Article

Response of Sheep Chondrocytes to Changes in Substrate Stiffness from 2 to 20 Pa: Effect of Cell Passaging

Authors:
  • ITRAMED (Instituto de Traumatologia y Medicina Regenerativa Avanzada)
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Abstract

Aim: The influence of culture substrate stiffness (in the kPa range) on chondrocyte behavior has been described. Here we describe the response to variations in substrate stiffness in a soft range (2-20 Pa), as it may play a role in understanding cartilage physiopathology. Methods: We developed a system for cell culture in substrates with different elastic moduli using collagen hydrogels and evaluated chondrocytes after 2, 4, and 7 days in monolayer and three-dimensional (3D) cultures. Experiments were performed in normoxia and hypoxia in order to describe the effect of a low oxygen environment on chondrocytes. Finally, we also evaluated if dedifferentiated cells preserve the capacity for mechanosensing. Results: Chondrocytes showed less proliferating activity when cultured in monolayer in the more compliant substrates. Expression of the cartilage markers Aggrecan (Acan), type II collagen (Col2a1), and Sox9 was upregulated in the less stiff gels (both in monolayer and in 3D culture). Stiffer gels induced an organization of the actin cytoskeleton that correlated with the loss of a chondrocyte phenotype. When cells were cultured in hypoxia, we observed changes in the cellular response that were mediated by HIF-1α. Results in 3D hypoxia cultures were opposite to those found in normoxia, but remained unchanged in monolayer hypoxic experiments. Similar results were found for dedifferentiated cells. Conclusions: Chondrocytes respond differently according to the stiffness of the substrate. This response depends greatly on the oxygen environment and on whether the chondrocyte is embedded or grown onto the hydrogel, since mechanosensing capacity was not lost with cell expansion.

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... In the case of hydrogels, effect of stiffness is much subtle, because different studies reported conflicting optimal stiffness values. Sanz-Ramos et al. [94] showed that collagen hydrogels with elastic modulus of * 5 Pa were superior in enhancing expression of aggrecan, type II collagen, and Sox9 than stiffer hydrogels (* 10 and * 20 Pa). On the other hand, Li et al. [27] described that stiff polyacrylamide gel (29.9 kPa), not the softer gels (3.8 or 17.1 kPa), promoted the redifferentiation of ACs and subsequent secretion activities. ...
... Pore sizes larger than 300 lm are advantageous to support the chondrogenic differentiation of MSCs and their subsequent production of cartilage-related matrices. Matsiko et al. [33] investigated chondrogenic differentiation of MSCs in collagen-based sponges at different pore sizes (94,130, and 300 lm) with relatively similar elastic modulus. The scaffold with the largest pore size at 300 lm was superior in supporting the chondrogenic differentiation of MSCs compared with the others on the basis of significantly higher expression of COL2, SOX9 and lower expression of COL1 for 28 days [33]. ...
... Effect of substrate stiffness (colored boxes) on gene transcription (italic name; Sox9, a-SMA, COL II, Col I, Col X, ACAN) and protein synthesize (underlined name; GAG/DNA, COLII) of ACs (blue and red colored) and MSCs (green and yellow colored). Blue and red boxes respectively indicate positive and negative changes of differentiated phenotype of ACs, as reported by Sanz-Ramoz et al.[94], Schuh et al.[26,95], Li et al.[27] and Lee et al.[83]. In a similar manner, green and yellow boxes are associated with changes of chondrogenic markers of MSCs as reported by Bian et al.[37] and Murphy et al.[31]. ...
Article
BACKGROUND: Cartilage tissue engineering (CTE) aims to obtain a structure mimicking native cartilage tissue through the combination of relevant cells, three-dimensional scaffolds, and extraneous signals. Implantation of ‘matured’ constructs is thus expected to provide solution for treating large injury of articular cartilage. Type I collagen is widely used as scaffolds for CTE products undergoing clinical trial, owing to its ubiquitous biocompatibility and vast clinical approval. However, the long-term performance of pure type I collagen scaffolds would suffer from its limited chondrogenic capacity and inferior mechanical properties. This paper aims to provide insights necessary for advancing type I collagen scaffolds in the CTE applications. METHODS: Initially, the interactions of type I/II collagen with CTE-relevant cells [i.e., articular chondrocytes (ACs) and mesenchymal stem cells (MSCs)] are discussed. Next, the physical features and chemical composition of the scaffolds crucial to support chondrogenic activities of AC and MSC are highlighted. Attempts to optimize the collagen scaffolds by blending with natural/synthetic polymers are described. Hybrid strategy in which collagen and structural polymers are combined in non-blending manner is detailed. RESULTS: Type I collagen is sufficient to support cellular activities of ACs and MSCs; however it shows limited chondrogenic performance than type II collagen. Nonetheless, type I collagen is the clinically feasible option since type II collagen shows arthritogenic potency. Physical features of scaffolds such as internal structure, pore size, stiffness, etc. are shown to be crucial in influencing the differentiation fate and secreting extracellular matrixes from ACs and MSCs. Collagen can be blended with native or synthetic polymer to improve the mechanical and bioactivities of final composites. However, the versatility of blending strategy is limited due to denaturation of type I collagen at harsh processing condition. Hybrid strategy is successful in maximizing bioactivity of collagen scaffolds and mechanical robustness of structural polymer. CONCLUSION: Considering the previous improvements of physical and compositional properties of collagen scaffolds and recent manufacturing developments of structural polymer, it is concluded that hybrid strategy is a promising approach to advance further collagen-based scaffolds in CTE.
... As discussed above, such findings are difficult to interpret, as the data can also be explained by an adhesion-independent mechanism, in which cells sense cell volume confinement in 3D culture [165]. Another study [222] used very soft hydrogels (2-20 Pa) to investigate the influence of stiffness in 2D and 3D environments on sheep CH (sCH) phenotype but no de-differentiation via serial passaging was performed. The study demonstrated that the softest collagen hydrogels, used as monolayer or 3D culture system, increased the expression of ACAN, collagen type II, and SOX9. ...
... The study demonstrated that the softest collagen hydrogels, used as monolayer or 3D culture system, increased the expression of ACAN, collagen type II, and SOX9. The loss of chondrogenic phenotype on stiffer hydrogels correlated with a diffuse organization of actin stress fibers [222]. Here, the 2D experimental results of sCH differentiation were comparable to the results of the 3D environment, as sCH phenotype, morphology and organization of cytoskeleton were comparable across both systems and, importantly, stiffness-mediated. ...
... Here, the 2D experimental results of sCH differentiation were comparable to the results of the 3D environment, as sCH phenotype, morphology and organization of cytoskeleton were comparable across both systems and, importantly, stiffness-mediated. Interestingly, the elastic moduli of these hydrogels used by Sanz-Ramos et al. [222] were much softer (2-20 Pa), compared to the other discussed studies (3.7 kPa vs. 53.2 kPa) [164,215], which did not find any association between chondrogenic mRNA expression and material stiffness. ...
Article
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Since material stiffness controls many cell functions, we reviewed the currently available knowledge on stiffness sensing and elucidated what is known in the context of clinical and experimental articular cartilage (AC) repair. Remarkably, no stiffness information on the various biomaterials for clinical AC repair was accessible. Using mRNA expression profiles and morphology as surrogate markers of stiffness-related effects, we deduced that the various clinically available biomaterials control chondrocyte (CH) phenotype well, but not to equal extents, and only in non-degenerative settings. Ample evidence demonstrates that multiple molecular aspects of CH and mesenchymal stromal cell (MSC) phenotype are susceptible to material stiffness, because proliferation, migration, lineage determination, shape, cytoskeletal properties, expression profiles, cell surface receptor composition, integrin subunit expression, and nuclear shape and composition of CHs and/or MSCs are stiffness-regulated. Moreover, material stiffness modulates MSC immuno-modulatory and angiogenic properties, transforming growth factor beta 1 (TGF-β1)-induced lineage determination, and CH re-differentiation/de-differentiation, collagen type II fragment production, and TGF-β1- and interleukin 1 beta (IL-1β)-induced changes in cell stiffness and traction force. We then integrated the available molecular signaling data into a stiffness-regulated CH phenotype model. Overall, we recommend using material stiffness for controlling cell phenotype, as this would be a promising design cornerstone for novel future-oriented, cell-instructive biomaterials for clinical high-quality AC repair tissue.
... This is characterized by a cessation in the expression of collagen type II, aggrecan and the transcription factor Sox9, among others. In our laboratory, we used collagen hydrogels that support the culture of chondrocytes on substrates with low mechanical properties (in the range of pascals) [6,7]. We achieved an efficient expansion method that slowed down the dedifferentiation process that takes place when cells are cultured onto plastic [8]. ...
... We can suggest that the changes occurring during expansion and prior to cell therapy applications may have special relevance for the final behavior of cells. Additionally, since one of the key factors implicated in these changes is the biomechanics of the culture system [6][7][8], we would like to open a new line of research in the epigenetic mechanisms that could be implicated in the sensing of matrix stiffness. In any case, our data point to a key role for the two receptors Alk-1 and Alk-5 in the physiology of chondrocytes. ...
Article
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We aim to demonstrate the role of Alk receptors in the response of hydrogel expansion. Chondrocytes from rat knees were cultured onto plastic and hydrogel surfaces. Alk-1 and Alk-5 were overexpressed or silenced and the effects on cells during expansion were tested and confirmed using peptide inhibitors for TGFβ. Overexpression of Alk-5 and silencing of Alk-1 led to a loss of the chondrocyte phenotype, proving that they are key regulators of chondrocyte mechanosensing. An analysis of the gene expression profile during the expansion of these modified cartilage cells in plastic showed a better maintenance of the chondrocyte phenotype, at least during the first passages. These passages were also assayed in a mouse model of intramuscular chondrogenesis. Our findings indicate that these two receptors are important mediators in the response of chondrocytes to changes in the mechanical environment, making them suitable targets for modulating chondrogenesis. Inhibition of TGFβ could also be effective in improving chondrocyte activity in aged or expanded cells that overexpress Alk-1.
... Substrate stiffness has been demonstrated to be able to control stem cell fate, 76,77 in particular neuronal, 94 chondrocyte, 95 cardiomyocyte, 96 dermal, 97 and limbal stem cells. 98 Recently, it has been shown that substrates of varying stiffness have different concentrations of protein anchorage sites that control stem cell fate through mechanical signals from cells attached to the anchorage proteins. ...
Article
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Traumatic soft tissue wounds present a significant reconstructive challenge. The adoption of closed circuit negative pressure wound therapy (NPWT) has enabled surgeons to temporize these wounds prior to reconstruction. Such systems use porous synthetic foam scaffolds as wound fillers at the interface between the negative pressure system and the wound bed. The idea of using a bespoke porous biomaterial that enhances wound healing as a filler for a NPWT system is attractive as it circumvents concerns regarding reconstructive delay and the need for dressing changes that are features of the current systems. Porous foam biomaterials are mechanically robust and able to synthesise in situ. Hence they exhibit potential to fulfil the niche for such a functionalised injectable material. Injectable scaffolds are currently in use for minimally invasive surgery but the design parameters for large volume expansive foams remain unclear. Candidate platforms include hydrogel systems, (particularly superabsorbent, superporous and nanocomposite systems) polyurethane based moisture cured foams and high internal phase emulsion polymer systems. The aim of this review is to discuss the design parameters for such future biomaterials and review potential candidate materials for further research into this up and coming field.
... Cell shape is well known to influence expression profile being associated with a particular cytoskeletal architecture. 16 In hydrogel culture, some cells die during the adaption to 3D conditions, due to the embedding and gel polymerization procedure as well as the burden of a cellular reorganization process. The density of alginate hydrogels depends on the content of alginate. ...
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In vitro-expanded intervertebral disc (IVD) cells could be a source for disc repair. However, IVD cell characterization still remains challenging and is demanded to detect phenotypical shifts. Therefore, the aim of the present study was to determine IVD cell expression profile during two- and three-dimensional culturing in direct comparison to in situ conditions. Human IVD tissue was analyzed immunohistologically and anulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated and characterized for cytoskeletal architecture and expression of typical markers (type I, II, and III collagens, aggrecan, decorin, cartilage oligomeric protein, the chondrogenic transcription factor sox9, the tendon markers scleraxis and tenascin C) during 6 monolayer passages using real-time detection polymerase chain reaction and/or immunolabellings. Cells were introduced in alginate and collagen hydrogels and cell morphology and viablility was determined after 7 days. In addition to typical extracellular matrix components, IVD tissue and isolated cells revealed scleraxis expression. In early passages of cell expansion, genes of sox9, scleraxis, and the small proteoglycan decorin were expressed higher, but type I and III collagen genes were expressed lower in NP cells compared with AF cells. However, in passage 6, actin stress fibers increased and the expression levels of sox9 were nearly similar in NP and AF cells. The immunolabeling indicated that the fibroblast marker tenascin C could only be detected in vitro in both cell types but not in situ. Decorin protein expression decreased in both cell types in vitro in passage 6. IVD cells survived in both hydrogel cultures, and some cells elongated in collagen gels.
... Therefore, it is possible that AFSCs were differentiated into various AF cells corresponding to the elasticity of PECUU substrates which resembled the different regions of native AF tissue. Such substrate elasticity-dependent gene expression echoes the findings from a number of previous studies [20,25,37]. After longer culture (two weeks), the expression of collagen-I gene in AFSCs continued to increase with the increase of substrate stiffness, yet the expression of collagen-II and aggrecan genes still decreased with the increase of substrate stiffness (see Fig. 4 in Ref. [35]). ...
Article
Statement of significance: Repairing the annulus fibrosus (AF) of intervertebral disc (IVD) is critical for the treatment of disc degeneration disease, but remains challenging due to the significant heterogeneity of AF tissue. Previously, we have identified rabbit AF-derived stem cells (AFSCs), which are AF tissue-specific and hold promise for AF regeneration. In this study, we synthesized a series of poly(ether carbonate urethane)ureas of various elasticity (or stiffness) and explored the potential of induced differentiation of AFSCs using electrospun PECUU scaffolds. This work has, for the first time, found that AFSCs are able to present different gene expression patterns simply as a result of the elasticity of scaffold material. Therefore, our findings will help supplement current knowledge of AF tissue regeneration and may benefit a diversified readership from scientific, engineering, and clinical settings whose work involves the biology and tissue engineering of IVD.
... In the hypoxia growth plate, HIF-1α maintains chondrocytes function as professional secretory cells by regulating collagen production [5]. HIF-1α not only inhibits the expression of COL1A1, COL1A2 and COL3A1 [6] but also simultaneously induces the expression of SOX9, COL2A1 and aggrecan [7]. HIF-1α inhibits cell proliferation [8] and is a survival factor for hypoxic chondrocytes [9]. ...
Article
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Background/aims: There have been many studies on the etiology of osteoarthritis (OA) with regard to the function of inflammatory cytokines, the process of cartilage degradation, the function of miR-146a, hypoxia stimulation and autophagy in OA chondrocytes, but there have been no reports on the relationship between miR-146a and autophagy in cartilage, especially under hypoxia. This study aimed to confirm the relationship of miR-146a and autophagy in cartilage under hypoxia. Methods: Chondrocytes were treated by hypoxia gradients, and the main factors including HIF-1α, HIF-2α, miR-146a and Bcl-2 and autophagy markers ULK-1, ATG-5 were detected by quantitative PCR (Q-PCR) and western blotting. The autophagy marker LC-3 was detected by immunofluorescence. The reciprocal effects between miR-146a and Bcl-2 were confirmed by several combinations of shRNAs and adenovirus-gene systems followed by Q-PCR and western blot detection. Results: Hypoxia maintained the chondrocytes phenotype and promoted autophagy and miR-146a expression via HIF-1α, but not HIF-2α, while miR-146a did not reversely affect HIF-1α. The autophagy induced by hypoxia through HIF-1α, miR-146a and Bcl-2. Simply, hypoxia induced HIF-1α, and HIF-1α increased miR-146a, but miR-146a suppressed Bcl-2, an autophagy inhibitor. While Bcl-2 affected neither HIF-1α nor miR-146a. The absence of both HIF-1α and miR-146a or Bcl-2 over-expression inhibited hypoxia-induced autophagy. Conclusion: HIF-1α, miR-146a and Bcl-2 play crucial roles during hypoxia-induced autophagy, Hypoxia, HIF-1α and miR-146a promote chondrocytes autophagy via depressing Bcl-2. We conclude that miR-146a may serve as a novel therapeutic target for protecting cartilage from degeneration in OA.
... It is well known that matrix stiffness regulates numerous cellular functions such as spreading, migration, proliferation and stem cell differentiation, influencing many major biological processes [1,2]. Chondrocyte physiology and biosynthetic activity are regulated by chemical and mechanical microenvironment cues, including matrix composition, soluble mediators, and interestingly physical cues, such as extracellular matrix (ECM) stiffness [3][4][5][6][7]. Adult articular cartilage is an avascular and nerveless tissue, and chondrocytes are the only cells of articular cartilage and are surrounded by a unique matrix region termed the pericellular matrix (PCM) which is wedged between the chondrocytes and ECM [8]. ...
Article
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The behavior of chondrocytes is regulated by multiple mechanical microenvironmental cues. During development and degenerative disease of articular cartilage, as an external signal, the extracellular matrix stiffness of chondrocytes changes significantly, but whether and how this biophysical cue affects biomechanical properties of chondrocytes remain elusive. In the present study, we designed supporting-biomaterials as mimics of native pericellular matrix to study the effect of matrix stiffness on chondrocyte morphology and F-actin distribution. Furthermore, the active mechanical behavior of chondrocytes during sensing and responding to different matrix stiffness was quantitatively investigated using atom force microscope technique and theoretical model. Our results indicated that stiffer matrix tends to increase the cell spreading area, the percentage of irregular cell shape distribution and mechanical parameters including elastic modulus (Eelastic), instantaneous modulus (E0), relaxed modulus (ER) and apparent viscosity (μ) of chondrocytes. Knowledge of matrix stiffness-dependent biomechanical behaviors of chondrocytes has important implications for optimizing matrix material and advancing chondrocyte-based applications for functional tissue engineering.
... Increasing substrate stiffness has been shown to reduce chondrogenic marker expression [50]. Conflictingly, as substrate stiffness decreases, cellular proliferation decreases [51]. Optimizing material stiffness to ideal levels thus requires fine, precise adjustments. ...
Article
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Background Cartilage is an important tissue found in a variety of anatomical locations. Damage to cartilage is particularly detrimental, owing to its intrinsically poor healing capacity. Current reconstructive options for cartilage repair are limited, and alternative approaches are required. Biomaterial science and Tissue engineering are multidisciplinary areas of research that integrate biological and engineering principles for the purpose of restoring premorbid tissue function. Biomaterial science traditionally focuses on the replacement of diseased or damaged tissue with implants. Conversely, tissue engineering utilizes porous biomimetic scaffolds, containing cells and bioactive molecules, to regenerate functional tissue. However, both paradigms feature several disadvantages. Faced with the increasing clinical burden of cartilage defects, attention has shifted towards the incorporation of Nanotechnology into these areas of regenerative medicine. Methods Searches were conducted on Pubmed using the terms “cartilage”, “reconstruction”, “nanotechnology”, “nanomaterials”, “tissue engineering” and “biomaterials”. Abstracts were examined to identify articles of relevance, and further papers were obtained from the citations within. Results The content of 96 articles was ultimately reviewed. The literature yielded no studies that have progressed beyond in vitro and in vivo experimentation. Several limitations to the use of nanomaterials to reconstruct damaged cartilage were identified in both the tissue engineering and biomaterial fields. Conclusion Nanomaterials have unique physicochemical properties that interact with biological systems in novel ways, potentially opening new avenues for the advancement of constructs used to repair cartilage. However, research into these technologies is in its infancy, and clinical translation remains elusive.
... 26 Studies have shown that chondrocytes respond differently to substrate stiffness in the range 2 to 20 kPa and the response to matrix elasticity depending on the 3D versus 2D culture environment. 27,28 But the chemical composition of a surface is also known to influence cellular responses. MSC differentiation on silane-modified surfaces with a range of chemical end groups was reported to control the phenotype of the attached MSCs. ...
Article
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Understanding the behavior of chondrocytes in contact with artificial culture surfaces is becoming increasingly important in attaining appropriate ex vivo culture conditions of chondrocytes in cartilage regeneration. Chondrocyte transplantation‐based cartilage repair requires efficiently expanded chondrocytes and the culture surface plays an important role in guiding the behavior of the cell. Micro‐ and nanoengineered surfaces make it possible to modulate cell behavior. We hypothesized that the combined influence of topography, substrate, and surface chemistry may affect the chondrocyte culturing in terms of proliferation and phenotypic means. Human chondrocytes were cultured on polystyrene fabricated microstructures, flat polydimethylsiloxane (PDMS) or polystyrene treated with fibronectin or oxygen plasma, and cultured for 1, 4, 7, and 10 days. The behavior of chondrocytes was evaluated by proliferation, viability, chondrogenic gene expression, and cell morphology. Contrary to our hypothesis, microstructures in polystyrene did not significantly influence the behavior of chondrocytes neither under normoxic‐ nor hypoxic conditions. However, changes in the substrate stiffness and surface chemistry were found to influence cell viability, gene expression and morphology of human chondrocytes. Oxygen plasma treatment was the most important parameter followed by the softer substrate type PDMS. The findings indicate the culture of human chondrocytes on softer substratum and surface activation by oxygen plasma may prevent dedifferentiation and may improve chondrocyte transplantation‐based cartilage repair. This article is protected by copyright. All rights reserved.
... However, those studies lacked measurements of the elastic modulus of the extracellular matrix used as a substrate, and their results would have been influenced by all of the stimuli present in the matrix (chemical as well as mechanical). In the present study, we attempted to focus solely on the mechanical stimuli by using a collagen substrate, as we showed in a previous study that coating of flasks with collagen had no significant effects on the differentiation stage of chondrocytes during expansion 30 . Taking all of these into account, our results showed a less effective response in the softest gels, partly as a result of the extremely malleable character of the substrate that makes it difficult to handle, suggesting that an optimal substrate for culture would have a stiffness of >22 Pa (but lower than that of plastic). ...
Article
Background: The use of autologous chondrocytes in cartilage repair is limited because of loss of the cartilage phenotype during expansion. The mechanosensing capacity of chondrocytes suggests evaluating the use of soft substrates for in vitro expansion. Our aim was to test the expansion of chondrocytes on collagen hydrogels to improve their capacity for chondrogenesis after a number of passages.METHODS: Rat cartilage cells were expanded on collagen hydrogels and on plastic, and the preservation of their chondrogenic capacity was tested both in vitro and in vivo. The expression of relevant markers during expansion on each surface was measured by real-time PCR (polymerase chain reaction). Expanded cells were then implanted in focal lesions in the medial femoral condyle of healthy sheep, and the newly formed tissue was analyzed by histomorphometry.RESULTS: Compared with cells cultured on plastic, cells cultured on hydrogels had better maintenance of the expression of the Sox9, Col2 (type-II collagen), FGFR3, and Alk-5 genes and decreased expression of Alk-1 and BMP-2. Pellets also showed increased expression of the cartilage marker genes aggrecan, Sox9, and Col2, and decreased expression of Col1 and Col10 (type-I and type-X collagen). ELISA (enzyme-linked immunosorbent assay) also showed a higher ratio of type-II to type-I collagen in pellets formed from cells expanded on hydrogels. When sheep chondrocytes were expanded and implanted in cartilage lesions in the femoral condyle of healthy sheep, hydrogel-expanded cells produced histologically better tissue compared with plastic-expanded cells.CONCLUSIONS: The expansion of chondrocytes on collagen hydrogels yielded cells with an improved chondrogenic capacity compared with cells expanded on plastic.CLINICAL RELEVANCE: The study results favor the use of hydrogel-expanded cells over the traditional plastic-expanded cells for autologous chondrocyte implantation.
... We found that on low modulus PECUU scaffolds, the expression of collagen-I gene in both AFSCs and tBMSCs was relatively low, whereas the expression of collagen-II and aggrecan genes was rel- atively high. These findings echo the results of a few previous studies [46,47]. In other words, on the soft PECUU scaffolds, both AFSCs and tBMSCs tended to differentiate into the cells which resembled the inner AF cells. ...
Article
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Tissue engineering has recently evolved into a promising approach for annulus fibrosus (AF) regeneration. However, selection of an ideal cell source, which can be readily differentiated into AF cells of various regions, remains challenging because of the heterogeneity of AF tissue. In this study, we set out to explore the feasibility of using transforming growth factor-β3-mediated bone marrow stem cells (tBMSCs) for AF tissue engineering. Since the differentiation of stem cells significantly relies on the stiffness of substrate, we fabricated nanofibrous scaffolds from a series of biodegradable poly(ether carbonate urethane)-urea (PECUU) materials whose elastic modulus approximated that of native AF tissue. We cultured tBMSCs on PECUU scaffolds and compared their gene expression profile to AF-derived stem cells (AFSCs), the newly identified AF tissue-specific stem cells. As predicted, the expression of collagen-I in both tBMSCs and AFSCs increased with scaffold stiffness, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. Interestingly, the expression of collagen-I, collagen-II and aggrecan genes in tBMSCs on PECUU scaffolds were consistently higher than those in AFSCs regardless of scaffold stiffness. In addition, the cell traction forces (CTFs) of both tBMSCs and AFSCs gradually decreased with scaffold stiffness, which is similar to the CTF change of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that tBMSCs had strong tendency to differentiate into various types of AF cells and presented gene expression profiles similar to AFSCs, thereby establishing a rationale for the use of tBMSCs in AF tissue engineering. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
... They noted that neurogenic differentiation was optimal at a stiffness of 0.1-1 kPa, myogenic differentiation at 8-17 kPa and osteogenic differentiation at 25-40 kPa. Several subsequent studies have shown how substrate stiffness affects several different cell types, including neuronal cells [134,135], chondrocytes [136], cardiomyocytes [137,138], dermal fibroblasts [139] and limbal stem cells [140]. Recently, Trappmann et al. [141] suggested that substrates of different stiffness have differing protein anchorage densities and configurations that regulate stem cell fate through the mechanical feedback of cells attached to anchored proteins [141]. ...
Article
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The development of hydrogel-based biomaterials represents a promising approach to generating new strategies for tissue engineering and regenerative medicine. In order to develop more sophisticated cell-seeded hydrogel constructs, it is important to understand how cells mechanically interact with hydrogels. In this paper, we review the mechanisms by which cells remodel hydrogels, the influence that the hydrogel mechanical and structural properties have on cell behaviour and the role of mechanical stimulation in cell-seeded hydrogels. Cell-mediated remodelling of hydrogels is directed by several cellular processes, including adhesion, migration, contraction, degradation and extracellular matrix deposition. Variations in hydrogel stiffness, density, composition, orientation and viscoelastic characteristics all affect cell activity and phenotype. The application of mechanical force on cells encapsulated in hydrogels can also instigate changes in cell behaviour. By improving our understanding of cell-material mechano-interactions in hydrogels, this should enable a new generation of regenerative medical therapies to be developed.
... When culturing on the DECM, articular chondrocytes maintained their original round morphology and the different stiffness between the DECM and TCPS might be responsible for the alternation of chondrocyte phenotype. It has been reported that the actin cytoskeleton of the adherent chondrocytes cultured on stiff gels was re-organized [26]. The cytoskeletal tension increased as the substrate stiffness decreased and chondrocytes on soft substrates showed high levels of COL II and aggrecan via inactivation of the RhoA/ROCK pathway [27]. ...
Article
Autologous chondrocyte implantation (ACI) is a promising approach to repair cartilage defects; however, the cartilage trauma-induced inflammatory environment compromises its clinical outcomes. Cell-derived decellularized extracellular matrix (DECM) has been used as a culture substrate for mesenchymal stem cells (MSCs) to improve the cell proliferation and lineage-specific differentiation. In this study, DECM deposited by synovium-derived MSCs was used as an in vitro expansion system for rabbit articular chondrocytes and the response of DECM-expanded chondrocytes to pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was evaluated. Compared with those grown on tissue culture polystyrene (TCPS), the proliferation rate was significantly improved in DECM-expanded chondrocytes. TCPS- and DECM-expanded chondrocytes were isolated and induced to redifferentiation in a high-density pellet culture. DECM-expanded chondrocytes exerted a stronger resistance to 1 ng/mL of IL-1β than TCPS-expanded cells, but the production of cartilage matrix in both groups was inhibited by 5 ng/mL of IL-1β. When exposed to 1 or 5 ng/mL of TNF-α, DECM-expanded chondrocytes showed higher levels of cartilage matrix synthesis than TCPS-expanded cells. In addition, the gene expression of IL-1β- or TNF-α-induced matrix degrading enzymes (MMP3, MMP9, MMP13, and ADAMTS5) was significantly lower in DECM-expanded chondrocytes than TCPS-expanded cells. Furthermore, we found that SIRT1 inhibition by nicotinamide completely counteracted the protective effect of DECM on chondrocytes in the presence of IL-1β or TNF-α, indicating that the SIRT1 signaling pathway was involved in the DECM-mediated enhancement of anti-inflammatory properties of chondrocytes. Taken together, this work suggests that stem cell-derived DECM is a superior culture substrate for in vitro chondrocyte expansion by improving proliferation and enhancing the anti-inflammatory properties of chondrocytes. DECM-expanded chondrocytes with enhanced anti-inflammatory properties hold great potential in clinically ACI-based cartilage repair.
... It is known that HIF-1α translocates to the nucleus in hypoxic environments and regulates the expression of SOX9, a master regulator in chondrocytes [10, 16]. Furthermore, a prior study found that the production of substrates, such as aggrecan and type 2 collagen, can be promoted via HIF-1α expression and by culturing chondrocytes in a hypoxic environment [17]. In this study, the histological examination performed on culture day 28 revealed that safranin O staining and substrate production were both increased in the group cultured under hypoxic conditions. ...
Preprint
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Background While cartilage can be formed from induced pluripotent stem cells (iPSCs), challenges such as long culture periods and compromised tissue purity remain. We aimed to determine whether cartilaginous tissue can be produced from iPSCs under hypoxia and to evaluate the effects on the cellular metabolism and purity of the tissue.Methods Human iPSCs (hiPSCs) were cultured for cartilage differentiation in monolayers under normoxia or hypoxia (5% O 2 ). We evaluated chondrocyte differentiation by real-time reverse transcription-polymerase chain reaction and fluorescence-activated cell sorting. Then, hiPSCs were cultured for cartilage differentiation in 3D culture under normoxia or hypoxia (5% O 2 ). We evaluated cartilage-like tissues on days 28 and 56 through histological analyses.ResultsHypoxia suppressed the expression of immature mesodermal markers T ( Brachyury ) and Forkhead box protein F1 ( FOXF1) and promoted the expression of the chondrogenic markers aggrecan and CD44 . Sex determining region Y- box (SOX) 9-positive cells were increased by culture under hypoxia. Percentages of safranin O-positive and type 2 collagen-positive tissues were increased under hypoxia. Moreover, upon hypoxia-inducible factor (HIF)-1α staining, the nuclear dyeability in tissues cultured under hypoxia was greater than that under normoxia.Conclusions Hypoxia not only led to enhanced cartilage matrix production but also improved cell purity by promoting the expression of HIF-1α . By applying this method, highly pure cartilaginous-like tissues may be produced more rapidly and conveniently.
... Therefore, no dedifferentiation and losing capacity of cartilage matrix were assured. Moreover, the cells were able to respond to the stiffness of scaffold in the three-dimensional environment, preserving their properties [34][35][36]. Conversely, the engineered scaffolds for both patients supported the attachment and proliferation of seeded chondrocytes when assessed by scanning electron microscopy. Worthy of note, the capability of the engineered silk fibroin-based scaffold in support of rabbit chondrocytes and human bone marrow-derived stem cells were demonstrated in our previous studies [24,27]. ...
... Therefore, no dedifferentiation and losing capacity of cartilage matrix were assured. Moreover, the cells were able to respond to the stiffness of scaffold in the three-dimensional environment, preserving their properties [34][35][36]. Conversely, the engineered scaffolds for both patients supported the attachment and proliferation of seeded chondrocytes when assessed by scanning electron microscopy. Worthy of note, the capability of the engineered silk fibroin-based scaffold in support of rabbit chondrocytes and human bone marrow-derived stem cells were demonstrated in our previous studies [24,27]. ...
... it is known that HiF-1α translocates to the nucleus in hypoxic environments and regulates the expression of SoX9 (10,17). Furthermore, a previous study found that the production of substrates, such as aggrecan and type 2 collagen, can be promoted via HiF-1α expression and by culturing chondrocytes in a hypoxic environment (18). in this study, HiF-1α expression increased as chondrogenic differentiation progressed from day 3 to day 14. The histological examination performed on culture day 28 revealed that safranin o staining and substrate production were both increased in the cells cultured under hypoxic conditions. ...
Article
While cartilage can be produced from induced pluripotent stem cells (iPSCs), challenges such as long culture periods and compromised tissue purity continue to prevail. The present study aimed to determine whether cartilaginous tissue could be produced from iPSCs under hypoxia and, if so, to evaluate its effects on cellular metabolism and purity of the produced tissue. Human iPSCs (hiPSCs) were cultured for cartilage differentiation in monolayers under normoxia or hypoxia (5% O2), and chondrocyte differentiation was evaluated using reverse transcription‑quantitative PCR and fluorescence‑activated cell sorting. Subsequently, cartilage differentiation of hiPSCs was conducted in 3D culture under normoxia or hypoxia (5% O2), and the formed cartilage‑like tissues were evaluated on days 28 and 56 using histological analyses. Hypoxia suppressed the expression levels of the immature mesodermal markers brachyury (T) and forkhead box protein F1; however, it promoted the expression of the chondrogenic markers Acan and CD44. The number of sex‑determining region Y‑box 9‑positive cells and the percentages of safranin O‑positive and type 2 collagen‑positive tissues increased under hypoxic conditions. Moreover, upon hypoxia‑inducible factor (HIF)‑1α staining, nuclei of tissues cultured under hypoxia stained more deeply compared with those of tissues cultured under normoxia. Overall, these findings indicated that hypoxia not only enhanced cartilage matrix production, but also improved tissue purity by promoting the expression of HIF‑1α gene. Potentially, pure cartilage‑like tissues could be produced rapidly and conveniently using this method.
... The increased stiffness and network density in GY785-enriched Si-HPMC may explain the enhancement of the chondrocyte phenotype in this hydrogel compared to Si-HPMC alone. Indeed, these mechanical parameters have previously been shown to influence chondrocyte phenotype (Brodkin et al., 2004;Sanz-Ramos et al., 2013) as well as direct mesenchymal stem cell differentiation (Discher et al., 2005;Engler et al., 2006). ...
Article
The development of biologically and mechanically competent hydrogels is a prerequisite in cartilage engineering. We recently demonstrated that a marine exopolysaccharide, GY785, stimulates the in vitro chondrogenesis of adipose stromal cells. In the present study, we thus hypothesized that enriching our silated hydroxypropyl methylcellulose hydrogel (Si-HPMC) with GY785 might offer new prospects in the development of scaffolds for cartilage regeneration. The interaction properties of GY785 with growth factors was tested by surface plasmon resonance (SPR). The biocompatibility of Si-HPMC/GY785 towards rabbit articular chondrocytes (RACs) and its ability to maintain and recover a chondrocytic phenotype were then evaluated in vitro by MTS assay, cell counting and qRT-PCR. Finally, we evaluated the potential of Si-HPMC/GY785 associated with RACs to form cartilaginous tissue in vivo by transplantation into the subcutis of nude mice for 3 weeks. Our SPR data indicated that GY785 was able to physically interact with BMP-2 and TGFβ. Our analyses also showed that three-dimensionally (3D)-cultured RACs into Si-HPMC/GY785 strongly expressed type II collagen (COL2) and aggrecan transcripts when compared to Si-HPMC alone. In addition, RACs also produced large amounts of extracellular matrix (ECM) containing glycosaminoglycans (GAG) and COL2. When dedifferentiated RACs were replaced in 3D in Si-HPMC/GY785, the expressions of COL2 and aggrecan transcripts were recovered and that of type I collagen decreased. Immunohistological analyses of Si-HPMC/GY785 constructs transplanted into nude mice revealed the production of a cartilage-like extracellular matrix (ECM) containing high amounts of GAG and COL2. These results indicate that GY785-enriched Si-HPMC appears to be a promising hydrogel for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
Article
Dedifferentiation of chondrocytes is common in culturing, and seriously affects the restorative efficacy of cartilage repair. The present study examines the effect of initial cell shapes on dedifferentiation of chondrocytes in vitro. The cell shape was controlled with a unique material micropatterning technique. With this technique, a series of microarrays of cell-adhesive peptide arginine-glycine-aspartate (RGD) were generated on a persistent non-fouling poly(ethylene glycol) (PEG) hydrogel. After culturing chondrocytes derived from rats on the micropatterned surfaces, the cell shapes were adapted by the geometries of adhesive microislands with pre-defined diameters (10, 15, 20 and 30 μm) for round ones and aspect ratios (1, 1.2, 1.5, 2, 4 and 6) for elliptical ones. After 10 days, collagen II staining was demonstrated to identify normal chondrocytes and dedifferentiated cells for those single cells on microislands. Furthermore, the gene expression of collagen II, collagen I, aggrecan and SOX9 were detected by qRT-PCR. The statistical results illustrated that dedifferentiation of chondrocytes happened more probably in the cases of larger sizes and higher aspect ratios. The conclusions stand under circumstances of both normoxia (21% O2) and hypoxia (5% O2) atmospheres.
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Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC-based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro-aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFβ3 added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFβ, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro-aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro-aggregates, under all TGFβ conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro-aggregates. Copyright © 2014 John Wiley & Sons, Ltd.
Article
The hallmark of OA is cartilage destruction, several factors such as catabolic enzymes and chondrocyte death include apoptosis and/or autophagy are considered for the pathogenesis. Articular cartilage is maintained in a low oxygen environment throughout life. Chondrocytes are therefore adapted to these hypoxic conditions. The increased HIF-1α and HIF-2α mediate the response of chondrocytes to hypoxia. HIF-1α regulates chondrogenesis by regulating SOX9 expression in the genetic level, HIF-1 also serves to regulate both autophagy and apoptosis. Therefore, HIF-1α may protect articular cartilage by promoting the chondrocyte phenotype, maintaining chondrocyte viability, and supporting metabolic adaptation to a hypoxic environment. In contrast with HIF-1α, HIF-2α is a catabolic factor in the osteoarthritic process. Although HIF-2α is essential for hypoxic induction of the human articular chondrocyte phenotype, HIF-2α directly induces the expression of catabolic factors in chondrocytes, and HIF-2α enhances Fas expression to mediate chondrocyte apoptosis and regulates autophagy in maturing chondrocytes. Taken together, manipulation of HIF-1α and HIF-2α could represent a promising approach to the treatment of OA. Further study should elucidate the exact machnism of HIF-1α and HIF-2α in cartilage and determine which is predominant in osteoarthritic process.
Article
Objective To clarify the role of YAP in modulating cartilage inflammation and degradation and the involvement of primary cilia and associated intraflagellar transport (IFT). Methods Isolated primary chondrocytes were cultured on substrates of different stiffness (6-1000kPa) or treated with YAP agonist lysophosphatidic acid (LPA) or YAP antagonist verteporfin (VP), or genetically modified by YAP siRNA, all ±IL1β. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were measured to monitor IL1β response. YAP activity was quantified by YAP nuclear/cytoplasmic ratio and percentage of YAP-positive cells. Mechanical properties of cartilage explants were tested to confirm cartilage degradation. The involvement of primary cilia and IFT was analysed using IFT88 siRNA and ORPK cells with hypomorphic mutation of IFT88. Results Treatment with LPA, or increasing PDMS substrate stiffness, activated YAP nuclear expression and inhibited IL1β-induced release of NO and PGE2, in isolated chondrocytes. Treatment with LPA also inhibited IL1β-mediated inflammatory signalling in cartilage explants and prevented matrix degradation and the loss of cartilage biomechanics. YAP activation reduced expression of primary cilia, knockdown of YAP in the absence of functional cilia/IFT failed to induce an inflammatory response. Conclusions We demonstrate that both pharmaceutical and mechanical activation of YAP blocks pro-inflammatory signalling induced by IL1β and prevents cartilage breakdown and the loss of biomechanical functionality. This is associated with reduced expression of primary cilia revealing a potential anti-inflammatory mechanism with novel therapeutic targets for treatment of OA.
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The development of novel hydrogels that possess adequate elasticity and toughness to withstand mechanically active environments, along with being biocompatible, remains a significant challenge in the design of materials for tissue engineering applications. In this study, a family of regenerated silk fibroin (RSF) based double network (DN) hydrogels were fabricated for the first time using a rapid one-pot method. The DN hydrogels combine the rigid covalently crosslinked RSF with the softer poly(N-vinylcaprolactam) (PVCL) through strong physical interactions. The formation of these DN hydrogels resulted in an improvement of the water uptake capacity, elasticity and toughness compared to the individual RSF hydrogel. The elasticity of the RSF/PVCL DN hydrogels was closer to that of native cartilage, which makes them promising materials for cartilage regeneration applications. An in vitro study on adhesion, proliferation and differentiation of a mouse pre-chondrocyte cell line (ATDC5) conducted using microscopic analysis, a cell proliferation assay and RT-PCR confirmed the cells cultured on the less stiff hydrogels demonstrated the most favourable chondrogenic response. Thus, this study demonstrates the potential of RSF-based hybrid hydrogels for cartilage tissue engineering applications.
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Tissue engineering holds great promise in the generation of cartilage analogues for cartilage injury repair and replacement. However, for a long time, a variety of issues have remained unsolved in articular cartilage tissue engineering concerning immunogenicity, stability, and mechanical strength, among others. One of the most remarkable reasons lies in the lack or insufficiency of recapitulating the chondrocyte biomechanical microenvironment (BME) in the articular cartilage tissue engineering. In recent years, an increasing number of studies have disclosed the crucial role of the BME in chondrocyte phenotype and cartilage functions, which has inspired more precise and individualized research in articular cartilage tissue engineering by engineering the chondrocyte BME. This review first takes an in-depth look into the chondrocyte BME and its crucial effects on chondrocytes and articular cartilage tissues. Then, as the core of this work, the principal strategies and their approaches of engineering the chondrocyte BME towards articular cartilage tissue engineering were comprehensively discussed, from the perspectives of simulating the main characteristics of chondrocyte BME including engineering the heterogeneous matrix and the dynamic mechanical stimulation. The current limitations in this emerging area and potential strategies were also proposed to shed some light on the future directions in this field. Although there are still challenges to obtaining engineered articular cartilages with desired performance, the road ahead is bright under the constant efforts in engineering the chondrocyte BME at higher levels towards articular cartilage tissue engineering.
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Reconstruction of native tissue's anatomical and biophysical milieu dictates the success of tissue engineered graft's cellular fate. Herein, we report a facile fabrication procedure to replicate the anatomical and biomechanical features of annulus fibrosus (AF) tissue. A seamless, full thickness disc-like angle-ply construct was fabricated using silk fibroin (SF) protein. To mimic the gradual transition of mechanical gradient from inner to outer region of native AF tissue, SF proteins from two different sources (namely Bombyx mori, BM SF as mulberry, and Antheraea assamensis, AA SF and Philosamia ricini, PR SF as non-mulberry) were blended that provided differential mechanical and cell binding properties. Fabricated constructs were physicochemically and biologically characterized. The seeded porcine AF cells were found to proliferate and align along the lamellar pores as visualized through staining. Gene expression study concluded higher expression of collagen-I with enhancement of mechanical properties, whereas an opposite trend was observed for both collagen-II and aggrecan. Overall, the angle-ply construct with tailored mechanical properties supported cellular alignment and proliferation, and modulated the extracellular matrix (ECM) deposition forming a functional AF tissue like construct, thus providing a robust foundation as an alternative tissue engineered strategy in intervertebral disc (IVD) regeneration for future replacement therapy.
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Matrix stiffness is known to alter cellular behaviors in various biological contexts. Previous investigations have shown that epithelial–mesenchymal transition (EMT) promotes the progression and invasion of tumor. Mechanical signaling is identified as a regulator of EMT. However, the molecular mechanisms underlying the influence exerted by matrix stiffness on EMT in osteosarcoma remains largely unknown. Using polyacrylamide hydrogel model, we investigate the effects of matrix stiffness on EMT and migration in osteosarcoma. Our data indicates that high matrix stiffness regulates cell morphology and promotes EMT and migration in osteosarcoma MG63 cell line in vitro. Notably, matrix stiffness promotes polymerization of actin and nuclear accumulation of myocardin-related transcription factor A (MRTF-A). Furthermore, inhibiting MRTF-A by CCG 203971 significantly reduces EMT and migration on rigid gels. These data suggest that matrix stiffness of the tumor microenvironment actively regulate osteosarcoma EMT and migration through cytoskeletal remodeling and translocation of MRTF-A, which may contribute to cancer progression.
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The optimal solution for articular cartilage repair has not yet been identified, in part because of the challenges in achieving integration with the host. Coatings have the potential to transform the adhesive features of surfaces, but their application to cartilage repair has been limited. Self-assembled monolayer of phosphonates (SAMPs) have been demonstrated to increase the adhesion of various immortalized cell types to metal and polymer surfaces, but their effect on primary chondrocyte adhesion has not been studied. The objective of this study was to investigate the response of primary chondrocytes to SAMP coatings. We hypothesized a SAMP terminated with an α,ω-bisphosphonic acid, in particular butane-1,4-diphosphonic acid, would increase the number of adherent primary chondrocytes to polyvinyl alcohol (PVA). To test our hypothesis, we first established our ability to successfully modify silicon dioxide (SiO2) surfaces to enable chondrocytes to attach to the surface, without substantial changes in gene expression. Secondly, we applied identical chemistry to PVA, and quantified chondrocyte adhesion. SAMP modification to SiO2 increased chondrocyte adhesion by 3x after 4 hr and 4.5x after 24 hr. PVA modification with SAMPs increased chondrocyte adhesion by at least 31x after 4 and 24 hours. Changes in cell morphology indicated that SAMP modification led to improved chondrocyte adhesion and spreading, without changes in gene expression. In summary, we modified SiO2 and PVA with SAMPs and observed an increase in the number of adherent primary bovine chondrocytes at 4 and 24 hr post-seeding. Mechanisms of chondrocyte interaction with SAMP-modified surfaces require further investigation.
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Cells are surrounded by extracellular matrix (ECM), which controls cellular functions through biological or physicochemical cues. In particular, cartilage tissue has abundant ECM that is viscoelastic and provides the necessary signals for the maintenance of chondrocyte activity and metabolism. The influence of ECM stiffness on chondrocyte functions has been broadly investigated using elastic hydrogels. However, it is not clear how viscosity impacts chondrocyte functions. In this study, a biphasic gelatin solution/hydrogel system was established for the three-dimensional culture of bovine articular chondrocytes (BACs) to investigate the influence of gelatin solution viscosity on chondrocyte proliferation, ECM secretion and the maintenance of the chondrocyte phenotype. Gelatin solutions of different viscosities supported chondrocyte proliferation and ECM production. However, the cell morphology, proliferation rate, secreted ECM quantity and gene expression levels were different, and this was dependent on the viscosity of the gelatin solutions. Low-viscosity solutions were more beneficial for proliferation, while high-viscosity solutions were more beneficial for ECM production and the expression of collagen type II and aggrecan. Chondrocytes had a more spread morphology in a low-viscosity gelatin solution than in a high-viscosity gelatin solution. The results suggested that high-viscosity was more beneficial for the maintenance of the chondrocyte phenotype, while low viscosity was more beneficial for cell expansion. Viscosity was demonstrated as one of the key parameters affecting cell morphology, proliferation and phenotype.
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This study adopts a combined computational and experimental approach to determine the mechanical, structural, and metabolic properties of isolated chondrocytes cultured within three-dimensional hydrogels. A series of linear elastic and hyperelastic finite-element models demonstrated that chondrocytes cultured for 24 h in gels for which the relaxation modulus is <5 kPa exhibit a cellular Young's modulus of ∼5 kPa. This is notably greater than that reported for isolated chondrocytes in suspension. The increase in cell modulus occurs over a 24-h period and is associated with an increase in the organization of the cortical actin cytoskeleton, which is known to regulate cell mechanics. However, there was a reduction in chromatin condensation, suggesting that changes in the nucleus mechanics may not be involved. Comparison of cells in 1% and 3% agarose showed that cells in the stiffer gels rapidly develop a higher Young's modulus of ∼20 kPa, sixfold greater than that observed in the softer gels. This was associated with higher levels of actin organization and chromatin condensation, but only after 24 h in culture. Further studies revealed that cells in stiffer gels synthesize less extracellular matrix over a 28-day culture period. Hence, this study demonstrates that the properties of the three-dimensional microenvironment regulate the mechanical, structural, and metabolic properties of living cells.
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Cells encounter physical cues such as extracellular matrix (ECM) stiffness in a microenvironment replete with biochemical cues. However, the mechanisms by which cells integrate physical and biochemical cues to guide cellular decision making are not well defined. Here we investigate mechanisms by which chondrocytes generate an integrated response to ECM stiffness and transforming growth factor β (TGFβ), a potent agonist of chondrocyte differentiation. Primary murine chondrocytes and ATDC5 cells grown on 0.5-MPa substrates deposit more proteoglycan and express more Sox9, Col2α1, and aggrecan mRNA relative to cells exposed to substrates of any other stiffness. The chondroinductive effect of this discrete stiffness, which falls within the range reported for articular cartilage, requires the stiffness-sensitive induction of TGFβ1. Smad3 phosphorylation, nuclear localization, and transcriptional activity are specifically increased in cells grown on 0.5-MPa substrates. ECM stiffness also primes cells for a synergistic response, such that the combination of ECM stiffness and exogenous TGFβ induces chondrocyte gene expression more robustly than either cue alone through a p38 mitogen-activated protein kinase-dependent mechanism. In this way, the ECM stiffness primes the TGFβ pathway to efficiently promote chondrocyte differentiation. This work reveals novel mechanisms by which cells integrate physical and biochemical cues to exert a coordinated response to their unique cellular microenvironment.
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Poly(1,8-octanediol-co-citrate) (POC) is a biocompatible, biodegradable elastomer with potential application for soft tissue applications such as cartilage. For chondrogenesis, permeability is a scaffold design target that may influence cartilage regeneration. Scaffold permeability is determined by many factors such as pore shape, pore size, pore interconnectivity, porosity, and so on. Our focus in this study was to examine the effects of pore shape and permeability of two different POC scaffold designs on matrix production, mRNA gene expression, and differentiation of chondrocytes in vitro and the consequent mechanical property changes of the scaffold/tissue constructs. Since type I collagen gel was used as a cell carrier in the POC scaffolds, we also examined the effects of collagen gel concentration on chondrogenesis. We found that lower collagen I gel concentration provides a favorable microenvironment for chondrocytes promoting better chondrogenic performance of chondrocytes. With regard to scaffold design, low permeability with a spherical pore shape better enhanced the chondrogenic performance of chondrocytes in terms of matrix production, and mRNA gene expressions in vitro compared to the highly permeable scaffold with a cubical pore shape.
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Hypoxia-inducible factor HIF-1 is responsible for radiation resistance and poor prognosis in cancer therapy. As part of our drug discovery program, a novel HIF inhibitor, LW6, was identified as a small compound that inhibits the accumulation of HIF-1alpha. We found that LW6 decreased HIF-1alpha protein expression without affecting HIF-1beta expression. MG132, a proteasome inhibitor, protected HIF-1alpha from LW6-induced proteasomal degradation, indicating that LW6 affects the stability of the HIF-1alpha protein. We found that LW6 promoted the degradation of wild type HIF-1alpha, but not of a DM-HIF-1alpha with modifications of P402A and P564A, at hydroxylation sites in the oxygen-dependent degradation domain (ODDD). LW6 did not affect the activity of prolyl hydroxylase (PHD), but induced the expression of von Hippel-Lindau (VHL), which interacts with prolyl-hydroxylated HIF-1alpha for proteasomal degradation. In the presence of LW6, knockdown of VHL did not abolish HIF-1alpha protein accumulation, indicating that LW6 degraded HIF-1alpha via regulation of VHL expression. In mice carrying xenografts of human colon cancer HCT116 cells, LW6 demonstrated strong anti-tumor efficacy in vivo and caused a decrease in HIF-1alpha expression in frozen-tissue immunohistochemical staining. These data suggest that LW6 may be valuable in the development of a HIF-1alpha inhibitor for cancer treatment.
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Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-alpha isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2alpha stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2alpha levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.
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Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are alpha E beta 4 (also called alpha 6 beta 4) and alpha 2 beta 1. The alpha E beta 4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas alpha 2 beta 1/alpha 3 beta 1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-beta 4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laminin, whereas anti-beta 1 antibodies were ineffective. Only anti-beta 4 antibodies were able to detach established keratinocyte colonies. These data suggest that alpha E beta 4 mediates keratinocyte adhesion to basal lamina, whereas the beta 1 subfamily is involved in cell-cell adhesion of keratinocytes.
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Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postmitotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.
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Several studies in the past year have shown that the cell cycle events typically attributed to a response to growth factors actually require signals provided by both growth factors and the extracellular matrix. Moreover, at least some of these matrix-based effects seem to involve matrix-dependent organization of the cytoskeleton rather than cell adhesion per se. Overall, these studies are providing new insights into the long-appreciated concepts of anchorage- and shape-dependent growth.
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Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.
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Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).
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Recent studies suggest that there are multiple regulatory pathways by which chondrocytes in articular cartilage sense and respond to mechanical stimuli, including upstream signaling pathways and mechanisms that may lead to direct changes at the level of transcription, translation, post-translational modifications, and cell-mediated extracellular assembly and degradation of the tissue matrix. This review focuses on the effects of mechanical loading on cartilage and the resulting chondrocyte-mediated biosynthesis, remodeling, degradation, and repair of this tissue. The effects of compression and tissue shear deformation are compared, and approaches to the study of mechanical regulation of gene expression are described. Of particular interest regarding dense connective tissues, recent experiments have shown that mechanotransduction is critically important in vivo in the cell-mediated feedback between physical stimuli, the molecular structure of newly synthesized matrix molecules, and the resulting macroscopic biomechanical properties of the tissue.
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Joint pain and loss of mobility are among the most common causes of impairment in middle-aged and older people36,134. In many instances, the degeneration of articular cartilage and alterations in other joint tissues that result from the loss of structure and function of articular cartilage cause the pain and the loss of motion28,46,47,85,118,150. This occurs most frequently in the clinical syndrome of idiopathic or primary osteoarthrosis, but it may also result from joint injury or from developmental, metabolic, and inflammatory disorders that destroy the articular surface, causing secondary osteoarthrosis28,46,118. An understanding of the degeneration of articular cartilage, osteoarthrosis, and the potential for restoring an articular surface depends to a large extent on an appreciation of the biological behavior and the responsiveness of articular cartilage to injury and disease. Of considerable importance is the observation, first reported centuries ago and confirmed by multiple investigators over the last fifty years, that adult articular cartilage does not have the capacity to repair structural damage resulting from injury or disease29,32,71. This observation has contributed to the view that adult articular cartilage is an inert bearing surface, like high-density polyethylene or metal, and that degeneration of the articular surface with age is the result of mechanical wear with inevitable, irreversible loss of structure and mechanical performance resulting from joint use62. The implication of this view is that, other than limiting joint use or loading, little or nothing can be done to prevent the degeneration of articular cartilage, and the most appropriate treatment for advanced degeneration of cartilage leading to the clinical syndrome of osteoarthrosis is replacement of the articular surface. Alternatively, if articular cartilage is …
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Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.
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Cartilage tissue engineering can provide functional cartilaginous constructs that can be used for controlled in vitro studies of chondrogenesis and potentially for in vivo articular cartilage repair. Ideally, engineered cartilage should be indistinguishable from native articular cartilage with respect to zonal organization, biochemical composition, and mechanical properties. In the model system presented here, chondrogenic cells are expanded in vitro as required, seeded onto three-dimensional polymeric scaffolds, and cultured in bioreactor vessels. During the course of in vitro cultivation, construct cellularity plateaus at a physiologic level, fractions of glycosaminoglycan and Type II collagen increase progressively, and the scaffold biodegrades. Construct structure (composition, morphology) and function (biosynthetic activity, mechanical properties) depend on cultivation conditions. This paper reviews recent studies of in vitro modulation of chondrogenesis by: (1) cell seeding density and source; (2) the tissue regeneration template; (3) biochemical regulatory signals; (4) mixing, mass transport and hydrodynamic forces; and (5) cultivation time. Key requirements and some of the critical research needs for successful cartilage tissue engineering are discussed.
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In the absence of in vivo measurements, the oxygen concentration within articular cartilage is calculated from the balance between cellular oxygen consumption and mass transfer. Current estimates of the oxygen tension within articular cartilage are based on oxygen consumption data from full-depth tissue samples. However, superficial and deep cell subpopulations of articular cartilage express intrinsic metabolic differences. We test the hypothesis that the subpopulations differ with respect to their intrinsic oxygen consumption rate. Chondrocytes from the full cartilage thickness demonstrate enhanced oxygen consumption when deprived of glucose, consistent with the Crabtree phenomena. Chondrocyte subpopulations differ in the prevailing availability of oxygen and glucose, which decrease with distance from the cartilage-synovial fluid interface. Thus, we tested the hypothesis that the oxygen consumption of each subpopulation is modulated by nutrient availability, by examining the expression of the Crabtree effect. The deep cells had a greater oxygen consumption than the superficial cells (V(max) of 6.6 compared to 3.2 fmol/cell/h), consistent with our observations of mitochondrial volume (mean values 52.0 vs. 36.4 microm(3)/cell). Both populations expressed the Crabtree phenomena, with oxygen consumption increasing approximately 2.5-fold in response to glycolytic inhibition by glucose deprivation or 2-deoxyglucose. Over 90% of this increase was oligomycin-sensitive and thus accounted for by oxidative phosphorylation. The data contributes towards our understanding of chondrocyte energy metabolism and provides information valuable for the accurate calculation of the oxygen concentration that the cells experience in vivo. The work has further application to the optimisation of bioreactor design and engineered tissues.
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Hunter in 174318 described ulcerations of articular cartilage as problems that will not heal. The clinical consequences of full-thickness articular cartilage defects of the knee are pain, swelling, mechanical symptoms, athletic and functional disability, and osteoarthritis (Fig. 1 ...
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Article
Objective Autologous chondrocyte implantation (ACI) is a form of tissue engineering that is being used increasingly to treat damaged articular cartilage. What happens at the graft site subsequent to the transplantation of chondrocytes beneath a periosteal flap has largely remained a matter of conjecture. We examined biopsy samples from the graft site using a panel of specific antibodies to investigate the cellular mechanisms involved and to determine whether remodeling of the matrix occurs.Methods Ten full-depth core biopsy samples were obtained from patients who had undergone ACI 9–30 months previously (ages 28–53 years), in addition to 6 “control” biopsy samples. Cryosections were evaluated by standard histologic examination using polarized light and immunohistochemistry. Antibodies specific for type II collagen (CIIC1) were used, as well as antibodies against the C-propeptide of type II collagen (R160) and its denaturation product (Col2-3/4m), as indicators of anabolism or catabolism. In addition, antibodies to the matrix proteinase–generated neoepitopes of the aggrecan core protein were used to demonstrate either aggrecanase (BC-3 and BC-13) or matrix metalloproteinase (MMP) (BC-4 and BC-14) activity.ResultsAll biopsy samples stained for type II collagen, even in areas of fibrocartilaginous morphology. There was evidence of newly synthesized type II collagen in addition to denatured collagen. MMP and aggrecanase activity on the proteoglycan population was evident, with aggrecanase being more active in fibrocartilaginous areas.Conclusion The findings of this study indicate that ACI is capable of not only cartilage repair but, in some cases, regeneration. This may be achieved by the turnover and remodeling of an initial fibrocartilaginous matrix via enzymatic degradation and synthesis of newly formed type II collagen.
Article
The aim of this work was to determine the pathways implicated in the mechanosensing of chondrocytes. Rat chondrocytes were cultured in collagen hydrogels of different stiffness (2-20 Pa) in normoxia and hypoxia, in monolayer and embedded inside hydrogels. First, chondrocyte were cultured on hydrogels in the presence of antibodies to block integrins. Second, custom RT-PCR array plates and western blot were used to detect changes in expression of genes implicated in downstream signalling pathways. The results allowed us to demonstrate the mechanosensing of chondrocytes for changes in stiffness in the range of Pascals. We also identified Non-Muscle Myosin II (NMMII) and integrins α1, β1 and β3 as participants in the mechanosensing, since their blockade inhibits the sensing of the stiffness, and they are up-regulated in the process. RT-PCR arrays and western blot detected up-regulation of Paxillin, RhoA, Fos, Jun and Sox9. We detected no expression of Src in the monolayer cultures, but we found a role for this protein in 3D. The expression of HIF-1α was not modified under normoxia but was found to participate under hypoxia. Focal Adhesion Kinase (FAK), showed a direct relationship with the expression of Aggrecan in hypoxia and an inverse one in normoxia. Finally, immunofluorescence analysis located the expression of factors AP-1, Sox-9 and HIF-1α inside the cell nuclei and RhoA, Src, Paxillin and FAK close to the cytoplasmic membrane. We determined here some of the genes that are up-regulated during the process of chondrocyte mechanosensing.
Article
Integrins are membrane integral glycoprotein heterodimers which were originally considered as cell-to-matrix adhesion receptors. Now evidence is growing that some integrins are also involved in cell-to-cell adhesion and share this role with other families of cell adhesion molecules like CAMs, cadherins, and selectins. This short review provides a summary of the roles of integrins in the organization and maintenance of tissues like the vascular endothelium and the epidermis which are formed by highly polarized cells. The early sorting of integrins to discrete membrane domains during development may be the key event in the organization of many tissues by inducing the conversion of non-polar migratory cells to polarized cells organized in monolayers. In cancer, a disturbance of cellular polarity may be crucial for the control of tumor invasive expansion and metastatic spreading.
Article
Cells actively probe the stiffness of their surrounding and respond to it. The authors recently found that maintenance of the chondrogenic phenotype was directly influenced by this property in 2D. Since studies about this process in 3D are still largely absent, this study aimed to transfer this knowledge into a 3D environment. Agarose was modified with RGD to allow active stiffness sensing or RGE as a control. Hydrogels with different mechanical properties were produced by using different concentrations of agarose. Primary chondrocytes were incorporated into the gel, cultured for up to two weeks, and then constructs were analyzed. Cells were surrounded by their own ECM from an early stage and maintained their chondrogenic phenotype, independent of substrate composition, as indicated by a high collagen type II and a lack of collagen type I production. However, softer gels showed higher DNA and GAG content and larger cell clusters than stiff gels in both RGD- and RGE-modified agarose. The authors hypothesize that matrix elasticity in the tested range does not influence the maintenance of the chondrogenic phenotype in 3D but rather the size of the formed cell ECM clusters. The deviation of these findings from previous results in 2D stresses the importance of moving towards 3D systems that more closely mimic in vivo conditions. Copyright © 2011 John Wiley & Sons, Ltd.
Article
To obtain sufficient cell numbers for cartilage tissue engineering with autologous chondrocytes, cells are typically expanded in monolayer culture. As a result, they lose their chondrogenic phenotype in a process called dedifferentiation, which can be reversed upon transfer into a 3D environment. We hypothesize that the properties of this 3D environment, namely adhesion site density and substrate elasticity, would influence this redifferentiation process. To test this hypothesis, chondrocytes were expanded in monolayer and their phenotypical transition was monitored. Agarose hydrogels manipulated to give different RGD adhesion site densities and mechanical properties were produced, cells were incorporated into the gels to induce redifferentiation, and constructs were analyzed to determine cell number and extracellular matrix production after 2 weeks of 3D culture. The availability of adhesion sites within the gels inhibited cellular redifferentiation. Glycosaminoglycan production per cell was diminished by RGD in a dose-dependent manner and cells incorporated into gels with the highest RGD density, remained positive for collagen type I and produced the least collagen type II. Substrate stiffness, in contrast, did not influence cellular redifferentiation, but softer gels contained higher cell numbers and ECM amounts after 2 weeks of culture. Our results indicate that adhesion site density but not stiffness influences the redifferentiation process of chondrocytes in 3D. This knowledge might be used to optimize the redifferentiation process of chondrocytes and thus the formation of cartilage-like tissue.
Article
Human mesenchymal stem cells (MSCs) reside under hypoxic conditions in vivo, between 4% and 7% oxygen. Differentiation of MSCs under hypoxic conditions results in inhibited osteogenesis, while chondrogenesis is unaffected. The reasons for these results may be associated with the inherent metabolism of the cells. The present investigation measured the oxygen consumption, glucose consumption and lactate production of MSCs during proliferation and subsequent differentiation towards the osteogenic and chondrogenic lineages. MSCs expanded under normoxia had an oxygen consumption rate of ∼98 fmol/cell/h, 75% of which was azide-sensitive, suggesting that these cells derive a significant proportion of ATP from oxidative phosphorylation in addition to glycolysis. By contrast, MSCs differentiated towards the chondrogenic lineage using pellet culture had significantly reduced oxygen consumption after 24 h in culture, falling to ∼12 fmol/cell/h after 21 days, indicating a shift towards a predominantly glycolytic metabolism. By comparison, MSCs retained an oxygen consumption rate of ∼98 fmol/cell/h over 21 days of osteogenic culture conditions, indicating that these cells had a more oxidative energy metabolism than the chondrogenic cultures. In conclusion, osteogenic and chondrogenic MSC cultures appear to adopt the balance of oxidative phosphorylation and glycolysis reported for the respective mature cell phenotypes. The addition of TGF-β to chondrogenic pellet cultures significantly enhanced glycosaminoglycan accumulation, but caused no significant effect on cellular oxygen consumption. Thus, the differences between the energy metabolism of chondrogenic and osteogenic cultures may be associated with the culture conditions and not necessarily their respective differentiation.
Article
Articular cartilage can tolerate a tremendous amount of intensive and repetitive physical stress. However, it manifests a striking inability to heal even the most minor injury. Both the remarkable functional characteristics and the healing limitations reflect the intricacies of its structure and biology. Cartilage is composed of chondrocytes embedded within an extracellular matrix of collagens, proteoglycans, and noncollagenous proteins. Together, these substances maintain the proper amount of water within the matrix, which confers its unique mechanical properties. The structure and composition of articular cartilage varies three-dimensionally, according to its distance from the surface and in relation to the distance from the cells. The stringent structural and biological requirements imply that any tissue capable of successful repair or replacement of damaged articular cartilage should be similarly constituted. The response of cartilage to injury differs from that of other tissues because of its avascularity, the immobility of chondrocytes, and the limited ability of mature chondrocytes to proliferate and alter their synthetic patterns. Therapeutic efforts have focused on bringing in new cells capable of chondrogenesis, and facilitating access to the vascular system. This review presents the basic science background and clinical experience with many of these methods and information on synthetic implants and biological adhesives. Although there are many exciting avenues of study that warrant enthusiasm, many questions remain. These issues need to be addressed by careful basic science investigations and both short- and long-term clinical trials using controlled, prospective, randomized study design.
Article
Analysis of cartilage from joints with osteoarthritis has demonstrated an increased number of cells in clones and evidence of DNA synthesis. Under circumstances of chronic injury chondrocytes are capable of mounting a significant repair response and can repudiate their DNA to form new cells. Evidence now exists also that articular chondrocytes from immature and adult animals can show either increased or decreased rates of proteoglycan synthesis in response to diverse physical and pathologic states. Thus the possibility exists of chondrocyte participation in the repair of articular cartilage. Articular cartilage is an avascular tissue, and thus does not respond as other body tissues do to trauma, i.e. by the phases of necrosis, inflammation, and repair. Necrosis certainly occurs, but due to avascularity no inflammation occurs, which considerably limits the number of cells that are available to respond to the trauma and the burden of repair falls on the existing chondrocytes. If the damage to the cartilage extends to the basal layers into the vascular subchondral cortex, all three phases of repair become possible. Lacerative injuries to articular cartilage that do not penetrate below the calcified zone evoke only a short-lived metabolic and enzymatic response which fails to provide sufficient numbers of new cells or matrix to repair even a minimum defect. These lesions remain unchanged for at least two years and do not progress to either the chondromalacia or osteoarthritic type of degenerative process. If the defect violates the underlying osseous plate, the base of the lesion heals with bone, but this new bone formation only fills the defect in the bone and stops at the old margin between calcified cartilage and osseous end-plate. The defect in the cartilage remains filled with the vascular fibrous tissue. The ultimate repair tissue closely resembles hyaline cartilage with diminished proteoglycans, but still retains the element of type-1 collagen, suggesting there is a mixture of fibrocartilage and hyaline cartilage. Lacerative injuries that do not violate the underlying bone for the most part remain stationary, neither healing nor progressing to osteoarthritis. Deep lacerations that penetrate the subchondral bone evoke an exuberant healing response from the subchondral bone which ultimately then becomes a form of hyaline cartilage with some fibrocartilaginous elements. Impactive injuries to cartilage that exceed a critical threshhold cause injury not only to chondrocytes but also to underlying bone and in experimental studies rapidly progress to an osteoarthritic lesion. (Weinstein, Iowa City, Iowa)
Article
Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.
Article
Stem cells transit along a variety of lineage-specific routes towards differentiated phenotypes. These fate decisions are dependent not just on the soluble chemical cues that are encountered or enforced in vivo and in vitro, but also on physical cues from the cellular microenvironment. These physical cues can consist of both nano- and micro-scale topographical features, as well as mechanical inputs provided passively (from the base properties of the materials to which they adhere) or actively (from extrinsic applied mechanical deformations). A suitable tool to investigate the coordination of these cues lies in nanofibrous scaffolds, which can both dictate cellular and cytoskeletal orientation and facilitate mechanical perturbation of seeded cells. Here, we demonstrate a coordinated influence of scaffold architecture (aligned vs. randomly organized fibers) and tensile deformation on nuclear shape and orientation. Sensitivity of nuclear morphology to scaffold architecture was more pronounced in stem cell populations than in terminally differentiated fibrochondrocytes. Tension applied to the scaffold elicited further alterations in nuclear morphology, greatest in stem cells, that were mediated by the filamentous actin cytoskeleton, but not the microtubule or intermediate filament network. Nuclear perturbations were time and direction dependent, suggesting that the modality and direction of loading influenced nuclear architecture. The present work may provide additional insight into the mechanisms by which the physical microenvironment influences cell fate decisions, and has specific application to the design of new materials for regenerative medicine applications with adult stem cells.
Article
In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage.
Article
We compared the morphology and cytoskeleton of chondrocytes seeded in monolayer or in agarose gels with those retained in situ i.e. within their extracellular matrix-the chondrocyte's natural habitat. Cartilage specimens were harvested from adult bovine femora. Chondrocytes were either enzymatically isolated to seed in both monolayer and agarose gel culture conditions or retained in situ. Full thickness cartilage on bone was sliced both parallel and perpendicular to the articular surface. After immunostaining, the morphology of chondrocytes and of their cytoskeletal organization, i.e. distribution of actin and vimentin, in chondrocytes seeded both in monolayer and 3D agarose and those retained in situ, were assessed using confocal laser scanning microscopy. The general cytoskeletal disposition of chondrocytes in situ was similar to that in agarose gel. Actin was seen to form stress fibres only in 2D culture, but not in 3D culture and in situ. In these latter conditions, actin showed a punctate staining pattern. The vimentin meshwork spanned the cytoplasm from the plasma membrane to the nuclear membrane in all culture conditions. However, the organization of the vimentin had a radiate organization in chondrocytes in monolayer and a more circumferential arrangement both in agarose gel and in situ. We further observed: (i) the prevalence of a bichondral configuration of chondrocytes in situ and (ii) the existence of a vimentin link joining some of the sister cells in situ. Bichondral configuration linked with cytoskeletal elements may potentially be significant for the normal function of the chondrocytes, and therefore have implications for approaches to tissue engineering of cartilage.
Article
Cartilaginous gene expression decreased when chondrocytes were expanded on cell-culture plates. Understanding the dedifferentiation mechanism may provide valuable insight into cartilage tissue engineering. Here, we demonstrated the relationship between the nuclear shape and gene expression during in vitro expansion culture of chondrocytes. Specifically, the projected nuclear area increased and cartilaginous gene expressions decreased during in vitro expansion culture. When the nuclear deformation was recovered by cytochalasin D treatment, aggrecan expression was up-regulated and type I collagen (Col1a2) expression was down-regulated. These results suggest that nuclear deformation may be one of the mechanisms for chondrocyte dedifferentiation during in vitro expansion culture.
Article
The development of ion selective microelectrodes has made it possible to measure the normal steady state in the pericellular environment together with ion fluxes in response to physiological or pathological disturbances. Combined intracellular and extracellular measurements indicate that there is a considerable range of ability between various types of cells in the efficiency with which they can tolerate changes in pericellular conditions. Macrophages are extremely tolerant while cells of the cerebral cortex require a very finely controlled local environment. Combination of ion selective probes with microelectrodes which measure substrate and oxygen availability extend the information which can be obtained about ionic composition of cellular environment and the factors which are important in its homostasis.
Article
The pathogenesis and clinical significance of articular cartilage lesions of the knee persist as topics of considerable interest among orthopedic surgeons. This study was designed to assess the association of articular cartilage degeneration with concomitant intraarticular abnormalities and to correlate the prevalence and severity of articular cartilage damage with preoperative historical and physical exam findings in patients presenting with knee pain. Twenty-six history and physical exam data points were prospectively collected from 192 patients (200 knees), consecutively undergoing arthroscopic knee surgery. During surgery, all articular cartilage lesions were recorded with respect to size, location, and character and were graded according to Oglivie-Harris et al. All concomitant knee joint abnormalities were simultaneously recorded. Of 200 knees examined arthroscopically, 12 knees revealed no demonstrable etiology for the presenting symptoms, 65 knees revealed assorted intraarticular pathology but no articular cartilage degeneration, and the remaining 123 knees revealed a total of 211 articular cartilage lesions (103 femoral, 72 patellar, 36 tibial); 7 femoral, 6 patellar and 0 tibial lesions were completely isolated (no concomitant knee joint pathology). The concomitance of femoral defects with tibial lesions was highly significant (p = 0.01). Femoral and tibial articular cartilage lesions were strikingly correlated with the presence of an unstable torn meniscus (p less than 0.001). Medial compartment articular cartilage lesions were significantly more common (p = 0.001), more closely associated with meniscal derangement, and appreciably more severe than lateral compartment lesions. In 75% of anterior cruciate ligament-deficient knees with concomitant articular cartilage degeneration, the duration from injury to surgery was greater than 9 months, and in each of these cases, a history of reinjury to the knee was elicited. From these data one can conclude that: (a) in some patients with painful knees, isolated articular cartilage lesions may be the only abnormality noted at arthroscopy; (b) unstable meniscal tears are significantly associated with destruction of articular cartilage; (c) the medial compartment is particularly susceptible to articular cartilage degeneration; and (d) in our series, anterior cruciate ligament tears were increasingly associated with articular cartilage destruction as the elapsed time from injury to arthroscopy increased.
Article
The relationship between cell shape, proliferation, and phenotypic expression was studied in human chondrocytes. Shape was controlled independent of serum concentration, anchorage, and cell density by alteration of substratum adhesiveness with poly(2-hydroxyethyl methacrylate) (poly[HEMA]). Cells that were held rounded displayed features of the chondrocyte phenotype; i.e., they were round, proliferated slowly, incorporated low levels of [3H]thymidine into DNA, and incorporated large amounts of 35SO4 into glycosaminoglycans. In contrast, cells that were held flat were fibroblast-like: they exhibited flattened morphology, more rapid growth, greater incorporation of [3H]thymidine, and less incorporation of 35SO4. These studies suggest that cell shape may play an important role in phenotypic expression in chondrocytes.
Article
The differentiated phenotype of rabbit articular chondrocytes consists primarily of type II collagen and cartilage-specific proteoglycan. During serial monolayer culture this phenotype is lost and replaced by a complex collagen phenotype consisting predominately of type I collagen and a low level of proteoglycan synthesis. Such dedifferentiated chondrocytes reexpress the differentiated phenotype during suspension culture in firm gels of 0.5% low Tm agarose. Approximately 80% of the cells survive this transition from the flattened morphology of anchorage-dependent culture to the spherical morphology of anchorage-independent culture and then deposit characteristic proteoglycan matrix domains. The rates of proteoglycan and collagen synthesis return to those of primary chondrocytes. Using SDS-polyacrylamide gel electrophoresis of intact collagen chains and two-dimensional cyanogen bromide peptide mapping, we demonstrated a complete return to the differentiated collagen phenotype. These results emphasize the primary role of cell shape in the modulation of the chondrocyte phenotype and demonstrate a reversible system for the study of gene expression.
Article
Determination of contact areas in diarthrodial joints is necessary for understanding the state of stress within the articular cartilage layers and the supporting bony structures. The present study describes the use of a stereophotogrammetry (SPG) system [Huiskes et al., J. Biomechanics 18, 559-570 (1985) and Ateshian et al., J. Biomechanics 24, 761-776 (1991)] for determining contact areas in diarthrodial joints, using a surface proximity concept similar to the one used by Scherrer et al. [ASME J. biomech. Engng 101, 271-278 (1979)]. This method consists of evaluating the proximity of the articular surfaces to determine joint contact areas using precise geometric models of the joint surfaces obtained from the SPG system, and precise kinematic data, also obtained from SPG. In this study, the SPG method for determining contact areas is compared to other commonly used methods such as dye staining, silicone rubber casting and Fuji film contact measurement techniques which have been often used and reported by other investigators. The bovine glenohumeral joint and the bovine lateral tibiofemoral articulation (without the meniscus) were used to represent congruent and incongruent joints, respectively. While all the methods yielded consistent contact patterns for the incongruent tibiofemoral articulations, the results for the congruent bovine glenohumeral joints showed that the SPG and Fuji film methods were in better agreement than those obtained from the dye staining and silicone rubber casting methods. The advantages of the new SPG method are that it can be used for intact joints, and used repeatedly and quickly thus making contact-area movement analyses possible [Soslowsky et al., J. orthop. Res. 10, 524-534 (1992)]. The results of this comparison study show that the SPG technique is a reliable and versatile method for determining contact areas in diarthrodial joints.
Article
Two computer models of nonuniform contact stress on the articular surface of the human hip were used to study the relationship between chronically excessive articular cartilage contact stress and long-term clinical outcome in a series of patients with congenital dislocation of the hip (CDH). The analyzed database consisted of 409 stylus digitized radiographs from 83 patients with unilateral CDH, who had been treated by closed reduction, and whose average follow-up time was 29.2 y. The first model (nonuniform Legal) involved a three-dimensional contact stress distribution function whose pole was coincident with the resultant force acting through the hip, and which acted over a contact area whose borders were determined soley by bony landmarks. In the second model (Brinckmann), the direction of the pole of the contact stress distribution function was initially unknown; one border of the contact region was determined by radiographic landmarks, while the other border depended upon the pole of the iteratively determined contact stress distribution function. In both models, the contact stress distributions were converted to area engagement histograms, corresponding to the fractional areas of cartilage experiencing specific ranges of stress (0.5 MPa increments). These histograms were integrated over time to calculate a cumulative contact stress overdose, which was then compared to clinical outcome. Reasonable correlations (Spearman rho = 0.63-0.66) with patient outcomes were obtained for optimally chosen damage thresholds, although these thresholds were appreciably different (2.0 versus 4.5 MPa) due to the respective modelling assumptions.
Article
The engineering of living tissues in vivo requires new concepts in cell culture technology. In contrast to conventional cell cultures, the development of tissues depends on a three-dimensional arrangement of cells and the formation or synthesis of an appropriate extracellular matrix. Special emphasis is given to the major role of the extracellular matrix and cell differentiation in an artificial tissue. New technical approaches of in vitro tissue engineering are compared to the natural development of tissues in vivo. Current methods using resorbable biomaterials, tissue encapsulation and perfusion culture are discussed. Major consideration is given to scaffold structures of biomaterials that define a three-dimensional shape of a tissue or guide matrix formation. The different goals of tissue engineering such as in vitro models and transplant production are taken into account in the described techniques. Practical concepts comprising cell multiplication and differentiation in subsequent steps for future clinical applications are outlined.
Article
The degeneration of articular cartilage as part of the clinical syndrome of osteoarthritis is one of the most common causes of pain and disability in middle-aged and older people. The strong correlation between increasing age and the prevalence of osteoarthritis, and recent evidence of important age-related changes in the function of chondrocytes, suggest that age-related changes in articular cartilage can contribute to the development and progression of osteoarthritis. Although the mechanisms responsible for osteoarthritis remain poorly understood lifelong moderate use of normal joints does not increase the risk. Thus, the degeneration of normal articular cartilage is not simply the result of aging and mechanical wear. However, high-impact and torsional loads may increase the risk of degeneration of normal joints, and individuals who have an abnormal joint anatomy, joint instability, disturbances of joint or muscle innervation, or inadequate muscle strength or endurance probably have a greater risk of degenerative joint disease. Recent work has shown the potential for the restoration of an articular surface. Currently, surgeons frequently debride joints and penetrate subchondral bone as well as perform osteotomies, with the intent of decreasing symptoms and restoring or maintaining a functional articular surface. The results of these procedures vary considerably among patients. Clinical and experimental work has shown the important influence of loading and motion on the healing of articular cartilage and joints. Experimental studies have revealed that transplantation of chondrocytes and mesenchymal stem cells; use of periosteal and perichondrial grafts, synthetic matrices, and growth factors: and other methods have the potential to stimulate the formation of a new articular surface. The long-term follow-up of small series of patients has shown that the transplantation of osteochondral autologous grafts and allografts can be effective for the treatment of focal defects of articular cartilage in selected patients. Thus far, none of these methods has been shown to predictably restore a durable articular surface to an osteoarthritic joint, and it is unlikely that any one of them will be uniformly successful. Rather, the available clinical and experimental evidence indicates that future optimum methods for the restoration of articular surfaces will begin with a detailed analysis of the structural and functional abnormalities of the involved joint and the patient's expectations for future use of the joint. On the basis of this analysis, the surgeon will develop a treatment plan that potentially combines correction of mechanical abnormalities (including malalignment, instability, and intra-articular causes of mechanical dysfunction), debridement that may or may not include hunted penetration of subchondral bone, and applications of growth factors of implants that may consist of a synthetic matrix that incorporates cells or growth factors or use of transplants followed by a postoperative course of controlled loading and motion.
Article
Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.
Article
Autologous chondrocyte transplantation (ACT) provides durable hyaline repair tissue in correctly selected patients; it is indicated for full thickness, weightbearing condyle injuries, and injuries to the trochlea of the femur. ACT results in reproducibly satisfactory results, with return to high level activities, including sports, in over 90% of these patients. Second look arthroscopies demonstrate macroscopic integrity of the grafts; and biopsies demonstrate hyaline cartilage repair, which is critical, as shown clinically, to giving durable results at two to nine years follow-up. Also discussed in this article is surgical technique which is especially important for complex reconstructions. As technical refinements and rehabilitation protocols improve, results for treating patellar and tibial injuries may improve; at this time, the response to treating bipolar focal chondral injuries is unknown and not recommended.
Article
Cartilage tissue engineering can provide functional cartilaginous constructs that can be used for controlled in vitro studies of chondrogenesis and potentially for in vivo articular cartilage repair. Ideally, engineered cartilage should be indistinguishable from native articular cartilage with respect to zonal organization, biochemical composition, and mechanical properties. In the model system presented here, chondrogenic cells are expanded in vitro as required, seeded onto three-dimensional polymeric scaffolds, and cultured in bioreactor vessels. During the course of in vitro cultivation, construct cellularity plateaus at a physiologic level, fractions of glycosaminoglycan and Type II collagen increase progressively, and the scaffold biodegrades. Construct structure (composition, morphology) and function (biosynthetic activity, mechanical properties) depend on cultivation conditions. This paper reviews recent studies of in vitro modulation of chondrogenesis by: (1) cell seeding density and source; (2) the tissue regeneration template; (3) biochemical regulatory signals; (4) mixing, mass transport and hydrodynamic forces; and (5) cultivation time. Key requirements and some of the critical research needs for successful cartilage tissue engineering are discussed.
Article
The deformational behavior of articular cartilage has been investigated in confined and unconfined compression experiments and indentation tests, but to date there exist no reliable data on the in situ deformation of the cartilage during static loading. The objective of the current study was to perform a systematic study into cartilage compression of intact human femoro-patellar joints under short- and long-term static loading with MR imaging. A non-metallic pneumatic pressure device was used to apply loads of 150% body weight to six joints within the extremity coil of an MRI scanner. The cartilage was delineated during the compression experiment with previously validated 2D and 3D fat-suppressed gradient echo sequences. We observed a mean (maximal) in situ deformation of 44% (57%) in patellar cartilage after 32 h of loading (mean contact pressure 3.6 MPa), the femoral cartilage showing a smaller amount of deformation than the patella. However, only around 7% of the final deformation (3% absolute deformation) occurred during the first minute of loading. A 43% fluid loss from the interstitial patellar matrix was recorded, the initial fluid flux being 0.217 +/- 0.083 microm/s, and a high inter-individual variability of the deformational behavior (coefficients of variation 11-38%). In conjunction with finite-element analyses, these data may be used to compute the load partitioning between the solid matrix and fluid phase, and to elucidate the etiologic factors relevant in mechanically induced osteoarthritis. They can also provide direct estimates of the mechanical strain to be encountered by cartilage transplants.
Article
The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0-50% static compression, and the transient and steady-state incorporation of [3H]proline and [35S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0-300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [3H]proline and [35S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of 125I-labeled IGF-I into cartilage explants. Static compression decreased both [3H]proline and [35S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [3H]proline and [35S]sulfate incorporation in a dose- dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (τ) for IGF-I stimulation of cartilage explants was approximately 24 h, while τ for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter τ for 125I-labeled IGF-I transport into cartilage but decreased the concentration of 125I-labeled IGF-I in the tissue at equilibrium.
Article
Articular cartilage in adults has a poor ability to self-repair after a substantial injury; however, it is not known whether there is a cartilage resurfacing technique superior to the existing techniques. It is not satisfactory that at the beginning of the new millennium, there still is a lack of randomized studies comparing different cartilage repair techniques and there still is little knowledge of the natural course of a cartilaginous lesion. To date, various articular cartilage resurfacing techniques have the potential to improve the repair of cartilage defects and reduce the patient's disability. One such cartilage repair technique is autologous chondrocyte transplantation combined with a periosteal graft. Since the first patient was operated on in 1987, much interest in cartilage repair and cell engineering has emerged. The experience with autologous chondrocyte transplantation during the past 13 years with in vitro chondrocyte expansion, cartilage harvest, and postoperative biopsy technique is discussed, and the latest followup of 213 consecutive patients in different subgroups with 2 to 10 years followup is presented. The technique gives stable long-term results with a high percentage of good to excellent results (84%-90%) in patients with different types of single femoral condyle lesions, whereas patients with other types of lesions have a lower degree of success (mean, 74%).
Article
Autologous chondrocyte implantation (ACI) is a form of tissue engineering that is being used increasingly to treat damaged articular cartilage. What happens at the graft site subsequent to the transplantation of chondrocytes beneath a periosteal flap has largely remained a matter of conjecture. We examined biopsy samples from the graft site using a panel of specific antibodies to investigate the cellular mechanisms involved and to determine whether remodeling of the matrix occurs. Ten full-depth core biopsy samples were obtained from patients who had undergone ACI 9-30 months previously (ages 28-53 years), in addition to 6 "control" biopsy samples. Cryosections were evaluated by standard histologic examination using polarized light and immunohistochemistry. Antibodies specific for type II collagen (CIIC1) were used, as well as antibodies against the C-propeptide of type II collagen (R160) and its denaturation product (Col2-3/4m), as indicators of anabolism or catabolism. In addition, antibodies to the matrix proteinase-generated neoepitopes of the aggrecan core protein were used to demonstrate either aggrecanase (BC-3 and BC-13) or matrix metalloproteinase (MMP) (BC-4 and BC-14) activity. All biopsy samples stained for type II collagen, even in areas of fibrocartilaginous morphology. There was evidence of newly synthesized type II collagen in addition to denatured collagen. MMP and aggrecanase activity on the proteoglycan population was evident, with aggrecanase being more active in fibrocartilaginous areas. The findings of this study indicate that ACI is capable of not only cartilage repair but, in some cases, regeneration. This may be achieved by the turnover and remodeling of an initial fibrocartilaginous matrix via enzymatic degradation and synthesis of newly formed type II collagen.
Article
The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.