Basic mechanisms behind the use of autologous
chondrocytes
for articular cartilage repair
Mats Brittberg, M.D., Ph.D.
Cartilage Research Unit, Department of Orthopaedics, Kungsbacka Hospital, S-434 40 Kungsbacka, Sweden
Introduction
Articular cartilage represents the only remnant of the foetal cartilage skeleton to persist throughout adult life and we have to be careful with this unique tissue while it has a poor ability to repair after injuries. The challenge to restore damaged articular surface has generated tremendous interest among research scientists, clinicians and many new articular cartilage repair techniques have emerged in the last 5-6 years most of which appear to be promising. One such therapy is to use in vitro expanded autologous chondrocytes in combination with a periosteal membrane to repair cartilage defects.
In a defect site, there is a cell density dependent differentiation occuring, meaning that the initial amount of cells that can take part in the repair events is of great importance.
Furthermore, there is a series of cellular transitions occuring4,10, a lineage which originates from a primitive cell, the so called mesenchymal stem cell and which ultimately could progress into the cells, the unique phenotype of the injured tissue4,10.
To repair a mesenchymal tissue, you are dependent on the local availability of those multipotential cells of the injured tissue. The goal for the orthopaedic surgeon is to try to deliver as high densities of the acquired cell-types as possible into the injured site, the cartilage lesion to achieve some sort of repair.
A fundamental feature of cartilage differentiation in the developing limb is the formation of a prechondrogeneic cell condensation, a blastema. In in vitro studies it has been shown that mesenchymal cell aggreggates must achieve a treshold size before chondrogenesis can proceed6.
The articular chondrocytes are responsible for the unique features of articular cartilage, they keep the cartilage alive, they alone maintain it and regulate it. Therefore it seems rational to use true committed chondrocytes to repair a cartilaginous defect. From a piece of arthro-scopically harvested cartilage, chondrocytes can be isolated by enzymatic digestion and in in vitro culture expanded 20-50 times the initial amount of cells.The cells are cultured in so called monolayer and during that time the cells dedifferentiate. The dedifferentiated chondrocytes have a similarity to primitive mesenchymal cells and an implantation of a high density of those in vitro expanded primitive immature chondrocytes could imitate the prechondrogeneic cell condensation and cartilage formation
In vitro experiments with chondrocytes directly seeded to cartilage explants shows a linear relationship between biosynthetic activity and the number of seeded chondrocytes which means that the number of seeded cells seems important5. In studies it has also been shown that a high proportion of synthesized proteoglycans could be lost to the medium after cell seeding and this suggests that for at least in early times , an overlying membrane is important5.
Rabbit model
In 1982 Lars Peterson and co-workers developed a rabbit model to treat cartilage defects in the rabbit patella with autologous chondrocytes15 and with that model using the knee joints of adult rabbits Grande and co-workers11 examined the effect of autologous chondrocytes grown in vitro on the healing rate of chondral defects not penetrating the subchondral bone plate. To determine whether any of the reconstituted cartilage resulted from the chondrocyte graft an experiment was conducted involving grafts with chondrocytes that had been labeled prior to grafting with a nuclear tracer. Results were evaluated using both qualitative and quantitative light microscopy. Macroscopic results from grafted specimens displayed a marked decrease in synovitis and other degenerative changes. In defects that had received transplants, a significant amount of cartilage was reconstituted (82%) compared to ungrafted controls (18%). Autoradiography on reconstituted cartilage showed that there were labelled cells incorporated into the repair matrix11.
The same rabbit model has since then been used and further developed by our group at the Göteborg University3. The cultured cells are injected into a premade cartilage defect in the patella of the rabbit and covered with a a flap of periosteum, functioning as a biological membrane. This method resulted in a high degree of healed rabbit patellar defects and the repair tissue had a similarity to the original cartilaginous tissue.
Hypothesis of induced repair with seeded chondrocytes:
We could regard the cartilage defect as a bioactive chamber. Repair could hypothetically come from the surrounding cartilage in the walls of the defect, from the calcified zone chondrocytes in the cryptae of the irregular subchondral bone plate. Cells could possibly migrate into the defect from the synovial fluid. We know that the cells in the adjacent cartilage show mitotic activity some time after injury but not enough for any significant repair .
The bioactive chamber theory is useful when alternative cell or tissue transplantation techniques in particular are discussed. Periosteal resurfacing, for instance, is almost always combined with an opening of the subchondral bone marrow space. The periosteal cells and bone marrow cells have dual phenotypic expression and are capable of differentiating to bone and/or cartilage depending on local environmental factors emanating from the vascular bone marrow and from the synovial fluid. However, in contrast to the cartilage nodule formation seen after pure chondrocyte implantation in the athymic mouse3, the implantation of periosteally derived cells into the subcutis of an athymic mouse will produce a nodule consisting of a central portion of cartilage which is slowly turned into bone via endochondral ossification, while the periphery develops into bone through intramembranous bone formation14. One theory is that the cells follow their initial programme of differentiation and that chondrocytes are committed to produce cartilage, while the periosteal and bone-marrow cells are committed to produce bone. This implies that the chondrocytes developed from both these cell types are pre-stage of osteoblasts. Low oxygen tension and chondrogenic factors will help to retain their chondrogenic status in the osteogenic lineage, but they may be more unstable than the purely committed chondrocytes and progress into hypertrophic chondrocytes and finally into bone. The phenotypic fate thus appears to determine the type of the final tissue.
The periosteum
Furthermore, in our rabbit work, the patellar defects were treated with autologous chondrocytes together with a covering periosteal graft on one side and the contra-lateral side was treated with periosteum alone. The defects were deep, reaching down to the calcified zone but with no opening of the subchondral space. In a defect of this type without any treatment, there was an intrinsic repair of 29% of the total defect area, primarily by what we call matrix flow from mitotic activity at the edges of the defect3. This level of repair should be compared with the mean repair area of 30% one year after periosteal grafting alone and significantly different from the 87% repair area with chondrocytes and periosteum3.In other reports where successful repair with periosteum has been reported, this repair has been combined with an opening of the subchondral space making it possible for repair-competent cells to invade the defect. These findings may all be explained within the previously mentioned concept of a bioactive chamber as a prerequisite for any cartilage repair .
We
have also studied the interaction between the periosteum and the cultured
chondrocytes and found that the periosteum stimulated the chondrocytes to
proliferate by exerting a paracrine effect on the chondrocyte cloning
efficiency2. Another important finding was that there were different
degrees of differentiation in the chondrocytes in the cultures which responded
differently to the periosteal stimulation2. The bioactive chamber
concept could be used to explain results without cell seeding but with an
opening of the subchondral space. In these cases, we can postulate that the
periosteum exerts the same paracrine effect on invading mesenchymal stem cells
from the bone marrow as it does on transplanted chondrocytes. In young
individuals, osteochondrogenic cells from the periosteal cambium layer could
directly certainly also contribute to the repair but perhaps even these cells
require information from another cell-type.
Clinical use of autologous chondrocytes:
In october 1987 the technique was first used to treat patients with chronic disabling symptoms of the knee joint with cultured cartilage cells from their own cartilage (Fig.1, Fig. 2-3). The first 23 patients (mean age 27) were presented in the New England Journal of Medicine in 19941.Those patients had local deep cartilage injuries that had been treated with conventional methods without any healing and 16 defects were located on the so called femoral surface and 7 on the patella. In all, 16 patients had ¨good¨or ¨excellent¨ knee function at mean three years postoperatively. The best results were found in the femoral group compared with the patella patients that were less successful. The technique appeared to be most successful in patients that had injuries on the femoral surfaces producing a single, localized deep cartilage lesion. This is important to note as opposed to the gradual wear and tear of advancing age. The disappointing outcome of the patella group might have resulted from mechanical misalignements of the patella that were not corrected at the time of transplant surgery. Since 1987 more than 850 patients have been treated with this technique and recently a new reexamination was presented by Lars Peterson at the 2nd International Cartilage Repair Society meeting in Boston in 1998 on 219 that underwent ACT between 1987 and 1996, 213 of whom were available to be assessed with 5 rating scales8,9. Forty-six patients agreed to be reexamined by arthroscopic second-look, 26 patients consented to a biopsy of tissue from the repair site, and 14 patients underwent biomechanical indentation probe testing. Defects evaluated were located on the femoral condyle (57), femoral condyle with anterior cruciate ligament (ACL) repair (27), osteochondritis dissecans (32), patella (32), trochlea (12), and multiple lesions (53). Patients averaged 2 previous surgeries to the affected knee. When the Cincinnati rating score was used, good to excellent results were achieved in 90% , with femoral condylar lesions, 74% in femoral defect with concomitant ACL reconstruction, 84% in osteochondritis dissecans, 69% in patellar lesions, 58% in trochlear lesions, and 75% in multiple lesions of patients.
Thirty-one patients graded good to excellent at 2 years were re-evaluated in the 5- to 10-year period (mean, 7.5 years), demonstrating 96% durability as measured by a rating of good/excellent maintained from 2 years to the mean of 7.5 years follow-up. 80% of the biopsies showed a hyaline-like appearance with at strong correlation between hyaline repair tissue and good/excellent results (Fig. 4).
In 1995 a randomized controlled multi-center study has started to compare multiple subchondral drilling of the cartilage injury with treatments with a periosteum with or without addition of the patients own cultured cartilage cells. In august 1997 in USA the Food and Drug Administration approved the cell technology that uses a patient’s own chondrocytes to repair cartilage injuries in the knee. This was the first type of cell technology that have been regulated for the industry of manipulated autologous structure guidance.
An international autologous chondrocyte implantation study has been started by Genzyme Tissue Repair and the study is monitored by a Registry Advisory Board and they have presented 12-month data on 249 patients and 24 month-data on 50 patients with overall patients and clinical scores improved significantly from baseline13.
In Germany, Lohnert et al. in 199812 presented 52 patients that had ben treated with autologous chondrocytes since 1996 and eleven patients were screened clinically and by MRI 18 months later. The biopsy specimen from the transplanted area showed formation of hyaline cartilage.
The Future
Future research must include randomized studies using the different clinical repair methods available. More research is needed using different cell sources, matrices, and on the durability of grafted areas. Chondrocytes are responsible for the unique features of cartilage, ensuring its continuing performance during the human lifespan. It is chondrocytes that keep the cartilage alive, and they alone maintain and regulate it. It therefore seems rational to use true committed chondrocytes to repair a local cartilaginous defect. More research is also required to determine the extent by which in vitro expanded autologous chondrocytes contribute to such a repair.
To date, a variety of articular cartilage resurfacing
techniques have the potential to improve the repair of cartilage defects
and reduce the patients
disability. Theoretically, articular cartilage resurfacing may also prevent the
progression of a local cartilage defect into osteoarthritis, but still we do
not know if leveling out a chondral defect will prevent such a development.
With a steadily increased co-operation between basic and clinical science we
may be able in the future to achieve a restoration of the injured cartilage;
from the small defect to the widespread cartilage loss of osteoarthritis7.
References
1. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New England Journal of Medicine 1994; 331:889-895.
2. Brittberg M. Cartilage repair. On cartilaginous tissue engineering with the emphasis on chondrocyte transplantation. PhD-thesis, Göteborg University, Göteborg,1996.
3. Brittberg M, Nilsson A, Lindahl A, Ohlsson C, Peterson L. Rabbit articular cartilage defects in the knee with autologous cultured chondrocytes. Clin Orthop Rel Res 1996; 326:270-283.
4. Caplan AI, Elyaderani M, Mochizuki Y, Wakitani S, Goldberg VM. Principles of cartilage repair and regeneration. Clin Orthop Rel Res 1997 ;342: 254-269
5. Chen AC, Nagrampa JP, Schinagl RM, Lottman LM, Sah R. Chondrocyte transplantation to articular cartilage in vitro. J Orthop Res 1997; 15: 791-802
6. Cottrill CP, Archer CW, Wolpert L. Cell sorting and chondrogenic aggregate formation in micromass culture. Dev Biol 1987 Aug;122(2):503-15
7. Doherty PJ, Zhang H, Tremblay L, Manolopoulos V, Marshall KW. Resurfacing of articular cartilage explants with genetically-modified human chondrocytes in vitro. Osteoarthritis Cartilage 1998 May;6(3):153-159
8. Gillogly SD. Autologous chondrocyte implantation: Current state-of-the-art. In: Knieinstabilität-knorpelschaden. Ed: Imhoff AB, Burkart A. Steinkopff Verlag, Darmstadt, Germany 1998:60-66.
9. Gillogly SD, Voight M, Blackburn T. Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation. J Orthop Sports Phys Ther 1998 Oct;28(4):241-251
10. Goldberg VM, Caplan AI. Cellular repair of articular cartilage. In: Osteoarthritic disorders. Kuettner KE, Goldberg VM (eds). Am Acad Orthop Surg, Monterrey, 1994: 357-364.
11. 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: 208-218.
12. Lohnert J. Regeneration of hyalin cartilage in the knee joint by treatment with autologous chondrocyte transplants--initial clinical results. Langenbecks Arch Chir Suppl Kongressbd 1998;115:1205-1207 (in german).
13. Mandelbaum BR, Browne JE, Fu F, Micheli L, Mosely JB, Erggelet C, Minas T, Peterson L. Articular cartilage lesions of the knee. Current concepts. Am J Sports Med 1998; 26: 853-861
14. Nakahara H, Bruder SP, Goldberg VM, Caplan AI. In vivo osteochondrogenic potential of cultured cells derived from the periosteum. Clin Ortop 1990; 259: 223-232.
15. Peterson L, Menche D, Grande D, et al: Chondrocyte transplantation- An experimental model in the rabbit. Trans Orthop Res Soc 9: 218, 1984.
Legends to Figures:
Fig 1.
1: The surgeon examines the knee joint with an arthroscope. A lesion on the medial femoral condyle is noted and a biopsy from a minor weight bearing area on the upper medial femoral condyle is harvested
2: The cartilage biopsy is taken to a cell laboratorium where the cartilage is enzymatically digested. The isolated chondrocytes are cultured in monolayer for two-three weeks during which time the cells multiple significantly, 20-50 times the initial amount of cells.
3: After two-three weeks the knee joint is opened and the cartilage lesion is debrided into stable vertical edges and with a clean bony bottom.
4: A piece of periosteum is harvested from the upper medial femoral condyle and is sutured over the prepared defect with resorbable, interrupted sutures
5: The cultured chondrocytes are finally implanted into the lesion beneath the periosteal membrane.
Fig. 2: A cartilage lesion situated in the trochlear area of a knee, suitable for treatment with autologous chondrocyte transplantation.
Fig. 3: The same lesion seen in Fig.2 after that the lesion has been covered with a flap of periosteum.
Fig. 4: Repair tissue seen after chondrocyte transplantation in combination with a periosteal graft. Note cells in cluster formation.