Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Ligand-immobilization to stents and vascular grafts is expected to promote endothelialization by capturing flowing endothelial progenitor cells (EPCs). However, the optimized ligand density and linker structure have not been fully elucidated. Here, we report that flowing EPCs were selectively captured by the REDV peptide conjugated with a short linker. The microchannel surface was modified with the REDV peptide via Gly-Gly-Gly (G3), (Gly-Gly-Gly)3 (G9), and diethylene glycol (diEG) linkers, and the moving velocity and captured ratio were evaluated. On the unmodified microchannels, the moving velocity of the cells exhibited a unimodal distribution similar to the liquid flow. The velocity of the endothelial cells and EPCs on the peptide-immobilized surface indicated a bimodal distribution, and approximately 20 to 30% of cells moved slower than the liquid flow, suggesting that the cells were captured and rolled on the surface. When the immobilized ligand density was lower than 1 molecule/nm2, selective cell capture was observed only in REDV with G3 and diEG linkers, but not in G9 linkers. An in silico study revealed that the G9 linker tends to form a bent structure, and the REDV peptide is oriented to the substrate side. These results indicated that REDV captured the flowing EPC in a sequence-specific manner, and that the short linker was more adequate.

DOI： 10.1016/j.msec.2021.112381

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGERNATURE  

Efficient topical delivery of antifungal drugs, such as lanoconazole (LCZ), is challenging due to the limited water solubility and required prolonged duration of treatment. In this study, LCZ-loaded emulsions stabilized with cellulose nanocrystals grafted with polyphosphoesters (LCZ-loaded CP-PEs) were developed to enhance the anti-inflammatory efficacy of LCZ on skin. A high drug-loading efficiency (>80%) of LCZ in CP-PEs with a small mean droplet size of 1.0-1.5 mu m was achieved. The sustained release of LCZ and superior skin permeation of the LCZ-loaded CP-PEs, likely due to the excellent stability and rigidity of oil droplets, assured prolonged local action. In addition, the excellent anti-inflammatory efficacy of the LCZ-loaded CP-PEs was clarified using a mouse ear model of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. Treatment with the LCZ-loaded CP-PEs significantly reduced auricular thickness compared to treatments with a commercial ointment and control solution containing LCZ. These results suggest that LCZ-loaded CP-PEs are a promising alternative for the treatment of inflammatory skin diseases, such as tinea pedis.We developed lanoconazole (LCZ)-loaded emulsions stabilized with cellulose nanocrystals grafted with polyphosphoesters (LCZ-loaded CP-PEs). The sustained release of LCZ and superior skin permeation of the LCZ-loaded CP-PEs, likely due to the excellent stability and rigidity of oil droplets, assured prolonged local action. The anti-inflammatory efficacy of the LCZ-loaded CP-PEs was confirmed using a mouse ear model of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation.

DOI： 10.1038/s41428-021-00548-1

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Biodegradable polyphosphoesters (PPEs) are of increasing interest due to their promising biomedical applications. Polyphosphodiesters (PPDEs), formed by polyphosphotriester (PPTE) dealkylation, have bone-targeting properties in vivo and therefore are promising polymer drug candidates for bone disease treatment. However, their effects on osteoblasts are still unclear. This study prepared two polymer structures, poly(methyl ethylene phosphate) (PMP) and poly(ethylene sodium phosphate) (PEP•Na), as PPTE and PPDE models, respectively, and investigated their effects on mouse osteoblastic cell (MC3T3-E1) differentiation. Results showed that PMP is inert toward osteoblast differentiation. In contrast, PEP•Na enhanced alkaline phosphatase (ALP) synthesis, matrix mineralization, and osteoblast-related gene expression. PEP•Na also enhanced Wnt signaling pathway–dependent Alp expression, in addition to its own internalization into the cytosol during 3 days of differentiation culture. These results showed that dealkylated PPEs are a polymer drug candidate for osteoporosis treatment, not only because of their bone-targeting properties but also because of the controllable effects of bone anabolism via osteoblast differentiation enhancement.

DOI： 10.1016/j.mtla.2020.100977

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOP Publishing  

Label-free cell separation is a promising method in the field of stem-cell research to obtain desired cell populations. Here, we report on phospholipid polymer-coated microfluidic channels with immobilized antibodies as devices for the capture of cells expressing target antigens in a label-free manner. We fabricated a microfluidic channel containing immobilized antibodies against vascular endothelial growth factor receptor 2 (Flk1), a potential marker for cardiac, angiogenic, and hematopoietic cell regeneration. A series of investigations was carried out to elucidate the effect of the immobilized antibodies on the adhesion behavior of the Flk1-expressing cell subpopulation derived from induced pluripotent stem cells. Increasing the immobilized antibody density (0.18-5.0 x 10(9) ligands mm(-2)) led to an increased number of cells adhering to the channel. The antibody-immobilized polymer-coated surface suppressed nonspecific cell adhesion, which was swept away by a weak shear flow, and captured Flk1-expressing cells under a wall shear stress of 1.7 Pa. Flk1 expression was 2.8-fold higher in the cells that adhered than in those that did not adhere. Therefore, an optimal antibody density and sweeping flow are required for effective label-free separation of Flk1-positive cells.

DOI： 10.1088/1361-6439/abe52a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Low-environmental-impact emulsion systems for transdermal drug delivery in topical treatment have gained increasing interest. However, low stability and adverse systemic side effects severely decrease their efficiency. This study proposed a stable oil-in-water (O/W) emulsion loaded with bifonazole (BFZ) as a lipophilic drug stabilized by poly(2-isopropoxy-2-oxo-1,3,2-dioxaphospholane)-modified cellulose nanocrystals (CNC-g-PIPP) as vehicles for topical delivery of lipophilic drugs. We fully characterized stability, BFZ-loaded particle-stabilized emulsions (PEs) for morphology, droplet size, and its distribution. In addition, we evaluated the in vitro drug-releasing capacity and in vitro skin permeation of BFZ in a porcine skin animal model using a side-bi-side® diffusion cell. An O/W BFZ-loaded emulsion stabilized with CNC-g-PIPP particles (BFZ-loaded CP-PE) with a small mean droplet size of 2.54 ± 1.39 μm was developed and was stable for > = 15 days without a significant change in droplet size. The BFZ-loading efficiency in PEs was 83.1 %. BFZ was slowly released over an extended period, and the releasing ratio from BFZ-loaded CP-PE was only 17 % after 48 h. The BFZ-loaded CP-PE showed a ∼4.4-fold increase in BFZ permeation and penetration compared to a conventional surfactant-stabilized emulsion and BFZ control solution. Fluorescence-labeling studies showed that BFZ-loaded CP-PE could well penetrate skin layers from the stratum corneum (SC) to the dermis. In addition, histopathology studies of porcine skin treated with the PE formulation showed an intact SC with unaltered adjacent structures and no observed signs of inflammation. Therefore, the proposed CP-PE shows great potential as a transdermal drug carrier for enhancing lipophilic drug permeation.

DOI： 10.1016/j.colsurfb.2020.111423

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Current chemotherapy methods have limited effectiveness in eliminating bone metastasis, which leads to a poor prognosis associated with severe bone disorders. To provide regional chemotherapy for this metastatic tumor, a bone-targeting drug carrier was produced by introducing the osteotropic bisphosphonate alendronate (ALN) units into an amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). The polymer can form nanoparticles with a diameter of less than 30 nm; ALN units were exposed to the outer layer of the particle. A simple mixing procedure was used to encapsulate a hydrophobic anticancer drug, known as docetaxel (DTX), in the polymer nanoparticle, providing a uniform solution of a polymer-DTX complex in the aqueous phase. The complex showed anticancer activities against several breast cancer cell lines, and the complex formation did not hamper the pharmacological effect of DTX. The fluorescence observations evaluated by an in vivo imaging system and fluorescence microscopy showed that the addition of ALN to the polymer-DTX complex enhanced bone accumulation. Bone-targeting phospholipid polymers are potential solubilizing excipients used to formulate DTX and deliver the hydrophobic drug to bone tissues by blood administration.

DOI： 10.1002/jbm.a.36968

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jbm.a.36968

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DOI： 10.1016/j.jiec.2019.03.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

Poly(ethylene sodium phosphate) (PEP·Na) showed excellent cytocompatibility and in vivo bone affinity. Moreover, PEP·Na did not interact with thrombin, which is a coagulation-related protein. Because immobilization of therapeutic agents and imaging probes on PEP·Na is easily performed, PEP·Na is a promising polymer for bone-targeted therapies.

DOI： 10.1039/C7BM00930E

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Cell-based therapies using self-beating cardiomyocytes have been attracting great attention for use in cardiac regeneration, although an effective procedure to improve cardiac differentiation and self-beating induction is required. The purpose of this study is to clarify the effect of the culture substrate on cardiac maturation by separately evaluating the cardiac differentiation step and the beating induction step in vitro. To this end, the well-studied cardiomyocyte-like progenitor cell line P19CL6 and neonatal cardiomyocytes (NCMs) were selected and cultured on substrates coated with collagen type I (Col-I), gelatin (Gel), fibronectin (FN), or poly-L-lysine (PLL). It was found that the cardiac differentiation step, which was assessed using cardiac marker gene expression (GATA-binding protein 4 (GATA4), myocyte-specific enhancer factor 2D (MEF2D), and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)) in the P19CL6 embryonal carcinoma cells, was greatly enhanced on Col-I, Gel, and PLL. In contrast, the spontaneous beating step, which was directly assessed by counting the beating colonies and measuring contractile protein gene expression (alpha-myosin heavy chain (alpha-MHC), troponin C type 1 (TnC1), and troponin T type 2 (TnT2)) in the rat NCMs, was enhanced on the FN and PLL surfaces. In the present study, for the first time, it was found that PLL enhances both the cardiac differentiation and the beating induction steps of cardiac maturation, which can aid in preparing beating cardiomyocytes for regenerative medicine. (C) 2017 Wiley Periodicals,Inc.

DOI： 10.1002/jbm.a.35977

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

When induced pluripotent stem cells (iPSCs), are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the immobilized-antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state.

DOI： 10.1021/acs.langmuir.6b04070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Large osteochondral defects have been difficult to repair via tissue engineering treatments due to the lack of a sufficient number of source cells for repairing the defect and to the severe mechanical stresses affecting the replacement tissue. In the present study, whole-area osteochondral defects of rabbit patella were covered and wrapped with a fibroin sponge containing chondrocytes, with or without Green Fluorescent Protein (GFP) transgenic marking, on the surface facing the osteochondral defect. Five of eight osteochondral defects that were covered with the chondrocyte-seeded fibroin sponges showed hyaline cartilage-like repair containing no fibroin fragments at 6 weeks after surgery. The repaired tissue showed a layer formation, which showed intensive safranin-O and toluidine blue staining, and which showed positive type II collagen immunostaining. The average surface coverage of the repaired cartilage was 53%. On average, 48% of the cells in the repaired tissue were derived from GFP transgenic chondrocytes, which had been seeded in the fibroin sponge. The fibroin-sponge covering had the potential to allow the early repair of large osteochondral defects. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1474-1482, 2016.

DOI： 10.1002/jbm.b.33656

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The effect of porous alpha-tricalcium phosphate (alpha-TCP) with immobilized basic fibroblast growth factor (bFGF) on bone regeneration was evaluated in a canine mandibular bone defect model. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous alpha-TCP with bFGF bound via heparin (bFGF group), whereas the other was filled with unmodified porous alpha-TCP (control group). Micro-computed tomography and histological evaluation were performed two, four and eight weeks after implantation. The bone mineral density of the bFGF group was higher than that of the control group at each time point (p < 0.05), and the bone mineral content of the bFGF group was higher than that of the control group at four and eight weeks (p < 0.05). Histological evaluation two weeks after implantation revealed that the porous alpha-TCP had degraded and bone had formed on the surface of alpha-TCP particles in the bFGF group. At eight weeks, continuous cortical bone with a Haversian structure covered the top of bone defects in the bFGF group. These findings demonstrate that porous alpha-TCP with immobilized bFGF can promote bone regeneration.

DOI： 10.3390/ma9100853

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

A quantitative analytical method was proposed for measuring cell co-migration, which was defined as two or more cells migrating together. To accurately identify and quantify this behavior, cell migration on fibroin substrates was analyzed with respect to intercellular distance. Specifically, cell size was characterized by major diameter, and then, based on these measurements and cell center data, a specific threshold distance for defining co-migration was determined after analyzing cell motion using the Voronoi diagram method. The results confirmed that co-migration occurrences of rounded cells were significantly more stable on fibroin than on ProNectin substrates under the present experimental conditions. The cell co-migration analysis method in this article was shown to be successful in evaluating the stability of cell co-migration and also suggested the presence of "critical distance" where two cells interact on fibroin substrates. With further research, the cell co-migration analysis method and "critical distance" may prove to be capable of identifying the aggregation behavior of other cells on different materials, making it a valuable tool that can be used in tissue engineering design.

DOI： 10.1089/ten.tec.2013.0344

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Physical Society  

Due to the coupling between the plasma membrane and the actin cytoskeleton, membrane molecules such as receptor proteins can become immobilized by binding to cytoskeletal structures. We investigate the effect of immobile membrane molecules on the diffusion of mobile ones by modeling the membrane as a two-dimensional (2D) fluid composed of hard particles and performing event-driven molecular dynamics simulations at a particle density where the system is in an isotropic liquid state. We show that the diffusion coefficient sharply decreases with increasing immobile fraction, dropping by a factor of ∼3 as the fraction of immobile particles increases from 0 to 0.1, in a system-size dependent manner. By combining our results with earlier calculations, we estimate that a factor-of-∼20 reduction in diffusion coefficients in live cell membranes, a puzzling finding in cell biology, can be accounted for when less than ∼22% of the particles in our model system is immobilized. Furthermore, we investigate the effects of confinement induced by a correlated distribution of immobile particles by calculating the distribution of the time it takes for particles to escape from a corral. In the regime where the particles can always escape from the corral, it is found that the escape times follow an exponential distribution, and the mean escape time grows exponentially with the density of obstacles at the corral boundary, increasing by a factor of 3-5 when immobile particles cover 50% of the boundary, and is approximately proportional to the area of the corral. We believe that our findings will be useful in interpreting (1) single molecule observations of membrane molecules and (2) results of particle based simulations that explore the effect of fluid dynamics on molecular transport in a 2D fluid. © 2014 American Physical Society.

DOI： 10.1103/PhysRevE.89.022724

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

Cell migration is one of the fundamental processes in histogenesis, and it is necessary to investigate such multicellular behavior quantitatively in cell regeneration studies. In this study, Voronoi diagram analysis was first confirmed in simulation testing, and then used to evaluate the multicellular behavior of chondrocytes on three different substrates: (1) wild-type fibroin (FIB); (2) L-RGDSx2 transgenic fibroin; (3) and collagen. The indices for the round factor average, round factor homogeneity, and area disorder (AD), calculated from Voronoi diagram analysis, were used to characterize the difference in spatiotemporal changes for the different chondrocyte populations, and a regression analysis of the AD index was used to measure the speed of cell aggregation. The results suggested that the arginine-glycine-aspartic acid-serine sequence affects aggregate formation of chondrocytes cultured on FIB. The Voronoi diagram analysis represents one of the promising quantitative analyses for cell regeneration studies.

DOI： 10.1089/ten.tec.2012.0424

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Cell migration plays important roles in natural processes involving embryonic development, inflammation, wound healing, cancer metastasis and angiogenesis. Cell migration on various biomaterials is also believed to improve the rate of wound healing and implant therapies in the tissue-engineering field. This study measured the distance traversed, or mileage, of mouse fibroblasts on a silk fibroin surface. Fibroblasts on the fibroin surface moved with better progress during 24 h than cells on collagen or fibronectin surfaces. Results obtained by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) revealed that fibroblasts on the fibroin surface expressed transforming growth factor -induced protein (TGFBI), which is an extracellular matrix (ECM) protein, stronger than on other surfaces in the early cell-culture stages. These results demonstrate that the fibroin surface shows higher potential to enhance cell migration and the production of ECM than a collagen or fibronectin surface. (C) Koninklijke Brill NV, Leiden, 2012

DOI： 10.1163/156856212X629025

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

The effects of substrate material on the spatio-temporal behavior of cells is an important issue. Although cell aggregation has been observed on various fibroin substrates, the mechanisms of this aggregation have yet to be fully clarified. In this study, cell aggregation behavior on fibroin substrates were evaluated, focusing on the distance between each cell and the direction of individual cell migration. Our results showed that on fibroin substrates cells did not attract each other. However cells stayed close to adjacent cells over 24 hours of cultivation.

DOI： 10.1109/EMBC.2013.6609522

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOS PRESS  

Condensation/aggregation process of rabbit-derived chondrocytes on a fibroin-coated patterned substrate was observed to estimate initial aggregation process in fibroin sponge. Chondrocytes were seeded on array of 160 mu m diameter pits in three densities: 5 cells/pit (2.5 x 10(4) cells/cm(2), LOW), 15 cells/pit (7.5 x 10(4) cells/cm(2), MID) and 25 cells/pit (12.5 x 10(4) cells/cm(2), HIGH). In the MID and HIGH groups, cells tended to form aggregates after 24 h after cell seeding. In the LOW group, cell aggregate were not seen in a majority of the pits. Observation of aggregates using confocal laser scanning microscope showed that the chondrocytes at the interface of the fibroin surface tended to extend to the surface, developing an extensive network of stress fibers throughout the cytoplasm. On the other hand, chondrocytes in the other part of the aggregates maintained spherical shape, and most of the actin was localized in the cell cortex as opposed to in stress fibers. These results suggest two functional structures in the aggregates, which may explain the good balance between the maintenance of their differentiated phenotype and proliferation rate in the fibroin sponge.

DOI： 10.3233/BME-2010-0615

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Several reports suggest that cell aggregation is important in the course of tissue regeneration or development. The cell aggregation is thought to be affected by direct contact such as cell-material interaction and/or cell-cell interaction and by indirect effects such as substance diffusion. In this study, we estimated whether cells in distant places have an effect on each other or not, and observed cartilage cell movement on fibroin and fibronectin by using time-lapse microscopic recordings. The cellular behavior was analyzed by comparing the relation between cell density distribution and direction of cell migration to the Moore neighborhood referring to the Bonnet's method. The results suggest that the direction of cartilage cell migration seems to have no relation to cell density distribution regardless of cell aggregation behavior. Judging from our previous report showing that aggregation rate and cell-cell adhesion rate of chondrocyte are much higher on fibroin than on fibronectin, the chondrocytic aggregate formation characteristically observed on the fibroin surface may be attributed to cell-cell adhesion.

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

We reported that we could treat the defect of patella by using fibroin which is from silkworm cocoon. And we also reported that we observed the rich cartilage matrixes and chondrocyte's aggregation in fibroin. So we focused on the chondrocyte's aggregation on fibroin and tried to develop the method of analyzing it quantitatively for evaluating the effect of cell-cell interaction on aggregated chondrocyte cultured on fibroin or other matrixes. First, we tried to define cell aggregation by comparing chondrocyte's aggregation on fibroin with random walk models. And second, we cultured chondrocytes on fibroin, fibronectin and collagen type I and on we evaluated the amount of GAG production and the number of chondrocytes cultured on each matrixes. As a result, at first we found that on fibroin the number of cells which are 10-20 |0.m away from other cells is especially high. And also, they keep cell-cell adhesion for more than an hour. And second, it is suggested that the chondrocyte on fibroin is easier to aggregate than that on fibronectin or collagen type I. What is more, the chondrocyte on fibroin has a higher ability to produce GAG than that on other matrixes. From these results it is suggested that the aggregation movement on fibroin is caused by cell-cell adhesion but also such cell-cell interaction is affected by cell-matrix interaction. In addition it is also suggested that the aggregation movement is related to the ability of chondrogenesis.

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