An Eco-Friendly Process to Extract Hydroxyapatite from Sheep Bones for Regenerative Medicine: Structural, Morphologic and Electrical Studies.

Hydroxyapatite (HA) promotes excellent bone regeneration in bone-tissue engineering, due to its similarity to bone mineral and its ability to connect to living tissues. These factors promote the osteointegration process. This process can be enhanced by the presence of electrical charges, stored in the HA. Furthermore, several ions can be added to the HA structure to promote specific biological responses, such as magnesium ions. The main objective of this work was to extract hydroxyapatite from sheep femur bones and to study their structural and electrical properties by adding different amounts of magnesium oxide. The thermal and structural characterizations were performed using DTA, XRD, density, Raman spectroscopy and FTIR analysis. The morphology was studied using SEM, and the electrical measurements were registered as a function of frequency and temperature. Results show that: (i) an increase of MgO amount indicates that the solubility of MgO is below 5%wt for heat treatments at 600 °C; (ii) the rise of MgO content increases the capacity for electrical charge storage; (iii) sheep hydroxyapatite presents itself as a natural source of hydroxyapatite, environmentally sustainable and low cost, and promising for applications in regenerative medicine.

Hydroxyapatite Thin Films of Marine Origin as Sustainable Candidates for Dental Implants.

Novel biomaterials with promising bone regeneration potential, derived from rich, renewable, and cheap sources, are reported. Thus, thin films were synthesized from marine-derived (i.e., from fish bones and seashells) hydroxyapatite (MdHA) by pulsed laser deposition (PLD) technique. Besides the physical-chemical and mechanical investigations, the deposited thin films were also evaluated in vitro using dedicated cytocompatibility and antimicrobial assays. The morphological examination of MdHA films revealed the fabrication of rough surfaces, which were shown to favor good cell adhesion, and furthermore could foster the in-situ anchorage of implants. The strong hydrophilic behavior of the thin films was evidenced by contact angle (CA) measurements, with values in the range of 15-18°. The inferred bonding strength adherence values were superior (i.e., ~49 MPa) to the threshold established by ISO regulation for high-load implant coatings. After immersion in biological fluids, the growth of an apatite-based layer was noted, which indicated the good mineralization capacity of the MdHA films. All PLD films exhibited low cytotoxicity on osteoblast, fibroblast, and epithelial cells. Moreover, a persistent protective effect against bacterial and fungal colonization (i.e., 1- to 3-log reduction of E. coli, E. faecalis, and C. albicans growth) was demonstrated after 48 h of incubation, with respect to the Ti control. The good cytocompatibility and effective antimicrobial activity, along with the reduced fabrication costs from sustainable sources (available in large quantities), should, therefore, recommend the MdHA materials proposed herein as innovative and viable solutions for the development of novel coatings for metallic dental implants.

Vitamin D3/vitamin K2/magnesium-loaded polylactic acid/tricalcium phosphate/polycaprolactone composite nanofibers demonstrated osteoinductive effect by increasing Runx2 via Wnt/ß-catenin pathway.

Vitamin D3, vitamin K2, and Mg (10%, 1.25%, and 5%, w/w, respectively)-loaded PLA (12%, w/v) (TCP (5%, w/v))/PCL (12%, w/v) 1:1 (v/v) composite nanofibers (DKMF) were produced by electrospinning method (ES) and their osteoinductive effects were investigated in cell culture test. Neither pure nanofibers nor DKMF caused a significant cytotoxic effect in fibroblasts. The induction of the stem cell differentiation into osteogenic cells was observed in the cell culture with both DKMF and pure nanofibers, separately. Vitamin D3, vitamin K2, and magnesium demonstrated to support the osteogenic differentiation of mesenchymal stem cells by expressing Runx2, BMP2, and osteopontin and suppressing PPAR-γ and Sox9. Therefore, the Wnt/ß-catenin signaling pathway was activated by DKMF. DKMF promoted large axonal sprouting and needle-like elongation of osteoblast cells and enhanced cellular functions such as migration, infiltration, proliferation, and differentiation after seven days of incubation using confocal laser scanning microscopy. The results showed that DKMF demonstrated sustained drug release for 144 h, tougher and stronger structure, higher tensile strength, increased water up-take capacity, decreased degradation ratio, and slightly lower Tm and Tg values compared to pure nanofibers. Consequently, DKMF is a promising treatment approach in bone tissue engineering due to its osteoinductive effects.

Investigation of 3D-Printed Polycaprolactone-/Polyvinylpyrrolidone-Based Constructs.

The aim of this study is to evaluate the mechanical and biological performance of cartilage-like constructs produced by 3D printing. During the investigation, poly(ε-caprolactone) (PCL) and polyvinylpyrrolidone (PVP) were used as a matrix polymer and low-molecular-weight chitosan (CS), hyaluronic acid (HA), and alginic acid sodium salt (SA) were integrated separately with the polymer matrix to fabricate the constructs. Thermal, mechanical, morphology, and chemical properties and swelling, degradation, and biocompatibility behaviors were evaluated in detail. With the addition of 3 fillers, the melting temperature of the matrix increased with the addition of fillers, and PCL/3wt.%PVP/1wt.%HA had the highest melting temperature value. Mechanical characterization results demonstrated that the printed PCL/3wt.%PVP/1wt.%CS displayed the highest compressive strength of around 9.51 MPa. The compressive strength difference between the PCL/3wt.%PVP and PCL/3wt.%PVP/1wt.%CS was 5.38 MPa. Biocompatibility properties of the constructs were tested by mitochondrial dehydrogenase activity, and in vitro studies showed that the PCL/3wt.%PVP/1wt.%HA composite construct had more cell viability than the other constructs by making use of the mesenchymal stem cell line.

Fabrication of tissue-engineered tympanic membrane patches using 3D-Printing technology.

In recent years, scaffolds produced in 3D printing technology have become more widespread tool due to providing more advantages than traditional methods in tissue engineering applications. In this research, it was aimed to produce patches for the treatment of tympanic membrane perforations which caused significant hearing loss by using 3D printing method. Polylactic acid(PLA) scaffolds with Chitosan(CS) and Sodium Alginate(SA) added in various ratios were prepared for artificial eardrum patches. Different amounts of chitosan and sodium alginate added to PLA increased the biocompatibility of the produced scaffolds. The created patches were designed by mimicking the thickness of the natural tympanic membrane thanks to the precision provided by the 3D printed method. The produced scaffolds were analyzed separately for chemical, morphological, mechanical and biocompatibility properties. Scanning electron microscope (SEM), Fourier-transform infrared (FT-IR) spectroscopy was performed to observe the surface morphology and chemical structure of the scaffolds. Mechanical, thermal and physical properties, swelling and degradation behaviors were examined to fully analyze whole characteristic features of the samples. Cell culture study was also performed to demonstrate the biocompatibility properties of the fabricated scaffolds with human adipose tissue-derived mesenchymal stem cells (hAD-MSCs). 15 wt % PLA was selected as the control group and among all concentrations of CS and SA, groups containing 3 wt% CS and 3 wt% SA showed significantly superior and favorable features in printing quality. The research continued with these two scaffolds (3 wt% CS, and 3 wt% SA), which showed improved print quality when added to PLA. Overall, these results show that PLA/CS and PLA/SA 3D printed artificial patches have the potential to tissue engineering solutions to repair tympanic membrane perforation for people with hearing loss.

Comparative Analysis of NF-κB in the MyD88-Mediated Pathway After Implantation of Titanium Alloy and Stainless Steel and the Role of Regulatory T Cells.

OBJECTIVE: Development of immunologically smart implants, integrated to biological systems, is a key aim to minimize the inflammatory response of the host to biomaterial implants. METHODS: The aim of this study is to investigate the influence of titanium alloy and stainless steel implants on immunological responses in rats by comparative analysis of nuclear factor kappa B (NF-κB) profiles in the activation of inflammatory signaling pathways and the role of CD4+CD25+Foxp3+. RESULTS: Both Ti alloy and stainless steel alloy group implantation affect Toll-like receptors-4 pathways and CD4+CD25+ regulatory T cells in different ways. CONCLUSIONS: Results show that NF-κB/p65 and NF-κB1/p50 possess potential as a therapeutic target in the prevention of adverse reactions to metal, especially for controlling inflammation after the implantation.

Development of Satureja cuneifolia-loaded sodium alginate/polyethylene glycol scaffolds produced by 3D-printing technology as a diabetic wound dressing material.

Acute wounds are a common health problem, with millions of people affected and decreased granulation tissue formation and vascularization, it is also a big challenge for wound care researchers to promote acute wound healing around the globe. This study aims to produce and characterize Satureja cuneifolia plant extract (SC)-blended with sodium alginate (SA) /polyethylene glycol (PEG) scaffolds for the potential treatment of diabetic ulcer. SA/PEG scaffolds were prepared by adding different concentrations (1, 3, and 5 wt%) of PEG to 9 wt% SA. The morphological and chemical composition of the resulting 3D printed composite scaffolds was determined using scanning electron microscopy (SEM) and Fourier transforms infrared spectroscopy (FTIR), respectively. Mechanical and thermal properties, swelling, and degradation behaviours were also investigated. The release kinetics of SC were performed. The antimicrobial analysis was evaluated against Escherichia coli and Staphylococcus aureus strains. 3D printed scaffolds have shown an excellent antibacterial effect, especially against gram-positive bacteria due to the antibacterial SC extract they contain. Furthermore, the cell viability of fibroblast (L929) cells on/within scaffolds were determined by the colourimetric MTT assay. The SA/PEG/SC scaffolds show a great promising potential candidate for diabetic wound healing and against bacterial infections.

Polycaprolactone/Gelatin/Hyaluronic Acid Electrospun Scaffolds to Mimic Glioblastoma Extracellular Matrix.

Glioblastoma (GBM), one of the most malignant types of human brain tumor, is resistant to conventional treatments and is associated with poor survival. Since the 3D extracellular matrix (ECM) of GBM microenvironment plays a significant role on the tumor behavior, the engineering of the ECM will help us to get more information on the tumor behavior and to define novel therapeutic strategies. In this study, polycaprolactone (PCL)/gelatin(Gel)/hyaluronic acid(HA) composite scaffolds with aligned and randomly oriented nanofibers were successfully fabricated by electrospinning for mimicking the extracellular matrix of GBM tumor. We investigated the effect of nanotopography and components of fibers on the mechanical, morphological, and hydrophilic properties of electrospun nanofiber as well as their biocompatibility properties. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) have been used to investigate possible interactions between components. The mean fiber diameter in the nanofiber matrix was increased with the presence of HA at low collector rotation speed. Moreover, the rotational velocity of the collector affected the fiber diameters as well as their homogenous distribution. Water contact angle measurements confirmed that hyaluronic acid-incorporated aligned nanofibers were more hydrophilic than that of random nanofibers. In addition, PCL/Gel/HA nanofibrous scaffold (7.9 MPa) exhibited a significant decrease in tensile strength compared to PCL/Gel nanofibrous mat (19.2 MPa). In-vitro biocompatibilities of nanofiber scaffolds were tested with glioblastoma cells (U251), and the PCL/Gel/HA scaffolds with random nanofiber showed improved cell adhesion and proliferation. On the other hand, PCL/Gel/HA scaffolds with aligned nanofiber were found suitable for enhancing axon growth and elongation supporting intracellular communication. Based on these results, PCL/Gel/HA composite scaffolds are excellent candidates as a biomimetic matrix for GBM and the study of the tumor.

Glioblastoma cell adhesion properties through bacterial cellulose nanocrystals in polycaprolactone/gelatin electrospun nanofibers.

Glioblastoma (GBM), the most common and extremely lethal type of brain tumor, is resistant to treatment and shows high recurrence rates. In the last decades, it is indicated that standard two-dimensional (2D) cell culture is inadequate to improve new therapeutic strategies and drug development. Hence, well-mimicked three-dimensional (3D) tumor platforms are needed to bridge the gap between in vitro and in vivo cancer models. In this study, bacterial cellulose nano-crystal (BCNC) containing polycaprolactone (PCL) /gelatin (Gel) nanofibrous composite scaffolds were successfully fabricated by electrospinning for mimicking the extracellular matrix of GBM tumor. The fiber diameters in the nanofibrous matrix were increased with an increased concentration of BCNC. Moreover, fiber morphology changed from the smooth formation to the beaded formation by increasing the concentration of the BCNC suspension. In-vitro biocompatibilities of nanofibrous scaffolds were tested with U251 MG glioblastoma cells and improved cell adhesion and proliferation was compared with PCL/Gel. PCL/Gel/BCNC were found suitable for enhancing axon growth and elongation supporting communication between tumor cells and the microenvironment, triggering the process of tumor recurrence. Based on these results, PCL/Gel/BCNC composite scaffolds are a good candidate for biomimetic GBM tumor platform.

Encapsulated melatonin in polycaprolactone (PCL) microparticles as a promising graft material.

Electrospraying assures many advantages with taking less time and costing less relatively to the other conventional particle production methods. In this research, we investigated the encapsulation of melatonin (MEL) hormone in polycaprolactone (PCL) microparticles by using electrospraying method. Morphology analysis of the produced particles completed with Scanning Electron Microscopy (SEM). SEM images demonstrated that micro-particles of 3 wt% PCL solution has the most suitable particle diameter size (2.3 ±â€¯0.64 µm) for melatonin encapsulation. According to the characterization of the particles, electrospraying parameters like optimal collecting distance, the flow rate of the solution and voltage of the system detected as 8 cm, 0.5 ml/h, and 10 kV respectively. For determining the chemical bonds of scaffold Fourier-Transform Infrared Spectroscopy (FTIR) were used and FTIR results showed that melatonin successfully loaded into PCL micro-particles. Drug release kinetics of the melatonin loaded particles indicated that melatonin released with a burst at the beginning and release behavior became sustainable over a period of 8 h with the encapsulation efficiency of about 73%. In addition, both in-vitro and in-vivo studies of the graft materials also completed. Primary human osteoblasts (HOB) cells and female Sprague Dawley rats were used in in-vitro and in-vivo studies. Test results demonstrate cell population, and bone volume of the rats grafted with composites has remarkably increased, this caused remodelling in bone structure. Overall, these findings indicate that encapsulation of melatonin in the PCL particles with electrospray method is optimum for new synthetic graft material.

Novel electrospun polycaprolactone/graphene oxide/Fe3O4 nanocomposites for biomedical applications.

In this study, one of the most promising methods of tailoring a composite scaffold material in nano sized diameters, electrospinning method were used to produce Polycaprolactone (PCL)/Graphene Oxide (GO)/Iron(II, III) Oxide (Fe3O4) nanocomposite fibers as biocompatible scaffolds for biomedical applications. Products were analyzed by scanning electron microscopy (SEM) for morphological analysis of the electrospun nanocomposites and Fourier Transform Infrared Spectroscopy (FTIR) was used to determine functional groups of the PCL, GO, and Fe3O4 materials in the electrospun nanocomposites. For physical properties, viscosity, density, permittivity, dielectric loss and liquid and solid state alternating current conductivity, measurements were done for each nanocomposite fibers. Effects of concentration percentage of GO on permittivity, dielectric loss and AC conductivity have been analyzed by using measured and calculated data. Trend lines have been drawn for permittivity, dielectric loss and conductivity via concentration percentage of GO. The relation between ac conductivity and frequency have been studied for each concentration percentage of GO and interpretations have been done by using the obtained results.

Chitosan/poly(ethylene glycol)/hyaluronic acid biocompatible patches obtained by electrospraying.

Electrospray is a promising technique to scale-up production of microparticles and nanoparticles. In this study, electrospraying was used in order to produce candidate biopatches (CPH) by using chitosan, poly(ethylene glycol) (PEG) and hyaluronic acid (HA). Four different ratios of polymer blend compositions (CPH1, CPH2, CPH3 and CPH4) were tested by dissolving in 2% acetic acid solution (Ac.A.). The HA amount in each blend was kept the same to designate the optimum surface with different chitosan/PEG ratios for electrospray process. Fourier-transform infrared (FTIR) microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) studies showed that obtained patches had highly adhesive surfaces with the aid of heterogeneously distributed micro- and nano-particles. Additionally, video images of FTIR microscopy and AFM images proved that all surfaces have similar heterogeneity except CPH2. The most homogenous surface was obtained by CPH3. Patches were directly subjected to antibacterial tests against ten different types of gram positive and gram negative bacteria using disc diffusion assay (Kirby-Bauer method). Extraordinarily there was no antibacterial property of patches coated with microparticles. Finally, biocompatibility studies were performed by using mouse fibroblast L929 cell lines (ATTC number CCL-1) to test cell adhesion and proliferation properties of the patches. Results of 72 h viability tests proved the electrospray of ternary blends had displayed good biocompatibility; in particular, CPH3 had the highest cell viability.

Development of Amoxicillin-Loaded Electrospun Polyurethane/Chitosan/ $\beta$ -Tricalcium Phosphate Scaffold for Bone Tissue Regeneration.

Biocompatible nanocomposite electrospun fibers containing Polyurethane/Chitosan/ $\beta $ -Tri calcium phosphate with diverse concentrations were designed and produced through the electrospinning process for bone tissue engineering applications. After the production process, density measurement, viscosity, electrical conductivity, and tensile strength measurement tests were carried out as physical analyses of blended solutions. The chemical structural characterization was scrutinized using Fourier transform infrared spectrometer (FTIR), and scanning electron microscopy (SEM) was used to observe the morphological details of developed electrospun scaffolds. Cell viability, attachment, and proliferation were performed using a L929 fibroblast cell line. Based on the physical, SEM, FTIR analysis, and cell culture studies, preferable nanofiber composition was selected for further studies. Amoxicillin (AMX) was loaded to that selected nanofiber composition for examination of the drug release. In comparison with other studies on similar AMX controlled products, higher drug loading and encapsulation efficiencies were obtained. It has been clearly found that the developed nanofiber composites have potential for bone tissue engineering applications.

Fabrication of naturel pumice/hydroxyapatite composite for biomedical engineering.

BACKGROUND: We evaluated the Bovine hydroxyapatite (BHA) structure. BHA powder was admixed with 5 and 10 wt% natural pumice (NP). Compression strength, Vickers micro hardness, Fourier transform infrared spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction studies were performed on the final NP-BHA composite products. The cells proliferation was investigated by MTT assay and SEM. Furthermore, the antimicrobial activity of NP-BHA samples was interrogated. RESULTS: Variances in the sintering temperature (for 5 wt% NP composites) between 1000 and 1300 °C, reveal about 700 % increase in the microhardness (~100 and 775 HV, respectively). Composites prepared at 1300 °C demonstrate the greatest compression strength with comparable result for 5 wt% NP content (87 MPa), which are significantly better than those for 10 wt% and those that do not include any NP (below 60 MPa, respectively). CONCLUSION: The results suggested the optimal parameters for the preparation of NP-BHA composites with increased mechanical properties and biocompatibility. Changes in micro-hardness and compression strength can be tailored by the tuning the NP concentration and sintering temperature. NP-BHA composites have demonstrated a remarkable potential for biomedical engineering applications such as bone graft and implant.

Histomorphometric evaluation of implants coated with enamel or dentine derived fluoride-substituted apatite.

OBJECTIVES: The aim of this study was to compare osseous healing characteristics of titanium implants coated with enamel-derived fluoride-substituted apatite (EFSA) or dentin-derived fluoride-substituted apatite (DFSA). METHODS: Fluoride-substituted apatite was derived from extracted human teeth with calcination method at 850 degrees C. DFSA and EFSA were separated and carefully ground with a blade grinder. Twenty-four titanium implants were prepared from a 99.99% pure titanium bar. EFSA and DFSA powders were sprayed separately on implants. As control group, unsprayed and sandblasted pure titanium implants were used. Eight adult rams were used in the study. One EFSA coated, 1 DFSA coated and 1 control implants were placed into right tibia of each rams. The rams were sacrificed after 6 months of healing. Undecalcified sections were prepared according to Donath's method and histomorphometric evaluation of implants was made. RESULTS: The mean bone contact percentage of DFSA-coated, EFSA-coated and control implants was 89.88%+/-2.34, 70.19%+/-13.11 and 53.12%+/-5.76 respectively. This study suggests that DFSA-coated implants achieved better bone contact than EFSA-coated implants (P<0.05). Also study groups presented better bone contact than control group (P<0.05). CONCLUSIONS: The results of this study show that although DFSA-coated implants achieved better bone contact, both DFSA and EFSA can be considered as appropriate coating materials.

Mechanical properties of bovine hydroxyapatite (BHA) composites doped with SiO2, MgO, Al2O3, and ZrO2.

Biologically derived hydroxyapatite from calcinated (at 850 degrees C) bovine bones (BHA) was doped with 5 wt% and 10 wt% of SiO(2), MgO, Al(2)O(3) and ZrO(2) (stabilized with 8% Y(2)O(3)). The aim was to improve the sintering ability and the mechanical properties (compression strength and hardness) of the resultant BHA-composites. Cylindrical samples were sintered at several temperatures between 1,000 and 1,300 degrees C for 4 h in air. The experimental results showed that sintering generally occurs at 1,200 degrees C. The BHA-MgO composites showed the best sintering performance. In the BHA-SiO(2) composites, extended formation of glassy phase occurred at 1,300 degrees C, resulting in structural degradation of the resultant samples. No sound reinforcement was achieved in the case of doping with Al(2)O(3) and zirconia probably due to the big gap between the optimum sintering temperatures of BHA and these two oxides.

Low-cost antibiotic loaded systems for developing countries.

Treatment of osteomyelitis with local antibiotic delivery systems has become a common practice in orthopaedic surgery. This study attempted to show that locally produced pure or bioglass reinforced plaster of Paris, hydroxyapatite and sodium alginate are promising biomaterials and mainly because of economical reasons and availability, may be an alternative in clinical practice, especially for developing countries. A total of 32 rabbits were divided into four groups (n:8). In group A, sodium alginate + cephazoline; in group B, plaster of Paris + bioglass + cephazoline; in group C, plaster of Paris + hydroxyapatite + cephazoline and in group D, plaster of Paris + cephazoline were used. The blood serum cephazoline concentrations were analyzed by high performance liquid chromatography on days 1 to 10 everyday and then at days 13, 17, 18, 24, 25 and 30. The mean values +/- standard deviations and median values of blood serum antibiotic concentrations for groups A, B, C and D were 1.45 +/- 0.40 (1.42) mcg/ml, 1.53 +/- 0.64 (1.31) mcg/ml, 1.92 +/- 0.39 mcg/ml (1.90) and 1.41 +/- 0.65 (1.25) mcg/ml, respectively. The detected antibiotic level was constantly over the minimum inhibitory concentration for Staphylococcus aureus. In conclusion, it can be stated that these materials are promising as a antibiotic delivery system even with simple production methods.