Stimulated Chondrogenesis via Chondrocytes Co-culturing | OMICS International
ISSN: 2153-0777
Journal of Bioengineering and Bioelectronics

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Stimulated Chondrogenesis via Chondrocytes Co-culturing

K. El Sayed 1 , H. Zreiqat2 , W. Ertel1 and G. Schulze-Tanzil1 *
1Department for Orthopaedic, Trauma and Reconstructive Surgery, Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany
2Tissue Engineering & Biomaterials Research Unit, Biomedical Engineering, School of AMMEJ07, University of Sydney, NSW 2006, Australia
Corresponding Author : Gundula Schulze-Tanzil, VMD
Department for Orthopaedic
Trauma and Reconstructive Surgery
Campus Benjamin Franklin, Berlin, Germany
Tel: +49-30–450-552-385/985
Fax: +49-30–450-552-985
E-mail: [email protected]
Received October 27, 2012; Accepted December 06, 2012; Published December 10, 2012
Citation: Sayed KE, Zreiqat H, Ertel W, Schulze-Tanzil G (2012) Stimulated Chondrogenesis via Chondrocytes Co-culturing. J Biochip Tissue chip S2:003. doi: 10.4172/2153-0777.S2-003
Copyright: © 2012 Sayed KE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Co-culture; Chondrogenesis; Chondrocytes; Mesenchymal stem cells; Cartilage repair
2D: Two-dimensional; 3D: Three-dimensional; AC: Articular Chondrocytes; ACI: Autologous Chondrocyte Implantation; ASC: Adipose Tissue derived Stem cells; ADAMTS: A Disintegrin and Metalloproteinase with Thrombospondin motifs; BMP2: Bone Morphogenetic Protein-2; CXCL: C-X-C motif chemokine Ligand; DF: Dermal Fibroblasts; ECM: Extracellular Cartilage Matrix; ESC: Embryonic Stem Cells; GAG: Glycosaminoglycans; I-CAM: Intercellular Adhesion Molecule; IGF-1: Insulin-like Growth Factor-1; IL: Interleukin; MACI: Matrix-assisted Autologous Chondrocyte Implantation; MMP: Matrix-metalloproteinase; MSC: Mesenchymal Stem Cells; OA: Osteoarthritis; OB: Osteoblasts; PCMO: Programmable Cells of Monocytic Origin; PDGF: Platelet Derived Growth Factors; PG: Proteoglycans; SF: Synovial Fibroblasts; SOX9: SRY (sex determining region Y)-box 9; TACE: TNF Converting Enzyme; TGFβ1: Transforming Growth Factor β1; TNF: Tumor Necrosis Factor; VCAM: Vascular Adhesion Molecule; VEGF: Vascular Endothelial Growth Factor.
Joint cartilage damage remains a major orthopedic challenge due to a very low intrinsic repair capacity of cartilage. Clinical therapeutic strategies available to treat cartilage defects include implantation of autologous osteochondral cartilage cylinders (mosaic plastic) [1], and Autologous Chondrocyte Implantation (ACI). For ACI, a small articular cartilage biopsy is excised, and isolated autologous chondrocytes are expanded in vitro for implantation. Chondrocytes can be implanted either alone [2], or guided and supported by a degradable biomaterial (Matrix-assisted Autologous Chondrocyte Implantation, MACI) [3,4]. However, drawbacks were encountered using this technique, whereby the cartilage biopsy, required for isolation of autologous chondrocytes, provides only a limited cell number; the isolated autologous chondrocytes dedifferentiate during in vitro expansion; and the damaged cartilage at the biopsy site may develop Osteoarthritis (OA). Hence, the interest increases in using Mesenchymal Stem Cells (MSC), capable for chondrogenic differentiation, or in using other chondrocyte species for cartilage repair. Attempts are made to utilize MSC from the subchondral bone using bioactive agents, which subsequently lead to the invasion of the MSC to the repair site, and their chondrogenic differentiation in situ to induce self-healing [5,6]. It is still unclear whether chondrogenic cells such as MSC acquire and maintain a stable differentiation state, when invaded or implanted into joint cartilage defects. In addition, an inflammatory microenvironment associated with an arising osteoarthritis might affect the differentiating/ redifferentiating chondrocytes, or MSC after implantation into the cartilage defect.
Concerns remain in using MACI and the fate of the cells used for implantation in cartilage defects. During articular cartilage development, intimate communication of several mutual differentiating joint tissue precursor cell populations [7,8] results in regular cartilage and joint formation. In response to joint diseases such as OA, phenotypic alterations of Articular Chondrocytes (AC) can be induced by neighboring cell types, such as sclerotic Osteoblasts (OB) [9]. For this reason, cell-cell communication in the joint is altered under disease conditions.
Co-culture in vitro systems provide a powerful tool for cartilage tissue engineering [10]. The mode of interaction of the different cell types during in vitro chondrogenesis remains a fascinating research field, reflected by the continued rise in published literature of in vitro co-culture studies. In the present review, we focused on the multifaceted types of co-culture systems used to improve chondrogenic differentiation of multi- or oligopotent cells. In addition, we describe some of the soluble factors which may play a role as putative mediators and cell-surface molecules, or receptors involved in cell-cell or cell- ECM interaction.
Results and Discussion
Cartilage healing and the cell types involved in the healing response
Untreated chondral defects are usually loosely covered by cells deriving from the synovial layer, probably by Synovial Fibroblasts (SF) [11]. Osteochondral defects allow the immigration of Bone Marrowderived MSC (BMSC), which represent a major source for defect coverage. A hyaline-like repair tissue can be produced by invading MSC, which undergo a chondrogenic differentiation. Blood-derived Programmable Cells of Monocytic Origin (PCMO) are another cell population capable of chondrogenic differentiation [12]. These cells can be released upon joint bleeding, associated with osteochondral cartilage breakdown, in response to traumatic injury. OB, osteocytes and osteoclasts might also contribute to modify the healing response, especially, when the subchondral bone plate is injured in osteochondral defects. However, irrespective of the origin of the precursor cell population, the resulting cartilaginous repair tissue remains fibrocartilage in nature, and is unable to sustain joint loading for a long time [13]. Much effort is currently undertaken to understand and optimise cartilage formation by chondrogenic precursor cells, with the hope to recruit them in future for cartilage repair.
Current approaches for improving chondrogenesis in vitro
Little is known about the factors stimulating chondrogenesis of precursor cells and dedifferentiated chondrocytes. Several approaches are used to provide natural chondrogenic mediators, including the use of conditioned media from differentiated AC, utilized to stabilise chondrogenic differentiation of MSC, or to stimulate redifferentiation of AC. The results of these studies strongly suggest the impact of soluble factors for chondrogenesis [14-17]. In other studies, synovial fluid was added to the culture medium to optimise chondrogenesis of BMSC [18]. In a similar manner, whole tissue chips, Extracellular Matrix (ECM) components and growth factors were applied to improve chondrogenesis [19-22]. Co-culturing of AC with different cell types (Table 1a and 1b), which are able to undergo chondrogenic differentiation has been utilised. These cells may stimulate each other in a paracrinic manner, and by providing direct cell-cell contacts to produce a plethora of trophic or chondrogenic mediators. These mediators are so far unknown in its detailed composition, but play a critical role in stabilising chondrogenic differentiation.
Tissues and cell types used for co-culture
Since the ECM of tissues is a complex reservoir of mediators and growth factors, whole tissue chips or ECM components have been applied to improve neo-cartilage formation by cultured chondrocytes and chondrocyte-like cells [23,24]. For example, nucleus pulposus tissue was cultured with hyaline cartilage tissue (fragments of the vertebral endplate), whereby an enhanced Proteoglycan (PG) retention by the chondrocyte-like nucleus pulposus cells could be observed, along with a downregulation of the expression of catabolic factors [23]. Grässel et al. [21] performed direct and indirect co-cultures consisting of periostal explants and AC pellet cultures. They found a paracrine interaction between chondrocyte pellets and periostal explants. An increased release of TGFβ1 was observed in the culture supernatant of direct, but not indirect co-cultures. In contrast, type I collagen expression was elevated in chondrocyte pellets in indirect, but suppressed in direct co-cultures, and was inversely regulated in periostal explants, compared with in pellets [21]. Bonasia et al. [24] conducted co-cultures by combining minced chondral fragements of adult and juvenile donors. They observed an increased PG content and a higher number of safranin O positive cells, after 6 weeks in vitro, compared with the adult monocultures.
In a co-culture system, usually two cell types are combined, one type providing the chondrogenic differentiation stimulus, the other representing the “therapeutic” progenitor cell population (Table 1a and 1b).
However, an intimate mutual interplay is discernible between these two cell populations, combined in the co-culture. For example, it has been reported that MSC might reciprocally affect the stimulusprovider, and that an enhanced AC proliferation could be detected as a trophic feeder effect, mediated by the MSC [25,26]. AC, either freshly isolated, dedifferentiated [27,28], osteoarthritic [17], or derived from juvenile or adult donors [29], but also heterotopic chondrocytes, isolated from nasoseptal, auricular, as well as costal cartilage [26,29-33], were used in various combinations in co-culture systems. Stem cells of different origin, e.g. embryonic (ESC), adult BMSC or isolated from adult adipose tissue (ASC) or muscle tissue, were differentiated in co-culture with AC (Table 1a and 1b). Furthermore, Induced Pluripotent Stem Cells (iPSC) were recently generated from osteoarthritic chondrocytes. These iPSC were indistinguishable from ESC, and able to undergo chondrogenic differentiation [34]. Other cell types such as OB and SF were also co-cultured with chondrocytes. Last but not least, a subpopulation of human Dermal Fibroblasts (DF) underwent chondrogenic differentiation, since cartilage-like structures with typical lacunae were formed by these cells, when co-cultured with chondrocytes (Table 1a). Therefore, one might hypothesise that the above mentioned chondrogenic cells represent human dermis-derived stem cells.
In view of the complex composition of the intervertebral disc consisting of hyaline endplate cartilage, the fibro-cartilaginous annulus fibrosus and the absolutely unique nucleus pulposus tissues, co-cultures of disc-derived cell types will not be thoroughly discussed in the present review, which is intended to focus on co-culture based strategies for articular cartilage defect repair.
Co-cultures with dedifferentiated AC or derived from different cartilage zones: The co-culturing of freshly isolated AC with passaged AC (up to the fourth passage) had a stimulatory effect on their redifferentiation capacity [27,28,35,36]. This finding suggests that the differentiated AC may provide a paracrinic inductive environment for facilitating the expression of cartilage-specific ECM components. Interestingly, the stimulatory effect observed in these co-cultures was not limited to the cell donor species [35], as a similar effect was detectable when human and bovine cells were combined. Animalderived chondrocytes, a cell source easy to harvest, have been considered for xenogeneic chondrocytes transplantation [37].
Native articular cartilage is a tissue with complex multizonal architecture, whereby chondrocytes of the different zones show slightly divergent phenotypes and hence, synthetic profiles in in vitro culture [38-42]. Co-culturing of chondrocytes from different articular cartilage zones resulted in modified characteristics of the built constructs. For example, AC of the deep joint cartilage zone, co-cultured with those of the superficial zone in a bilayered three-dimensional (3D) culture system, revealed a reduced cell proliferation and an increased ECM synthesis within the construct, compared to deep zone derived AC, cultured alone. Cartilage constructs produced by co-culturing possessed an enhanced shear and compressive strength [43]. Monolayer coculture of superficial- and middle zone-derived chondrocytes resulted in a significant upregulation of lubricin secretion, and a simultaneous downregulation of Glycosaminoglycans (GAG) [44]. Co-culture of superficial and deep zone chondrocytes actively suppressed the ECM mineralisation of the deep zone chondrocytes [45]. In most of the cartilage tissue engineering studies, cartilage samples with less defined zones origin were used for chondrocyte isolation. Whether these chondrocyte populations of mixed/undefined zonal origin are the reason for the differences in the outcome of co-culture studies, is unclear. However, the above mentioned experiments together underline the impact of zonal-dependent cellular interactions of AC. It should be further investigated, whether chondrocytes of defined articular cartilage zones are usable as a more specific tool for co-culture based cartilage tissue engineering.
Co-culture with stem cells: Compared with chondrocytes, MSC are easier to harvest, lacking major donor site morbidity. The high proliferative activity of MSC bears the advantage that their in vitro expansion can be rapidly performed. Key problems with chondrogenic differentiation of stem cells are that (1) it is a time-consuming process, (2) it remains instable, (3) the chondrogenic capacity of stem cells is strongly donor, age and source dependent [46-48], and (4) it can lead to cartilage hypertrophy [49-51]. These drawbacks may be addressed by using co-culture systems.
Whereas the applications of human ESC are restricted for ethical reasons, multiple adult stem cell sources are explored for chondrogenic differentiation, e.g. bone marrow, adipose tissue, synovium, or muscle derived stem cells (Table 1b). Several studies suggested a strong chondrogenic effect of both direct [52,53], as well as indirect co-cultures of AC with ESC [54]. Nearly in every tissue, stem cell niches have been detected, but the particular differentiation potential of these adult MSC differs depending on their origin [46,55]. It is up to now unclear, which adult MSC source, the most promising one for chondrogenesis and cartilage repair is.
Meanwhile, many studies have shown that AC combined either in direct [56], or in indirect co-culture with MSC [5,17], stimulated their chondrogenesis (Table 1 a and 1b). The fact that cartilage hypertrophy is impaired in chondrocyte/MSC co-cultures compared with MSC monocultures is also supported by several studies [17,32,51]. In these co-cultures, both the deposition of type X collagen which is an indicator of hypertrophic chondrocytes and the expression of the osteogenic transcription factor RUNX2 decreased. Fischer et al. [57] hypothesised that this anti-hypertrophic effect was probably mediated by secretion of parathyroid hormone related protein.
Taken together, the above mentioned studies suggest that chondrocytic cells provide a strong stimulus for MSC of several sources to undergo chondrogenesis, and that the time-consuming in vitro differentiation of MSC might be shortened and stabilised by coculturing (Table 1).
Co-culture with synovial and dermal fibroblasts: In contrast to MSC, synovial fibroblasts (synoviocytes type B) are a natural part of the immediate joint compartment. They provide the key components of the synovial fluid for nutrition of articular cartilage. Synovial fibroblasts have been found as an interesting source for cartilage repair–untreated chondral defects are usually covered by a tissue layer produced by synovium-derived cells [11]. Moreover, a stem cell niche exists within the synovium; presenting an interesting approach for coculture experiments [58]. Rabbit-derived synovial MSC co-cultured with AC overexpressing the growth factor TGFβ3, lead to an improved ECM expression [59]. Moreover, D’Àndrea et al. [60] found cell-cell coupling between SF and AC via gap junctions in a direct co-culture of both cell types. Kurz et al. [61] reported in 1999 that chondrocytes exhibit protective effects in co-culture with synoviocytes, because the phorbol 12-myristate 13-acetate, Fe2++, and ascorbic acid induced lipid peroxidation was inhibited. Steinhagen et al. [62] established a co-culture perfusion system as a novel tool to investigate interactions of different cell types with less artificial interferences. Their results suggested that SF and their culture supernatants modulate the biosynthetic activity and the ECM deposition of chondrocytes, as well as the susceptibility to radical attack of reactive oxygen species. However, SF have also been reported to undergo an inflammatory interplay, in direct proximity with chondrocytes and hence, AC/SF co-cultures were often used as a testing system to analyse their inflammatory interplay [63,64]. It remains unclear which factors determine whether SF interact in a stimulatory or suppressive manner, when co-cultured with AC.
In contrast to SF, DF can be harvested with low donor site morbidity, representing an exhaustless cell source in the human body. Similar to SF, a DF subpopulation can exhibit progenitor cell properties suggesting that it possesses a stem cell character [65,66]. DF co-cultured with AC resulted in a certain fibroblast subpopulation undergoing chondrogenesis [65] (Table 1a).
Co-cultures with osteoblasts: Articular cartilage interacts with the subchondral bone in the joint compartment in-vivo and studies have demonstrated a pathological interplay, when normal AC were co-cultured with osteoarthritic OB [9,67-71]. Under these conditions, the AC became to a higher degree hypertrophic and its ECM calcified. This interaction led to the activation of ERK1/2 and suppression of p38 pathways [71,72]. Co-culturing of AC with OB plays a major role, e.g. to produce biphasic implants for osteochondral defect repair [73], and the distinct mutual interaction between both cell types becomes highly relevant.
Taken together, many of the available co-culture systems using various cell types provided a stimulatory effect for chondrogenesis (Table 1), whereby the underlying mechanisms remain mainly enigmatic.
Impact of co-culturing on cells, chondrogenesis and ECM quality
It is known that the effects observed in response to co-culturing of different cell types are divergent. Some commonly observed aspects are briefly summarised below.
Cell proliferation can be influenced by co-culturing. The coculturing of AC and osteoarthritic AC embedded in alginate beads with dedifferentiated AC increased their proliferation capacity. This effect seemed to be mediated by soluble factors [74]. Further, it has been shown that trophic effects of MSC, probably independent of their origin, increased chondrocytes proliferation in co-culture systems, combining chondrocytes or chondrocyte-like cells and various MSC types, e.g. ASC, BMSC, synovial membrane and muscle derived stem cells [25,32,75,76].
Further, the ECM deposition is modulated by co-culturing. Tissueengineered neo-cartilage matrix built by co-cultured cells should resemble the natural cartilage ECM, in regard to quantity, composition, distribution and organisation of their constituents. Typical components of cartilage ECM are type II collagen and cartilage PG, especially aggrecan. The expression of some of them is regulated by the master chondrogenic transcription factor SOX9. The type II/I collagen ratio is expected to be high, since type I collagen is not present in natural healthy articular cartilage, but is up-regulated in culture during chondrocyte dedifferentiation. The content of type X collagen, typical for hypertrophic chondrocytes, should be low. An elevated PG and collagen expression was observed in AC constructs co-cultured with a feeder monolayer consisting of AC [33]. Upregulation of cartilagespecific ECM components has also been observed in a multitude of other AC/MSC co-culturing studies, as summarised in table 1b.
Suppression of inflammatory and catabolic factors can occur in coculture. The ECM balance is affected by catabolic and inflammatory factors. Co-culturing of chondrocyte-like cells with native cartilage tissue led to the suppression of the gene expression of inflammatory factors; Matrix degrading enzymes such as Matrix-metalloproteinase (MMP)3, MMP13, a disintegrin and metalloproteinase with thrombospondin motif (ADAM-TS)5, the pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α and TNF-α Converting Enzyme (TACE), which activates TNF-α, were downregulated [23]. Altogether, the above mentioned factors mediate ECM degradation in a direct and indirect manner and hence, impair ECM quality and strength.
Biomechanical properties of the ECM are influenced by coculturing. Biomechanical competence of cartilage is the result of sufficient ECM deposition, balanced composition, organisation and maturation (possibly by cross-linking of collagen fibers, or forming of collagen/PG composites), and reduced ECM degradation. Biomechanical competence clearly characterises cartilage functionality, and is critical for healthy joint cartilage and tissue-engineered neocartilage constructs. Functional properties were improved when constructs were produced by co-culturing of AC or auricular chondrocytes with BMSC [32,51], or when AC embedded in a hydrogel were combined with a feeder layer of AC [33]. Modulating the cell to cell ratio of two co-cultured connective tissue cell types can allow the design of a desired ECM quality and appropriate biomechanics [77]. Adequate biomechanical stimuli are important for chondrogenesis. Intermittent loading and some shear stress are the natural impulses for joint cartilage homeostasis. Dynamic culturing provides chondrogenic biomechanical stimuli [78,79]. A low oxygen environment determinates chondrocyte synthetic profile and improves chondrogenesis of BMSC [80-83]. Therefore, mechanostimulation and low oxygen tension might further improve the chondrogenesis in AC/MSC co-cultures.
Soluble/paracrine mediators in co-culture: Soluble mediators may play a pivotal role in cell-cell interactions in co-cultures. Although some authors stated that these mediators are more important for chondroinduction in co-culture than direct cell-cell contacts [84], other studies suggested that direct cell-cell interactions are preferentially responsible for improved chondrogenesis [26,33,85]. This divergence could be explained by the selection of different investigation points of time and culture conditions.
A release of growth factors such as TGFβ1, IGF-I, BMP2 was detected in the supernatants of co-cultured BMSC and AC [84]. The above-mentioned factors, among others, stimulate MSC migration and proliferation [5], however, the signaling pathways activated in the coculture system remain largely unknown. Enhanced gene expression of several growth factors such as TGFβ1,-2,-3, BMP4,-5, IGF-I, EGF, VEGF, FGF2 and some of their receptors was reported in a MSC cell line (Kp-hMSC) co-cultured with chondrocytes by Chen et al. [86], Growth factor associated signaling proteins, the Smads, were found to be upregulated. In view of the well-known chondrogenic effect of TGFβ1, iPSC were lentivirally transduced with TGFβ1 by Wei et al. [34] to improve ECM synthesis. MSC are also known to exhibit immunomodulatory effects by the release of cytokines and other factors such as IL-10, PGE2, indoleamine 2,3 dioxygenase, as shown for human amniotic membrane-derived mesenchymal stem cells by Kang et al. [87]. Acharya et al. [26] suggested that, particularly IL-10, which was released in AC/BMSC co-cultures, could be involved in chondro-induction. It was, in contrast to the other detected soluble factors, initially upregulated (at the third day of co-culture), and later (after 14 days) downregulated [26]. Soluble ECM components might also contribute to chondro-induction in co-cultures.
Conditioned medium: using chondrocytes culture extracts for cultivation of MSC?: Conditioned media are a versatile tool to stimulate progenitor cells, particularly, if the stimulatory factors and their levels produced by chondrocytes are undefined. In 1973, Soloursh and Meier [14] reported that supernatants from cultured chondrocytes improved their own differentiation. Endochondral cartilage growth was stimulated in avian tibiotarsal organ cultures by conditioned media from articular perichondral cells and hypertrophic chondrocytes. Interestingly, the use of conditioned media from other cell types can lead to suppressive effects [15]. Lee and Im [16] showed that conditioned media from chondrocyte culture induced chondrogenesis of ASC. When MSC received conditioned media from OB, their chondrogenesis was enhanced [88]. However, Aung et al. [17] found no stimulatory effect when co-culturing AC and the MSC cell line p7043L. Media volumes per cell and incubation time periods are important contributing factors for an effective concentration of mediators in conditioned media.
In summary, conditioned media can be a suitable tool to stimulate chondrogenesis of precursor cells. Further efforts should be undertaken to identify the active soluble mediators in these culture supernatants, and other components which might be counterproductive.
Co-culture systems
Direct and indirect co-culture: In mature articular cartilage, individual chondrocytes are separated from each other by their ECM, which is only permeable for small particles (60 nm), restricted by the collagen fiber meshes [89]. However, during early chondrogenesis, intimate cell-cell contacts are important for chondrogenic differentiation of mesenchymal precursor cells. In the native cartilage, some cell-cell contacts are still present in the superficial layer, since the exchange of a fluorescent dye clearly indicated cell-cell communications [90]. Varying types of co-culture systems have been used, which allow either direct cell-cell contacts, or are proposed to study separately the effects of soluble mediators (Table 1, figure 1).
Direct co-cultures allow both cell-cell contacts, as well as the exchange of soluble mediators. During cartilage development and joint cavity formation, condensations of mesenchymal precursor cells and intimate cell-cell interactions are the starting point for chondrogenesis and hence, direct cell-cell contacts might contribute to improved chondrogenesis in co-culture systems [91,92]. Comparable processes proceed in endochondral ossification during fracture healing, within the callus. Direct co-cultures, allowing multiple cell-cell contacts and intimate interactions, can be performed by mixing both cell types, using different cell to cell ratios, whereby diverse culture systems (monolayer, 3D scaffold-free high-density, hydrogel or scaffold-associated) can be used. Some cell-cell adhesion molecules found on chondrocytes surface [93], e.g. vascular (V)-Cell Adhesion Molecule (CAM)1, V-CAM2, intercellular (I)-CAM, and the gap junction proteins connexins, can be involved in the differentiation processes [94]. However, the particular mechanisms/targets of contact formation remain unknown. Direct cellcell contacts via connexins have been shown in direct co-culture of SF and AC [60]. Gap junctions consisting of connexins have been shown to play a role in chondrocyte differentiation in vitro [94]. The role of gap junctions has not been studied in MSC co-cultured with AC which undergoes chondrogenic differentiation, but in human fetal amniotic membrane MSC co-cultured with cardiomyocytes, heterocellular gap junction coupling could be detected [95]. Moreover, the bidirectional exchange of membrane components has recently been observed in BMSC co-cultured with chondrocyte-like nucleus pulposus cells [96].
Some evidence in the literature suggests that the presence of some ECM components or intact tissue, e.g. a porcine cartilage ECM scaffold, stimulated and stabilised chondrogenic differentiation of BMSC [97]. β1-integrins acting as essential cell matrix receptors play a substantial role in chondrocytes differentiation, as well as in co-cultures of AC and ESC, where β1-integrin activating antibodies could amplify pellet sizes [86,98]. An indirect co-culture experiment of MSC with another cell type, endothelial cells, indicated the stimulatory role of a via cadherin/ β-catenin mediated pathway [99]. Co-culturing of BMSC with vascular smooth muscle cells elucidated another mechanism of direct cell-cell coupling via formation of so called tunnelling nanotubes between BMSC and the smooth muscle cells [100].
Combining cells with different growth rates and nutritional requirements in co-culture might bear some problems, e.g. the overgrowth of one cell type caused by different growth rates. Not only MSC, but also chondrocytes are able to migrate in culture [5,101], where they may sort each other to form an organised zonal tissue, as observed during chondrogenesis with ESC [102]. Adverse effects such as phagocytosis or cytotoxicity should be considered, since some cells used in co-cultures, e.g. chondrocyte-like nucleur pulposus cells, are capable for phagocytosis [103]. Bian et al. [51] concluded that a close proximity between the co-cultured cell types is necessary. When MSC and AC were co-cultured in the same chamber, but trapped within separate gels, no stimulatory effect was evident.
In indirect co-cultures, where no cell-cell contacts were possible, no effect could be detected [84,85]. On the contrary, Liu et al. [84] reported that cell-cell contacts alone are insufficient to improve the chondrogenesis of the co-cultured cells. Indirect co-cultures are a suitable tool to assess solely the effect of soluble mediators. One can assume that cell-cell adhesion plays only a minor role in mature intact cartilage in situ, where chondrocytes are surrounded by an abundant dense ECM, mostly impermeable for cells. Hence, soluble factors are of high importance for cartilage homeostasis. The impact of soluble mediators (e.g. growth factors, cytokines, chemokines, but also soluble ECM components) can be distinguished using an indirect co-culture system. Indirect co-cultures can be performed using two chamber systems [29], or by immobilising the cell populations using e.g. hydrogels. However, it is well known that some chondrocytes might emigrate from hydrogels, e.g. alginate [104,105]. Some chemotactic factors are known to attract and recruit stem cells [101,106]. Chemotactic agents attracting chondrocytes are less well known and it is still unclear, whether these factors have an effect on chondrocytes. Serum and Platelet Derived Growth Factor (PDGF) have been shown by Mishima and Lotz [101] to attract human AC. Serum, PDGF, VEGF, IGF-I, IL-8, BMP4, BMP7, but also CXCL-10 and -11 induced a chemotactic response in BMSC [101]. These factors might be valuable to recruit MSC in vivo, but might also play a role in co-cultures.
In summary, it remains a controversy, whether direct cell-cell contacts or soluble mediators are more inductive for chondrogenesis. It might be dependent on the cell types combined in a co-culture system and the phase of chondrogenesis.
Influence of culture systems used for co-culturing and culture time: Monolayer (2D) culture is the simplest form of chondrocyte culture; however, the risk of irreversible dedifferentiation of chondrocytes in 2D culture is well known [107-109]. 3D cultured AC has also been combined with chondrocytes in monolayer culture, acting as a feeder cell layer [33]. In view of the fact that super-confluence is reached rapidly in chondrocyte monolayer culture, chondrocytes have to be passaged and this enzymatic cell detachment might disturb the interactive milieu in co-culture. Hence, most co-cultures are performed in 3D culture systems including alginate, agarose, or other hydrogels [30,110], alginate/chitosan microcapsules [111], PGA felts [31,65,112], high-density or pellet culture or as a “side by side culture” using a insert systems [27], or tube chamber system [36] (Table 1, figure 1).
The influence of the particular co-culture system setting on the quality of chondrogenesis has not been systematically investigated. 3D cultures provide divergent possibilities for cell-cell and cell-matrix contacts, e.g. with a high contact area in pellet cultures, and a limited contact when both cell types were trapped within the same hydrogels. The permeation of soluble mediators might be lower in pellet systems because of the high cell and ECM density, compared with monolayer and hydrogel cultures. To allow a continuous nutrient supply, bioreactor systems have a strong influence on the chondrogenesis in in vitro produced cartilage constructs [113]. Whether such effects would enhance chondrogenesis in co-culture is yet to be verified. Pound et al. [111] found a beneficial effect by using a rotating wall reactor, as well as a perfusion system, compared with a static cultivation by using cocultures consisting of chondrocytes and BMSC.
Co-culturing time and time courses of analysis differed substantially between studies, contributing to the lack of established cell culture parameters, necessary for effective chondrogenesis. In the study of Tan et al. [33], the stimulatory effect produced by co-culturing of dedifferentiating and differentiated chondrocytes was only transient and disappeared later. Giovannini et al. [114] reported that rather the early neo-chondrogenesis was stimulated by AC/BMSC co-culture. For this reason, it is important to undertake further studies to detect the sensitive phases of co-culturing, which might be specific for the cell types combined, and each culture system applied.
Influence of the cell to cell ratio of co-cultured cells: Although Hildner et al. [115] could not discriminate the influence of the cell to cell ratio, they observed a chondro-inductive effect (e.g. higher SOX9 gene expression) at all chosen AC/ASC cell ratios (1:4, 1:10, 1:20), using three different biomaterial associated direct co-cultures. They concluded that the cell donor heterogeneity in their study could be a possible reason for the lack of defining an optimal cell to cell ratio. On the contrary, other studies revealed a clear dependency between particular cell to cell ratios in co-culture and the efficacy of chondrogenesis. In the study of Qing et al. [116], the optimal cell to cell ratio (AC:BMSC) appeared to be 2:1, when 4:1, 2:1, 1:1, 1:2, 1:4 ratios were tested. Acharya et al. [26] combined AC or nasoseptal chondrocytes with BMSC or ASC in co-culture and observed a stronger chondro-induction at a cell to cell ratio of 1:3, when compared with 1:9. Bian et al. [51] found a robust chondro-induction co-culturing the AC and MSC at a ratio of 1:4. Auricular chondrocytes and BMSC were combined at a ratio of 1:1 in the study of Kang et al. [32], leading to a strong chondro-induction.
Summarising the results of co-culture studies using different cell to cell ratios does not allow a uniform proposal, applicable to all cell types commonly co-cultured with AC. Optimal cell to cell ratios might depend on the cell types combined and should be defined for each cell type and culturing conditions separately.
However, the most economic concept would be to combine a smaller number of human primary AC with progenitor cells that can be obtained in high numbers, and are easy to harvest and expand to promote chondrogenesis in co-culture [17]. Another strong argument for this approach is the observation of Acharya et al. [26] that during AC/BMSC co-culturing, the number of BMSC continuously decreased.
Effects impairing chondrogenesis and influence of donor species
Phagocytosis and cytotoxicity might arise, when different cell types were combined. Chondrocyte-like nucleus pulposus cells are capable to phagocytise, if induced [103]. Whether AC or MSC are also able to phagocytise, remains unclear. Human AC can present antigens to autologous cytotoxic T-cells, indicating that AC interacts with immune cells. Cell death, cell lysis via complement activation, granzyme B or necrosis might also be observed in co-cultures [117]. Chondrocytes have been shown to exhibit cytotoxicity after pretreated with granzyme B [118]. Hence, it should be carefully recognized that suppressive effects might occur when chondrocytes were co-cultured with other cell types. Co-culture of juvenile and adult chondrocytes revealed also a suppressive effect on the ECM synthesis of the juvenile cells [24]. The authors hypothesised that the adult cell population developed an increased catabolic activity.
Hence, reciprocal effects have to be considered when using coculture models–whereby both cell types have to be analysed for their specific response. It is rather complex to assess chondrogenesis, particularly, in direct co-cultures, since it is difficult to visualise exactly the survival of both cell types combined, and to analyse which cell type is responsible for ECM formation, or the release of soluble mediators.
For this reason, cell types derived from different donor species or genders are combined in co-culture to allow the distinction between them and their synthetic products using species-specific analysis tools, such as primers or antibodies [26]. Nevertheless, immunological interactions between the cell types might be misleading. However, species-independent effects were observed in co-cultures, where cells of different species were combined [26,35,67]. For instance, rat OB were stimulated with bovine chondrocytes in the study of Nakaoka et al. [67], or porcine AC induced human DF, to undergo chondrogenic differentiation [65]. Bovine chondrocytes were utilised to stimulate the osteogenic differentiation of rat BMSC. The interspecial nature of these co-cultures did not appear to significantly affect the response of rat BMSC to the osteoinductive influence of bovine chondrocytes [119].
Taken together, cells of different species can be combined for in vitro analyses without major problems. However, in vitro and in vivo xenotransplantation studies suggest that an immune response may occur, if chondrocytes deriving from different species were combined [37,120]. TNF-α release and CD86 and VCAM-1 expressed on the chondrocytes might be involved in these processes [37].
In vivo experiments utilising co-cultures
In the face of the bulk of in vitro co-culture studies, in vivo studies are very rare. Several studies could consistently demonstrate that BMSC underwent ectopic chondrogenesis in the subcutaneous milieu of immuno-comprimised mice, when co-transplanted with mature chondrocytes [32,84,111,121]. Co-cultures should be transferred in future in in vivo cartilage repair models.
Conclusion and Outlook
Co-cultures, performed by combining chondrocytes with various chondrogenic progenitor cells improve chondrogenesis by stimulating chondrocyte proliferation, increasing cartilage ECM synthesis, its biomechanical stability, and by limiting the degradation of the neomatrix. Additionally, AC/MSC co-cultures seem to be a tool to give MSC differentiation, a more stabilised chondrogenic direction impairing the risk of cartilage hypertrophy.
Chondro-induction can be mediated by soluble mediators. These mediators are up to now insufficiently characterized. They should be studied in ongoing experiments and the signaling pathways engaged have to be explored. However, a bulk of studies suggested that chondro-induction is not exclusively mediated by soluble mediators, but a multitude of mostly unknown cell-cell and cell-ECM interactions seem to be involved, which requires further attention.
In addition, low oxygen tension and mechanostimulation are well-known impulses to improve cartilage-specific ECM synthesis and chondrogenesis of MSC. However, up to now, most of the co-culture experiments have been performed under normoxic conditions, without any mechanostimulation. These stimuli should be utilised in future, hopefully, amplifying chondro-inductive effects of co-culturing.
The donor age of both cell types combined in a co-culture should be kept in mind as an influencing factor.
Last but not least, the available knowledge extracted from the huge bulk of literature concerning co-cultures has to be transferred into ongoing in vivo experiments in the joint milieu. The joint milieu is influenced by systemic “extrinsic” factors, which affect chondrogenesis. These extrinsic factors should also be defined in future in vivo.
The authors would like to acknowledge the support of the ENDO Foundation.

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