The biocompatibility of novel starch-based polymers and composites: in vitro studies

Studies with biodegradable starch-based polymers have recently demonstrated that these materials have a range of properties. which make them suitable for use in several biomedical applications, ranging from bone plates and screws to drug delivery carriers and tissue engineering scaffolds. The aim of this study was to screen the cytotoxicity and evaluate starch-based polymers and composites as potential biomaterials. The biocompatibility of two different blends of corn-starch, starch ethylene vinyl alcohol (SEVA-C) and starch cellulose acetate (SCA) and their respective composites with hydroxyapatite (HA) was assessed by cytotoxicity and cell adhesion tests. The MTT assay was performed with the extracts of the materials in order to evaluate the short-term effect of the degradation products. The cell morphology of L929 mouse fibroblast cell line was also analysed after direct contact with polymers and composites for different time periods and the number of cells adhered to the surface of the polymers was determined by quantification of the cytosolic lactate dehydrogenase (LDH) activity. Both types of starch-based polymers exhibit a cytocompatibility that might allow for their use as biomaterials. SEVA-C blends were found to be the less cytotoxic for the tested cell line, although cells adhere better to SCA surface. The cytotoxicity test also revealed that SCA and SEVA-C composites have a similar response to the one obtained for SCA polymer. Scanning electron microscopy (SEM) analysis showed that cells were much more spread on the SCA polymer and LDH measurements showed a higher number of cells on this surface.     

Biocompatibility testing of novel starch-based materials with potential application in orthopaedic surgery: a preliminary study.

This paper describes an extensive biocompatibility evaluation of biodegradable starch-based materials aimed at orthopaedic applications as temporary bone replacement/fixation implants. For that purpose, a polymer (starch/ethylene vinyl alcohol blend, SEVA-C) and a composite of SEVA-C reinforced with hydroxyapatite (HA) particles, were evaluated in both in vitro and in vivo assays. For the in vitro analysis cell culture methods were used. The in vivo tissue reactions were evaluated in an intramuscular and intracortical bone implantation model on goats, using light and scanning electron microscopy. A computerized image analysis system was used to obtain histomorphometric data regarding bone contact and remodelling after 6 and 12 weeks of implantation. In both in vitro and in vivo models, the SEVA-C-based materials did not induce adverse reactions, which in addition to their bone-matching mechanical properties makes them promising materials for bone replacement fixation.     

Biodegradable polymer/hydroxyapatite composites: surface analysis and initial attachment of human osteoblasts

Biodegradable polymer/hydroxyapatite (HA) composites have potential application as bone graft substitutes. Thin films of polymer/HA composites were produced, and the initial attachment of primary human osteoblasts (HOBs) was assessed to investigate the biocompatibility of the materials. Poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLA) were used as matrix materials for two types of HA particles, 50-microm sintered and submicron nonsintered. Using ESEM, cell morphology on the surfaces of samples was investigated after 90 min, 4 h, and 24 h of cell culture. Cell activity and viability were assessed after 24 h of cell culture using Alamar blue and DNA assays. Surface morphology of the polymer/HA composites and HA exposure were investigated using ESEM and EDXA, respectively. ESEM enabled investigation of both cell and material surface morphology in the hydrated condition. Combined with EDXA it permitted chemical and visual examination of the composite. Differences in HA exposure were observed on the different composite surfaces that affected the morphology of attached cells. In the first 4 h of cell culture, the cells were spread to a higher degree on exposed HA regions of the composites and on PLA than they were on PCL. After 24 h the cells were spread equally on all the samples. The cell activity after 24 h was significantly higher on the polymer/HA composites than on the polymer films. There was no significant difference in the activity of the cells on the various composite materials. However, cells on PCL showed higher activity compared to those on PLA. A polymer surface exhibiting "point exposure" of HA appeared to provide a novel and favorable substrate for primary cell attachment. The cell morphology and activity results indicate a favorable cell/material interaction and suggest that PLA and PCL and their composites with HA may be candidate materials for the reconstruction of bony tissue. Further investigations regarding long-term biomaterial/cell interactions and the effects of acidic degradation products from the biodegradable polymers are required to confirm their utility.     

Tissue engineering of electrically responsive tissues using polyaniline based polymers: A review

Conducting polymers have found numerous applications as biomaterial components serving to effectively deliver electrical signals from an external source to the seeded cells. Several cell types including cardiomyocytes, neurons, and osteoblasts respond to electrical signals by improving their functional outcomes. Although a wide variety of conducting polymers are available, polyaniline (PANI) has emerged as a popular choice due to its attractive properties such as ease of synthesis, tunable conductivity, environmental stability, and biocompatibility. PANI in its pure form has exhibited biocompatibility both in vitro and in vivo, and has been combined with a host of biodegradable polymers to form composites having a range of mechanical, electrical, and surface properties. Moreover, recent studies in literature report on the functionalization of polyaniline oligomers with end segments that make it biodegradable and improve its biocompatibility, two properties which make these materials highly desirable for applications in tissue engineering. This review will discuss the features and properties of PANI based composites that make them effective biomaterials, and it provides a comprehensive summary of studies where the use of PANI as a biomaterial component has enhanced cellular function and behavior. We also discuss recent studies utilizing functionalized PANI oligomers, and conclude that electroactive PANI and its derivatives show great promise in eliciting favorable responses from various cell lines that respond to electrical stimuli, and are therefore effective biomaterials for the engineering of electrically responsive biological tissues and organs.     

聚乳酸及其复合材料在骨组织工程方面的研究进展

聚乳酸（PLA）是具有良好的生物相容性和生物降解特性的聚合物,是FDA认可的一类可植入体内的生物材料。本文综述了PLA、羟基磷灰石（HA）／PLA复合材料及生物活性有机成分／PLA复合材料在骨组织工程中应用的研究进展。     

Patterning Methods for Polymers in Cell and Tissue Engineering

Polymers provide a versatile platform for mimicking various aspects of physiological extracellular matrix properties such as chemical composition, rigidity, and topography for use in cell and tissue engineering applications. In this review, we provide a brief overview of patterning methods of various polymers with a particular focus on biocompatibility and processability. The materials highlighted here are widely used polymers including thermally curable polydimethyl siloxane, ultraviolet-curable polyurethane acrylate and polyethylene glycol, thermo-sensitive poly(N-isopropylacrylamide) and thermoplastic and conductive polymers. We also discuss how micro- and nanofabricated polymeric substrates of tunable elastic modulus can be used to engineer cell and tissue structure and function. Such synergistic effect of topography and rigidity of polymers may be able to contribute to constructing more physiologically relevant microenvironment.     

Tensile properties,tension-tension fatigue and biological response of polyetheretherketone-hydroxyapatite composites for load-bearing orthopedic implants

Polyetheretherketone-hydroxyapatite composites were developed as alternative materials for load-bearing orthopedic applications. The amount of hydroxyapatite (HA) incorporated into the polyetheretherketone (PEEK) polymer matrix ranges from 5 to 40 vol% and these materials were successfully fabricated by injection molding. This study presents the mechanical and biological behavior of the composite materials developed. It was found that the amount of HA in the composite influenced the tensile properties. Dynamic behavior under tension-tension fatigue revealed that the fatigue-life of PEEK-HA composites were dependent on the HA content as well as the applied load. The biological responses of PEEK-HA composites carried out in vivo verified the biocompatibility and bioactive nature of the composite materials.     

Molecular dynamics simulations on the interaction between polymers and hydroxyapatite with and without coupling agents

Molecular dynamics (MD) simulations were employed to study hydroxyapatite/biopolymer interface interactions in composites for biomedical applications. The study analyzed the binding energies between hydroxyapatite (HA) and three polymers: polyethylene (PE), polyamide (PA) and polylactic acid (PLA). The interactions of polymers on HA crystallographic planes (0 0 1), (1 0 0) and (1 1 0) were simulated. The effects of the silane coupling agent (A174) on interfacial binding energies were also examined. The results show that HA (1 1 0) has the highest binding energy with these polymers because of its higher planar atom density than that of HA (0 0 1) and (1 0 0). The binding energies of PA/HA and PLA/HA are much higher than that of PE/HA, which might be attributed to large number of polar groups in PA and PLA chains. The silane coupling agent A174 increases the binding energy between PE and HA, but not for the PA/HA and PLA/HA systems. The MD results can be used to guide the design of polymer/HA composites and to select proper coupling agents.     

Review paper: progress in the field of conducting polymers for tissue engineering applications

This review focuses on one of the most exciting applications area of conjugated conducting polymers, which is tissue engineering. Strategies used for the biocompatibility improvement of this class of polymers (including biomolecules' entrapment or covalent grafting) and also the integrated novel technologies for smart scaffolds generation such as micropatterning, electrospinning, self-assembling are emphasized. These processing alternatives afford the electroconducting polymers nanostructures, the most appropriate forms of the materials that closely mimic the critical features of the natural extracellular matrix. Due to their capability to electronically control a range of physical and chemical properties, conducting polymers such as polyaniline, polypyrrole, and polythiophene and/or their derivatives and composites provide compatible substrates which promote cell growth, adhesion, and proliferation at the polymer-tissue interface through electrical stimulation. The activities of different types of cells on these materials are also presented in detail. Specific cell responses depend on polymers surface characteristics like roughness, surface free energy, topography, chemistry, charge, and other properties as electrical conductivity or mechanical actuation, which depend on the employed synthesis conditions. The biological functions of cells can be dramatically enhanced by biomaterials with controlled organizations at the nanometer scale and in the case of conducting polymers, by the electrical stimulation. The advantages of using biocompatible nanostructures of conducting polymers (nanofibers, nanotubes, nanoparticles, and nanofilaments) in tissue engineering are also highlighted.     

HA/PDLLA复合材料的体内成骨过程研究

目的研究HA／PDLLA复合材料植入体内后与细胞、组织的相互作用，探讨HA／PDLLA复合材料在体内的成骨过程，为其临床应用及设计具有生物功能的人工骨替换材料和骨组织工程支架材料提供依据。方法采用液相吸附法制备了HA／PDLLA复合材料，以纯PDLLA和空白组进行对照，进行体内植入实验，通过组织学观察和四环素标记考察其成骨过程。结果HA／PDLLA复合材料植入机体后，体内的无菌性炎症轻微，新骨形成速率高于PDLLA材料。HA微粒的存在，加强了复合材料的机械强度，使之可以避免过早的丧失力学强度。第24w时，材料被组织分隔包裹，新生骨组织长入材料，骨愈合情况良好。结论HA／PDLLA复合材料具有良好的生物相容性、生物降解性能、生物学活性和骨传导性能。     

Shape memory properties of poly(D,L-lactide)/hydroxyapatite composites

Poly(D,L-lactide) (PDLLA) and Hydroxyapatite (HA) are compounded, which possess biodegradation, biocompatibility and shape memory properties. In the paper, we prepared serial imposing shape memory composites with different shapes, composite ratios and sample thicknesses. Scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) were carried out to examine surface morphology, glass transition temperature (Tg), dynamic mechanical properties, and shape memory effect of PDLLA/HA composites, respectively. Moreover, some interesting shape memory behaviors were investigated. The results show that the better disperse morphology of HA grains using the experiment methods, and PDLLA/HA composites at a definite range of compound ratio have much better shape memory effect than pure PDLLA polymer. It indicates that HA particles can improve shape memory effect and PDLLA/HA composites are potential for biomedical applications.     

Adhesion between biodegradable polymers and hydroxyapatite: Relevance to synthetic bone-like materials and tissue engineering scaffolds

Many studies are currently underway on the quest to make synthetic bone-like materials with composites of polymeric materials and hydroxyapatite (HA). In the present work, we use wetting experiments and surface tension measurements to determine the work of adhesion between biodegradable polymers and HA, with specific reference to the role of humid environments. All the polymers are found to exhibit low contact angles (60 degrees ) on the ceramic with work of adhesion values ranging between 48Jm(-2) for poly(epsilon-caprolactone) and 63Jm(-2) for polylactide; these values are associated with physical bonding across the organic/inorganic interface. The corresponding mechanical fracture strengths, measured using four-point bending tests of HA-polymer-HA bonds, scale directly with the results from the wetting experiments. Short-time aging (up to 30h) in a humid environment, however, has a dramatic influence on such HA/polymer interfacial strengths; specifically, water diffusion through the organic/inorganic interface and degradation of the polymer results in a marked decrease, by some 80-90%, in the bond strengths. These results cast doubt on the use of biodegradable polymers/ceramic composites for load-bearing synthetic bone-like materials, as desired optimal mechanical properties are unlikely to be met in realistic physiological environments.     

Evaluation of biomimetic scaffold of gelatin-hydroxyapatite crosslink as a novel scaffold for tissue engineering: biocompatibility evaluation with human PDL fibroblasts, human mesenchymal stromal cells, and primary bone cells

Biomimetic gelatin (gel)-hydroxyapatite (HA) composites have been prepared for studying hard tissue engineering scaffolds. However, the biocompatibility test of this form of material using these three cell types, which are periodontal ligament (PDL) fibroblast cells, human mesenchymal stromal cells (HMSc) and primary cells from human hip bone (HBc) has never been evaluated. The objective of this article is to prepare and evaluate the biocompatibility of gel-HA crosslinked scaffold for tissue engineering. Two different scaffolds were prepared: preparation (1), 2.5% gel/2.5% HA; preparation (2), 2.5% gel/5% HA. Three cell types including PDL, HMSc, and HBc were used. Assessment of biocompatibility and osteoblastic cellular responses was evaluated using a three-dimensional cell culture method and scanning electron microscopy (SEM). From SEM, it was observed that scaffold (1) exhibits stable porous formation with well-blended and dispersed HA powder. All three cell types were able to proliferate in both scaffolds. The HMSc and HBc got attached to the scaffolds to a significantly higher degree and subsequently proliferated more than PDL. The alkaline phosphatase (ALP) activities of HMSc and HBc were stronger when cultured in scaffold (S1) than (S2). It was seen that the two scaffold preparations show good biocompatibility with all three cell types tested. The better cellular responses with scaffold (S1) than (S2) might be due to the different structural and morphological characteristics, that is, scaffold (S1) retained more small-sized apatite crystals and a better developed pore configuration than scaffold (S2). Based on these findings, the biomimetically synthesized composite scaffolds have the potential to be used in hard tissue regeneration and tissue engineering fields.     

Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications

New tissue engineering technologies will rely on biomaterials that physically support tissue growth and stimulate specific cell functions. The goal of this study was to create a biomaterial that combines inherent biological properties which can specifically trigger desired cellular responses (e.g., angiogenesis) with electrical properties which have been shown to improve the regeneration of several tissues including bone and nerve. To this end, composites of the biologically active polysaccharide hyaluronic acid (HA) and the electrically conducting polymer polypyrrole (PP) were synthesized and characterized. Electrical conductivity of the composite biomaterial (PP/HA) was measured by a four-point probe technique, scanning electron microscopy was used to characterize surface topography, X-ray photoelectron spectroscopy and reflectance infrared spectroscopy were used to evaluate surface and bulk chemistry, and an assay with biotinylated hyaluronic acid binding protein was used to determine surface HA content. PP/HA materials were also evaluated for in vitro cell compatibility and tissue response in rats. Smooth, conductive, HA-containing PP films were produced; these films retained HA on their surfaces for several days in vitro and promoted vascularization in vivo. PP/HA composite biomaterials are promising candidates for tissue engineering and wound-healing applications that may benefit from both electrical stimulation and enhanced vascularization.     

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。     

In vitro evaluation of novel bioactive composites based on Bioglass-filled polylactide foams for bone tissue engineering scaffolds

Highly porous poly(DL-lactic acid) (PDLLA) foams and Bioglass-filled PDLLA composite foams were characterized and evaluated in vitro as bone tissue engineering scaffolds. The hypothesis was that the combination of PDLLA with Bioglass in a porous structure would result in a bioresorbable and bioactive composite, capable of supporting osteoblast adhesion, spreading and viability. Composite and unfilled foams were incubated in simulated body fluid (SBF) at 37 degrees C to study the in vitro degradation of the polymer and to detect hydroxyapatite (HA) formation, which is a measure of the materials' in vitro bioactivity. HA was detected on all the composite samples after incubation in SBF for just 3 days. After 28 days immersion the foams filled with 40 wt % Bioglass developed a continuous layer of HA. The formation of HA for the 5 wt % Bioglass-filled foams was localized to the Bioglass particles. Cell culture studies using a commercially available (ECACC) human osteosarcoma cell line (MG-63) were conducted to assess the biocompatibility of the foams and cell attachment to the porous substrates. The osteoblast cell infiltration study showed that the cells were able to migrate through the porous network and colonize the deeper regions within the foam, indicating that the composition of the foams and the pore structures are able to support osteoblast attachment, spreading, and viability. Rapid formation of HA on the composites and the attachment of MG-63 cells within the porous network of the composite foams confirms the high in vitro bioactivity and biocompatibility of these materials and their potential to be used as scaffolds in bone tissue engineering and repair.     

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。     

Preparation and characterization of collagen-hydroxyapatite composite used for bone tissue engineering scaffold

In this study, highly porous collagen-HA scaffolds were prepared by solid-liquid phase separation method. Microstructure of the composites was characterized by SEM, TEM and XRD. The results show that collagen-HA scaffolds are porous with three-dimension interconnected fiber microstructure, pore sizes are 50-150 microm, and HA particles are dispersed evenly among collagen fiber. Compared with pure collagen, the mechanical property of collagen-HA composite improves significantly. To gain further insight into cell growth throughout 3D scaffolds, the cell proliferation and attachment on the scaffold in vitro was investigated. The collagen-HA composite has good biocompatibility, and adding HA does not affect the histocompatibility of the scaffold materials. The porous collagen-HA composite is suitable as scaffold used for bone tissue engineering.     

Development of biodegradable polyurethane and bioactive glass nanoparticles scaffolds for bone tissue engineering applications

The development of polymer/bioactive glass has been recognized as a strategy to improve the mechanical behavior of bioactive glass-based materials. Several studies have reported systems based on bioactive glass/biopolymer composites. In this study, we developed a composite system based on bioactive glass nanoparticles (BGNP), obtained by a modified St?ber method. We also developed a new chemical route to obtain aqueous dispersive biodegradable polyurethane. The production of polyurethane/BGNP scaffolds intending to combine biocompatibility, mechanical, and physical properties in a material designed for tissue engineering applications. The composites obtained were characterized by structural, biological, and mechanical tests. The films presented 350% of deformation and the foams presented pore structure and mechanical properties adequate to support cell growth and proliferation. The materials presented good cell viability and hydroxyapatite layer formation upon immersion in simulated body fluid.     

纳米羟基磷灰石骨修复复合材料的研究进展

羟基磷灰石（Hydroxyapatite，HA）是一种性能良好的骨修复材料，但是由于脆性而限制了它在承力部位的应用。天然骨本身是纳米羟基磷灰石（Nanohydroxyapatite，n．HA）和胶原的复合材料，从仿生的角度看，n—HA与其它材料复合可以提高生物相容性和力学性能。目前研究的纳米羟基磷灰石复合材料可分为两类：非降解的纳米羟基磷灰石复合材料和可降解的纳米羟基磷灰石复合材料。前者包括n—HA/聚乙烯、n-HA／尼龙以及n—HA／聚丙烯酸。后者主要有n—HA与胶原、明胶、壳聚糖、聚乳酸和聚酸酐等的复合材料。本文详细综述了近年来纳米羟基磷灰石复合材料的制备、力学性能以及生物学性能的研究进展。