HEIDELBERG, Germany—Joint cartilage has little ability to repair itself, but chondrocytes taken from relatively healthy areas of cartilage can be expanded through in vitro culture and reimplanted to repair cartilage defects with some degree of success. Because this involves possible morbidity at the donor site, and because articular chondrocytes lose their special characteristics the more times they divide in vitro, researchers turned to adult mesenchymal stem cells (MSCs) as a possible alternate source for transplantable chondrocytes. The hope was that MSCs could become stable chondrocytes, providing large amounts of vat-grown cartilage that could be used to repair even large tears and defects. However, Karoliina Pelttari and colleagues at the Orthopaedic University Hospital of Heidelberg in Germany discovered while comparing cultured bone marrow MSCs to cultured articular chondrocytes that the MSCs did not reliably form and maintain hyaline cartilage. The data Ms. Pelttari reports in Arthritis & Rheumatism show that under the culture and in vivo conditions tested, the MSCs were disappointing.¹

"An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID [severe combined immunodeficiency] mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype," Ms. Pelttari writes.

Mesenchymal stem cells compared to autologous chondrocytes

The investigators asked whether human MSCs derived from bone marrow would achieve the normal stages of chondrogenesis when cultured in vitro and whether, when implanted into SCID mice, pellets of such cells could develop into stable cartilage. If so, MSCs might offer an attractive source of cells that could be used to grow cartilage patches for damaged joints. They hypothesized that "upon chondrogenic stimulation, MSCs would recapitulate natural embryonic differentiation pathways in which articular, but also hypertrophic, phenotypes may be reached."

The MSCs were harvested from bone marrow samples obtained from nine subjects undergoing total hip replacement of iliac bone graft harvest. Articular cartilage samples were taken from the tibial plateaus of nine patients with knee osteoarthritis, from areas without evident degeneration, and from the knee of one normal subject.

The MSC cells were fractionated by density-gradient centrifugation and seeded in culture flasks in MSC expansion medium; nonadherent cells were removed, and colonies of adherent MSCs were expanded to about 16–18 population doublings in culture. The cartilage samples were digested with collagenase B and hyaluronidase, and human articular chondrocytes (HACs) were seeded either in low-glucose Dulbecco's modified Eagle's medium (DMDM) or in MSC expansion medium for two to six population doublings.

Pellets of MSCs or expanded HACs were induced to differentiate into cartilage by culture in a chondrogenic medium for up to 7 weeks. Pellets of freshly isolated chondrocytes, chondrocytes expanded for two population doublings, or MSCs were then transplanted into subcutaneous pouches on the backs of SCID mice for 4–6 weeks, then harvested and examined.

Little cartilage formed from MSCs

The investigators found that during the in vitro expansions, hypertrophy-associated genes were prematurely upregulated in the MSCs but not in the HSCs. Immunostaining showed that in the MSC pellets in vitro, type X collagen was deposited prior to type II collagen, and alkaline phosphatase activity was upregulated. The HACs down-regulated cartilage differentiation markers during culture, but apparently did not "forget" their history as stable articular chondrocytes.

After the cartilage-like pellets of the various types of cells had been transplanted into SCID mice for 4 weeks, ectopic stable cartilage had formed from all of the chondrocyte-derived pellets, but the MSC-derived pellets  either remained fibrous, disappeared, or were maintained as fibrocartilage-like tissue that stained for both type II and type I collagen. MSC pellets also showed vascular invasion and the formation of minute ossicles.

Pelttari et al conclude, "On the basis of these observations, we had to reject the generally assumed hypothesis that natural differentiation cascades of chondrogenesis can be recapitulated by MSCs in this in vitro model."

The single-step induction protocol for chondrogenesis used in in vitro models is probably too simple to mimic the natural conditions for cartilage differentiation or to induce permanent articular chondrocytes for repair when needed. "[W]e suggest that common protocols of in vitro chondrogenesis be improved so that permanent chondrocyte differentiation can be induced in the absence of hypertrophy," Pelttari says.

In an editorial that accompanies this article, Rocky S. Tuan, MD, of the Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, in Bethesda, Maryland, writes, "Application of autologous chondrocyte implantation has already garnered an extensive history, thus providing a sound basis for the future development of cell-based cartilage repair and regeneration." Dr. Tuan points out, however, that although the results of autologous chondrocyte implantation are generally considered acceptable for small lesions, the effectiveness of the procedure is under debate. ²

"The challenge in using MSCs as a cell source for articular cartilage tissue engineering is therefore to maintain the MSC-derived chondrocytes in the prehypertrophic state and prevent them from undergoing terminal differentiation as seen in the growth plate," Dr. Tuan writes. He points out that the MSCs were used after up to 18 population doublings, while the HACs were used after only two to six doublings. A more appropriate comparison would be between cells of similar passage history. Dr Tuan also suggests that implanting into the subcutaneous pouch "clearly does not provide sufficient mechanobiologic stimulation to the developing cartilage and could contribute to the premature hypertrophy observed in these cultures compared with cultures derived from articular chondrocytes that have a history in the weight-bearing joint."

Study is not the last word on MSCs

David S. Pisetsky, MD, PhD, chief of the division of rheumatology, Duke University Medical Center, Durham, North Carolina, told CIAOMed that the results of this study are disappointing but not the last word on whether adult mesenchymal stem cells are worth pursuing.

"This paper does not show that this approach won't work, only that it won't work under these particular culture and implantation conditions," Dr. Pisetsky said. "Stem cells for cartilage repair will be difficult to develop. Development varies depending on context and probably on other things such as cytokines. This study tells us that we need more in the way of control of the culture conditions and that we need more refined assays."

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