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.

Fibrous polymeric composites based on alginate fibres and fibres made of poly-ε-caprolactone and dibutyryl chitin for use in regenerative medicine.

This work concerns the production of fibrous composite materials based on biodegradable polymers such as alginate, dibutyryl chitin (DBC) and poly-ε-caprolactone (PCL). For the production of fibres from these polymers, various spinning methods were used in order to obtain composite materials of different composition and structure. In the case of alginate fibres containing the nanoadditive tricalcium phosphate (TCP), the traditional method of forming fibres wet from solution was used. However in the case of the other two polymers the electrospinning method was used. Two model systems were tested for biocompatibility. The physicochemical and basic biological tests carried out show that the submicron fibres produced using PCL and DBC have good biocompatibility. The proposed hybrid systems composed of micrometric fibres (zinc and calcium alginates containing TCP) and submicron fibres (DBC and PCL) meet the requirements of regenerative medicine. The biomimetic fibre system, the presence of TCP nanoadditive, and the use of polymers with different resorption times provide a framework with specific properties on which bone cells are able to settle and proliferate.

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.

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.

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.

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.

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.

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.

The membrane with polylactide and hyaluronic fibers for skin substitute.

PURPOSE: Skin substitutes are heterogeneous group of scaffolds (natural or synthetic) and cells. We hypothesize that nanofibers with layer composition made of polylactide (PLA) and sodium hyaluronate (HA) obtained using electrospinning method are a good matrix for cell adhesion and proliferation. METHODS: Optimal conditions of electrospinning of PLA and HA nanofibers to create layered compositions (PLA membrane covered with HA nonwovens) were determined by modifying parameters such as the appropriate amount of solvents, polymer concentration, mixing temperature and electrospinning process conditions. By changing the parameters, it was possible to control the diameter and properties of both polymer fibers. The spinning solution were characterized by surface tension and rheology. A scanning electron microscope (SEM) was used to determine the morphology and fiber diameters: PLA and HA. Structure of the PLA/HA nonwoven was analyzed using spectroscopy (FTIR/ATR). Biocompatibility of the nonwoven with fibroblasts (ECM producers) was assessed in the in vitro conditions. RESULTS: The results showed that stable conditions for the formation of submicron PLA fibers were obtained using a 13% wt. solution of the polymer, dissolved in a 3:1 mixture of DCM:DMF at 45 °C. The hyaluronic fibers were prepared from a 12% wt. solution of the polymer dissolved in a 2:1 mixture of ammonia water and ethyl alcohol. All materials were biocompatible but to a different degree. CONCLUSIONS: The proposed laminate scaffold was characterized by a hydrophobic-hydrophilic domain surface with a maintained fiber size of both layers. The material positively underwent biocompatibility testing in contact with fibroblasts.

Biodegradable ceramic-polymer composites for biomedical applications: A review.

The present work focuses on the state-of-the-art of biodegradable ceramic-polymer composites with particular emphasis on influence of various types of ceramic fillers on properties of the composites. First, the general needs to create composite materials for medical applications are briefly introduced. Second, various types of polymeric materials used as matrices of ceramic-containing composites and their properties are reviewed. Third, silica nanocomposites and their material as well as biological characteristics are presented. Fourth, different types of glass fillers including silicate, borate and phosphate glasses and their effect on a number of properties of the composites are described. Fifth, wollastonite as a composite modifier and its effect on composite characteristics are discussed. Sixth, composites containing calcium phosphate ceramics, namely hydroxyapatite, tricalcium phosphate and biphasic calcium phosphate are presented. Finally, general possibilities for control of properties of composite materials are highlighted.

Osteochondral Repair Using Porous Three-dimensional Nanocomposite Scaffolds in a Rabbit Model.

AIM: To evaluate the utility of a novel nanocomposite biomaterial consisting of poly-L/D-lactide, and hydroxyapatite bioceramics, enriched with sodium alginate in articular cartilage defect treatment. MATERIALS AND METHODS: The biomaterial was prepared using the method of solvent casting and particle leaching. The study was conducted on 20 New Zealand White rabbits. Experimental osteochondral defects were created in the femoral trochlear grooves and filled with biomaterials. In control groups, the defects were left to spontaneously heal. The quality of newly-formed tissue was evaluated on the basis of macroscopic and histological assessment. Additionally the level of osteogenic and cartilage degradation markers were measured. RESULTS: The majority of the defects from the treatment group were covered with tissue similar in structure and colour to healthy cartilage, whereas in the control group, tissue was uneven, and not integrated into the surrounding cartilage. CONCLUSION: The results obtained validate the choice of biomaterial used in this study as well as the method of its application.

Non-penetrating very deep sclerectomy with a hydrophobic polymer implant in a rabbit model.

PURPOSE: The aim of the study was to evaluate the influence of an implant made of a terpolymer (PTFE-PVDF-PP) on the condition of rabbit eyes during a one year observation period. METHODS: The implant in the shape of an equilateral triangle (3 mm side length) was manufactured from a thin hydrophobic porous membrane. There were evaluated 40 eyes of 20 rabbits. The animals had non-penetrating very deep sclerectomy (NPVDS) performed, with insertion of an implant in the form of a triangular thin membrane. The control group consisted of 20 eyes where the animals had NPVDS performed without implant insertion. The evaluations included the study of the anterior part of the eye together with photographic documentation. Histopathological examination of the eyes 52 weeks after NPVDS procedure has been made. The process of wound healing was comparable in both groups. RESULTS: The evaluation of the rabbits did not reveal any acute process of intraocular inflammation. After 12 month period of observation, no statistically significant differences in the process of wound healing or status of eyes were found between the groups. An analysis of fibrous connective tissue attachment to the implant showed that its layer was not thick and did not differ significantly from the control. The procedure of very deep sclerectomy and insertion of a polymer implant were well tolerated by the rabbit eyes. CONCLUSIONS: The in vivo results indicate that the hydrophobic implant in the form of a membrane can serve as a sclera implant after further study.

Effect of the preparation methods on architecture, crystallinity, hydrolytic degradation, bioactivity, and biocompatibility of PCL/bioglass composite scaffolds.

In this study, two different composition gel derived silica-rich (S2) or calcium-rich (A2) bioactive glasses (SBG) from a basic CaO-P2 O5 -SiO2 system were incorporated into poly(ε-caprolactone) (PCL) matrix to obtain novel bioactive composite scaffolds for bone tissue engineering applications. The composites were fabricated in the form of highly porous 3D scaffolds using following preparation methods: solvent casting particulate leaching (SCPL), solid-liquid phase separation, phase inversion (PI). Scaffolds containing 21% vol. of each bioactive glass were characterized for architecture, crystallinity, hydrolytic degradation, surface bioactivity, and cellular response. Results indicated that the use of different preparation methods leads to obtain highly porous (60-90%) materials with differentiated morphology: pore shape, size, and distributions. Thermal analysis (DSC) showed that the preparation method of materials and addition of bioactive glass particles into polymer matrix induced the changes of PCL crystallinity. Composites obtained by SCPL and PI method containing A2 SBG rapidly formed a hydroxyapatite calcium phosphate surface layer after incubation in SBF. Bioactive glasses used as filler in composite scaffolds could neutralize the released acidic by-products of the polymer degradation. Preliminary in vitro biological studies of the composites in contact with osteoblastic cells showed good biocompatibility of the obtained materials. Addition of bioactive glass into the PCL matrix promotes mineralization estimated on the basis of the ALP activity. These results suggest that through a process of selection appropriate methods of preparation and bioglass composition it is possible to design and obtain porous materials with suitable properties for regeneration of bone tissue.

Histopathological Evaluation of a Hydrophobic Terpolymer (PTFE-PVD-PP) as an Implant Material for Nonpenetrating Very Deep Sclerectomy.

PURPOSE: The purpose of the study was to assess the biocompatibility of porous terpolymer (polytetrafluoroethylene-co-polyvinylidene fluoride-co-polypropylene, PTFE-PVDF-PP) membranes as an implant material to be placed during nonpenetrating very deep sclerectomy (NPVDS). Another study objective was to determine whether the polymer membrane under investigation could be used to manufacture a new-generation implant, which would actively delay the process of fistula closure and facilitate aqueous humor drainage. METHODS: Histological response and tissue tolerance of the implant material were assessed. The study was performed on 38 eyeballs of 19 New Zealand white rabbits (19 implanted, 19 control). Histological assessment was carried out between 2 and 52 weeks after surgery. We routinely assessed inflammatory infiltrate, neovascularization, hemorrhage, and stromal edema as well as connective tissue attachment to the implant and adjacent tissues. RESULTS: At 52 weeks of observation, a statistically significant difference was revealed between the study and control groups in terms of resorptive granulation, tissue, and the inflammatory infiltrate. No features of acute inflammatory response to the implant were observed, and there was an absence of histological features of acute inflammatory infiltrates and subsidence of chronic inflammatory infiltrates and resorptive granulation over time. CONCLUSIONS: Slight fibrotic response and insignificant changes in neighboring eye tissues all indicate good tolerance to bioimplant materials. This allows for some optimism regarding the use of hydrophobic terpolymer in the construction of new intrascleral implants. However, the ultimate decision regarding its usefulness and safety in the treatment of glaucoma requires further investigation.