Synergistic performance of collagen-g-chitosan-glucan fiber biohybrid scaffold with tunable properties.

Hybrid biocomposite scaffolds (HBS) that serve as a carrier for cell proliferation and differentiation are increasingly used for tissue regeneration. 3D hybrid scaffold based on collagen-grafted-chitosan-glucan fiber (CO-g-CGF-HBS) was prepared by freeze-drying technique. The swelling percentage, hydrolytic stability, and modulus of elasticity of HBS were enhanced after the chemical modification of CO with CGF. Pore size and porosity of HBS were decreased with an increased CGF ratio. HBS exhibits a higher reduction rate against different types of bacteria compared with a control sample. Thus, chemical modification of CO with different ratios of CGF significantly improved the physicochemical, antibacterial properties of HBS.

Chitosan-glucan complex hollow fibers reinforced collagen wound dressing embedded with aloe vera. II. Multifunctional properties to promote cutaneous wound healing.

This study presents an innovative multifunctional system in fabricating new functional wound dressing (FWD) products that could be used for skin regeneration, especially in cases of infected chronic wounds and ulcers. The innovation is based on the extraction, characterization, and application of collagen (CO)/chitosan-glucan complex hollow fibers (CSGC)/aloe vera (AV) as a novel FWS. For the first time, specific hollow fibers were extracted with controlled inner (500-900 nm)/outer (2-3 µm) diameters from mycelium of Schizophyllum commune. Further on, research and evaluation of morphology, hydrolytic stability, and swelling characteristics of CO/CSGC@AV were carried out. The obtained FWS showed high hydrolytic stability with enhanced swelling characteristics compared to native collagen. The hemostatic effect of FWS increased significantly in the presence of CSGC, compared to native CO and displayed excellent biocompatibility which was tested by using normal human dermal fibroblast (NHDF). The FWS showed high antibacterial activity against different types of bacteria (positive/negative grams). From in vivo measurements, the novel FWS increased the percentage of wound closure after one week of treatment. All these results imply that the new CO/CSGC@AV-FWD has the potential for clinical skin regeneration and applying for controlled drug release.

Hyaluronan biofilms reinforced with partially deacetylated chitin nanowhiskers: Extraction, fabrication, in-vitro and antibacterial properties of advanced nanocomposites.

Fabrication of nanocomposite biofilms with enhanced mechanical and antibacterial properties was successfully achieved from hyaluronan (HA) and partially deacetylated chitin nanowhiskers (ChNWs) by a casting-evaporation method. The hydrolysis process of chitin showed an important role in the dimensions, stability, and the crystallinity of extracted ChNWs in a time-dependent manner. The volume fraction of ChNWs nanofiller varying from (0.001 to 0.5) exhibited a great influence on the mechanical properties of the biofilms (young modulus, strength) was enhanced by the high load-bearing capacity of NWs compared with net HA film. The antibacterial activity of the nanocomposite biofilms exhibited significant bactericidal activity against different types of bacteria (-/+ gram). HA/ChNWs Nanocomposite biofilms did not show any toxicity against normal human dermal fibroblasts (NHDF) and human primary osteogenic sarcoma (Saos-2) cell lines. The new biofilms with unique properties like edibleness, environmental friendliness, high mechanical properties, antibacterial performance, and non-cytotoxicity that could be used in skin tissue regenerations, and drug delivery applications.

Comparative study of chitosan and silk fibroin staple microfibers on removal of chromium (VI): Fabrication, kinetics and thermodynamic studies.

Short staple microfibers (SSM) based on chitosan (CS) or silk fibroin (SF) were fabricated via the wet-rotate-spinning technique and employed to adsorb hexavalent chromium from aqueous solution. Adsorption efficiencies, physicochemical and morphological properties of CS and SF-SSM were systematically investigated and evaluated before and after adsorption of Cr(VI) using different techniques like ATR-FTIR, TGA, XRD, XPS, and SEM. CS and SF-SSM showed removal efficiency (>90 %) toward Cr(VI) ions. Pseudo-second order kinetic and Langmuir isotherm models could describe the Cr(VI) ions uptake process. Considering the inexpensive, sustainability and higher adsorption capacity of CS and SF-SSM hold great promising applications as natural adsorbent materials for removing different hazardous metals from aqueous medium.

Chitosan-glucan complex hollow fibers reinforced collagen wound dressing embedded with aloe vera. Part I: Preparation and characterization.

Collagen (CO)/chitosan-glucan complex (CSGC) hollow fibers encapsulated aloe vera (AV) dressing scaffold (CO/CSGC@AV) were fabricated for the first time by the freeze-dried process. Extraction process, morphology, mechanical properties, pore size, porosity, swelling ability, and degradation behavior of composites scaffold were investigated. CSGC hollow fibers were extracted from mycelium of Schizophyllum commune CSGC hollow fiber exhibited inner diameter of (600 ±â€¯250 nm) and outer fiber diameter of (2.5 ±â€¯0.5 µm). The results of swelling and hydrolytic degradation studies demonstrated that the physicochemical of CO/CSGC@AV was significantly enhanced by CSGC in a concentration-dependent manner. The mechanical property of the CO/CSGC@AV was improved after encapsulated AV into CSGC hollow fibers compared with native CO. The pore size and porosity of the CO/CSGC@AV were slightly decreased in the presence of AV. All these results suggested that the new dressing scaffold has a potential for clinical skin regeneration, particularly for infected chronic wounds and ulcers.

Electrospinning of hyaluronan/polyvinyl alcohol in presence of in-situ silver nanoparticles: Preparation and characterization.

Novel nanofibers mats were fabricated by electrospinning of polyvinyl alcohol/hyaluronan (PVA/HA) solutions in the presence of silver nanoparticles (AgNPs). The AgNPs were synthesized by in-situ chemical reduction of silver ions (Ag+) using HA as a reducing and stabilizing agent. Narrow size distribution and spherical shape of AgNPs were achieved by optimizing the initial silver nitrate concentration (0.01 to 1 M) and reaction time (10-60 min). HA-AgNPs nanocomposite and PVA/HA-AgNPs nanofibrous mats were fabricated by electrospinning technique from aqueous solution containing a different mass ratio of PVA and HA-AgNPs and characterized by UV/Vis spectroscopy, SEM, TEM, DLS, XRD, TGA, and ATR-FTIR. Mechanical and rheological properties were also investigated and discussed. The novel nanofibrous mats show great potential in skin regeneration and drug carrier applications.

A novel in situ silver/hyaluronan bio-nanocomposite fabrics for wound and chronic ulcer dressing: In vitro and in vivo evaluations.

In-situ formed hyaluronan/silver (HA/Ag) nanoparticles (NPs) were used to prepare composite fibers/fabrics for the first time. Different concentrations of silver nitrate (1, 2mg/100ml) were added at ambient temperature to sodium hyaluronate solution (40mg/ml), then the pH was increased to 8 by adding sodium hydroxide. The in-situ formed HA/Ag-NPs were used to prepare fibers/nonwoven fabrics by wet-dry-spinning technique (WDST). UV/vis spectroscopy, SEM, TEM, DLS, XPS, XRD and TGA were employed to characterize the structure and composition of the nanocomposite, surface morphology of fiber/fabrics, particle size of Ag-NPs, chemical interactions of Ag0 and HA functional groups, crystallinity and thermal stability of the wound dressing, respectively. The resultant HA/Ag-NPs1 and HA/Ag-NPs2 composite showed uniformly dispersed throughout HA fiber/fabrics (SEM), an excellent distribution of Ag-NPs with 25±2, nm size (TEM, DLS) and acceptable mechanical properties. The XRD analysis showed that the in-situ preparation of Ag-NPs increased the crystallinity of the resultant fabrics as well as the thermal stability. The antibacterial performance of medical HA/Ag-NPs fabrics was evaluated against gram negative bacteria E. coli K12, exhibiting significant bactericidal activity. The fibers did not show any cytotoxicity against human keratinocyte cell line (HaCaT). In-vivo animal tests indicated that the prepared wound dressing has strong healing efficacy (non-diabetics/diabetics rat model) compared to the plain HA fabrics and greatly accelerated the healing process. Based on our results, the new HA/Ag-NPs-2mg nonwoven wound dressing fabrics can be used in treating wounds and chronic ulcers as well as cell carrier in different biological research and tissue engineering.

Preparation and characterization of alginate/silver/nicotinamide nanocomposites for treating diabetic wounds.

Silver/Alginate/Nicotinamide nanoparticles composite (Ag/ALG/Nic) was prepared and used for the first time to fabricate wound dressing material. Sodium alginate (ALG) was used as reducing and stabilizing agents for preparation of silver nanoparticles (Ag-NPs). Effect of concentrations of alginate (ALG) on the particle size of silver were studied and confirmed by different techniques like UV/vis spectroscopy, transmission electron microscope (TEM) and dynamic light scattering (DLS). Nonwoven viscous fabrics were used as a carrier for silver/alginate/nanoparticles composite by impregnated the nonwoven fabrics as per the padding-curing technique. Nicotinamide (Nic) as anti-inflammatory drug was entrapped into Ag-NPS/ALG/nonwoven fabrics. Scanning electron microscope and energy dispersive x-ray (SEM-EDX) were used to evaluate the presence of Ag/ALG/Nic nanoparticles composite anchored the nonwoven fabrics. The antibacterial activity of the Ag/ALG/Nic wound dressing material was evaluated against Escherichia coli (E. coli) and Staphylococcus Aureus (St. Aureus). The wound healing and histological studied were evaluated by using burn diabetic rat animals.

Novel chitin/chitosan-glucan wound dressing: Isolation, characterization, antibacterial activity and wound healing properties.

Chitin/chitosan-glucan complex (ChCsGC) was isolated from Schizophyllum commune (S. commune) and dissolved for the first time in precooled (-15°C) 8wt.% urea/6wt.% NaOH aqueous solution. Novel nonwoven microfiber mats were fabricated by wet-dry-spinning technique and evaluated the mechanical of fabrics mats and surface morphology. Isolated and nonwoven mat were characterized employing FTIR-ATR, Optical microscope, TGA, DSC, H/C NMR, SEM and XRD techniques. According to the physical/chemical characterization measurements we can assumed that, the net and the novel dressing mats have the same chemical structure with slightly changes in the thermal stability for the dressing mats.The biological activity of the nonwoven ChCsGC fabric was tested against different types of bacteria exhibiting excellent antibacterial activity. Cell viability of the plain complex and nonwovens mats were evaluated utilizing mouse fibroblast cell line varying concentrations and treatment time. ChCsGC did not show any cytotoxicity against mouse fibroblast cells and the cell-fabrics interaction was also investigated using fluorescence microscope. The novel ChCsGC nonwovens exhibited excellent surgical wound healing ability when tested using rat models.

Wound healing of different molecular weight of hyaluronan; in-vivo study.

Recruitment of cells and mediators is altered during impaired wound healing, thereby delaying this process. To overcome this problem, the correlation of wound healing in older rats, and the impact of different molecular weight of hyaluronan without silver nanoparticles; (low-HA1), (High-HA2), (Medium- HA3) and with silver nanoparticles (High-HA4) is investigated. The superior HA were selected to be further investigated onto diabetic wounds. Our results pointed to a marked deficiency in wounds granulation in older rats, which was accompanied with impairment of healing process. In older rats group treated with HA2 or HA4, granulation and dermal construction were improved. Furthermore, the number of pathogenic bacteria on wounds was declined throughout the first 24h by HA2 and HA4. The wound size in HA4-treated older rats was significantly smaller than that in other HA1, HA2 or HA3-treated older ones. Also, diabetes impaired the level of inflammatory cytokine, in diabetic model. On contrary, HA4 was found to normalize the level of inflammatory cytokine, in the diabetic model. Furthermore, HA4 was found to recover all oxidative and toxicity markers in diabetic models. This data confirms the critical role of HA4 to improve granulation and inflammatory mediators in impaired older and diabetic rat wound healing.

Wound dressing based on chitosan/hyaluronan/nonwoven fabrics: Preparation, characterization and medical applications.

Thin layers of chitosan (positively charged)/sodium hyaluronate (negatively charged)/nonwoven fabrics were constructed by polyelectrolyte multilayer pad-dry-cure technique. Pure chitosan (CS) was isolated from shrimp shell and immobilized onto nonwoven fabrics (NWFs) using citric acid (CTA) as cross linker and solvent agents through a pad-dry-cure method. The prepared thin layer of chitosan citrate/nonwoven fabrics (CSCTA/NWFs) were consequently impregnated with hyaluronan (CSCTA/HA/NWFs) in the second path through a pad-dry-cure method. Chitosan/hyaluronan/nonwoven fabrics wound dressing was characterized by different techniques such as FTIR-ATR, TGA and SEM. The antibacterial activity and the cytotoxicity of the dressing sheets were evaluated against Escherichia coli (E. coli) and Streptococcus aureus (S. aureus), mouse fibroblast (NIH-3T3) and keratinocytes (HaCaT) cell lines, respectively. The cell-fabrics interaction was also investigated using fluorescence microscope, based on live/dead staining assay of 3T3 cells. The healing properties of the new wound dressing were evaluated and compared with the control sample.

Collagen/hydroxyapatite scaffold enriched with polycaprolactone nanofibers, thrombocyte-rich solution and mesenchymal stem cells promotes regeneration in large bone defect in vivo.

A three-dimensional scaffold of type I collagen and hydroxyapatite enriched with polycaprolactone nanofibers (Coll/HA/PCL), autologous mesenchymal stem cells (MSCs) in osteogenic media, and thrombocyte-rich solution (TRS) was an optimal implant for bone regeneration in vivo in white rabbits. Nanofibers optimized the viscoelastic properties of the Coll/HA scaffold for bone regeneration. MSCs and TRS in the composite scaffold improved bone regeneration. Three types of Coll/HA/PCL scaffold were prepared: an MSC-enriched scaffold, a TRS-enriched scaffold, and a scaffold enriched with both MSCs and TRS. These scaffolds were implanted into femoral condyle defects 6 mm in diameter and 10-mm deep. Untreated defects were used as a control. Macroscopic and histological analyses of the regenerated tissue from all groups were performed 12 weeks after implantation. The highest volume and most uniform distribution of newly formed bone occurred in defects treated with scaffolds enriched with both MSCs and TRS compared with that in defects treated with scaffolds enriched by either component alone. The modulus of elasticity in compressive testing was significantly higher in the Coll/HA/PCL scaffold than those without nanofibers. The composite Coll scaffold functionalized with PCL nanofibers and enriched with MSCs and TRS appears to be a novel treatment for bone defects.

Preparation, characterization and cytotoxicity of schizophyllan/silver nanoparticle composite.

Silver nanoparticles (Ag-NPs) have been successfully prepared with a simple and "green" chemical reduction method. Triple helical schizophyllan (SPG) was used for the first time as reducing and stabilizing agents. The effect of temperature, silver nitrate/schizophyllan concentrations, pH of the reactions medium and the reaction time were investigated. The obtained schizophyllan/Ag-NP was characterized by UV-vis spectroscopy, TEM, DLS, X-ray diffraction, TGA, and ATR-FTIR. The results revealed that, Ag-NPs attached to SPG through a strong non-covalent interaction, leading to good dispersion of Ag-NPs with a diameter of 6 nm within the biopolymer matrix. By increasing the pH of the reaction medium, the triple helical structure of SPG was partially broken. The SPG/AgNP nanocomposite was non-toxic for mouse fibroblast line (NIH-3T3) and human keratinocyte cell line (HaCaT).

Synthesis, characterization and antibacterial activity of new fluorescent chitosan derivatives.

The present work aims to the development of innovative new derivatives of chitosan that can be used for medical applications. This innovation is based on the synthesis and characterization of chitosan-g-aminoanthracene derivatives. Thus, N-(anthracen-9-yl)-4,6-dichloro-[1,3,5]-triazin-2-amine (AT) reacted with chitosan by the following steps: at first, cyanuric chloride reacted with 9-aminoanthracene to obtain N-(anthracen-9-yl)-4,6-dichloro-[1,3,5]-triazin-2-amine (AT), then the AT reacted with chitosan to obtain (CH-g-AT). The final product of CH-g-AT was separated, purified and re-crystallized by dioxane. The structure of the prepared chitosan derivatives was confirmed by FTIR-ATR, solid-NMR, TGA, X-RD, and DSC. The new chitosan derivatives showed fluorescence spectra in liquid and in solid state as well. CH-g-AT showed also high antibacterial activity against gram -ve species (Escherichia coli).

Optimized conditions for mesenchymal stem cells to differentiate into osteoblasts on a collagen/hydroxyapatite matrix.

Collagen/hydroxyapatite (HA) composite scaffolds are known to be suitable scaffolds for seeding with mesenchymal stem cells (MSCs) differentiated into osteoblasts and for the in vitro production of artificial bones. However, the optimal collagen/HA ratio remains unclear. Our study confirmed that a higher collagen content increased scaffold stiffness but that a greater stiffness was not sufficient for bone tissue formation, a complex process evidently also dependent on scaffold porosity. We found that the scaffold pore diameter was dependent on the concentration of collagen and HA and that it could play a key role in cell seeding. In conclusion, the optimal scaffold for new bone formation and cell proliferation was found to be a composite scaffold formed from 50 wt % HA in 0.5 wt % collagen I solution.

Quality of newly formed cartilaginous tissue in defects of articular surface after transplantation of mesenchymal stem cells in a composite scaffold based on collagen I with chitosan micro- and nanofibres.

The aim of this study was to evaluate macroscopically, histologically and immunohistochemically the quality of newly formed tissue in iatrogenic defects of articular cartilage of the femur condyle in miniature pigs treated with the clinically used method of microfractures in comparison with the transplantation of a combination of a composite scaffold with allogeneic mesenchymal stem cells (MSCs) or the composite scaffold alone. The newly formed cartilaginous tissue filling the defects of articular cartilage after transplantation of the scaffold with MSCs (Group A) had in 60 % of cases a macroscopically smooth surface. In all lesions after the transplantation of the scaffold alone (Group B) or after the method of microfractures (Group C), erosions/fissures or osteophytes were found on the surface. The results of histological and immunohistochemical examination using the modified scoring system according to O'Driscoll were as follows: 14.7+/-3.82 points after transplantations of the scaffold with MSCs (Group A); 5.3+/-2.88 points after transplantations of the scaffold alone (Group B); and 5.2+/-0.64 points after treatment with microfractures (Group C). The O'Driscoll score in animals of Group A was significantly higher than in animals of Group B or Group C (p<0.0005 both). No significant difference was found in the O'Driscoll score between Groups B and C. The treatment of iatrogenic lesions of the articular cartilage surface on the condyles of femur in miniature pigs using transplantation of MSCs in the composite scaffold led to the filling of defects by a tissue of the appearance of hyaline cartilage. Lesions treated by implantation of the scaffold alone or by the method of microfractures were filled with fibrous cartilage with worse macroscopic, histological and immunohistochemical indicators.

Use of allogenic stem cells for the prevention of bone bridge formation in miniature pigs.

This study appears from an experiment previously carried out in New Zealand white rabbits. Allogenic mesenchymal stem cells (MSCs) were transplanted into an iatrogenically-created defect in the lateral section of the distal physis of the left femur in 10 miniature pigs. The right femur with the same defect served as a control. To transfer MSCs, a freshly prepared porous scaffold was used, based on collagen and chitosan, constituting a compact tube into which MSCs were implanted. The pigs were euthanized four months after the transplantation. On average, the left femur with transplanted MSCs grew more in length (0.56+/-0.14 cm) compared with right femurs with physeal defect without transplanted MSCs (0.14+/-0.3 cm). The average angular (valgus) deformity of the left femur had an angle point of 0.78 degrees , following measurement and X-ray examination, whereas in the right femur without transplantation it was 3.7 degrees. The initial results indicate that preventive transplantation of MSCs into a physeal defect may prevent valgus deformity formation and probably also reduce disorders of the longitudinal bone growth. This part of our experiment is significant in the effort to advance MSCs application in human medicine by using pig as a model, which is the next step after experimenting on rabbits.

Subcutaneous nodule after vaccination with an aluminum-containing vaccine.

Persistent subcutaneous nodules may arise after vaccination or allergen desensitization. The swelling might appear as a result of a specific histiocytic reaction to aluminum, which is used in many preparations to hasten immune response. A wide range of such vaccines are used in national childhood vaccination programs. Such nodules are frequently itchy or painful with local skin alterations. The condition tends to resolve spontaneously, although long-term observation is recommended. We describe the clinical history of a 10-year-old girl who presented with an itchy subcutaneous nodule that appeared five months after her second DiTe revaccination.

Use of 3D geometry modeling of osteochondrosis-like iatrogenic lesions as a template for press-and-fit scaffold seeded with mesenchymal stem cells.

Computed tomography (CT) is an effective diagnostic modality for three-dimensional imaging of bone structures, including the geometry of their defects. The aim of the study was to create and optimize 3D geometrical and real plastic models of the distal femoral component of the knee with joint surface defects. Input data included CT images of stifle joints in twenty miniature pigs with iatrogenic osteochondrosis-like lesions in medial femoral condyle of the left knee. The animals were examined eight and sixteen weeks after surgery. Philips MX 8000 MX and View workstation were used for scanning parallel plane cross section slices and Cartesian discrete volume creation. On the average, 100 slices were performed in each stifle joint. Slice matrices size was 512 x 512 with slice thickness of 1 mm. Pixel (voxel) size in the slice plane was 0.5 mm (with average accuracy of +/-0.5 mm and typical volume size 512 x 512 x 100 voxels). Three-dimensional processing of CT data and 3D geometrical modelling, using interactive computer graphic system MediTools formerly developed here, consisted of tissue segmentation (raster based method combination and 5 % of manual correction), vectorization by the marching-cubes method, smoothing and decimation. Stifle- joint CT images of three individuals of different body size (small, medium and large) were selected to make the real plastic models of their distal femurs from plaster composite using rapid prototyping technology of Zcorporation. Accuracy of the modeling was +/- 0.5 mm. The real plastic models of distal femurs can be used as a template for developing custom made press and fit scaffold implants seeded with mesenchymal stem cells that might be subsequently implanted into iatrogenic joint surface defects for articular cartilage-repair enhancement.

Mechanical response of porous scaffolds for cartilage engineering.

Mechanical properties of scaffolds seeded with mesenchymal stem cells used for cartilage repair seem to be one of the critical factors in possible joint resurfacing. In this paper, the effect of adding hyaluronic acid, hydroxyapatite nanoparticles or chitosan nanofibers into the cross-linked collagen I on the mechanical response of the lyophilized porous scaffold has been investigated in the dry state at 37 oC under tensile loading. Statistical significance of the results was evaluated using ANOVA analysis. The results showed that the addition of hyaluronic acid significantly (p<<0.05) reduced the tensile elastic modulus and enhanced the strength and deformation to failure of the modified cross-linked collagen I under the used test conditions. On the other hand, addition of hydroxyapatite nanoparticles and chitosan nanofibers, respectively, increased the elastic modulus of the modified collagen ten-fold and four-fold, respectively. Hydroxyapatite caused significant reduction in the ultimate deformation at break while chitosan nanofibers enhanced the ultimate deformation under tensile loading substantially (p<<0.05). The ultimate tensile deformation was significantly (p<<0.05) increased by addition of the chitosan nanofibers. The enhanced elastic modulus of the scaffold was translated into enhanced resistance of the porous scaffolds against mechanical load compared to scaffolds based on cross-linked neat collagen or collagen with hyaluronic acid with similar porosity. It can be concluded that enhancing the rigidity of the compact scaffold material by adding rigid chitosan nanofibers can improve the resistance of the porous scaffolds against compressive loading, which can provide more structural protection to the seeded mesenchymal stem cells when the construct is implanted into a lesion. Moreover, scaffolds with chitosan nanofibers seemed to enhance cell growth compared to the neat collagen I when tested in vitro as well as the scaffold stability, extending its resorption to more than 10 weeks.