Commentary & Perspective

Commentary & Perspective on
"Chondrocyte Transplantation into Articular Cartilage Defects with Use of Calcium Alginate: The Fate of the Cells"
by Cay M. Mierisch, MD, et al.

Commentary & Perspective by
Tom Minas, MD, MS*,
Cartilage Repair Center, Brigham and Women's Hospital, Harvard Medical School, Boston MA

Tissue engineering represents the future of musculoskeletal repair. Our hope as clinicians and scientists is that the restoration of anatomy will resolve pain and restore function for our patients. To this end, academic institutions and industry and specialty societies are researching biomaterials, growth factors, and cell therapies as well as combinations of these with regard to promoting tissue repair. These therapies are costly.

If cell therapy is to be assessed as a repair technology, then labeling technologies, animal models, and end-point assessments by histological, biochemical, and mechanical modalities will need to be validated and standardized to demonstrate proof of principle prior to or simultaneously with clinical application.

This paper demonstrates the development of a sophisticated gene-vector cell-labeling technique applicable to chondrocyte labeling. It also confirms the committed potential of the chondrocyte by showing the dedifferentation-redifferentiation phenomenon of chondrocytes with gene expression of cartilage markers dependent on their physical environment in monolayer culture or three-dimensional calcium alginate suspension. Finally, a rabbit model is used to assess articular repair and cell source in an osteochondral model.

The initial stages of this study demonstrate that allogeneic-labeled rib chondrocytes, when placed in a three-dimensional alginate-well system, reproducibly up-regulate their gene expression to produce aggrecan and type-II collagen and down-regulate type-I collagen with the results obtained from a control monolayer cell culture. The cells remain committed to their phenotype, dependent on their physical environment. This confirms the results reported in previous studies^1,2 and has been a useful in-vitro model for demonstrating cartilage markers expressed by chondrocytes, presumably in vivo. The authors have gone on to demonstrate that a murine retrovirus reproducibly labels chondrocytes to express enhanced green fluorescent protein (EGFP) and that these cells can be concentrated to >90% with the use of fluorescence-activated cell sorter analysis.

The authors also demonstrate that allogeneic rib chondrocytes, genetically labeled, delivered in a calcium alginate medium, and implanted in an osteochondral defect, can survive in a rabbit model for six weeks after implantation. In particular, the cells do not contribute to the repair tissue; the repair tissue arises from host-derived marrow cells. This raises the question of the usefulness of implanted chondrocytes.

Prior chondrocyte research has utilized models with a chondral rather than an osteochondral defect and has made use of autologous rather than allogeneic cells. Grande et al.³ demonstrated that tritium-labeled autologous articular chondrocytes contributed to the repair tissue of a full-thickness chondral defect of the patella in a rabbit model. Breinan et al.⁴ utilized a full-thickness chondral defect of the femoral sulcus in a canine model. These authors demonstrated that implanted autologous articular chondrocytes, labeled with a b-galactosidase reporter gene in a retrovirus, contributed to the repair tissue. Brittberg et al.⁵ demonstrated excellent repair tissue derived from autologous cultured articular chondrocytes in a rabbit patella defect model in which the cells were not labeled. There was no marrow penetration that would enlist host-derived cellular repair, and the control defects in periosteum alone showed poor repair compared with that seen in the implanted chondrocytes. These three studies suggest that it was the implanted cells that contributed to the cellular repair.

Autologous chondrocyte implantation has been successful clinically, with more than 10,000 patients treated worldwide. There is now more than ten years of follow-up study to demonstrate durable biomechanical performance, and histological studies of biopsy specimens have demonstrated hyaline cartilage⁶. However, there are many clinical factors that influence clinical outcome.

The variable of allogeneic cell source as opposed to autologous cell source warrants discussion. Mature osteoarticular allografts have been successful in the clinical setting. It is believed that the environment of the chondrocyte in the matrix of the tissue protects human leukocyte antigen (HLA) chondrocyte cell receptors from circulating immune mechanisms. However, this may not be the case with allogeneic chondrocytes in a cell suspension or gel delivery system. Was the lack of repair tissue from implanted cells due to HLA-related incompatibility?

The in vivo model used to assess the fate of implanted rib chondrocytes is an osteochondral allogeneic rabbit model on the femoral sulcus. Both knees are treated. The control chosen was culture time before implantation to assess the effect of "concentrating" the label by preimplantation culturing time. To assess the quality of the repair tissue in addition to the fate of the cells, controls of interest could have also included an empty osteochondral defect, an osteochondral defect with agarose gel alone, or an osteochondral defect with labeled cells alone without gel.

As can be seen from this collaborative effort of scientists with expertise in molecular and gene biology, veterinarians, and clinicians, these studies are expensive, necessary, and require continued funding. This study emphasizes the need for a good animal model to assess the clinical problem of treating a full-thickness chondral defect. Are such defects best treated with biomaterials, growth factors, cell therapies, or with a combination of all three (a so-called tissue-engineered construct)? Standardized animal models, cell labeling, and end-point analyses are required and are being developed by societies such as the International Cartilage Repair Society. It is our responsibility to demonstrate proof of principle as best as can be performed prior to implantation in humans.

I congratulate the authors. This labeling technique is fascinating. The murine retrovirus with EGFP labeling appears to be stable and reproducible and would be useful to demonstrate the fate of the implanted chondrocytes. Perhaps repeating the in vivo experiment with articular chondrocytes in a chondral defect model with elimination of the host-derived cell source and the aforementioned controls would be useful with both autologous and allogeneic cells. This would allow us to understand whether the use of allogeneic cells is supported and whether calcium alginate is a useful carrier.

*The author did not receive grants or outside funding in support of his research or preparation of this manuscript. The author received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (Honoraria from Genzyme Biosurgery for presentations on chondrocyte transplantation). In addition, a commercial entity (Genzyme Biosurgery) provides funding for fellowship training at the author's institution.

References

1. Gagne TA, Chappell-Afonso K, Johnson JL, McPherson JM, Oldham CA, Tubo RA, Vaccaro C, and Vasios GW. Enhanced proliferation and differentiation of human articular chondrocytes when seeded at low cell densities in alginate in vitro. J Orthop Research. 2000;18:882-90.
2. Haudenschild DR, McPherson JM, Tubo R, Binette F. Differential expression of multiple genes during articular chondrocyte redifferentiation. Anat Rec. 2001;263:91-8.
3. Grande DA, Pitman MI, Peterson L, Menche D, Klein M. The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation. J Orthop Res. 1989;7;208-18.
4. Breinan HA, Minas T, Barone L, Tubo R, Hsu HP, Shortkroff S, Nehrer S, Sledge CB, Spector M. Histological evaluation of the course of healing of canine articular cartilage defects treated with cultured autologous chondrocytes. Tissue Engineering. 1998;4:101-14.
5. Brittberg M, Nilsson A, Lindahl A, Ohlsson C, Peterson L. Rabbit articular cartilage defects treated with autologous cultured chondrocytes. Clin Orthop. 1996;326;270-83.
6. Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A. Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med. 2002;30:2-12.