Biomimetic Nanocomposite Scaffold Research Articles

Preparation and In Vitro Osteogenic Evaluation of Biomimetic Hybrid Nanocomposite Scaffolds Based on Gelatin/Plasma Rich in Growth Factors (PRGF) and Lithium-Doped 45s5 Bioactive Glass Nanoparticles.

Bone tissue engineering is an emerging technique for repairing large bone lesions. Biomimetic techniques expand the use of organic-inorganic spongy-like nanocomposite scaffolds and platelet concentrates. In this study, a biomimetic nanocomposite scaffold was prepared using lithium-doped bioactive-glass nanoparticles and gelatin/PRGF. First, sol-gel method was used to prepare bioactive-glass nanoparticles that contain 0, 1, 3, and 5%wt lithium. The lithium content was then optimized based on antibacterial and MTT testing. By freeze-drying, hybrid scaffolds comprising 5, 10, and 20% bioglass were made. On the scaffolds, human endometrial stem cells (hEnSCs) were cultured for adhesion (SEM), survival, and osteogenic differentiation. Alkaline phosphatase activity and osteopontin, osteocalcin, and Runx2 gene expression were measured. The effect of bioactive-glass nanoparticles and PRGF on nanocomposites' mechanical characteristics and glass-transition temperature (Tg) was also studied. An optimal lithium content in bioactive glass structure was found to be 3% wt. Nanoparticle SEM examination indicated grain deformation due to different sizes of lithium and sodium ions. Results showed up to 10% wt bioactive-glass and PRGF increased survival and cell adhesion. Also, Hybrid scaffolds revealed higher ALP-activity and OP, OC, and Runx2 gene expression. Furthermore, bioactive-glass has mainly increased ALP-activity and Runx2 expression, whereas PRGF increases the expression of OP and OC genes. Bioactive-glass increases scaffold modulus and Tg continuously. Hence, the presence of both bioactive-glass and nanocomposite scaffold improves the expression of osteogenic differentiation biomarkers. Subsequently, it seems that hybrid scaffolds based on biopolymers, Li-doped bioactive-glass, and platelet extracts can be a good strategy for bone repair.

A novel gelatin/carboxymethyl chitosan/nano-hydroxyapatite/β-tricalcium phosphate biomimetic nanocomposite scaffold for bone tissue engineering applications.

Hydroxyapatite (HA) and tricalcium phosphate (TCP) constitute 60% of the content of the bone, and their combination has a better effect on bone tissue engineering than either single element. This study demonstrates a new degradable gelatin/carboxymethyl chitosan (CMC) bone scaffold loaded with both nano-HA and β-TCP (hereinafter referred to as HCP), and freeze drying combined with stir foaming was used to obtain highly connected macropores. Only a few studies have used these components to synthesize a four-component osteogenic scaffold. The aim of this study was to comprehensively assess the biocompatibility and osteoinductivity of the nanocomposites. Three HCP/CMC/gelatin scaffolds were made with different HCP contents: group A (10 wt% HCP), group B (30 wt% HCP), and group C (50 wt% HCP) (the ratio of nano-HA and β-TCP was fixed at 3:2). The scaffolds were macroporous with a high porosity and pore connectivity, as observed by morphological analysis by scanning electron microscopy. Additionally, the pore size of groups A and B was more homogeneous than that of group C. There were no significant differences in physicochemical characterization among the three groups. The Fourier-transform infrared (FTIR) spectroscopy test indicated that the scaffold contained active groups, such as hydroxyl, amino, or peptide bonds, corresponding to gelatin and CMC. The XRD results showed that the phase structures of HA and β-TCP did not change in the nanocomposite. The scaffolds had biodegradation potential and an appreciable swelling ratio, as demonstrated with the in vitro test. The scaffolds were cultured in vitro with MC3T3-E1 cells, showing that osteoinduction and osteoconduction increased with the HCP content. None of the scaffolds showed cytotoxicity. However, cell adhesion and growth in group B were better than those in group A and group C. Therefore, freeze drying combined with a stir foaming method may have a solid component limit. This study demonstrates a novel four-component scaffold via a simple manufacturing process. Group B (30% HCP) had the best characteristics for bone scaffold materials.

A porcine cholecystic extracellular matrix conductive scaffold for cardiac tissue repair.

Cardiac tissue engineering using cells, scaffolds or signaling molecules is a promising approach for replacement or repair of damaged myocardium. This study addressed the contemporary need for a conductive biomimetic nanocomposite scaffold for cardiac tissue engineering by examining the use of a gold nanoparticle-incorporated porcine cholecystic extracellular matrix for the same. The scaffold had an electrical conductivity (0.74±0.03S/m) within the range of native myocardium. It was a suitable substrate for the growth and differentiation of cardiomyoblast (H9c2) as well as rat mesenchymal stem cells to cardiomyocyte-like cells. Moreover, as an epicardial patch, the scaffold promoted neovascularisation and cell proliferation in infarcted myocardium of rats. It was concluded that the gold nanoparticle coated cholecystic extracellular matrix is a prospective biomaterial for cardiac tissue engineering.

Biomimetic development of chitosan and sodium alginate-based nanocomposites contains zirconia for tissue engineering applications.

Nanostructured materials possess unique structural and functional properties that play a crucial position in tissue engineering applications. Present investigation is aimed to synthesize chitosan-sodium alginate (CS) nanocomposite using hydrothermally prepared zirconia nanoparticles. In this, three different weight percentages of (0.5, 1, and 1.5) zirconia nanoparticles are utilized for the preparation of biomimetic nanocomposite scaffolds (CSZ) employing 4wt% of CS by a solvent casting technique. Physico-chemical and thermal behavior of the prepared nanoparticles and their CSZ scaffolds are comprehensively characterized. Bioactivity of the prepared zirconia nanoparticles and CSZ scaffolds are explored in terms of in vitro biocompatibility, protein absorption in simulated body fluid (SBF), and phosphate buffered saline (PBS). Agar disc diffusion method is employed to identify the antibacterial property against Staphylococcus aureus and Escherichia coli. In vitro cytotoxicity of zirconia nanoparticles and CSZ scaffolds is identified against human urothelial carcinoma (UC6) and osteosarcoma (MG-63) cells. These studies explore that zirconia nanoparticles are suitable for biomedical applications while it is interacted with chitosan and sodium alginate (CS) due to their promising biocompatibility. Biomimetically obtained chitosan/sodium alginate scaffold contain 1wt% zirconia nanoparticles show higher biocompatibility amenable for tissue engineering applications.

Preparation and in vitro characterization of electrospun scaffolds composed of chitosan, gelatin and 58S bioactive glass nanoparticles for skin tissue engineering

Background and aims: The presence of an appropriate scaffold at the wound site could significantly improve the healing process. In this study, we aimed to prepare a biomimetic nanocomposite scaffold composed of chitosan, gelatin, and 58S bioglass nanoparticles for skin tissue engineering. Methods: The nanocomposite scaffolds composed of chitosan, gelatin, and 58S bioglass nanoparticles were fabricated through electrospinning process. Then the cell viability assay was performed in order to evaluate the biological properties of the membranes. The optimum concentration of bioglass nanoparticles was determined for further studies. In vitro characterization was also performed to evaluate physicochemical properties of the scaffolds. Results: The chitosan/gelatin scaffold containing 2% of 58S bioglass nanoparticles showed no cell toxicity, and the dermal fibroblasts were found capable of proliferation on the membrane. The in vitro results obtained from the scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and porosity tests demonstrated the appropriate properties of the membrane as a scaffold for skin regeneration. Conclusions: It was concluded that a chitosan-gelatin membrane containing 2% of 58S bioglass nanoparticles had the potential to function as a scaffold to accelerate wound healing due to its suitable properties, such as high porosity, high surface/volume ratio, and excellent biocompatibility.

Repair of Osteoporotic Bone Defects Using Adipose-Derived Stromal Cells and Umbilical Vein Endothelial Cells Seeded in Chitosan/Nanohydroxyapatite-P24 Nanocomposite Scaffolds

Background. The cell regeneration and blood supply of bone defect lesions are restricted under osteoporotic pathological conditions, which make the healing of bone defect of osteoporosis still a great challenge. The current therapeutic strategies that mainly inhibit bone resorption are not always satisfactory for osteoporotic bone defects, which make the development of new therapies an urgent need. Methods. Previously, we prepared chitosan/nanohydroxyapatite (CS/nHA) biomimetic nanocomposite scaffolds for controlled delivery of bone morphogenetic protein 2-derived peptide (P24). In this study, we determined the effect of coculturing adipose-derived stromal cells (ADSCs) and human umbilical vein endothelial cells (HUVECs) with the CS-P24/nHA nanocomposite scaffolds on osteoporotic bone defect healing. In vitro mixed coculture models were employed to assess the direct effects of coculture. Results. ADSCs cocultured with HUVECs showed significantly greater osteogenic differentiation and mineralization compared with ADSCs or HUVECs alone. The CS-P24/nHA scaffold cocultured with ADSCs and HUVECs was more effective in inducing osteoporotic bone repair, as demonstrated by micro-computed tomography and histology of critical-sized calvariae defects in ovariectomized rats. Calvariae defects treated with the CS-P24/nHA nanocomposite scaffold plus ADSC/HUVEC coculture had a greater area of repair and better reconstitution of osseous structures compared with defects treated with the scaffold plus ADSCs or the scaffold plus HUVECs after 4 and 8 weeks. Conclusion. Taken together, coculture of ADSCs and HUVECs with the CS-P24/nHA nanocomposite scaffold is an effective combination to repair osteoporotic bone defects.

Biomimetic TiO2-chitosan/sodium alginate blended nanocomposite scaffolds for tissue engineering applications.

The study is aimed to synthesize potent metal oxide based biomimetic nanocomposites to overcome the risk associated with artificial bone tissue engineering. High purity TiO2 nanoparticles are synthesized via hydrothermal route. A biomimetic nanocomposite scaffolds containing chitosan-sodium alginate (4: 4) blended with three different (0.5, 1, and 1.5wt%) concentrations of hydrothermally synthesized TiO2 nanoparticles are obtained by solvent casting technique. The physico-chemical and thermal degradation properties of as-synthesized TiO2 nanoparticles and their nanocomposite scaffolds are analyzed. In-vitro cytotoxicity and biocompatibility of the prepared TiO2 nanoparticles and nanocomposites are tested against human bladder tumor (UC6) and osteosarcoma (MG-63) cell lines. Antibacterial property is tested against Escherichia coli and Staphylococcus aureus. These studies reveal that TiO2 nanoparticles and polymeric nanocomposites contain good physico-chemical and mechanical properties for enhanced in-vitro biocompatibility suitable for biomedical applications. Biomimetically prepared chitosan-sodium alginate scaffold containing TiO2 nanoparticles (1wt%) is found to exhibit superior biocompatibility for bone tissue engineering applications.

Biomimetic nanocomposite scaffolds based on surface modified PCL-nanofibers containing curcumin embedded in chitosan/gelatin for skin regeneration

Recently, nanofibrous-hydrogel composites are luring attention for tissue regeneration applications as they mimic soft-tissues' microstructure. Mostly, the electrospun nanofibers based on synthetic polymers such as Polycaprolactone (PCL) are placed in a crosslinked hydrogels. Due to hydrophobic nature of PCL, integration of these nanofibers with the hydrophilic hydrogels of matrix is not sufficient. In this study, we applied Poly(ethylene glycol) methyl ether methacrylate (PEGMA)-surface-modified PCL nanofibers within chitosan-gelatin hydrogels for skin regeneration applications. In addition, curcumin was loaded into PCL nanofibers due to its great impact on skin regeneration process. Fabricated nanofibrous-hydrogel scaffolds were characterized using scanning electron microscopy (SEM), porosimetery, Fourier transform infrared spectroscopy (FTIR), mechanical compression test, and water uptake studies. Curcumin release was investigated using UV/Vis spectrophotometry. In order to study biocompatibility of scaffolds MTT assay and cell culture was performed using L929 cells. FTIR spectra confirmed PEGMA modification of PCL nanofibers. Results of mechanical test determined that surface modified PCL nanofibers improved mechanical strength and modulus of scaffolds. Porosimetery studies showed proper porosity of scaffolds for skin regeneration from 90.43 to 71.48% and pore size of 101–256 μm. Biological test confirmed proper biocompatibility and good cell attachment to the scaffolds. Taken together, the chitosan/gelatin hydrogel incorporating PEGMA modified PCL nanofibers containing curcumin shows great potential for skin regeneration.

Dual release of growth factor from nanocomposite fibrous scaffold promotes vascularisation and bone regeneration in rat critical sized calvarial defect

A promising strategy for augmenting bone formation involves the local delivery of multiple osteoinductive and vasculogenic growth factors. However, success depends on sustained growth factor release and its appropriate combination to induce stem cells and osteogenic cells at the bony site. Herein, we have developed a nanocomposite fibrous scaffold loaded with fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) and its ability to promote vascularisation and bone regeneration in critical sized calvarial defect was compared to the scaffold with VEGF + BMP2 and FGF2 + BMP2. Simple loading of growth factors on the scaffold could provide a differential release pattern, both in vitro and in vivo (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks). Among all the groups, dual growth factor loaded scaffold (VEGF + BMP2 & FGF2 + BMP2) enhanced vascularisation and new bone formation, but there was no difference between FGF2 and VEGF loaded scaffolds although its release pattern was different. FGF2 mainly promoted stem cell migration, whereas VEGF augmented new blood vessel formation at the defect site. This study suggests that biomimetic nanocomposite scaffold is a promising growth factor delivery vehicle to improve bone regeneration in critical sized bone defects. Statement of SignificanceMany studies have shown the effect of growth factors like VEGF-BMP2 or FGF2-BMP2 in enhancing bone formation in critical sized defects, but there are no reports that demonstrate the direct comparison of VEGF-BMP2 and FGF2-BMP2. In this study, we have developed a nanocomposite fibrous scaffold that could differentially release growth factors like VEGF, BMP2 and FGF2 (VEGF release for 1 week where as BMP2 and FGF2 release for 3 weeks), which in turn promoted neovascularisation and new bone formation in critical sized defects. There was no difference in vascularisation and bone formation induced by VEGF + BMP2 or FGF2 + BMP2. The growth factor was loaded in a simple manner, which would ensure ease of use for the end-user, especially for the surgeons treating a patient in an operating room.

Screening of Nanocomposite Scaffolds Arrays Using Superhydrophobic‐Wettable Micropatterns

Platforms containing multiple arrays for high‐throughput screening are demanded in the development of biomaterial libraries. Here, an array platform for the combinatorial analysis of cellular interactions and 3D porous biomaterials is described. Using a novel method based on computer‐aided manufacturing, wettable regions are printed on superhydrophobic surfaces, generating isolated spots. This freestanding benchtop array is used as a tool to deposit naturally derived polymers, chitosan and hyaluronic acid, with bioactive glass nanoparticles (BGNPs) to obtain a scaffold matrix. The effect of fibronectin adsorption on the scaffolds is also tested. The biomimetic nanocomposite scaffolds are shown to be osteoconductive, non‐cytotoxic, promote cell adhesion, and regulate osteogenic commitment. The method proves to be suitable for screening of biomaterials in 3D cell cultures as it can recreate a multitude of combinations on a single platform and identify the optimal composition that drives to desired cell responses. The platforms are fully compatible with commercially routine cell culture labware and established characterization methods, allowing for a standard control and easy adaptability to the cell culture environment. This study shows the value of 3D structured array platforms to decode the combinatorial interactions at play in cell microenvironments.

Biomimetic composite scaffolds based on mineralization of hydroxyapatite on electrospun poly(ɛ-caprolactone)/nanocellulose fibers

A biomimetic nanocomposite scaffold with HA formation on the electrospun poly(ɛ-caprolactone) (PCL)/nanocellulose (NC) fibrous matrix was developed in this study. The electrospun PCL/NC fiber mat was built and then biomineralized by treatment in simulated body fluid (SBF). Using such a rapid and effective procedure, a continuous biomimetic crystalline HA layer could be successfully formed without the need of any additional chemical modification of the substrate surface. The results showed that the introduction of NC into composite fibers is an effective approach to induce the deposition of HA nucleus as well as to improve their distribution and growth of a crystalline HA layer on the fibrous scaffolds. The water contact angle (WCA) of the PCL/NC/HA scaffolds decreases with increasing NC content and mineralization time, resulting in the enhancement of their hydrophilicity. These results indicated that HA-mineralized on PCL/NC fiber can be prepared directly by simply using SBF immersion.

Sub-nanoscale free volume and local elastic modulus of chitosan-carbon nanotube biomimetic nanocomposite scaffold-materials.

Future progress in materials for tissue engineering and 3D cell cultures applications requires control of two key physical properties: nanoscale mechanical properties and mass transport. These requirements remain uncontrolled partly due to a lack of physical parameters and quantitative measurements. Using chitosan scaffolds as a model system in close-to-physiological conditions and a combination of experimental techniques and theory, we link structure with local nanomechanical properties. Additionally we introduce a parameter, the free volume, to predict variations in transport properties. By fabricating nanocomposites with single walled carbon nanotubes (SWNTs) we are able to test our approach: incorporation of acid-treated, soluble, ∼80 nm SWNTs in a chitosan matrix leads to a 2 fold increase in mean local elastic modulus and a decrease of 3% of the free volume available for oxygen diffusion. Inclusion of hydrophobic, ∼800 nm SWNTs leads to a 100 fold increase of elastic modulus and doubles the voids percentage available for the transport of glucose.

Biomimetic composite scaffolds based mineralization of hydroxyapatite on electrospun calcium-containing poly(vinyl alcohol) nanofibers

Nanocomposite materials consisting of polymer matrix and inorganic salts in the form of nanocrystals of hydroxyapatite (HA) are regarded as superior candidates for bone treatment. A biomimetic nanocomposite scaffold with HA formation on the electrospun poly(vinyl alcohol) (PVA) nanofibrous structure by employing a Ca–P alternate soaking method was developed in this work. The calcium-containing PVA nanofibers were prepared by adding calcium nitrate to the starting solution prior to electrospinning, and then mineralized by Ca–P treatment in incubation solution. With this rapid and effective procedure, a continuous biomimetic crystalline HA layer could be formed successfully without the need of a prior chemical modification of the substrate surface under very mild reaction conditions. Moreover, the HA formed with a relatively accelerated growth had a carbonated and poor crystalline structure, resembling biological apatite in the bone mineral. The introduction of calcium ions in nanofibers by electrospinning was a favorable approach to induce the deposition of calcium phosphate and improve the distribution, nucleation, and growth of crystalline HA layer on nanofibrous scaffolds. Bioactivity tests revealed that these mineralized PVA/HA composite scaffolds improved the biocompatibility. The porous polymer/HA composite scaffolds produced in the present study might have potential applications in bone tissue engineering.

In vitro evaluation of biomimetic nanocomposite scaffold using endometrial stem cell derived osteoblast-like cells

Current study encourages the differentiation of human endometrial stem cells into osteoblast-like cells using osteogenic media for potential bone tissue engineering purposes. A biomimetic nanocomposite scaffolds based on GEL/calcium phosphate were fabricated and behavior of differentiated osteoblast cells was evaluated after seeding on this scaffold. Prepared scaffolds were assessed in terms of attachment, alkaline phosphatase activity, gene expression and proliferation of osteoblast cells. The matrix mineralization was approved by alizarin red and the treated cultures with osteogenic media and BMP2 were positive for osteopontin and osteocalcin antibodies. RT-PCR confirmed presence of osteopontin, osteonectin and alkaline phosphatase mRNA after differentiation in EnSCs-derived osteoblast-like cells. Also, it has been shown that the biomimetic nanocomposites possess appropriate chemical and physical properties to support the attachment and proliferation of differentiated osteoblast cells.

Mineralization of nanohydroxyapatite on electrospun poly(l-lactic acid)/gelatin by an alternate soaking process: A biomimetic scaffold for bone regeneration

Biomimetic nanocomposite scaffolds were fabricated by electrospinning poly(l-lactic acid) and a blend of poly(L-lactic acid)/gelatin to eliminate the use of collagen. The scaffolds were mineralized via alternate soaking in calcium and phosphate solutions, whereby 66.8% nanohydroxyapatite formation was successfully induced which is similar to that of native human bone (60%). The poly(L-lactic acid)/gelatin scaffolds had uniform nanohydroxyapatite formation throughout the scaffold. The mineralization enhanced the tensile modulus and tensile strength without increasing the brittleness. The in vitro biocompatibility of scaffolds was evaluated with murine adipose tissue–derived stem cells. The scaffolds with nanohydroxyapatite aided cell attachment and promoted cell–cell interaction. The mineralization and osteocalcin expression of the murine adipose tissue–derived stem cells were maximum in the poly(L-lactic acid)/gelatin/nanohydroxyapatite scaffold. Therefore, the gelatin and nanohydroxyapatite in poly(L-lactic acid)/gelatin/nanohydroxyapatite scaffolds provided cues for the differentiation of murine adipose tissue–derived stem cells. The biochemical nature of poly(L-lactic acid)/gelatin/nanohydroxyapatite scaffold accelerated osteogenic differentiation and could be a potential candidate for bone regeneration.

Preparation of a biomimetic nanocomposite scaffold for bone tissue engineering via mineralization of gelatin hydrogel and study of mineral transformation in simulated body fluid

In this study, double diffusion method in a physiologically relevant environment was used to prepare a biomimetic gelatin-amorphous calcium phosphate nanocomposite scaffold. The precipitated calcium phosphate within gelatin as well as produced nanocomposite scaffolds were characterized by the commonly used bulk techniques. The results showed that nanocomposite scaffolds were porous with three-dimensionally interconnected microstructure, pore size ranging from 150 to 350 μm. Porosity was about 82% and nanocrystalline precipitated minerals were dispersed evenly among gelatin fibers. A mineral containing amorphous calcium phosphate and brushite precipitate was formed within the gelatin matrix at 4°C. After incubation in SBF solution at 37°C for 5 days, the mineral phase was transformed to nanocrystalline hydroxyapatite. It should be noted that precursor phases inside a scaffold implanted into the body can result in biomimetic conversion of precursors to hydroxyapatite that is very similar to the bone mineral and has a profound level of biocompatibility. Thus, our results highlight the potential use of engineered biomimetic bone tissue scaffolds in the bone tissue repair process.