Preparation and characterization of new materials based on silk fibroin,chitosan and nanohydroxyapatite

Abstract

In this paper, a series of porous nanohydroxyapatite/silk fibroin/chitosan (nHA/SF/CTS) scaffolds were successfully prepared using the freeze-drying method. The biomaterials were characterized by attenuated total reflection Fourier transform infrared spectroscopy, and mechanical testing and thermogravimetric analysis. Moreover, studies of porosity, pore size, swelling properties and in vitro degradation test were performed. Research has proved that micro-structure, porosity, water adsorption and compressive strength were greatly affected by the components’ concentration, in particular the content of silk fibroin. SEM observations showed that the scaffolds of nHA/SF/CTS are highly porous, with pore size in wide range from 25 to 300?µm which is suitable for cell growth. nHA/SF/CTS scaffolds have sufficient mechanical integrity to resist handling during implantation and in vivo loading. Both, the compressive modulus and compressive strength of the scaffold, decrease with the increase in silk fibroin content.     

Biocompatibility In-vitro of Gel/HA Composite Scaffolds Containing Nano-Bioactive Glass for Tissue Engineering

Designing and fabricating nanocomposite scaffolds for bone regeneration from different biodegradable polymers and bioactive materials are an essential step to engineer tissues. In this study, the composite scaffold of gelatin/hyaluronic acid (Gel/HA) containing nano-bioactive glass (NBG) was prepared by using freeze-drying method. The biocompatibilities in-vitro of the Gel-HA/NBG composite scaffolds, including MTT assay, ALP activity, von Kossa staining and tetracycline staining, were investigated. The SEM observations revealed that the prepared scaffolds were porous with three-dimensional (3D) and interconnected microstructure, agglomerated NBG particles were uniformly dispersed in the matrix. MTT results indicated that the tested materials didn't show any cytotoxicity. The presence of NBG in the composite scaffold further enhanced the ALP activity in comparison with the pure Gel/HA scaffold. The von Kossa staining and tetracycline staining results also indicated that the NBG may improve the cell response. Therefore, the results indicated the nanocomposite scaffold made from Gel, HA and NBG particles could be considered as a potential bone tissue engineering implant.     

Improving mechanical and biological properties of macroporous HA scaffolds through composite coatings

Interconnected porous hydroxyapatite (HA) scaffolds are widely used for bone repair and replacement, owing to their ability to support the adhesion, transfer, proliferation and differentiation of cells. In the present study, the polymer impregnation approach was adopted to produce porous HA scaffolds with three-dimensional (3D) porous structures. These scaffolds have an advantage of highly interconnected porosity (≈85%) but a drawback of poor mechanical strength. Therefore, the as-prepared HA scaffolds were lined with composite polymer coatings in order to improve the mechanical properties and retain its good bioactivity and biocompatibility at the same time. The composite coatings were based on poly(d,l-lactide) (PDLLA) polymer solutions, and contained single component or combination of HA, calcium sulfate (CS) and chondroitin sulfate (ChS) powders. The effects of composite coatings on scaffold porosity, microstructure, mechanical property, in vitro mineralizing behavior, and cell attachment of the resultant scaffolds were investigated. The results showed that the scaffolds with composite coatings resulted in significant improvement in both mechanical and biological properties while retaining the 3D interconnected porous structure. The in vitro mineralizing behaviors were mainly related to the compositions of CS and ChS powders in the composite coatings. Excellent cell attachments were observed on the pure HA scaffold as well as the three types of composite scaffolds. These composite scaffolds with improved mechanical properties and bioactivities are promising bone substitutes in tissue engineering fields.     

Biocompatibility properties of composite scaffolds based on 1,4-butanediamine modified poly(lactide-co-glycolide) and nanobioceramics

Three-dimensional biodegradable porous scaffolds play an important role in tissue engineering. The degradable scaffold material, based on 1,4-butanediamine-modified poly(lactide-co-glycolide) (BMPLGA), nano-bioactive glass (NBAG), and nano-β-tricalciumphosphate (β-TCP), was prepared by a solution-casting/salt-leaching method. The biological properties were studied by using cell cytotoxicity, von Kossa staining, alkaline phosphatase activity, hemolytic test, acute toxicity, and genetic toxicity test. The MTT results indicated that the BMPLGA/NBAG-β-TCP materials did not show any cytotoxicity. The result of von Kossa staining showed that the introduction of the NBAG and β-TCP promoted fibroblastic differentiation and improved the mineral deposition of the BMPLGA matrix. In addition, the presence of NBAG and β-TCP in the composite further enhanced the ALP activity in comparison with the sole BMPLGA material. The hemolytic potential showed that the nanocomposite scaffolds were non-hemolytic. The BMPLGA/NBAG-β-TCP scaffolds showed no acute systemic toxicity or mutagenic action. Therefore, the results indicated the BMPLGA/NBAG-β-TCP nanocomposite scaffold could be considered as a potential bone tissue engineering implant.     

In-Vitro Biocompatibility Evaluation of Collagen-Hyaluronic Acid/Bioactive Glass Nanocomposite Scaffold

A nano-structured scaffold was designed for bone repair using collagen, hyaluronic acid (HYA) and nano-bioactive glass (NBaG) as its main components. The collagen-HYA/NBaG scaffold was prepared by using a freeze-drying technique and characterized by scanning electron microscopy (SEM). Osteoblastls were seeded on these scaffolds and their proliferation rate, alkaline phosphatase (ALP) activity and ability to form mineralized bone nodules were compared with those osteoblasts grown on cell culture plastic surfaces. The cross-section morphology shows that the collagen-HYA/NBaG scaffold possessed a three-dimensional (3D) interconnected homogenous porous structure. The results obtained from biological assessment show that this scaffold did not negatively affect osteoblasts proliferation rate and improves osteoblasts function as shown by increasing the ALP activity and calcium deposition and formation of mineralized bone nodules. Therefore, the composite scaffolds could provide a favorable environment for initial cell adhesion, maintained cell viability and cell proliferation, and had good in-vitro biocompatibility.     

Fabrication of chitosan/collagen/hydroxyapatite scaffolds with encapsulated Cissus quadrangularis extract

Here, we demonstrated the fabrication of a composite scaffold (chitosan [CS], collagen [Col], and hydroxyapatite [HA]) with the incorporation of encapsulated Cissus quadrangularis (CQ) extract for tissue engineering applications. First, the crude extract of CQ loaded nanoparticles were synthesized via double emulsion technique using polycaprolactone (PCL) and polyvinyl alcohol (PVA) as oil and aqueous phases, respectively. Both PCL (20, 40, and 80 mg/mL) and PVA (0.5%, 1%, and 3% w/v) concentrations were varied to determine the optimum concentrations for CQ‐loaded nanoparticle preparation. The CQ‐loaded PCL nanoparticles (CQ‐PCL NPs), prepared with 20 mg/mL PCL and 0.5% (w/v) PVA, exhibited the smallest size of 334.22 ± 43.21 nm with 95.54 ± 1.49% encapsulation efficiency. Then, the CQ‐PCL NPs were incorporated into the CS/Col/HA scaffolds. These scaffolds were also studied for their ultrastructure, pore sizes, chemical composition, compressive modulus, water swelling, weight loss, and biocompatibility. The results showed that the addition of CQ‐PCL NPs into the scaffolds did not dramatically alter the ultrastructure and properties of the scaffolds, compared to CS/Col/HA scaffolds alone. However, incorporation of CQ‐PCL NPs in the scaffolds improved the release profile of CQ by preventing the initial burst release and prolonging the release rate of CQ. In addition, the CQ‐PCL NPs‐loaded CS/Col/HA scaffolds supported the attachment and proliferation of MC3T3‐E1 osteoblast cells.     

Nano‐hydroxyapatite/polyamide66 composite tissue‐engineering scaffolds with anisotropy in morphology and mechanical behaviors

Based on a biomimetic conception, nano‐hydroxyapatite (n‐HA)/polyamide66 (PA66) composite scaffolds were prepared with anisotropic properties both in morphology and mechanical behavior. A novel improved thermally induced phase separation (TIPS) technique was developed to generate orientation‐structured scaffolds for tissue engineering. The physiochemical, morphological, and mechanical properties of the resultant scaffolds were evaluated. According to the results, the improved TIPS method exhibited good processability and reproducibility and enabled the composite scaffolds to have a high content of inorganic fillers. The morphological study proved that the n‐HA/PA66 scaffolds exhibited unidirectional microtubular architecture with high porosity (ca. 80–85%) and an optimal pore size ranging from 200 to 500 μm. Besides, the effect of n‐HA content on the morphology of the scaffolds was studied, and the results indicated that the obtained scaffolds presented an improvement in anisotropic morphology with increase of n‐HA content. The anisotropy was also evaluated in the mechanical properties of the scaffolds, that is, the longitudinal compressive strength and modulus were ～1.5 times of the transverse ones. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 658–669, 2009     

Fabrication of poly(lactide‐co‐glycolide) scaffold embedded spatially with hydroxyapatite particles on pore walls for bone tissue engineering

Poly(lactide‐co‐glycolide) (PLGA) scaffolds embedded spatially with hydroxyapatite (HA) particles on the pore walls (PLGA/HA‐S) were fabricated by using HA‐coated paraffin spheres as porogens, which were prepared by Pickering emulsion. For comparisons, PLGA scaffolds loaded with same amount of HA particles (2%) in the matrix (PLGA/HA‐M) and pure PLGA scaffolds were prepared by using pure paraffin spheres as porogens. Although the three types of scaffolds had same pore size (450–600 µm) and similar porosity (90%–93%), the PLGA/HA‐S showed the highest compression modulus. The embedment of the HA particles on the pore walls endow the PLGA/HA‐S scaffold with a stronger ability of protein adsorption and mineralization as well as a larger mechanical strength against compression. In vitro culture of rat bone marrow stem cells revealed that cell morphology and proliferation ability were similar on all the scaffolds. However, the alkaline phosphatase activity was significantly improved for the cells cultured on the PLGA/HA‐S scaffolds. Therefore, the method for fabricating scaffolds with spatially embedded nanoparticles provides a new way to obtain the bioactive scaffolds for tissue engineering. Copyright © 2011 John Wiley & Sons, Ltd.     

HPMC crosslinked chitosan/hydroxyapatite scaffolds containing Lemongrass oil for potential bone tissue engineering applications

The chitosan (CS), hydroxypropyl methyl cellulose (HPMC), hydroxyapatite (HAp and Lemon grass oil (LGO) based scaffolds was prepared by freeze gelation method. The composite formation was confirmed by FTIR (Fourier-transform infrared spectroscopy) analysis and surface morphology was evaluated by SEM (Scanning Electron Microscopy) analysis. The mechanical strength, biodegradation, swelling, porosity and antibacterial activity were evaluated on the basis of LGO contents. The scaffold structure was porous and the mechanical strength was enhanced as a function of LGO contents. The scaffold properties analysis revealed the biodegradation nature and swelling behavior of CS-HPMC-HAp-LGO was also affected significantly as a function of LGO contents. The cytotoxicity of CS-HPMC-HAp-LGO was studied against MC3T3-E1 cells and based on cell viability, no toxic sign was observed. The antimicrobial activity was evaluated against S. aureus and CS-HPMC-HAp-LGO scaffolds showed promising activity, which was varied as a function of LGO contents. The findings revealed that the CS-HPMC-HAp-LGO are biocompatible and have potential for bone tissue engineering.     

Preparation and modification of collagen-based porous scaffold for tissue engineering

In the effort to generate cartilage tissues using mesenchymal stem cells, porous scaffolds with prescribed biomechanical properties were prepared. Scaffolds with interconnected pores were prepared via lyophilisation of frozen hydrogels made from collagen modified with chitosan nanofibres, hyaluronic acid, copolymers based on poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), and itaconic acid (ITA), and hydroxyapatite nanoparticles. The modified collagen compositions were cross-linked using N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) combined with N-hydroxysuccinimide (NHS) in water solution. Basic physicochemical and mechanical properties were measured and an attempt to relate these properties to the molecular and supermolecular structure of the modified collagen compositions was carried out. Scaffolds containing hydrophilic chitosan nanofibres showed the highest swelling ratio (SR = 20–25) of all the materials investigated, while collagen modified with an amphiphilic PLGA-PEG-PLGA copolymer or functionalised with ITA exhibited the lowest swelling ratio (SR = 5–8). The best resistance to hydrolytic degradation was obtained for hydroxyapatite containing scaffolds. On the other hand, the fastest degradation rate was observed for synthetic copolymer-containing scaffolds. The results showed that the addition of hydroxyapatite or hyaluronic acid to the collagen matrix increases the rigidity in comparison to the collagen-chitosan scaffold. Collagen scaffold modified with hyaluronic acid presented reduced deformation at break while the presence of hydroxypatatite enhanced the scaffold deformation under tensile loading. The tensile elastic modulus of chitosan nanofibre collagen scaffold was the lowest but closest to the articular cartilage; however, the strength and deformation to failure increased up to 200 %. Presented at the 1st Bratislava Young Polymer Scientists Workshop, Bratislava, 20–23 August 2007.     

In vitro growth and activity of chondrocytes on three dimensional polycaprolactone/chitosan scaffolds

Porous polycaprolactone/chitosan blend scaffolds with various compositional proportions were prepared using a particulate‐leaching method. The pore parameters of resultant scaffolds were found to be mainly modulated by porogen. The compressive mechanical properties and hydrophilicity of scaffolds were examined by measuring their compressive modulus and stress strength as well as swelling index. Selected chondrocytes isolated from articular cartilage of knee joints of rabbits were seeded on these scaffolds, and further in vitro cultured for various periods. The growth and activity of seeded cells were estimated by counting numbers of cells proliferated on scaffolds and measuring the amounts of proteoglycans and type II collagen synthesized by the seeded cells. It was found that some scaffolds composed of proper component ratios and having appropriate pore parameters exhibited promising characteristics for the adhesion and proliferation of seeded cells while maintaining the phenotype and activity of the cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Collagen-based scaffolds enriched with glycosaminoglycans isolated from skin of Salmo salar fish

In this study both collagen and glycosaminoglycans were isolated from biodegradable waste. Namely collagen was isolated from rat tail tendons and glycosaminoglycans (GAGs) from fish skin. Porous materials were then obtained based on the isolated collagen with 1 or 5% addition of GAGs by freeze-drying process. The scaffolds were studied by infrared spectroscopy, mechanical testing and examined for the porosity and density. The scaffolds structure was observed by scanning electron microscope. The adhesion and proliferation of human osteosarcoma SaOS-2 cells was examined on prepared scaffolds to assess their biocompability.The results showed that the addition of glycosaminoglycans improves the properties of collagen-based scaffolds. Mechanical strength was increased by GAGs addition as well as the porosity of studied materials. Each scaffold with and without GAGs displayed porous structure with interconnected regular shaped pores. The attachment of cells was better for pure collagen scaffold, however, GAGs additive promoted the cells proliferation on the scaffold.     

Preparation of porous nanocomposite scaffolds with honeycomb monolith structure by one phase solution freezedrying method

Biodegradable porous nanocomposite scaffolds of poly（lactide-co-glycolide）（PLGA） and L-lactic acid（LAc） oligomer surface-grafted hydroxyapatite nanoparticles（op-HA） with a honeycomb monolith structure were fabricated with the single-phase solution freeze-drying method.The effects of different freezing temperatures on the properties of the scaffolds,such as microstructures,compressive strength,cell penetration and cell proliferation were studied.The highly porous and well interconnected scaffolds with a tunable pore structure were obtained.The effect of different freezing temperature（4℃,-20℃,-80℃and -196℃） was investigated in relation to the scaffold morphology,the porosity varied from 91.2%to 83.0%and the average pore diameter varied from（167.2±62.6）μm to（11.9±4.2）μm while theσ₁₀ increased significantly.The cell proliferation were decreased and associated with the above-mentioned properties.Uniform distribution of op-HA particles and homogeneous roughness of pore wall surfaces were found in the 4℃frozen scaffold.The 4℃frozen scaffold exhibited better cell penetration and increased cell proliferation because of its larger pore size,higher porosity and interconnection.The microstructures described here provide a new approach for the design and fabrication of op-HA/PLGA based scaffold materials with potentially broad applicability for replacement of bone defects.     

Fabrication and Evaluation of Alginate/Bacterial Cellulose Nanocrystals–Chitosan–Gelatin Composite Scaffolds

It is common knowledge that pure alginate hydrogel is more likely to have weak mechanical strength, a lack of cell recognition sites, extensive swelling and uncontrolled degradation, and thus be unable to satisfy the demands of the ideal scaffold. To address these problems, we attempted to fabricate alginate/bacterial cellulose nanocrystals-chitosan-gelatin (Alg/BCNs-CS-GT) composite scaffolds using the combined method involving the incorporation of BCNs in the alginate matrix, internal gelation through the hydroxyapatite-d-glucono-δ-lactone (HAP-GDL) complex, and layer-by-layer (LBL) electrostatic assembly of polyelectrolytes. Meanwhile, the effect of various contents of BCNs on the scaffold morphology, porosity, mechanical properties, and swelling and degradation behavior was investigated. The experimental results showed that the fabricated Alg/BCNs-CS-GT composite scaffolds exhibited regular 3D morphologies and well-developed pore structures. With the increase in BCNs content, the pore size of Alg/BCNs-CS-GT composite scaffolds was gradually reduced from 200 μm to 70 μm. Furthermore, BCNs were fully embedded in the alginate matrix through the intermolecular hydrogen bond with alginate. Moreover, the addition of BCNs could effectively control the swelling and biodegradation of the Alg/BCNs-CS-GT composite scaffolds. Furthermore, the in vitro cytotoxicity studies indicated that the porous fiber network of BCNs could fully mimic the extracellular matrix structure, which promoted the adhesion and spreading of MG63 cells and MC3T3-E1 cells on the Alg/BCNs-CS-GT composite scaffolds. In addition, these cells could grow in the 3D-porous structure of composite scaffolds, which exhibited good proliferative viability. Based on the effect of BCNs on the cytocompatibility of composite scaffolds, the optimum BCNs content for the Alg/BCNs-CS-GT composite scaffolds was 0.2% (w/v). On the basis of good merits, such as regular 3D morphology, well-developed pore structure, controlled swelling and biodegradation behavior, and good cytocompatibility, the Alg/BCNs-CS-GT composite scaffolds may exhibit great potential as the ideal scaffold in the bone tissue engineering field.     

Synthesis of bioceramic scaffolds for bone tissue engineering by rapid prototyping technique

This study presents a novel rapid prototyping process to fabricate silicate/hydroxyapatite (HA) bone scaffolds for tissue engineering applications. The HA particles are embedded in the gelled silica matrix to form a green part of bioceramic bone scaffold after processing by selective laser sintering. The composition of the bioceramic scaffold is in the series of SiO₂·P₂O₅·CaO. Results indicate that the proposed process could fabricate a multilayer hollow shell structure with brittle property but sufficient integrity for handling prior to post-processing. The fabricated bone scaffold models had a surface finish of 25 μm, a dimensional shrinkage of 16 %, a maximum bending strength of 4.7 MPa, and an apparent porosity of 28 % under the laser energy density of 1.5 J/mm². In vitro bioactivity evaluation by optical density value using a microculture tetrazolium test assay revealed that the bioceramic bone scaffolds were suitable for cell culture, demonstrating their application in tissue engineering.     

Effects of Laminin For Osteogenesis in Porous Hydroxyapatite

Summery: As a tooth is composed of hard tissue covering pulp, it may be suitable for tooth regeneration to use porous cylindrical hydroxyapatite (HA) scaffolds with a hollow center. Generally, in vivo examination, bone marrow cell suspension for osteogenesis in cell/HA composite scaffold without subculture is prepared at a density of 1 × 10⁷ cells/ml or higher. In dentistry, stem cells would be obtained from tooth pulp. For dentine formation, a smaller number of stem cells must be used. In this study, a suspension of rat bone marrow cells at 1 × 10⁶ cells/ml of density was prepared to estimate the adhesive effect of laminin. After immersion of HA scaffold in laminin solution, bone marrow cells were seeded in the pores of the HA scaffolds by immersion in the cell suspension for preparing the cell/HA composite scaffolds. The specimens were respectively implanted in the dorsal subcutis of 7-week-old male Fischer 344 rats for 4 weeks for histological examination. Comparing with the results of in vivo examination, alkaline phosphatase activity of bone marrow cells on laminin-coated plate with and without dexamethasone cultured for 2 weeks was measured in vitro. It was considered that laminin contributed to bone formation in pores of a scaffold.     

Cellulose nanocrystals as biobased nucleation agents in poly-l-lactide scaffold: Crystallization behavior and mechanical properties

The mechanical strength of polymer scaffold is closely related to its crystallinity. In this work, cellulose nanocrystals (CNC) were incorporated into poly-l-lactide (PLLA) scaffold which was fabricated by selective laser sintering, aiming to improve the mechanical properties. CNC possesses numerous hydroxyl groups which might form hydrogen bond with PLLA molecular chains. The hydrogen bond induces the ordered arrangement of PLLA chain by using CNC as heterogeneous nucleating agent, thereby increasing crystallization rate and crystallinity. Results showed that PLLA scaffolds with 3 wt% CNC resulted in 191%, 351%, 34%, 83.5%, 56% increase in compressive strength, compressive modulus, tensile strength, tensile modulus and Vickers hardness, respectively. Encouragingly, with the incorporation of hydrophilic CNC, the PLLA/CNC scaffolds showed not only better hydrophilicity, but also faster degradation than PLLA. In vitro cell culture studies proved that the PLLA/CNC scaffolds were biocompatible and capable of supporting cell adhesion, proliferation and differentiation. The above results indicated that the PLLA/CNC scaffolds may therefore be a potential replacement in bone repair.     

Surface Modification of GTA Crosslinked Collagen‐based Composite Scaffolds with Low Temperature Plasma Technology

In this study for preparing the better performance scaffold materials for peripheral nerve repairing, the collagen‐based composite scaffolds are crosslinked with glutaraldehyde and their structure and performance are investigated. The results of FTIR indicated that the collagen and chitosan are certainly crosslinked through GTA without any significant change in the chemical property. It was observed under a scanning electron microscope (SEM) that the crosslinked collagen‐based composite scaffolds had a porous three‐dimensional cross‐linked structure. The experiments showed that the biostability of the scaffold is greatly enhanced, but the GTA crosslinking induces the potential cytotoxicity and poor hydrophilic nature. To overcome these disadvantages, the low temperature plasma technology is utilized to modify the surface of the cross‐linked collagen‐based composite scaffolds in this study. Measurements of water contact angle showed that hydrophilic nature of surface of the scaffolds was improved after low temperature plasma technology modification. The cell proliferation experiments revealed that the modified collagen‐based composite scaffolds still kept their bioactivity and benefited the proliferation.     

Enhanced mechanical performance and biological evaluation of a PLGA coated β-TCP composite scaffold for load-bearing applications

Porous β-tricalcium phosphate (β-TCP) has been used for bone repair and replacement in clinics due to its excellent biocompatibility, osteoconductivity, and biodegradability. However, the application of β-TCP has been limited by its brittleness. Here, we demonstrated that an interconnected porous β-TCP scaffold infiltrated with a thin layer of poly(lactic-co-glycolic acid) (PLGA) polymer showed improved mechanical performance compared to an uncoated β-TCP scaffold while retaining its excellent interconnectivity and biocompatibility. The infiltration of PLGA significantly increased the compressive strength of β-TCP scaffolds from 2.90 to 4.19 MPa, bending strength from 1.46 to 2.41 MPa, and toughness from 0.17 to 1.44 MPa, while retaining an interconnected porous structure with a porosity of 80.65%. These remarkable improvements in the mechanical properties of PLGA-coated β-TCP scaffolds are due to the combination of the systematic coating of struts, interpenetrating structural characteristics, and crack bridging. The in vitro biological evaluation demonstrated that rat bone marrow stromal cells (rBMSCs) adhered well, proliferated, and expressed alkaline phosphatase (ALP) activity on both the PLGA-coated β-TCP and the β-TCP. These results suggest a new strategy for fabricating interconnected macroporous scaffolds with significantly enhanced mechanical strength for potential load-bearing bone tissue regeneration.