposites with the nanometric silica inducted differentiation of mesenchymal cells into osteoblasts at a proliferation stage in in vitro conditions. A higher concentration of alkaline phosphatase (ALP) was observed on the material modified with hss-SiO2 silica.

Multifunctional biodegradable polymer/clay nanocomposites with antibacterial properties in drug delivery systems.

PURPOSE: The aim of this study was to investigate the possibility of intercalation of gentamicin and neomycin in montmorillonite (MMT) nanofillers, as well as to study the in vitro antimicrobial properties of nanocomposite films containing a small amount of thus obtained nanofillers. METHODS: The polylactide matrix (PLA) nanocomposite films with drug-intercalated montmorillonite fillers were obtained by casting after intercalation of drugs in aqueous solutions. The efficiency of intercalation has been confirmed by X-ray diffraction (XRD) and Zeta potential measurements. The materials were studied for surface wettability, roughness and mechanical properties during 6 weeks of incubation in phosphate buffer saline, and their bactericidal activity was tested against Escherichia coli bacteria before and after 6 weeks of incubation in distilled water at 37 °C. The presence of antibiotics during the incubation was monitored by conductivity and pH measurements. RESULTS: The results indicate that nanocomposite polylactide films with montmorillonite filler intercalated with gentamicin and neomycin tend to degrade faster that their counterparts with non-intercalated fillers, which affects their mechanical properties. However, drug intercalation provided an antibacterial activity, which was confirmed by the presence of zones inhibiting the growth of Gram-negative bacteria for both antibiotics. It was also confirmed that the interaction of antibiotics with clay and polymer matrix did not adversely affect this bactericidal effect. CONCLUSIONS: Montmorillonite can be successfully intercalated with both gentamicin and neomycin, and then used as active filler for polylactide films having very good antibacterial properties, therefore their use in biomedical applications can be significantly expanded.

Porous poly(lactic acid) based fibres as drug carriers in active dressings.

PURPOSE: The polymeric porous surface of fibres (PLA) may influence the kinetics of release of biologically active compounds (gentamicin, G and ethacridine lactate, R) affecting development of a bacterial biofilm. METHODS: The porous fibres with different morphology were manufactured by the electrospinning method from ternary systems composed of PLA and selected solvents. Fibres morphology was examined using a scanning electron microscopy (SEM), their structure was analyzed by FT-IR ATR spectroscopy and differential scanning calorimetry (DSC). Changes in the drug release profile were measured using ICP/UV-Vis methods and the resulting bactericidal or bacteriostatic properties were tested by two-layer disk diffusion test in relation to various drug incorporation methods. RESULTS: The porous fibres can be applied to produce drug-bearing membranes. The spectroscopic studies confirmed incorporation of gentamicin into the fibres and the presence of ethacridine lactate on their surface. Bimodal fibres distribution (P3) promoted faster release of gentamicin and ethacridine lactate from P3G and P3R materials. The electrospinning process coupled with the vapor induced phase separation influenced the glass transition temperature of the porous polymer fibres. The pre/post-electrospinning modification influenced the glass transition, maximum temperature of cold crystallization and melting point of the porous membrane, compared to the neat polymer. The polylactide fibres with gentamicin showed strong bactericidal effect on Gram-positive bacteria, while fibres with ethacridine lactate were bacteriostatic. CONCLUSIONS: The obtained fibres with complex surface morphology can be used as a membrane in active dressings as they make it possible to control the release profile of the active compounds.

Conjugates of Copper Alginate with Arginine-Glycine-Aspartic Acid (RGD) for Potential Use in Regenerative Medicine.

Current restrictions on the use of antibiotics, associated with increases in bacterial resistance, require new solutions, including materials with antibacterial properties. In this study, copper alginate fibers obtained using the classic wet method were used to make nonwovens which were modified with arginine-glycine-aspartic acid (RGD) derivatives. Stable polysaccharide-peptide conjugates formed by coupling with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-), and materials with physically embedded RGD derivatives, were obtained. The materials were found to be characterized by very high antibacterial activity against S. aureus and K. pneumoniae. Cytotoxicity studies confirmed that the materials are not cytotoxic. Copper alginate conjugates with RGD peptides have strong potential for use in regenerative medicine, due to their biocompatibility and innate antibacterial activity.

Bioresorbable Stent in Anterior Cruciate Ligament Reconstruction.

The exact causes of failure of anterior cruciate ligament (ACL) reconstruction are still unknown. A key to successful ACL reconstruction is the prevention of bone tunnel enlargement (BTE). In this study, a new strategy to improve the outcome of ACL reconstruction was analyzed using a bioresorbable polylactide (PLA) stent as a catalyst for the healing process. The study included 24 sheep with 12 months of age. The animals were randomized to the PLA group (n = 16) and control group (n = 8), subjected to the ACL reconstruction with and without the implantation of the PLA tube, respectively. The sheep were sacrificed 6 or 12 weeks post-procedure, and their knee joints were evaluated by X-ray microcomputed tomography with a 50 µm resolution. While the analysis of tibial and femoral tunnel diameters and volumes demonstrated the presence of BTE in both groups, the enlargement was less evident in the PLA group. Also, the microstructural parameters of the bone adjacent to the tunnels tended to be better in the PLA group. This suggested that the implantation of a bioresorbable PLA tube might facilitate osteointegration of the tendon graft after the ACL reconstruction. The beneficial effects of the stent were likely associated with osteogenic and osteoconductive properties of polylactide.

Impact of a pulsed magnetic field on selected polymer implant materials.

PURPOSE: Physiotherapy with the use of pulsed magnetic fields is one of the methods of activating the processes of bone healing and regeneration. Exposing materials serving as membranes in guided bone regeneration (GBR) or guided tissue regeneration (GTR) to magnetic fields is an effective model that allows to monitor changes in the material under the influence of the magnetic field. METHODS: Materials engineering methods were used to verify the extent of material degradation resulting from magnetic field exposure in an aqueous environment. Changes in surface morphology were observed under an optical microscope and a scanning electron microscope (SEM). Changes in surface wettability were analysed in relation to the direct contact angle. Chemical structural changes were verified with the use of infrared spectroscopy (FTIR-ATR). RESULTS: The PCL-based membrane materials underwent relatively moderate surface degradation (altered contact angle, changes in surface morphology), but the absence of observable FTIR-ATR spectral shifts evidenced material stability under the influence of magnetic field. More extensive degradation processes were observed in the case of PLDLA-based materials, whose surface character changed from hydrophilic to hydrophobic. The spectra revealed enhanced intensity of the chain terminal groups, provided that modifiers (nanometric SiO2 and TCP (water reservoir)) were present in the polymer matrix. CONCLUSIONS: The extent degradation in the polymer membrane was primarily dependent on the presence of aqueous environment, while the influence of the magnetic field on the analysed membrane materials was negligible. Therefore, GBR/GTR membrane implants can be considered to remain stable during rehabilitation with the use of alternating magnetic field.

Study on the Materials Formed by Self-Assembling Hydrophobic, Aromatic Peptides Dedicated to Be Used for Regenerative Medicine.

The aims of this study were to identify the short aromatic peptides which are able to form highly ordered amyloid-like structures in self-assembling processes, to test the influence of length of hydrophobic peptides on tendency to aggregation, and to check if aggregated peptides fulfill requirements expected for materials useful for scaffolding. All tested hydrophobic peptides were prepared on solid phase by using DMT/NMM/TsO- as a coupling reagent. The progress of aggregation was studied by set of independent tests. All aggregated peptides were found stable under in vitro conditions. All fibrous material formed by self-assembling of peptides does not show any cytotoxic effects on L929 fibroblast cells. Peptides containing tyrosine and tryptophan residues even effectively accelerated the proliferation and stimulated the activity of L929 fibroblasts.

Surface-Potential-Controlled Cell Proliferation and Collagen Mineralization on Electrospun Polyvinylidene Fluoride (PVDF) Fiber Scaffolds for Bone Regeneration.

This study represents the unique analysis of the electrospun scaffolds with the controlled and stable surface potential without any additional biochemical modifications for bone tissue regeneration. We controlled surface potential of polyvinylidene fluoride (PVDF) fibers with applied positive and negative voltage polarities during electrospinning, to obtain two types of scaffolds PVDF(+) and, PVDF(-). The cells' attachments to PVDF scaffolds were imaged in great details with advanced scanning electron microscopy (SEM) and 3D tomography based on focus ion beam (FIB-SEM). We presented the distinct variations in cells shapes and in filopodia and lamellipodia formation according to the surface potential of PVDF fibers that was verified with Kelvin probe force microscopy (KPFM). Notable, cells usually reach their maximum spread area through increased proliferation, suggesting the stronger adhesion, which was indeed double for PVDF(-) scaffolds having surface potential of -95 mV. Moreover, by tuning the surface potential of PVDF fibers, we were able to enhance collagen mineralization for possible use in bone regeneration. The scaffolds built of PVDF(-) fibers demonstrated the greater potential for bone regeneration than PVDF(+), showing after 7 days in osteoblasts culture produce well-mineralized osteoid required for bone nodules. The collagen mineralization was confirmed with energy dispersive X-ray spectroscopy (EDX) and Sirius Red staining, additionally the cells proliferation with fluorescence microscopy and Alamar Blue assays. The scaffolds made of PVDF fibers with the similar surface potential to the cell membranes promoting bone growth for next-generation tissue scaffolds, which are on a high demand in bone regenerative medicine.

Effects of Polylactide Copolymer Implants and Platelet-Rich Plasma on Bone Regeneration within a Large Calvarial Defect in Sheep.

The aim of this study was to verify whether L-lactide/DL-lactide copolymer 80/20 (PLDLLA) and platelet-rich plasma (PRP) trigger bone formation within critical-sized calvarial defects in adult sheep (n = 6). Two craniectomies, each ca. 3 cm in diameter, were created in each animal. The first craniectomy was protected with an inner polylactide membrane, filled with PRP-polylactide granules, and covered with outer polylactide membrane. The second control craniectomy was left untreated. The animals were euthanized at 6, 7, 17, 19, 33, and 34 weeks after surgery, and the quality and the rate of reossification were assessed histomorphometrically and microtomographically. The study demonstrated that application of implants made of PLDLLA 80/20 combined with an osteopromotive substance (e.g., PRP) may promote bone healing in large calvarial defect in sheep. These promising proof-of-concept studies need to be verified in the future on a larger cohort of animals and over a longer period of time in order to draw definitive conclusions.

Search for Fibrous Aggregates Potentially Useful in Regenerative Medicine Formed under Physiological Conditions by Self-Assembling Short Peptides Containing Two Identical Aromatic Amino Acid Residues.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H-dPhedPhe-OH, H-dCysdPhedPhe-OH, H-CysTyrTyr-OH, H-dPhedPhedCys-OH, H-TyrTyrMet-OH, and H-TyrMetTyr-OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.