- Email: [email protected]
Elastic Cartilage Reconstruction by Transplantation of Cultured Hyaline Cartilage–Derived Chondrocytes
Elastic Cartilage Reconstruction by Transplantation of Cultured Hyaline CartilageeDerived Chondrocytes M. Mizunoa, T. Takebea,b,c, S. Kobayashia,d, S. Kimuraa, M. Masutania, S. Leee, Y.H. Joe, J.I. Leee, and H. Taniguchia,b,* a Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan; bProject Leader of Advanced Medical Research Center, Yokohama City University, Yokohama, Japan; cPRESTO, Japan Science and Technology Agency, Kawaguchi, Japan; dDepartment of Plastic and Reconstructive Surgery, Kanagawa Children’s Medical Center, Yokohama, Japan; and e Regenerative Medicine Laboratory, Center for Stem Cell Research, Department of Biomedical Science and Technology, Institute of Biomedical Science and Technology (IBST), Konkuk University, Seoul, Republic of Korea
ABSTRACT Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilageederived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilageederived chondrocytes are a possible cell source for elastic cartilage reconstruction.
A
UTOLOGOUS tissue transplantation is a current standard treatment for surgical reconstruction of craniofacial injuries or abnormalities, but it puts a highly invasive burden on patients and causes adverse events, such as an inflammation, extrusion, calcification, and abnormal skin. To overcome the limitations of current therapies, the use of reconstructed elastic cartilage from in vitro expanded chondrocytes is expected to be one possible alternative approach [1,2]. The source of chondrocytes for elastic cartilage reconstruction so far relies on elastic cartilage grafted from the auricle, which made it difficult to isolate a large amount of cells owing to donor site morbidity. This limitation highlights the need for identifying other autologous chondrocyte sources, such as articular or costal cartilage, for elastic cartilage reconstruction [3].
Elastic cartilage, such as auricular cartilage, has abundant elastic fibers, characterized by a highly elastic mechanical property. Hyaline cartilage, including articular, costal, and
Funding: Grants-in-Aid from 1) the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT) to T. Takebe (no 24659785), 2) Health and Labor Sciences Research Grants to T. Takebe (no.12103252) and S. Kobayashi (Research on intractable disease no. 2011-164) and 3) Japan Korea Basic Scientific Cooperation Program by Japan Society for the Promotion of Science (JSPS) to T. Takebe. *Address correspondence to Dr Hideki Taniguchi, Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-004, Japan. E-mail: [email protected]
Crown Copyright ª 2014 Published by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 1217e1221 (2014)
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.006 1217
1218
nasal cartilage is totally different from the nature of elastic cartilage. It has rich extracellular matrix and high mechanical strength, which is responsible for protection of the articular surface or for maintenance of the tissue structure. The availability of hyaline cartilageederived chondrocytes for a distinctive type of (elastic) cartilage reconstruction remains to be elucidated. Here, we examined whether canine articular chondrocytes could be a possible candidate of cell source for elastic cartilage reconstruction.
MIZUNO, TAKEBE, KOBAYASHI ET AL mice (Sankyo Laboratory, Tokyo, Japan). The mice were bred and maintained in accordance with our institutional guidelines for the use of laboratory animals.
Histochemical and Immunohistochemical Analysis We stained the sections of primary tissue and reconstructed cartilage with hematoxylin/eosin (HE), Alcian blue, Elastica van Gieson (Muto Pure Chemicals Industries, Osaka, Japan), or collagen type II (clone no 6B3; Millipore), as previously described [4].
Statistical Analysis MATERIALS AND METHODS Isolation and Cultivation of Canine Articular and Auricular Chondrocytes We obtained canine cartilage samples from 3 male Toyo beagle dogs that were 5 months old and weighed 11.3 1.11 kg (Oriental Kobo, Osaka, Japan), following the approved guidelines set by the Ethical Committee at Yokohama City University (approval no 11-126). The cartilages were harvested microscopically. The canine cells were cultured in Dulbecco modified Eagle and Ham F-12 media (DMEM/ F-12; Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies Co) and 1% antibiotic antimycotic solution (Sigma) in 5% CO2 at 37 C, as previously described [4].
Growth Curve and Colony Forming Assay To evaluate the growth curve, canine articular and auricular chondrocytes were seeded in 6-well plates. Cells were rinsed twice with phosphate-buffered saline solution and fixed with cold acetone/methanol fixation solution after 1, 3, 5, 7, and 10 days. The number of cells was photographed and calculated by IN Cell Analyzer 2000 (GE Healthcare). For the assessment of colony formation, those cells were cultured in our standard culture medium. The colonies were counted 14 days after seeding and were stained with Giemsa in methanol. The number of colonies per well was counted.
Gene Expression Analysis Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was referenced to an internal control gene as canine glyceraldehyde-3-phosphate dehydrogenase (Gap-dh, forward 50 ccccacccccaatgtatcag-30 and reverse 50 -cgaaggtggaagagtgggtg-30 ) with the use of Sybr gene expression assays with Sybr Premix Ex Taq II (Takara Bio, Shiga, Japan).
Protein Production Analysis We quantitatively determined the chondrogenic potential of canine articular/auricular chondrocytes by measuring sulfated glycosaminoglycan (sGAG) and a-elastin production. We evaluated extracellular and intracellular protein from the cell culture dish with the use of Blyscan and Fastin assays according to the manufacturer’s protocol (Biocolor, Belfast, UK).
In Vitro Chondrogenic Induction and in Vivo Transplantation To induce chondrogenic differentiation, canine cells were cultured for 21 days with the use of chondrogenic medium. The detailed method was described previously [4]. To estimate elastic cartilage reconstruction capability, chondrogenic induced cells were collected into a 2.5-mL syringe (Terumo, Tokyo, Japan). An appropriate volume of the culture was subcutaneously injected into NOD/SCID
Data are expressed as mean SD from 3 independent experiments. Differences between articular and auricular cartilage were analyzed with post hoc comparisons using Mann-Whitney’s U test with Bonferroni correction. Two-tailed P values of <.05 were considered to be significant.
RESULTS
Histologic analysis showed that canine articular (Fig 1A) and auricular (Fig 1B) cartilage were especially different regarding elastic fiber deposition. Harvested tissues were digested, cultivated, and analyzed for proliferative capacity. Both types of cultivated canine chondrocytes exhibited indistinguishable cell morphology (Fig 1C). Under low-density culture condition, canine articular and auricular chondrocytes formed 3.8 (2.2) and 9.5 (4.9) colonies, respectively (Fig 1E and F). These results showed that auricular chondrocytes proliferated more robustly than articular chondrocytes. Gene expression of canine collagen 1 alpha 1 (Col1a1) and versican/chondroitin sulfate proteoglycan 2 (Vcan/Cspg2), which are 2 of the genes that produce the extracellular matrix, was similar in articular and auricular chondrocytes (Fig 2A and B). Protein production analysis revealed that sGAG/DNA of canine articular chondrocytes was 2.46 (0.09) mg/mg, and this value was equivalent to that of auricular chondrocytes, 2.38 (0.37) mg/mg (Fig 2C). In contrast, gene expression of canine elastin (Eln), which is a major gene in composing the elastic fiber, was significantly higher in auricular chondrocytes than in articular chondrocytes (Fig 2D). The protein production analysis also demonstrated that the amount of a-elastin in canine articular chondrocytes (44.36 [19.15] mg/well) was significantly lower than in auricular chondrocytes (59.86 [17.78] mg/well; Fig 2E). Next, to evaluate the elastic cartilage reconstruction capacity, in vitro expanded dedifferentiated cells redifferentiated into mature chondrocytes under the previously reported chondrogenic differentiation condition [1]. Redifferentiated canine chondrocytes were subcutaneously transplanted into immunodeficient mice. Two months later, both chondrocytes reconstructed cartilage-like tissues (Fig 3A). Histologic analysis confirmed the formation of cartilage that contained adequate glycosaminoglycan (Fig 3B and C). Protein production analysis also suggested that in cartilage reconstruction derived from canine articular cartilage, sGAG/DNA was 25.16 (2.79) mg/mg, and this value was equivalent to that of auricular chondrocytesederived reconstructed cartilage, 21.49 (2.97) mg/mg (Fig 3D and E). Notably, canine articular chondrocytesederived
ELASTIC CARTILAGE RECONSTRUCTION
1219
Fig 1. Comparison of the articular/auricular cartilage and chondrocytes. (A, B) Histologic characteristics of articular and auricular cartilage. Scale bars, 50 mm. (C, D) Time-dependent changes and proliferative rates of articular and auricular chondrocytes. Data are shown as the means SD (n ¼ 9). *P < .05. (E, F) Colony formation of articular and auricular chondrocytes. Data are presented as mean SD (n ¼ 5e8). *P < .05. HE, hematoxylin and eosin.
1220
MIZUNO, TAKEBE, KOBAYASHI ET AL
Fig 2. Gene expression and protein production of the articular and auricular chondrocytes. (A, B, C) Gene expressions of articular and auricular cartilage. Data are presented as mean SD (n ¼ 3e5). *P < .05. (D, E) Protein production of articular and auricular cartilage. Data are presented as mean SD (n ¼ 6 e10). NS, not significant. *P < .05.
tissues contained rich elastic fibers according to Elastica van Gieson staining, suggesting that reconstructed cartilage seemed to be elastic cartilage (Fig 3F). DISCUSSION
In the present study, we have demonstrated the usefulness of hyaline cartilageederived chondrocytes as alternative cell sources for reconstruction of elastic cartilage. The proliferative capacity of canine articular chondrocytes is inferior to that of auricular chondrocytes, although the canine articular chondrocytes had similar gene expression profiles to auricular chondrocytes in the expression of cartilage matrix productionerelated genes and the production of sGAG. Consistent with earlier reports, articular chondrocytes had significantly less expression of elastin than auricular chondrocytes. Nevertheless, elastic cartilage could be reconstructed from cultured articular chondrocytes after subcutaneous transplantation in vivo. We speculated that both types of dedifferentiated chondrocytes have a marked propensity to differentiate into mature chondrocytes of elastic cartilage
except for transplanting under physically and/or chemically defined environmental conditions such as articular (joint) cartilage. Our recent report supports this hypothesis that the contribution environmental factors instruct the fate of immature cells of elastic cartilage into chondrocytes of hyaline cartilage by transplantation into injured knee cartilage [4]. Further studies are needed to elucidate the critical external factors including the extracellular matrix or mechanical stimulation, and this will lead to establish a novel culture method to direct the cell fate into a desired chondrocyte type. Craniofacial injuries and abnormalities affect millions of patients in the world [5]. There are serious limitations associated with current surgical approaches for these diseases. Although costal cartilage transplantation is performed on craniofacial defects, in some cases the volume is too small to cover whole parts of larger lesions [1,2]. We proposed a possible approach as follows. First, chondrocytes are isolated from a tiny piece of hyaline (costal) cartilage concurrently with costal cartilage grafting procedures. Then, cells are extensively expanded in culture to obtain a sufficient quantity. Finally, redifferentiated cells
ELASTIC CARTILAGE RECONSTRUCTION
1221
Fig 3. Cartilage reconstructive capability of the articular and auricular chondrocytes. (A) Gross morphology of reconstructed cartilage derived from articular and auricular chondrocytes. Scale bars, 1 mm. (B, C, D) Histologic analysis of reconstructed cartilage. Scale bars, 100 mm. (E, F) Extracellular matrix amounts of the reconstructed cartilage. Data are presented as mean SD (n ¼ 3e4). HE, hematoxylin and eosin; NS, not significant.
were transplanted back to the patient’s deformed lesion, which could not be repaired by conventional surgical treatment. Patients who have complicated and large craniofacial defects would especially benefit from this procedure. Taken together, though further studies are needed to determine the availability of costal (hyaline) cartilage, this study poses a new possible regenerative approach to reconstructing elastic cartilage. REFERENCES [1] Kobayashi S, Takebe T, Inui M, et al. Reconstruction of human elastic cartilage by a CD44þ CD90þ stem cell in the
ear perichondrium. Proc Natl Acad Sci USA 2011;108: 14479e84. [2] Takebe T, Kobayashi S, Kan H, et al. Human elastic cartilage engineering from cartilage progenitor cells using rotating wall vessel bioreactor. Transplant Proc 2012;44:1158e61. [3] Naumann A, Dennis JE, Awadallah A, et al. Immunochemical and mechanical characterization of cartilage subtypes in rabbit. J Histochem Cytochem 2002;50:1049e58. [4] Mizuno M, Kobayashi S, Takebe T, et al. Reconstruction of joint hyaline cartilage by autologous progenitor cells derived from ear elastic cartilage. Stem Cells. Stem cells 2014;32:816e21. [5] Chang SC, Tobias G, Roy AK, et al. Tissue engineering of autologous cartilage for craniofacial reconstruction by injection molding. Plast Reconstr Surg 2003;112(3):793e9. discussion 800e1.
ABSTRACT Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilageederived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilageederived chondrocytes are a possible cell source for elastic cartilage reconstruction.
A
UTOLOGOUS tissue transplantation is a current standard treatment for surgical reconstruction of craniofacial injuries or abnormalities, but it puts a highly invasive burden on patients and causes adverse events, such as an inflammation, extrusion, calcification, and abnormal skin. To overcome the limitations of current therapies, the use of reconstructed elastic cartilage from in vitro expanded chondrocytes is expected to be one possible alternative approach [1,2]. The source of chondrocytes for elastic cartilage reconstruction so far relies on elastic cartilage grafted from the auricle, which made it difficult to isolate a large amount of cells owing to donor site morbidity. This limitation highlights the need for identifying other autologous chondrocyte sources, such as articular or costal cartilage, for elastic cartilage reconstruction [3].
Elastic cartilage, such as auricular cartilage, has abundant elastic fibers, characterized by a highly elastic mechanical property. Hyaline cartilage, including articular, costal, and
Funding: Grants-in-Aid from 1) the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT) to T. Takebe (no 24659785), 2) Health and Labor Sciences Research Grants to T. Takebe (no.12103252) and S. Kobayashi (Research on intractable disease no. 2011-164) and 3) Japan Korea Basic Scientific Cooperation Program by Japan Society for the Promotion of Science (JSPS) to T. Takebe. *Address correspondence to Dr Hideki Taniguchi, Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-004, Japan. E-mail: [email protected]
Crown Copyright ª 2014 Published by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 1217e1221 (2014)
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.006 1217
1218
nasal cartilage is totally different from the nature of elastic cartilage. It has rich extracellular matrix and high mechanical strength, which is responsible for protection of the articular surface or for maintenance of the tissue structure. The availability of hyaline cartilageederived chondrocytes for a distinctive type of (elastic) cartilage reconstruction remains to be elucidated. Here, we examined whether canine articular chondrocytes could be a possible candidate of cell source for elastic cartilage reconstruction.
MIZUNO, TAKEBE, KOBAYASHI ET AL mice (Sankyo Laboratory, Tokyo, Japan). The mice were bred and maintained in accordance with our institutional guidelines for the use of laboratory animals.
Histochemical and Immunohistochemical Analysis We stained the sections of primary tissue and reconstructed cartilage with hematoxylin/eosin (HE), Alcian blue, Elastica van Gieson (Muto Pure Chemicals Industries, Osaka, Japan), or collagen type II (clone no 6B3; Millipore), as previously described [4].
Statistical Analysis MATERIALS AND METHODS Isolation and Cultivation of Canine Articular and Auricular Chondrocytes We obtained canine cartilage samples from 3 male Toyo beagle dogs that were 5 months old and weighed 11.3 1.11 kg (Oriental Kobo, Osaka, Japan), following the approved guidelines set by the Ethical Committee at Yokohama City University (approval no 11-126). The cartilages were harvested microscopically. The canine cells were cultured in Dulbecco modified Eagle and Ham F-12 media (DMEM/ F-12; Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies Co) and 1% antibiotic antimycotic solution (Sigma) in 5% CO2 at 37 C, as previously described [4].
Growth Curve and Colony Forming Assay To evaluate the growth curve, canine articular and auricular chondrocytes were seeded in 6-well plates. Cells were rinsed twice with phosphate-buffered saline solution and fixed with cold acetone/methanol fixation solution after 1, 3, 5, 7, and 10 days. The number of cells was photographed and calculated by IN Cell Analyzer 2000 (GE Healthcare). For the assessment of colony formation, those cells were cultured in our standard culture medium. The colonies were counted 14 days after seeding and were stained with Giemsa in methanol. The number of colonies per well was counted.
Gene Expression Analysis Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was referenced to an internal control gene as canine glyceraldehyde-3-phosphate dehydrogenase (Gap-dh, forward 50 ccccacccccaatgtatcag-30 and reverse 50 -cgaaggtggaagagtgggtg-30 ) with the use of Sybr gene expression assays with Sybr Premix Ex Taq II (Takara Bio, Shiga, Japan).
Protein Production Analysis We quantitatively determined the chondrogenic potential of canine articular/auricular chondrocytes by measuring sulfated glycosaminoglycan (sGAG) and a-elastin production. We evaluated extracellular and intracellular protein from the cell culture dish with the use of Blyscan and Fastin assays according to the manufacturer’s protocol (Biocolor, Belfast, UK).
In Vitro Chondrogenic Induction and in Vivo Transplantation To induce chondrogenic differentiation, canine cells were cultured for 21 days with the use of chondrogenic medium. The detailed method was described previously [4]. To estimate elastic cartilage reconstruction capability, chondrogenic induced cells were collected into a 2.5-mL syringe (Terumo, Tokyo, Japan). An appropriate volume of the culture was subcutaneously injected into NOD/SCID
Data are expressed as mean SD from 3 independent experiments. Differences between articular and auricular cartilage were analyzed with post hoc comparisons using Mann-Whitney’s U test with Bonferroni correction. Two-tailed P values of <.05 were considered to be significant.
RESULTS
Histologic analysis showed that canine articular (Fig 1A) and auricular (Fig 1B) cartilage were especially different regarding elastic fiber deposition. Harvested tissues were digested, cultivated, and analyzed for proliferative capacity. Both types of cultivated canine chondrocytes exhibited indistinguishable cell morphology (Fig 1C). Under low-density culture condition, canine articular and auricular chondrocytes formed 3.8 (2.2) and 9.5 (4.9) colonies, respectively (Fig 1E and F). These results showed that auricular chondrocytes proliferated more robustly than articular chondrocytes. Gene expression of canine collagen 1 alpha 1 (Col1a1) and versican/chondroitin sulfate proteoglycan 2 (Vcan/Cspg2), which are 2 of the genes that produce the extracellular matrix, was similar in articular and auricular chondrocytes (Fig 2A and B). Protein production analysis revealed that sGAG/DNA of canine articular chondrocytes was 2.46 (0.09) mg/mg, and this value was equivalent to that of auricular chondrocytes, 2.38 (0.37) mg/mg (Fig 2C). In contrast, gene expression of canine elastin (Eln), which is a major gene in composing the elastic fiber, was significantly higher in auricular chondrocytes than in articular chondrocytes (Fig 2D). The protein production analysis also demonstrated that the amount of a-elastin in canine articular chondrocytes (44.36 [19.15] mg/well) was significantly lower than in auricular chondrocytes (59.86 [17.78] mg/well; Fig 2E). Next, to evaluate the elastic cartilage reconstruction capacity, in vitro expanded dedifferentiated cells redifferentiated into mature chondrocytes under the previously reported chondrogenic differentiation condition [1]. Redifferentiated canine chondrocytes were subcutaneously transplanted into immunodeficient mice. Two months later, both chondrocytes reconstructed cartilage-like tissues (Fig 3A). Histologic analysis confirmed the formation of cartilage that contained adequate glycosaminoglycan (Fig 3B and C). Protein production analysis also suggested that in cartilage reconstruction derived from canine articular cartilage, sGAG/DNA was 25.16 (2.79) mg/mg, and this value was equivalent to that of auricular chondrocytesederived reconstructed cartilage, 21.49 (2.97) mg/mg (Fig 3D and E). Notably, canine articular chondrocytesederived
ELASTIC CARTILAGE RECONSTRUCTION
1219
Fig 1. Comparison of the articular/auricular cartilage and chondrocytes. (A, B) Histologic characteristics of articular and auricular cartilage. Scale bars, 50 mm. (C, D) Time-dependent changes and proliferative rates of articular and auricular chondrocytes. Data are shown as the means SD (n ¼ 9). *P < .05. (E, F) Colony formation of articular and auricular chondrocytes. Data are presented as mean SD (n ¼ 5e8). *P < .05. HE, hematoxylin and eosin.
1220
MIZUNO, TAKEBE, KOBAYASHI ET AL
Fig 2. Gene expression and protein production of the articular and auricular chondrocytes. (A, B, C) Gene expressions of articular and auricular cartilage. Data are presented as mean SD (n ¼ 3e5). *P < .05. (D, E) Protein production of articular and auricular cartilage. Data are presented as mean SD (n ¼ 6 e10). NS, not significant. *P < .05.
tissues contained rich elastic fibers according to Elastica van Gieson staining, suggesting that reconstructed cartilage seemed to be elastic cartilage (Fig 3F). DISCUSSION
In the present study, we have demonstrated the usefulness of hyaline cartilageederived chondrocytes as alternative cell sources for reconstruction of elastic cartilage. The proliferative capacity of canine articular chondrocytes is inferior to that of auricular chondrocytes, although the canine articular chondrocytes had similar gene expression profiles to auricular chondrocytes in the expression of cartilage matrix productionerelated genes and the production of sGAG. Consistent with earlier reports, articular chondrocytes had significantly less expression of elastin than auricular chondrocytes. Nevertheless, elastic cartilage could be reconstructed from cultured articular chondrocytes after subcutaneous transplantation in vivo. We speculated that both types of dedifferentiated chondrocytes have a marked propensity to differentiate into mature chondrocytes of elastic cartilage
except for transplanting under physically and/or chemically defined environmental conditions such as articular (joint) cartilage. Our recent report supports this hypothesis that the contribution environmental factors instruct the fate of immature cells of elastic cartilage into chondrocytes of hyaline cartilage by transplantation into injured knee cartilage [4]. Further studies are needed to elucidate the critical external factors including the extracellular matrix or mechanical stimulation, and this will lead to establish a novel culture method to direct the cell fate into a desired chondrocyte type. Craniofacial injuries and abnormalities affect millions of patients in the world [5]. There are serious limitations associated with current surgical approaches for these diseases. Although costal cartilage transplantation is performed on craniofacial defects, in some cases the volume is too small to cover whole parts of larger lesions [1,2]. We proposed a possible approach as follows. First, chondrocytes are isolated from a tiny piece of hyaline (costal) cartilage concurrently with costal cartilage grafting procedures. Then, cells are extensively expanded in culture to obtain a sufficient quantity. Finally, redifferentiated cells
ELASTIC CARTILAGE RECONSTRUCTION
1221
Fig 3. Cartilage reconstructive capability of the articular and auricular chondrocytes. (A) Gross morphology of reconstructed cartilage derived from articular and auricular chondrocytes. Scale bars, 1 mm. (B, C, D) Histologic analysis of reconstructed cartilage. Scale bars, 100 mm. (E, F) Extracellular matrix amounts of the reconstructed cartilage. Data are presented as mean SD (n ¼ 3e4). HE, hematoxylin and eosin; NS, not significant.
were transplanted back to the patient’s deformed lesion, which could not be repaired by conventional surgical treatment. Patients who have complicated and large craniofacial defects would especially benefit from this procedure. Taken together, though further studies are needed to determine the availability of costal (hyaline) cartilage, this study poses a new possible regenerative approach to reconstructing elastic cartilage. REFERENCES [1] Kobayashi S, Takebe T, Inui M, et al. Reconstruction of human elastic cartilage by a CD44þ CD90þ stem cell in the
ear perichondrium. Proc Natl Acad Sci USA 2011;108: 14479e84. [2] Takebe T, Kobayashi S, Kan H, et al. Human elastic cartilage engineering from cartilage progenitor cells using rotating wall vessel bioreactor. Transplant Proc 2012;44:1158e61. [3] Naumann A, Dennis JE, Awadallah A, et al. Immunochemical and mechanical characterization of cartilage subtypes in rabbit. J Histochem Cytochem 2002;50:1049e58. [4] Mizuno M, Kobayashi S, Takebe T, et al. Reconstruction of joint hyaline cartilage by autologous progenitor cells derived from ear elastic cartilage. Stem Cells. Stem cells 2014;32:816e21. [5] Chang SC, Tobias G, Roy AK, et al. Tissue engineering of autologous cartilage for craniofacial reconstruction by injection molding. Plast Reconstr Surg 2003;112(3):793e9. discussion 800e1.