Gelatin-chondroitin-hyaluronan tri-copolymer scaffold for cartilage tissue engineering

The mechanism by which the cell synthesizes and secretes extracellular matrix (ECM) and is, in turn, regulated by the ECM is termed dynamic reciprocity. The aim of the present work was to produce a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer to mimic natural cartilage matrix for use as a scaffold for cartilage tissue engineering. The scaffold produced had a uniform pore size of about 180 microm and adequate porosity of 75%. Porcine chondrocytes were seeded onto the tri-copolymer scaffold and cultured in Petri dishes or spinner flasks for 2, 3, 4, or 5 weeks. Chondrocytes were uniformly distributed in the scaffold in the spinner flask cultures, but less so in the Petri dish cultures. Secretion of ECM was found under histology examination. In spinner flask cultures, chondrocytes retained their phenotype for at least 5 weeks, as shown immunohistochemically, and synthesized type II collagen. These results show that gelatin/chondroitin sulfate/hyaluronan tri-copolymer has potential for use as a cartilage tissue engineering scaffold.     

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.     

Rabbit articular chondrocytes seeded on collagen-chitosan-GAG scaffold for cartilage tissue engineering in vivo

In this study, we prepared a tri-copolymer porous matrices by natural polymer, collagen (Col), Chitosan (Chi) and Chondroitin (CS). Rabbit articular chondrocytes were isolated from the shoulder articular joints of a rabbit, seeded in Col-Chi-CS scaffold, and implanted subcutaneously in the dorsum of athymic nude mice to tissue engineer articular cartilage in vivo. In vitro studies show that Chondrocytes adhered to the scaffold, where they proliferated and secreted extracellular matrices with time, filling the space within the scaffold. The results of hematoxylin and eosin staining scanning electron microscopy revealed that most of the chondrocytes maintained their typically rounded morphology. After 28 days of culture within Col-Chi-CS scaffold in vitro, the results of histological staining showed forming of cartilage-specific morphological appearance and structural characteristics such as lacunae. Subcutaneous implantation studies in nude mice demonstrated that a homogeneous cartilaginous tissue, which was similar to those of natural cartilage, formed when chondrocytes were seeded in Col-Chi-CS matrix after implant 12 weeks. The tri-copolymer matrix could therefore have potential applications as a three-dimensional scaffold for cartilage tissue engineering.     

Synthesis and characterization of collagen/hyaluronan/chitosan composite sponges for potential biomedical applications

Cells, scaffolds and growth factors are three main components of a tissue-engineered construct. Collagen type I, a major protein of the extracellular matrix (ECM) in mammals, is a suitable scaffold material for regeneration. Another important constituent of the ECM, hyaluronic acid (hyaluronan, HA), has been used for medical purposes due to its hydrogel properties and biodegradability. Chitosan is a linear polysaccharide comprised of β1- to β4-linked d-glucosamine residues, and its potential as a biomaterial is based on its cationic nature and high charge density in solution. This study was conducted to evaluate the characteristics of scaffolds composed of different ratios of type I comb collagen and chitosan with added HA in order to obtain the optimum conditions for the manufacture of collagen–hyaluronan–chitosan (Col–HA–Ch; comprising collagen, HA and chitosan mixed in different ratios: 10:1:0, Col10HACh0; 9:1:1, Col9HACh1; 8:1:2, Col8HACh2; 7:1:3, Col7HACh3; 6:1:4, Col6HACh4; and 5:1:5, Col5HACh5) composite porous scaffolds. Microstructural observation of the composite scaffolds was performed using scanning electron microscopy. The mean pore diameters ranged from 120 to 182 μm and decreased as the chitosan composition increased. All scaffolds showed high pore interconnectivity. Swelling ratio measurements showed that all specimens could bind 35- to 40-fold of physiological fluid and still maintain their form and stability. For tensile strength, the optimal ratio of collagen and chitosan was 9:1. Thermal stability was investigated using a differential scanning calorimeter and showed that Col5HACh5 and Col6HACh4 were significantly more stable than the other groups. In enzymatic sensitivity, a steady increase in the biostability of the scaffolds was achieved as the chitosan concentration was increased. In biocompatibility testing, the proliferation of the fibroblasts cultured in Co-HA-Ch tri-copolymer scaffolds was high. Overall, we observed the 9:1:1 mixing ratio of collagen, hyaluronan and chitosan to be optimal for the manufacture of complex scaffolds. Furthermore, Col–HA–Ch tri-polymer scaffolds, especially Col9HACh1, could be developed as a suitable scaffold material for tissue engineering applications.     

Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering

To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.     

Tissue engineering-based cartilage repair with allogenous chondrocytes and gelatin-chondroitin-hyaluronan tri-copolymer scaffold: a porcine model assessed at 18, 24, and 36 weeks

We previously showed that cartilage tissue can be engineered in vitro with porcine chondrocytes and gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer which mimic natural cartilage matrix for use as a scaffold. In this animal study, 15 miniature pigs were used in a randomized control study to compare tissue engineering with allogenous chondrocytes, autogenous osteochondral (OC) transplantation, and spontaneous repair for OC articular defects. In another study, 6 pigs were used as external controls in which full thickness (FT) and OC defects were either allowed to heal spontaneously or were filled with scaffold alone. After exclusion of cases with infection and secondary arthritis, the best results were obtained with autogenous OC transplantation, except that integration into host cartilage was poor. The results for the tissue engineering-treated group were satisfactory, the repair tissue being hyaline cartilage and/or fibrocartilage. Spontaneous healing and filling with scaffold alone did not result in good repair. With OC defects, the subchondral bone plate was not restored by cartilage tissue engineering. These results show that tri-copolymer can be used in in vivo cartilage tissue engineering for the treatment of FT articular defects.     

纳米羟基磷灰石/Ⅰ型胶原/壳聚糖复合支架材料的制备与优化

目的制备适合于骨组织工程的高强度纳米羟基磷灰石/Ⅰ型胶原/壳聚糖复合支架材料。方法用原位合成法代替传统的直接分散法,以胶原和壳聚糖为模板原位合成羟基磷灰石,再用冷冻干燥法使材料成型,制成可用于骨组织工程的多孔支架材料。结果制备的材料孔隙率高,孔的连通性好,材料中羟基磷灰石结晶度更小,表面能大,与有机物基底结合紧密,也能为成骨细胞的粘附提供更多的活性位点。结论用紫外辐照对材料进行处理,能使其抗压性能得到提高。制备的支架材料适用于骨组织工程。     

Developing an alginate/chitosan hybrid fiber scaffold for annulus fibrosus cells

A fibrous scaffold made of alginate or alginate/chitosan was fabricated for annulus fibrosus (AF) cell culture using a wet-spinning and lyophilization technique. The scaffolds were evaluated using several in vitro tests. Scanning electron microscopy showed the scaffold fibers generally aligned in one direction with individual fiber diameters varying between 40-100 microm. The alginate/chitosan hybrid scaffold exhibited a slower degradation rate, while both scaffold types did not display any cyto-toxicity to 3T3 fibroblasts and could maintain canine AF cell growth. The AF cells retained their spherical shape within the fibrous scaffold at the beginning of the culture period and formed into cell clusters at later times. Specific extracellular matrix molecules, including collagen I, collagen II, and aggrecan, could be detected in the AF cell clusters. These results demonstrate the feasibility of using this hybrid alginate/chitosan scaffold for AF cell culture, and the potential application for intervertebral disc tissue engineering.     

Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffolds

Porous scaffold materials that can provide a framework for the cells to adhere, proliferate, and create extracellular matrix are considered to be suitable materials for bone regeneration. Interconnected porous chitosan scaffolds were prepared by freeze-drying method, and were mineralized by calcium and phosphate solution by double-diffusion method to form nanoapatite in chitosan matrix. The mineralized chitosan scaffold contains hydroxyapatite nanocrystals on the surface and also within the pore channels of the scaffold. To assess the effect of apatite and porosity of the scaffolds on cells, human osteoblast (SaOS-2) cells were cultured on unmineralized and mineralized chitosan scaffolds. The cell growth on the mineralized scaffolds and on the pure chitosan scaffold shows a similar growth trend. The total protein content and alkaline phosphatase enzyme activity of the cells grown on scaffolds were quantified, and were found to increase over time in mineralized scaffold after 1 and 3 weeks of culture. The electron microscopy of the cell-seeded scaffolds showed that most of the outer macropores became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed extra cellular matrix, consisting mainly of collagen in mineralized scaffolds. The hydroxyproline content also confirmed the formation of the collagen matrix by cells in mineralized scaffolds. This study demonstrated that the presence of apatite nanocrystals in chitosan scaffolds does not significantly influence the growth of cells, but does induce the formation of extracellular matrix and therefore has the potential to serve for bone tissue engineering.     

Preparation and characterization of a collagen/chitosan/heparin matrix for an implantable bioartificial liver

A new type of collagen/chitosan/heparin matrix, fabricated by gelation of collagen/chitosan with heparin sodium containing ammonia, was produced to construct livers by tissue engineering and regenerative engineering. The obtained collagen/chitosan/heparin matrix was found to be highly porous, swelled rapidly in PBS solution and was stable in vitro for at least 60 days in collagenase/lysozyme containing buffered aqueous solution (PBS, pH 7.4) at 37°C. The collagen/chitosan/heparin matrix resulted in a superior blood compatibility compared to the ammonia-treated collagen and collagen/chitosan matrices. The morphology and behavior of the cells on the collagen/chitosan/heparin membrane were found to be similar to those on the collagen membrane but different from those on the collagen/chitosan membrane. Hepatocytes cultured on the collagen/chitosan/heparin matrices exhibited highest urea and triglyceride secretion functions 25 days post seeding. These results suggest that this collagen/chitosan/heparin matrix is a potential candidate for liver tissue engineering.     

Synergistic Stimuli by Hydrodynamic Pressure and Hydrophilic Coating on PLGA Scaffolds for Extracellular Matrix Synthesis of Engineered Cartilage

Abstract

Scaffold surface properties and mechanical stimuli have been known to have a critical influence on cell proliferation and extracellular matrix synthesis in cultured cells for tissue engineering. Hydrophilic surface and hydrodynamic pressure (HP) stimulation have been shown to have a beneficial effect on chondrocyte activity. The aim of this study was, thus, to assess the synergic influences of HP and hydrophilic coating on cell activity using primary porcine chondrocytes inoculated in hydrophilic-coated poly(lactide-co-glycolide) (PLGA) sponge scaffolds. The natural materials hyaluronic acid (HA), chitosan and HA/chitosan were cross-linked on porous PLGA as a hydrophilic surface modification. HP was applied to scaffolds at an amplitude of 2.24 MPa and a frequency of 0.1 Hz for 30 min per day, twice a week, over a period of 28 days. Cell activities were determined by the MTS assay and the dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Our results displayed that PLGA coated with both HA and chitosan had the best hydrophilicity (contact angle 49.46°) and initial compressive modulus (1.10 ± 0.13 MPa) among the tested scaffold groups. Additionally, HP stimulation enhanced cell proliferation as well as GAG production (up to 3-fold in culture medium and 15-fold in scaffolds at 28 days compared to static culture of PLGA alone in the scaffold group) in the hydrophilic-coated scaffold groups. The synergistic benefit from hydrophilic coating and HP stimulation may be imperative in regenerating engineered cartilage in the long-term.     

Comparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis

Combining bovine collagen with chitosan followed by freeze-drying has been shown to produce porous scaffolds suitable for skin and connective tissue engineering applications. In this study collagen extracted from porcine and avian skin was compared with bovine collagen for the production of tissue engineered scaffolds. A similar purity of the collagen extracts was shown by electrophoresis, confirming the reliability of the extraction process. Collagen was solubilized, cross-linked by adding chitosan to the solution and freeze-dried to generate a porous structure suitable for tissue engineering applications. Scaffold porosity and pore morphology were shown to be source dependant, with bovine collagen and avian collagen resulting into the smallest and largest pores, respectively. Scaffolds were seeded with dermal fibroblasts and cultured for 35 days to evaluate the suitability of the different collagen–chitosan scaffolds for long-term tissue engineered dermal substitute maturation in vitro. Cell proliferation and scaffold biocompatibility were found to be similar for all the collagen–chitosan scaffolds, demonstrating their capability to support long-term cell adhesion and growth. The scaffolds contents was assessed by immunohistochemistry and showed increased deposition of extracellular matrix by the cells as a function of time. These results correlate with measurements of the mechanical properties of the scaffolds, since both the ultimate tensile strength and tensile modulus of the cell seeded scaffolds had increased by the end of the culture period. This experiment demonstrates that porcine and avian collagen could be used as an alternative to bovine collagen in the production of collagen–chitosan scaffolding materials.     

Preparation and characterization of a collagen/chitosan/heparin matrix for an implantable bioartificial liver

A new type of collagen/chitosan/heparin matrix, fabricated by gelation of collagen/ chitosan with heparin sodium containing ammonia, was produced to construct livers by tissue engineering and regenerative engineering. The obtained collagen/chitosan/heparin matrix was found to be highly porous, swelled rapidly in PBS solution and was stable in vitro for at least 60 days in collagenase/lysozyme containing buffered aqueous solution (PBS, pH 7.4) at 37 degrees C. The collagen/chitosan/heparin matrix resulted in a superior blood compatibility compared to the ammonia-treated collagen and collagen/chitosan matrices. The morphology and behavior of the cells on the collagen/chitosan/heparin membrane were found to be similar to those on the collagen membrane but different from those on the collagen/chitosan membrane. Hepatocytes cultured on the collagen/chitosan/heparin matrices exhibited highest urea and triglyceride secretion functions 25 days post seeding. These results suggest that this collagen/chitosan/heparin matrix is a potential candidate for liver tissue engineering.     

A novel collagen/hydroxyapatite/poly(lactide-co-ε-caprolactone) biodegradable and bioactive 3D porous scaffold for bone regeneration

The goal of this study was to design a nontoxic scaffold with both composition and microstructure suitable for bone engineering using collagen (Coll), hydroxyapatite (HA), and poly(lactide-co-ε-caprolactone) (PLCL). Mineralized type I Coll was produced by direct nucleation of HA particles inside self-assembled Coll fibers to obtain a Coll/HA complex, which was then added to dissolved PLCL (70:30) in 1,4-dioxane. A 3D porous Coll/HA/PLCL scaffold was subsequently produced through freeze-drying/lyophilization and salt-leaching procedures. The resulting Coll/HA/PLCL scaffold displayed a high uniform porosity and highly interconnected pores. X-ray photoelectron spectrometer and Fourier transform infrared analyses revealed the presence of both collagen and HA particles on the surface of the Coll/HA/PLCL scaffold. Proliferation assay, microscopic observations, and gene analysis with quantitative RT-PCR showed that osteoblast cells were able to attach, proliferate, and maintain an osteoblastlike phenotype when cultured on the Coll/HA/PLCL scaffold. In summary, we produced a nontoxic scaffold that contains natural polymers (Coll and HA) and synthetic polymer (PLCL). Through its chemical composition and porous morphology, this scaffold may be useful for osteoblast growth, differentiation, and bone tissue formation.     

Construction of chitosan-gelatin-hyaluronic acid artificial skin in vitro

To further enhance the properties of chitosan (Cs)-gelatin (Gel) scaffolds for skin tissue engineering, hyaluronic acid (HA) is introduced to the Cs-gel complex. Porous scaffolds composed of Cs, Gel, and HA are prepared using the freeze-drying method. The scaffold has an interconnected pore structure with two different pore size layers. The water uptake ability, flexibility, and biocompatibility of the scaffold are greatly increased with the incorporation of HA. To construct an artificial skin in vitro, fibroblasts and keratinocytes are co-cultured in Cs-Gel-HA scaffolds at an air-liquid interface. After 2 weeks of co-culture, the epithelial layer becomes progressively stratiform, including cubic perpendicularly oriented cells and a superficial layer of flattened cells. Immunohistochemical analyses confirmed the presence of laminin and type IV collagen, typical molecules of the basement membrane. The results of this study suggest that it is possible to construct a functional artificial skin in vitro and the Cs-Gel-HA scaffold is a promising matrix for skin tissue engineering.     

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。     

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.     

Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

透明质酸/壳聚糖复合支架的制备及其力学性能评价

目的　模拟天然骨组织的结构和成分,寻找适合骨组织工程的新型支架材料。 方法 以透明质酸、壳聚糖为基质材料,在微酸性环境中以一定配比与氯化钙和磷酸二氢钠混合,冷冻干燥得到多孔复合支架材料。然后在乙醇/水/尿素环境中分别陈化0、2、4、8、12和24 h,以生成产物钙磷盐前驱体转变为羟基磷灰石,最终制备出一种深度矿化的透明质酸/壳聚糖复合支架。并通过SEM、EDS等对支架进行表征,研究支架的形貌、成分及力学强度等性能。 结果 SEM观察显示,支架材料具有比较均匀的多孔结构,孔径大小为100~200 μm。EDS结果表明,复合支架在一次冻干之后形成的是磷酸氢钙（DCPD）,随着陈化时间的延长,DCPD逐渐向羟基磷灰石（HAP）转化。而压缩强度则表明经过原位矿化的支架力学性能显著提高。 结论 通过该法得到的透明质酸/壳聚糖复合支架可作为骨组织工程的新型支架材料。     

Novel gene-activated matrix with embedded chitosan/plasmid DNA nanoparticles encoding PDGF for periodontal tissue engineering

Recently, much attention has been paid to tissue engineering and local gene delivery system in periodontal tissue regeneration. Gene-activated matrix (GAM) blends these two strategies, serving as a local bioreactor with therapeutic gene expression and providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In this study, we designed a novel GAM with embedded chitosan/plasmid nanoparticles encoding platelet derived growth factor (PDGF) based on porous chitosan/collagen composite scaffold. The chitosan/collagen scaffold acted as three-dimensional carrier and chitosan nanoparticles condensed plasmid DNA. The plasmid DNA entrapped in the scaffolds showed a sustained and steady release over 6 weeks and could be effectively protected by chitosan nanoparticles. MTT assay demonstrated that periodontal ligament cells (PDLCs) cultured into the novel GAM achieved high proliferation. Luciferase reporter gene assay displayed that the novel GAM could express 1.07 x 10(4) LU/mg protein after 1 week and 8.97 x 10(3) LU/mg protein after 2 weeks. The histological results confirmed that PDLCs maintained a fibroblast figure and the periodontal connective tissue-like structure formed in the scaffolds after 2 weeks. Semi-quantitative immunohistochemical results suggested that PDGF protein expressed at a relatively high level after 2 weeks. From this study, it can be concluded that the novel GAM had potential in the application of periodontal tissue engineering.