Autologous chondrocyte implantation represents a paradigm in the biologic repair of joint surface defects (1). Despite the promising clinical results (2, 3), the limitations of this procedure may be related to the source and the in vitro expansion of the cells. Chondrocytes are obtained by articular cartilage biopsy of an “uninvolved” and minor load-bearing area of the same joint. This results in additional injury to the joint surface, creating a potential locus minoris resistentiae and, possibly, an increased risk of developing osteoarthritis, especially in genetically predisposed individuals. Moreover, since the clinical indications may extend to joints with early osteoarthritis, the availability of healthy articular cartilage may be a limiting factor.
Chondrocyte expansion is essential for obtaining a sufficient number of cells for implantation. In vitro expansion of mature articular chondrocytes poses its challenges. The expandability of chondrocytes, as with most somatic cells, is limited by the occurrence of cell senescence (4). More importantly, chondrocyte expansion in monolayer cultures results in dedifferentiation (5) and loss of the capacity to form stable hyaline cartilage in vivo that is resistant to vascular invasion and endochondral ossification (6). This may account for some reported variability of autologous chondrocyte implantation and, more particularly, may affect the quality of the repair tissue and the long-term outcome of this treatment. Indeed, in some patients, the repair tissue consists of poorly differentiated and disorganized fibrocartilage (2).
Mesenchymal stem cells (MSCs) can be obtained without irreversible damage, are easily expandable with limited senescence, and are “phenotypically stable” throughout expansion and after storage in liquid nitrogen, as assessed by a number of molecular markers (7–9). The use of MSCs is restricted by insufficient knowledge of the long-term stability of the repair tissue and by their tendency to differentiate into other cell lineages, with the associated risk of heterotopic tissue formation (10). Autologous chondrocyte implantation can indeed be performed using cells that are stably committed to the appropriate phenotype and naturally resistant to vascular invasion, mineralization, and ossification (6).
We previously described an in vivo assay for measuring the capacity of adult human articular cartilage–derived cells (ACDCs) to form ectopic stable cartilage in vivo when injected intramuscularly into nude mice (6). In the same study, we identified molecular markers that reproducibly predict the outcome of this in vivo assay, independently of donor age. Stable ACDCs, as defined by the expression of these markers, were capable of forming ectopic stable cartilage in a per se nonchondrogenic environment, the skeletal muscle (6), and thereby partially overriding environmental signals (11). This marker set included type II collagen, specifically, α1(II) collagen (COL2A1), fibroblast growth factor receptor 3 (FGFR-3), and bone morphogenetic protein 2 (BMP-2) as positive markers, and activin receptor-like kinase 1 (ALK-1) as a negative marker (6). We have also characterized a population of adult human MSCs from the synovial membrane (SM) that are capable of differentiation into cartilage, bone, skeletal muscle, and adipose tissue in vitro (9). Subsequently, we demonstrated that SM MSCs can differentiate into skeletal muscle in vivo when injected intramuscularly into nude mice (12).
In the present study, we investigated whether human SM MSCs can acquire in vitro the expression of the reported markers of the stable-chondrocyte phenotype of ACDCs (6). We also examined whether this is associated with the capacity to form ectopic stable cartilage in vivo.
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- MATERIALS AND METHODS
Autologous chondrocyte implantation is becoming a generally accepted technique for the repair of focal joint surface defects and has proved superior to mosaicplasty in a prospective controlled clinical trial (3). However, several constraints, including those related to the source and the manufacture of the cellular product, represent important limitations for daily clinical practice.
SM MSCs are a promising alternative to ACDCs for the repair of joint surface defects. A small SM biopsy specimen represents an easily accessible source of autologous MSCs in the context of an explorative, diagnostic, or therapeutic arthroscopy. Expanded human SM MSCs maintain a stable molecular profile, as assessed by a number of markers, and retain the capacity to form a cartilage-like tissue in vitro (under specific conditions) over at least 10 passages as well as after cryopreservation (9). These properties of SM MSCs represent an opportunity to increase the consistency, reproducibility, and flexibility of the autologous cell implantation procedure. Yet, the use of multipotent MSCs implies the risk of unforeseen events, such as heterotopic tissue formation (10). Predifferentiation has been suggested as a useful step for inducing proper lineage commitment and reducing the risk of undesired tissue formation by restricting alternate differentiation pathways. However, it is not clear to what extent the phenotype acquired in vitro is maintained once the cells are implanted in vivo.
In this study, we investigated the stability of the chondrocyte-like phenotype obtained in SM MSCs by combining micromass culture and TGFβ1 treatment, using the nude mouse assay for stable-cartilage formation in vivo in an ectopic site validated with ACDCs and the associated molecular markers (6). We demonstrated that SM MSC–derived chondrocyte-like cell populations obtained after 3 weeks of TGFβ1 treatment in micromass culture acquired the expression of the markers associated with the stable-chondrocyte phenotype at levels comparable with those of stable ACDC populations. However, the chondrocyte-like phenotype of SM MSCs was transient in vitro upon removing the chondrogenic conditions and replating the released cells in monolayer. In addition, despite the expression of the stable-chondrocyte markers, cells released from TGFβ1-treated micromasses failed to form stable cartilage when injected intramuscularly into nude mice, suggesting that the loss of the chondrocyte-like phenotype is not solely related to the monolayer culture conditions.
The cell fate of the SM MSCs differed in the two nude mouse assays adopted in this study. In TGFβ1-treated micromasses implanted subcutaneously, we observed cell death and neoangiogenesis within the cartilage-like tissue. Although the experiments were performed in immunodeficient nude mice, it cannot be excluded that some form of inflammatory/immunologic reactions might have contributed to the death of the human cells after micromass implantation. However, no obvious inflammatory infiltrates or foreign-body reactions were documented at histologic examination at all time points examined. Alternatively, these data could be interpreted as showing the formation of a transient cartilage tissue in the context of endochondral ossification. Indeed, during endochondral ossification, the cartilage mold is invaded by blood vessels, undergoes apoptosis, and is replaced by bone (16, 17). However, we were unable to detect bone formation up to day 40, in contrast to bone seen in cartilage tissue obtained by implantation of epiphyseal chondrocytes (ref. 18 and Dell'Accio F, et al: unpublished observations). It is possible that bone did not form because of either an absence of competent osteoprogenitors within the implanted human micromasses or a failure in their recruitment from the mouse host.
Although we have proved that human SM MSCs are multipotent at the single-cell level, and are therefore also osteogenic, we cannot exclude that micromass culture and TGFβ1 treatment may have limited their differentiation potential. Also, when implanted within the skeletal muscle of the thigh of nude mice, TGFβ1-treated micromasses did not give rise to any retrievable cartilage or bone tissues, suggesting that the lack of stability of the implanted cartilage-like tissue and the absence of bone are not related to the site of implantation. When the cells were released and injected intramuscularly, we were unable to retrieve any cartilage-like tissue, although human cells were detected for at least 40 days. In contrast, after subcutaneous implantation of the TGFβ1-treated micromasses, human cells were not retrieved at time points later than 15 days. The released cells, but not the cells aggregated in micromasses, displayed the capacity to survive and to respond to environmental signals. RT-PCR detection of human MyHC-IIx/d at 3 weeks suggests muscle differentiation of the intramuscularly injected released cells. SM MSCs are known to be competent for myogenesis after transplantation into skeletal muscle (12). The acquisition of a muscle phenotype seems to indicate that myogenic environmental signals were sufficient to override the induced chondrocyte-like phenotype of the injected cell population.
To respond to inductive signals, competent cells must be surrounded by similar cells (19). This phenomenon, called the “community effect,” has been described in the mammalian embryo as a prerequisite for mesodermal cells to undergo differentiation and maturation (20) and has been shown to be important for the maintenance of a stable-cartilage phenotype by ACDCs (11). The capacity to form cartilage in vivo by stable ACDC populations is dependent on the number of cells, since injection of 5 × 105 cells did not result in the formation of any retrievable cartilage, and although cells persisted in the muscle, as determined by in situ hybridization for human Alu repeats, no cartilage tissue was detected in serially sectioned injected muscles (ref. 11 and Dell'Accio F, et al: unpublished observations). This suggests that specific threshold levels and, possibly, autocrine/paracrine mechanisms are required for in vivo cartilage tissue formation by stable ACDCs. A threshold might also be required for SM MSC–derived chondrocyte-like cell populations and could be higher than the threshold for stable ACDCs, thereby explaining, at least in part, the failure to form stable cartilage in this assay.
Two lines of evidence, however, plead against this possibility. First, 10 × 106 viable cells from TGFβ1-treated SM MSC micromasses proved to be insufficient to form cartilage in vivo (data not shown). Second, undigested intact micromasses, in which the SM MSC–derived chondrocyte-like cells remained tightly clustered together, also failed to retain their cartilage-like phenotype when implanted in vivo. It remains to be investigated whether the same cell populations would make durable cartilage tissue when implanted into a cartilage-inducing/maintaining environment, such as an articular cartilage defect. Moreover, the proportion of cells with a stable-chondrocyte phenotype and the expression of each marker at the single-cell level might differ between the stable ACDC population and the SM MSC–derived chondrocyte-like cell population. The aim of this study was to compare the SM MSC–derived chondrocyte-like cells with the stable ACDCs as whole cell populations in terms of the expression of the molecular markers reported to be associated with the stable-chondrocyte phenotype of ACDCs and in terms of the related capacity to form ectopic stable cartilage in vivo (6). A phenotype analysis at the single-cell level is an ongoing effort in our laboratory.
We previously found that when injected into regenerating anterior tibialis muscles of nude mice, stable ACDC populations did not form a retrievable cartilage pellet, but maintained their expression of type II collagen in vivo for at least 4 weeks (11). In the present study, SM MSC–derived chondrocyte-like cells did not retain the expression of type II collagen (undetectable at 3 weeks). This might be due to either selective cell death or nuclear reprogramming with, possibly, a shift from the chondrocyte-like phenotype to the myocyte-like phenotype. The data obtained in vitro demonstrate that the chondrocyte-like phenotype of SM MSCs is transient and is rapidly lost upon removal of the chondrogenic conditions. Whether the chondrocyte-like cells dedifferentiate to the original phenotype of expanded SM MSCs, undergo cell death, or are overgrown by the undifferentiated cells remains to be addressed. The absence of detectable senescence, cell death, or proliferation would lend support to a rapid dedifferentiation process, making the cells competent to respond to myogenic signals. This is not surprising, and it is especially not specific to SM MSCs, since human ACDCs also undergo a myogenic fate in this assay after they have lost their phenotypic stability due to serial passaging (11). We recently reported that myogenesis of SM MSCs injected into regenerating skeletal muscle takes place in the absence of nuclear fusion in a multistep process where myogenic commitment happens very early and before integration into host myofibers (12). However, we cannot rule out the possibility that under different experimental conditions, the expression of human MyHC-IIx/d may be the consequence of reprogramming of human nuclei secondary to fusion into host muscle fibers (21).
The molecular markers identified so far as being predictive of the in vivo stable-cartilage–forming capacity are specific to ACDC populations (6) and, under our experimental conditions, appear not to be directly applicable to other chondrogenic cell populations, such as SM MSCs. This emphasizes the basic question related to the identification of the signaling pathways that are necessary and sufficient to obtain and maintain the stable-chondrocyte phenotype (22).
The clinical relevance of our findings pertains to the use of MSCs for articular cartilage repair. Although SM MSCs can be converted into a cartilage phenotype (9), our data indicate that predifferentiation in vitro is not sufficient to guarantee stable lineage commitment and restriction of differentiation. Indeed, the cartilage-like phenotype induced in vitro was not stable in nonchondrogenic environments and did not prevent muscle differentiation when the cells were implanted into skeletal muscle. The stabilization of cell lineage commitment is a challenge in modern tissue engineering and can be achieved by means of signaling molecules, genetic manipulations, biomechanics, and the implantation of cells within 3-dimensional (bioactive) scaffolds (23). Finally, we are aware that the mouse assay for stable-cartilage formation is not representative of the joint environment. Clearly, studies in animal models of joint surface defect repair (24) are necessary to evaluate the potential use of SM MSCs and their phenotypic behavior within the articular cartilage microenvironment.