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.     

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.     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

Regulation of cellular responses to macroporous inorganic films prepared by the inverse-opal method

Regenerative medicine for repairing damaged body tissues has recently become critically important. Cell culture scaffolds are required for the control of cell attachment, proliferation, and differentiation in in vitro cell cultures. A new strategy to control cell adhesion, morphology, and proliferation was developed by culturing mouse osteoblast-like MC3T3-E1 cells on novel cell culture scaffolds fabricated using ordered nanometer-sized pores (100, 300, 500, and 1000 nm). Results of this study indicate that after 72 h of incubation, the number of cells cultured on a silica film with a pore size of 1000 nm was similar to or slightly lower than that cultured on a non-porous control silica film. Films with 100-500 nm pore sizes, however, resulted in the cell growth inhibition. Morphology of the cultured cells revealed increased elongation and the formation of actin stress fibers was virtually absent on macroporous silica films with 100-500 nm pore size. Vinculin molecules expressed in cells cultured on the non-porous silica films showed many clear focal adhesions, whereas focal contacts were insufficiently formed in cells cultured on macroporous films. The influence of hydroxyapatite (HAp) and alumina scaffolds on the behavior of MC3T3-E1 cells was also evaluated. The proliferation rate of MC3T3-E1 cells cultured on HAp films with 1000 nm pore size was increased to approximately 20% above than that obtained of cells cultured on non-porous HAp films. These results demonstrate that the pore size and constituents of films play a role in controlling the morphology and proliferation rate of MC3T3-E1 cells.     

Modifying the Pores of an Inverse Opal Scaffold With Chitosan Microstructures for Truly Three‐Dimensional Cell Culture

Inverse opal scaffolds have recently emerged as a novel class of scaffolds with uniform and controllable pore sizes for tissue engineering to provide better nutrient transport, a uniform cell distribution, and an adjustable microenvironment for cell differentiation. However, when the pore size of the scaffold is much larger than the dimension of a cell, the cell actually encounters a local 2D environment and the void space associated with the pore can not be efficiently utilized. Here, we demonstrate that a truly 3D microenvironment can be created inside a pore by further functionalizing the as‐prepared inverse opal scaffold with a second polymer by freeze‐drying. The resultant inverse opal scaffold with hierarchically structured pores can enhance both cell proliferation and tissue infiltration.     

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.     

Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds

Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.     

Nanofibrous poly(lactic acid)/hydroxyapatite composite scaffolds for guided tissue regeneration

The production of nanofibrous PLA/HA composite scaffolds is described. The morphological, mechanical, surface, and thermal properties of the composites were extensively investigated. The results show that the mixture of PLA and HA formed smooth nanofibers without lumps. The incorporation of HA increased the mechanical strength of the nanofibers and changed the morphology, increasing the mean fiber diameter and pore size. Surface and internal properties confirmed that HA was homogeneously distributed inside the nanofibers and oriented towards their surface. The nanofiber composites allowed the adhesion and proliferation of pre-osteoblasts for up to 3 weeks.     

Atmospheric plasma treatment of porous polymer constructs for tissue engineering applications

Porous 3D polymer scaffolds prepared by TIPS from PLGA (53:47) and PS are intrinsically hydrophobic which prohibits the wetting of such porous media by water. This limits the application of these materials for the fabrication of scaffolds as supports for cell adhesion/spreading. Here we demonstrate that the interior surfaces of polymer scaffolds can be effectively modified using atmospheric air plasma (AP). Polymer films (2D) were also modified as control. The surface properties of wet 2D and 3D scaffolds were characterised using zeta-potential and wettability measurements. These techniques were used as the primary screening methods to assess surface chemistry and the wettability of wet polymer constructs prior and after the surface treatment. The surfaces of the original polymers are rather hydrophobic as highlighted but contain acidic functional groups. Increased exposure to AP improved the water wetting of the treated surfaces because of the formation of a variety of oxygen and nitrogen containing functions. The morphology and pore structure was assessed using SEM and a liquid displacement test. The PLGA and PS foam samples have central regions which are open porous interconnected networks with maximum pore diameters of 49 microm for PLGA and 73 microm for PS foams.     

Fabrication of open‐porous PCL/PLA tissue engineering scaffolds and the relationship of foaming process,morphology, and mechanical behavior

Tissue engineering scaffolds should provide a suitable porous structure and proper mechanical strength, which is beneficial for the delivery of growth factor and regulation of cells. In this study, the open‐porous polycaprolactone (PCL)/poly (lactic acid) (PLA) tissue engineering scaffolds with suitable porous scale were fabricated using different ratios of PCL/PLA blends. At the same time, the relationship of foaming process, morphology, and mechanical behavior in the optimized batch microcellular foaming process were studied based on the single‐factor experiment method. The porous structures and mechanical strength of the scaffolds were optimized by adjusting foaming parameters, including the temperature, pressure, and CO₂ dissolution time. The results indicated that the foaming parameters influence the cell morphology, further determine the mechanical behavior of PCL/PLA blends. When the PCL content is high, with the increase of temperature and time, the cell diameter and the elastic modulus increased, and the tensile strength and elastic modulus increased with the increase of the average cell size, and decreased as the increase of the cell density. While when the PLA content was high, the cell diameter showed the same trend, and the tensile strength and elastic modulus were higher, and the elongation at break was lower, and tensile strength and elastic modulus decreased with the increase of the average cell size and increased with the increase of cell density. This work successfully fabricated optimized porous PCL/PLA scaffolds with excellent suitable mechanical properties, pore sizes, and high interconnectivity, indicating the effectiveness of modulating the batch foaming process parameters.     

Aerogel microspheres based on cellulose nanofibrils as potential cell culture scaffolds

Crosslinked poly(vinyl alcohol) (PVA)/cellulose nanofibril (CNF) hybrid aerogel micro-spheres with two different particle sizes were fabricated via a combination of the water-in-oil (W/O) emulsification process and the freeze-drying process. The aerogel micro-spheres were highly porous with a bulk density as low as 0.0047 g/cm³ for the large microspheres. The pore size of the microspheres ranged from nano- to micro-meters. Preliminary biocompatibility assays of the aerogel microspheres were investigated with NIH 3T3 cells to explore their potential application as cell culture scaffolds. The highly crosslinked aerogel microspheres were robust and were able to maintain their shape during the cell culture process. The live/dead assay showed that the cells could be seeded, attached, and proliferated on the surface of PVA/CNF aerogel microspheres. The fluorescence images showed that some of the cells migrated into the inner pores of the microspheres. Moreover, the large microspheres with larger average pore sizes had a higher cell count than that of the small microspheres. This study confirms that the PVA/CNF aerogel microspheres fabricated in this work are nontoxic and biocompatible. Furthermore, the interconnected, highly porous nanofibrous structure of the microspheres can successfully facilitate cell attachment, differentiation, and proliferation.     

明胶/聚乳酸复合纤维膜的制备、表征及作为角膜细胞载体的评价

利用静电纺丝技术制备了明胶与聚乳酸的复合纤维膜, 研究了组分配比对复合膜的表面性能、孔隙结构和力学性能的影响, 并以复合膜为组织工程支架进行兔角膜上皮细胞的体外培养. 采用扫描电子显微镜、免疫荧光染色和噻唑蓝四氮唑溴化物(MTT)比色法综合评价了细胞在支架表面的黏附与增殖能力. 结果表明, 纺丝溶液的组分对纤维的直径分布和表面亲水性有显著影响, 不同组分配比的复合纤维膜均具有高孔隙率的通孔结构; 以明胶为基材可维持复合膜的细胞黏附性; 与聚乳酸复合可以明显提高复合膜的力学性能.     

Biopolymer/Glycopolypeptide‐Blended Scaffolds: Synthesis,Characterization and Cellular Interactions

Three‐dimensional (3D) scaffolds formed from natural biopolymers gelatin and chitosan that are chemically modified by galactose have shown improved hepatocyte adhesion, spheroid geometry and functions of the hepatocytes. Galactose specifically binds to the hepatocytes via the asialoglycoprotein receptor (ASGPR) and an increase in galactose density further improves the hepatocyte proliferation and functions. In this work, we aimed to increase the galactose density within the biopolymeric scaffold by physically blending the biopolymers chitosan and gelatin with an amphiphlic β‐galactose polypeptide (PPO‐GP). PPO‐GP, is a di‐block copolymer with PPO and β‐galactose polypeptide, exhibits lower critical solution temperature and is entrapped within the scaffold through hydrophobic interactions. The uniform distribution of PPO‐GP within the scaffold was confirmed by fluorescence microscopy. SEM and mechanical testing of the hybrid scaffolds indicated pore size, inter connectivity and compression modulus similar to the scaffolds made from 100 % biopolymer. The presence of the PPO‐GP on the surface of the scaffold was tested monitoring the interaction of an analogous mannose containing PPO‐GP scaffold and the mannose binding lectin Con‐A. In vitro cell culture experiments with HepG2 cells were performed on GLN‐GP and CTS‐GP and their cellular response was compared with GLN and CTS scaffolds for a period of seven days. Within three days of culture the Hep G2 cells formed multicellular spheroids on GLN‐GP and CTS‐GP more efficiently than on the GLN and CTS scaffolds. The multicellular spheroids were also found to infiltrate more in GLN‐GP and CTS‐GP scaffolds and able to maintain their round morphology as observed by live/dead and SEM imaging.     

Biomimetic polymer/apatite composite scaffolds for mineralized tissue engineering

The material surface must be considered in the design of scaffolds for bone tissue engineering so that it supports bone cells adhesion, proliferation and differentiation. A biomimetic approach has been developed as a 3D surface modification technique to grow partially carbonated hydroxyapatite (the bonelike mineral) in prefabricated, porous, polymer scaffolds using a simulated body fluid in our lab. For the rational design of scaffolding materials and optimization of the biomimetic process, this work focused on various materials and processing parameters in relation to apatite formation on 3D polymer scaffolds. The apatite nucleation and growth in the internal pores of poly(L-lactide) and poly(D,L-lactide) scaffolds were significantly faster than in those of poly(lactide-co-glycolide) scaffolds in simulated body fluids. The apatite distribution was significantly more uniform in the poly(L-lactide) scaffolds than in the poly(lactide-co-glycolide) scaffolds. After incubation in a simulated body fluid for 30 d, the mass of poly(L-lactide) scaffolds increased approximately 40%, whereas the mass of the poly(lactide-co-glycolide) scaffolds increased by about 15% (see Figure). A higher ionic concentration and higher pH value of the simulated body fluid enhanced apatite formation. The effects of surface functional groups on apatite nucleation and growth were found to be more complex in 3D scaffolds than on 2D films. Surprisingly enough, it was found that carboxyl groups significantly reduced the apatite formation, especially on the internal pore surfaces of 3D scaffolds. These findings are critically important in the rational selection of materials and surface design of 3D scaffolds for mineralized tissue engineering and may contribute to the understanding of biomineralization as well.SEM micrograph of a poly(L-lactide) 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,Crosslinking and in vitro Biocompatibility of a Novel Degradable Nano-structure Urethral Tubular Scaffold

A degradable poly(lactic-co-glycolic acid, LA:GA=80:20)(PLGA) urethral tubular scaffold was fabricated by electrospinning. In order to enhance the mechanical properties, the scaffold was crosslinked with glutaraldehyde. The structure and properties of the crosslinked scaffolds were investigated by the mechanical property testing, scanning electron microscopy(SEM), degradability test in vitro and 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-diphenytetrazo- liumromide(MTT). The results show that the scaffold has the nano-structure. The pore size and the porosity are suitable for cell seeding, growth and extracellular matrix production. Although influenced by the crosslinking slightly, the pore size and the porosity could still support cell proliferation and tissuse formation. The mechanical properties are remarkably increased by the crosslinking of glutaraldehyde, and it could meet the demands of a urethral stent. The scaffold could completely collapse within 70 d. The results of the biocompatibility test show that the PLGA scaffold had no cytotoxicity.     

Electrospun collagen-chitosan-TPU nanofibrous scaffolds for tissue engineered tubular grafts

The objective of this study is to design a novel kind of scaffolds for blood vessel and nerve repairs. Random and aligned nanofibrous scaffolds based on collagen-chitosan-thermoplastic polyurethane (TPU) blends were electrospun to mimic the componential and structural aspects of the native extracellular matrix, while an optimal proportion was found to keep the balance between biocompatibility and mechanical strength. The scaffolds were crosslinked by glutaraldehyde (GTA) vapor to prevent them from being dissolved in the culture medium. Fiber morphology was characterized using scanning electron microscopy (SEM) and atomic-force microscopy (AFM). Fourier transform infrared spectroscopy (FTIR) showed that the three-material system exhibits no significant differences before and after crosslinking, whereas pore size of crosslinked scaffolds decreased drastically. The mechanical properties of the scaffolds were found to be flexible with a high tensile strength. Cell viability studies with endothelial cells and Schwann cells demonstrated that the blended nanofibrous scaffolds formed by electrospinning process had good biocompatibility and aligned fibers could regulate cell morphology by inducing cell orientation. Vascular grafts and nerve conduits were electrospun or sutured based on the nanofibrous scaffolds and the results indicated that collagen-chitosan-TPU blended nanofibrous scaffolds might be a potential candidate for vascular repair and nerve regeneration.     

Cell adhesive and growth behavior on electrospun nanofibrous scaffolds by designed multifunctional composites

Nanostructured biocomposite scaffolds of poly(l-lactide) (PLLA) blended with collagen (coll) or hydroxyapatite (HA), or both for tissue engineering application, were fabricated by electrospinning. The electrospun scaffolds were characterized for the morphology, chemical and tensile properties by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), Fourier transform infrared (FTIR) measurement, and tensile testing. Electrospun biocomposite scaffolds of PLLA and collagen or (and) HA in the diameter range of 200-700 nm mimic the nanoscale structure of the extracellular matrix (ECM) with a well-interconnection pore network structure. The presence of collagen in the scaffolds increased their hydrophility, and enhanced cell attachment and proliferation, while HA improved the tensile properties of the scaffolds. The biocompatibility of the electrospun scaffolds and the viability of contacting cells were evaluated by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and by fluorescein diacetate (FDA) and propidium iodide (PI) double staining methods. The results support the conclusion that 293T cells grew well on composite scaffolds. Compared with pure PLLA scaffolds a greater density of viable cells was seen on the composites, especially the PLLA/HA/collagen scaffolds.     

The fabrication and characterization of poly(lactic acid) scaffolds for tissue engineering by improved solid–liquid phase separation

In this article a new technique was developed to fabricate scaffolds with a unique microstructure by solid–liquid separation in combination with particulate-leaching. Firstly, the effects of polymer concentration, quenching temperature on the porous morphology and the mechanical property of obtained scaffolds during solid–liquid separation have been investigated. Then, salt granules as porogen were introduced into the solid–liquid phase separation to produce the unique pore structure of the scaffold. The pore diameter of the scaffold could be controlled with the particulate size and the wall of pores possessed special microstructure, which enhanced the pore interconnectivity. The cell culture results confirmed that a good interconnectivity of the scaffold prepared by the improved solid–liquid separation was useful for nutrition transportation and cell proliferation. Copyright © 2003 John Wiley & Sons, Ltd.