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Development of Articular Cartilage Grafts Using Organoid Formation Techniques
Development of Articular Cartilage Grafts Using Organoid Formation Techniques Y. Irie, H. Mizumoto, S. Fujino, and T. Kajiwara ABSTRACT In order to develop articular cartilage grafts, one must control shape and safety. We have developed scaffold-free culture methods in which the cells form multicellular aggregates (organoids). In this study, we applied the organoid culture method to chondrocytes attempting to reconstitute articular cartilage grafts. Primary rat costal chondrocytes and subcultured human articular chondrocytes were immobilized in hollow fibers by centrifugation at a density of 3 ⫻ 108 cells/cm3 to induce the formation of cylindrical-shaped organoids. To improve convenience, we developed a culture device to form sheet-shaped organoids (organoid-sheet). Primary bovine articular chondrocytes were cultured in this device. These organoids were evaluated by histological and gene expression analyses. In the primary rat culture system, chondrocytes formed cylindrical organoids in hollow fibers. Histochemical analysis revealed the presence of extracellular matrix (collagen and proteoglycan). The organoid maintained cartilage-specific gene expression (type II collagen, aggrecan) for 1 month of culture. In the subcultured human chondrocyte system, the organoid regained the decreased cartilage-specific gene expression. In the primary bovine culture system, the cells formed a 300 m thickness organoid-sheet including abundant extracellular matrix. In conclusion, our organoid formation method was effective to form cartilage-like tissue. This result suggested that the technique may be applicable for the development of an articular cartilage graft.
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RTICULAR CARTILAGE is a quite durable tissue that has high load-bearing ability. However, once the tissue has been damaged, its capacity for regeneration is limited. Recently, the reconstruction of cartilage-like tissue using chondrocytes has attracted attention as a treatment for cartilage defects. Until now, many researchers have studied how to make the tissue with both chondrocytes and several scaffold materials (eg, collagen, polylactic acid).1 However, several problems remain: cell distribution in the scaffold, connection to the native cartilage, and safety of the scaffold.2 A scaffold-free culture method, ie, pellet culture that forms multicellular aggregates (organoids), would solve many of these problems. It has been reported that organoid culture could assist in the production of extracellular matrix, and the organoid could be integrated with in vivo cartilage.3 However, this approach is inadequate for direct clinical application for treatment of cartilage defects, because of difficulties in controlling organoid shape. Additionally, if the size of the organoid is too thick, necrotic cells arise as a result of nutrient exhaustion inside the organoid. © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 631– 633 (2008)
Until now, we have developed original organoid culture methods that control organoid shape.4,5 In this study, we applied these culture methods to chondrocytes attempting to reconstitute articular cartilage grafts. MATERIALS AND METHODS Hollow Fiber/Chondrocyte-Organoid Culture We made a hollow fiber (HF) bundle composed of 4 HFs (polyethylene HFs coated with ethylene vinyl alcohol; inner diameter, 330 m; outer diameter, 430 m; pore size, about 0.3 m; Asahi Kasei Medical Co, Ltd) as a culture device for organoid formation. From the Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka, Japan. This study was supported in part by a Grant-in-Aid for Scientific Research (B): 19360375 and a Grant-in-Aid for Scientific Research (A) (2): 14205119 from the Japan Society for the Promotion of Science. Address reprint requests to Toshihisa Kajiwara, PhD, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. E-mail: [email protected] 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.024 631
632 In this study, we used primary rat costal chondrocytes and subcultured human articular chondrocytes. Primary rat costal chondrocytes were isolated from the ribs of 7-week-old rats by enzyme treatment. The purchased subcultured human articular chondrocytes (Cambrex Co) were further subcultured 3 times. When they reached 80% to 90% confluence, the cells were harvested by trypsin/EDTA digestion. Primary rat chondrocytes were suspended in culture medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 0.4 mmol/L proline, 0.05 mg/mL ascorbic acid, and 0.1 mmol/L nonessential amino acid. Subcultured human chondrocytes were suspended in culture medium consisting of DMEM/F12 supplemented with 10% fetal bovine serum, 0.025 mg/mL ascorbic acid. The suspension was injected into HFs at a density of 3 ⫻ 108 cells/cm3. The bundle was centrifuged (rat chondrocytes: 660g for 300 seconds; human chondrocytes: 220g for 300 seconds) to induce formation of cylindrical-shaped organoids in the lumen of the HFs. The bundle containing the chondrocytes was placed in a 35 mm diameter culture dish with 2 mL of culture medium for rotation culture at 45 rpm on a shaker for 1 month. For histological analysis of proteoglycan and collagen, the chondrocytes cultured in HFs were fixed in neutral buffered formalin. Cross-sections were stained with toluidine blue or Masson Trichrome on days 14 and 28. For gene expression analysis, total RNA was obtained from chondrocytes cultured in HFs on days 0, 14, and 28. As a control, RNA of the cells cultured in monolayer was obtained in the same way. For quantitative assessment of cell differentiation status, we measured extracellular matrix gene expression (collagen type I and II and aggrecan) by real-time polymerase chain reaction (PCR).
IRIE, MIZUMOTO, FUJINO ET AL
Fig 2. Change in gene expression of collagen type II in the primary rat culture system.
stuck on both sides. The thickness of the spacer was set at 300 m so that oxygen deprivation would not arise inside the organoidsheets. The cells were seeded inside the cell immobilization space on the same principle as HF/organoid culture. In this study, we used primary bovine articular chondrocytes obtained from the knee joint by enzyme treatment.6 The cells were centrifuged at 600g for 300 seconds before seeding.
Sheet-Shaped Organoid (Organoid-Sheet) Culture Next, to improve the convenience as cartilage grafts, we developed a culture device that formed a sheet-shaped organoid. The organoid formation device consisted of a polycarbonate spacer to control the thickness of the cell immobilization space, and a polycarbonate porous membrane (pore diameter, 3 m; porosity, 14%; thickness, 9 m). A square hole (1.9 ⫻ 0.4 cm, cell immobilization space) in the center of the spacer had a porous membrane
Fig 1. Histological analysis of rat chondrocyte organoid cultured in HF for 28 days (Masson Trichrome staining).
RESULTS Hollow Fiber/Chondrocyte-Organoid Culture
In the primary rat culture system, chondrocytes packed into HFs formed cylindrical organoids. During the culture, extracellular matrix accumulated around the cells in the lumen of HFs. At low cellular density a tissue like native cartilage, containing abundant extracellular matrix, was formed by day 28. Toluidine blue staining indicated that proteoglycan accumulated inside the organoid. Masson Trichrome staining confirmed the accumulation of collagen as well as proteoglycan (Fig 1). For quantitative assessment of cell differentiation, the extracellular matrix gene was measured by real-time PCR. In organoid culture, expression of collagen type II (Fig 2) and aggrecan in the organoid cells on days 7, 14, 21, and 28 was maintained at the same level as in vivo chondrocytes for 1 month of culture. Collagen type I expression was up-regulated. Meanwhile, in monolayer culture collagen type II and aggrecan expression was rapidly down-regulated at the beginning of the culture (Fig 2). In the subcultured human culture system, collagen type II (Fig 3) and aggrecan expression increased with the duration of the organoid culture. This indicated the possibility of redifferentiation of dedifferentiated cells. However, collagen type I expression was also up-regulated. Meanwhile, collagen type II expression of the cells in monolayer was less up-regulated than in the organoid culture (Fig 3).
DEVELOPMENT OF ARTICULAR CARTILAGE GRAFTS
633
gen and proteoglycan) existed in the organoid-sheet. From these results, this device seemed to control the organoid shape and reconstitute cartilage-like tissue. DISCUSSION
The organoid-sheet device needs improvement. In cartilage regeneration, it is important to reconstitute the sizable cartilage-like tissue with a limited cell number. However, this organoid culture method in a closed space blocks the spontaneous enlargement of the organoid by accumulation of extracellular matrix. Therefore, it will be necessary to convert the closed space culture technique to an open space technique during culture. REFERENCES
Fig 3. Change in gene expression of collagen type II in the subcultured human culture system.
From these results, our organoid formation method by centrifugation was effective for both primary and subcultured chondrocytes. Organoid-Sheet Culture
The cells immobilized in the device formed a 300 m thickness organoid-sheet, which regulated the shape of the organoid by a spacer. From the results of histological analysis on day 7, an abundant extracellular matrix (colla-
1. Frenkel SR, DiCesare PE: Scaffolds for articular cartilage repair. Ann Biomed Eng 32:26, 2004 2. Sittinger M, Reitzel D, Dauner M, et al: Resorbable polyesters in cartilage engineering: affinity and biocompatibility of polymer fiber structures to chondrocytes. J Biomed Mater Res 33:57, 1998 3. Anderer U, Libera J: In vitro engineering of human autogenous cartilage. J Bone Miner Res 17:1420, 2002 4. Fukuda J, Mizumoto H, Nakazawa K, et al: Hepatocyte organoid culture in elliptic hollow fibers to develop a hybrid artificial liver. Int J Artif Organs 27:1091, 2004 5. Ishihara K, Mizumoto H, Nakazawa K, et al: Formation of a sheet-shaped organoid using rat primary hepatocytes for long-term maintenance of liver-specific functions. Int J Artif Organs 29:318, 2006 6. Sawae Y, Shelton JC, Bader DL, et al: Confocal analysis of local and cellular strains in chondrocyte–agarose constructs subjected to mechanical shear. Ann Biomed Eng 32:860, 2004
A
RTICULAR CARTILAGE is a quite durable tissue that has high load-bearing ability. However, once the tissue has been damaged, its capacity for regeneration is limited. Recently, the reconstruction of cartilage-like tissue using chondrocytes has attracted attention as a treatment for cartilage defects. Until now, many researchers have studied how to make the tissue with both chondrocytes and several scaffold materials (eg, collagen, polylactic acid).1 However, several problems remain: cell distribution in the scaffold, connection to the native cartilage, and safety of the scaffold.2 A scaffold-free culture method, ie, pellet culture that forms multicellular aggregates (organoids), would solve many of these problems. It has been reported that organoid culture could assist in the production of extracellular matrix, and the organoid could be integrated with in vivo cartilage.3 However, this approach is inadequate for direct clinical application for treatment of cartilage defects, because of difficulties in controlling organoid shape. Additionally, if the size of the organoid is too thick, necrotic cells arise as a result of nutrient exhaustion inside the organoid. © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 631– 633 (2008)
Until now, we have developed original organoid culture methods that control organoid shape.4,5 In this study, we applied these culture methods to chondrocytes attempting to reconstitute articular cartilage grafts. MATERIALS AND METHODS Hollow Fiber/Chondrocyte-Organoid Culture We made a hollow fiber (HF) bundle composed of 4 HFs (polyethylene HFs coated with ethylene vinyl alcohol; inner diameter, 330 m; outer diameter, 430 m; pore size, about 0.3 m; Asahi Kasei Medical Co, Ltd) as a culture device for organoid formation. From the Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka, Japan. This study was supported in part by a Grant-in-Aid for Scientific Research (B): 19360375 and a Grant-in-Aid for Scientific Research (A) (2): 14205119 from the Japan Society for the Promotion of Science. Address reprint requests to Toshihisa Kajiwara, PhD, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. E-mail: [email protected] 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.024 631
632 In this study, we used primary rat costal chondrocytes and subcultured human articular chondrocytes. Primary rat costal chondrocytes were isolated from the ribs of 7-week-old rats by enzyme treatment. The purchased subcultured human articular chondrocytes (Cambrex Co) were further subcultured 3 times. When they reached 80% to 90% confluence, the cells were harvested by trypsin/EDTA digestion. Primary rat chondrocytes were suspended in culture medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 0.4 mmol/L proline, 0.05 mg/mL ascorbic acid, and 0.1 mmol/L nonessential amino acid. Subcultured human chondrocytes were suspended in culture medium consisting of DMEM/F12 supplemented with 10% fetal bovine serum, 0.025 mg/mL ascorbic acid. The suspension was injected into HFs at a density of 3 ⫻ 108 cells/cm3. The bundle was centrifuged (rat chondrocytes: 660g for 300 seconds; human chondrocytes: 220g for 300 seconds) to induce formation of cylindrical-shaped organoids in the lumen of the HFs. The bundle containing the chondrocytes was placed in a 35 mm diameter culture dish with 2 mL of culture medium for rotation culture at 45 rpm on a shaker for 1 month. For histological analysis of proteoglycan and collagen, the chondrocytes cultured in HFs were fixed in neutral buffered formalin. Cross-sections were stained with toluidine blue or Masson Trichrome on days 14 and 28. For gene expression analysis, total RNA was obtained from chondrocytes cultured in HFs on days 0, 14, and 28. As a control, RNA of the cells cultured in monolayer was obtained in the same way. For quantitative assessment of cell differentiation status, we measured extracellular matrix gene expression (collagen type I and II and aggrecan) by real-time polymerase chain reaction (PCR).
IRIE, MIZUMOTO, FUJINO ET AL
Fig 2. Change in gene expression of collagen type II in the primary rat culture system.
stuck on both sides. The thickness of the spacer was set at 300 m so that oxygen deprivation would not arise inside the organoidsheets. The cells were seeded inside the cell immobilization space on the same principle as HF/organoid culture. In this study, we used primary bovine articular chondrocytes obtained from the knee joint by enzyme treatment.6 The cells were centrifuged at 600g for 300 seconds before seeding.
Sheet-Shaped Organoid (Organoid-Sheet) Culture Next, to improve the convenience as cartilage grafts, we developed a culture device that formed a sheet-shaped organoid. The organoid formation device consisted of a polycarbonate spacer to control the thickness of the cell immobilization space, and a polycarbonate porous membrane (pore diameter, 3 m; porosity, 14%; thickness, 9 m). A square hole (1.9 ⫻ 0.4 cm, cell immobilization space) in the center of the spacer had a porous membrane
Fig 1. Histological analysis of rat chondrocyte organoid cultured in HF for 28 days (Masson Trichrome staining).
RESULTS Hollow Fiber/Chondrocyte-Organoid Culture
In the primary rat culture system, chondrocytes packed into HFs formed cylindrical organoids. During the culture, extracellular matrix accumulated around the cells in the lumen of HFs. At low cellular density a tissue like native cartilage, containing abundant extracellular matrix, was formed by day 28. Toluidine blue staining indicated that proteoglycan accumulated inside the organoid. Masson Trichrome staining confirmed the accumulation of collagen as well as proteoglycan (Fig 1). For quantitative assessment of cell differentiation, the extracellular matrix gene was measured by real-time PCR. In organoid culture, expression of collagen type II (Fig 2) and aggrecan in the organoid cells on days 7, 14, 21, and 28 was maintained at the same level as in vivo chondrocytes for 1 month of culture. Collagen type I expression was up-regulated. Meanwhile, in monolayer culture collagen type II and aggrecan expression was rapidly down-regulated at the beginning of the culture (Fig 2). In the subcultured human culture system, collagen type II (Fig 3) and aggrecan expression increased with the duration of the organoid culture. This indicated the possibility of redifferentiation of dedifferentiated cells. However, collagen type I expression was also up-regulated. Meanwhile, collagen type II expression of the cells in monolayer was less up-regulated than in the organoid culture (Fig 3).
DEVELOPMENT OF ARTICULAR CARTILAGE GRAFTS
633
gen and proteoglycan) existed in the organoid-sheet. From these results, this device seemed to control the organoid shape and reconstitute cartilage-like tissue. DISCUSSION
The organoid-sheet device needs improvement. In cartilage regeneration, it is important to reconstitute the sizable cartilage-like tissue with a limited cell number. However, this organoid culture method in a closed space blocks the spontaneous enlargement of the organoid by accumulation of extracellular matrix. Therefore, it will be necessary to convert the closed space culture technique to an open space technique during culture. REFERENCES
Fig 3. Change in gene expression of collagen type II in the subcultured human culture system.
From these results, our organoid formation method by centrifugation was effective for both primary and subcultured chondrocytes. Organoid-Sheet Culture
The cells immobilized in the device formed a 300 m thickness organoid-sheet, which regulated the shape of the organoid by a spacer. From the results of histological analysis on day 7, an abundant extracellular matrix (colla-
1. Frenkel SR, DiCesare PE: Scaffolds for articular cartilage repair. Ann Biomed Eng 32:26, 2004 2. Sittinger M, Reitzel D, Dauner M, et al: Resorbable polyesters in cartilage engineering: affinity and biocompatibility of polymer fiber structures to chondrocytes. J Biomed Mater Res 33:57, 1998 3. Anderer U, Libera J: In vitro engineering of human autogenous cartilage. J Bone Miner Res 17:1420, 2002 4. Fukuda J, Mizumoto H, Nakazawa K, et al: Hepatocyte organoid culture in elliptic hollow fibers to develop a hybrid artificial liver. Int J Artif Organs 27:1091, 2004 5. Ishihara K, Mizumoto H, Nakazawa K, et al: Formation of a sheet-shaped organoid using rat primary hepatocytes for long-term maintenance of liver-specific functions. Int J Artif Organs 29:318, 2006 6. Sawae Y, Shelton JC, Bader DL, et al: Confocal analysis of local and cellular strains in chondrocyte–agarose constructs subjected to mechanical shear. Ann Biomed Eng 32:860, 2004