Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro.

Rapid oriented fibril formation of fish scale collagen facilitates early osteoblastic differentiation of human mesenchymal stem cells.

We studied the effect of fibril formation of fish scale collagen on the osteoblastic differentiation of human mesenchymal stem cells (hMSCs). We found that hMSCs adhered easily to tilapia scale collagen, which remarkably accelerated the early stage of osteoblastic differentiation in hMSCs during in vitro cell culture. Osteoblastic markers such as ALP activity, osteopontin, and bone morphogenetic protein 2 were markedly upregulated when the hMSCs were cultured on a tilapia collagen surface, especially in the early osteoblastic differentiation stage. We hypothesized that this phenomenon occurs due to specific fibril formation of tilapia collagen. Thus, we examined the time course of collagen fibril formation using high-speed atomic force microscopy. Moreover, to elucidate the effect of the orientation of fibril formation on the differentiation of hMSCs, we measured ALP activity of hMSCs cultured on two types of tilapia scale collagen membranes with different degrees of fibril formation. The ALP activity in hMSCs cultured on a fibrous collagen membrane was significantly higher than on a non-fibrous collagen membrane even before adding osteoblastic differentiation medium. These results showed that the degree of the fibril formation of tilapia collagen was essential for the osteoblastic differentiation of hMSCs.

Synthesis of luminescent nanoporous silica spheres functionalized with folic acid for targeting to cancer cells.

Luminescent europium(III)-doped nanoporous silica nanospheres (Eu:NPS) were successfully synthesized, and a folate N-hydroxysuccinimidyl ester (FA-NHS) molecule as a targeting ligand for cancer cells was immobilized on the nanosphere surfaces through mediation of the 3-aminopropyltriethoxysilane (APTES) adlayer. The ordered nanopores were preserved by the immobilization, and the specific surface area decreased only with the APTES immobilization, suggesting that the FA-NHS was predominantly immobilized on the outer surface of the nanopores. The photoluminescence of the nanospheres functionalized with folic acid (FA) exhibited a characteristic peak due to the interactions (e.g., energy transfer) between FA and Eu(3+), and further the orange luminescence could be clearly detected by fluorescence microscopy in air and water. Furthermore, the nanospheres highly dispersed in cell culture medium exhibited nontoxicity in the cellular proliferation stages of the Hela cancer cells and NIH3T3 fibroblasts and specifically bind to the Hela cells. The nanospheres after the binding and uptake also showed intense luminescence from the outer/inner cell surfaces for the culture time of 4 days. Therefore, the luminescent FA-functionalized Eu:NPS nanospheres could be used for specific targeting and imaging abilities for cancer cells.

Evaluation of calcium alginate gel as electrode material for alternating current iontophoresis of lidocaine using excised rat skin.

Iontophoresis (IOP) is a noninvasive method of delivering medication transcutaneously through the skin. The electrodes used in this method should tightly fit to rough and irregular surfaces and be biologically safe, easy to handle and prepare, and cost-effective. To satisfy these requirements, calcium alginate gel can be a candidate electrode for IOP. Using calcium alginate gel electrodes, we examined whether lidocaine can be effectively transported across an excised rat skin by squarewave alternating current (AC) application. A squarewave AC with either a 70% or 80% duty cycle was continuously applied to 0.5% calcium alginate gel electrodes containing 10% lidocaine at 10 V and 1 kHz for 60 min. Lidocaine concentration was measured using a spectrophotometer and the temperature of the gel was determined. The lidocaine concentrations for AC-IOP at the 70% and 80% duty cycles were significantly higher than that without AC-IOP. Furthermore, the group with the 80% duty cycle showed higher lidocaine concentrations than the group with the 70% duty cycle. The temperatures of all the groups were lower than 28 °C throughout the procedure. In conclusion, the calcium alginate gel can be used as a possible matrix for IOP electrodes.

Photoluminescence and doping mechanism of theranostic Eu3+/Fe3+ dual-doped hydroxyapatite nanoparticles.

Theranostic nanoparticles currently have been regarded as an emerging concept of 'personalized medicine' with diagnostic and therapeutic dual-functions. Eu3+ doped hydroxyapatite (HAp) has been regarded as a promising fluorescent probe for in vivo imaging applications. Additionally, substitution of Ca2+ with Fe3+ in HAp crystal may endow the capability of producing heat upon exposure to a magnetic field. Here we report a preliminary study of doping mechanism and photoluminescence of Eu3+ and Fe3+ doped HAp nanoparticles (Eu/Fe:HAp). HAp with varied concentration of Eu3+ and Fe3+ doping are presented as Eu(10 mol%):HAp, Eu(7 mol%)-Fe(3 mol%):HAp, Eu(5 mol%)-Fe(5 mol%):HAp, Eu(3 mol%)-Fe(7 mol%):HAp, and Fe(10 mol%):HAp in the study. The results showed that the HAp particles, in nano-size with rod-like morphology, were successfully doped with Eu3+ and Fe3+, and the particles can be well suspended in cell culture medium. Photoluminescence analysis revealed that particles have prominent emissions at 536 nm, 590 nm, 615 nm, 650 nm and 695 nm upon excitation at a wavelength of 397 nm. Moreover, these Eu/Fe:HAp nanoparticles belonged to B-type carbonated HAp, which has been considered an effective biodegradable and biocompatible drug/gene carrier in biological applications.

ERK-dependent downregulation of Skp2 reduces Myc activity with HGF, leading to inhibition of cell proliferation through a decrease in Id1 expression.

UNLABELLED: Hepatocyte growth factor (HGF) has an inhibitory effect on human HepG2 hepatoma cell proliferation. Previously, it was shown that HGF treatment downregulated Id1 and upregulated p16(INK4a) in an ERK-dependent manner, leading to the inhibition of cellular proliferation. Here, new insight suggests that Skp2, an SCF complex component and potential prognosticator in cancer, is downregulated by injection of HGF into established HepG2 xenograft tumors. The downregulation was evident at both the mRNA and protein level and in an ERK-dependent manner. Critically, high expression of Skp2 restored HGF-inhibited cell proliferation, indicating that the inhibitory effect of HGF required the downregulation of Skp2. However, downregulation was not involved in the HGF-induced upregulation of a CDK inhibitor, p27(Kip1), a known SCF-Skp2 target. Instead, data revealed that Skp2 regulated Myc activity, which has oncogenic potential in the generation of hepatocellular carcinoma. Elevated expression of Skp2 or a mutant that is unable to associate with the SCF complex was capable of activating Myc, suggesting that Skp2 does not act on Myc as a component of the SCF complex, and thus functions as an activator of Myc independent of its role in ubiquitination. Furthermore, Skp2 regulated Id1 expression by regulating Myc activity, and the regulation of Skp2 is involved in the activity of p16 promoter through regulation of Id1 expression. Overall, these mechanistic findings provide the first evidence that ERK-dependent downregulation of Skp2 reduced Myc activity, leading to HGF-induced inhibition of cell proliferation through decreased Id1 expression. IMPLICATIONS: This study elucidates the molecular details of HGF-induced inhibition of cellular proliferation in liver cancer cells.

The generation of pancreatic ß-cell spheroids in a simulated microgravity culture system.

Islet transplantation can induce a substantial improvement in the treatment of type 1 diabetes mellitus. However, the clinical application of islet transplantation is severely limited by the shortage of donor organs. It is thus essential to improve the engraftment rate to achieve the expected outcome in the treatment of diabetes mellitus using a limited amount of donor islets. In this manuscript, we describe the generation of ß-cell spheroids using mouse insulinoma cells (MIN6) as a model of ß-cells. We established a 3D culture system that simulates microgravity using a 3D clinostat. Using this method, we were able to produce 100 spheroids per mL of culture media. The optimization of the culture conditions in the clinostat produced spheroids with a size of approximately 250 µm, which is a size that is known to induce good graft survival after islet transplantation. The spheroids produced in the clinostat expressed several ß-cell signature genes at higher levels than the levels that were found in MIN6 cells that were cultured in a standard 2D culture dish (MIN6-2D). The transplantation of the spheroids into the portal vein of streptozotocin-induced diabetic mice ameliorates hyperglycemia, whereas the transplantation of the equivalent number of 2D-cultured cells failed to cure diabetes. These results indicate that the clinostat culture provides a new method for the reconstitution of a large number of functional ß-cell spheroids for diabetes treatment.

Construction and characterization of a tissue-engineered oral mucosa equivalent based on a chitosan-fish scale collagen composite.

This study was designed to (1) assess the in vitro biocompatibility of a chitosan-collagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm® (EVPOME-A), BioMend® Extend™ (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the ß1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine.

In vitro evaluation of calcium alginate gels as matrix for iontophoresis electrodes.

Calcium alginate gel has some unique properties, such as the capability to keep the drugs, bioadhesiveness, safety, and low cost. The purpose of this study is to determine whether calcium alginate gel can be used as a matrix of electrodes for iontophoresis (IOP). We measured the concentration of lidocaine transported from calcium alginate gels with various concentrations of alginic acid using an in vitro experimental cell with square-wave alternating current (AC) application. Temperature and pH changes were also determined during AC-IOP. The results revealed that lidocaine was released from calcium alginate gels at concentrations nearly 1.71-fold larger at 5 V, 60 min after AC application than in the case of passive diffusion. Lidocaine transport depended on the alginic acid concentration in the gels. Although there were slight increases in temperature and pH, chemical and thermal burns were not severe enough to be a concern. In conclusion, the calcium alginate gel can be used as a possible matrix for IOP electrodes.

Synthesis and luminescence properties of Eu(III)-doped nanoporous silica spheres.

Europium (III) (Eu(3+))-doped nanoporous silica spheres were synthesized, and the states of Eu(3+) ions in the silica framework structure were investigated. The ordered nanopores were preserved with the doping at the Eu(3+) molar concentration to Si up to 10 mol%, and the O-Si-O and Si-OH groups in the structures were clearly rearranged with the doping, indicating the interaction of Eu(3+) with the O atoms. The significant morphological changes in the spheres were observed with the doping. The photoluminescence spectral shapes due to the transitions of (5)D(0)-(7)F(1) and (5)D(0)-(7)F(2) were indicative of the presence of the Eu(3+) in an environment of a low symmetry. It was found that the Eu(3+) was located inside the silica framework to electrostatically interact with the environmental O atoms, which would prevent the aggregation among Eu(3+) ions to show the efficient luminescence. Therefore, the interactions between the Eu(3+) ions and silica framework structures in the spheres were successfully clarified.

Surface plasmon resonance biosensor with high anti-fouling ability for the detection of cardiac marker troponin T.

Designing a surface recognition layer with high anti-fouling ability, high affinity, and high specificity is an important issue to produce high sensitivity biosensing transducers. In this study, a self-assembled monolayer (SAM) consisting of a homogeneous mixture of oligo(ethylene glycol) (OEG)-terminated alkanethiolate and mercaptohexadecanoic acid (MHDA) on Au was employed for immobilizing troponin T antibody and applied in detecting cardiac troponin T by using surface plasmon resonance (SPR). The mixed SAM showed no phase segregation and exhibited human serum albumin resistance, particularly with an antibody-immobilized surface. X-ray photoemission spectra revealed that the chemical composition ratio of OEG to the mixed SAM was 69% and the OEG packing density was 82%. The specific binding of troponin T on the designed surface indicated a good linear correlation (R=0.991, P<0.0009) at concentrations lower than 50 µgmL(-1) with the limit of detection of 100 ngmL(-1) using a SPR measuring instrument. It is concluded that the mixed SAM functions as designed since it has high detection capability, high accuracy and reproducibility, as well as shows strong potential to be applied in rapid clinical diagnosis for label-free detection within 2 min.

Immobilization of folic acid on Eu3+-doped nanoporous silica spheres.

Folic acid (FA) was immobilized on Eu(3+)-doped nanoporous silica spheres (Eu:NPSs) through mediation of the 3-aminopropyltriethoxysilane adlayer. The ordered nanopores of Eu:NPS were preserved by the immobilization. The FA-immobilized Eu:NPSs showed the characteristic photoluminescence peak due to interactions between the FA molecules and Eu(3+) ions, and highly dispersed stability in phosphate buffered saline.

Effect of interfacial proteins on osteoblast-like cell adhesion to hydroxyapatite nanocrystals.

A quartz crystal microbalance with dissipation (QCM-D) technique was employed to detecting the protein adsorption and subsequent osteoblast-like cell adhesion to hydroxyapatite (HAp) nanocrystals. The interfacial phenomena with the preadsorption of three proteins (albumin (BSA), fibronectin (Fn), and collagen (Col)), the subsequent adsorption of fetal bovine serum (FBS), and the adhesion of the cells were investigated. The QCM-D measured the frequency shift (Δf) and dissipation energy shift (ΔD), and the viscoelastic properties of the adlayers were evaluated using ΔD-Δf plot and Voigt-based viscoelastic model. The Col adsorption significantly showed higher Δf, ΔD, elasticity, and viscosity values as compared to the BSA and Fn adsorption, and the subsequent FBS adsorption depended on the preadsorbed proteins. The ΔD-Δf plot of the cell adhesion also showed a different behavior depending on the surfaces, and the Fn- and Col-modified surfaces showed the rapid mass and ΔD changes by forming the viscous interfacial layers with cell adhesion, indicating that the processes were affected by the cellular reaction through the extracellular matrix (ECM) proteins. The confocal laser scanning microscope images of adherent cells showed a different morphology and pseudopod on the surfaces. The cells adhered to the surfaces modified with the Fn and Col had significantly uniaxially expanded shapes and fibrous pseudopods, and those modified with the BSA had a round shape. Therefore, the different cell-protein interactions would cause the arrangement of the ECM and the cytoskeleton changes at the interfaces, and these phenomena were successfully detected by the QCM-D and Voigt-based model.

Detection of interfacial phenomena with osteoblast-like cell adhesion on hydroxyapatite and oxidized polystyrene by the quartz crystal microbalance with dissipation.

The adhesion process of osteoblast-like cells on hydroxyapatite (HAp) and oxidized polystyrene (PSox) was investigated using a quartz crystal microbalance with dissipation (QCM-D), confocal laser scanning microscope (CLSM), and atomic force microscope (AFM) techniques in order to clarify the interfacial phenomena between the surfaces and cells. The interfacial viscoelastic properties (shear viscosity (η(ad)), elastic shear modulus (µ(ad)), and tan δ) of the preadsorbed protein layer and the interface layer between the surfaces and cells were estimated using a Voigt-based viscoelastic model from the measured frequency (Δf) and dissipation shift (ΔD) curves. In the ΔD-Δf plots, the cell adhesion process on HAp was classified as (1) a mass increase only, (2) increases in both mass and ΔD, and (3) slight decreases in mass and ΔD. On PSox, only ΔD increases were observed, indicating that the adhesion behavior depended on the surface properties. The interfacial µ(ad) value between the material surfaces and cells increased with the number of adherent cells, whereas η(ad) and tanδ decreased slightly, irrespective of the surface. Thus, the interfacial layer changed the elasticity to viscosity with an increase in the number. The tan δ values on HAp were higher than those on PSox and exceeded 1.0. Furthermore, the pseudopod-like structures of the cells on HAp had periodic stripe patterns stained with a type I collagen antibody, whereas those on PSox had cell-membrane-like structures unstained with type I collagen. These results indicate that the interfacial layers on PSox and HAp exhibit elasticity and viscosity, respectively, indicating that the rearrangements of the extracellular matrix and cytoskeleton changes cause different cell-surface interactions. Therefore, the different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully monitored in situ and evaluated by the QCM-D technique combined with other techniques.

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor.

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

Effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of hydroxyapatite-collagen composites as artificial bone materials.

The aim of this study was to evaluate the effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of porous hydroxyapatite (HAp)-collagen composites as artificial bone materials. Seven types of porous HAp-collagen composites were prepared from HAp nanocrystals and dense collagen fibrils. Their densities and HAp/collagen weight ratios ranged from 122 to 331 mg cm⁻³ and from 20/80 to 80/20, respectively. The flexural modulus and strength increased with an increase in density, reaching 2.46 ± 0.48 and 0.651 ± 0.103 MPa, respectively. The porous composites with a higher collagen-matrix density exhibited much higher mechanical properties at the same densities, suggesting that increasing the collagen-matrix density is an effective way of improving the mechanical properties. It was also suggested that other structural factors in addition to collagen-matrix density are required to achieve bone-like mechanical properties. The in vivo absorbability of the composites was investigated in bone defects of rabbit femurs, demonstrating that the absorption rate decreased with increases in the composite density. An exhaustive increase in density is probably limited by decreases in absorbability as artificial bones.

Competitive adsorption of fibronectin and albumin on hydroxyapatite nanocrystals.

Competitive adsorption of two-component solutions containing fibronectin (Fn) and albumin (Ab) on hydroxyapatite (HAp) nanocrystals was analyzed in situ using the quartz crystal microbalance with dissipation (QCM-D) technique. Adsorption of the one-component protein (Fn or Ab) and the two-component proteins adjusted to different molar ratios of Fn to Ab at a fixed Fn concentration was investigated. The frequency shift (Δf; Hz) and the dissipation energy shift (ΔD) were measured with the QCM-D technique, and the viscoelastic changes of adlayers were evaluated by the saturated ΔD/Δf value and the Voigt-based viscoelastic model. For the adsorption of the one-component protein, the Fn adlayer showed a larger mass and higher viscoelasticity than the Ab adlayer, indicating the higher affinity of Fn on HAp. For the adsorption of the two-component proteins, the viscoelastic properties of the adlayers became elastic with increase in Ab concentration, whereas the adsorption mass was similar to that of Fn in the one-component solution regardless of the Ab concentration. The specific binding mass of the Ab antibody to the adlayers increased with increase in Ab concentration, whereas that of the Fn antibody decreased. Therefore, Fn preferentially adsorbs on HAp and Ab subsequently interacts with the adlayers, indicating that the interfacial viscoelasticity of the adlayers was dominated by the interaction between Fn and Ab.

Reusable hydroxyapatite nanocrystal sensors for protein adsorption.

The repeatability of the adsorption and removal of fibrinogen and fetal bovine serum on hydroxyapatite (HAp) nanocrystal sensors was investigated by Fourier transform infrared (FTIR) spectroscopy and quartz crystal microbalance with dissipation (QCM-D) monitoring technique. The HAp nanocrystals were coated on a gold-coated quartz sensor by electrophoretic deposition. Proteins adsorbed on the HAp sensors were removed by (i) ammonia/hydrogen peroxide mixture (APM), (ii) ultraviolet light (UV), (iii) UV/APM, (iv) APM/UV and (v) sodium dodecyl sulfate (SDS) treatments. FTIR spectra of the reused surfaces revealed that the APM and SDS treatments left peptide fragments or the proteins adsorbed on the surfaces, whereas the other methods successfully removed the proteins. The QCM-D measurements indicated that in the removal treatments, fibrinogen was slowly adsorbed in the first cycle because of the change in surface wettability revealed by contact angle measurements. The SDS treatment was not effective in removing proteins. The APM or UV treatment decreased the frequency shifts for the reused HAp sensors. The UV/APM treatment did not induce the frequency shifts but decreased the dissipation shifts. Therefore, we conclude that the APM/UV treatment is the most useful method for reproducing protein adsorption behavior on HAp sensors.

Amelioration of anemia in the ICGN mouse, a renal anemia model, with a subcutaneous bolus injection of erythropoietin adsorbed to hydroxyapatite matrix.

The recombinant human erythropoietin (rhEPO) is used for the treatment of patients with renal anemia. However, rhEPO should be administered subcutaneously or intravenously three times a week. The repetitive injections of rhEPO result in burdens to patients. To resolve this problem, we investigated the sustaining release methods using an rhEPO-hydroxyapatite (HAp) made by spray-drying technique as the drug delivery system. Two types of rhEPO-HAp formulations were prepared; zinc (Zn) formulation and Zn and poly-L-lactic acid (PLA) formulation. These formulations were examined in genetically anemic model, ICGN (ICR-derived glomerulonephritis) mice. According to in vivo release test of rhEPO from HAp in ICGN mice, elevated plasma concentration of rhEPO could be maintained for more than 7 days. These mice showed the amelioration of anemia for more than 3 weeks post-administration without causing any side effect. In conclusion, Zn or Zn/PLA formulation of HAp was considered to be one of the useful carriers of rhEPO for long-term improvement of anemia.

A collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite as a scaffold for cartilage tissue engineering.

Because cartilage has limited potential for self-repair, tissue engineering is expected to replace the present therapies for damaged cartilage, such as total knee arthroplasty. However, scaffolds suitable for cartilage tissue engineering have not been established. We synthesized a novel porous scaffold, a collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite (pCol-HAp/ChS), containing materials which resemble extracellular matrices in bone and cartilage tissues. In this report, the physical, mechanical and biological properties of the scaffold are compared with those of a collagen sponge (pCol) and pCol incorporating a hydroxyapatite composite (pCol-HAp). HAp/ChS had smaller crystals and a larger total surface area than HAp. SEM images of the three materials showed pCol-HAp/ChS to have the roughest surface. The mechanical properties suggest that pCol-HAp/ChS and pCol/HAp are similar, and superior to pCol. Seeding experiments showed a uniform distribution of mesenchymal stem cells (MSCs) in pCol-HAp/ChS and pCol/HAp. Histochemical staining after 2 weeks of culture revealed pCol-HAp/ChS to be the most chondrogenic. From these results, pCol-HAp/ChS is expected to be a candidate for a scaffold for cartilage tissue engineering in place of collagen sponge.