γ辐射和EDC/NHS改性对胶原壳聚糖支架性能的影响EI北大核心CSCD

以5%梯度制备11组壳聚糖质量分数为0%~50%的胶原壳聚糖支架。使用γ辐射和EDC/NHS分别改性处理各组胶原壳聚糖支架,采用傅里叶变换红外光谱仪(FTIR)和扫描电镜(SEM)分析支架内部结构,利用吸水率、孔隙率、降解率和力学性能等指标对其性能进行检测,研究γ辐射和EDC/NHS改性对胶原壳聚糖支架性能的影响。结果表明:γ辐射和EDC/NHS改性均能使胶原与壳聚糖产生交联,壳聚糖的加入改善了γ辐射对支架分子结构的损伤;EDC/NHS改性支架的微结构好于γ辐射支架;两种改性支架壳聚糖较优,质量分数均为25%;γ辐射和EDC/NHS改性均能使支架产生取向结构。     

双交联明胶-壳聚糖复合人工真皮支架的制备及其性能研究

采用1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC.HCl)和三聚磷酸钠(STPP)作为共价-离子双交联剂交联制备了明胶-壳聚糖(gel-CS)复合人工真皮支架。通过FT-IR和SEM对复合多孔支架结构及形貌进行表征,同时对复合多孔支架的孔隙率、平均孔径、溶胀性能和降解性能进行了研究。研究结果表明,明胶和壳聚糖两相间成功地发生了交联反应,形成了具有贯通孔结构和良好孔隙率的复合多孔支架。该支架平均孔径在136.1～182.9μm之间,且随着壳聚糖含量的增加,孔径及孔隙率随之降低;溶胀性能研究发现,相较于单一共价交联方法,采用双交联法制备的支架溶胀率有所降低,再次印证双交联法制备的支架其交联度较单一共价交联的高;酶降解实验表明,固定STPP含量,随着EDC含量增加,复合支架的降解性能越好,而EDC含量一定,则STPP含量为0.5%时,复合支架具有最好的降解性能。     

Facile fabrication of the glutaraldehyde cross-linked collagen/chitosan porous scaffold for skin tissue engineering

Porous scaffold is one of the key factors in skin tissue engineering. In this study, a facile method was developed to prepare the glutaraldehyde (GA) cross-linked collagen/chitosan porous scaffold (S2). The properties of S2 were compared with the scaffolds prepared by the traditional method (S1). Compared to the rough surface and collapsed inner structure of S1, S2 showed a smooth surface and controlled size. After treated by GA with same concentration, S1 and S2 showed the similar swelling ratios, which are big enough to ensure the nutrient supply in the early stage of wound healing. The effects of the fabrication methods as well as the GA concentration on the cross-linking degree and in vitro degradation degree of the scaffolds were studied. It was found that the cross-linking degree of S2-0.25% was much higher than that of S1. Investigation of the tensile and compression properties of the scaffolds found that the mechanical property of S2-0.04% is closest to that of S1. High performance liquid chromatography (HPLC) was applied to determine the residual GA. The results proved that, compared to water rinse, oven drying is a feasible and effective method to remove the residual GA. Finally, the cytocompatibility of S2 was evaluated by in vitro culture of fibroblasts. The results of cell morphology and cell viability proved that S2-0.04% could retain the original good cytocompatibility of S1 to accelerate cell infiltration and proliferation effectively. All these results indicate that it is a feasible method to prepare the GA cross-linked collagen/chitosan scaffold.     

Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility

To construct a novel scaffold for nucleus pulposus (NP) tissue engineering, The porous type II collagen (CII)/hyaluronate (HyA)–chondroitin-6-sulfate (6-CS) scaffold was prepared using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking system. The physico-chemical properties and biocompatibility of CII/HyA–CS scaffolds were evaluated. The results suggested CII/HyA–CS scaffolds have a highly porous structure (porosity: 94.8 ± 1.5%), high water-binding capacity (79.2 ± 2.8%) and significantly improved mechanical stability by EDC/NHS crosslinking (denaturation temperature: 74.6 ± 1.8 and 58.1 ± 2.6°C, respectively, for the crosslinked scaffolds and the non-crosslinked; collagenase degradation rate: 39.5 ± 3.4 and 63.5 ± 2.0%, respectively, for the crosslinked scaffolds and the non-crosslinked). The CII/HyA–CS scaffolds also showed satisfactory cytocompatibility and histocompatibility as well as low immunogenicity. These results indicate CII/HyA–CS scaffolds may be an alternative material for NP tissue engineering due to the similarity of its composition and physico-chemical properties to those of the extracellular matrices (ECM) of native NP.     

The effect of cross-linking on the microstructure,mechanical properties and biocompatibility of electrospun polycaprolactone–gelatin/PLGA–gelatin/PLGA–chitosan hybrid composite

Abstract

In this study, multilayered scaffolds composed of polycaprolactone (PCL)–gelatin/poly(lactic-co-glycolic acid) (PLGA)–gelatin/PLGA–chitosan artificial blood vessels were fabricated using a double-ejection electrospinning system. The mixed fibers from individual materials were observed by scanning electron microscopy. The effects of the cross-linking process on the microstructure, mechanical properties and biocompatibility of the fibers were examined. The tensile stress and liquid strength of the cross-linked artificial blood vessels were 2.3 MPa and 340 mmHg, respectively, and were significantly higher than for the non-cross-linked vessel (2.0 MPa and 120 mmHg). The biocompatibility of the cross-linked artificial blood vessel scaffold was examined using the MTT assay and by evaluating cell attachment and cell proliferation. The cross-linked PCL–gelatin/PLGA–gelatin/PLGA–chitosan artificial blood vessel scaffold displayed excellent flexibility, was able to withstand high pressures and promoted cell growth; thus, this novel material holds great promise for eventual use in artificial blood vessels.     

碳化二亚胺改性胶原纤维支架材料的特性表征

用碳化二亚胺(EDC)对胶原纤维支架材料(CFSM)进行改性,评价了改性前后的胶原纤维支架材料的理化及生物降解性能.结果表明:胶原的羧基与EDC的氨基发生了酰胺化反应,且EDC改性未破坏胶原的三股螺旋结构.随着EDC用量增加,EDC-CFSM的热变性温度升高明显,耐降解性显著提高.用浓度为14mmol/L的EDC改性得到的EDC-CFSM表现出较优的生物学性能,其热变性温度达96.7℃,耐降解性最优,孔隙率为80.91%,孔径约为70～180μm.     

Fabrication and characterization of chitosan microspheres agglomerated scaffolds for bone tissue engineering

In the presented paper authors describe a method for bone scaffolds fabrication. The technique is based on the agglomeration of chitosan microspheres. The fabrication process is complex and consists of a few steps: chitosan spheres extrusion, scaffold formation by compression followed by the spheres agglomeration and bonding with cross-linking agent (STPP, sodium tripolyphosphate). The described method allows manufacturing of porous materials with controllable shape, pore size distribution and their interconnectivity. In this technique 3D scaffold porosity can be regulated by altering spheres diameter. Authors studied influence of cross-linker concentrations and time of cross-linking process on the scaffold morphology, mechanical properties, enzymatic degradation rate (in the presence of lysozyme) and human osteoblasts response. Surface morphology and topography were evaluated by SEM. Porosity and pore interconnectivity were observed via μCT scanning. Mechanical tests showed that chitosan scaffolds perform compression characteristic (Young Modulus) similar to natural bone. Cytotoxicity established by XTT assay confirmed that most of the developed composite materials do not show toxic properties. Osteoblast adhesion and morphology were analyzed by SEM and optical microscopy.     

Preparation and comparative characterization of keratin–chitosan and keratin–gelatin composite scaffolds for tissue engineering applications

We report fabrication of three dimensional scaffolds with well interconnected matrix of high porosity using keratin, chitosan and gelatin for tissue engineering and other biomedical applications. Scaffolds were fabricated using porous Keratin–Gelatin (KG), Keratin–Chitosan (KC) composites. The morphology of both KG and KC was investigated using SEM. The scaffolds showed high porosity with interconnected pores in the range of 20–100 μm. They were further tested by FTIR, DSC, CD, tensile strength measurement, water uptake and swelling behavior. In vitro cell adhesion and cell proliferation tests were carried out to study the biocompatibility behavior and their application as an artificial skin substitute. Both KG and KC composite scaffolds showed similar properties and patterns for cell proliferation. Due to rapid degradation of gelatin in KG, we found that it has limited application as compared to KC scaffold. We conclude that KC scaffold owing to its slow degradation and antibacterial properties would be a better substrate for tissue engineering and other biomedical application.     

Fabrication and development of artificial osteochondral constructs based on cancellous bone/hydrogel hybrid scaffold

Using tissue engineering techniques, an artificial osteochondral construct was successfully fabricated to treat large osteochondral defects. In this study, porcine cancellous bones and chitosan/gelatin hydrogel scaffolds were used as substitutes to mimic bone and cartilage, respectively. The porosity and distribution of pore size in porcine bone was measured and the degradation ratio and swelling ratio for chitosan/gelatin hydrogel scaffolds was also determined in vitro. Surface morphology was analyzed with the scanning electron microscope (SEM). The physicochemical properties and the composition were tested by using an infrared instrument. A double layer composite scaffold was constructed via seeding adipose-derived stem cells (ADSCs) induced to chondrocytes and osteoblasts, followed by inoculation in cancellous bones and hydrogel scaffolds. Cell proliferation was assessed through Dead/Live staining and cellular activity was analyzed with IpWin5 software. Cell growth, adhesion and formation of extracellular matrix in composite scaffolds blank cancellous bones or hydrogel scaffolds were also analyzed. SEM analysis revealed a super porous internal structure of cancellous bone scaffolds and pore size was measured at an average of 410 ± 59 μm while porosity was recorded at 70.6 ± 1.7 %. In the hydrogel scaffold, the average pore size was measured at 117 ± 21 μm and the porosity and swelling rate were recorded at 83.4 ± 0.8 % and 362.0 ± 2.4 %, respectively. Furthermore, the remaining hydrogel weighed 80.76 ± 1.6 % of the original dry weight after hydration in PBS for 6 weeks. In summary, the cancellous bone and hydrogel composite scaffold is a promising biomaterial which shows an essential physical performance and strength with excellent osteochondral tissue interaction in situ. ADSCs are a suitable cell source for osteochondral composite reconstruction. Moreover, the bi-layered scaffold significantly enhanced cell proliferation compared to the cells seeded on either single scaffold. Therefore, a bi-layered composite scaffold is an appropriate candidate for fabrication of osteochondral tissue.     

Evaluation of cross-linked gelatin membranes as delivery carriers for retinal sheets

The delivery of intact sheet transplants to the subretinal space can prevent cell loss that is generally associated with the injection of cell suspensions or cell aggregates. The aim of this study was to develop chemically modified gelatin matrices that enhance the delivery efficiency and analyze whether the gelatin membranes cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) can be considered as potential carriers for retinal sheets. The characteristics of EDC cross-linked gelatin membranes were determined by mechanical and in vitro degradation tests, melting point measurements, cell proliferation assays, cytokine expression analyses, and tissue delivery studies. Gelatin membranes without cross-linking and glutaraldehyde cross-linked gelatin samples were used for comparison. Results of this study indicated that introduction of cross-links is capable of rendering the gelatin network more stable against mechanical stresses and deformations as well as rapid hydrolysis during intraocular delivery of delicate tissue sheets. In comparison with the glutaraldehyde treated samples, the EDC cross-linked gelatin membranes showed a better degradation profile and a relatively higher cytocompatibility. In addition, after EDC cross-linking, the gelatin matrices having an acceptable melting point could be used for the fabrication of a sandwich-like carrier with a high transfer and encapsulation efficiency. These findings suggest that the cross-linking agent type gives an influence on delivery functionality of gelatin membranes. In summary, the EDC cross-linked gelatin is an ideal candidate for use as a carrier material in retinal sheet delivery applications.     

Influence of solvent composition on the performance of carbodiimide cross-linked gelatin carriers for retinal sheet delivery

Gelatin is a protein molecule that displays bioaffinity and provides a template to guide retinal pigment epithelial (RPE) cell organization and growth. We have recently demonstrated that the carbodiimide cross-linked gelatin membranes can be used as retinal sheet carriers. The purpose of this work was to further determine the role of solvent composition in the tissue delivery performance of chemically modified biopolymer matrices. The gelatin molecules were treated with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in the presence of binary ethanol/water mixtures with varying ethanol concentrations (70–95 vol%) to obtain the carriers with different cross-linking efficiencies and mechanical properties. Results of melting point measurements and in vitro degradation tests showed that when the cross-linking index reached a high level of around 45 %, the EDC cross-linked gelatin materials have sufficient thermal stability and resistance to enzymatic degradation, indicating their suitability for the development of carriers for retinal sheet delivery. Irrespective of the solvent composition, the chemically modified gelatin samples are compatible toward human RPE cells without causing toxicity and inflammation. In particular, the membrane carriers prepared by the cross-linking in the presence of solvent mixtures containing 80–90 vol% of ethanol have no impact on the proliferative capacity of ARPE-19 cultures and possess good efficiency in transferring and encapsulating the retinal tissues. It is concluded that, except for cell viability and pro-inflammatory cytokine expression, the retinal sheet delivery performance strongly depends on the solvent composition for EDC cross-linking of gelatin molecules.     

A study on the in vitro degradation of poly(L-lactide)/chitosan microspheres scaffolds

Recent research shows that the addition of chitosan microspheres (CMs) to poly(L-lactide) (PLLA) can result in a composite scaffold material with improved biocompatibility and mechanical properties for tissue engineering applications. However, research regarding the influence of CMs on scaffold degradation is absent in the literature. This paper presents a study on the in vitro degradation of scaffolds made from PLLA with CMs. In this study, the PLLA/CMs scaffolds with a 25% ratio of CMs to PLLA were immersed in phosphate-buffered saline (PBS) solution at 37°C for 8 weeks. The in vitro degradation of the scaffolds was investigated using micro-computed tomography (μCT), weight loss analysis, Raman spectroscopy, and differential scanning calorimetry (DSC). Microstructure changes during degradation were monitored using μCT. The μCT results were consistent with the results obtained from Raman spectra and DSC analysis, which reflected that adding CMs into PLLA can decrease the degradation rate compared with pure PLLA scaffolds. The results suggest that PLLA/CMs scaffold degradation can be regulated and controlled to meet requirements imposed a given tissue engineering application.     

3D Bioplotting of Gelatin/Alginate Scaffolds for Tissue Engineering:Influence of Crosslinking Degree and Pore Architecture on Physicochemical Properties

Gelatin/Alginate hydrogels were engineered for bioplotting in tissue engineering. One major drawback of hydrogel scaffolds is the lack of adequate mechanical properties. In this study, using a bioplotter, we constructed the scaffolds with different pore architectures by deposition of gelatin/alginate hydrogels layerby-layer. The scaffolds with different crosslinking degree were obtained by post-crosslinking methods.Their physicochemical properties, as well as cell viability, were assessed. Different crosslinking methods had little influence on scaffold architecture, porosity, pore size and distribution. By contrast, the water absorption ability, degradation rate and mechanical properties of the scaffolds were dramatically affected by treatment with various concentrations of crosslinking agent(glutaraldehyde). The crosslinking process using glutaraldehyde markedly improved the stability and mechanical strength of the hydrogel scaffolds. Besides the post-processing methods, the pore architecture can also evidently affect the mechanical properties of the scaffolds. The crosslinked gelatin/alginate scaffolds showed a good potential to encapsulate cells or drugs.     

Effects of different cross-linking conditions on the properties of genipin-cross-linked chitosan/collagen scaffolds for cartilage tissue engineering

A cross-linking reagent is required to improve mechanical strength and degradation properties of biopolymers for tissue engineering. To find the optimal preparative method, we prepared diverse genipin-cross-linked chitosan/collagen scaffolds using different genipin concentrations and various cross-linking temperatures and cross-linking times. The compressive strength increased with the increasing of genipin concentration from 0.1 to 1.0%, but when concentration exceeded 1.0%, the compressive strength decreased. Similarly, the compressive strength increased with the increasing of temperature from 4 to 20°C, but when temperature reached 37°C, the compressive strength decreased. Showing a different trend from the above two factors, the effect of cross-linking time on the compressive strength had a single increasing tendency. The other results also demonstrated that the pore size, degradation rate and swelling ratio changed significantly with different cross-linking conditions. Based on our study, 1.0% genipin concentration, 20°C cross-linking temperature and longer cross-linking time are recommended.     

纳米羟基磷灰石/壳聚糖-羧甲基纤维素复合支架材料的研究

用冷冻干燥法制备了不同比例的纳米羟基磷灰石/壳聚糖-羧甲基纤维素(n-HA/CS-CMC)无机/有机复合多孔支架材料, 并探讨了其复合机理及无机组分n-HA对复合支架的结构形貌、力学性能、体外降解性能的影响. 结果表明, 其复合支架主要是通过无机组分n-HA均匀分散充填在CS-CMC聚电解质有机网络结构中形成的, 且三组分间有较强的化学键合. 无机组分n-HA的加入使孔结构变得不规则, 孔隙率略有减小, 使复合支架的抗压缩强度提高, 并且可使其体外降解速度减慢. 无机组分n-HA含量为40\%复合支架材料的性能最佳, 有望用作骨组织工程支架材料.     

Optimization strategies on the structural modeling of gelatin/chitosan scaffolds to mimic human meniscus tissue

Meniscus lesions are frequently occurring injuries with poor ability to heal. Typical treatment procedure includes removal of damaged regions, which can lead to sub-optimal knee biomechanics and early onset of osteoarthritis. Some of the drawbacks of current treatment approach present an opportunity for a tissue engineering solution. In this study, gelatin (G)/chitosan (Cs) scaffolds were synthesized via gel casting method and cross-linked with naturally derived cross-linker, genipin, through scaffold cross-linking method. Based on the characteristics of native meniscus tissue microstructure and function, three different layers were chosen to design the macroporous multilayered scaffolds. The multi-layered scaffolds were investigated for their ability to support human-derived meniscus cells by evaluating their morphology and proliferation using MTT assay at various time points. Based on structural, mechanical and cell compatibility considerations, laminated scaffolds composed of G60/Cs40, G80/Cs20 and G40/Cs60 samples, for the first, second and third layers, respectively, could be an appropriate combination for meniscus tissue engineering applications.     

Silk fibroin/chitosan scaffold: preparation,characterization, and culture with HepG2 cell

Tissue engineering requires the development of three-dimensional water-stable scaffolds. In this study, silk fibroin/chitosan (SFCS) scaffold was successfully prepared by freeze-drying method. The scaffold is water-stable, only swelling to a limited extent depending on its composition. Fourier Transform Infrared (FTIR) spectra and X-Ray diffraction curves confirmed the different structure of SFCS scaffolds from both chitosan and silk fibroin. The homogeneous porous structure, together with nano-scale compatibility of the two naturally derived polymers, gives rise to the controllable mechanical properties of SFCS scaffolds. By varying the composition, both the compressive modulus and compressive strength of SFCS scaffolds can be controlled. The porosity of SFCS scaffolds is above 95% when the total concentration of silk fibroin and chitosan is below 6 wt%. The pore sizes of the SFCS scaffolds range from 100 μm to 150 μm, which can be regulated by changing the total concentration. MTT assay showed that SFCS scaffolds can promote the proliferation of HepG2 cells (human hepatoma cell line) significantly. All these results make SFCS scaffold a suitable candidate for tissue engineering.     

Fabrication and characterization of PCL/gelatin/chitosan ternary nanofibrous composite scaffold for tissue engineering applications

In the present study, we have fabricated a ternary composite nanofibrous scaffold from PCL/gelatin/chitosan, by electrospinning technique, using a solvent system—chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin and chitosan, for tissue engineering applications. Field emission scanning electron microscopy (FE-SEM) was used to investigate the fiber morphology of the scaffold and it was found that the fiber morphology was influenced by the concentrations of PCL, gelatin, and chitosan in polymer solution during electrospinning. X-ray diffraction, Fourier transform infrared, and thermogravimetric (TG) analysis results showed some interactions among the molecules of PCL, gelatin, and chitosan within the scaffold. In-vitro cell culture studies were done by seeding L929 mouse fibroblasts on fabricated composite scaffold, which confirmed the cell viability, high cell proliferation rate, and cell adhesion on composite scaffold as indicated by MTT assay, DNA quantification, and FE-SEM analysis of cell-scaffold construct. Thus, the ternary composite scaffold made from the combination of PCL (synthetic polymer), gelatin, and chitosan (natural polymer) may find potential application in tissue engineering.     

Preparation and bioactivity evaluation of hydroxyapatite-titania/chitosan-gelatin polymeric biocomposites

Biocomposites consisting of hydroxyapatite (HA) and natural polymers such as collagen, chitosan, chitin,and gelatin have been extensively investigated. However, studies on the combination of HA and titania with chitosan and gelatin have not been conducted yet. Novel biodegradable hydroxyapatite-titania/chitosan-gelatin polymeric composites were fabricated. In this work, our results are concerning with the preparation and characterization of HA powder and HA filler containing titania powder (10 and 30%) with a chitosan and gelatin copolymer matrix. The present research focuses on characterizing the structure of this novel class of biocomposites. Thermogravimetric analysis (TGA), X-ray diffraction (XRD), and Fourier Transformed Infrared Spectroscopy (FT-IR), Scanning electron microscopy (SEM-EDAX) were employed to assess the produced composites. The mechanical properties in terms of compressive strength and hardness test were also investigated. The in vitro study in simulated body fluid (SBF) was performed to assess the bioactivity of composites. The results proved that apatite resembling natural bone are formed faster and greater in the case the composite of HA containing 10% titania into chitosan-gelatin polymeric matrix when they are soaked in a simulated body fluid (SBF) than the composite containing 30% titania. The biocomposites containing HA with 10% titania are expected to be attractive for bioapplications as bone substitutes and scaffolds for tissue engineering in future.     

Comparative study on the role of gelatin,chitosan and their combination as tissue engineered scaffolds on healing and regeneration of critical sized bone defects: an in vivo study

Gelatin and chitosan are natural polymers that have extensively been used in tissue engineering applications. The present study aimed to evaluate the effectiveness of chitosan and gelatin or combination of the two biopolymers (chitosan–gelatin) as bone scaffold on bone regeneration process in an experimentally induced critical sized radial bone defect model in rats. Fifty radial bone defects were bilaterally created in 25 Wistar rats. The defects were randomly filled with chitosan, gelatin and chitosan–gelatin and autograft or left empty without any treatment (n?=?10 in each group). The animals were examined by radiology and clinical evaluation before euthanasia. After 8?weeks, the rats were euthanized and their harvested healing bone samples were evaluated by radiology, CT-scan, biomechanical testing, gross pathology, histopathology, histomorphometry and scanning electron microscopy. Gelatin was biocompatible and biodegradable in vivo and showed superior biodegradation and biocompatibility when compared with chitosan and chitosan–gelatin scaffolds. Implantation of both the gelatin and chitosan–gelatin scaffolds in bone defects significantly increased new bone formation and mechanical properties compared with the untreated defects (P?<?0.05). Combination of the gelatin and chitosan considerably increased structural and functional properties of the healing bones when compared to chitosan scaffold (P?<?0.05). However, no significant differences were observed between the gelatin and gelatin–chitosan groups in these regards (P?>?0.05). In conclusion, application of the gelatin alone or its combination with chitosan had beneficial effects on bone regeneration and could be considered as good options for bone tissue engineering strategies. However, chitosan alone was not able to promote considerable new bone formation in the experimentally induced critical-size radial bone defects.