AUTOLOGOUS CHONDROCYTE IMPLANTATION FOR CHONDRAL KNEE DAMAGE B.A. Jalba 1, C.S. Jalba 2, F. Gherghina 3, M. Cruce 3

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1 AUTOLOGOUS CHONDROCYTE IMPLANTATION FOR CHONDRAL KNEE DAMAGE B.A. Jalba 1, C.S. Jalba 2, F. Gherghina 3, M. Cruce 3 1-EMERGENCY CLINICAL HOSPITAL FLOREASCA BUCHAREST 2-EMERGENCY CLINICAL HOSPITAL SFANTUL PANTELIMON BUCHAREST 3-UNIVERSITY OF MEDICINE AND PHARMACY, CRAIOVA Summary Autologous chondrocyte transplantation (ACT) is a method for the repair of focal cartilage lesions in the knee based on the cultivation of chondrocytes in vitro. It consists of three stages: the harvesting and growth of chondrocytes in the laboratory, open-knee surgery for the implantation of the cultured cells, and the rehabilitation period. 16 patients were submitted to ACT since 2004 in our hospital. Mean Lysholm score of the remaining 15 patients increased from 43 before treatment to 85 at 1 year post-surgery. 18 of these (90%) had an improved knee score, (paired t-test, p = 8x10-6 ). 7 of the 9 patients who have been followed up for 2 years retained this improvement or had a further increase in knee score. Two patients who have reached 3 years post surgery had knee scores of 95 and 100 respectively. 82% patients were pleased or extremely pleased with the outcome at most recent follow-up and several have returned to competitive sport. No statistically significant correlation between the patients age and the success of the ACT procedure was found. While autologous chondrocyte transplantation appears to be a promising alternative to standard approaches for managing cartilage defects of the knee, the efficacy of this procedure has not been firmly established and the long-term functional outcome is unknown. At the present time, autologous chondrocyte transplantation is a reasonable therapeutic alternative only for carefully selected patients. Key words: Autologous chondrocyte implantation, knee, chartilage. bogdan_jalba@yahoo.com Introduction The cartilage is a form of extracellular matrix secreted by specialized cells called chondrocytes (Minas T, Nehrer S, 1997). Articular cartilage covers the end of the femur, the top of the tibia, and the back of the patella. It serves as a shock absorber and is essentially frictionless, providing a smooth surface for the contact and movement of the bones of the knee joint. Meniscal cartilage is on the tibia and serves mostly as a shock absorber. Autologous chondrocyte implantation is used to repair defects to the articular cartilage (Buckwalter JA, Mankin HJ, 1998; Curl WW, et al., 1997). 155 Chondrocytes are one of the few cell types that can survive without being close to a blood supply (Newman AP, 1998). However, when the cartilage is damaged, the chondrocytes' distance from a blood supply means that they lack the ability to regenerate themselves, a process that requires an increased amount of nutrients and access through the blood stream to other cells and proteins that stimulate the regeneration (O'Driscoll SW, 1998). Available strategies for managing cartilage defects include debridement or abrasion arthroplasty, subchondral drilling, and microfracture (Suh J-K et al., 1995). These treatments are not always effective and, when they are, the benefits may not be long-lasting.

2 Autologous chondrocyte transplantation (ACT) is a method for the repair of focal cartilage lesions in the knee based on the cultivation of chondrocytes in vitro (Brittberg M et al., 1994; Richardson JB, et al., 1999; Gillogly SD et al., 1998; Garrett JC, 1986). It consists of three stages: the harvesting and growth of chondrocytes in the laboratory, open-knee surgery for the implantation of the cultured cells, and the rehabilitation period (Peterson, L. et al., 2000; Drobnic, M et al., 2001). In order to use ACT to treat patients, an orthopaedic surgeon needs skills in the assessment and treatment of knee injuries, including arthroscopic surgery. In addition, special training is required in the techniques of ACT (Minas T, 1999). This technique has been also studied in our hospital, Emergecy Clinical Hospital Floreasca in Bucharest, where since 2004 until now 16 procedures of autologous chondrocyte transplantation have been made. Our goal is to analyze the outcome of ACT treatment of the cartilage lesions in the knee. Material and methods ACI is generally applied to patients between the ages of 15 and 55, with little or no additional damage to the knee joint (Nelson FRT, Wagner J, 1998). These are patients who do not have enough knee damage to need a total knee replacement, but who are experiencing considerable pain that may be impairing their quality of life. Clinically appropriate patients are identified through traditional diagnostic methods, such as MRI and x-ray evaluation (Menche DS et al., 1998). Final decisions are made at the discretion of the orthopedic physician with the aid of an arthroscopic examination. 16 patients were submitted to ACT since 2004 in our hospital (see table I). The injury was caused by trauma in 12 cases (acute 4 cases, chronic 8 cases) and 4 cases were diagnosed as osteochondritis dissecans (Farnworth L, 2000). Duration of 156 clinical symptoms before surgery was days. Average size of cartilage defect was 2.5 cm 2, reaching from 1.5 to 4.2 cm 2. Chondral defect was localised on the weight-bearing area of medial femoral condyle in 10 cases, and on the weightbearing area of the lateral condyle in 6 cases. We observed concomitant injuries of the soft knee: rupture of anterior crucial ligament in 8 patients, lesion of medial meniscus in 5 patients, lesion of lateral meniscus in 3 patients, patellar chondromalacia up to degree II.a. in 2 patients. ACI procedure Once a patient is determined to be eligible for this procedure, during an arthroscopic examination, we took a biopsy of the patient's articular cartilage. This sample, between 200 mg and 300 mg, was removed from an area of the knee that is not weight-bearing, usually the femoral condyle (Chen FS et al., 1997). Chondrocytes are separated from their surrounding cartilage and cultured for four to five weeks, generating between 5 and 10 million cells. Table I. Patients submitted to ACT Sex Male 8 Female 8 Mean age ± SD (range) 35.45±9.21 (18-53) Localizing Medial femoral condyle 10 Lateral femoral condyle 6 Mechanism Trauma acute 6 - chronic 8 Osteochondritis dissecans 2 Duration of clinical symptoms before surgery Average size of cartilage defect 2.5 cm 2 ( cm 2 ) Concomitant injuries of the soft knee rupture of anterior crucial ligament 8 lesion of medial meniscus 5 lesion of lateral meniscus 3 patellar chondromalacia 2

3 The procedure to implant the cells is a surgical procedure in which the patient's joint is exposed by the orthopedic surgeon. The defect area is prepared by removing dead cartilage and smoothing the surrounding living cartilage (Chen FS et al., 1999a; Chen FS et al., 1999b)(figure 1). A piece of periosteum, the membrane which covers bone, is taken from the patient's tibia and sutured over the prepared defect (figure 2). The cultured chondrocytes are injected by the surgeon under the periosteum, where they will grown and mature over time. Figure 1. Image of the defect area after removing dead cartilage Figure 2. Transfer of the periosteum over the prepared defect. Other repairs to the knee joint, such as joint realignment or ligament repair, may also be performed during the implantation 157 of the new chondrocytes. It will be between ten and twelve weeks before the patient can put his full weight on the knee. Cartilage heals slowly and maturation and tempering do not occur before one year. All our patients were submitted to this standard surgical procedure. Rehabilitation So as to give the implanted chondrocytes the stimulus to adapt themselves to their natural function, the motor splint is brought into use as early as possible after the implantation. Compression and decompression forces can stimulate the chondrocytes to syntehsia the correct matrix molecules. To underpin this maturation process a carefully planned programme of progresive rehabilitation is ncessary. For the first 6 to 12 weeks, the knee which has been operated upon must be relieved of weight bearing by the use of walking crutches, and exercise training must be started. Stepwise increases in weight bearing and in muscle and extension training follow in the subsequent postoperative months. Although the cartilage defect may well have been filled up with hyaline-type cartilage tissue within a few weeks, it is advisable not to commence even gentle weight-bearing activities such as bicycling before six months and more stressful weight-bearing activities such as tennis before twelve months, the reason being that the definitve maturation and hardening of the newformed cartilage will not be complete until months have elapsed. All patients have been subject to annual follow-up for a period of 1 to 3 years during which a self-assessment functional knee score (Lysholm knee score, max=100) (Table II) and the patient s satisfaction with the outcome (extremely pleased, pleased, no difference, worse, much worse) was recorded. Associated risks Autologous chondrocytes are human cells, harvested aseptically from cartilage that is processed to isolate and propagate

4 these specific cell types for aseptic implantation into the same person. The cells are aseptically packaged in unit dose containers and transported in secondary packaging validated for shipment that maintains the cellular integrity for a specificed time under defined conditions. Precautions and warnings are issued by the manufacturer in the Directions for Use. Care must be taken to ensure that the microbial integrity of both the biopsy specimen and cultured chondrocytes is assured. Precautions should be taken in using autoogous chondrocyte in patients with known histories of anaphylaxis to gentamycin. Currently, Genzyme Tissue Repair, the only company to provide cultured autologous chondrocytes, indicates that the biopsy medium used with chondrocytes (biopsy source material) contains DMEM with gentamycin. However, the transport medium for the cultured cells does not contain gentamycin. Table II. Lysholm Knee Scale 158

5 Intra-operative and postoperative complications resulting from any open knee procedure may occur as a result of cultured autologous chondrocyte implantation. While no serious adverse events definitely related to the use of autologous chondrocytes have been reported, risks from the surgical procedure including infection and inflammatory responses must be taken under consideration. Results and discussion One patient who had a pre-operative Lysholm knee score of 46 and a chondral defect of 4cm 2 following 2 previous debridements elected to have a total knee replacement 5 months post-operative. Mean Lysholm score of the remaining 15 patients increased from 43 before treatment to 85 at 1 year postsurgery. 18 of these (90%) had an improved knee score, (paired t-test, p=8x10-6 ). 7 of the 9 patients who have been followed up for 2 years retained this improvement or had a further increase in knee score. Two patients who have reached 3 years post surgery had knee scores of 95 and 100 respectively. 82% patients were pleased or extremely pleased with the outcome at most recent follow-up and several have returned to competitive sport. No statistically significant correlation between the patients age and the success of the ACT procedure was found. Second look arthroscopy was provided in four patients 3 5 months after ACT. In two patients very good healing was documented. During second-look arthroscopy samples for conventional light and electron microscopy were obtained. Microscopic controls documented presence of hyalinelike cartilage in the healing defect (presence of typical spherical chondrocytes, extracellular collagenous filaments, formation of typical isogenetic cellular groups) (figures 3, 4 and 5). 159 Figure 3. Presence of typical spherical chondrocytes samples obtained by second look arthroscopy. Figure 4. Formation of typical isogenetic cellular groups samples obtained by second look arthroscopy. Figure 5. Presence of extracellular collagenous filaments samples obtained by second look arthroscopy.

6 Conclusion While autologous chondrocyte transplantation appears to be a promising alternative to standard approaches for managing cartilage defects of the knee, the efficacy of this procedure has not been firmly established and the long-term functional outcome is unknown. Well-designed, randomized, controlled trials are needed to prove efficacy, define patient selection criteria, and determine long-term benefits relative to alternative strategies. At the present time, autologous chondrocyte transplantation is a reasonable therapeutic alternative only for carefully selected patients. Future developments include allografts. Allograft transplantation of articular cartilage has been performed for many years but is limited by tissue availability, possibility of disease transmission, and chondrocyte viability within preserved specimens. Allografts are osteochondral composites, and the bone in these grafts can provoke an immunogenic response. Thus, rejection is also possible. References Brittberg M, Lindahl A, Nilsson A, et al: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med; 331(14): , Buckwalter JA, Mankin HJ: Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect; 47: , Chen FS, Frenkel SR, Di Cesare PE: Chondrocyte transplantation and experimental treatment options for articular cartilage defects. Am J Orthop; 26(6): , 1997 Chen FS, Frenkel SR, Di Cesare PE: Repair of articular cartilage defects, part 2: treatment options. Am J Orthop; 28(2):88-96, 1999b Chen FS, Frenkel SR, Di Cesare PE: Repair of articular cartilage defects, part 1: basic science of cartilage healing. Am J Orthop; 28(1):31-33, 1999a Curl WW, Krome J, Gordon ES, et al: Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy; 13(4): , Drobnic, M., Radosavljevic, D., Koritnik, B., Gorens ek, M., Kregar-Velikonja, N., Jeras, M. and Knez evi, M. Five years of autologous chondrocyte transplantation (ACT) for the knee cartilage lesions. In: Cartilage Weekend. The 2nd symposium of recent advances in cartilage repair and tissue engineering. (Koritnik, B., Radosavljevic, D., Gorens ek, M. Eds.) Ljubljana: Orthopaedic Clinic, Clinical Centre, 48, Farnworth L: Osteochondral defects of the knee. Orthopedics; 23(2): , Garrett JC: Treatment of osteochondral defects of the distal femur with fresh osteochondral allografts: a preliminary report. Arthroscopy;2(4): , Gillogly SD, Voight M, Blackburn T: Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation. J Orthop Sports Phys Ther;28(4): , Menche DS, Vangsness CT Jr, Pitman M: The treatment of isolated articular cartilage lesions in the young individual. Instr Course Lect; 47: , Minas T, Nehrer S: Current concepts in the treatment of articular cartilage defects. Orthopedics; 20(6): , Minas T, Peterson L: Advanced techniques in autologous chondrocyte transplantation. Clin Sports Med; 18(1):13-44, v-vi, Minas T: The role of cartilage repair techniques, including chondrocyte transplantation, in focal chondral knee damage. Instr Course Lect; 48: , Nelson FRT, Wagner J: An update on articular cartilage transplantation. J Musculoskel Med; 15(2):56-66, Newman AP: Articular cartilage repair. Am J Sports Med 1998;26(2): O'Driscoll SW: The healing and regeneration of articular cartilage. J Bone Joint Surg (Am); 80(12): , Peterson, L., Minas, T., Brittberg, M., Nilsson, A., Sjogren-Jansson, E. and Lindhal, A. Two- to 9- year outcome after autologous chondrocyte transplantation of the knee. Clin. Ortop. 374, , Richardson JB, Caterson B, Evans EH, et al: Repair of human articular cartilage after implantation of autologous chondrocytes. J Bone Joint Surg (Br); 81(6): , 1999 Suh J-K, Scherping S, Marui T, et al: Basic science of articular cartilage injury and repair. Operative Techniques Sports Med;3(2):78-86, 1995.