You are on page 1of 12

Acta Biomaterialia 33 (2016) 1–12

Contents lists available at ScienceDirect

Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Review article

Differentiation of mesenchymal stem cells for cartilage tissue
engineering: Individual and synergetic effects of three-dimensional
environment and mechanical loading
J.A. Panadero a,b,⇑, S. Lanceros-Mendez a,c, J.L. Gomez Ribelles b,d
a

Center/Departament of Physics, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Center for Biomaterials and Tissue Engineering, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain
BCMaterials, Parque Científico y Tecnológico de Bizkaia, 48160-Derio, Spain
d
Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Valencia, Spain
b
c

a r t i c l e

i n f o

Article history:
Received 15 July 2015
Received in revised form 17 December 2015
Accepted 25 January 2016
Available online 27 January 2016
Keywords:
Mesenchymal stem cells
Chondrogenic differentiation
Cell adhesion
Mechanotransduction
Mechanical loading

a b s t r a c t
Chondrogenesis of dedifferentiated chondrocytes and mesenchymal stem cells is influenced not only by
soluble molecules like growth factors, but also by the cell environment itself. The latter is achieved
through both mechanical cues – which act as stimulation factor and influences nutrient transport –
and adhesion to extracellular matrix cues – which determine cell shape. Although the effects of soluble
molecules and cell environment have been intensively addressed, few observations and conclusions
about the interaction between the two have been achieved. In this work, we review the state of the
art on the single effects between mechanical and biochemical cues, as well as on the combination of
the two. Furthermore, we provide a discussion on the techniques currently used to determine the
mechanical properties of materials and tissues generated in vitro, their limitations and the future
research needs to properly address the identified problems.
Statement of Significance
The importance of biomechanical cues in chondrogenesis is well known. This paper reviews the existing
literature on the effect of mechanical stimulation on chondrogenic differentiation of mesenchymal stem
cells in order to regenerate hyaline cartilage. Contradictory results found with respect to the effect of different modes of external loading can be explained by the different properties of the scaffolding system
that holds the cells, which determine cell adhesion and morphology and spatial distribution of cells, as
well as the stress transmission to the cells. Thus, this review seeks to provide an insight into the interplay
between external loading program and scaffold properties during chondrogenic differentiation. The
review of the literature reveals an important gap in the knowledge in this field and encourages new
experimental studies. The main issue is that in each of the few cases in which the interplay is investigated, just two groups of scaffolds are compared, leaving intermediate adhesion conditions out of study.
The authors propose broader studies implementing new high-throughput techniques for mechanical
characterization of tissue engineering constructs and the inclusion of fatigue analysis as support
methodology to more exhaustive mechanical characterization.
Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Differentiation in 3D culture without mechanical stimulus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
3D structure of cartilage in vivo, formation of cartilage during development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Differentiation effects of 3D structures in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding author at: Center/Departament of Physics, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
E-mail address: ex2323@fisica.uminho.pt (J.A. Panadero).
http://dx.doi.org/10.1016/j.actbio.2016.01.037
1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

2
2
2
3

. . whose molecules. . . . . . . . The main features of cell response when cultured in a chondrogenic medium in a 3D environment in the absence of mechanical stimulus (individual effect of 3D structure) as well as the general effects of mechanical loading have been described. . . . 2. . . . . . . . . . . . . . . 4. . . . . . . . . . . . 3. . 5. . . . . . . Threedimensional scaffold materials. . . . . . . . chondrocytes at the site of the growth plate become hypertrophic. . . . . . . . . As differentiation progresses. . 2. . . . . decorin. growth factor supply. . . . . . is also enriched in some components that in the bulk ECM are less predominant. . . . is reduced. hypoxia. . . . . . . Interaction of mechanical loading with 3D structure and cell adhesion . like the proteoglycan tenascin. . . . . . . . . . . . . and fibromodulin). . . Mechanotransduction signaling . . . . . . . . . . . . . . . .2. . Encapsulation and scaffolding materials . there is still lack of high-throughput techniques for mechanical characterization of tissue engineering constructs. . . . . . . the ECM and the PCM drive the three-dimensional conformation to their components by integrin-based cell adhesion. . such as fatigue analysis. . . . . . . . . . . . . . . . . . . the expression of many focal adhesion receptors to early pericellular matrix molecules [11. . . The 3 3 4 4 5 5 6 7 8 9 9 undifferentiated MSCs migrate to the sites of the developing cartilage and condense through a specific combination of precartilage matrix and cell adhesion molecules. . . . . . . . . . . . . . Pellets and micromass culture . . . . . . . . . Differentiation in 3D culture without mechanical stimulus 2. . . . . . . . 2. . . . . . . the chondrocytes remain with the mature phenotype for the rest of the lifespan of the organism. . . . 3D structure is a regulator of cell shape and chondrogenic differentiation during development in limb primordia.2 J. . such as collagen type VI [24]. . . . . . . . . . . . . . . . . . link protein. whose negatively charged glycosaminoglycans are responsible for swelling [20. . . . . . The chondrogenic capacity of mature chondrocytes dedifferentiating in monolayer expansion and mesenchymal stem cells has been demonstrated. . . . . . . . . . as well as implementation of other relevant assays for the evaluation of mechanical properties. . . . . . . . . . . . morphological changes take place in the chondrogenic progenitors. Finally. The same can be stated about other culture parameters. . . . . Although high-throughput techniques have been implemented for the identification of extracellular matrix components produced in chondrogenic differentiation assays. . . . . . . . . Then. . upregulating the synthesis of transcription factors (such as Sox5. . . In vivo. . . . . . . . . . . . . . . . . . . . . . . . . the differentiated chondrocytes start to be isolated by the ECM. . . . . . . . from their fibroblastic-like shape to the more spherical morphology of hyaline chondrocytes. it is known that the inhibition of N-cadherin and the route Wnt is a necessary step to differentiate into cartilage after condensation [21]. . . . . . there are two possible fates for hyaline chondrocytes: (a) in endochondral bone development of long bones until adolescence. . . . . . and even the spatial organization of them vary with the zone of cartilage [23]. . . . . . . . . . . . . . formation of cartilage during development In vivo. . . . . . . . . . . . . . . .1. . . . . thus. . . Mechanical loading in vitro . . . . . . which regulate the transcription and transduction of cartilage-specific extra-cellular matrix (ECM) molecules such as collagen types II. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV.2. . . . . . . The ECM produced by the chondrocytes consists of 45–50% collagens (90% of which is collagen type II) and 20–25% proteoglycans (predominantly aggrecan. . . . . . . . . . . . This issue also demands new methodological approaches to identify and understand that interplay and to manage the large quantity of generated information. . . . Mechanical loading in vivo . . . . . . . . . . This review focusses on the interplay between mechanical stimuli and the 3D environment for chondrogenesis from mesenchymal stem cells. . . . . . . . . . . . . . . . . . . . . . . Conclusions and open questions . . . . . . . . . . . mainly N-cadherine and NCAM [10]. . . . . . . . . . . The simultaneous influence of many external factors on cell fate sometimes prevents reaching such conclusions. . . . . 3D structure of cartilage in vivo. . . . .12]. fibronectin and laminin [25–27]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cell–material interaction. . . . . . . . . . / Acta Biomaterialia 33 (2016) 1–12 3. . . In mature hyaline cartilage. . . . . Moreover. . . . . . . . . . . . A discussion on these methodological needs and possible future solutions is also provided in this work. . . . . 1. . . . . . . . . . . . . . . . . . . For instance. . . . . . . . . . . . . . . . . . each lacuna acts as an individual functional unit responsible for maintaining the ECM metabolically. . . . . . . . . . . the interplay between mechanical loading and biochemical cues of the substrate is explored. . . . . . . . . . . . . . 3. . .18] of membrane receptors to molecules of the matrix like fibronectin. mechanical stimulus and co-culture with mature cells have been emphasized as important factors driving differentiation to the hyaline cartilage phenotype. The relative values of these components. . . . . . . . . . . . . . . . . . . .A. as it demands more exhaustive studies comparing materials with different cell adhesion patterns. . . . References . . . produce alkaline phosphatase and collagen type X and are eventually reabsorbed while new bone is formed. . . . . . . . . . which is useful to identify their role in mechanotransduction processes. . . . . . . . The importance of 3D environment configuration for chondrogenesis is such that even MSCs cultured in decellularized cartilage . . . . . . . . At this stage of development. . . . . 3. . . . . thus also being called transient chondrocytes [19] and (b) in the articular hyaline tissues. . . . . . . . . These morphological changes initiate a re-organization of cytoskeleton [13] organization. . . . . . . . . . . . or pericellular matrix (PCM). . . . . . . . . . Consequently. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . This condensation allows essential cell–cell surface interactions and signaling events that conclude in differentiation to hyaline chondrocytes. . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . simultaneous analysis of different studies carried out with particular supporting materials seems to be necessary to get general conclusions about this feature. . IX. . . . . . Differentiation in 3D: mechanical loading effects . . . chondrogenesis in vitro being ruled by the characteristics of the environment in which pluripotential cells are cultured. . . . Sox6 and specially Sox9 [14]). There are nevertheless important drawbacks in the literature that hinder reaching conclusions with respect to the influence of specific culture parameters on cell differentiation or cell biology. . . . . . . . . . the immediately surrounding matrix of the chondrocytes. . . . . . .2. . .23]. . . . . . . . Panadero et al. . . . . . . . and XI [15] and the highly-sulfated proteoglycan aggrecan [16]. . . . . Mechanical measurements in cartilage tissue engineering: a necessity to complement the biochemical data . . . . . . . . . . .4. . . . . .22. . . . . . . . . hyaluronan or biglycan. . . . . . .1. Although the mechanisms determining the two different fates remain unclear. .3. . . . . . . . . . 3. . . . . . . . .1. . . . Introduction The literature about cartilage engineering is large and some reviews can be found in references [1–9]. . . . . . . . . . . . . . . . . . . . . . . are separated in lacunae – formed by a highly hydrated extracellular matrix (ECM) – and maintain the ECM of persistent hyaline cartilage [20]. . . . . . . . . . . favor the rounded shape of chondrocytes by steric hindrance [17. . . the effect of mechanical stimulation is dependent on the scaffolding material in which cells are immersed. . . . . . . . . .2. . . . . . . . . like fibronectin. . . . . . . . . . . . . . . . . . . . . . .

In micromass cultures. After 24 h. Panadero et al. As cell shape is a factor of differentiation. In bone-marrow-derived MSCs it is shown that a more rounded nuclear shape was associated to the larger expression of molecular chondrogenic markers [52].J. Hydrogels. semicrystalline polymers providing a broader set of structures due to the extended plethora of processing methods [81]. These polymers [69] include hydrophobous biodegradable polyesters. It has been reported that micromass culture systems enhance chondrogenesis more than standard 3 pellet systems [49].78] among others [79].2. Differentiation effects of 3D structures in vitro 2. hinder linking of integrin-binding with fibronectin and regulation of mRNA transcription in the nucleus [40]). poly(vinyl alcohol) [68] and others.2. Pellet cultures consist of centrifuging the MSC suspension in a conical tube and then incubation.2. 2. flattened morphology and develop a fibrous phenotype. Both micromass and pellet cultures have been useful to show that cell and nuclear shape are strong regulators of cell growth and physiology also in vitro. Chondrogenic differentiation is only promoted by agents like cytochalasin D [33]. biodegradable poly(ether ester) multiblock copolymers [75]. resulting in the control of protein expression and posttranslational modifications. MSCs are capable of chondrogenic differentiation in pellet culture using serum-free medium containing glucocorticoids and TGF-b family [45]. for example fibrin with PCL [80]. However. As indicated above. and in a lesser extent. In vivo.1. which is a negative actin cytoskeleton-regulating protein. the interactions of the cells with their matrix involve intracellular signaling beyond just the morphology of the cells. The first strategies for MSC differentiation toward chondrogenic in 3D were designed to mimic the conditions during stem cell condensation: pellet and micromass cultures.50. polyglicolide [71]. After 24 h in culture.1. in the micromasses the cartilage-like tissue is more homogenous and enriched in collagen II. which is an advantage for surgery solutions. Hydrogels are formed by cross-linking of chain molecules. to intermediate filaments and microtubules [16. that. the inhibition of the other downstream metabolite in this pathway. mainly semicrystalline polymers. the morphological change to the rounded shape and the subsequent reorganization of the cytoskeleton is a key component in the initiation of chondrogenic differentiation. Both can surpass some limitations of micromass and pellet systems. synthetic hydrogels as crosslinked poly(ethylene glycol) [67]. Encapsulation and scaffolding materials Although micromass culture being a suitable as model is able to mimic some of the conditions for chondrogenesis during development. resembling. it is hard to obtain a satisfactory cartilage in pellet culture. as shown by upregulation of collagen X [47]. as well as their copolymers such as polylactide [70]. Thus. 2D configuration enhances the natural tendency of MSCs to express relatively more collagen type I [44]. decreasing the expression of fibrocartilage collagen and collagen X. A broad set of different materials have been used to produce hydrogels for cartilage tissue engineering: proteins as collagen type I or collagen type II [53–55]. chondroitin sulfate [61]. All chondrogenic cultures are performed for more than 21 days. scaffolds prevent the diffusion of transplanted cells and can improve integration. These membrane receptors to the matrix are linked to the three cytoskeletal networks: largely to actin microfilaments. which usually lead to necrotized tissue. Further. which can result in cells remaining in pre-cartilage stage. These scaffolds must be evaluated first in vitro to comprehend their effects in differentiation. elastin-like polypeptides [59]. fibrin [56–58]. Disruption of tubulin (microtubules) with chemical agents like colcemid and nocodazole does not lead to chondrogenic differentiation of stem cells [38]. because their viscoelastic behavior can be suitable for the mechanical environment of the cartilage. in adults disappears when the lacunae are formed. The interactions determine the polymerization and organization of the cytoskeletal filaments. / Acta Biomaterialia 33 (2016) 1–12 in vitro are capable of chondrogenic differentiation without addition of other exogenous factors [28–30]. Rho-associated protein kinase (ROCK). than GAG and collagen II. Two kinds of biomaterial supports are employed: hydrogels. poly (3-hydroxybutyrateco-3-hydroxyhexanoate) [76]. it has two main problems as a therapy: it is not suitable for implantation and produce necrotic problems. MSCs take a spread. a droplet of cell suspension is carefully placed in the center of each well of a multiwell plate. polycaprolactone [72–74]. The nutrients and the oxygen reach the central zone of the micromasses easier than the pellets. 2. Hydrogels show interesting characteristics such as the possibility of obtaining highly swollen structures approaching similar conditions than cartilage natural ECM and also the possibility of obtaining cell homogeneous distributions. However. polysaccharides as hyaluronic acid [60–62].2. 3D systems for in vitro culture provide more surface for cell adhesion and proliferation.2. and the dissociation of many receptor–matrix complexes in cartilage dissipates partially the isotonic tension between the contractile forces of the cytoskeleton and the resistive force of the matrix [36]. 2. non-specific of cartilage. This tension produces a signal propagation from adhesion sites and transmit signals by linking to the nucleus [37]. which disrupt actin cytoskeleton through direct effects in several parts of the network (capping of the endings of actin microfilaments [39].43]. and it is known that mature chondrocytes de-differentiate [42.83]. the cells aggregate and form a round cell pellet.51]. in particular. followed by the addition of chondrogenic medium. One reason for the existence of collagen type I and type X can be the lack of time regulation of the adhesion molecules typical from condensation. Many combinations of these hydrogels with polymer scaffolds can exist. the effect of scaffolds on chondrogenesis is mainly regulated by cell interactions with the matrix and the morphologic configuration acquired as a consequence [82. at the most.33–35]. and also biostable acrylic polymers [77. Inhibition of regulatory metabolites in some pathways like RhoA. Additionally. Cells are allowed to adhere at 37 °C for some hours. agar gel. Cells are often found undifferentiated or necrotized in the central region of the pellet and only the outside layer cells undergo chondrogenic differentiation [46– 48].32]. In adherent 2D surfaces that . the cells in every droplet merge and form a spherical mass. Thus. which can simulate the biomechanical environment existing in vivo. the latter attributed to too close contact. only the chondrocytes of the articular surface. does not affect chondrogenesis.2.A. and the decrease in the interaction bindings with other matrix components like fibronectin. MSCs in the pellet culture show induced fibrocartilage-like features such as expression of collagen I and hypertrophy. and macroporous scaffolds. the most common solution is to embed the cell suspension in a surrounding environment capable to retain the cells. are responsible for the round shape. Other non–hydrogel scaffolding systems are produced for cartilage tissue engineering. suggesting that there are unknown alternative pathways [41]. When compared with pellet cultures. normally encapsulating the cells at the beginning of the culture. gellan gum [66]. differentiation of adult or embryonic stem cells into a chondrocytic phenotype requires a rounded cell shape [45. chitosan [63–65]. Pellets and micromass culture Cell culture in 2D does not resemble the in vivo situation. enhances chondrogenesis. The biophysical interaction between integrins with the collagens and proteoglycans in the cartilage matrix [31. formed by suspension of cells within a solution that encapsulates them. as it should be reminded.

physically or covalently. may mask the effects of the 3D conformation. GAG) or analog molecules provide direct adhesion properties to cell ligands. Reducing fiber diameter to nanoscale can potentially enhance the chondrogenic differentiation of mesenchymal stem when compared with the same material as a sponge of thicker fibers [103. This is the only zone where the interstitial fluid can flow out of the cartilage when it squeezes.102]. and can be suitable for chondrogenic differentiation in specific medium [85. surfaces can be modified to attach specific proteins in desired conformations. Nevertheless. In this zone. such as fibrin or disperse patterned PDMS. Fibrin. which present a lower cell adhesion degree than fibrin [93]. usually this layer formation is controlled in some manner through functionalization with specific proteins and allowing non-specific attaching before cell culture [101]. The expression and synthesis of chondrogenic markers in hyaluronan gels is also dependent of the macromere density and cross-linking of the gel [97]. In the deep zone. the differences in these pathways between the two systems have not been identified yet [92].112].86]. this zone sustains shear stress resulting from angular displacement of the two sides of the joint. Macroporous scaffolds.4 J. through the surface. such as PEG.2. The explanation can be that more binding points can be established by increasing porosity. but rather through an interfacial layer formed on material surface once it is in contact with a physiological environment. Thus. encapsulation of MSCs in non-adhesive hydrogels. parameters that influence differentiation through fluid flow. Hyaluronic acid is the natural backbone of the proteoglycans of hyaline cartilage. fibrin) in terms of chondrogenic marker expression. / Acta Biomaterialia 33 (2016) 1–12 favor integrin binding. 2. similarly to pellet and micromass systems and cell condensation. values of . from this zone to the lower parts. although more data are needed to reach consistent conclusions. For example in bovine. laminin and vitronectin. However. Some comparisons between fibrin and agarose have been carried out for chondrogenic differentiation of MSCs. such as RGD sequences [82]. a recent study [88] shows no differences between the two types (alginate vs. is largely employed. such a layer is created as result of nonspecific adsorption of pericellular matrix proteins. Also. migration and differentiation [100]. hydrophilicity. Few experiments compare directly an adherent gel vs. when pore diameters are significantly larger than the cell diameter (more than 100 lm). the creation and characteristics of this layer can be guided. like soluble growth factors. This difference in loads results in anisotropic mechanical properties through the cartilage. Thus. Therefore. mainly integrins and cadherins. This environment improves chondrogenesis of MSCs compared to PEG gels in the same conditions [95]. through grafting specific peptide ligands. cell migration. due to differences in composition and structure. by tuning these parameters through suitable physicochemical modifications. being also the most resistant zone to shear stress. In 3D. processing methods for these materials are more limited and few structures can be designed. the highest strains can be found (up to 50%) as well as the highest fluid flow. a hydrogel with a high adhesive internal surface. organization of collagen. in polyurethane scaffolds. In the superficial zone. Signaling complexity in 3D also increases with respect to 2D.g. water uptake by GAG and other matrix components provides mechanical stiffness able to resist mechanical loading caused by joint movement and weight bearing. Mechanical loading is thus essential for proper musculoskeletal development [113]. which provide at the same time the mechanical properties of cartilage. with the elastic modulus of hyaline cartilage increasing with depth. unlike in in vivo. The mechanical forces acting in the knee are varying within the articular zones. In no defined culture media. and the hydrostatic pressure is the lowest of all cartilages. Differentiation in 3D: mechanical loading effects 3. being more adhesive than agarose or PEG. Fibrin properties are highly dependent of the used concentrations of fibrinogen and thrombin [89–91]. interconnectivity and permeability. as subchondral bone and adjacent tissue confine to these zones. In the middle zone the strains range between 10% and 20% and there is less fluid flow and. is permissive to chondrogenesis because the cells adopt a rounded shape. allowing both interactions with the matrix and cell–cell contact [96]. Only in a simulated osteochondral environment the agarose showed a superior response. in less adherent 2D surfaces that promote more spherical morphologies. which favors expression of chondrogenic markers by direct cell–cell contact. In native hyaline cartilage. Scaffold chemistry influences surface properties such as morphology.2. but with interactions in some points through CD44 cell receptors [94]. The discrepancies among experiments could be caused by the difference in fibrin composition. However. 3. Hyaluronic acid gels are common gels used for chondrogenic differentiation. the surface presented to cells ranges in the micrometric scale and the cells adhere in a manner resembling cell adhesion on 2D substrates [82. Macroporous sponges fabricated from molecules found in ECMs (e. While one of them [87] shows fewer accumulation of GAG and collagen type II for adherent gels (fibrin) with respect to non-adherent gels. but they also regulate the stress transmission to the cells. uniform compressive normal stresses ranging from 5 to 10 MPa [109] and frequencies between 0. For example. Moreover. other non-bioactive materials are also used and the cell response to their surface is not mediated by a direct contact. the strains are 0–5% and practically fluid movement does not exist. cell–cell contact and nutrient and soluble factors local concentration [97. In these sponges. surface energy and charge. Not only the mechanical properties of ECM allow the cartilage to accomplish its function.105–108].1 and 10 Hz [110]. MSCs adopt spread morphology and differentiate spontaneously toward myocites [84] in chondrogenic medium. It could be expected that the differences in adhesion could lead to differences in the commitment of MSCs toward chondrogenic lineage. the higher hydrostatic pressures are found [114]. the cartilage behaves as a viscoelastic material that deforms easily at small strains but stiffens while strain is increased [111.A. the knowledge on mechanical properties of tissue environment and the ways in which loadings are transmitted to cells is fundamental. but the different works are not conclusive.1. 3D adherent gels can be potentially inductive of chondrogenesis by mimicking the interaction of the isolated chondrocytes with the components of the pericellular matrix of adult cartilage. This finding can indicate that other environmental conditions in vitro. it is limited to inside the cartilage matrix. This protein deposition not only is fundamental for cell adhesion [99]. collagens. Panadero et al. which interact with surface integrins [98]. There are several metabolic pathways involved in the differentiation in both 2D and 3D systems. nonadherent. encapsulating the MSCs in fibrin inside the scaffolds results in higher GAG and collagen type II than seeding the cells directly to the pore walls [86].104]. but also influences posterior cell events like proliferation. There is a feedback situation between loading and matrix synthesis.2. Mechanical loading in vivo Mechanical loading is also an important factor regulating and maintaining the chondrocyte phenotype. From a materials science point of view. which control this protein adsorption [69]. For chondrogenesis. because the loads that must sustain the matrix act as signaling factor to the expression and production of its components. MSCs differentiate toward chondrocytes in the same medium. alginate or agarose. like fibronectin.

from all the lineage commitments. However. leading mature hyaline cartilage phenotype.J. The biochemical transduction is mediated through the cytoskeleton also. An immotile primary cilium on human mesenchymal stem cells [118].123]. first in micromass cultures [139] and hydrogels with mature chondrocytes and more recently with mesenchymal stem cells. activated by a variety of nucleosides. activating calcium signaling via ATP binding to purine P2 receptors [136] and chondrogenic differentiation of MSCs.08 MPa at the surface and 2. For example. which bind with the pericellular matrix. Each one of these mechanical solicitations simulate one or some of the different components of mechanical stress in vivo and each one matches more closely to the conditions given in the different zones of cartilage. intermittent or dynamic application of mechanical stresses [141. the molecular mechanisms still remain unclear. which dephosphorylates cyclic adenosine monophosphate (cAMP) to generate extracellular phosphate and adenosine. Success of application of dynamic loads depends on the frequency of application [144]. Purinergic receptors (ATP) can be found also in the primary cilium of chondrocytes [138]. it has been shown that the primary cilium length depends on the strain applied over the cells. which are subdivided in ligand-gated ion channels (P2X)-activated by adenosine triphosphate (ATP). Dynamic loading can also trigger ATP release to the extracellular environment. and TGF/BMP [126].38 MPa [115]. but also in the 5 chondrogenic differentiation of MSCs [133]. The loadings in the hyaline cartilage are a complex combination of tensile. the primary cilium in chondrocytes also contains integrins [121]. the purinergic response involves activation of connexin hemichannels and unidentified mechanisms. The apparent modulus of the whole cartilage is 0.135]. but has been used only recently [154] as it is hard to perform for cell culture. until it finally shortens too much and becomes osteoarthritic. external mechanical stresses have been applied through bioreactor devices. Effects of compression and hydrostatic pressure have been extensively studied for chondrogenic differentiation. and P2 receptor. 3. which is directly associated with many of the mechanoreceptors mentioned previously[120].1 MPa in the deep zone. / Acta Biomaterialia 33 (2016) 1–12 modulus are 0. the cilium length is reduced by loading. Moreover. Mechanotransduction signaling Although the macroscopic effects of mechanical loading in vivo are well established [113].157–159] produces higher expressions of collagen II and proteoglycan expression than static loading [160. it remains unclear how mechanical loading produces the changes in the length of primary cilium in MSCs. composed of two families: P1 receptors. di. the route is activated.3. It seems that dynamic loading downregulates CD73 (50 -nucleotidase). In vitro. specifically to strain deformations [117]. altogether indicating that the combination of loading modes prevents hypertrophy. the primary cilium is related to its differentiation potential and its inhibition reduces the expression of differentiation markers [118]. Unconfined compression resembles the conditions found in the upper zones of the cartilage with higher medium flow.2. but with dynamic compression. and how it regulates their chondrogenic differentiation. Direct shear stress can be combined with unconfined compression. The calcium release is activated by purinergic receptors. The purinergic calcium signaling is a broad regulation mechanism of many biological processes and has been recently identified also as a mechanotransduction pathway taking part not only in the phenotype regulation of mature chondrocytes. like TRPV4 [116]. the absence of cilium also activates this route [124. semi-confined compression would resemble a gradient of all the zones. which has also been verified in vitro. purines or pyrimidines. as integrins interact with focal adhesion kinase and the cytoskeleton.A. Recent research is unraveling that the mechanotransduction in chondrocytes in vivo is mediated through mechanoreceptors in the plasma membrane. The primary cilium has also been proposed to transduce the mechanical loading through the PKA route. In mature chondrocytes. the cilium is disgregated with mechanical loading. the hypertrophic markers (col X) did not increase and the GAG liberation is constant. and therefore also have a role in mechanotransduction [122. there is still no evidence. activated preferentially by adenosine.or triphosphates. and compressive stresses and strains. If this proposed mechanism could be corroborated. shear.and G-protein coupled receptors (P2Y)-activated by nucleotides. vimentin intermediate filaments are thought to play a role in mechanosensing. As a possible mechanism. mainly integrins. mechanical loading is an important signaling factor for the correct regulation of ECM in vivo. However. Some systems have combined compression and shear stress [155]. Ideally. leading to chondrogenic differentiation [134. direct shear stress [144–146] and perfusion (shear stress) [147–149] and hydrostatic pressure [150– 153]. If the cilium is not disrupted. To investigate its effects in vitro. integrins are binding proteins to the pericellular matrix. because of the different mechanical environment in each zone. vesicular release mechanisms [137] have been identified. It suggests the hypothesis that only a range of length is optimal for non-hypertrophic chondrogenesis. 3.151. the chondrogenic potential is the least affected by this inhibition.161] and better mechanical properties in mature chondrocytes in explants and hydrogels. which is a recent route of mechanical transduction. Although the influence of surface topography in primary cilium length has been studied in MSCs [131]. On the other hand. In mesenchymal stem cells.150. after the initial stages of cartilage development. Furthermore. The length seems to be a determinant factor of activation of the Hedgehog route. and this combination enhances chondrogenic matrix production in MSCs with respect to each single loading mode [156]. results in osteoarthritic cartilage [129]. the exact mechanisms are not elucidated yet. associated with stretch-activated ion channels and voltage-gated calcium channel. it would explain the role of the cilium in mechanotransduction during differentiation. When MSCs are subjected to oscillatory fluid flow. It is known that for both compression and hydrostatic pressure in hydrogels. In reference [128]. As indicated before. Receptors from both families have been found to participate in chondrogenic mechanotransduction. arthritic chondroprogenitor cells (CPCs) and chondrocytes [119] has been also identified. The mean length of the cilium is different depending on the zone of the cartilage [127]. requiring a permeable load plate over the load zone and impermeable boundaries around the rest of the scaffold [114].130]. Primary cilium transduces mechanical loading and regulates metabolic routes such as Hedgehog [124]. [140]: unconfined uniaxial compression [141–143]. because ATP release can occur by different routes. Hydrostatic pressure resembles better the condition in the deeper zones. as well as the adenosine A2a receptor from the P1 family. and finally. suggesting additional mechanisms of regulation. typically 1 Hz and the addition of . low hydrostatic pressure. high stresses (in displacement control) or low strains (in force control). Wnt [125]. which play an important role in the transmission of loading [24]. the ways of providing mechanical stresses are based in the reduction to single mechanical components – thoroughly reviewed by O’Conor et al. Panadero et al. through a cascade resulting in the activation of sox9 transcription factor [132]. which is an antagonist route of chondrogenesis that if expressed in chondrogenic precursors leads them to a phenotype similar to the hypertrophic phenotype of the end-stage differentiation in the growth plate. Mechanical loading in vitro As previously stated. because the levels of sox9 are the least reduced. However.

The period of continuous cycling loading in knee rarely exceeds 1 h and is dispersed through all day with resting periods. Although MSC cyclic loadings are capable to express chondrogenic markers and to produce ECM in non-adherent hydrogels. formed by the cells or artificially recreated with coating. the opposite to non-adherent gels. This indicates that fibrin may not be acting with the same function as a pericellular matrix generated in a predifferentiation step. The graph shows the results in base of the increasing trend of the gels to form cell–matrix bindings. In fibrin gels. which reach at least 16 h [114]. the difference between adherent and non-adherent gels is not as clear. the loading from early times (3 days) did not hinder chondrogenesis [168]. Only the cases in which the cyclic loading has been applied from the beginning of cell culture in differentiation medium have been taken into consideration. this could Fig. the disparity of the adhesion degree (non-adherent vs. is mandatory for the differentiation of MSCs. However. If a pericellular matrix is allowed to be generated for at least 2 weeks. highly-adherent gels) produces different responses. like hyaluronic acid gels. dynamic compression improves GAG and collagen deposition after 70 days despite the expression levels remaining statistically similar to free-swelling conditions. With respect to adherent hydrogels under cyclic hydrostatic pressure. General description of the effects of the different types of cyclic loadings on MSC chondrogenic differentiation. due to the absence of quantitative determination of the level of adhesion in the reviewed cases. they need a pre-differentiation step and some initial pericellular matrix in order to sustain the same dynamic loads than differentiated chondrocytes for cultures until 21–28 days [141. suggesting that the adhesion may provide a better environment for mechanotransduction. higher expression and matrix synthesis is obtained. Panadero et al. In a direct comparison between agarose and fibrin gels [87]. but in long-term culture. In any case. In less adhesive gels. although it is not clear if the effects over chondrogenesis are caused by the cell morphology conformation. 3. the differences between hyaluronan and fibrin could suggest that the degree of this adhesion also influences the response to cyclic compression.164]. the presence of a pericellular matrix. cyclic compression from day 0 inhibits GAG and total collagen accumulation in both gels in short-term. which remains higher if the compression is performed from the beginning. or because of hindrance of hydrodynamic and transport of nutrients [86]. MSCs first keep un-differentiated traits. 1.4. / Acta Biomaterialia 33 (2016) 1–12 repose periods is necessary. depending on the grade of adhesion provided by the encapsulating gel and according to the data of comparative studies between gels. the differences are found in mid-term adhesive gels. For the cases in which direct cyclic compression is applied to MSCs. 1). they increase expression of chondrogenic markers compared to unloaded gels and inhibit myogenic markers even when loads are applied from the first day. Unlike agarose hydrogels [167]. under cyclic hydrostatic pressure. MSCs in fibrin accumulate more GAG and reduce collagen type I under the effects of dynamic hydrostatic pressure than non-adherent hydrogels.6 J. and it does not improve early chondrogenic differentiation over agarose. leaving non-adherent and highly-adherent gels with similar responses (Fig. Despite the lack of studies about the necessity of this pericellular matrix under cyclic compression. in which levels of markers can drop below free-swelling conditions [165]. . it seems that the MSC responds positively to this loading even if it is applied from the first days.A. If the differences in chondrogenic differentiation are not caused by other indirect factors like nutrient diffusion. Recapitulating. This effect is more noticeable in cultures until 42 days with daily loadings. Interaction of mechanical loading with 3D structure and cell adhesion In non-adherent hydrogels under cyclic hydrostatic pressure [162]. in which hydrostatic pressure has little effect [162]. while under direct cyclic compression. the findings about the resistance to non-cyclic compression of this matrix [163] suggest that it could show the same effect under cyclic compression. but the accumulation is enhanced after 42 days. fibrin gels support chondrogenic differentiation under cyclic compression [166]. when cyclic loading is applied. the delay of the compression regime in fibrin only enhances accumulation of GAG and not of collagen type II. Nevertheless.

the expression of N-cadherin is necessary at the beginning but it is reduced as 7 chondrogenesis progresses. The cytoskeleton. Without mechanical loading. The results reviewed can be the first steps into unraveling these interactions. Mechanical measurements in cartilage tissue engineering: a necessity to complement the biochemical data Traditionally. It would be interesting to study the effect of the adhesion in other types of cyclic loadings like the combination of uniaxial compression and shear stress. in the experiments for chondrogenic differentiation. which adds the difficulty for working with a high number of samples. it is reasonable to think that MSCs should sustain mechanical loadings in different ways. Nevertheless. but it would mean that chondrogenesis would be induced by skipping the condensation step. The analysis of the cell-ECM-scaffold constructs is mandatory. in which the depletion of the expression of these genes leads to loss of function. it promotes chondrogenesis of MSCs but only for the early days [96]. which determine its functionality as medical substitutes only if they are capable to resist the mechanical loading and transmit its signals properly. whose contribution to cartilage development is completely necessary. It is necessary to indicate that. However.J. the role of cytoskeleton in mechanotransduction of mature chondrocytes may be different to the cytoskeleton of undifferentiated MSCs. and fibrin for instance only retains 30–50% of GAGs [144]. Actually. However. the presence of an adherent hydrogel from the beginning could make unnecessary a pre-differentiation step. The first gap is that more efforts are needed to develop nondestructive and high-throughput screening techniques for mechanical properties. thus in an in vitro culture the expression should be reduced at the end. the mechanical properties of the constructs should be defined by the cell surrounding environment. 4. spreading increases N-cadherin expression. matrix and mechanical loading – and to categorize each one as cause or consequence of the other. in order to produce a pericellular matrix [170]. under the same mechanical and surrounding structure conditions. Thus. It should be noted that in native cartilage. like indentation [181]. for cyclic hydrostatic pressure. as a consequence of the stronger cytoskeleton [172]. this hypothesis failed to be valid for cyclic compression [87]. which plays an important role in the adhesion. If this is correct. it is difficult to identify all the relations among the three parameters – cell shape. MSCs respond better with lower concentrations of TGF-b than in non-loaded cultures [173]. The response of MSCs to mechanical loading seems also conditioned by other factors. suggest the fact that actin cytoskeleton of mature chondrocytes undergoes remodeling under mechanical loading [171]. it could explain the lack of differentiation response of MSCs to myogenic and chondrogenic lineage at the beginning of the culture. or simulating the final steps of condensation to mature cartilage. However. Like in vivo. if the samples are employed for mechanical measurements with a non-destructive method. because a total correlation among mechanical and biochemical data has not been found yet [142. could suggest that the response of mesenchymal stem cells with a pre-differentiation step is due to the generation of some pericellular matrix that could be acting as an adherent hydrogel. . it has been concluded that mesenchymal stem cells need a pre-differentiation step before applying the dynamic loading. the effect of a rounded cell shape can resemble the situation of MSCs in the pre-condensation step. . the pericellular matrix plays an important role in retention of the synthesized GAG. the morphology and the chondrogenic differentiation in the absence of mechanical cues. It has been suggested that dynamic compression can inhibit N-cadherin activity. In the last years. However. chondrocytes in the different zones show different morphology [169]: The typical round shape found in the literature corresponds to the chondrocytes from the middle and deep radial zone. and because of it. However. as seen in previous sections.) were analyzed. we consider that. Despite the importance of mechanical properties. but in the superficial zone. . The usual techniques for mechanical analysis – summarized in Table 1 – are also destructive. the main binding protein during condensation [87]. The analysis of mechanical properties complements the data obtained from biochemical assays of matrix expression and deposition. Generally. as could be seen in knock-out mice [174. no one has found a direct relation between the cytoskeleton state (which determines cell shape) and the response to mechanical loading. The findings described in our review. and other components as collagens IX. Moreover. Thus. only the expression and translation predominant ECM markers (collagen type II. Panadero et al. all the cases that support this finding have been performed with non-adherent hydrogels. leading to the up-regulation of myogenic genes. a stage of the development when the mechanical cues are not present. higher concentration of TGF-b masks the effects of mechanical loading. which make it difficult to interpret the specific relations among data. measurements of mechanical properties in individual cells find that undifferentiated MSCs have a higher elastic modulus than mature chondrocytes. because from a structural point of view. However. The techniques for –omics analysis require destruction of the samples. These approaches serve to obtain more accurate data of the MSC signaling and more detailed information about the ECM composition after differentiation. For instance. aggrecan. Without loading. comparing with the advance in the tools that have been developed for ECM screening. the integration of these data in a coherent model is defying. Although there have been efforts to separate the effects of matrix components in cartilage by bottom-up approaches of measuring single ECM components [180]. sox9. If cadherin is added to HA hydrogels. these techniques generate huge amounts of data. due to the feed-back nature of the interactions in cartilage. in any case. / Acta Biomaterialia 33 (2016) 1–12 suggest that the mechanosensing of MSCs behaves differently when the loading is homogeneous (hydrostatic pressure) than when it is anisotropic (uniaxial compression). in cell cultures that minimally require times of 28 days. the –omics have a great potential to describe complete profiles of the ECM and soluble components produced and their role in chondrogenic differentiation. and not directly by the state of the cells.179]. Thus. the pericellular matrix could still be necessary in chondrogenic differentiation for other reasons beyond a loading shelter effect. they can be used . the different loading profiles in the different zones and the varying matrix composition and structure may be related to the different morphology through different pathways of mechanotransduction.175]. Although there is no evidence that dynamic loading transforms cell shape in vitro. the chondrocytes are more spread. what suggests that mechanical loading acts via similar pathways. These spreading chondrocytes can express collagen type I and lower amounts of collagen type II relative to proteoglycans than the round chondrocytes. can also be involved in the response of chondrocytes to mechanical loading. although more evidence needs to be found to determine the optimal initial conditions in chondrogenic differentiation of MSCs under mechanical stimuli. the resources to describe chondrogenic differentiation have been expanded through the use of the ‘‘omics” high-throughput screening techniques: transcriptomics (with microarray technology) [176–178] and proteomics (with mass spectrometry) [179]. there are still gaps in the implementation of mechanical measurements of constructs after culture in vitro or after transplant in vivo. and one cannot be inferred from the other.A. XI were neglected.

requires seeding and culturing in 3D configurations. the application of dynamic loading. it is still an opening discipline that needs to be improved. it has a strong potential if combined with –omics approaches. Before a correlation between all relevant components of ECM under mechanical loading is found. However.and micro-pores in scaffolds themselves. despite that the initial elastic modulus is the same. widening the performance parameters (frequency. Conclusions and open questions The differentiation of mesenchymal stem cells to obtain hyalinelike structures in vitro. However. The 3D configuration determines the cell commitment toward chondrogenic differentiation. an epigenetical signaling also necessary as a stimulus. But keeping the sensors in good working conditions in the harsh oxidating conditions of cell culture media is challenging. Technique Description Strengths Extensiometry Two grips hold the material and tensile force is applied by its movement – Immediate obtaining of Young’s modulus. It is not clear why this phenomenon happens. However. – Only materials with shape of strips or rings. a phenomenon appearing in all materials when they are submitted to cyclic loading [183]. Fatigue analysis has the potential to model predictions of the mechanical resistance of a construct that would be implanted in the knee. Fatigue in material sciences is understood as the irreversible structural damage produced in a material that is subjected to cyclic loading. – Only uniaxial strains – Destructive – The material requires a flat surface – Destructive – Potential leakage of the sample – Difficulty to control applied pressure – Dissolved air becoming trapped in the solution – Requires finite – The election of tip geometry plays an important role in determining the materials mechanical strength Fracture-related failure has been assessed in cartilage and in some hydrogels with techniques that evaluate resistance to a crack generated in purpose. the study of mechanical properties uses parameters common in materials science (Table 2). Panadero et al. the material fabrication processes. and the loss modulus is a measure of the energy converted to heat. in cyclical motions of strain and stress Ratio of stress to strain under vibratory conditions Limitations m ¼  eetrans long mÞ Ha ¼ ½ð1þEð1 mÞð12mÞ G ¼ sc E ¼ E0 þ iE00 for other purposes. increasing the scaffolding materials tested and including other extracellular matrices and native cartilage explants. it is necessary to create specific models more statistically suitable for viscoelastic materials. as ratio of the stress along an axis over the strain along that axis in the range of stress in which Hooke’s law is valid Negative ratio of transverse to axial strain Stiffness when fluid has stopped flowing Ratio of the shear stress to shear strain E ¼ re Poisson ratio (m) Aggregate modulus (Ha) Shear modulus (G) Storage and loss modulus (E0 and E00 ) Dynamic compressive modulus (E⁄) The storage modulus is a measure of the energy stored elastically during deformation. With respect to high throughput screening mechanical analysis. only recently a device has been developed for this purpose [182]. yield strength and ultimate tensile strength Compression test (unconfined) Bulge test Two plates compress the material in between – Not limited by geometric shape Air pressure inflates the material through a window in the substrate. strain.). Compression tests could be non-destructive with the possibility of performing them at the same time of mechanical stimulation. . can alter the way the 3D configuration determines differentiation. elastic modulus.8 J. fatigue behavior is different for Poly-e-caprolactone (PCL) scaffolds with chondrogenic precursors seeded inside their pores – which produce ECM – that for the same scaffolds with other filler. via adding force sensors to the actuators of the bioreactors. it is important to ensure the proper knowledge on the mechanical properties of the biomaterials and their relationship to overall cell response.186]. The second gap is that the traditionally measured properties may not be sufficient to fully characterize mechanically materials that are submitted to cyclic loading like in the knee. 5. Compression to failure in hydrogels or other biomaterials has been barely studied [187] and never under cyclic loading. if an independent displacement or force control could be added. where cyclic loadings and permanent deformation can occur. that grows depending on the test. introduce inherent imperfections that can act as stress concentration points [185. albeit. The displacement can be measured using a CCD camera or a laser – Able to characterize the residual stress. because the model used in fatigue analysis in that work is a model developed for metals. granted that sterile and clean conditions are kept. Furthermore. capable to measure the compressive equilibrium modulus of up to 48 samples simultaneously. It still has a limitation of disparity on precision (larger standard deviations compared to sampleby-sample testing). number of cycles. and other important parameters such as yield strength and fracture toughness – Best suitable for thin films Microindentation (and nano) A tip indents the material at a single point to a predetermined displacement depth and measuring the reaction force required – – – – – Non-destructive Allows quick and real time measurements of materials It can be used to measure localized mechanical strength at different points on a material surface Table 2 Common mechanical parameters measured in hydrogels and macroporous scaffolds before and after cell culture in vitro for cartilage tissue engineering. but it could be related with the boundaries between the filler. analogous to a series of standard methodology in material science [184]. / Acta Biomaterialia 33 (2016) 1–12 Table 1 Common tests to measure mechanical properties in hydrogels and macroporous scaffolds before and after cell culture in vitro for cartilage tissue engineering. Furthermore. Some preliminary works show that fatigue in scaffolds with in vitro generated ECM provides information that other common measurements miss. has not been discussed properly for biomaterials and ECM yet. which can adopt many forms depending on the geometry and the physico-chemical characteristics of the surrounding cells.A. Parameter Physical meaning Formula Young modulus or elastic modulus (E) Stiffness. that can act also as different stress concentration points than other discontinuities. and the macro. . which can initiate fatigue effects. . . Hitherto. However fatigue. In a pioneering work [188]. this device could serve as loading bioreactor and data collector at the same time.

[28] N. C. Portugal).B. Turnbull.-H. 418 (2012) 500–505. Biochem.X. Betensky. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011. Am. Houston.A. S. B. The authors also thank the financial support from the Basque Government Industry Department under the ELKARTEK Program. T. H. Richter. Le. J. W. Hoffman. Karperien. R. Weston. 22 (1999) 85–89. Stott. Daniels.G. Chuon. Zhang.B. Biophys. 4 (1988) 487–525. Osteochondral defects: present situation and tissue engineering approaches. J. Cell Biol. Z. Cao. Cheng. Articular cartilage repair: basic science and clinical progress. Papageorgiou. Tenascin interferes with fibronectin action. Y. 209 (2011) 139–151. [10] A. J. F. Hayes. J. Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy. A. [23] R. NJ) 137 (2000) 359– 375.-H. Cytoskeletal changes of mesenchymal stem cells during differentiation. Csaki. Temenoff. Besides. [2] Z. R. Rev. Stringa. Anat.B. H.S. H. Modulation of chondrogenesis by the cytoskeleton and extracellular matrix. [38] G. cells and cell produced ECM should be obtained. N.R. Tuan. D.M. Alexopoulos. Acad. Athanasiou.J.J. 85-A (Suppl 2) (2003) 124–132. Cell Biol.L. J. [27] N. K. Ingber. Wendt.R. Anat. Regulation of the chondrogenic phenotype in culture. J. Velasquillo. Chen. S. Ibarra.A. J. Seawright. England) 113 (1991) 327–337. Huang. References [1] W. M. Chiquet. M. Cheng. Hussain. / Acta Biomaterialia 33 (2016) 1–12 reversing the effect of adherent and non-adherent surroundings. Jakob. J. A review of the current status and prospects. T. Jefferies. [41] M. [7] I. Cell-cycle control and the cartilage growth plate. Post. [5] C. [26] C.A. J. Lee.A. Acknowledgments This work is funded by European Regional Development Fund through the ‘‘Programa Operacional Fatores de Competitividade – COMPETE” and by portuguese national funds arranged by FCT (Foundation for the Science and Technology. E. cytoskeletal mechanics.D. Poole. Fath. Biochim. C: Embryo Today 87 (2009) 351–371. Anat. typically not considered.T. Commun. E. Cell Biol. Embryonic limb mesenchyme micromass culture as an in vitro model for chondrogenesis and cartilage maturation. Georgoulis. Chem. [11] Y. Genet. Shakibaei. Flachsbart. Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton. I. Poole. Tuan.A. Chiquet-Ehrismann. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. 1 (2007) 261–273. Immunolocalization of type VI collagen in the pericellular capsule of isolated canine tibial chondrons. Genipin-crosslinked cartilagederived matrix as a scaffold for human adipose-derived stem cell chondrogenesis. Kresse. Jin. Horwitz. M. Ruoslahti. J. Youn. by measuring also relevant parameters. Senentxu Lanceros-Méndez also thanks the Diputación de Bizkaia (Spain) for financial support under the Bizkaia Talent program. Wang. K.M.-M. Goetz. 8 (1998) 19–32. A. Cell shape. Upton. Regenerative Medicine and Tissue Engineering. [29] C. more complete profiles of the mechanical properties of the constructs formed by scaffolds. Disassembly of the vimentin cytoskeleton disrupts articular cartilage chondrocyte homeostasis. 100 (Pt 2) (1991) 249–254. Physiol. Tissue Eng. Osteoarthritis Res. Burridge. Beier. Articular cartilage bioreactors and bioprocesses. Estes. Expression of cell-cycle-dependent genes in serum stimulated cells whose entry into S phase is blocked by cytochalasin D. [8] J. Diverse roles of integrin receptors in articular cartilage. Underhill. Kalla. Review: tissue engineering for regeneration of articular cartilage. Biomech. Review.J. (Clifton. Lykissas. Biophys. C. Orthop. S. Wu.M. K. Guilak. Beris. T.S. Deng.S. Gene expression profiling of dedifferentiated human articular chondrocytes in monolayer culture. J.E.D. Light and electron microscopical immunohistochemical localization of the small proteoglycan core proteins decorin and biglycan in human knee joint cartilage. 2013.M. R. 26 (1994) 939–945. Solursh. Widelitz. ASAIO J. Choi.A. Jiang.C. Mao.F. Chondrons from articular cartilage: I. More evidence about the interaction between mechanical loading and 3D configuration is needed. Bone 25 (1999) 405–412. S. Guilak.J. [39] M. Schultz.C. Liao. Development (Cambridge. Awad. J. Kim. 99 (1984) 115–123. Takasuka.J. P.S.M. [22] C. 202 (2005) 1–8. 36 (2007) 539–568.N. [18] R. Matrix Biol. [20] B. Sun. Oberlender. Histochem. L. Cell 53 (1988) 383–390. Cells Mater. [40] T. Y. Chung. function and failure.M. Cartilage tissue engineering: the role of extracellular matrix (ECM) and novel strategies. Cell. Med. Wang. Molecular mechanisms regulating chondroblast differentiation. The pericellular matrix as a transducer of biomechanical and biochemical signals in articular cartilage. R.A. W. 197 (2008) 1–60. [36] K. W. L.A. Osteoarthritis Res. [17] E. The authors also thank support from the COST Action MP1206 ‘‘Electrospun Nano-fibres for bio inspired composite materials and innovative industrial applications” and MP1301 ‘‘New Generation Biomimetic and Customized Implants for Bone Engineering”.C. Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix. M. [13] K. B. J.). Schliwa. Cell. [32] M. Guimond. Consolider Program. Ayad. Kim. [33] N. C. Guilak. Methods Mol. Down-regulation of the chicken alpha 5 beta 1 integrin fibronectin receptor during development. J.-H. Mano. Woodward.H. M. Miot. Duance. Articular cartilage chondrons: form. van Blitterswijk. Injury 36 (Suppl. A. Osteoarthritis Cartilage/OARS. G. [35] E. Garciadiego-Cázares. Part A 19 (2013) 484–496.A.Y. Darling. Beck. V. Bi.F.S. DeLise. Woods.J. Intern. Turner. and cell cycle control in angiogenesis. Soc. Bobick.E. Med. such as mechanical fatigue. Ide. Spatiotemporal profile of N-cadherin expression in the developing limb mesenchyme. = Anatomischer Anzeiger 190 (2008) 395–412. C. J. Csaki. Wang. Biomech. Schneider. Rev. Martin. Part A 15 (2009) 231–241. Lester. J. M. Bone Joint Surg. Blain. Kip. N. Embryol. project reference PEST-C/FIS/UI607/2014. D. D. J. J.E.M. [42] B. Annu. Cell Adhes. B. Behringer. S. Bader. Cell Sci. A correlation between as much parameters of mechanical behavior as possible with cell phenotype and the histological characteristics of new-formed tissue would be a crucial step beyond the state of the art. A.A. probably because of loading transmission effects. 266 (2009) 390–405. 30 (2012) 393–400. J. C. . Zanetti. J. Estes. J. R.A. S. [21] S. [6] E. A. Y. [25] C. M. C. Commun. B. [30] N. Mikos. in: Jose A.S. Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions. Young. F. Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells. J. Gilbert. L. (American Society for Artificial Internal Organs: 1992) 53 (2007) 219–228. Tissue Eng. [15] L. Regener. 60 (2008) 243–262. Reis. Mesenchymal stem cells and cartilage in situ regeneration. M. A. Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction. Castro Carmona. F. M. [19] F. A. C. Soc. M. Tuan. Chang. Panadero et al. Physiol.A. F. Beier.H. Nat. [37] D. T. Kim. C. Adv. A.D. [9] E. Drug Deliv. 21 (2013) 599–603. Ishibashi. Ann. E. Herken.J. R. Schulz. 191 (1997) 1–13.L.C. Barbero. A. Hsu. Fang.-C. Osteochondral tissue engineering. Parra-Cid.T. J. 92 (1982) 79–91.H. 2 (1994) 521–537. [31] S. Miosge. Solursh. Sonn. Acta 909 (1987) 161–164. Res. A. Z. Haider.L. Leijten. Ann.S. 28 (1995) 1471–1484. de Crombrugghe. C. Eur. M. This work was also funded by the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2013-46467-C4-1-R (including the FEDER financial support). Chen. Tissue Eng. Andrades (Ed. Pearson. 9 [12] J. Yourek.-C. Aguilar-Gaytán. Kim. K.K. 10 (2002) 432–463. Juan Alberto Panadero thanks the FCT for the SFRH/ BD/64586/2009 fellowship grant. G. 9 (2003) 9–26. Advances in articular cartilage repair. CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. J. Nuckolls.-W. The additionally obtained relevant information can help to understand the mechanical processes of the scaffolds in the knee cartilage loaded environment. D. 25 (2006) 398–408. [4] A. J.G. [34] C. Osteoarthritis Cartilage/OARS. 264 (1989) 13369–13372. Iniciativa Ingenio 2010. J. T. Biomaterials 21 (2000) 431–440. Biophys. Tissue Eng. Prusty. C. J. R. L. Res. [16] A. Muschler. C. [24] F. Cell Sci. Birth Defects Res. P. Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes. likewise works comparing the response of undifferentiated mesenchymal stem cells to different cell adhesion patterns in hydrogels and scaffolds. Biol. [3] J.R. Mobasheri. Hunziker. R. S. Engineering cartilage tissue. Action of cytochalasin D on cytoskeletal networks. Shakibaei. Schofield. M. H. 4) (2005) S14–S23. Adv. García-Carvajal. 90 (Pt 4) (1988) 635–643. [14] W.Y. N. Extracellular matrix and cell signalling: the dynamic cooperation of integrin. Burdick. 213 (2007) 1–8. 40 (2007) 750–765. Mello. Cell Biol.A. D.G. Farquharson.-H. Ma. Mesenchymal stem cells as a potential pool for cartilage tissue engineering. Setton. Early events during precartilage condensation in limb bud micromass cultures. Biol. 1068 (2006) 498–512. proteoglycan and growth factor receptor. Kelly. Sci. Endocrinol. Sox9 is required for cartilage formation. Proteoglycans in cell regulation.

J. European Society of Biomechanics S.L. H. [87] S. Athanasiou. M.M. Steward. [91] M. Structural origins of fibrin clot rheology. Awad. Chondrogenesis from mesenchymal stem cells derived from adipose tissue on the fibrin scaffold.M. The effects of dynamic and threedimensional environments on chondrogenic differentiation of bone marrow stromal cells. Guilak. Res. Ohio) 25 (2007) 2786–2796. J. Gomez Ribelles. Hollander. 12 (2006) 1345–1355. Guilak. Panadero et al. Expert Rev. Chiellini. 56 (2007) 177–187. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.A. Staudenmaier.A. Grady. Xu. Ribelles. Tissue Eng. Garcia. Tissue Eng. M. F.B. W.F.J.M.A. Schulze-Tanzil. D. Biodegradable PCL scaffolds with an interconnected spherical pore network for tissue engineering. N. Dare. Hering. Wiese. Biomaterials 28 (2007) 55–65. Gomez-Ribelles. E. Biomed. J. J. Randolph. Biomed. C. Lorand. L. Knudson. Stem Cells (Dayton. T. Koevoet. Bader. R. Hui. S.L. R. Neves. A. Mistry. Ohio) 28 (2010) 564–572.G. Reis. Gorkun. [68] S.A. Weigel. Part A 83 (2007) 145–155. Bencherif. [70] J.W. Huebsch. Mano. López-Ruiz. Randolph. Delgado-Martínez. J.M. Han. [77] J.P. Lett. L. J. Reis. S. Anat. Thomas. K. D. C. Zhang.A. [71] E. 3 (2007) 59–67.S. [49] L.W.T. Bellingham. Malafaya. M. Huang. Nanotechnology 22 (2011) 212001. Izquierdo. Shvartsman. Roy. Chen. R.J. M. J. Acrylic scaffolds with interconnected spherical pores and controlled hydrophilicity for tissue engineering. Olmedilla. Checa. L. Diego. Puppi. Chondrogenic differentiation of human mesenchymal stem cells using a thermosensitive poly(N-isopropylacrylamide) and water-soluble chitosan copolymer.R. Prog.H. Barry. D. Cool. M. Lee. Wang. Baumann. Monllau. J. Mano. M. Commun. A. Mao.R. Chen. A.V. Arthritis Rheum. T.B.. J. L. J.A.S.J.A. Feijen. L. [60] C. Res. [86] Y.E. C. C.E. K. Steinert. 104 (2007) 1475– 1481. Franklin.Y. Alini. Mueller. Acta Biomater.M. Biomech. Hutmacher. Kelly. Caplan. Salmeron-Sanchez.G. Polymeric materials for bone and cartilage repair. [76] Y. Falvo.A.A. C. Myers. Howdle. Wilson.S. Part A 93 (2010) 852–863.C. B. Spector. Riesle. Gomes.W. Noth. B. Mano. A. Suay.Q. Scotchford.H.. Kim. M. R.T. J.T. Biomaterials 29 (2008) 2858–2868. Bian. J. K. Eglin. S. Linse. Kafienah. Picciochi. J.C. Eur. M. [75] J. Picón. McMahon. Sanchez. M. Goepferich. A. Lendlein. Campbell. R. J.C... V. J. G. Appl. A. Biotechnol. Sci.J. [61] L. D.10 J. J. J. Akens.B. A. Sa-Lima.A. Ho. Farhadi.G. Aroca. ERK1/2 and PI3K in growth factor-induced chondrogenic differentiation of mesenchymal stem cells.A. Martins. Chen. McBeath. J. Reis. 6 (2010) 2108–2115. [80] S. Lee. Caceres. Gao. D. Sims. J. Nogues. Chung.W. Suay. J.M. Caceres. A. Jung. Res. Keeley. Biomater. Brandl. Warren. Learmonth. H. Tissue Eng. Ali. [82] I. F. J. [62] H. D. E. [51] G. Karperien. Appel. Biomaterials 32 (2011) 1327–1338. 77 (1999) 2813–2826. E.A. [90] E. Eulert. Wenzel. J. [52] S. Burdick. Nat. (2004) S152–S162. A. Perán.M.J. T.L. Heymer. [88] M. Su. Y. Part A 14 (2008) 1573–1580. K. Part A 85 (2008) 25–35. Bustamante. K. M. Nehls. microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis. Biomaterials 30 (2009) 2544–2551. J. [73] J. Kops. Mater. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. M. M.S. Biomaterials 31 (2010) 38–47. Garcia-Giralt. [78] R. A comparison of the involvement of p38. van der Vloodt. J. M. J. Assessment of biocompatibility and initial evaluation of genipin cross-linked elastin-like polypeptides in the treatment of an osteochondral knee defect in rabbits. P. Martens. Novel poly(L-lactic acid)/hyaluronic acid macroporous hybrid scaffolds: characterization and assessment of cytotoxicity. Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. J. [58] G. M. Tan. E. Nanoscale tissue engineering: spatial control over cell-materials interactions. J.S.B. Verhaar. Hyaluronan and CD44: modulators of chondrocyte metabolism.A. The external mechanical environment can override the influence of local substrate in determining stem cell fate.M. R. N. Bryant. Dijkstra. Hoben.T. F. Kirchhoff. R. Biophys.S.S. M. Bick. Park. Biomed. 9 (2010) 518– 526. Gellan gum: a new biomaterial for cartilage tissue engineering applications. Z. R.M.P. J. Chondrogenic differentiation of human mesenchymal stem cells in collagen type I hydrogels. Stem Cells (Dayton. P. A. J. T. W. Mater. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. I. Kim. J. Res. Thornton. S. Orthopaedic Res. Zscharnack. Tihaya. K.S. Fibrochondrogenesis in two embryonic stem cell lines: effects of differentiation timelines. Eyrich. 16 (2005) 693–698.S. Gomez-Ribelles. C. McBride. Sánchez. M.M. Lee. Tissue Eng. Maher. Y. [46] W. [56] T.L.D. Noh. Porous methacrylate scaffolds: supercritical fluid fabrication and in vitro chondrocyte responses. J.M. Zhong. M. / Acta Biomaterialia 33 (2016) 1–12 [43] G. Weber.H. 50 (1995) 311–322.L. Med. N. Gimble. J. Perren Award. van Osch. Diego. 91 (2009) 277–286. Garcia-Giralt. Goldberg. M. M. G. Xu. A. [81] T. van Blitterswijk. Activation and dedifferentiation of chondrocytes: implications in cartilage injury and repair. Yaremchuk. A. T. J. J. Mater. Polym. Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: generation of scaffold-free cartilage. Cell Res. Kim.T. Chondrocytes extract from patients with osteoarthritis induces chondrogenesis in infrapatellar fat pad-derived stem cells. Peretti. T.A.D. Effects of cross-linking type II collagenGAG scaffolds on chondrogenesis in vitro: dynamic pore reduction promotes cartilage formation. Part A 83 (2007) 626–635. Anseth. Squitieri.-I. Buckley. Perez-Olmedilla. Mater. Influence of gel properties on neocartilage formation by auricular chondrocytes photoencapsulated in hyaluronic acid networks.C. R. A.M. 152 (2010) 15–20. J. Rivera-Feliciano. [63] J. Review of injectable cartilage engineering using fibrin gel in mice and swine models. D. Ryan. Wu. Thorpe.M. Res. Khademhosseini. Gidda. 368 (2008) 990–995. W. Chondrogenic differentiation of human mesenchymal stem cells: a comparison between micromass and pellet culture systems.M. J. R. 12 (2006) 1151–1168. P. [85] G. [94] C. O. Hrabchak. A. Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in a simulated osteochondral environment is hydrogel dependent.H. J. K. Control of stem cell fate by physical interactions with the extracellular matrix. I. England) 4 (2009) 055009. 27 (2014) 112–123 (discussion 23). [50] G. Johnstone. [74] R. Soria. Soft Matter 6 (2010) 5184–5195.A.L. M. J. Jabbari.F. S. Design and preparation of polymeric scaffolds for tissue engineering. Res.L. P. [72] N. A. M. Stimuli-responsive chitosan-starch injectable hydrogels combined with encapsulated adipose-derived stromal cells for articular cartilage regeneration.R. Mockros. Galle.R. Res. Tramper.H. S. Res.V. Nguyen.I. M.A. Bader.B. V. Sci.M. B. F. Pradas. [92] L.M. Biochem. Soria. Modulation of stem cell shape and fate B: mechanical modulation of cell shape and gene expression. S. Pore collapse during the fabrication process of rubber-like polymer scaffolds. R. The influence of fibrin based hydrogels on the chondrogenic differentiation of human bone marrow stromal cells. D. Fibrin: a versatile scaffold for tissue engineering applications. Kudva. [66] J. G. S. [84] L. Wheeldon. S. F.J. Ribelles. J.J. Yaremchuk. C. Weisel. W.M. Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilage. = Anatomischer Anzeiger 191 (2009) 325–338. M.A. G. Knothe Tate. Biomaterials 26 (2005) 63–72. M. Microvasc. Yoo.H. J. E. Part A 77 (2006) 518–525. M. J. J. . Acta Biomater. Biomed. Payne. 58 (2008) 1377–1388. Devices 3 (2006) 835–851. S. Murdoch.S. Erickson. [69] D. 5 (2005) 438–443. Benito. Mater. M. 35 (2010) 403–440. J. [45] B.L. Ferrer. A. Part B: Rev. Low oxygen expansion improves subsequent chondrogenesis of ovine bone-marrow-derived mesenchymal stem cells in collagen type I hydrogel. C. Biomaterials 30 (2009) 2499–2506. Rackwitz.P.. P. Oliveira. X. S. Res. S. D.. D. S. J. J. Mano.C.E. S. Exp. The molecular origins of the mechanical properties of fibrin. Woodhouse. [64] R. C. Cells Tissues Organs 190 (2009) 81–93. [55] M. H. Liedtke. J. [44] N. E. Chem. C.A.M.I. Yang. Commun.K. Hincke. G. Biophys. E. Biomed. Gimble. Mooney.M. Vickers.Z. J. S. Biomed. 238 (1998) 265–272. Sci. Biophys. Ahmed. Lord. J. Biochem. Koay.J. D.W. Rodriguez. J. R. Biophys.F. Biomaterials 25 (2004) 5743–5751. M. Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits.B.J.J. Polym. Ohio) 26 (2008) 422–430. The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage.A. Mater. Stem Cells (Dayton. Murphy. Curr. Stegemann.A. J. Woodfield. M. Jung. Schutze. Nondegradable hydrogels for the treatment of focal cartilage defects. 32 (2010) 1339–1346. Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering. Appl. Doty. Monleon. 22 (2004) 1143– 1149. Dennis. Marra. Izquierdo. [83] F. H. Caridade.A. J. Proliferation and differentiation of goat bone marrow stromal cells in 3D scaffolds with tunable hydrophilicity. Dickinson. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Antunes. Jin. Montañez. A. (Bristol. W.B. Arthritis Rheum. Im. Estes. [65] H. Chu. Biomed. M. [47] M. Guckert.L. Monllau. Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels. [89] S. Ann.M.C. J. Cell Stem Cell 5 (2009) 17–26. Moss. Blunk. M.M. T. [57] D. Relat. Piras. Jiménez. Moreira Teixeira. Rouleau. Yu. Kim. Hernández-Lamas. C. Knudson. D. Tuan. N. Jakob. J.D. Reis. [53] U. P.U. 290 (2002) 763–769. Biomaterials 25 (2004) 3559–3568. Sousa.L. Chiellini. Poesel. Part A 94 (2010) 856–869.L. Hardingham.D.F. Clin. Oliveira. [54] S. G.H. Schinkel. Mater. Rice. L. Mater.H. L.L. Drenckhahn. Res. B Appl. Med. N. Prendergast. Orthop.. [67] S. A novel. van Blitterswijk. [93] V. P.L. Mater.F. Mater. Phys. Yang. Cells Mater.D. Ablett. Mesa. S. T. Torzilli. Marchal.T. Bonassar. M.G. [48] A. Arrebola. de Vries-van Melle. Crosslinking density influences the morphology of chondrocytes photoencapsulated in PEG hydrogels during the application of compressive strain. Meadows. C. Lowman.L. Pradas. Ribelles. L. Ivirico. Maier. Biomed. [59] C. F.A. Cohen. Cobo-Molinos. A porous PCL scaffold promotes the human chondrocytes redifferentiation and hyalinespecific extracellular matrix protein synthesis. [79] R.H. Mater.S. Rowe. Biomed. K. Injectable chitosan-based hydrogels for cartilage tissue engineering. E. F. Res. Part A 85 (2008) 1082–1089. J.M.L. Mater.L. 2012 (45) (2012) 2483–2492.J. Long-term stable fibrin gels for cartilage engineering.M. Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin. Malda.J.S. Cho.G.J. J.D. Arany. Katopodi.P.E. Res. Y. Osteoarthritis Cartilage 21 (2013) 246–258. 14 (2008) 199–215. O. A. Q. Yee. Wright. Falls.

Byers. [110] B. O’Conor. D. Osteoarthritis Cartilage/OARS. Mauck. Depth-dependent confined compression modulus of full-thickness bovine articular cartilage. Tissue Eng.S. Part A 81 (2007) 586–593. Thompson. [111] S.L. Duda. Tissue Eng. Natl. C. M. Majima.A.A. Wong. M. F. Osteoarthritis Cartilage/ OARS. C.R. 3 (2013) 312–319. M. J. Pankov. R. Primary cilia mediate mechanotransduction through control of ATPinduced Ca2+ signaling in compressed chondrocytes. Med. Marsano. M. Biomater. Cell-matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels. Ramaswamy. 18 (2000) 739–748. Ho. J. Alini. Takacs. Alman. L. Schinagl.L. Jacobs. Ali. Geiger.J. Schipper. [103] J. M. Mittelstaedt.C.L. Bryant. Gomez-Ribelles. Poole. McGlashan. R. Stoddart.F. [107] T. Jacobs. Kramer.R. U. Biomed. Muhammad. Mol. 25 (2013) 37–47. Cluett.S. Ferrer. Agrawal. 54 (2006) 1005–1014. Henriques de Jesus. A. Kaps. T. R.L.M. J. Mol.. Serra. The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin. Clement.M.J. Hunziker. Mauck. Panadero et al. 110 (2013) 10117–10122. Kaebisch. C. Z. L. Hoemann. Streuli. tubulin.B. Treppo. O’Conor. J. Gadkanski. Differentiation 83 (2012) S49–S55. R. Gamboa-Martínez. Leddy. Biomolecular surface coating to enhance orthopaedic tissue healing and integration. Ferrer. Schagemann. Votta. scaffold-free cartilage. H. D. Mol. C. Ornan. [112] J. I. Get a grip: integrins in cell-biomaterial interactions. Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes. S. Inversin. Liedtke.J. Andersen. J. McMurray. Karacsonyi. Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures. J.J. [98] B. J. J. L.A. Castro. Weigel.J. L. The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation. Huang. Guvendiren.J. [99] A. Vunjak-Nokavic. N. S. C.G. Birth Defects Res. Yao. D. D. Pedersen. The primary cilium as a dual sensor of mechanochemical signals in chondrocytes. R. Chang. 27 (2012) 299–309. Soria. L. Articular cartilage functional histomorphology and mechanobiology: a research perspective. Stem Cell Res. Biomaterials 26 (2005) 7525–7529. Mater. 22 (2007) 589–600. Bioeng.J. M. [122] D.G. Taylor. C. Eng. Chondrocytes cultured in an adhesive macroporous scaffold subjected to stirred flow bioreactor behave like in static culture.A. E.Y. W. Riddle. S. Orthopaedic Res. Nebot. Natl. K.R. A.D. S.G. S.L. / Acta Biomaterialia 33 (2016) 1–12 [95] C. Hsu. Nanotechnology 24 (2013) 365102. Grodzinsky. R. [105] J. Cuddihy. Cell Sci. S. Orthopaedic Res. Elder. E. Harada. Primary cilia disassembly downregulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes.J. Macromer density influences mesenchymal stem cell chondrogenesis and maturation in photocrosslinked hyaluronic acid hydrogels.M. Cole. Mechanical regulation of chondrogenesis. A. G. T. [116] C. [114] M. Osteoarthritis Res. Ng. K.A. Biomech.A. Burdick. 237 (2008) 2013–2020. J. Mater. architecture. N. Reyes. Primary cilia attenuate hedgehog signalling in neoplastic chondrocytes. Arner. Kasahara. Khoury. Osteoarthritis Res.C. 18 (2010) 249. J.J.D. Transmembrane crosstalk between the extracellular matrix and the cytoskeleton. C. Ali. B. 3 (2013) 1806–1814. K. G. R. Rep. Influence of 3D hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis. B.. Lee. Kuettner. R. Biomed. 69 (2012) 2101–2107. Ode. Athanasiou. Garcia. 23 (2012) 425–440. Biomater. Proc. A. Griggs.T. J. [96] L.A. . Ivirico. K. Eur.J.C. Erickson.D. Suetterlin. Biomaterials 28 (2007) 3228–3235. Signal.H. 15 (2013) e1. Kim. Eng. R. Levy.S..L. A. N.K. Vunjak-Novakovic. Ann. W. 15 (2009) 1421–1425. Monde. Mechanical anisotropy of the human knee articular cartilage in compression.C. Langelier. M. Li. Bershadsky. Tummala. The beneficial effect of delayed compressive loading on tissueengineered cartilage constructs cultured with TGF-beta3.N. Kandel. 9 (2013) 858–872. U. Case. Ribelles. Lienkamp. Kumar. J. S. Jensen. Lima.T. Histochem. Part B: Rev.J. Buschmann.A. P.G. Keidar.J. A. Aebi. Comparison of biomechanical and biochemical properties of cartilage from human knee and ankle pairs. Leddy. Res.C. Matta. Mol.E. Somogyi. Bian. McGlashan. A. The effects of hydrostatic pressure on matrix synthesis in articular cartilage. [109] A. I. J.H. M. M. Tissue Eng. Part A 101 (2013) 1620–1628. J. S. Y. Sah. H.P.G. M.L. Wann. 15 (2009) 43–53. Elder. T.E. H. Expert Rev. increased Hedgehog signaling.M. R. Haycraft. Wann. Long-term dynamic loading improves the mechanical properties of chondrogenic mesenchymal stem cell-laden hydrogel.L.M.A. Burdick. [117] E. Guilak.A. Sci. N. [104] B. Localization of extracellular matrix receptors on the chondrocyte primary cilium. Poole. [101] C.S.R. N.H.A. Koefoed. K. Geissler. Guilak. Bartoszko. Res.D. E.E. The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells. 15 (1997) 499–506. Bryant. Histochem.E. Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds. Biomed.L.G. R. A. Gurskis. C: Embryo Today 90 (2010) 75–85.H. G. Gaetjen. S. 32 (2004) 1728–1743. 2 (2001) 793–805. Acta Biomater. 13 (2015) 75–84. Liedtke. C. Oncogene 32 (2013) 5388–5396. Lebourg.C. Whetstone. Biomed. J. Knight. Part A 16 (2010) 575–584. Walz. R. Modulating hedgehog signaling can attenuate the severity of osteoarthritis. Chai. [H] 217 (2003) 215–219. ATP release mediates fluid flow-induced proliferation of human bone marrow stromal cells. S. C. [106] S. Cell Rep. Part B: Rev.J. S.K. Donahue.M. A. 9 (1991) 1–10. Burdick. A. McGlashan. and vimentin filaments. Nat. Orthopaedic Res. Diffusion in musculoskeletal tissue engineering scaffolds: design issues related to porosity.A. Al-Jazrawe. 15 (2007) 1025–1033. S. permeability. Cooley. CD73/ 50 -ecto-nucleotidase acts as a regulatory factor in osteo-/chondrogenic differentiation of mechanically stimulated mesenchymal stromal cells. Y. J. Giralt.A. C. Reinholz. G. Connelly.F. J. Mesenchymal stem cells produce functional cartilage matrix in three-dimensional culture in regions of optimal nutrient supply. [108] M. Hung.J. T. Acad. Schoon.D.L. Mauck. S. K. R.R. Katona. U. Rev. M.B. Li. Farrell. Gergely. J. Kestle. Juhasz. H. S. [115] R.A. S. J.J. Proc. Minami. Lebourg. Garcia. J. Yamada.A. Nat. N. Rais. Struct. Ode. A. P.M. Karande.P. D. D. G. S. Comput.R. Osteoarthritis Res. Carroll. Benmerah. Zakany.J.J. Soc. R.E.F. 60 (2009) 3028–3037. Y. C. D. Res. J. Ho. Osteoarthritis Cartilage/OARS.C. Lin.A. E. Acad. Xie. Phan. Christensen. J. Αv and b1 integrins regulate dynamic compression-induced proteoglycan synthesis in 3D gel [123] [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] [145] [146] [147] 11 culture by distinct complementary pathways. A. Holmes. F. Three-dimensional cell culture matrices: state of the art. B. Purinergic responses of chondrogenic stem cells to dynamic loading. J. Mauck. Z. 20 (2012) 152–161. R. Ródenas-Rochina. Appl. M. Osteoarthritis Res. Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-epsilon-caprolactone scaffolds. Alman. Osteoarthritis Cartilage/OARS.C.P. [120] M.G. 41 (2008) 1528–1536. Wnt signaling and primary cilia. J. Wunder. Stem Cell Rev. [121] S. R. E. 78 (2013) 1865–1874. C. Serb.A. and symptoms of early osteoarthritis. M. C. G. N. Soc. Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis. Mech. A. A. Mauck. Arthritis Rheum. Comeau. The role of purinergic receptors in stem cell differentiation.M. Urban. J.H. A. Cytochem.J. Tuan.L. Gehl. Soc. Benefield.S. [102] M. I. C. H.L. Surface topography regulates Wnt signaling through control of primary cilia structure in mesenchymal stem cells.S. Schwartz. E.G. 17 (2009) 1639–1648. M.L. Jurvelin. Nishimura.S.H. 22 (2014) 490–498. K.M. Kotov. 48 (2000) 1307–1320. N.A. I. A. In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold. 14 (2008) 61–86. Paul. M. Ribelles. Buschmann. Eur. M.L.T. J. Soha. Kelly. Casper. Fehr.P. S. A. J. C. Part A 100 (2012) 2330–2341. J. E. R.G. K. Ong.J. Cytochem. Osteoarthritis Res. Chapple. Farrell. Yamane. A. Kurtz. E. 3 (2013). Chondrogenesis of human bone marrow mesenchymal stem cells in fibrin-polyurethane composites is modulated by frequency and amplitude of dynamic compression and shear stress. Cell. Dev.B. Primary cilia in osteoarthritic chondrocytes: from chondrons to clusters. 3 (2010) 207–212. J. Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers. Miosge. Knight. 26 (2014) 468–482. Osteoarthritis Cartilage/OARS. M. Tortosa. Hall.J. Sci. Cells Mater. Zhang. G. M.J. M. In vitro 3D culture of human chondrocytes using modified epsilon-caprolactone scaffolds with varying hydrophilicity and porosity. Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration. C.R. P. J.A. Zhang. C.J. Jensen. L. [113] D. Veland. C.A. [97] I. Babczyk. J. C. J. Bian. Ajbro.A. Lopez. Rogers.A. F. Olmedilla.C. Ther.C. L. M.D. Knight. Cell. Inst. Villanueva.B.W. Tissue Eng. TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading. Ateshian. Sanchez. J. M. and nutrient mixing. Soc. C.C. Rohwedel. M. Cell. TGF-beta signaling is associated with endocytosis at the pocket region of the primary cilium. Sci. Nicodemus. 122 (2009) 171– 177.R. Burns. 19 (2010) 72–85. Salmeron-Sanchez. Guilak. A. C.G. Thompson. Bone 33 (2003) 1–13. J. Koepp. Differentiation of mesenchymal stem cells to osteoblasts and chondrocytes: a focus on adenosine receptors. J. FASEB J. 5 (2009) 2832–2846. C. Soc. Cserhati. Lipshutz. Lee. Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes. Chen. Tran.G. Proc. Grodzinsky. E. Jensen. B. Huang. Dyn. Cells Mater. Cell Biol. E.W.H. G. H. M. Zuo. Zhang. T. Carter.R. S.D. C. C. K. Quan. Cells Mater. Part A 15 (2009) 243– 254. Larsen. [119] H.S. 111 (2014) 1316–1321.A. Life Sci. Ravid. Eur. 4 (2013) 61. Garcia Cruz. Szody. Sengupta. Depletion of primary cilia in articular chondrocytes results in reduced Gli3 repressor to activator ratio. Med. 108 (2011) 1421–1429. H. Soc. The role of primary cilia in mesenchymal stem cell differentiation: a pivotal switch in guiding lineage commitment. Z. B. G. I. Biotechnol. Tobiasch. K. Effect of a mechanical stimulation bioreactor on tissue engineered. Iwasaki. C.A. M. Vestergaard. Petrie. Chung. Poole. [100] C. K. A. Mater. Chem.N. Biotechnol. Integrins and cell-fate determination. Szabo. E. [118] P.S. S. Bone Mineral Res.A. Bako. Tissue Eng. Bioeng. J. Seeto. G. Ganner. 26 (2012) 1663–1671. Arnsdorf.J.A.

Biomech. 20 (2010) 175–181. Biochem. The proteoglycan metabolism of mature bovine articular cartilage explants superimposed to continuously applied cyclic mechanical loading. Chandran. M. Kelly. [171] M. Thorpe. Expression of chondrogenic genes by undifferentiated vs.R.L. Hierarchically designed agarose and poly(ethylene glycol) interpenetrating network hydrogels for cartilage tissue engineering. D. S. D. Bioeng.G. Pasrija. . [185] D. Eggli. Vinardell.D. mechanical properties. Farrell.S. Fatigue and Durability of Structural Materials. Mathieu. J. M.C. S.M. Mizrahi.H.R. 6 (2007) 113–125. J. B: Appl. Biomech. G. Probst.H. Tissue Eng. The response of bone marrow-derived mesenchymal stem cells to dynamic compression following TGF-b3 induced chondrogenic differentiation. [152] S. Elisseeff. S. Cell Mater. Terraciano. Gomez Ribelles. P. Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse. Frisbie.E. Bian. Tissue Eng. M. Alini. Technol. D. C.A. L. and differentiation down osteogenic.M. Pelaez. [166] D. 12 (2006) 1419–1428. J. Mauck. Anat. [179] J. Mauck.S. G. Chondrocyte biosynthesis correlates with local tissue strain in statically compressed adult articular cartilage. Yoo.E. Mechanobiol.A. Friis. Smith. Biorheology 46 (2009) 45–55. 22 (2011) 214–225. Lv.J. M. Part B: Rev. C. K. Eur. Dynamic compression stimulates proteoglycan synthesis by mesenchymal stem cells in the absence of chondrogenic cytokines. Quantitation of structural features characterizing weight. Pastor. Y. [168] L. Mahmoudifar. Dodge. Tissue Eng. G. J. H. G. C. Kim. P. Tissue Eng. J.J. Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose. Eng. Matrix Biol. Biomech. Zhao. Res. A. E. Gehrke.L. Weis.J.H.H. Huang. [183] S. and chondrogenic pathways. adipogenic. Jepsen. Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.Y.J. R. Buschmann.R. DeKosky. A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells.A.J. Lee. Y. Park. Zhai.S. Mol. Z. Part B: Rev.T.S. Sencadas. Zeiter. Biomaterials 31 (2010) 3858–3867. Stein. 240 (1997) 216–221. 36 (2008) 813–820. [155] A. Acta Biomater. [150] P.T. 26 (2007) 85–95. Cosgrove. Cao. Longaker.L.F. Salzmann. [176] C. Part A 18 (2012) 715–724. J. The application of comparative proteomic analysis to screen proteins associated with mechanical properties of engineered cartilage: a preliminary study. B. Mater. X. J. Mauck. J. Grodzinsky. Wagner.H. R. 47 (2014) 2130–2136. Knue. L. H. S. Schatti.P. O’Brien.H. 377 (2008) 458–462.W.C. T. Schenk. J.Y. W.J. Yao.R. Effect of flow perfusion conditions in the chondrogenic differentiation of bone marrow stromal cells cultured onto starch based biodegradable scaffolds.B. Smith. BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells.J. Proc.R. Cytoskeletal and focal adhesion influences on mesenchymal stem cell shape. Song. Grifka. Goncalves.D.J. Seliktar. Silva. Campbell.L. [172] P. T. J. P. Tummala. 12 (2006) 147–155. Hwang. P. S. Campbell. Cells Mater. C. differentiated human mesenchymal stem cells using array technology. J. Lindsey. 3. Lanceros-Mendez.L.A. R. Yoon. Meloni. C. Sliding contact loading enhances the tensile properties of mesenchymal stem cell-seeded hydrogels. Doran. Scott. S.R. Zhang. R. Lee. Smith. [160] J. Stoddart.D.D. Ingavle. Biomed. Cell. G.S.L. 21 (2003) 451–457. [186] D. [173] Z.S. T. Eur. Li. 15 (1997) 189–196. R. Mechanical fatigue performance of PCLchondroprogenitor constructs after cell culture under bioreactor mechanical stimulus. Park. X. H. Vinardell. Kelly. 18 (2012) 436–444. Schubert. J. Ratzinger. Carter. Zou. Ito. Grodzinsky. Beaupre. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro. Kisiday. Res. V. Pinzano.W. Grassel.D. Biochem. Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice. Carter. Liu. S. Nanomechanics of the cartilage extracellular matrix. Mechanical indentation tester designed to control and measure in real time the microhardness process. Thakore. [162] A. G. Hunziker. M. M. Wagner. Panadero et al. S. Manson.J. [169] P. [154] S. R. Martin. Knight. Johnstone. D. E. Mauck. Biomech. Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells. Kelly. Eyre. Goldhahn. R. Buckley. The influence of microstructural stiffness changes on the stress concentration factor of porous polymer materials. The effect of loading direction and pores distribution mode on porous polymer material stress concentration. [149] N. Stem Cells Dev. S. P. 26 (2007) 597–603. J. Zeleniakiene. 11 (2005) 123–128.D. S.W. Thorpe. [163] Á. Cheung.J. Rev. D. Gomes. S.L. D. J. Stem Cells (Dayton.C. R. Huang. Thorpe. J.J. Role of pericellular matrix in mesenchymal stem cell deformation during chondrogenic differentiation. J. Vinardell. Gillet. R. Lindsey. Panadero. Thorpe. J. T. Netter. Stoddart.R. Zhang. Sci. B. Yuan.K. B. Gehrke. Beaupre. Commun. Spiegel. Eggli. Li.and less-weight-bearing regions in articular cartilage: a stereological analysis of medial femoral condyles in young adult rabbits. 2 (1991) 740. T. [178] A. Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels. Sci. H. Detamore. F.C. S.R. Biomed. [157] R. [165] V.J. Steward. Dormer. Liukaitis.L. D. J. [188] J. Hou. [158] J. Huang. [164] S. Tissue Eng.T. Rec. Chondrogenic differentiation of human adipose-derived stem cells in polyglycolic acid mesh scaffolds under dynamic culture conditions. R. Q. Bruckner. Ortiz. G.S. A. Mohanraj. Carroll. Mater. D. [184] Y.B. E. Li. 43 (2010) 128–136. W. Gene expression profile during chondrogenesis in human bone marrow derived mesenchymal stem cells using a cDNA microarray.H. G. [182] B.J. Biomater.S. 24 (2012) 29–45. Kupcsik. Acad.A.A. Wong. Ohio) 25 (2007) 2730–2738. 3 (2010) 387–397. V. V. Liu. E. 19 (2013) 403–412. Modulating gradients in regulatory signals within mesenchymal stem cell seeded hydrogels: a novel strategy to engineer zonal articular cartilage.M.C. E. V.H. A. M. Wuethrich. 39 (2006) 1547–1551. [174] A. D. S. Yan. M. Tuan. T. Bensoussan. D. Buckley. K. M.M. B. A. P. Y-L. [181] J.L.P. Biophys.Y. Annu. 41 (2011) 133–168. Matrix Biol. 14 (2010) 1338–1346.J. Zhang. Model.R. Res. M. Henrionnet. Fernandes. [167] S. Schmidt. [161] M. Kleveckas. Seegmiller. Orthopaedic Res. Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels. 14 (2015) 283–296. Biomed. I.Y. M. [151] K. Opolka. Cell Mol. Gillet. 18 (2009) 93–102. E. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1. Hunziker. Burdick. [170] A. Chen. [187] B. Res. Y. P. Cell–matrix interactions regulate mesenchymal stem cell response to hydrostatic pressure. N.A. A. Ding. K. Mauck. N. Sci. O’Brien. C. F. [175] R. Part C: Methods 16 (2010) 1533–1542.L. Toyoda. D. M. Mechanical testing of hydrogels in cartilage tissue engineering: beyond the compressive modulus. [156] O. E. Zhao. McIlwraith. Halford. Byers. Biophys. Ann. Eng. M. R. L.U. C. Kelly. S. Lee. Part A 16 (2010) 2699–2708. Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway. [153] Y-H. R. Commun. Mansour. G. E. Costa.J. Lomakin.U. 2006 (chapters 1. F. Evaluation of the complex transcriptional topography of mesenchymal stem cell chondrogenesis for cartilage tissue engineering. Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. 38 (2010) 2896–2909. Mater. Reis. Baker. B. 222 (1988) 217–227. Grad.L. Detamore.E.D. Dias. Merino. Yoo.L. Z. Moroni.B. Zhang. Part A 15 (2009) 2817– 2824. Correia. Y-J. Goodman. Huang. Bio-Med. Loboa. Li.12 J. Roatch. C. Galois. Stem Cell Res. Mainard. M. Zhonghua zheng xing wai ke za zhi 29 (2013) 49–54. P. Steinmeyer. Meas.W. Ateshian. Gama. PLoS One 8 (2013) e60764. J. D. Schurman. Tissue Eng. Ribeiro. Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. D. Nagel. Vigfúsdóttir. P. [180] L. A high throughput mechanical screening device for cartilage tissue engineering. P. Rodrigues. Angele. M. D. 7 (2011) 1644–1652.J. Effect of TGF beta1.L. T. Med. [177] H. Zeleniakiene. D. Hsieh. [159] D. S.D. Buckley.J. Bader. 26 (2011) 851–858. Sci. Mauck. Xiao. (2015). 10 and 12). Orthopaedic Res. M. Liu. Acta Biomater. Trindade. 8 (2012) 2153–2159. A.F.S. Miyanishi. Est. Nerlich. Eng. Han. C. / Acta Biomaterialia 33 (2016) 1–12 [148] A.T.M. Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.A. Roeder. Mater. J. Ye.B. R. Alini. Zhou. D. M. Lezuo. Korean Med. Ann. C. H. D. C.P.S. Mechanics and mechanobiology of mesenchymal stem cell-based engineered cartilage. Tissue Eng.A. D. D.