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.     

Effects of boron derivatives on extracellular matrix formation

Boric acid solution (3%) dramatically improves wound healing through action on the extracellular matrix, a finding that has been obtained in vitro. Consequently, investigations are presently underway to produce boronated compounds having a therapeutical effectiveness similar to that of boric acid. On the basis of experimental results obtained with boric acid, we examined the effects of boron derivatives on extracellular matrix formation and degradation and analyzed their potential toxicity by using two biological models (chick embryo cartilage and human fibroblasts). The four boron derivatives tested in this study (triethanolamine borate; N-diethyl-phosphoramidate-propylboronique acid; 2,2 dimethylhexyl-1,3-propanediol-aminopropylboronate and 1,2 propanediol-aminopropylboronate) mimicked the effects of boric acid. They induced a decrease of intracellular concentrations in extracellular matrix macromolecules (proteoglycans, proteins)-associated with an increase of their release in culture medium and stimulated the activity of intra- and extracellular proteases. Similarly to boric acid, these actions occurred after exposure of the cells to concentrations of all boron derivatives without apparent toxic effects. The compounds were found to be more toxic than boric acid itself when concentrations were calculated according to their molecular weight. Nevertheless, these in vitro preliminary results demonstrate effects of boron derivatives that may be of therapeutic benefit in wound repair.     

Preservation of the chondrocyte's pericellular matrix improves cell‐induced cartilage formation

The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real‐time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross‐linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50‐fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell‐induced cartilage production. J. Cell. Biochem. 110: 260–271, 2010. © 2010 Wiley‐Liss, Inc.     

The presence of extracellular matrix alters the chondrocyte response to endoplasmic reticulum stress

The objective of this study was to test the hypothesis that extracellular matrix (ECM) would alter the endoplasmic reticulum (ER) stress response of chondrocytes. Chondrocytes were isolated from calf knees and maintained in monolayer culture or suspended in collagen I to form spot cultures (SCs). Our laboratory has shown that bovine chondrocytes form cartilage with properties similar to native cartilage after 2-4 weeks in SCs. Monolayer cultures treated with ER stressors glucose withdrawal (-Glu), tunicamycin (TN), or thapsigargin (TG) up-regulated Grp78 and Gadd153, demonstrating a complete ER stress response. SCs were grown at specific times from 1 day to 6 weeks before treatment with ER stressors. Additionally, SCs grown for 1, 2, or 6 weeks were treated with increasing concentrations of TN or TG. Western blotting of SCs for Grp78 indicated that increased ECM accumulation results in delayed expression; however, Grp78 mRNA is up-regulated in response to ER stressors even after 6 weeks in culture. SCs treated with ER stressors did not up-regulate Gadd153, suggesting that the cells experienced ER stress but would not undergo apoptosis. In fact, SCs undergo apoptosis upon ER stress treatment after 0-1 day of growth; however, after 4 days and to 6 weeks, apoptosis in treated samples was not different than controls. Pro-survival molecules Bcl-2 and Bag-1 were up-regulated upon ER stress in SCs. These results suggest that presence of ECM confers protection from ER stressors. Future studies involving chondrocyte physiology should focus on responses in conditions more closely mimicking the in vivo cartilage environment.     

Articular cartilage and changes in Arthritis: Noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage

Cartilage contains numerous noncollagenous proteins in its extracellular matrix, including proteoglycans. At least 40 such molecules have been identified, differing greatly in structure, distribution, and function. Some are present in only selected cartilages or cartilage zones, some vary in their presence with a person's development and age, and others are more universal in their expression. Some may not even be made by the chondrocytes, but may arise by absorption from the synovial fluid. In many cases, the molecules' function is unclear, but the importance of others is illustrated by their involvement in genetic disorders. This review provides a selective survey of these molecules and discusses their structure, function, and involvement in inherited and arthritic disorders.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

Comparative proteomic profiling of human osteoblast‐derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.     

Real‐time measurements of human chondrocyte heat production during in vitro proliferation

Isothermal microcalorimeters (IMC) are highly sensitive instruments that allow the measurement of heat flow in the microwatt range. Due to their detection of minute thermal heat, IMC techniques have been used in numerous biological applications, including the study of fermentation processes, pharmaceutical development, and cell metabolism. In this work, with the ultimate goal of establishing a rapid and real‐time method to predict the proliferative capacity of human articular chondrocytes (HAC), we explored to use of IMC to characterize one of the crucial steps within the process of cartilage tissue engineering, namely the in vitro expansion of HAC. We first established an IMC‐based model for the real‐time monitoring of heat flow generated by HAC during proliferation. Profiles of the heat and heat flow curves obtained were consistent with those previously shown for other cell types. The average heat flow per HAC was determined to be 22.0 ± 5.3 pW. We next demonstrated that HAC proliferation within the IMC‐based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. HAC growth and HAC heat over time appeared correlated for cells derived from particular donors. However, based on the results from 12 independent donors, no predictive correlation could be established between the growth rate and the heat increase rate of HAC. This was likely due to variability in the biological function of HAC derived from different donors, combined with the complexity of tightly couple metabolic processes beyond proliferation. In conclusion, IMC appears to be a promising technique to characterize cell proliferation. However, studies with more reproducible cell sources (e.g., cell lines) could be used before adding the complexity associated with primary human cells. Biotechnol. Bioeng. 2011;108: 3019–3024. © 2011 Wiley Periodicals, Inc.     

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.     

A computational model of in vitro angiogenesis based on extracellular matrix fibre orientation

Recent interest in the process of vascularisation within the biomedical community has motivated numerous new research efforts focusing on the process of angiogenesis. Although the role of chemical factors during angiogenesis has been well documented, the role of mechanical factors, such as the interaction between angiogenic vessels and the extracellular matrix, remains poorly understood. In vitro methods for studying angiogenesis exist; however, measurements available using such techniques often suffer from limited spatial and temporal resolutions. For this reason, computational models have been extensively employed to investigate various aspects of angiogenesis. This paper outlines the formulation and validation of a simple and robust computational model developed to accurately simulate angiogenesis based on length, branching and orientation morphometrics collected from vascularised tissue constructs. Microvessels were represented as a series of connected line segments. The morphology of the vessels was determined by a linear combination of the collagen fibre orientation, the vessel density gradient and a random walk component. Excellent agreement was observed between computational and experimental morphometric data over time. Computational predictions of microvessel orientation within an anisotropic matrix correlated well with experimental data. The accuracy of this modelling approach makes it a valuable platform for investigating the role of mechanical interactions during angiogenesis.     

Kinetics of chondrocyte growth in cell-polymer implants

In vitro cultivation of cartilage cells (chondrocytes) on biodegradable polyglycolic acid (PGA) scaffolds resulted in implants which could potentially be used to repair damaged joint cartilage or for reconstructive surgery. Cell growth kinetics were studied to define conditions under which the cellularity of implants made from isolated calf chondrocytes reached that of the parent calf cartilage. In static cultures, condrocyte growth rates decreased as either implant thickness or implant cell density increased. Over 4 weeks of cultivation, implant permeability to glucose decreased to 3% that of the plain polymer scaffold; this effect was attributed to the decrease in effective implant porosity associated with cartilage tissue regeneration.In a well-mixed culture, implants 1 cm in diameter by 0.3 cm thick maintained high cell growth rates over 7 weeks and hard normal cell densities. Regenerated cartilage with these dimensions is large enough to resurface small joints such as the trapezium bone at the base of the human thumb. Such implants could not be grown statically, since cell growth stopped at 3-4 weeks and cell densities remained below normal. Optimization of the tissue culture environment is thus essential in order to cultivate clinically useful cartilage implants in vitro. (c) 1994 John Wiley & Sons, Inc.     

Increased rate of chondrocyte aggregation in a wavy-walled bioreactor

A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension. Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors. Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time. The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture. At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors. During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks. The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate. Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps. Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment. These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage. Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage. Characterizing the wavy-walled bioreactor's hydrodynamic environment and its effects on cartilage cell/tissue culture can help establish direct relationships between hydrodynamic forces and engineered tissue properties.     

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.     

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     

Synergistic action of fibroblast growth factor-2 and transforming growth factor-beta1 enhances bioprinted human neocartilage formation

Bioprinting as a promising but unexplored approach for cartilage tissue engineering has the advantages of high throughput, digital control, and highly accurate placement of cells and biomaterial scaffold to the targeted 3D locations with simultaneous polymerization. This study tested feasibility of using bioprinting for cartilage engineering and examined the influence of cell density, growth, and differentiation factors. Human articular chondrocytes were printed at various densities, stimulated transiently with growth factors and subsequently with chondrogenic factors. Samples were cultured for up to 4 weeks to evaluate cell proliferation and viability, mechanical properties, mass swelling ratio, water content, gene expression, ECM production, DNA content, and histology. Bioprinted samples treated with FGF-2/TGF-β1 had the best chondrogenic properties among all groups apparently due to synergistic stimulation of cell proliferation and chondrogenic phenotype. ECM production per chondrocyte in low cell density was much higher than that in high cell seeding density. This finding was also verified by mechanical testing and histology. In conclusion, cell seeding density that is feasible for bioprinting also appears optimal for human neocartilage formation when combined with appropriate growth and differentiation factors.     

脱细胞基质生物墨水制备方法及应用进展

组织器官脱细胞后制备成的脱细胞基质 (Decellularized extracellular matrix,dECM) 含有许多蛋白质和生长因子,不仅能够为细胞提供三维支架还能够调控细胞再生,是目前最具有生物结构的生物材料。3D生物打印可以层层打印dECM和自体细胞的组合,构建载细胞组织结构。文中综述了不同来源的组织器官脱细胞基质生物墨水制备方法,包括脱细胞、交联等,以及脱细胞基质生物墨水在生物打印中的应用,并展望了其未来的应用前景。