Evaluation of RGD- or EGF-immobilized chitosan scaffolds for chondrogenic activity

Chitosan scaffolds were prepared by freeze-drying method and modified with Arg-Gly-Asp (RGD) sequence of fibronectin or epidermal growth factor (EGF) by covalent immobilization. The results obtained from FTIR-ATR, fluorescence visualization and quantitative measurements showed that biosignal molecules, RGD and EGF, were successfully immobilized on chitosan scaffolds. ATDC5 murine chondrogenic cells were seeded on both type of scaffolds, chitosan-RGD and chitosan-EGF, and cultured for 28 days in stationary conditions. According to the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) test, considerable increase in cell proliferation was only detected on chitosan-EGF scaffolds. Biochemical analysis of the chondrocyte seeded scaffolds showed that glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA) content of the scaffolds increases with time. In conclusion, EGF-modified chitosan scaffolds (containing 1.83 microg EGF/3 mg dry scaffold) have been proposed to promote chondrogenesis and to have potential for reticular cartilage regeneration.     

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.     

Effects of co‐cultures of meniscus cells and articular chondrocytes on PLLA scaffolds

The knee meniscus, a fibrocartilaginous tissue located in the knee joint, is characterized by heterogeneity in extracellular matrix and biomechanical properties. To recreate these properties using a tissue engineering approach, co‐cultures of meniscus cells (MCs) and articular chondrocytes (ACs) were seeded in varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) on poly‐L ‐lactic acid (PLLA) scaffolds and cultured in serum‐free medium for 4 weeks. Histological, biochemical, and biomechanical tests were used to assess constructs at the end time point. Strong staining for collagen and glycosaminoglycan (GAG) was observed in all groups. Constructs with 100% MCs were positive for collagen I and constructs cultured with 100% ACs were positive for collagen II, while a mixture of collagen I and II was observed in other co‐culture groups. Total collagen and GAG per construct increased as the percentage of ACs increased (27 ± 8 µg, 0% AC to 45 ± 8 µg, 100% ACs for collagen and 12 ± 4 µg, 0% ACs to 40 ± 5 µg, 100% ACs for GAG). Compressive modulus (instantaneous and relaxation modulus) of the constructs was significantly higher in the 100% ACs group (63 ± 12 and 22 ± 9 kPa, respectively) when compared to groups with higher percentage of MCs. No differences in tensile properties were noted among groups. Specific co‐culture ratios were identified mimicking the GAG/DW of the inner (0:100, 25:75, and 50:50) and outer regions (100:0) of the meniscus. Overall, it was demonstrated that co‐culturing MCs and ACs on PLLA scaffolds results in functional tissue engineered meniscus constructs with a spectrum of biochemical and biomechanical properties. Biotechnol. Bioeng. 2009;103: 808–816. © 2009 Wiley Periodicals, Inc.     

Perfusion affects the tissue developmental patterns of human mesenchymal stem cells in 3D scaffolds

Human mesenchymal stem cells (hMSCs) developed in three‐dimensional (3D) scaffolds are significantly affected by culture conditions. We hypothesized that the hydrodynamic forces generated in perfusion bioreactors significantly affected hMSC functionality in 3D scaffolds by shaping the extracellular matrix (ECM) proteins. In this study, hMSCs were grown in 3D poly(ethylene terephthalate) (PET) scaffolds in static and a parallel perfusion system under similar initial conditions for up to 35 days. Results demonstrated that even at very low media velocities (O [10^?4 cm/sec]), perfusion cultures affected the ability of hMSCs to form an organized ECM network as illustrated by the immunostaining of collagen I and laminin fibrous structure. The change in the ECM microenvironment consequently influenced the nuclear shape. The hMSCs grown at the lower surface of static culture displayed a 15.2 times higher nuclear elongation than those at the upper surface, whereas cells grown in the perfusion bioreactor displayed uniform spherical nuclei on both surfaces. The difference in ECM organization and nuclear morphology associated with gene expression and differentiation characteristics of hMSCs. The cells exhibited lower CFU‐F colony forming ability and decreased expressions of stem‐cell genes of Rex‐1 and Oct‐4, implying a less primitive stem‐cell phenotype was maintained in the perfusion culture relative to the static culture conditions. The significantly higher expression level of osteonectin gene in the perfusion culture at day 28 indicated an upregulation of osteogenic ability of hMSCs. The study highlights the critical role of dynamic culture conditions on 3D hMSC construct development and properties. J. Cell. Physiol. 219: 421–429, 2009. © 2009 Wiley‐Liss, Inc.     

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor

It has been widely demonstrated that perfusion bioreactors improve in vitro three‐dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C₂C₁₂ muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G₂/M) owing to medium perfusion in long‐term cultures. After 7–10 days, the percentage of proliferating cells was 8–12% for dynamic cultures and 3–5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Mechanical response of porous scaffolds for cartilage engineering

Mechanical properties of scaffolds seeded with mesenchymal stem cells used for cartilage repair seem to be one of the critical factors in possible joint resurfacing. In this paper, the effect of adding hyaluronic acid, hydroxyapatite nanoparticles or chitosan nanofibers into the cross-linked collagen I on the mechanical response of the lyophilized porous scaffold has been investigated in the dry state at 37 oC under tensile loading. Statistical significance of the results was evaluated using ANOVA analysis. The results showed that the addition of hyaluronic acid significantly (p<0.05) reduced the tensile elastic modulus and enhanced the strength and deformation to failure of the modified cross-linked collagen I under the used test conditions. On the other hand, addition of hydroxyapatite nanoparticles and chitosan nanofibers, respectively, increased the elastic modulus of the modified collagen ten-fold and four-fold, respectively. Hydroxyapatite caused significant reduction in the ultimate deformation at break while chitosan nanofibers enhanced the ultimate deformation under tensile loading substantially (p<0.05). The ultimate tensile deformation was significantly (p<0.05) increased by addition of the chitosan nanofibers. The enhanced elastic modulus of the scaffold was translated into enhanced resistance of the porous scaffolds against mechanical load compared to scaffolds based on cross-linked neat collagen or collagen with hyaluronic acid with similar porosity. It can be concluded that enhancing the rigidity of the compact scaffold material by adding rigid chitosan nanofibers can improve the resistance of the porous scaffolds against compressive loading, which can provide more structural protection to the seeded mesenchymal stem cells when the construct is implanted into a lesion. Moreover, scaffolds with chitosan nanofibers seemed to enhance cell growth compared to the neat collagen I when tested in vitro as well as the scaffold stability, extending its resorption to more than 10 weeks.     

Composition of cell-polymer cartilage implants

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.     

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

Application of polyethyleneimine‐modified scaffolds to the regeneration of cartilaginous tissue

In this study, we analyzed the physicochemical and biophysical properties of three‐dimensional scaffolds modified using polyethyleneimine (PEI) and applied these scaffolds to the cultivation of bovine knee chondrocytes (BKCs). PEI was crosslinked in the bulk or on the surface of the ternary scaffolds comprising polyethylene oxide, chitin and chitosan. The results revealed that when the concentration of PEI was less than 300 μg/mL, the cytotoxicity of a scaffold was on the same order in the two method of modification. An increase in the concentration of PEI favored the adhesion of BKCs. When the amount of PEI in scaffolds is fixed, the surface‐modified scaffolds exhibited a higher adhesion efficiency of BKCs than the bulk‐modified scaffolds. For the regeneration of cartilaginous components, a higher amount of PEI in a scaffold yielded larger amounts of proliferated BKCs, secreted glycosaminoglycans, and produced collagen. In addition, the formation of neocartilage in the surface‐modified scaffolds was more effective than that in the bulk‐modified scaffolds. These tissue‐engineered scaffolds, modified by an appropriate concentration of PEI, can be potentially applied to cartilage repair in clinical trials. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z®RP platform

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three‐dimensional functional tissue‐like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three‐dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram®. Furthermore, hydroxyapatite coated Sponceram® was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP‐expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram® compared to hydroxyapatite coated Sponceram®. With this study, we present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cultivation of cell-polymer tissue constructs in simulated microgravity

Tissue-engineered cartilage was cultivated under conditions of simulated microgravity using rotating bioreactors. Rotation randomized the effects of gravity on inoculated cells (chondrocytes) and permitted their attachment to three-dimensional (3D) synthetic, biodegradable polymer scaffolds that were freely suspended within the vessel. After 1 week of cultivation, the cells regenerated a cartilaginous extracellular matrix (ECM) consisting of glycosaminoglycan (GAG) and collagen types I and II. Tissue constructs grown in simulated microgravity had higher GAG contents and thinner outer capsules than control constructs grown in turbulent spinner flasks. Two fluid dynamic regimes of simulated microgravity were identified, depending on the vessel rotation speed: (i) a settling regime in which the constructs were maintained in a state of continuous free-fall close to a stationary point within the vessel and (ii) an orbiting regime in which the constructs orbited around the vessel spin axis. In the settling regime, the numerically calculated relative fluid-construct velocity was comparable to the experimentally measured construct settling velocity (2-3 cm/s). A simple mathematical model was used in conjunction with measured construct physical properties to determine the hydrodynamic drag force and to estimate the hydrodynamic stress at the construct surface (1.5 dyn/cm(2)). Rotating bioreactors thus provide a powerful research tool for cultivating tissue-engineered cartilage and studying 3D tissue morphogenesis under well-defined fluid dynamic conditions. (c) 1995 John Wiley & Sons, Inc.     

Microgravity tissue engineering

Summary Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter × 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.     

Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation

Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein-2 (BMP-2). Chondrocytes expanded without FGF-2 exhibited high intensity immunostaining for smooth muscle alpha-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds.     

Role of glycosaminoglycans of biglycan in BMP-2 signaling

Recently we have reported that biglycan (BGN) promotes osteoblast differentiation and that this function is due in part to its ability to positively modulate bone morphogenetic protein (BMP) functions. In this study we investigated the role of glycosaminoglycans (GAGs) of BGN in this function using in vitro and in vivo models. C2C12 myogenic cells were treated or untreated with BMP-2 alone or in combination with glycanated, partially glycanated or de-glycanated BGN, and the effects on BMP signaling and function were assessed by Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the effect of de-glycanation of BGN on BMP-2 induced osteogenesis was investigated employing a rat mandible defect model. The defects were filled with collagen scaffolds loaded with glycanated or de-glycanated BGN alone or in combination with a sub-optimal dose of BMP-2 (subBMP). In in vitro experiments, BMP signaling and function were the greatest when BMP-2 was combined with de-glycanated BGN among the groups tested. In the rat mandible experiments, μCT analyses revealed that the newly formed bone was significantly increased only when subBMP was combined with de-glycanated BGN. The data indicate that the GAG component of BGN functions as a suppressor for the BGN-assisted BMP function.     

Dynamic compressive loading of image-guided tissue engineered meniscal constructs

This study investigated the hypothesis that dynamic compression loading enhances tissue formation and increases mechanical properties of anatomically shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2%w/v alginate, crosslinked with CaSO(4), injected into μCT based molds, and post crosslinked with CaCl(2). Samples were loaded via a custom bioreactor with loading platens specifically designed to load anatomically shaped constructs in unconfined compression. Based on the results of finite element simulations, constructs were loaded under sinusoidal displacement to yield physiological strain levels. Constructs were loaded 3 times a week for 1 h followed by 1 h of rest and loaded again for 1 h. Constructs were dynamically loaded for up to 6 weeks. After 2 weeks of culture, loaded samples had 2-3.2 fold increases in the extracellular matrix (ECM) content and 1.8-2.5 fold increases in the compressive modulus compared with static controls. After 6 weeks of loading, glycosaminoglycan (GAG) content and compressive modulus both decreased compared with 2 week cultures by 2.3-2.7 and 1.5-1.7 fold, respectively, whereas collagen content increased by 1.8-2.2 fold. Prolonged loading of engineered constructs could have altered alginate scaffold degradation rate and/or initiated a catabolic cellular response, indicated by significantly decreased ECM retention at 6 weeks compared with 2 weeks. However, the data indicates that dynamic loading had a strikingly positive effect on ECM accumulation and mechanical properties in short term culture.     

Serum‐free,chemically defined medium with TGF‐β3 enhances functional properties of nucleus pulposus cell‐laden carboxymethylcellulose hydrogel constructs

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum‐containing medium formulation was compared to a chemically defined, serum‐free medium to determine the effect on matrix elaboration and functional properties of NP cell‐laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor‐beta 3 (TGF‐β₃). Glycosaminoglycan (GAG) content increased in both TGF‐β₃‐treated groups and was highest for treated, serum‐free constructs (9.46 ± 1.51 µg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum‐containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum‐free constructs. Type II collagen content was greater in both serum‐free groups and highest in treated, serum‐free constructs. The equilibrium Young's modulus was highest in serum‐free samples supplemented with TGF‐β₃ (18.54 ± 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 ± 0.46 vs. 19.94 ± 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum‐free, TGF‐β₃ supplemented medium, indicating the importance of medium formulation in NP construct development. Biotechnol. Bioeng. 2010; 105: 384–395. © 2009 Wiley Periodicals, Inc.     

Novel chitosan/collagen scaffold containing transforming growth factor-beta1 DNA for periodontal tissue engineering

The current rapid progression in tissue engineering and local gene delivery system has enhanced our applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-beta1 (TGF-beta1). These scaffolds were evaluated in vitro by analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen scaffold. The scaffold containing Ad-TGF-beta1 exhibited the highest proliferation rate, and the expression of type I and type III collagen up-regulated in Ad-TGF-beta1 scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-TGF-beta1 as a good substrate candidate in periodontal tissue engineering.