Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action

Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1.     

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.     

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7

Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.     

Localization of transforming growth factor beta receptor II interacting protein-1 in bone and teeth: implications in matrix mineralization

Transforming growth factor beta receptor II (TGFβR-II) interacting protein 1 (TRIP-1) is a WD-40 protein that binds to the cytoplasmic domain of the TGF-β type II receptor in a kinase-dependent manner. To investigate the role of TRIP-1 in mineralized tissues, we examined its pattern of expression in cartilage, bone, and teeth and analyzed the relationship between TRIP-1 overexpression and mineralized matrix formation. Results demonstrate that TRIP-1 was predominantly expressed by osteoblasts, odontoblasts, and chondrocytes in these tissues. Interestingly, TRIP-1 was also localized in the extracellular matrix of bone and at the mineralization front in dentin, suggesting that TRIP-1 is secreted by nonclassical secretory mechanisms, as it is devoid of a signal peptide. In vitro nucleation studies demonstrate a role for TRIP-1 in nucleating calcium phosphate polymorphs. Overexpression of TRIP-1 favored osteoblast differentiation of undifferentiated mesenchymal cells with an increase in mineralized matrix formation. These data indicate an unexpected role for TRIP-1 during development of bone, teeth, and cartilage.     

Protein engineering of a fibroblast growth factor-1 fusion protein with cell adhesive activity

Fibroblast growth factor-1 （FGF1） is one of the most potent angiogenic growth factors, and also plays an important role in regulating cellular functions including cell proliferation, motility, differentiation, survival, and tissue regeneration processes. Here we described a novel fusion protein that was designed by combining the cell adhesion sequence from fibronectin with FGF1. The F1-Fn fusion protein functions as a minimized protein that directs integrin-dependent cell adhesion and stimulates cellular responses including cell proliferation and differentiation. Moreover, our results indicate that Fn-mediated signaling synergizes with signals from FGF1 in promoting cellular adhesion, proliferation, and differentiation in MG63 cells.     

Engineering and expression of a recombinant fusion protein possessing fibroblast growth factor-2 and fibronectin fragment

There is a synergistic effect between fibronectin and fibroblast growth factor 2 (FGF-2) on osteoblast cell adhesion through an extracellular, signal-regulated kinase pathway. Here we describe the engineering of a fusion protein containing fibronectin fragment (FNIII9-10) connected to the COOH-terminus of FGF-2. Purified FGF2-FNIII9-10 fusion protein exhibited a significant increase of cell adhesion and proliferation of MG63 cells compared with FNIII9-10 alone (p < 0.05).     

Stimulation of normal human fibroblast collagen production and processing by transforming growth factor-beta

Transforming growth factor-beta (TGF beta) a growth factor with diverse effects on cellular growth and metabolism, caused dramatic stimulation of total protein and collagen synthesis by confluent normal human dermal fibroblasts in culture in a dose-dependent manner. Gel electrophoresis of the newly synthesized macromolecules from the culture media of TGF beta treated cultures demonstrated accelerated procollagen processing. These results indicate that TGF beta is capable of qualitatively and quantitatively influencing the biosynthesis of matrix molecules by fibroblasts, and raise the possibility that TGF may play a role in the development of normal and pathologic fibrogenesis.     

Fibronectin-mediated adhesion rescues cell cycle arrest induced by fibroblast growth factor-1 by decreased expression of P21cip/waf in human chondrocytes

Summary In chondrocytes, fibroblast growth factors (FGFs) inhibit chondrocytes proliferation by upregulation of the cell cycle inhibitor p21^cip/waf. In this report, we first investigated the roles of fibronectin (FN)-mediated cell adhesion in the modulation of FGF-1's antiproliferative function in chondrocytes. In this study, we found that FN-mediated signaling could rescue cell cycle arrest induced by FGF-1 in primary human chondrocytes. This prevention of cell cycle arrest induced by FGF-1 was due to the suppression of the cell cycle inhibitor p21^cip/waf expression on adhesion to FN and its downstream activation of signaling pathways. Finally, we showed that this rescue induced by FN-mediated adhesion is dependent on the extracellular regulated kinase (ERK) signaling pathway. Taken together, these studies support that, despite FGF-FGF receptor's growth-inhibitory function, the FN-mediated signaling can collaborate to compensate for its negative effect on chondrocytes proliferation, providing evidence for cross talk between signals emerging from these cell surface molecules in chondrocyte.     

Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel: a preliminary study

Extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold. Five multi‐step methods (methods A–E) were used to generate acellular ECM from porcine skeletal muscle [rinsing in SDS, trypsin, ethylenediaminetetraacetic acid (EDTA), Triton X‐100 and/or sodium deoxycholate at 4–37°C]. The resulting ECM was evaluated using haematoxylin and eosin, 4‐6‐diamidino‐2‐phenylindole (DAPI) staining, and DNA quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed in vivo and in vitro. ECM components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while DAPI staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% EDTA, 0.5% Triton X‐100, and 1% Triton X‐100/0.2% sodium deoxycholate was nuclei‐free and produced a viscous solution that formed a structurally stable white jelly‐like hydrogel. The residual DNA content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (P < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.     

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.     

The effect of continuous culture on the growth and structure of tissue‐engineered cartilage

The use of bioreactors for cartilage tissue engineering has become increasingly important as traditional batch‐fed culture is not optimal for in vitro tissue growth. Most tissue engineering bioreactors rely on convection as the primary means to provide mass transfer; however, convective transport can also impart potentially unwanted and/or uncontrollable mechanical stimuli to the cells resident in the construct. The reliance on diffusive transport may not necessarily be ineffectual as previous studies have observed improved cartilaginous tissue growth when the constructs were cultured in elevated volumes of media. In this study, to approximate an infinite reservoir of media, we investigated the effect of continuous culture on cartilaginous tissue growth in vitro. Isolated bovine articular chondrocytes were seeded in high density, 3D culture on Millicell? filters. After two weeks of preculture, the constructs were cultivated with or without continuous media flow (5–10 μL/min) for a period of one week. Tissue engineered cartilage constructs grown under continuous media flow significantly accumulated more collagen and proteoglycans (increased by 50–70%). These changes were similar in magnitude to the reported effect of through‐thickness perfusion without the need for large volumetric flow rates (5–10μL/min as opposed to 240–800 μL/min). Additionally, tissues grown in the reactor displayed some evidence of the stratified morphology of native cartilage as well as containing stores of intracellular glycogen. Future studies will investigate the effect of long‐term continuous culture in terms of extracellular matrix accumulation and subsequent changes in mechanical function. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin-like growth factor-1 and transforming growth factor-beta, and analysis of fibroblast growth factors in rat kidney.

Growth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose-dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot replace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin-like growth factor 1 (IGF-1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2-1.0 microgram/ml) of insulin required for cell growth can be replaced by a low concentration of IGF-1 (10-20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF-1 receptor. Transforming growth factor-beta 1 (TGF-beta 1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration-dependent manner. Northern blot analysis shows that mRNA for TGF-beta 1, IGF-1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF-like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF-beta, FGFs may also be important kidney-derived regulators of proximal tubule epithelial cell growth in vivo and in vitro.     

Enhanced matrix synthesis in de novo, scaffold free cartilage-like tissue subjected to compression and shear

Production of a de novo cartilage-like tissue construct is a goal for the repair of traumatic chondral defects. We aimed to enhance the matrix synthesis within a scaffold free, de novo cartilage-like tissue construct by way of mechanical load. A novel loading machine that enables the application of shear, as well as compression, was used to subject tissue engineered cartilage-like tissue to mechanical stress. The machine, which applies the load through a roller mechanism, can load up to 20 constructs with four different loading patterns simultaneously. The expression of mRNA encoding matrix products, and subsequent changes in matrix protein content, were analyzed after various loading regimes. The force applied to the immature tissue had a direct bearing on the short-term (first 4 h) response. A load of 0.5 N caused an increase in collagen II and aggrecan mRNA within an hour, with a peak at 2 h. This increased mRNA expression was translated into an increase of up to 60% in the glycosaminoglycan content of the optimally loaded constructs after 4 days of intermittent cyclical loading. Introducing pauses between load cycles reproducibly lead to an increase in GAG/DNA. In contrast, constant cyclical load, with no pause, lead to a decrease in the final glycosaminoglycan content compared with unloaded controls. Our data suggest that a protocol of mechanical stimulation, simulating in vivo conditions and involving shear and compression, may be a useful mechanism to enhance the properties of tissue engineered tissue prior to implantation.     

Effects of extracellular matrices derived from different cell sources on chondrocyte functions

Cell-derived extracellular matrices (ECMs) are a key factor in regulating cell functions in tissue engineering and regenerative medicine. The fact that cells are surrounded by their specific ECM in vivo elicits the need to elucidate the effects of ECM derived from different cell sources on cell functions. Here, three types of ECM were prepared by decellularizing cultured chondrocytes, fibroblasts, and mesenchymal stem cells (MSC) and used for chondrocyte culture to compare their effects on chondrocyte adhesion, proliferation, and differentiation. Chondrocyte adhesion to the chondrocyte-derived ECM was greater than those to the fibroblast- and MSC-derived ECM. Chondrocyte proliferation on the chondrocyte-derived ECM was lower than those on the fibroblast- and MSC-derived ECM. The ECM showed no evident effect on chondrocyte differentiation. The effects of ECM on cell functions depended on the cell source used to prepare the ECM.