Hyaluronic acid/corn silk extract based injectable nanocomposite: A biomimetic antibacterial scaffold for bone tissue regeneration.

Injectable hydrogels have revealed the great potential for use as scaffolds in cartilage and bone tissue engineering. Here, thermosensitive and injectable hydrogels containing ß-tricalcium phosphate, hyaluronic acid, and corn silk extract-nanosilver (CSE-Ag NPs) were synthesized for their potential use in bone tissue regeneration applications. Spherical nanoparticles of silver were biosynthesized through microwave-assisted green approach using CSE in organic solvent-free medium. Rheological experiments demonstrated that the thermosensitive hydrogels have gelification temperature (Tgel) close to body temperature. The samples containing Ag NPs showed antibacterial activity toward gram-positive (Bacillus Subtilis, Staphylococcus Aureus) and gram-negative (Pseudomonas Aeruginosa, Escherichia Coli) bacteria along without cytotoxicity after 24 h. Mesenchymal stem cells seeded in the nanocomposite exhibited high bone differentiation which indicate that thay could be a good candidate as a potential scaffold for bone tissue regeneration.

Biosynthesis and characterization of antibacterial thermosensitive hydrogels based on corn silk extract, hyaluronic acid and nanosilver for potential wound healing.

The wounds closure after physical injury or surgery is of significant clinical and research importance. In this study, thermosensitive and injectable hydrogels based on hyaluronic acid (HA), corn silk extract (CSE) and nanosilver were prepared and their potential use as a wound care material was investigated. Silver nanoparticles (Ag NPs) were biosynthesized by a microwave-assisted green technique using corn silk extract in an organic solvent-free medium. Rheological analysis demonstrated that the nanocomposites have good mechanical properties with gelation temperature close to the body temperature; hence, they can be easily administrated locally on wounded skins. The samples exhibited antibacterial activity toward gram-positive and gram-negative bacteria. Cytotoxicity assay showed that the hydrogels have good biocompatibility. Interestingly, an in-vitro model of wound healing revealed that the nanocomposites allow faster wound closure and repair, compared to the control. The obtained results highlight the potential application of these novel injectable hydrogels as wound dressing.

Thermosensitive chitosan/phosphate hydrogel-composites fortified with Ag versus Ag@Pd for biomedical applications.

INTRODUCTION: Thermo-responsive hydrogels are promising biomedical systems as their gelation is triggered by temperature changes. Greenly synthesized noble metallic nanoparticles are a growing research area assessing their potential applications in nanomedicine. MATERIALS AND METHODS: Chitosan/phosphate thermosensitive gels were successfully achieved. The developed composite scaffolds were functionalized with the greenly synthesized Ag or Ag@Pd targeting improved bactericidal activity and biocompatibility performance. The physicochemical characterization was assessed through TGA, DSC, FESEM, HRTEM, XRD and FTIR. Bactericidal activities were tested against gram- positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa. Their biodegradability upon DMEM immersion was followed up to seven days through measuring ionic concentrations of Ca, P, Ag and Pd successively. KEY FINDINGS: The newly developed phosphatic layers over the scaffold surfaces post-immersion assessed their osteogenic ability. Further, their promising and differentiated bactericidal activities due to the noble metals incorporation were proved. Cytotoxicity assessment demonstrated their high biocompatibility since no toxic effect was recorded. SIGNIFICANCE: Consequently, they can be successfully and directly applied in biomedical and dental surgeries.

Regeneration of periapical lesions post-endodontic treatment and periapical surgeries in experimental animals utilizing thermo-responsive nano-ß-tricalcium phosphate/chitosan hydrogel: a proof of concept.

Using phosphate nanoparticles/polymeric hydrogels presents an interesting approach, especially concerning the reduced particle migration and enhanced biocompatibility. The current work aims to achieve a proof of concept for the development of a thermo-sensitive nano ß-tricalcium phosphate (ß-TCP)/chitosan (Cs)/glycerophosphate (Gl)/glyoxal (Gly) hydrogel to be applied in periapical surgeries post endodontic treatment. Physicochemical characterization using x-ray powder diffraction, Fourier transform infrared, TEM and SEM was performed. Bone formation efficiency of the achieved ß-TCP/Cs/Gl/Gly hydrogel was followed. The composite gels were tested in vivo in dogs in comparison with the commercially available and surgically applied Klipdent-PL® up to three months. Radiographic examinations were performed. Histological evaluations were achieved through histomorphological criteria being apical cementum surface, bone tissue resorption, apical PDL thickness, the intensity of inflammatory reaction and osseous repair. The cytotoxicity results proved the safety of the developed hydrogel. The thermo-sensitive hydrogel possessed comparable enhanced biocompatibility with anti-inflammatory activity. New bone formation was clearly enhanced in the infected teeth. Therefore, it can be directly applied in specific non-invasive dental surgeries.

Synthesis of biogenic Ag@Pd Core-shell nanoparticles having anti-cancer/anti-microbial functions.

Biogenic Ag@Pd core-shell nanoparticles were greenly synthesized within two plant extracts aiming at enhanced anticancer/bactericidal functions. These functions were verified for the two Pd@Ag biogenic core-shell nanoparticles (BCSnp) with constant Pd to several Ag contents. BCSnp were synthesized within two extracts of Almond nuts and Black Berry fruits, four samples each, through simple, low cost and echo friendly microwave route. The BCSnp Surface Plasmon Resonance (SPR) was detected via UV/visible spectrophotometer. Their morphology was assessed using High-Resolution Transmission Electron Microscope and Field Emission Scanning Electron Microscope supplemented with EDAX. Particle size/zeta potential of the achieved nanoparticles was measured. The active reducing groups were depicted by FTIR while XRD assessed nanoparticles crystallinity. The enhanced particle size distribution as proved by UV and band gap energies, imparted better functionality by the Almond extract compared to the berry one due to its protein content. Cytotoxicity against human breast cancer (MCF7) and liver cancer (HEPG2) cell lines were followed and compared to the normal Wish cells. The antimicrobial impact against gram-negative (G-veo) E. coli, gram-positive (G+ve) S. aureus bacteria and mycotic strain C. albicans species were verified and compared to antibiotics. A significant inhibition of cancer cell growth of MCF 7 and HEPG2 compared to Wish normal cells and doxorubicin is assessed. A discriminative effect was recorded for G-ve compared to G+ve, along with Mycotic strain C. albicans is achieved. The obtained BCSnp are proposed for cancer therapy and bactericidal applications with improved efficiency applying the nanomedicine approach. Tailorable properties can be obtained by tuning the individual structures.

Influence of the protocol of fibroin extraction on the antibiotic activities of the constructed composites.

The effect of the solvents for silk fibroin (SF) extraction on its antimicrobial activity was studied. Extraction protocols were performed using LiBr (SFL) and Ajisawa's reagent (CaCl2:ethanol:H2O) (SFC). The morphological and structural characteristics of the extracted SF and their composites were assessed. Corresponding bactericidal activities against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Pseudomonas aeroginosa (ATCC 27853) were performed. The resulting solutions were either casted into films or individually incorporated into composites of silver nanoparticles (NS) embedded into chitosan fragments (Cs) through γ-irradiation. Films of SF, obtained by using the two solvents, as well as the final prepared composites of SF, NS and Cs were analyzed using XRD, FTIR, SEM, TEM and zeta potential at several pH values. The band gap values were calculated. The results proved that, although SFC consumed shorter gelation time, yet SFL exerted higher antibiotic activity against the tested microorganisms. Moreover, the final composites had the ability to significantly reduce the growth of these medically relevant bacteria and are, therefore, recommended as a novel natural antibacterial biomaterial for several biomedical applications.

Tailoring the properties and functions of phosphate/silk/Ag/chitosan scaffolds.

Two novel silk composites of phosphatic phases with nanosilver/chitosan having enhanced biocompatibility were achieved. Hydroxyapatite and octa calcium phosphates were synthesized in situ within silk fibroin/chitosan/nanosilver composites recently studied. Thermo-gravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC) verified their thermal behavior. The structural aspects were characterized applying X-ray diffraction analysis (XRD), transmission electron microscopy (TEM) and field emission scanning electron microscope (FESEM) with EDAX. Additionally X-ray Photoelectron Spectroscopy (XPS) and Fourier Transform Infrared spectroscopy (FTIR) were applied. Mercury porosimeter was used to verify the pore size distribution. The in vitro degradation was followed in D-MEM for 48 h in a cumulative manner for five successive periods. Biochemical analyses of Ca, P and total protein using relevant chemical kits and atomic absorption for silver were performed. ANOVA statistics was carried out. Phosphatic crystalline phases along with the presence of silk, chitosan and nano-silver were developed. The diameters of hydroxyapatite and octa calcium phosphate particles were ~8-17 nm and 15-22 nm respectively. Comparatively higher degradation of Octa composite possessing higher porosity proved in turn more osteoinduction with in situ apatitic development.

Alternative technique for calcium phosphate coating on titanium alloy implants.

As an alternative technique for calcium phosphate coating on titanium alloys, we propose to functionalize the metal surface with anionic bath containing chlorides of palladium or silver as activators. This new deposition route has several advantages such as controlled conditions, applicability to complex shapes, no adverse effect of heating, and cost effectiveness. A mixture of hydroxyapatite and calcium phosphate hydrate is deposited on the surface of Ti-6Al-4V. Calcium phosphate coating is built faster compared with the one by Simulated Body Fluid. Cell morphology and density are comparable to the control one; and the results prove no toxic compound is released into the medium during the previous seven days of immersion. Moreover, the cell viability is comparable with cells cultivated with the virgin medium. These experimental treatments allowed producing cytocompatible materials potentially applicable to manufacture implantable devices for orthopedic and oral surgeries.

Novel biomimetic thermosensitive ß-tricalcium phosphate/chitosan-based hydrogels for bone tissue engineering.

Among the less invasive surgical procedures for tissue engineering application, injectable in situ gelling systems have gained great attention. In this contest, this article is aimed to realize thermosensitive chitosan-based hydrogels, crosslinked with ß-glycerophosphate and reinforced via physical interactions with ß-tricalcium phosphate. The kinetics of sol-gel transition and the composite hydrogel properties were investigated by rheological analysis. The hydrogels were also characterized by Fourier transform infrared study, X-ray diffraction, scanning electron microscopy, transmission electron microscopy analysis, and thermal and biological studies. The hydrogels exhibit a gel-phase transition at body temperature, and a three-dimensional network with typical rheological properties of a strong gel. The presence of the inorganic phase, made up of nanocrystals, provides a structure with chemico-physical composition that mimics natural bone tissue, favoring cellular activity. These findings suggest the potential of the materials as promising candidates for hard tissue regeneration.

An efficient biomimetic coating methodology for a prosthetic alloy.

The combination of the load-bearing metallic implants with the bioactive materials in the design of synthetic implants is an important aspect in the biomaterials research. Biomimetic coating of bioinert alloys with calcium phosphate phases provides a good alternative to the prerequisite for the continual replacement of implants because of the failure of bone-implant integration. We attempted to accelerate the biomimetic coating process of stainless steel alloy (316L) with biomimetic apatite. In addition, we investigated the incorporation of functioning minerals such as strontianite and smithsonite into the deposited layer. In order to develop a highly mature apatite coating, our method requires soaking of the pre-treated alloy in highly concentrated synthetic body fluid for only few hours. Surface characterizations were performed by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS). Also, the deposited apatitic layers were analysed by powder diffraction X-ray analysis (XRD). 316L surface showed the growth of highly crystalline, low carbonated hydroxyapatite, after only 6h of the whole soaking process.

Nano-beta-tricalcium phosphates synthesis and biodegradation: 2. Biodegradation and apatite layer formation on nabo-ß-TCP synthesized via microwave treatment.

The degradation and/or apatite layer precipitation ability of porous ß-tricalcium phosphate(ß-TCP) samples treated and untreated with microwave radiation during synthesis is investigated. Microwave heating was used to accelerate the formation of CDHA with the Ca/P ratio 1.5 in a shorter processing time which later forms ß-TCP at around 650 ◦C. Soaking in simulated body fluid (SBF) for several periods (4, 8, 12, 24, 36, 48, 60 and 72 h) is performed in a cumulative manner. The deposition of an apatite layer is followed through diffuse reflected FT-IR, SEM and EDS. A microwave-treated sample having a smaller particle size than its parent induces the formation of a homogeneous carbonated apatite layer on its surface.On the other hand, the parent ß-TCP sample exhibited less ability to induce Ca­P formation after being soaked in SBF. The formation of an apatite layer is attributed to the increase in surface area consequent to reduced particle and grain sizes besides the presence of a minor amount of hydroxyapatite phase in the microwave-treated ß-TCP sample. The results prove that it is possible to control the biodegradation and apatite layer formation on sintered ß-TCP porous disks through controlling the particle size.

Chitosan-poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: in vitro degradation and in vivo bone regeneration studies.

Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation.

Functionalization of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering.

Scaffolds exhibiting biological recognition and specificity play an important role in tissue engineering and regenerative medicine. The bioactivity of scaffolds in turn influences, directs, or manipulates cellular responses. In this study, chitosan/poly(lactic acid-co-glycolic acid) (chitosan/PLAGA) sintered microsphere scaffolds were functionalized via heparin immobilization. Heparin was successfully immobilized on chitosan/PLAGA scaffolds with controllable loading efficiency. Mechanical testing showed that heparinization of chitosan/PLAGA scaffolds did not significantly alter the mechanical properties and porous structures. In addition, the heparinized chitosan/PLAGA scaffolds possessed a compressive modulus of 403.98 +/- 19.53 MPa and a compressive strength of 9.83 +/- 0.94 MPa, which are in the range of human trabecular bone. Furthermore, the heparinized chitosan/PLAGA scaffolds had an interconnected porous structure with a total pore volume of 30.93 +/- 0.90% and a median pore size of 172.33 +/- 5.89 mum. The effect of immobilized heparin on osteoblast-like MC3T3-E1 cell growth was investigated. MC3T3-E1 cells proliferated three dimensionally throughout the porous structure of the scaffolds. Heparinized chitosan/PLAGA scaffolds with low heparin loading (1.7 microg/scaffold) were shown to be capable of stimulating MC3T3-E1 cell proliferation by MTS assay and cell differentiation as evidenced by elevated osteocalcin expression when compared with nonheparinized chitosan/PLAGA scaffold and chitosan/PLAGA scaffold with high heparin loading (14.1 microg/scaffold). This study demonstrated the potential of functionalizing chitosan/PLAGA scaffolds via heparinization with improved cell functions for bone tissue engineering applications.

Nano-beta-tricalcium phosphates synthesis and biodegradation: 1. Effect of microwave and SO(4)(2-) ions on beta-TCP synthesis and its characterization.

Nano-sized calcium deficient hydroxyapatite (CDHA) powders with an average particle size less than 100 nm were prepared by a co-precipitation method at low temperature. The initial Ca/P molar ratio was chosen to be less than the stoichiometric ratio of beta-TCP (1.5). Additionally, lowering the temperature and pH values accelerated HPO(4)(2-) incorporation in the CDHA structure. HPO(4)(2-) is considered as an essential source for beta-TCP formation. Sulfate ion doping during the maturation period is proved to be an effective step to eliminate the pyrophosphate P(2)O(7)(2-) phase that results during the calcination of CDHA with Ca/P < 1.5. Furthermore, the heating effect of microwave irradiation resulted in an increase in Ca ion concentration and lowered the CDHA deficiency which affected beta-TCP purity despite its ability to reduce the particle size. A purity of 99.32% beta-TCP with respect to the P(2)O(7)(2-) phase was achieved by increasing the sulfate ion concentration from 2% to 3% and the calcination temperatures from 900 degrees C to 1100 degrees C.

Synthesis, characterization of chitosans and fabrication of sintered chitosan microsphere matrices for bone tissue engineering.

The objective of the present study was to synthesize and characterize chitosans with different degrees of deacetylation (DDA%), prepare chitosan microspheres with controlled chemistry and geometry, and fabricate three-dimensional (3-D) chitosan matrices based on microspheres with appropriate pore size, porosity and mechanical properties suitable for bone tissue engineering applications. Chitosans with three DDA% of 69%, 79% and 97% were obtained using a thermomechanochemical technique by varying the applied pressure and NaOH solution concentration. The prepared chitosans were comprehensively characterized by proton nuclear magnetic resonance, elemental analysis, viscosity measurements, thermal analyses and X-ray diffraction. In addition, chitosan microspheres were prepared using an ionotropic gelation method. Three-dimensional chitosan matrices were fabricated via a sintered microsphere technique. Scanning electron microscopy revealed rough surfaces of the prepared chitosan microspheres. Mercury intrusion porosimetry revealed a porosity of 19.2% and a median pore diameter of 199.62microm of the fabricated 3-D matrix. The compressive modulus of the sintered microsphere matrix (662.26+/-54.53MPa) was in the range of human cancellous bone (10-2000MPa), making it suitable for bone tissue engineering applications.

In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering.

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170-200microm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.