Cellulose-based scaffold materials for cartilage tissue engineering

Non-woven cellulose II fabrics were used as scaffolds for in vitro cartilage tissue engineering. The scaffolds were activated in a saturated Ca(OH)(2) solution and subsequently coated with a calcium phosphate layer precipitated from a supersaturated physiological solution. Chondrocyte cell response and cartilage development were investigated. The cell adherence was significantly improved compared to untreated cellulose fabrics, and the proliferation and vitality of the adhered chondrocytes were excellent, indicating the biocompatibility of these materials. A homogeneous distribution of the seeded cells was possible and the development of cartilageous tissue could be proved. In contact with a physiological chondrocyte solution, calcium is expected to be leached out from the precipitated layer, which might lead to a microenvironment that triggers the development of cartilage in a way similar to cartilage repair in the vicinity of subchondral bone.     

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.     

Potential use of chitosan as a cell scaffold material for cartilage tissue engineering

One of the most important factors in any tissue-engineering application is the cell substrate. The purpose of this study was the initial evaluation of chitosan, a derivative of the abundant, naturally occurring biopolymer chitin, as a cell scaffold for cartilage tissue engineering. Chitosan scaffolds having an interconnecting porous structure were easily fabricated by simple freezing and lyophilization of a chitosan solution. After rehydration of scaffolds, porcine chondrocytes were seeded onto scaffolds and cultured for up to 28 days in a rotating-wall bioreactor. Chitosan scaffolds supported cell attachment and maintenance of a rounded cell morphology. After 18 days, cells within the scaffolds had synthesized extracellular matrix in which proteoglycan and type II collagen were detected by toluidine blue staining and immunohistochemistry, respectively. Abundant extracellular matrix was found almost exclusively in the periphery of the scaffolds, as scaffold microstructure prevented cells from penetrating to interior regions. Nonetheless, the results suggest that chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Poly(lactide-co-glycolide) microspheres as a moldable scaffold for cartilage tissue engineering

This study demonstrates the use of biodegradable poly(lactide-co-glycolide) (PLG) microspheres as a moldable scaffold for cartilage tissue engineering. Chondrocytes were delivered to a cylindrical mold with or without PLG microspheres and cultured in vitro for up to 8 weeks. Cartilagenous tissue formed using chondrocytes and microspheres maintained thickness, shape, and chondrocyte collagen type II phenotype, as indicated by type II collagen staining. The presence of microspheres further enhanced total tissue mass and the amount of glycosaminoglycan that accumulated. Evaluation of microsphere composition demonstrated effects of polymer molecular weight, end group chemistry, and buffer inclusion on tissue-engineered cartilage growth. Higher molecular weight PLG resulted in a larger mass of cartilage-like tissue formed and a higher content of proteoglycans. Cartilage-like tissue formed using microspheres made from low molecular weight and free carboxylic acid end groups did not display increases in tissue mass, yet a modest increased proteoglycan accumulation was detected. Microspheres comprised of PLG with methyl ester end groups yielded a steady increase in tissue mass, with no real increase in matrix accumulation. The microencapsulation of Mg(OH)(2) had negative effects on tissue mass and matrix accumulation. The data herein reflect the potential utility of a moldable PLG-chondrocyte system for tissue-engineering applications.     

Alginate as a chondrocyte-delivery substance in combination with a non-woven scaffold for cartilage tissue engineering

For tissue engineering of cartilage, chondrocytes can be seeded in a scaffold and stimulated to produce a cartilage-like matrix. In the present study, we investigated the effect of alginate as a chondrocyte-delivery substance for the construction of cartilage grafts. E210 (a non-woven fleece of polyglactin) was used as a scaffold. When bare' E210 (without alginate and without chondrocytes) was implanted subcutaneously in nude mice for 8 weeks. the explanted tissue consisted of fat and fibrous tissue only. When E210 with alginate but without chondrocytes was implanted in nude mice, small areas of newly formed cartilage were found. Alginate seems to stimulate chondrogenesis of ingrowing cells. When chondrocytes were seeded in E210, large amounts of cartilage were found, independent of the use of alginate. This was expressed by a high concentration of glycosaminoglycans (30 microg/mg w.w.) and the presence of collagen type II (1.5 microg/mg w.w.). Macroscopically the grafts of E210 without alginate were shrunk and warped, whereas the grafts with alginate had kept their original shape during the 8 weeks of implantation. The use of alginate did not lead to inflammatory reactions nor increased capsule formation. In conclusion, the use of alginate to seed chondrocytes in E210 does not influence the amount of cartilage matrix proteins produced per tissue wet weight. However, it provides retention of the graft shape.     

组织工程软骨支架材料的应用选择

背景：软骨损伤后几乎不可能完全修复,近年来采用组织工程学方法构建软骨复合组织已成为软骨修复方面新的研究领域。其中支架材料在软骨修复过程中起着至关重要的作用,支架材料的选择也就影响着整个修复过程。 目的：全面了解组织工程软骨支架材料的优缺点,并对其选择进行综述。 方法：由第一作者于2010-11在CBM、PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed)、万方数据库(http://www. wanfangdata.com.cn)及google学术网检索组织工程软骨支架材料方面的内容,检索时限为1990/2010,英文检索词为“cartilage, tissue engineering, scaffold materials, bone mesenchymal stem cell”,中文检索词为“软骨,组织工程,支架材料,骨髓间充质干细胞”,排除重复性研究。 结果与结论：计算机初检得到1 185篇文献,阅读标题和摘要进行初筛,排除因研究目的与本文无关及内容重复的研究1 142篇,共保留其中的43篇进行归纳总结,最终引入文献29篇。结果提示,目前应用于组织工程的支架不论是天然的还是人工合成的,都存在一定的缺陷,如体内降解速度过快或过慢,生物相容性不佳、引起炎症等问题。最重要的是组织工程软骨支架距临床应用仍有很大差距,而未来组织工程支架材料的研究重点是改进现有材料和制备工艺,研制复合材料、仿生材料、纳米材料及改性天然材料等。     

Potential use of collagen-chitosan-hyaluronan tri-copolymer scaffold for cartilage tissue engineering

A three-dimensional biodegradable porous scaffold plays a vital role in a tissue engineering approach. Collagen, chitosan and hyaluronan (HA) are natural extracellular matrix (ECM) or similarity, and may provide appropriate environment for the generation of cartilage-like tissue. In this study, we prepared a collagen/chitosan/HA tri-copolymer porous scaffold by freezing and lyophilization to evaluate physico-chemical properties of the tri-copolymer scaffold and its capacity to sustain chondrocytes proliferation and differentiation in vitro. The results show that the mechanical strength, the resistance to enzymatic degradation, and the waterblinding capacity were improved when chitosan and hyaluronan were incorporated into a collagen scaffold. After 21 days of culture, the porous scaffold had been surfaced with cartilaginous tissue. DNA and glycosaminoglycan (GAG) contents were significantly higher during culture periods in collagen/ chitosan/hyaluronan matrix compared to collagen alone matrix, and most seeded cells preserved the chondrocytic phenotype during culture within the scaffold. The collagen/chitosan/hyaluronan tri-copolymer scaffold has potential applications in a cartilage tissue engineering scaffold field.     

Decellularized extracellular matrix derived from human adipose tissue as a potential scaffold for allograft tissue engineering

Decellularized tissues composed of extracellular matrix (ECM) have been clinically used to support the regeneration of various human tissues and organs. Most decellularized tissues so far have been derived from animals or cadavers. Therefore, despite the many advantages of decellularized tissue, there are concerns about the potential for immunogenicity and the possible presence of infectious agents. Herein, we present a biomaterial composed of ECM derived from human adipose tissue, the most prevalent, expendable, and safely harvested tissue in the human body. The ECM was extracted by successive physical, chemical, and enzymatic treatments of human adipose tissue isolated by liposuction. Cellular components including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. Major ECM components were quantified, including acid/pepsin-soluble collagen, sulfated glycosaminoglycan (GAG), and soluble elastin. In an in vivo experiment using mice, the decellularized ECM graft exhibited good compatibility to surrounding tissues. Overall results suggest that the decellularized ECM containing biological and chemical cues of native human ECM could be an ideal scaffold material not only for autologous but also for allograft tissue engineering.     

Microporous bacterial cellulose as a potential scaffold for bone regeneration

Nanoporous cellulose biosynthesized by bacteria is an attractive biomaterial scaffold for tissue engineering due to its biocompatibility and good mechanical properties. However, for bone applications a microscopic pore structure is needed to facilitate osteoblast ingrowth and formation of a mineralized tissue. Therefore, in this study microporous bacterial cellulose (BC) scaffolds were prepared by incorporating 300–500 μm paraffin wax microspheres into the fermentation process. The paraffin wax microspheres were subsequently removed, and scanning electron microscopy confirmed a microporous surface of the scaffolds while Fourier transform infrared spectroscopy verified the elimination of paraffin and tensile measurements showed a Young’s modulus of approximately 1.6 MPa. Microporous BC and nanoporous (control) BC scaffolds were seeded with MC3T3-E1 osteoprogenitor cells, and examined by confocal microscopy and histology for cell distribution and mineral deposition. Cells clustered within the pores of microporous BC, and formed denser mineral deposits than cells grown on control BC surfaces. This work shows that microporous BC is a promising biomaterial for bone tissue engineering applications.     

Poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for cartilage tissue engineering

Injectable scaffold has raised great interest for tissue regeneration in vivo, because it allows easy filling of irregularly shaped defects and the implantation of cells through minimally invasive surgical procedures. In this study, we evaluated poly(lactic-co-glycolic acid) (PLGA) microsphere as an injectable scaffold for in vivo cartilage tissue engineering. PLGA microspheres (30-80 microm in diameter) were injectable through various gauges of needles, as the microspheres did not obstruct the needles and microsphere size exclusion was not observed at injection. The culture of chondrocytes on PLGA microspheres in vitro showed that the microspheres were permissive for chondrocyte adhesion to the microsphere surface. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into athymic mouse subcutaneous sites. Chondrocyte transplantation without PLGA microspheres and PLGA microsphere implantation without chondrocytes served as controls. Four and 9 weeks after implantation, chondrocytes implanted with PLGA microspheres formed solid, white cartilaginous tissues, whereas no gross evidence of cartilage tissue formation was noted in the control groups. Histological analysis of the implants by hematoxylin and eosin staining showed mature and well-formed cartilage. Alcian blue/safranin O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosaminoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for in vivo cartilage tissue engineering. This scaffold may be useful to regenerate cartilaginous tissues through minimally invasive surgical procedures in orthopedic, maxillofacial, and urologic applications.     

软骨组织工程中支架材料的文献回顾*

背景：目前报道的软骨组织工程支架材料,大体分为天然高分子材料、人工合成可降解材料、天然材料与合成高分子材料复合构造的新型生物材料和纳米材料4大类。 目的：总结近年来国内外关于组织工程支架材料的文献,对其进行简要综述,并探讨目前存在的问题和应用前景。 方法：由第一作者应用计算机检索PubMed数据库及中国期刊网全文数据库1997-01/2011-01有关软骨组织工程支架材料的文章,英文检索词为“natural polymer materials,synthetic materials,new biological materials,nanometer materials,scaffold materials,cartilage tissue engineering”,中文检索词为“天然高分子材料,人工合成材料,新型生物材料,纳米材料,支架材料,软骨组织工程”。排除重复性研究,共保留33篇文献进行综述。 结果与结论：软骨组织工程支架已由先前的单一材料逐渐向复合材料转变,其孔隙率更高、抗原性更低、组织相容性更好,软骨细胞的黏附及增殖更好。结合计算机辅助设计和三维打印快速成形技术,将生物可降解材料预制加工成精确形状,通过降解速率较慢的内支撑支架,维持材料支架的精确外形,研发具有一定机械强度、适当孔径和精确外观形状的可降解生物支架材料是未来的发展方向。     

Chitosan-RGDSGGC conjugate as a scaffold material for musculoskeletal tissue engineering

In the present study, we have developed a novel and versatile method for the preparation of chitosan-peptide complex based on the selective reaction of chitosan with 2-iminothiolane. The new type of SH-chitosan derivative showed an excellent solubility to aqueous solution even in the alkaline conditions. This characteristic greatly facilitated further modification study of chitosan with a variety of bioactive substances. A synthetic peptide, RGDSGGC containing RGDS moiety that is known as one of the most important cell adhesive peptides, was readily coupled by disulfide bonds formation with sulfhydryl groups of SH-chitosan in the presence of dimethyl sulfoxide. Next, the effect of the introduction of RGDSGGC moiety to chitosan on cell adhesion and proliferation activity of chondrocytes and fibroblasts were evaluated. As a result, it was suggested that this polysaccharide-peptide conjugate exhibited excellent capacities for both cell adhesion and cell proliferation of chondrocytes and fibroblasts. Considering the growing importance of the biocompatible scaffolds in the recent tailored tissue engineering technique, these results indicate that the present strategy of 2-iminothiolane-based conjugation of polysaccharides with biologically active peptides will become a key and potential technology to develop desirable scaffold materials for the tissue regenerations.     

Transglutaminase crosslinked gelatin as a tissue engineering scaffold

Gelatin is one of the most commonly used biomaterials for creating cellular scaffolds due to its innocuous nature. In order to create stable gelatin hydrogels at physiological temperatures (37 degrees C), chemical crosslinking agents such as glutaraldehyde are typically used. To circumvent potential problems with residual amounts of these crosslinkers in vivo and create scaffolds that are both physiologically robust and biocompatible, a microbial transglutaminase (mTG) was used in this study to enzymatically crosslink gelatin solutions. HEK293 cells encapsulated in mTG-crosslinked gelatin proliferated at a rate of 0.03 day(-1). When released via proteolytic degradation with trypsin, the cells were able to recolonize tissue culture flasks, suggesting that cells for therapeutic purposes could be delivered in vivo using an mTG-crosslinked gelatin construct. Upon submersion in a saline solution at 37 degrees C, the mTG-crosslinked gelatin exhibited no mass loss, within experimental error, indicating that the material is thermally stable. The proteolytic degradation rate of mTG-crosslinked gelatin at RT was slightly faster than that of thermally-cooled (physically-crosslinked) gelatin. Thermally-cooled gelatin that was subsequently crosslinked with mTG resulted in hydrogels that were more resistant to proteolysis. Degradation rates were found to be tunable with gelatin content, an attribute that may be useful for either long-time cell encapsulation or time-released regenerative cell delivery. Further investigation showed that proteolytic degradation was controlled by surface erosion.     

A collagen-phosphophoryn sponge as a scaffold for bone tissue engineering

Non-collagenous phosphoproteins that interact with a type-I collagen are thought to nucleate bone mineral into collagen networks of mineralized tissues. Previously, phosphophoryn cross-linked to type-I collagen was reported to be an effective nucleator of appatite. However, free phosphophoryn molecules inhibit the formation of apatite in vitro. On the basis of the above study, we expected a collagen-phosphophoryn sponge to be a good scaffold for bone-tissue engineering and examined the formation of bone in orthotopically transplanted composites of the sponge and bone marrow osteoblasts in vivo in Fischer rats. Osteoblastic primary cells were obtained from the bone shaft of femorae of Fisher rats, according to the method of Maniatopoulous et al. A suspension of marrow cells was distributed through a flask with standard culture medium and incubated at 37 degrees C. When cultures were nearly confluent after 10 days, they were concentrated by centrifugation to 10(6) cells/ml and subcultured onto the synthesized collagen-phosphophoryn sponge and a collagen sponge (control). After 14 days, the composites of collagen-phosphophoryn and osteoblastic cells as well as control composites were transplanted into bone-defect sites of Fisher rats (holes 2 mm in diameter) and then the wounds were sutured. The composites were harvested at 1-8 weeks after implantation, and stained with hematoxylin and eosin. It was found that more bone was formed in the composites of collagen-phosphophoryn sponge and osteoblasts than control composites from 1 week to 8 weeks, suggesting that the collagen-phosphophoryn sponge is a good candidate as a scaffold for bone-tissue engineering.     

丝素蛋白作为组织工程材料应用于角膜的研究进展

背景：丝素蛋白纤维材料具有透明性、结构可塑性、成分单一性、力学强韧性及生物相容性等特点。 目的：综述国内外丝素蛋白应用于角膜组织工程的研究进展。 方法：由第一作者在标题和摘要中以“silk fibroin, corneal, ocular”或“丝素,角膜”为检索词,检索1980至2011年PubMed及1990至2011年CNKI数据库中关于丝素蛋白角膜的文章。 结果与结论：从天然蚕丝中提取的高分子丝素蛋白,因其良好的生物相容性、独特的力学性能、光学透明性及降解速率可控性,既可以单独应用于角膜组织结构的重建,又可与其他组织材料联合应用,成为角膜组织工程学应用的理想材料。现已证明多种角膜细胞可在丝素纤维膜上良好生长,但体外培养的细胞应用于动物模型的相关研究较少;此外丝素蛋白材料植入角膜内对其产生何种影响的研究数据较缺乏,这些均是亟待解决的问题。     

Pulsed electric field as a potential new method for microbial inactivation in scaffold materials for tissue engineering: the effect on collagen as a scaffold

Hybrid scaffolds for tissue engineering are becoming increasingly complex through incorporation of biologically active biomacromolecules. There is a need to develop a compatible sterilization method that is capable of killing microorganisms, without adversely affecting the labile scaffold biomaterials or biomacromolecular components. Pulsed electric field (PEF) treatment has been successful as a nonthermal microbial inactivation-pasteurization method within the food industry. We have previously demonstrated that PEF treatment inactivates E. coli seeded in collagen gels. Here, we show that PEF treatment does not affect the structural integrity of the collagen molecule or its adsorption to polystyrene and hydroxyapatite surfaces. Moreover, osteoblast cells cultured on PEF-treated collagen, which was coated onto two- and three-dimensional scaffolds, retained their normal morphology, growth rate, and functionality. PEF treatment, therefore, shows great potential to be used as a sterilization method for collagen-based biomaterials.     

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.     

支架材料在软骨组织工程中的应用进展

正关节软骨属于透明软骨,为终末分化组织,缺乏神经和血管滋养,一旦损伤很难修复,常诱发骨关节炎等退行性疾病。组织工程是目前修复软骨损伤最具潜力的方法,其中支架技术的本质是在体外构建软骨细胞外基质替代物,为种子细胞提供与天然细胞外基质相似的微环境。支架材料应具备的条件包括:(1)良好的组织相容性和表面活性;(2)一定机械强度;(3)可控降解性;(4)支架及降解产物的无毒性;(5)三维结构和高     

CD146阳性亚群有作为软骨组织工程种子细胞的应用潜力

文题释义：CD146：也称为MUC18,MCAM,Mel-CAM或S-Endo1,是一种跨膜糖蛋白,同时属于免疫球蛋白超级家族的一员。在人体组织中,CD146阳性细胞被认为是微血管的壁细胞,其具有较强的增殖、迁移及自我更新能力。 组织工程种子细胞：组织工程包括3个关键要素——种子细胞、支架和活性因子,其中种子细胞应具有良好的细胞活性、增殖能力、分化及合成基质等生物功能。 背景：再生医学的发展、组织工程技术的出现为软骨缺损重建提供了新的解决思路。在组织工程中,间充质干细胞是应用广泛的种子细胞,然而干细胞作为一个异质性的细胞群体其不同亚群发挥着不同的功能。因此,应用间充质干细胞关键功能亚群进行软骨修复具有广泛应用前景。 目的：从人脂肪间充质干细胞中分离出CD146阳性亚群细胞,验证其生物学特性及其作为软骨组织工程种子细胞的潜力。 方法：人脂肪间充质干细胞由浙江金时代生物技术有限公司提供,通过流式细胞术对人脂肪间充质干细胞表面标志物进行鉴定,应用免疫磁珠分选方法从人脂肪间充质干细胞中分选出CD146阳性表达的细胞亚群。通过基因芯片检测技术及生物信息学分析技术揭示2种细胞的分子特性;体外诱导2种细胞成软骨分化并进行鉴定;冻存复苏前后检测2种细胞的细胞活性及凋亡情况。 结果与结论：①人脂肪间充质干细胞表达高水平干细胞相关标志物CD73、CD90,不表达造血干细胞相关标志物CD34、CD45、HLA-DR;②生物信息分析结果表明CD146阳性亚群细胞与脂肪间充质干细胞相比在炎症通路及骨骼肌肉系统疾病有不同功能;③CD146阳性亚群细胞能够成球软骨分化,并且其成软骨分化能力要优于人脂肪间充质干细胞;④CD146阳性亚群细胞复苏后凋亡情况和活性均要优于人脂肪间充质干细胞;⑤结果表明,CD146阳性亚群细胞具有良好的成软骨分化潜力,是一种具有前景的软骨组织工程种子细胞。 ORCID: 0000-0003-4210-4708(眭翔) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Chitosan-alginate as scaffolding material for cartilage tissue engineering

Tissue compatibility of chitosan-alginate scaffolds was studied in vitro in terms of cell morphology, proliferation, and functionality using HTB-94 cells. The scaffold has an interconnected 3D porous structure, and was fabricated by thermally induced phase separation followed by freeze drying. Cell proliferation on the chitosan-alginate scaffold was found to be faster than on a pure chitosan scaffold. After cell culture for 2 weeks in vitro, the cells on the chitosan scaffold gradually assumed a fibroblast-like morphology while the cells on the chitosan-alginate scaffold retained their spherical morphology throughout the period of study. SDS-PAGE electrophoresis and Western blot assays for proteins extracted from cells grown on scaffolds indicated that production of cartilage-specific collagen type II, a marker for chondrocytic phenotype, increased from week 2 to week 3 on the chitosan-alginate scaffold but decreased on the chitosan scaffold. This study suggested that chitosan-alginate scaffolds promote cell proliferation, enhance phenotype expression of HTB-94 chondrocytes, and may potentially serve as an improved alternative to chitosan scaffolds for cartilage tissue engineering.