Electrospun gelatin/nylon‐6 composite nanofibers for biomedical applications

We describe the preparation and characterization of gelatin‐containing nylon‐6 electrospun fibers and their potential use as a bioactive scaffold for tissue engineering. The physicochemical properties of gelatin/nylon‐6 composite nanofibers were analyzed using field emission scanning electron microscopy (FE‐SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, TGA and contact angle and tensile measurements. FE‐SEM and TEM images revealed that the nanofibers were well oriented and showed a good incorporation of gelatin. FTIR spectroscopy and TGA also revealed that there was good interaction between the two polymers at the molecular level. The adhesion, viability and proliferation properties of osteoblast cells on the gelatin/nylon‐6 composite nanofibers were analyzed by an in vitro cell compatibility test. Our results suggest that the incorporation of gelatin can increase the cell compatibility of nylon‐6 and therefore the composite mat obtained has great potential in hard tissue engineering. © 2012 Society of Chemical Industry     

Glutaraldehyde crosslinked gelatin/hydroxyapatite nanocomposite scaffold,engineered via compound techniques

In this study, a scaffold was designed to be used in bone tissue repair and the effect of glutaraldehyde (GA) concentration as crosslinking agent was investigated. To mimic the mineral and organic component of natural bone, hydroxyapatite (HAp) and gelatin (GEL) were used as the main components of this composite. Nanopowders of HAp were synthesized and also used together with GEL to engineer a three‐dimensional nanocomposite scaffold. The results show that GEL/HAp nanocomposite is porous with three‐dimensional interconnected structure, pore sizes ranging from 300 to 500 μm, and about 85% porosity. In addition, increasing GA concentration provokes the enhancement of compressive strength until 1 w/v% GA solution followed by a reduction to 2.5%, whereas it causes work fracture to decrease. It was concluded that optimum concentration for crosslinking GEL matrix for this purpose is 1 w/v% GA solution. A specific combination of commonly used techniques applied to engineer a scaffold with almost ideal properties intended for the bone tissue engineering is introduced. In addition, scaffolds that are prepared via this compound process has the potential to be used in the solid free form applications and so being formed in any dimension and geometry relevant to the defect size and shape. POLYM. COMPOS., 31:2112–2120, 2010. © 2010 Society of Plastics Engineers     

Preparation,properties and in vitro cellular response of multi-walled carbon nanotubes/bioactive glass/poly(etheretherketone) biocomposite for bone tissue engineering

Desired bone repair biomaterial must have good biocompatibility and suitable mechanical properties that are equivalent to those of human bones. In this study, multi-walled carbon nanotubes (MWCNTS) was designed to incorporate into bioactive glass/poly(etheretherketone) to fabricate a composite of multi-walled carbon nanotubes/bioactive glass/poly(etheretherketone) (MWCNTS/BG/PEEK) through a compounding and injection-molding process. The microstructures, mechanical properties, thermal stability and bioactivity of the ternary biocomposite, as well as preliminary cell responses of MC3T3-E1 osteoblast cells to this biomaterial, were investigated. The mechanical performance of ternary MWCNTS/BG/PEEK composite was vastly superior to binary BG/PEEK composite. More importantly, cell culture tests showed that cell adhesion, viability and differentiation of MC3T3-E1 cells were significantly promoted on the MWCNTS/BG/PEEK composite. Moreover, it was found that MWCNTS in composite further promoted cell metabolic vitality and osteogenic differentiation of osteoblast cells. Hence, this MWCNTS/BG/PEEK biomaterial may be used as a promising bone graft scaffold in dental and orthopedic applications.     

Development of a novel glucosamine/silk fibroin–chitosan blend porous scaffold for cartilage tissue engineering applications

Silk fibroin–chitosan blend is reported to be an attractive scaffold material for tissue engineering applications. In our earlier study, we developed a scaffold having an optimal silk fibroin–chitosan blend ratio of 80:20 and proved its potentiality for cartilage tissue engineering applications. Glucosamine is one of the major structural components of cartilage tissue. The present work investigates the effect of glucosamine components on the physicochemical and biocompatibility properties of this scaffold. To this end, varied amounts of glucosamine were added to silk fibroin–chitosan blend with the aim of improving various scaffold properties. The addition of glucosamine components did not show any significant change in physicochemical properties of silk fibroin–chitosan blend scaffolds. The composite scaffold showed an open pore structure with desired pore size and porosity. However, cell culture study using human mesenchymal stem cells derived from umbilical cord blood revealed an overall increase in cell supportive properties of glucosamine-added scaffolds. Cell viability, cell proliferation and glycosaminoglycan assays confirmed enhanced cell viability and proliferation of mesenchymal stem cells. Thus, this study demonstrated the beneficial effect of glucosamine on improving the cell supportive property of silk fibroin–chitosan blend scaffolds making it more potential for cartilage tissue regeneration. To the best of our knowledge, this is the first report on the study of glucosamine-added silk fibroin–chitosan blend porous scaffolds seeded with mesenchymal stem cells derived from umbilical cord blood.     

Preparation of chitosan-sodium alginate/bioactive glass composite cartilage scaffolds with high cell activity and bioactivity

Chitosan-sodium alginate/bioactive glass (CSB) composite cartilage scaffold with outstanding in vitro mineralization property and cytocompatibility is synthesized by freeze drying method. The effect of bioactive glass (BG) addition on the microstructure, porosity, swelling/degradation ratio, in vitro mineralization property and cytocompatibility of CSB scaffold is investigated by the characterization techniques of SEM, XRD, FTIR and BET. Results showed that CSB composite cartilage scaffold had a three-dimensional (3D) porous structure, and both porosity and average pore size met the requirements of cartilage tissue repair. Among, the typical CSB-1.0 had the largest overall pore size and lowest compressive modulus (1.083 ± 0.002 MPa). As the amount of BG increased, pore volume and porosity of CSB scaffolds gradually decreased, and the swelling and degradation ratios gradually reduced. After immersing in SBF for 3 d, cauliflower like hydroxyapatite (HA) was formed on CSB surface, indicating that the scaffold had good in vitro mineralization property. Moreover, the introduction of BG into the composite scaffold can improve the relative cell viability of MC3T3-E1 cells, and CSB-1.0 has the strongest ability to promote the proliferation of cells. Therefore, the as-obtained CSB scaffold can be used as a strong candidate for cartilage tissue engineering scaffold to meet clinical needs.     

Antibacterial and Cellular Behaviors of Novel Zinc-Doped Hydroxyapatite/Graphene Nanocomposite for Bone Tissue Engineering

Bacteria are one of the significant causes of infection in the body after scaffold implantation. Effective use of nanotechnology to overcome this problem is an exciting and practical solution. Nanoparticles can cause bacterial degradation by the electrostatic interaction with receptors and cell walls. Simultaneously, the incorporation of antibacterial materials such as zinc and graphene in nanoparticles can further enhance bacterial degradation. In the present study, zinc-doped hydroxyapatite/graphene was synthesized and characterized as a nanocomposite material possessing both antibacterial and bioactive properties for bone tissue engineering. After synthesizing the zinc-doped hydroxyapatite nanoparticles using a mechanochemical process, they were composited with reduced graphene oxide. The nanoparticles and nanocomposite samples were extensively investigated by transmission electron microscopy, X-ray diffraction, and Raman spectroscopy. Their antibacterial behaviors against Escherichia coli and Staphylococcus aureus were studied. The antibacterial properties of hydroxyapatite nanoparticles were found to be improved more than 2.7 and 3.4 times after zinc doping and further compositing with graphene, respectively. In vitro cell assessment was investigated by a cell viability test and alkaline phosphatase activity using mesenchymal stem cells, and the results showed that hydroxyapatite nanoparticles in the culture medium, in addition to non-toxicity, led to enhanced proliferation of bone marrow stem cells. Furthermore, zinc doping in combination with graphene significantly increased alkaline phosphatase activity and proliferation of mesenchymal stem cells. The antibacterial activity along with cell biocompatibility/bioactivity of zinc-doped hydroxyapatite/graphene nanocomposite are the highly desirable and suitable biological properties for bone tissue engineering successfully achieved in this work.     

In Vitro Biocompatibility and Antibacterial Activity of Electrospun Ag Doped HAp/PHBV Composite Nanofibers

The authors report herein in vitro antibacterial property and osteoblast biocompatibility of electrospun Ag doped HAp/PHBV (Ag-HAp/PHBV) composite nanofibers as an osteoconductive and antibacterial material for bone tissue engineering applications. Ag-HAp powders were synthesized and stable composite suspensions of Ag-HAp/PHBV were prepared with the aid of a cationic surfactant DTAB for the electrospinning process. Continuous and uniform composite nanofibers were generated within a diameter range of 400–900 nm. Obtained nanocomposite scaffolds provide a favorable environment for bone mineralization, SaOS-2 osteoblastic cell attachment and growth as well as they present antibacterial activity against E. coli and S. aureus bacteria without any noticeable cytotoxic effect.     

Chitosan-gelatin/hydroxyapatite-based scaffold associated with mesenchymal stem cells differentiate into osteoblasts improves the surface of the bone lesion in mice C57BL/6J

Extensive injuries to bone tissue are still considered a significant clinical challenge; therefore, developing new bone tissue engineering (BTE) strategies is still necessary. This work aims to construct and characterize a chitosan-gelatin/hydroxyapatite-based (CG/H) scaffold to provide well-design support for mesenchymal stem cell (MSC) growth and differentiation to osteoblasts. First, the CG/H scaffolds are construct by freeze-drying. Then, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, x-ray diffraction, water uptake, and degradation profiles evaluate the material's surface. In addition, the CG/H morphological, biochemical, and MSC adhesion processes and growth behavior are also assess, indicating reasonable adhesion rates to the surface, low material cytotoxicity, and excellent alkaline phosphatase activity compared to control on the cellular framework. Based on these results, we obtain a highly biocompatible scaffold and that can support osteoblast differentiation. Finally, the in vivo studies demonstrate the CG/H scaffold with MSC adhere is capable of differentiating into osteoblasts, and the application of this scaffold is able to significantly enhance the closure of the bone lesion. Therefore, the CG/H scaffold has potential clinical application for bone regeneration.     

Physical and mechanical properties of a poly-3-hydroxybutyrate-coated nanocrystalline hydroxyapatite scaffold for bone tissue engineering

A major challenge for tissue engineers is the design of scaffolds with appropriate physical and mechanical properties. The present research discusses the formation of ceramic scaffolding in tissue engineering. Hydroxyapatite (HAp) powder was made from bovine bone by thermal treatment at 900?°C; 40, 50 and 60%wt porous HAp was then produced using the polyurethane sponge replication method. Scaffolds were coated with poly-3-hydroxybutyrate (P3HB) for 30?s and 1?min in order to increase the scaffold??s mechanical properties. XRD, SEM and FT-IR were used to study phase structure, morphology and agent groups, respectively. In XRD and FT-IR data, established hydrogen bands between polymer and ceramic matrix confirm that the scaffold is formed as a composite. The scaffold obtained with 50%wt HAp and a 30?s coating was 90% porous, with an average diameter of 100?C400???m, and demonstrated a compressive strength and modulus of 1.46 and 21.27?MPa, respectively. Based on these results, this scaffold is optimised for the aforementioned properties and can be utilised in bone tissue engineering.     

The odontogenic differentiation of human dental pulp stem cells on hydroxyapatite-coated biodegradable nanofibrous scaffolds

The authors aimed to design nanofibrous (NF) scaffolds that facilitate odontogenic and osteogenic differentiation of human dental pulp-derived mesenchymal stem cells (DPSCs) in vitro. For this purpose, hydroxyapatite (HA)–loaded poly (L-lactic acid)/poly (?-caprolactone) (PLLA:PCL 2;1) blend NFs were prepared using the electrospinning method. Alizarin red activity and cell viability were evaluated by MTT assay, and SEM revealed the proliferation properties of NF scaffolds. QRT-PCR results demonstrated that HA-loaded PLLA/PCL can lead to osteoblast/odontoblast differentiation in DPSCs through the up-regulation of related genes, thus indicating that electrospun biodegradable PCL/PLA/HA has remarkable prospects as scaffolds for bone and tooth tissue engineering.     

The Osteogenesis Effect and Underlying Mechanisms of Local Delivery of gAPN in Extraction Sockets of Beagle Dogs

A plastic and biodegradable bone substitute consists of poly (l-lactic-co-glycolic) acid and 30 wt % β-tricalcium phosphate has been previously fabricated, but its osteogenic capability required further improvement. We investigated the use of globular adiponectin (gAPN) as an anabolic agent for tissue-engineered bone using this scaffold. A qualitative analysis of the bone regeneration process was carried out using μCT and histological analysis 12 weeks after implantation. CBCT (Cone Beam Computed Tomography) superimposition was used to characterise the effect of the different treatments on bone formation. In this study, we also explored adiponectin’s (APN) influence on primary cultured human jaw bone marrow mesenchymal stem cells gene expressions involved in the osteogenesis. We found that composite scaffolds loaded with gAPN or bone morphogenetic protein 2 (BMP2) exhibited significantly increased bone formation and mineralisation following 12 weeks in the extraction sockets of beagle dogs, as well as enhanced expression of osteogenic markers. In vitro investigation revealed that APN also promoted osteoblast differentiation of primary cultured human jaw bone marrow mesenchymal stem cells (h-JBMMSCs), accompanied by increased activity of alkaline phosphatase, greater mineralisation, and production of the osteoblast-differentiated genes osteocalcin, bone sialoprotein and collagen type I, which was reversed by APPL1 siRNA. Therefore, the composite scaffold loaded with APN exhibited superior activity for guided bone regeneration compared with blank control or Bio-Oss^® (a commercially available product). The composite scaffold with APN has significant potential for clinical applications in bone tissue engineering.     

Direct ink writing of vancomycin-loaded polycaprolactone/ polyethylene oxide/ hydroxyapatite 3D scaffolds

Novel inks were formulated by dissolving polycaprolactone (PCL), a hydrophobic polymer, in organic solvent systems; polyethylene oxide (PEO) was incorporated to extend the range of hydrophilicity of the system. Hydroxyapatite (HAp) with a weight ratio of 55–85% was added to the polymer-based solution to mimic the material composition of natural bone tissue. The direct ink writing (DIW) technique was applied to extrude the formulated inks to fabricate the predesigned tissue scaffold structures; the influence of HAp concentration was investigated. The results indicate that in comparison to other inks containing HAp (55%, 75%, and 85%w/w), the ink containing 65% w/w HAp had faster ink recovery behavior; the fabricated scaffold had a rougher surface as well as better mechanical properties and wettability. It is noted that the 65% w/w HAp concentration is similar to the inorganic composition of natural bone tissue. The elastic modulus values of PCL/PEO/HAp scaffolds were in the range of 4–12 MPa; the values were dependent on the HAp concentration. Furthermore, vancomycin as a model drug was successfully encapsulated in the PCL/PEO/HAp composite scaffold for drug release applications. This paper presents novel drug-loaded PCL/PEO/HAp inks for 3D scaffold fabrication using the DIW printing technique for potential bone scaffold applications.     

Development of Electrospun Composite Fibers in Multiscale Structure and Investigating the Performance on Proliferation and Osteogenic Differentiation of ADSCs

Electrospun composite membranes in multiscale structures are developed for bone tissue engineering. Aligned polycaprolactone (PCL) fibers entrapping CA‐HAp microparticles (containing CaCO₃, hydroxyapatite, and casein in a hierarchical organization) are electrospun to find whether synergistic effects of fiber alignment and CA‐HAp microparticles on improving osteogenic differentiation can be obtained. CA‐HAp microparticles are in a spherical morphology of 1.42 ± 0.26 µm. Their presence increases fiber diameter and does not significantly affect fiber alignment. On all membranes, adipose derived stem cells (ADSCs) from humans spread very well. On a random group, cells distribute randomly and the presence of CA‐HAp microparticles facilitates cell proliferation, especially for the one at CA‐HAp/PCL 50 wt%; the one at CA‐HAp/PCL 20 wt% shows significantly much higher alkaline phosphatase (ALP) activity (112.0% higher) than the pure PCL membrane. On aligned samples, cells align along fibers and expression of ALP is enhanced. However, at the same composition (CA‐HAp/PCL 20 wt%), the random sample has much higher ALP activity than the aligned sample. The expressions of osteogenic marker genes are also evaluated. Combining the results and the applicability of membranes together, the random membrane at CA‐HAp/PCL 20 wt% is the best candidate for bone tissue engineering.     

Fabrication and evaluation of nanofibrous polyhydroxybutyrate valerate scaffolds containing hydroxyapatite particles for bone tissue engineering

Tissue engineering is a new approach for regeneration of damaged tissues. The current clinical methods such as autograft and allograft transplantation are not effective for repairing bone damages, mainly due to the limited available sources and the donor-site side effects. In this research, the nanocomposite poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/nano hydroxyapatite (nHA) scaffolds with different nHA ratios for bone regeneration were utilized. The diameter and porosity of scaffolds were approximately 200?nm and 74%, respectively. The degradability test of the scaffolds suggests a low degradation rate with total degradation of 30% after 3 months. Cytotoxicity result showed that cultured osteoblast cells (MC3T3) on nanocomposite scaffolds had superiority in terms of higher proliferation and attachment in comparison with PHBV scaffold. The protein expression of alkaline phosphatase illustrated that nanofibrous scaffold containing hydroxyapatite had the highest alkaline phosphatase activities as a result of better proliferation. These results recommend that PHBV/nHA scaffolds are suitable candidates for bone tissue engineering.     

Fabrication of MgAl2O4 Spinel Scaffolds and Sonochemical Synthesis and Deposition of Hydroxyapatite Nanorods

Hydroxyapatite (HAp) is one of the most emerging biocompatible ceramic widely used as scaffolds in various biomedical applications such as orthopedics and dentistry. In spite of the superior properties for biomedical applications, they exhibit poor mechanical properties. This has lead to the concept of fabricating composite out of bioactive and bioinert material to derive bioactivity in combination with desirable mechanical properties. In this study, bioinert magnesium aluminate (MgAl₂O₄) spinel scaffolds are prepared through polymeric foam replication process followed by sintering. Sintered foams have exhibited good structural integrity and the stress–strain curves recorded during the uniaxial compression have shown a plateau indicating high‐energy absorption capability. Sonochemical process has been employed for the simultaneous synthesis and deposition of HAp formulation from the stoichiometric solution of precursors on the spinel scaffolds. Sonochemical process has resulted in the formation of phase pure HAp with unique morphology of nanorods coated throughout the three‐dimensional foam structures. Cytotoxicity evaluation of this new scaffold material has not shown any alteration in the viability, growth, and morphology of the cells. The new scaffold material thus developed is expected to have high potential for biomedical applications.     

Biomimetic biphasic 3‐D nanocomposite scaffold for osteochondral regeneration

Scaffold‐based interfacial tissue engineering aims to not only provide the structural and mechanical framework for cellular growth and tissue regeneration, but also direct cell behavior. Due to the disparity in composition of the osteochondral (cartilage and bone) interface, this work has developed a novel biomimetic biphasic nanocomposite scaffold integrating two biocompatible polymers containing tissue‐specific growth factor‐encapsulated core–shell nanospheres. Specifically, a poly(caprolactone) (PCL)‐based bone layer was successfully integrated with a poly(ethylene glycol) (PEG) hydrogel cartilage layer. In addition, a novel nanosphere fabrication technique for efficient growth factor encapsulation and sustained delivery via a wet coaxial electrospray technique was developed. Human bone marrow mesenchymal stem cell (hMSC) adhesion, osteogenic, and chondrogenic differentiation were evaluated. Our in vitro results showed significantly improved hMSC adhesion and differentiation in bone and cartilage layers, respectively. Studies have demonstrated promising results with novel biphasic nanocomposite scaffold for osteochondral tissue regeneration, thus, warranting further studies. © 2013 American Institute of Chemical Engineers AIChE J 60: 432–442, 2014     

Development of bone scaffold using Puntius conchonius fish scale derived hydroxyapatite: Physico-mechanical and bioactivity evaluations

Naturally derived Hydroxyapatite (HAp) from fish scale is finding wide applications in the development of bone scaffold to promote bone regeneration. But porous HAp scaffold is fragile in nature making it unsuitable for bone repair or replacement applications. Thus, it is essential to improve the mechanical property of HAp scaffolds while retaining the interconnected porous structure for tissue ingrowth in vivo. In this study solvent casting particulate leaching technique is used to develop novel Puntius conchonius fish scale derived HAp bone scaffold by varying the wt.% of the HAp from 60 to 80% in PMMA matrix. Physico-chemical, mechanical, structural and bioactive properties of the developed scaffolds are investigated. The obtained results indicate that HAp-PMMA scaffold at 70?wt % HAp loading shows optimal properties with 7.26?±?0.45?MPa compressive strength, 75?±?0.8% porosity, 8.0?±?0.68% degradation and 190?±?11% water absorption. The obtained results of the scaffold can meet the physiological demands to guide bone regeneration. Moreover, in vitro bioactivity analysis also confirms the formation of bone like apatite in the scaffold surface after 28 days of SBF immersion. Thus, the developed scaffold has the potential to be effectively used in bone tissue engineering applications.     

The role of nanostructured mesoporous silicon in discriminating in vitro calcification for electrospun composite tissue engineering scaffolds

The impact of mesoporous silicon (PSi) particles-embedded either on the surface, or totally encapsulated within electrospun poly (ε-caprolactone) (PCL) fibers-on its properties as a tissue engineering scaffold is assessed. Our findings suggest that the resorbable porous silicon component can sensitively accelerate the necessary calcification process in such composites. Calcium phosphate deposition on the scaffolds was measured via in vitro calcification assays both at acellular and cellular levels. Extensive attachment of fibroblasts, human adult mesenchymal stem cells, and mouse stromal cells to the scaffold were observed. Complementary cell differentiation assays and ultrastructural measurements were also carried out; the levels of alkaline phosphatase expression, a specific biomarker for mesenchymal stem cell differentiation, show that the scaffolds have the ability to mediate such processes, and that the location of the Si plays a key role in levels of expression.     

Effect of ZnO reinforcement on the compressive properties,in vitro bioactivity,biodegradability and cytocompatibility of bone scaffold developed from bovine bone-derived HAp and PMMA

In this study, the effect of zinc oxide (ZnO) incorporation on the properties of Hydroxyapatite (HAp)/Poly(methyl methacrylate) (PMMA)/ZnO based composite bone scaffold is investigated. HAp is derived from calcination of bovine bone bio-waste and ZnO is synthesized by direct precipitation technique. Porous scaffolds are developed by gas foaming process using ammonium bicarbonate as the foaming agent and adding ZnO nanoparticles (NPs) at 2.5, 5, 7.5 and 10% (w/w) respectively. Incorporation of ZnO up to 5% (w/w) is found to significantly enhance the porosity, compressive strength, thermal stability and swelling properties of the developed scaffolds. In-vitro bioactivity and biodegradability assessment using simulated body fluid (SBF) show improved results of 5% ZnO loaded scaffolds. Furthermore, the composite scaffold show enhanced cytocompatibility during the in vitro cytotoxicity test performed using XTT assay. A comprehensive study on the scaffold properties shows that 5% ZnO composite scaffold exhibits the best-optimized properties suitable for bone tissue engineering applications.