Characterization of scaffolds based on chitosan and collagen with glycosaminoglycans

Scaffolds based on chitosan (CTS), collagen (Coll), and glycosaminoglycans (GAGs) cross-linked by N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mixture were obtained with the use of the freeze-drying method. They were characterized by different analyses, e.g. mechanical and swelling tests, porosity, and density measurement. Moreover, the scaffolds behavior in cell culture was examined with human osteosarcoma SaOS-2 cells. The results showed that the scaffolds based on CTS, Coll, and GAGs cross-linked by EDC/NHS present physicochemical properties appropriate for biomedical purposes. They show porosity above 90% and are highly swellable. The increasing GAGs content improves the attachment and survival of cells on the obtained scaffolds. It can be assumed that scaffolds based on CTS and Coll, GAGs-enriched and cross-linked by EDC/NHS addition are biocompatible, and have properties appropriate for the tissue engineering purposes.     

3D Plotted PCL Scaffolds for Stem Cell Based Bone Tissue Engineering

The ability to control the architecture and strength of a bone tissue engineering scaffold is critical to achieve a harmony between the scaffold and the host tissue. Rapid prototyping (RP) technique is applied to tissue engineering to satisfy this need and to create a scaffold directly from the scanned and digitized image of the defect site. Design and construction of complex structures with different shapes and sizes, at micro and macro scale, with fully interconnected pore structure and appropriate mechanical properties are possible by using RP techniques. In this study, RP was used for the production of poly(ε-caprolactone) (PCL) scaffolds. Scaffolds with four different architectures were produced by using different configurations of the fibers (basic, basic-offset, crossed and crossed-offset) within the architecture of the scaffold. The structure of the prepared scaffolds were examined by scanning electron microscopy (SEM), porosity and its distribution were analyzed by micro-computed tomography (µ-CT), stiffness and modulus values were determined by dynamic mechanical analysis (DMA). It was observed that the scaffolds had very ordered structures with mean porosities about 60%, and having storage modulus values about 1 × 10⁷ Pa. These structures were then seeded with rat bone marrow origin mesenchymal stem cells (MSCs) in order to investigate the effect of scaffold structure on the cell behavior; the proliferation and differentiation of the cells on the scaffolds were studied. It was observed that cell proliferation was higher on offset scaffolds (262000 vs 235000 for basic, 287000 vs 222000 for crossed structure) and stainings for actin filaments of the cells reveal successful attachment and spreading at the surfaces of the fibers. Alkaline phosphatase (ALP) activity results were higher for the samples with lower cell proliferation, as expected. Highest MSC differentiation was observed for crossed scaffolds indicating the influence of scaffold structure on cellular activities.     

The comparison of physic-chemical properties of chitosan/collagen/hyaluronic acid composites with nano-hydroxyapatite cross-linked by dialdehyde starch and tannic acid

Scaffolds based on chitosan, collagen and hyaluronic acid, cross-linked by dialdehyde starch with hydroxyapatite were obtained with the use of the freeze-drying method. Scaffolds were cross-linked by tannic acid or dialdehyde starch addition. Composites were characterized by different analyses, e.g. SEM images, porosity, density, liquid uptake, and mechanical tests. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells were examined on the obtained scaffolds.The results showed that the properties of the scaffolds based on chitosan, collagen, and hyaluronic acid can be modified by cross-linkers addition. The compressive modulus for the scaffolds cross-linked by dialdehyde starch was higher than for those cross-linked by tannic acid. The porosity of scaffolds cross-linked by starch was higher than those in which tannic acid was applied. However, the former presented lower density. SEM images showed the homogeneous scaffold structure with interconnected pores. Scaffolds cross-linked by tannic acid exhibited higher biocompatibility than those cross-linked by dialdehyde starch. However, the results showed that both scaffolds, cross-linked by dialdehyde starch and by tannic acid can provide the support required in tissue engineering and regenerative medicine. The scaffolds presented here may be easily operated to fit such small bone defects without causing adverse reactions.     

Surface modification with peptide for enhancing chondrocyte adhesion and cartilage regeneration in porous scaffolds

The present study presents the regeneration of cartilage in hybrid scaffolds comprising polyethylene oxide (PEO) and chitosan with surface CDPGYIGSR. This surface peptide was grafted via crosslinking onto the scaffolds. The pores in the scaffolds were interconnected and uniformly distributed with an average diameter about 200-250 μm. A high weight percentage of PEO in the matrix yielded a rugged topography of the pore surfaces. The adhesion of bovine knee chondrocytes (BKCs) in the peptide-grafted scaffolds was more efficient than that in the peptide-free scaffolds. In addition, the constructs with surface peptide could stimulate chondrogenesis with enhanced quantities of BKCs, glycosaminoglycans (GAGs), and collagen over cultivation. The histological staining of the proliferated BKCs and secreted GAGs indicated that the surface peptide favored the production of neocartilage in the constructs. Moreover, the immunochemical staining against type II collagen demonstrated the maintenance of phenotypic chondeocytes on the peptide-grafted surfaces. The peptide-grafted PEO/chitosan scaffolds can be applied to the treatment for injured cartilage in preclinical trials.     

Heparin-conjugated scaffolds with pore structure of inverted colloidal crystals for cartilage regeneration

A uniform de novo production of neocartilage is a critical issue in the fabrication of tissue-engineered diarthrodial substitutes. The aim of this work is to develop homogeneous chondrogenesis in heparinized scaffolds with pores of inverted colloidal crystal (ICC) geometry. Monodispersed polystyrene microspheres were self-assembled by floating in the medium containing ethylene glycol, dried, annealed and infiltrated with heparin/chitin/chitosan gels. The results indicated that the colloidal template was in a structure of hexagonal arrays. In addition, the regularity of the organized pores in the scaffolds reduced when the concentration of ethylene glycol decreased. An increase in the weight percentage of heparin enhanced the viability of bovine knee chondrocytes (BKCs) in ICC matrices. Over 4 weeks of cultivation, the amount of cartilaginous components including BKCs, glycosaminoglycans (GAGs) and collagen enhanced with time. Moreover, an increase in the weight percentage of heparin promoted the production of BKCs, GAGs and collagen in ICC constructs. Histological and immunochemical staining of the cultured ICC constructs revealed minor differences in BKCs, GAGs and type II collagen between the peripheral and core regions. Therefore, the ordered pores in the heparinized ICC constructs could favor the chondrocyte culture to regenerate a uniform distribution of cartilage.     

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.     

与胶原特异性结合的BMP2模拟肽/PLGA 3D打印复合支架的制备及成骨诱导活性

将胶原绑定结构域(CBD)多肽序列与骨形态发生蛋白2模拟肽(BMP2-MP)序列连接制备具有胶原绑定能力的CBD-BMP2-MP, 再将CBD-BMP2-MP与聚丙交酯-乙交酯/胶原(PLGA/COL)3D打印支架相结合, 以支架表面的胶原成分为媒介, 将CBD-BMP2-MP更有效地固定于骨修复材料上, 达到对其进行改性的目的. 利用扫描电子显微镜(SEM)、 电子万能试验机和接触角测量仪对复合支架表面形貌、 力学强度和亲水性等材料学性能进行评价. 用荧光成像法评测 CBD-BMP2-MP及BMP2-MP与支架材料的结合能力. 在各组支架材料表面接种MC3T3-E1细胞进行体外培养, 采用CCK-8、 鬼笔环肽荧光染色、 茜素红染色及qPCR综合评价细胞在材料表面的黏附、 增殖和成骨分化等细胞行为, 研究CBD-BMP2-MP修饰的3D多孔PLGA/COL复合支架的生物学性能. 研究结果表明, 利用3D打印技术制备的多孔支架具有形貌可控的孔隙结构, 为细胞生长创造更有利的细胞微环境, 支架表面胶原成分的加入提高了支架材料的亲水性, 同时对支架材料本身的力学性能无任何影响, 提高了复合支架本身的生物相容性. 与普通BMP2-MP相比, CBD-BMP2-MP具有更好的胶原绑定能力, 与复合支架的结合更稳定, 提高了PLGA/COL复合支架对BMP2-MP的负载能力. 支架表面负载CBD-BMP2-MP后具有极强的促细胞成骨分化能力. MC3T3-E1细胞表现出更高的钙沉积能力, 并且成骨分化相关基因Runx2, ALP, COL-I及OPN等水平也有了明显提升. 表明CBD-BMP2-MP多孔复合支架具有良好的生物相容性和成骨诱导活性, 在骨组织修复领域具有良好的应用前景.     

Preparation and characterization of collagen/chitosan poly (ethylene glycol)/nanohydroxyapatite composite scaffolds

Porous three‐dimensional collagen/chitosan scaffolds combined with poly (ethylene glycol) (PEG) and hydroxyapatite were obtained through a freeze‐drying method. Physical cross‐linking was examined by dehydrothermal treatment. The prepared materials were characterized by different analyses, eg, scanning electron microscopy (SEM), measurements of porosity and swelling, mechanical properties, and resistance to enzymatic degradation. The porosity of scaffolds and their swelling ratio decreased with the addition of hydroxyapatite. Moreover, after exposure to collagenase, the collagen/chitosan matrices containing PEG showed much faster degradation rate than matrices with the addition of hydroxyapatite. The results indicated that the addition of hydroxyapatite led to improvement of stiffness. The highest degree of porosity and swelling were demonstrated by collagen/chitosan/PEG matrices without hydroxyapatite.     

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.     

Polyester scaffolds with bimodal pore size distribution for tissue engineering

This paper presents a method for the preparation of porous poly(L-lactide)/poly[(L-lactide)-co-glycolide] scaffolds for tissue engineering. Scaffolds were prepared by a mold pressing-salt leaching technique from structured microparticles. The total porosity was in the range 70-85%. The pore size distribution was bimodal. Large pores, susceptible for osteoblasts growth and proliferation had the dimensions 50-400 microm. Small pores, dedicated to the diffusion of nutrients or/and metabolites of bone forming cells, as well as the products of hydrolysis of polyesters from the walls of the scaffold, had sizes in the range 2 nm-5 microm. The scaffolds had good mechanical strength (compressive modulus equal to 41 MPa and a strength of 1.64 MPa for 74% porosity). Scaffolds were tested in vitro with human osteoblast-like cells (MG-63). It was found that the viability of cells seeded within the scaffolds obtained using the mold pressing-salt leaching technique from structured microparticles was better when compared to cells cultured in scaffolds obtained by traditional methods. After 34 d of culture, cells within the tested scaffolds were organized in a tissue-like structure. Photos of section of macro- and mesoporous PLLA/PLGA scaffold containing 50 wt.-% of PLGA microspheres after 34 d of culture. Dark spots mark MG-63 cells, white areas belong to the scaffold. The specimen was stained with haematoxylin/eosin. Bar = 100 microm.     

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.     

Fabrication and Properties of Porous Collagen/Hyaluronic Composite Scaffolds Containing Nano-Bioactive Glass for Tissue Engineering

In this work, nano-structured scaffolds were designed for tissue engineering using collagen, hyaluronic acid (HA) and nano-bioactive glass (NBAG) as their main components. The scaffold was prepared via freeze-drying method and the properties including morphology, porosity, compressive strength, swelling ratio and cytotoxicity in-vitro, were also evaluated. The composite scaffolds showed well interconnected macropores with the pore size of ranging from 100 to 500 μm. The porosity percent and swelling ability were decreased with the introduction of NBAG into the collagen/HA hydrogel; however, the compressive strength was enhanced. The cytotoxicity in-vitro study shows that the collagen-HA/NBAG scaffolds have good biocompatibility with improving effect on fibroblastic cells growth. It could be concluded that this scaffold fulfills the main requirements to be considered as a bone substitute.     

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 cellulose-based scaffold with microarchitecture using a leaching technique for biomedical applications

Providing a conclusive microenvironment for cell growth, proliferation and differentiation is a major developmental strategy in the tissue engineering and regenerative medicine. This is usually achieved in the laboratory by culturing cells in three-dimensional polymer-based scaffolding materials. Here, we describe the fabrication of a cellulose scaffold for tissue engineering purposes from cellulose fiber using a salt leaching method. The 1-n-allyl-3-methylimidazolium chloride (AmimCl) IL was used as a solvent for cellulose. The leaching methodology used in this study offers the unique advantage of providing effective control of scaffold porosity by simply varying cellulose concentration. Morphologic testing of the scaffolds produced revealed pore sizes of 200–500 μm. In addition, the scaffolds had high water adsorption rates and slow degradation rates. To further investigate the suitability of these scaffolds for tissue engineering applications, biocompatibility was checked using an MTT assay and confirmed by Live/Dead^® viability testing. In addition, scanning electron microscopy and DAPI studies and in vivo experiment demonstrated the ability of cells to attach to scaffold surfaces, and a biocompatibility of matrices with cells, respectively. The authors describe the environmentally friendly fabrication of a novel cellulose-based tissue engineering scaffold.     

Synthesis and distribution of glycosaminoglycans in human leukemic B- and T-cells and monocytes studied using specific enzymic treatments and high-performance liquid chromatography

Identification of glycosaminoglycans (GAGs) synthesized by three human leukaemic cell lines-Jurkat (T-cell leukaemia), Daudi (Burkitt's lymphoma, B-cell leukaemia) and THP-1 (acute monocytic leukemia)-and normal peripheral blood mononuclear cells (PBMC) and their distribution among cell membrane and culture medium were studied. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and their composition and fine chemical structure were studied using high-performance liquid chromatography with radiochemical detection. All cell lines synthesize chondroitin sulphate (CS) and heparan sulphate (HS) in both cell membrane and culture medium. No hyaluronan was detected using treatment with specific lyases and highly sensitive HPLC methodology. CS is the major secreted GAG in all cell lines tested and the major cell retained GAG in Jurkat and Daudi. HS is the major GAG in the cell membrane of THP-1. The amounts of distinct GAGs synthesized by all cancer cell lines differ from those produced by normal PBML indicating a major role of GAGs in malignant transformation of human lymphocytes and monocytes.     

海藻酸壳聚糖可塑性支架材料的制备及表征

利用海藻酸钠和壳聚糖2种原料, 采用阴阳离子静电复合原理, 通过滴注法层层自组装成可搭载药物的缓释微球, 再按一定比例与海藻酸钠-壳聚糖溶液混合制成缓释微球型支架材料, 将缓释微球结构嵌入疏松多孔海绵状结构中. 研究了缓释微球的组分比对缓释微球型支架材料的孔隙率、 收缩率、 亲水性及降解性能的影响; 扫描电子显微镜照片显示, 微球结构相对完整, 多孔海绵状结构孔径为140~200 μm; 支架浸出液细胞毒性检测实验组对照组未见差异. 缓释微球体积所占比例即组分比为10%的缓释微球型支架材料孔隙率最高为68.2%~70.8%, 亲水性最好, 收缩率最低为4.4%~5.2%; 支架降解速率随缓释微球组分比升高而减慢, 组分比为20%的缓释微球型支架材料综合性能更优; 缓释微球型支架材料冻干成型前为液态, 具有良好可塑性. 缓释微球型支架材料为缓释系统与多孔支架材料有机结合提供了新思路.     

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.     

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.     

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.