具有生物活性的高导复合支架材料制备表征及其生物相容性☆

背景：组织工程新材料必须既能支持组织生长,又能激发理想的细胞反应(如血管发生),而导电性则可促进包括神经在内的组织再生。 目的：构建一种新型材料,能将导电性和生物活性有效的结合起来,为组织工程提供一种理想的备选材料。 方法：将具有生物活性的透明质酸多糖和导电聚合物聚吡咯进行结合,制备具有生物活性的导电聚合物复合材料。 结果与结论：含有透明质酸多糖的聚吡咯/透明质酸双层膜具有光滑的表面形貌和良好的导电性。体外细胞相容性实验显示这种复合材料可显著促进神经轴突的延伸。体内实验显示该复合材料还具有良好的惰性和促血管生成效应,是一种较为理想的组织工程备选材料。     

Characterization of a bovine collagen-hydroxyapatite composite scaffold for bone tissue engineering

Different biomaterials have been used as scaffolds for bone tissue engineering. Here we characterize a biomaterial composed of sintered (1100 degrees C) and powdered hydroxyapatite (HA) and type I collagen (Coll), both of bovine origin, designed for osteoconductive and osteoinductive scaffolds. Coll/HA proportions were 1/2.6 and 1/1 (wet weight), and particles sizes varied from 200 to 400 microm. Vv (volume density) and Sv (surface to volume density) for the HA particles in the composite ranged from 0.48 +/- 0.06 to 0.55 +/- 0.02 and 5.090 +/- 0.545 to 6.366 +/- 0.289 microm(-1), respectively. Due to the relatively small changes in Vv and Sv, a macroporosity could be characterized for the biocomposite. X-ray diffraction and infrared spectroscopy showed that the sintered bone was composed essentially of HA with minimum additional groups such as surface calcium hydroxide, surface and crystal water, free carbon dioxide and possibly brushite. Mass spectrometry detected carbonates at A and B sites of HA, and weakly bound to the structure. Human osteoblasts adhered and spread on both the HA particle surface and the collagen fibers, which seemed to guide cells between adjacent particles. The biocomposite studied has several characteristics considered as ideal for its use as a scaffold for osteoconduction and osteoinduction.     

Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15 days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.     

Differential functional effects of biomaterials on dendritic cell maturation

The immunological outcome of dendritic cell (DC) treatment with different biomaterials was assessed to demonstrate the range of DC phenotypes induced by biomaterials commonly used in combination products. Immature DCs (iDCs) were derived from human peripheral blood monocytes, and treated with different biomaterial films of alginate, agarose, chitosan, hyaluronic acid (HA), or 75:25 poly(lactic-co-glycolic acid) (PLGA) and a comprehensive battery of phenotypic functional outcomes was assessed. Different levels of functional changes in DC phenotype were observed depending on the type of biomaterial films used to treat the DCs. Treatment of DCs with PLGA or chitosan films supported DC maturation, with higher levels of DC allostimulatory capacity, pro-inflammatory cytokine release, and expression of CD80, CD86, CD83, HLA-DQ and CD44 compared with iDCs, and lower endocytic ability compared with iDCs. Alginate film induced pro-inflammatory cytokine release from DCs at levels higher than from iDCs. Dendritic cells treated with HA film expressed lower levels of CD40, CD80, CD86 and HLA-DR compared with iDCs. They also exhibited lower endocytic ability and CD44 expression than iDCs, possibly due to an insolubilized (cross-linked) form of high molecular weight HA. Interestingly, treatment of DCs with agarose film maintained the DC functional phenotype at levels similar to iDCs except for CD44 expression, which was lower than that of iDCs. Taken together, these results can provide selection criteria for biomaterials to be used in immunomodulating applications and can inform potential outcomes of biomaterials within combination products on associated immune responses as desired by the application.     

Reduction of abdominal adhesions using composite collagen-GAG implants for ventral hernia repair

Structural biomaterials can restore abdominal wall integrity but may cause adhesions to the underlying viscera. Collagen-glycosaminoglycan (CG) matrices induce the formation of connective tissue and may reduce adhesion formation to permanent biomaterials such as polypropylene (PP) mesh. Composite implants were created by interposing PP mesh within a porous CG matrix created composite implants. The implants were cross-linked with glutaraldehyde one group (CG-G/PP) or left untreated (CG-nG/PP) and compared to PP mesh. At 4 weeks, the abdominal wall was assessed for the degree of adhesions. The composite implants developed a nascent connective tissue-like structure that reduced adhesions to the bowel. The thickest connective tissue developed in the CG-G/PP group (0.7 +/- 0.1 mm) and thinnest in the PP mesh (0.05 +/- 0.01 mm). The surface area covered with adhesions was greatest in the PP group (72 +/- 17%) compared with the CG-G/PP group (28 +/- 15%) or the CG-nG/PP group (21 +/- 8%). Bowel preferentially adhered to the PP mesh, whereas omentum had some adherence to all constructs. Integrating a biodegradable extracellular matrix analog with a permanent structural biomaterial reduced adhesions in this animal model. Alterations in cross-linking of the CG matrix altered the biological response. This technology may be useful in reconstructive surgery by reducing adhesion formation, while maintaining the strength of permanent structural biomaterials.     

胶原-纳米羟基磷灰石复合支架的细胞相容性

背景：观察成骨细胞在生物材料上的形态、增殖和分化等项目,可评估生物支架材料的生物相容性。 目的：观察复合支架材料纳米羟基磷灰石/胶原对成骨细胞增殖、分化的影响。 方法：取新生24 h内Wistar大鼠的颅盖骨,采用改良胶原酶消化法进行成骨细胞原代培养,取第3代细胞与纳米羟基磷灰石/胶原支架或普通羟基磷灰石材料体外复合培养。培养3,6,9 d后,观察材料周边的细胞形态及支架材料对细胞分化、增殖的影响。 结果与结论：纳米羟基磷灰石/胶原材料较普通的羟基磷灰石材料更有利于成骨细胞的黏附、生长、分化、增殖,证实其生物相容性更好,有望成为一种新型的骨组织工程支架材料。     

Differential levels of dendritic cell maturation on different biomaterials used in combination products

Immature dendritic cells (iDCs) were derived from human peripheral blood monocytes, and treated with films of biomaterials commonly used in combination products (e.g., tissue engineered constructs or vaccines) to assess the resultant dendritic cell (DC) maturation compared to positive control of lipopolysaccharide (LPS) treatment for DC maturation or negative control of untreated iDCs. The following biomaterials were tested: alginate, agarose, chitosan, hyaluronic acid, 75:25 poly(lactic-co-glycolic acid) (PLGA). The effect of DC culture on these films was undertaken to identify biomaterials which support DC maturation and those biomaterials that did not. Dendritic cells treated with chitosan or PLGA (agarose to a lesser extent) films increased expression levels of CD86, CD40, and HLA-DQ, compared to control iDCs, similar to LPS-matured DCs, whereas DCs treated with alginate or hyaluronic acid films decreased their expression levels of these same molecules. In summary, a differential effect of the biomaterial on which iDCs were cultured was observed as far as the extent of induced DC maturation. The effect of biomaterials on DC maturation, and the associated adjuvant effect, is a novel biocompatibility selection and design criteria for biomaterials to be used in combination products in which immune consequences are potential complications or outcomes.     

The characterisation of human respiratory epithelial cells cultured on resorbable scaffolds: first steps towards a tissue engineered tracheal replacement

In this study we have used lectin histochemistry and scanning electron microscopy (SEM) to assess the growth and characterise the differentiation of human respiratory epithelial cells (REC) cultured on two biomaterial scaffolds. The first scaffold, based on a hyaluronic acid derivative, was observed to be non-adhesive for REC. This lack of adhesion was found to be unrelated to the presence of the hyaluronic acid binding domain on the surface of isolated REC. The other scaffold, consisting of equine collagen. was observed to encourage REC spreading and adhesion. Positive Ulex Europaeus agglutinin (UEA) lectin staining of this preparation indicated the presence of ciliated REC on the scaffold surface. However, the marked decrease in peanut agglutinin (PNA) positive staining, relative to that of control cultures and native tissue, indicates a dedifferentiation of the secretory cells of the REC monolayer. SEM analysis of REC cultured on the collagen scaffold confirmed the presence of ciliated cells thereby validating the UEA positive staining. The presence of both established and developing cilia was also verified. This study indicates that collagen biomaterials are appropriate for the tissue engineering of REC. Furthermore, that UEA and PNA staining is a useful tool in the characterisation of cells cultured on biomaterials, therefore helpful in identifying biomaterials that are suitable for specific tissue engineering purposes.     

HA/TCP compounding of a porous CaP biomaterial improves bone formation and scaffold degradation--a long-term histological study

In the present study, two biphasic calcium phosphate biomaterials (BCP) with HA/TCP ratios of 50/50 and 30/70 were obtained from a pure HA biomaterial. The biomaterials which showed the same three-dimensional geometry were implanted into corticocancellous costal defects of sheep. In the specimens of all three biomaterials, abundant bone formation, mineral dissolution from the biomaterial scaffolds, and active cellular resorption of the scaffolds was present after 6 and 12 months. Backscattered electron microscopy showed bone invasion into the pores of the scaffolds and micromechanical interlocking at the bone/biomaterial interface without intervening soft tissue. The pattern of bone formation and scaffold resorption was different for cortical and cancellous bone. No time-based effect, however, was observed. Overall, the BCP biomaterials had formed significantly more bone than the HA biomaterial. Also, scaffold resorption, which was followed by a replacement with newly formed bone, was significantly higher in the BCP biomaterials. Although no significant differences were observed between both BCP biomaterials, the present study had confirmed the assumption that HA/TCP compounding was suitable to improve bone formation and scaffold resorption in the investigated biomaterials and at the same time maintain the osteoconductive properties of the scaffolds.     

HA/β-TCP支架材料的研究进展

随着骨修复材料需求的增多,HA/β-TCP复合支架作为一种兼具良好的生物相容性、生物降解性、骨传导性以及骨诱导性的生物材料受到了人们的广泛关注。本文关注于web of science上的相关文献,主要论述以下几个方面内容:HA/β-TCP组成比例对复合支架的性能影响;HA/β-TCP复合支架与其他主流材料的复合;新制作工艺的出现与结构改进。在技术推进和研究深入的背景下,对作为骨修复工程中较为理想的生物材料HA/β-TCP复合支架的发展现状进行介绍。     

神经干细胞与透明质酸水凝胶材料生物相容性的研究

目的:评价神经干细胞与改性透明质酸水凝胶支架新材料的生物相容性,研究该透明质酸水凝胶支架作为中枢神经组织工程载体材料的可行性,为用组织工程及干细胞技术治疗中枢神经系统损伤提供基础。方法:以冷冻干燥法制备透明质酸水凝胶材料支架,通过化学接枝法将抗Nogo受体抗体(Anti-NogoR)和多聚赖氨酸(poly-l-lysine,PLL)分子接枝到水凝胶上对其改性,制成新的支架材料。体外培养胚胎13.5d大鼠前脑泡神经干细胞.将神经干细胞与生物支架共培养,通过扫描电镜观察透明质酸水凝胶的内部结构及神经干细胞在支架材料上的粘附与生长情况,通过细胞免疫组织化学技术观察神经干细胞在透明质酸水凝胶材料上的存活与分化情况。结果:制备的透明质酸水凝胶材料具有疏松的三维多孔结构,神经干细胞在支架材料上能够粘附并且有突起长出,生长良好。神经干细胞在支架材料上能够分化。结论:神经干细胞与经过改性的透明质酸水凝胶新材料有很好的生物相容性,能够在材料上存活分化。该新透明质酸水凝胶材料有望作为修复中枢神经损伤的组织工程载体。     

In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane) composite scaffold for bone tissue engineering

Scaffolds for bone tissue engineering should provide an osteoconductive surface to promote the ingrowth of new bone after implantation into bone defects. This may be achieved by hydroxyapatite loading of distinct scaffold biomaterials. Herein, we analyzed the in vitro and in vivo properties of a novel nanosize hydroxyapatite particles/poly(ester-urethane) (nHA/PU) composite scaffold which was prepared by a salt leaching–phase inverse process. Microtomography, scanning electron microscopy and X-ray spectroscopy analyses demonstrated the capability of the material processing to create a three-dimensional porous PU scaffold with nHA on the surface. Compared to nHA-free PU scaffolds (control), this modified scaffold type induced a significant increase in in vitro adsorption of model proteins. In vivo analysis of the inflammatory and angiogenic host tissue response to implanted nHA/PU scaffolds in the dorsal skinfold chamber model indicated that the incorporation of nHA particles into the scaffold material did not affect biocompatibility and vascularization when compared to control scaffolds. Thus, nHA/PU composite scaffolds represent a promising new type of scaffold for bone tissue engineering, combining the flexible material properties of PU with the advantage of an osteoconductive surface.     

Review. Hyaluronan: a powerful tissue engineering tool

Hyaluronan (HA) is a versatile molecular tool with considerable potential for tissue engineering applications. The inclusion of HA has created biocompatible biomaterials and engineered tissues that can be crosslinked or degraded controllably and can facilitate angiogenesis, osteointegration, and cell phenotype preservation. The utility of HA in tissue engineering has been broadened further by the recently identified HA synthases, which can be manipulated to stimulate the endogenous production of HA by cells seeded within biomaterial scaffolds. Overall, HA shows great promise in the development of engineered tissues and biomaterials for a variety of biomedical needs including orthopedic, cardiovascular, pharmacologic, and oncologic applications.     

Biocompatibility of biodegradable semiconducting melanin films for nerve tissue engineering

The advancement of tissue engineering is contingent upon the development and implementation of advanced biomaterials. Conductive polymers have demonstrated potential for use as a medium for electrical stimulation, which has shown to be beneficial in many regenerative medicine strategies including neural and cardiac tissue engineering. Melanins are naturally occurring pigments that have previously been shown to exhibit unique electrical properties. This study evaluates the potential use of melanin films as a semiconducting material for tissue engineering applications. Melanin thin films were produced by solution processing and the physical properties were characterized. Films were molecularly smooth with a roughness (R_ms) of 0.341 nm and a conductivity of 7.00 ± 1.10 × 10^?5 S cm^?1 in the hydrated state. In vitro biocompatibility was evaluated by Schwann cell attachment and growth as well as neurite extension in PC12 cells. In vivo histology was evaluated by examining the biomaterial–tissue response of melanin implants placed in close proximity to peripheral nerve tissue. Melanin thin films enhanced Schwann cell growth and neurite extension compared to collagen films in vitro. Melanin films induced an inflammation response that was comparable to silicone implants in vivo. Furthermore, melanin implants were significantly resorbed after 8 weeks. These results suggest that solution-processed melanin thin films have the potential for use as a biodegradable semiconducting biomaterial for use in tissue engineering applications.     

Comparison of Chondroitin Sulfate and Hyaluronic Acid Doped Conductive Polypyrrole Films for Adipose Stem Cells

Polypyrrole (PPy) is a conductive polymer that has aroused interest due to its biocompatibility with several cell types and high tailorability as an electroconductive scaffold coating. This study compares the effect of hyaluronic acid (HA) and chondroitin sulfate (CS) doped PPy films on human adipose stem cells (hASCs) under electrical stimulation. The PPy films were synthetized electrochemically. The surface morphology of PPy–HA and PPy–CS was characterized by an atomic force microscope. A pulsed biphasic electric current (BEC) was applied via PPy films non-stimulated samples acting as controls. Viability, attachment, proliferation and osteogenic differentiation of hASCs were evaluated by live/dead staining, DNA content, Alkaline phosphatase activity and mineralization assays. Human ASCs grew as a homogenous cell sheet on PPy–CS surfaces, whereas on PPy–HA cells clustered into small spherical structures. PPy–CS supported hASC proliferation significantly better than PPy–HA at the 7 day time point. Both substrates equally triggered early osteogenic differentiation of hASCs, although mineralization was significantly induced on PPy–CS compared to PPy–HA under BEC. These differences may be due to different surface morphologies originating from the CS and HA dopants. Our results suggest that PPy–CS in particular is a potential osteogenic scaffold coating for bone tissue engineering.     

The use of layer by layer self-assembled coatings of hyaluronic acid and cationized gelatin to improve the biocompatibility of poly(ethylene terephthalate) artificial ligaments for reconstruction of the anterior cruciate ligament

In this study layer by layer (LBL) self-assembled coatings of hyaluronic acid (HA) and cationized gelatin (CG) were used to modify polyethylene terephthalate (PET) artificial ligament grafts. Changes in the surface properties were characterized by scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and contact angle and biomechanical measurements. The cell compatibility of this HA–CG coating was investigated in vitro on PET films seeded with human foreskin dermal fibroblasts over 7 days. The results of our in vitro studies demonstrated that the HA–CG coating significantly enhanced cell adhesion, facilitated cell growth, and suppressed the expression of inflammation-related genes relative to a pure PET graft. Furthermore, rabbit and porcine anterior cruciate ligament reconstruction models were used to evaluate the effect of this LBL coating in vivo. The animal experiment results proved that this LBL coating significantly inhibited inflammatory cell infiltration and promoted new ligament tissue regeneration among the graft fibers. In addition, the formation of type I collagen in the HA–CG coating group was much higher than in the control group. Based on these results we conclude that PET grafts coated with HA–CG have considerable potential as substitutes for ligament reconstruction.     

Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering

In this study, we hypothesized that hyaluronic acid could provide superior biological effects on the chondrocytes in a three-dimensional culture system. To test this hypothesis, we investigated the in vitro behavior of rabbit chondrocytes on a novel chitosan-based hyaluronic acid hybrid polymer fiber. The goal of the current study was to show the superiority of this novel fiber as a scaffold biomaterial for cartilage tissue engineering. Chitosan polymer fibers (chitosan group) and chitosan-based hyaluronic acid hybrid polymer fibers (HA 0.04% and HA 0.07% groups, chitosan coated with hyaluronic acid 0.04% and 0.07%, respectively) were originally developed by the wetspinning method. Articular chondrocytes were isolated from Japanese white rabbits and cultured in the sheets consisting of each polymer fiber. The effects of each polymer fiber on cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were analyzed by quantitative a cell attachment test, DNA quantification, light and scanning electron microscopy, semi-quantitative RT-PCR, and immunohistochemical analysis. Cell adhesivity, proliferation and the synthesis of aggrecan were significantly higher in the hybrid fiber (HA 0.04% and 0.07%) groups than in the chitosan group. On the cultured hybrid polymer materials, scanning electron microscopic observation showed that chondrocytes proliferated while maintaining their morphological phenotype and with a rich extracellular matrix synthesis around the cells. Immunohistochemical staining with an anti-type II collagen antibody demonstrated rich production of the type II collagen in the pericellular matrix from the chondrocytes. The chitosan-based hyaluronic acid hybrid polymer fibers show great potential as a desirable biomaterial for cartilaginous tissue scaffolds.     

基于丝素/胶原/羟基磷灰石仿生骨材料的多维结构及其性能

目的 体外构建丝素蛋白（silk fibroin,SF）、I型胶原(type I collagen,Col-I)和羟基磷灰石(hydroxyapatite, HA)共混体系制备二维复合膜和三维仿生支架,研究其理化性质和生物相容性,探讨其在组织工程支架材料中应用的可行性。方法 通过在细胞培养小室底部共混SF/Col-I/HA以及低温3D打印结合真空冷冻干燥法制备二维复合膜及三维支架。通过机械性能测试、电子显微镜和Micro-CT检测材料的理化性质,检测细胞的增殖评估其生物相容性。结果 通过共混和低温3D打印获得稳定的二维复合膜及三维多孔结构支架;力学性能具有较好的一致性,孔径、吸水率、孔隙率和弹性模量均符合构建组织工程骨的要求;支架为网格状的白色立方体,内部孔隙连通性较好; HA均匀分布在复合膜中,细胞黏附在复合膜上,呈扁平状;细胞分布在支架孔壁周围,呈梭形状,生长及增殖良好。结论 利用SF/Col-I/HA共混体系成功制备复合膜及三维支架,具有较好的孔连通性与孔结构,有利于细胞和组织的生长以及营养输送,其理化性能以及生物相容性符合骨组织工程生物材料的要求。     

Development of photocrosslinkable hyaluronic acid-polyethylene glycol-peptide composite hydrogels for soft tissue engineering

Hyaluronic acid (HA; also called hyaluronan) is a naturally derived, nonimmunogenic, nonadhesive glycosaminoglycan that has important roles in several wound-healing processes. In previous work, we created photocrosslinkable glycidyl methacrylate-HA (GMHA) hydrogel biomaterials that were cytocompatible, biologically active, and had a decreased rate of hyaluronidase degradation compared with native HA. The goal of the studies presented herein was to explore peptide conjugation techniques to further adjust the material and biological properties of the GMHA hydrogels. We conjugated GMHA with acrylated forms of polyethylene glycol (PEG) and PEG-peptides to yield GMHA-PEG-peptide composite hydrogels. By varying the reactant concentrations, we created stable hydrogels with high peptide conjugation efficiencies (up to 80%), controllable peptide concentrations (in the range of 1-6 micromol peptide per milliliter of hydrogel), and defined physicochemical properties (e.g., swelling ratio, enzymatic degradation rate). These composite hydrogels may prove to be a promising scaffolding biomaterial for a variety of soft tissue engineering applications.     

Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering

Injectable, biodegradable scaffolds are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural polysaccharides are ideal scaffolds as they resemble the extracellular matrices of tissues comprised of various glycosaminoglycans (GAGs). Here, we report a new class of biocompatible and biodegradable composite hydrogels derived from water-soluble chitosan and oxidized hyaluronic acid upon mixing, without the addition of a chemical crosslinking agent. The gelation is attributed to the Schiff base reaction between amino and aldehyde groups of polysaccharide derivatives. In the current work, N-succinyl-chitosan (S-CS) and aldehyde hyaluronic acid (A-HA) were synthesized for preparation of the composite hydrogels. The polysaccharide derivatives and composite hydrogels were characterized by FTIR spectroscopy. The effect of the ratio of S-CS and A-HA on the gelation time, microstructure, surface morphology, equilibrium swelling, compressive modulus, and in vitro degradation of composite hydrogels was examined. The potential of the composite hydrogel as an injectable scaffold was demonstrated by the encapsulation of bovine articular chondrocytes within the composite hydrogel matrix in vitro. The results demonstrated that the composite hydrogel supported cell survival and the cells retained chondrocytic morphology. These characteristics provide a potential opportunity to use the injectable, composite hydrogels in tissue engineering applications.