Abstract. 1. Introduction CLINICAL CASE STUDY

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1 JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE J Tissue Eng Regen Med 2007; 1: Published online in Wiley InterScience ( CLINICAL CASE STUDY Repair of articular cartilage defects in the patello-femoral joint with autologous bone marrow mesenchymal cell transplantation: three case reports involving nine defects in five knees Shigeyuki Wakitani 1 *, Masashi Nawata 2,KeijiTensho 2, Takahiro Okabe 2,HirokoMachida 3 and Hajime Ohgushi 3 1 Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan 2 Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Japan 3 Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Amagasaki, Japan Abstract To investigate the effectiveness of autologous culture-expanded bone marrow mesenchymal cell transplantation for repairing articular cartilage defects, we transplanted autologous cultureexpanded bone marrow mesenchymal cells into nine full-thickness articular cartilage defects of the patello-femoral joints (including two kissing lesions) in the knees of three patients, a 31 yearold female, a 44 year-old male and a 45 year-old male. Three weeks before transplantation, bone marrow blood was aspirated from the iliac crest. Adherent cells were cultured with media containing autologous serum. Single-passaged cells were collected, embedded in a collagen solution ( cells/ml), placed on a collagen sheet, gelated, transplanted into the defect and covered with autologous periosteum or synovium. Six months after transplantation, the patients clinical symptoms had improved and the improvements have been maintained over the follow-up periods (17 27 months). Histology of the first patient 12 months after the transplantation revealed that the defect had been repaired with the fibrocartilaginous tissue. Magnetic resonance imaging of the second patient 1 year after transplantation revealed complete coverage of the defect, but we were unable to determine whether or not the material that covered the defects was hyaline cartilage. Autologous bone marrow mesenchymal cells transplantation may be an effective approach to promote the repair of articular cartilage defects. Copyright 2007 John Wiley & Sons, Ltd. Received 27 November 2006; Revised 11 January 2007; Accepted 30 January 2007 Keywords kissing lesion; progenitor cells; regeneration; periosteal flap; synovial flap 1. Introduction The patello-femoral (PF) joint consists of the patella and femoral patellar groove. Articular cartilage injury in this joint disrupts the articulation mechanism and causes clinical symptoms, such as pain and click on motion. In some cases, when both sides of the joint are injured, *Correspondence to: Shigeyuki Wakitani, Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, Asahi, Abeno-ku, Osaka , Japan. wakitani@med.osaka-cu.ac.jp the term kissing lesion is used. This disease is sometimes treated by decompression of the joint, such as elevation of the anterior tibial tubercle, although this procedure does not usually lead to biological repair of the damaged articular cartilage. If possible, the articular cartilage defect itself should be repaired. To repair articular cartilage defects, tissue engineering techniques have been explored. Although many kinds of cells have been tried in animal experiments, few are available for clinical use. Autologous chondrocyte implantation (ACI) was reported to be effective clinically (Brittberg et al., 1994) and has been widely used all over Copyright 2007 John Wiley & Sons, Ltd.

2 Cartilage repair withautologouscultured bone marrow mesenchymal cells 75 the world. However, the effectiveness of this method is still controversial (Horas et al., 2003; Bentley et al., 2003; Knutsen et al., 2004; Jakobsen et al., 2005). In the case of ACI, difficulties in obtaining a sufficient number of chondrocytes and donor site morbidity are also another clear limitation of this method. New cell sources have been explored. We reported previously that transplantation of autologous culture-expanded bone marrow mesenchymal cells (BMMC) could result in the repair of articular cartilage defects in rabbit (Wakitani et al., 1994), in human osteoarthritic knees (Wakitani et al., 2002) and in articular cartilage defects in the human patella (Wakitani et al., 2004). Clinically, autologous BMMC transplantation is more straightforward to perform than ACI. The cell collection procedure for BMMC is much less invasive than that of ACI, and can be performed under local anaesthesia in the outpatient clinic. This also means that surgery is required only once, while ACI requires two surgeries. Furthermore, in the case of ACI, cell harvesting results in damage of the normal articular surface and the proliferation capacity of BMMC is greater than that of chondrocytes. BMMC can be harvested without impairing normal articular cartilage, and has excellent proliferation (Wakitani et al., 1994; Ohgushi and Caplan, 1999) as well as chondrogenic differentiation capacity (Kotobuki et al., 2004; Indrawattana et al., 2004). The purpose of this study was to evaluate the clinical results of autologous BMMC transplantation for the treatment of articular cartilage defect in the PF joint, or in the case of patients with kissing lesions, defects on both joint surfaces (Andereya et al., 2004). 2. Patients and methods 2.1. Cell preparation All three patients were treated in Shinshu University Hospital. Introductory reports of these patients were briefly depicted in our previous paper (Wakitani et al., 2006). Cell culture was performed at the cell-processing centre (CPC) of the National Institute of Advanced Industrial Science and Technology in Amagasaki, a city which is almost 400 km away from Shinshu University Hospital. The CPC facility maintains a good manufacturing practice level (ISO certified facility), with three clean rooms of class The main part of the culture procedure was performed under appropriate standard operative procedures. Heparinized bone marrow (15 ml, i.e. 3 ml five times) was aspirated from the left iliac crest and placed in tubes containing heparinized phosphate-buffered saline. This was carried to the CPC on ice by one of the authors by train, and the culture was started within 6 h. At the CPC, the cells from the 3 ml fresh marrow were put into two 75 cm 2 plastic culture flasks and cultured with changes of the medium three times/week. Because we had 15 ml bone marrow blood in total, we used 10 flasks. When the culture medium was changed, non-adherent haematopoietic cells were removed, leaving only adherent cells in the dish. After about 10 days, the number of adherent cells grew and reached several million. The cells were collected with trypsinization (first passage) and sub-cultured in additional flasks for approximately 10 days. At this point in culture the cells had a fibroblastic appearance and were negative for haematopoietic markers (CD14, CD34) and HLA-DR but positive for markers present in the stromal cells (CD29, CD44, and CD105) (Kotobuki et al., 2005). These findings indicate that the adherent cultured cells were of a stromal type (Ohgushi and Caplan, 1999). The culture medium used was α-minimum essential medium, supplemented with 15% autologous serum. The subcultured cells were collected and embedded in 1% acid soluble type I collagen from bovine skin (Koken, Tokyo, Japan; final cell density, cells/ml), placed onto a collagen sheet from porcine tendon (Gunze, Kyoto, Japan) and gelated. This gel cell composite was further cultured for a couple of days. A portion of the culture of expanded BMMCs was used for contamination check and the remaining cells were frozen. We used a conventional culture method for bacterial and fungal contamination but also used the BACT/ALERT 3D microbial detection system (biomerieux, Durham, NC, USA). We performed these tests at first at the beginning of the culture when we prepared the culture medium with the patient s serum, and lastly when we performed the final medium change. At the time of the first tests, part of the medium was sent to a company for endotoxin analysis (Sumika Chemical Analysis Service Ltd, Osaka, Japan). At the time of the last tests, we also checked mycoplasma contamination using a PCR mycoplasma detection kit (Takara Bio Inc., Otsu, Japan). All the tests except for the endotoxin test were performed at the CPC. After confirming that there was no contamination, the composite was carried back to the hospital and transplanted. To evaluate clinical symptoms, we used the International Knee Documentation Committee Subjective Knee Evaluation Form (2000) (IKDC score), which ranged from 0 (worst) to 100 (best). This study was approved by the institutional review board of each institution and in accordance with the Helsinki Declaration of 1975, as revised in Case 1 The first case involved a 32 year-old female patient. At age 29 years she began to feel pain in her left knee without any proximate cause. At age 30 years she underwent arthroscopic surgery and an articular cartilage defect was found in the femoral patellar groove. Multiple perforations were performed on the defect to try to alleviate the pain. After the surgery, she continued to feel pain and click in the left knee joint and also in the right knee. At this point, she came to our hospital. Physical examination established crepitation in the PF joint and anterior knee pain in both knees. The range of

3 76 S. Wakitani et al. motion was not restricted in either knee. X-rays revealed no abnormality but magnetic resonance imaging (MRI) showed a deficiency of the patellar and femoral articular cartilage in the PF joint of both knees. After thorough examination of her knees, we concluded that the other parts of the knee were normal and that the knee pain was due to the injured articular cartilage of the PF joint. A couple of months of conservative treatment (medication with non-steroidal anti-inflammatory drugs, quadriceps muscle training and thermo-therapy) did not result in pain reduction. At this point, we decided to perform autologous transplantation of culture-expanded BMMC to repair the articular cartilage defect in the patella and femoral articular cartilage of her left knee. We also decided to perform arthroscopic multiple perforations simultaneously in her right knee because the symptoms were mild in comparison with the left knee. The transplantation surgery for the left knee took place. After a medial parapatellar approach, we monitored the PF joint and found articular cartilage defects on both sides. The subchondral bone at the defect sites was exposed and multiple perforations using K-wire (1.5 mm diameter) were made to facilitate bleeding. The areas of the fullthickness cartilage defects in the femur and patella were 1.6 and 1.0 cm 2, respectively. The composite was then put in place and covered with autologous periosteum taken from the anterior surface of the tibia, with the cambium layer facing the bone marrow. The flap was sutured to the surrounding rim of the normal cartilage or soft tissue with interrupted absorbable sutures. Arthroscopic multiple perforations in the patient s right knee were performed simultaneously. Continuous passive motion was initiated 3 days after surgery but otherwise the knee was immobilized with a brace for 3 weeks. Full weight bearing was started 3 weeks after the operation. The pain in the knee diminished and the patient was able to walk without crutches or a cane. A full knee range of motion was achieved 6 months after the operation. Although arthroscopic multiple perforations were made in her right knee, the clinical symptoms did not improve. As a result, we decided to perform BMMC transplantation for the right knee. The transplantation surgery took place 6 months after the first surgery, using the approach we had taken for the left knee. The areas of the full-thickness cartilage defects in the femur and patella were 3.1 and 1.7 cm 2, respectively (Figure 1). The clinical symptoms improved and 7 months after the operation she returned to work (nursing) after 1 year on medical leave because of her knee problem. The time course of the IKDC scores for both knees is shown in Figure 2. The height and weight before surgery were 153 cm and 45 kg, respectively, and the body mass index (BMI) was Her weight has not changed and is now 45 kg now. Arthroscopic observation 11 months after the second surgery showed that the patellar articular surface was completely covered with cartilage-like tissue, which appeared to have a smooth surface with elastic properties, as determined by probing. Histological examination of a A B C Figure 1. Macroscopic appearance of the patello-femoral joint of the right knee from Case 1. (A) Damaged articular cartilage of the femoral patellar groove; (B) damaged articular cartilage of the patella; (C) appearance following suturing of autologous periosteum in both lesions tissue specimen taken from the surface of the articular cartilage in the graft area in the right knee demonstrated the presence of an extracellular matrix with strong metachromatic staining. We concluded that the area was covered with cartilaginous matrix but not typical hyaline cartilage (Figure 3).

4 Cartilage repair withautologouscultured bone marrow mesenchymal cells 77 bus) after 1 year and 9 months on medical leave. The IKDC scores before surgery were 30 for the right knee and 11 for the left knee. Seven months after the surgery, they improved to 74 and 67, respectively, and they were at the same level at the last observation 20 months after the surgery. His height and weight before surgery were 161 cm and 66 kg, respectively, and the BMI was His weight has remained almost the same and is now 66 kg. One year after transplantation, MRI was performed and revealed complete coverage of the defect. However, we were unable to determine whether or not the material that covered the defects was hyaline cartilage (Figure 4) Case 3 Figure 2. Time course of clinical symptoms, assessed using the International Knee Documentation Committee Subjective Knee Evaluation Form (2000); ž, right knee;, left knee. At first, the scorefortheleftkneewasinferiortothatfortherightknee. After the transplantation in the left knee, the score for the left knee improved. After the transplantation was performed on the right knee, the scores for both knees deteriorated transiently but then improved with time 2.3. Case 2 The second case involved a 43 year-old male patient. At age 41 years he began to feel pain in his left knee without any proximate cause. Aspiration of the joint revealed an accumulation of about 50 ml of fluid with debris. The aspiration procedure was repeated three times. Arthroscopic debridement of injured articular cartilage in the femoral patellar groove of the left knee was performed 1 month after the onset of pain. Three months later, he felt pain in his right knee and four aspirations were performed. Arthroscopic debridement of the injured articular cartilage was performed on the femoral patellar groove of the right knee 4 months after the onset of pain. Because there was no improvement, he came to us 3 months after the second arthroscopy and indicated that he was experiencing click and pain in the PF joint of both knees. Physical examination established patello-femoral crepitation and anterior knee pain. The range of motion of the knees was not restricted. The X-ray findings of both knees showed no abnormality but MRI showed a deficiency of the femoral articular cartilage in the PF joint of both knees. In the same way as Case 1, transplantation surgery for both knees was undertaken as a simultaneous procedure. The articular cartilage of the patellar groove in each knee was injured, and in the left knee the area of the defect was 4.2 cm 2. The areas in the right knee were 0.7 and 1.3 cm 2. In this case we used a synovial membrane, taken from the same knee, to cover implants placed on the joint side facing the joint space, because the defects were large. The postoperative physiotherapy programme was the same as for Case 1 except for the omission of immobilization. The clinical symptoms improved and 7 months after the operation, the patient returned to work (driving a The third case involved a 45 year-old male patient. At age 39 years (in 1989), he injured his right knee in a fall while skiing. In 2004, he injured his right knee again and began to feel dull pain. One month after the onset of pain, arthroscopy was performed and an articular cartilage injury in the patella was found. Multiple drilling was performed, but the patient s clinical symptoms did not improve significantly. He was introduced to us as a potential candidate for BMMC transplantation. MRI showed degeneration of the articular cartilage in the patella of the right knees. In the same way as the patients in the two cases reported above, transplantation surgery was performed 1 year after arthroscopy. The areas of the defects in the right patella were 1.1 and 1.0 cm 2.Weused synovial membrane to cover the implant in the same way as in Case 2. The postoperative physiotherapy programme was also the same as for Case 2. The clinical symptoms improved and 8 months after the operation, the patient returned to desk work as a member of the Self-defence Force. The IKDC score before surgery was 64, while that 6 months after surgery was 77. The score was the same at the last observation, 18 months after the surgery. The patient s height and weight before surgery were 178 cm and 87 kg, respectively, and the BMI was His weight has remained almost the same and is now 90 kg (BMI = 28.4). 3. Results and Discussion This paper describes the application of autologous cultureexpanded BMMC embedded in collagen gels and sheets covered with periosteum or synovium transplantation procedures for the treatment of nine defects in five knees of three patients. Clinical symptoms were reduced dramatically and remained satisfactory for the duration of our observation periods, although longer followup is needed. As the patents body weights had not changed from surgery until the last observation, their improvement of clinical symptoms was not due to body weight change. We accessed the repair tissues with MRI and/or arthroscopy. For the patient in Case 1, we confirmed by arthroscopy that cartilage-like tissue

5 78 S. Wakitani et al. A B Figure 3. Microscopic appearance of the repaired tissue of the right knee in Case 1 at 11 months after transplantation. (A) Haematoxylin and eosin staining; (B) toluidine blue staining (bar indicates 1 mm 20). Strong metachromatic staining with fibrous appearance and scattered chondrocytes were observed in repaired cartilage covered the defect, and by histological examination that the regenerated tissue was comprised of a cartilaginous matrix. We previously reported that BMMC transplantation was effective in repairing articular cartilage defects (Wakitani et al., 2002, 2004). In the present study, we transplanted BMMC cells into articular cartilage defects in the femoral patellar groove and/or patella, which included kissing lesions. The latter are defects in facing articular cartilages, which are thought to be difficult to repair (Andereya et al., 2004). In repairing kissing lesions by cell transplantation, adhesion of the facing covers (periosteum or synovium) may occur. However, as shown in Case 1 above, the result was not inferior to the outcome achieved in the other cases. Early onset of continuous passive motion is thought to be effective, so kissing lesions might be repaired successfully by taking this procedure. Conventional marrow stimulation technique, such as microfracture, might be considered to provide satisfactory results (Steadman et al., 2003). However, it cannot do so in some cases, such as Cases 1 and 3 in this report. In such cases, alternative salvage procedures are wanted. BMMC transplantation can be one of the candidates for these. In Case 1, we used autologous periosteum to cover the implants but in Case 2 and 3, we used autologous synovial membrane. Part of the reason for this is that the chondrogenic potential of periosteum decreases with age and may be very limited in patients aged 40 years and older (O Driscoll et al., 2001). Furthermore, cells from the synovial membrane have chondrogenic potential (Sakaguchi et al., 2005), and it is much easier to collect a larger patch from the synovial membrane than from the periosteum, because it is not necessary to make another skin incision to collect synovial membrane. Thus, we used

6 Cartilage repair withautologouscultured bone marrow mesenchymal cells 79 Figure 4. Magnetic resonance imaging of the transplanted area of the Case 2 patient 1 year after transplantation. The defect in the patellar groove of the right femur was filled with white to grey material to the level of the surrounding cartilage, the intensity of which was not so high as that of normal cartilage and irregular. Arrows indicate the repaired tissue synovial membrane instead of periosteum and it was shown to be effective. We used collagen solution and collagen sheets, which have been approved for clinical use by the Japanese Ministry of Health, Labour and Welfare. The collagen solution is for smoothing skin wrinkles in plastic surgery, and collagen sheet is an artificial skin. There have been no reports of obvious complications. This method is clinically straightforward to perform because autologous cells can be readily harvested and expanded in culture, and they have a robust capacity to differentiate into chondrocytes. For these reasons, autologous BMMC transplantation can be considered to be a highly promising method for the repair of articular cartilage defects. Acknowledgements This study was supported in part by a Grant-in Aid for Scientific Research (No ) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. References Andereya S, Mumme T, Muller-Rath R, Schneider U. 2004; What factors influence the results of ACT when the indication limits are exceeded? Z Orthop Ihre Grenzgeb 142: Bentley G, Biant LC, Carrington RW, et al. 2003; A prospective, randomised comparison of autologous chondrocyte implantation versus mosaicplasty for osteochondral defects in the knee. JBone Joint Surg (Br) 85: Brittberg M, Lindahl A, Nilsson A, et al. 1994; Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. NEnglJMed331: HorasU,PelinkovicD,HerrG,et al. 2003; Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective, comparative trial. J Bone Joint Surg (Am) 85: Indrawattana N, Chen G, Tadokoro M, et al. 2004; Growth factor combination for chondrogenic induction from human mesenchymal stem cells. Biochaem Biophys Res Commun 320: Jakobsen RB, Engebretsen L, Slauterbeck JR. 2005; An analysis of the quality of cartilage repair studies. J Bone Joint Surg (Am) 87: Knutsen G, Engebretsen L, Ludvigsen TC, et al. 2004; Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial. J Bone Joint Surg (Am) 86: Kotobuki N, Hirose M, Takakura Y, Ohgushi H. 2004; Cultures autologous human cells for hard tissue regeneration: preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 28: Kotobuki N, Katsube Y, Hirose M, et al. 2005; Surface marker expressions of human mesenchymal stem cells used for regenerative medicine. Proceedings of the International Federation for Medical and Biological Engineering 10: O Driscoll SW, Saris DB, Ito Y, Fitzimmons JS. 2001; The chondrogenic potential of periosteum decreases with age. JOrthop Res 19: Ohgushi H, Caplan AI. 1999; Stem cells therapy and bioceramics. From cell to gene engineering. J Biomed Mater Res 48: Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. 2005; Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source, Arthritis Rheum 52: Steadman JR, Briggs KK, Rodrigo JJ, et al. 2003; Outcomes of microfracture for traumatic chondral defects of the knee: average 11-year follow-up. Arthroscopy 19: Wakitani S, Goto T, Pineda SJ, et al. 1994; Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. JBone Joint Surg (Am) 76: Wakitani S, Imoto K, Yamamoto T, et al. 2002; Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthr Cart 10: Wakitani S, Mitsuoka T, Nakamura N, et al. 2004; Autologous bone marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in human patellae: two case reports. Cell Transpl 13: Wakitani S, Ohgushi H, Machida H, et al. 2006; Autologous culture expanded bone marrow stromal cell transplantation for cartilage repair. In Bone Marrow Transplantation. New Research, Davidson DF (ed.). Nova Science Publishers: New York; Alford JW, Cole BJ. 2004; Cartilage restoration, part 2: techniques, outcomes, and future directions. Am J Sports Med 33: