- Email: [email protected]
Expression of Transcription Factor Sox9 in Cartilage Formation from Grafted Periosteal Cells
Ueno, Kagawa, Kanou, et al
Asian J Oral Maxillofac Surg 2006;18(1):35-40. ORIGINAL RESEARCH
Expression of Transcription Factor Sox9 in Cartilage Formation from Grafted Periosteal Cells Takaaki Ueno, Toshimasa Kagawa, Miwa Kanou, Nobuhisa Ishida, Yoshiro Sakata, Takashi Fujii, Hideaki Imura, Seiji Kondou, Nobuyoshi Mizukawa, Toshio Sugahara Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Abstract Objective: To investigate the expression of Sox9 protein in the process of cartilage formation after periosteal grafting using the rat periosteum graft model. Materials and Methods: Periostea taken from twenty 7-week-old rats were immediately grafted into the suprahyoid muscles. Grafted tissues were harvested 0, 7, 14, and 21 days after grafting and examined by microscopy and immunohistochemistry using CD44, type 2 collagen, and Sox9 antibodies as markers. Results: Periosteal cells expressed CD44 after 7 days and Sox9 after 14 days. The Sox9-positive cells showed chondrogenic differentiation with type 2 collagen expression by 21 days after grafting. Endochondral ossification was also seen 21 days after grafting. Sox9 was not detected in hypertrophic chondrocytes. Conclusion: These findings suggested that Sox9 may be related to chondrogenic differentiation of periosteal cells in grafted periosteum. Key words: Periosteum, Sox-9 transcription factor
Introduction Recently, the regulatation of stem cell differentiation for tissue repair after surgical resection of tumours, physical trauma, or craniomaxillofacial lesions has become an important research subject. In the field of cartilage and bone repair, considerable attention has been paid to the application of osteoprogenitor cells in bone marrow and periosteum.1,2 These authors have previously demonstrated the potential of chondrogenic differentiation of the periosteal cell in the rat tibial periosteal graft model and additionally used the periosteal graft for the repair of mandibular condyle defects.3 The periosteal cell has bipotential of chondrogenic and osteogenic differentiation, where these differentiation processes are regulated by several growth factors such as transforming growth factor–1, insulin-like growth factor–1, and bone morphogenetic protein–2.4-6 Regulation or control of Correspondence: Takaaki Ueno, Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata, Okayama 7008525, Japan. Tel: (81 86) 235 6697; Fax: (81 86) 235 6699; E-mail: [email protected]
© Asian 2006J Asian Oral Maxillofac Association Surg of Oral Vol 18, andNo Maxillofacial 1, 2006 Surgeons.
chondrogenic cell differentiation of periosteal cells would enable determination of whether the periosteal cell is a suitable material for the repair of damaged cartilage such as articular cartilage damaged by mandibular condyle diseases. Sox9 plays a crucial role in chondrogenesis. Sox9 encodes a high-mobility group (HMG)-domain transcription factor that activates an enhancer in the gene for type 2 collagen (Col2a1), a principal cartilage matrix protein. Sox proteins are involved in several processes during embryogenesis. Based on the amino acid sequence of the HMG domain, Sox proteins can be divided into 10 subgroups. Subgroup E consists of 3 members (Sox8, Sox9, and Sox10) that are expressed in several developing tissues.7 In frog embryos, morpholino-mediated depletion of Sox9 results in loss of cranial neural crest progenitors.8 Bi et al9 and Bell et al10 described the relation of Sox9 to chondrogenic cell differentiation in the development of secondary cartilage in chick articular cartilage. Aigner et al detected Sox9 immunohistologically in normal adult human articular chondrocytes and 35
Immunoexpression of Sox9 in the Periosteum
described decreases in the level of Sox9 mRNA in osteoarthritic cartilage.11 These researchers further stated that Sox9 is involved in chondrocytic phenotype. To improve the effective induction of chondrogenic cell differentiation from periosteal cells, the role of Sox9 should be analysed in the process of periosteal bone and cartilage formation. However, to date, there is no report on Sox9 expression in the periosteal graft model. The aim of this study was to investigate the expression of Sox9 protein in the process of cartilage formation occurring at an early stage after periosteal grafting.
Materials and Methods Animal Care and Surgery Twenty 7-week-old Sprague-Dawley rats were purchased from Charles River (Osaka, Japan). They were housed at 25ºC and fed a standard animal diet (Oriental Co, Osaka, Japan) with water provided ad libitum for 1 week prior to the grafting operation. All surgical procedures were performed under anaesthesia with sodium pentobarbital 10 mg/kg body weight. The grafting procedure used in the present study has been described previously.5 Briefly, a section of periosteum (7 x 5 mm) from the tibia was carefully stripped then folded to be sutured with 5-0 nylon to form a cylinder with the osteogenic layer facing inward. The harvested periosteum was immediately grafted into the suprahyoid muscles. The grafted periostea were removed at 0, 7, 14, and 21 days after grafting (n = 5 in each group). The study protocol was approved by the Okayama University Dental School Committee of Animal Research. All animals were cared for in accordance with the Guidelines for Animal Research of Okayama University Dental School and with the principles of the Declaration of Helsinki. Observation by Light Microscopy Grafted tissue was removed and fixed in 10% neutral buffered formalin before decalcification in 5% trichloroacetic acid for 7 days. Tissue samples were subsequently dehydrated using a graded ethanol series and embedded in paraffin. The 6-mm sections were stained with haematoxylin and eosin for light microscopic observation. 36
Immunohistochemical Observation Specimens were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4ºC for 24 hours, then were decalcified in 5% ethylene diaminetetraacetic acid in 0.1 M phosphate buffer (pH 7.4) for 2 days. After dehydration with a graded ethanol series and embedding in paraffin, sections cut to a thickness of 5 μm were mounted on glass slides and rehydrated. The slides were immersed in 0.3% hydrogen peroxide solution for 15 minutes to inhibit endogenous peroxidase before blocking with 10% bovine serum in phosphate buffered saline (PBS) for 30 minutes at room temperature. Slides for detection of CD44, as a marker of osteo/chondrogenic precurser cells, were exposed to mouse anti-CD44 antibody (BD Bioscience, San Jose, USA). Slides for type 2 collagen detection, as a marker of chondrogenic cells, were exposed to rabbit anti-type 2 collagen antibody (Cosmo Bio Co, Ltd, Tokyo, Japan). To detect Sox9, slides were exposed to mouse anti-Sox9 antibody (Chemicom International, Inc, Temeula USA) diluted 1:100 in PBS containing 3% bovine serum albumin (BSA) at 4ºC overnight. After incubation, the slides were exposed to horseradish peroxidase (HRP)-labelled anti-mouse rat immunoglobulin G diluted 1:100 in PBS containing 3% bovine serum albumin for 60 minutes at room temperature. After washing in PBS, slides were incubated for 5 minutes at room temperature in a solution containing 0.05% 3,3' diaminobenzidine tetrahydrochloride, 0.01% hydrogen peroxide and 0.05 M TrisHCl (pH 7.6) to visualise the immune complexes. The sections were counter-stained with methyl green or haematoxylin. As a positive control, rabbit knee joints were used to confirm immunoreaction. For a negative control, some slides were incubated with 3% BSA in PBS instead of the primary antibody.
Results Histological Observations In the immediately grafted tissue on day 0 after grafting, periosteal cells showed a fibroblastic appearance with surrounding connective tissue. Periosteal cells were scarce. At this stage, periosteal cells did not express any osteo/chondrogenic marker (Figure 1). By day 7 after grafting, the number of periosteal cells increased remarkably with CD44 expression. Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
Ueno, Kagawa, Kanou, et al
a
b
c
Figure 1. Histological findings of immediately harvested periosteum. (a) Fibroblastic cells (arrows) in the harvested periosteum (haematoxylin and eosin; original magnification, x 60); (b) fibroblastic cells show no immunoreaction to CD44 (counter-stained with haematoxylin, original magnification, x 60); and (c) fibroblastic cells show no immunoreaction to Sox9 (counter-stained with haematoxylin, original magnification, x 60).
Most of these cells started to express Sox9. The Sox9positive cells became larger in size and were of chondroblastic appearance. However, some cells expressed no Sox9 (Figure 2). Fourteen days after grafting, grafted periosteum formed cartilage with type 2 collagen production. Periosteal cells with Sox9 immunoreaction showed chondrogenic differentiation. At this stage, some Sox9-negative cells showed osteogenic cell differentiation producing woven bone with vascular invasion from the surrounding connective tissue (Figure 3). Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
By day 21 after grafting, cartilage in the periosteum developed and partial endochondral ossification with blood invasion was observed. Sox9 was not detected in hypertrophic chondrocytes (data not shown).
Discussion Although periosteal grafts show chondrogenic potential and have been used to repair defects in articular cartilage, the immunolocalisation of Sox9 in the endochondral ossification from grafted periosteum has not been well documented. In the rat periosteal graft model described here, periosteal cells 37
Immunoexpression of Sox9 in the Periosteum
a
b
c
d
Figure 2. Histological findings of grafted periosteum 7 days after grafting. (a) The number of periosteal cells is remarkably increased in the grafted tissue and proliferating cells become round in shape (haematoxylin and eosin; original magnification, x 60). (b) Most proliferating periosteal cells show CD44 immunoreaction (arrows) [counter-stained with haematoxylin; original magnification, x 60]; and (c) Sox9 immunoreaction (arrows) [counter-stained with haematoxylin; original magnification, x 60]. (d) Some periosteal cells present at the periphery of the graft show no immunoreaction to Sox9 (counter-stained with haematoxylin; original magnification, x 60).
that differentiated to chondrocytes showed Sox9 immunoexpression. This finding suggested that Sox9 was related to the chondrogenic differentiation of periosteal cells in the grafted condition.
results of these studies agree with the results of this study and suggest the relationship of Sox9 with chondrogenic differentiation of osteo/chondrogenic precurser cells in the periosteum.
Although some reports suggested that Sox9 was related to chondrogenic differentiation,12 the exact role of Sox9 has not been clarified. Bi et al suggested that the major function of Sox9 in foetal growth cartilage was the maintenance of the chondrocytic phenotype as well as inhibition of hypertrophic chondrocyte differentiation.9 Kulyk et al used micromass culture of an embryonic cartilage differentiation model to examine the temporal pattern of Sox9 and demonstrated activation of Sox9 RNA expression in the prechondrogenic phase of culture. 13 The
In the present study, Sox9 immunoreaction was detected in the rat periosteal graft model. We used CD44 as a marker of chondrogenic cells at the proliferating stage in the grafted periosteum in accordance with Gilson et al’s work.14 CD44 is a cell surface hyaluronan binding protein. CD44 is commonly found on the surface of several different cell types, including lymphocytes, epithelial cells, and melanoma cells. Recently, CD44 has been identified on the surface of cells to have a capability to differentiate osteocytes, osteoclasts, and chondrogenic periosteal cells.
38
Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
Ueno, Kagawa, Kanou, et al
a
b
c
d
Figure 3. Histological findings of grafted periosteum 14 days after grafting. (a) Periosteal graft forms cartilage (thick arrows) and there is little bone formation (thin arrows) in the peripheral area (haematoxylin and eosin; original magnification, x 40). Most periosteal cells have differentiated into chondrocytes and show (b) type 2 collagen (counter-stained with haematoxylin; original magnification, x 100); and (c) Sox9 (counter-stained with haematoxylin; original magnification, x 100). (d) Osteoblasts differentiated from periosteal cells show no Sox9 immunoreaction (counter-stained with haematoxylin; original magnification, x 100). Abbreviations: Car = cartilage induced from grafted periosteum; Bo = directly formed new bone.
Periosteal cell in grafted periosteum Sox9-positive
Sox9-negative
Direct bone formation Chondrogenic differentiation
Endochondral ossification
Figure 4. The role of Sox9 in the process of cartilage formation in the grafted periosteum. Sox9-positive cells differentiate into chondrocytes in the grafted periosteum. Sox9-negative cells directly differentiate into osteoblasts. Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
In the present study, CD44-positive chondrogenic periosteal cells showed expression of Sox9 prior to cartilage formation, as shown in Figure 2. However, some periosteal cells without Sox9 expression in grafted tissue directly differentiated into osteoblasts. These results suggest that Sox9 is related to the regulation of chondrogenic differentiation of grafted periosteal cells (Figure 4). Recent research has suggested the efficacy of the periosteum graft for the repair of articular cartilage. O’Driscoll and Recklies reported cartilage formation from cultured rabbit tibial periosteum with transforming growth factor-β and emphasised the efficacy of the periosteal graft for the repair of the damaged surface of articular cartilage.1 The authors of this study have also reported the efficacy of the periosteum graft for the repair of damaged mandibular head in the rabbit and demonstrated 39
Immunoexpression of Sox9 in the Periosteum
histologically the regeneration of the articular cartilage surface.3 However, wide cartilage defects cannot be fully repaired by these conventional periosteal grafts. Recently, gene inductive therapy using bone growth factor and transcription factor has been attempted in the field of hard tissue engineering. Kishimoto et al enhanced bone formation by BMP-4 gene transfer using in vivo electroporation.6 Sox9 is possibly an important transcription factor of chondrogenic cell differentiation, and these results may contribute to the development of gene inductive therapy for the repair of bone and cartilage defects. The findings of this study suggest the relation of Sox9 to chondrogenic differentiation of periosteal cells. However, further studies using additional techniques such as double staining with both type 2 collagen and Sox9 are required in order to confirm the exact role of Sox9.
References 1. O’Driscoll SW, Recklies AD. Chondrogenesis in periosteal explants. J Bone Joint Surg Am 1994;76:1042-1051. 2. Honda MJ, Yada T, Ueda M, Kimata K. Cartilage formation by serial passaged cultured chondrocytes in a new scaffold: hybrid 75:25 poly (L-Lactide-e-Caprolactone) sponge. J Oral Maxillofac Surg 2004;62:1510-1516. 3. Ueno T, Kagawa T, Fujii T, Kanou M. Regeneration of mandibular head from grafted periosteum. Ann Plast Surg 2003;51:77-83. 4. Hanada K, Solchaga LA, Caplan AI. BMP-2 induction and TGF-β1 modulation of rat periosteal cell chondrogenesis. J Cell Biochem 2001;81:284-294.
40
5. Ueno T, Mizukawa N, Sugahara T. Experimental study of bone formation from autogenous periosteal graft following insulin-like growth factor-one administration. J Craniomaxillofac Surg 1999;27:308-313. 6. Kishimoto K, Watanabe Y, Nakamura H, Kokubun S. Ectopic bone formation by electroporat transfer of bone morphogenetic protein-4 gene. Bone 2002;31:340-347. 7. Cheung M, Briscoe J. Neural crest development is regulated by the transcription factor Sox9. Development 2003;130: 5681-5693. 8. Spokony RF, Aoki Y, Saint-Germain N, Magner-Fink E, Saint-Jeannet JP. The transcription factor Sox9 is required for cranial neutral crest. Development 2001;129:421-432. 9. Bi W, Huang W, Whitworth DJ, Min DJ, Zhang Z, Behringer RR. Haploinsufficiency of Sox9 results in defective cartilage primordial and premature skeletal mineralization. Proc Natl Acad Sci USA 2001;98:66986703. 10. Bell DM, Leung KK, Wheatley SC, Ng LJ, Zhou S, Ling K, Sham W, Koopman P, Tam PP, Cheah KS. Sox9 directly regulate the type two collagen gene. Nat Genet 1997;16: 174-178. 11. Aigner T, Gebhard PM, Schmid E, Bau B, Harley V, Poschl E. Sox9 expression does not correlate with type two collagen expression in adult articular chondrocytes. Matrix Biol 2003;22:363-372. 12. Ikeda T, Kawaguchi S, Kamekura S. Distinct roles of Sox5. Sox6, and Sox9 in differentiation of chondrogenic differentiation. J Bone Miner Metab 2005;23:337-340. 13. Kulyk WM, Franklin JL, Hoffman LM. Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme. Exp Cell Res 2000;255:327-332. 14. Gilson R, Mcculloch CA, Zohar R. Stromal mesenchymal progenitor cells. Leuko Lymphoma 1999;32:211-221.
Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
Asian J Oral Maxillofac Surg 2006;18(1):35-40. ORIGINAL RESEARCH
Expression of Transcription Factor Sox9 in Cartilage Formation from Grafted Periosteal Cells Takaaki Ueno, Toshimasa Kagawa, Miwa Kanou, Nobuhisa Ishida, Yoshiro Sakata, Takashi Fujii, Hideaki Imura, Seiji Kondou, Nobuyoshi Mizukawa, Toshio Sugahara Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Abstract Objective: To investigate the expression of Sox9 protein in the process of cartilage formation after periosteal grafting using the rat periosteum graft model. Materials and Methods: Periostea taken from twenty 7-week-old rats were immediately grafted into the suprahyoid muscles. Grafted tissues were harvested 0, 7, 14, and 21 days after grafting and examined by microscopy and immunohistochemistry using CD44, type 2 collagen, and Sox9 antibodies as markers. Results: Periosteal cells expressed CD44 after 7 days and Sox9 after 14 days. The Sox9-positive cells showed chondrogenic differentiation with type 2 collagen expression by 21 days after grafting. Endochondral ossification was also seen 21 days after grafting. Sox9 was not detected in hypertrophic chondrocytes. Conclusion: These findings suggested that Sox9 may be related to chondrogenic differentiation of periosteal cells in grafted periosteum. Key words: Periosteum, Sox-9 transcription factor
Introduction Recently, the regulatation of stem cell differentiation for tissue repair after surgical resection of tumours, physical trauma, or craniomaxillofacial lesions has become an important research subject. In the field of cartilage and bone repair, considerable attention has been paid to the application of osteoprogenitor cells in bone marrow and periosteum.1,2 These authors have previously demonstrated the potential of chondrogenic differentiation of the periosteal cell in the rat tibial periosteal graft model and additionally used the periosteal graft for the repair of mandibular condyle defects.3 The periosteal cell has bipotential of chondrogenic and osteogenic differentiation, where these differentiation processes are regulated by several growth factors such as transforming growth factor–1, insulin-like growth factor–1, and bone morphogenetic protein–2.4-6 Regulation or control of Correspondence: Takaaki Ueno, Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata, Okayama 7008525, Japan. Tel: (81 86) 235 6697; Fax: (81 86) 235 6699; E-mail: [email protected]
© Asian 2006J Asian Oral Maxillofac Association Surg of Oral Vol 18, andNo Maxillofacial 1, 2006 Surgeons.
chondrogenic cell differentiation of periosteal cells would enable determination of whether the periosteal cell is a suitable material for the repair of damaged cartilage such as articular cartilage damaged by mandibular condyle diseases. Sox9 plays a crucial role in chondrogenesis. Sox9 encodes a high-mobility group (HMG)-domain transcription factor that activates an enhancer in the gene for type 2 collagen (Col2a1), a principal cartilage matrix protein. Sox proteins are involved in several processes during embryogenesis. Based on the amino acid sequence of the HMG domain, Sox proteins can be divided into 10 subgroups. Subgroup E consists of 3 members (Sox8, Sox9, and Sox10) that are expressed in several developing tissues.7 In frog embryos, morpholino-mediated depletion of Sox9 results in loss of cranial neural crest progenitors.8 Bi et al9 and Bell et al10 described the relation of Sox9 to chondrogenic cell differentiation in the development of secondary cartilage in chick articular cartilage. Aigner et al detected Sox9 immunohistologically in normal adult human articular chondrocytes and 35
Immunoexpression of Sox9 in the Periosteum
described decreases in the level of Sox9 mRNA in osteoarthritic cartilage.11 These researchers further stated that Sox9 is involved in chondrocytic phenotype. To improve the effective induction of chondrogenic cell differentiation from periosteal cells, the role of Sox9 should be analysed in the process of periosteal bone and cartilage formation. However, to date, there is no report on Sox9 expression in the periosteal graft model. The aim of this study was to investigate the expression of Sox9 protein in the process of cartilage formation occurring at an early stage after periosteal grafting.
Materials and Methods Animal Care and Surgery Twenty 7-week-old Sprague-Dawley rats were purchased from Charles River (Osaka, Japan). They were housed at 25ºC and fed a standard animal diet (Oriental Co, Osaka, Japan) with water provided ad libitum for 1 week prior to the grafting operation. All surgical procedures were performed under anaesthesia with sodium pentobarbital 10 mg/kg body weight. The grafting procedure used in the present study has been described previously.5 Briefly, a section of periosteum (7 x 5 mm) from the tibia was carefully stripped then folded to be sutured with 5-0 nylon to form a cylinder with the osteogenic layer facing inward. The harvested periosteum was immediately grafted into the suprahyoid muscles. The grafted periostea were removed at 0, 7, 14, and 21 days after grafting (n = 5 in each group). The study protocol was approved by the Okayama University Dental School Committee of Animal Research. All animals were cared for in accordance with the Guidelines for Animal Research of Okayama University Dental School and with the principles of the Declaration of Helsinki. Observation by Light Microscopy Grafted tissue was removed and fixed in 10% neutral buffered formalin before decalcification in 5% trichloroacetic acid for 7 days. Tissue samples were subsequently dehydrated using a graded ethanol series and embedded in paraffin. The 6-mm sections were stained with haematoxylin and eosin for light microscopic observation. 36
Immunohistochemical Observation Specimens were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4ºC for 24 hours, then were decalcified in 5% ethylene diaminetetraacetic acid in 0.1 M phosphate buffer (pH 7.4) for 2 days. After dehydration with a graded ethanol series and embedding in paraffin, sections cut to a thickness of 5 μm were mounted on glass slides and rehydrated. The slides were immersed in 0.3% hydrogen peroxide solution for 15 minutes to inhibit endogenous peroxidase before blocking with 10% bovine serum in phosphate buffered saline (PBS) for 30 minutes at room temperature. Slides for detection of CD44, as a marker of osteo/chondrogenic precurser cells, were exposed to mouse anti-CD44 antibody (BD Bioscience, San Jose, USA). Slides for type 2 collagen detection, as a marker of chondrogenic cells, were exposed to rabbit anti-type 2 collagen antibody (Cosmo Bio Co, Ltd, Tokyo, Japan). To detect Sox9, slides were exposed to mouse anti-Sox9 antibody (Chemicom International, Inc, Temeula USA) diluted 1:100 in PBS containing 3% bovine serum albumin (BSA) at 4ºC overnight. After incubation, the slides were exposed to horseradish peroxidase (HRP)-labelled anti-mouse rat immunoglobulin G diluted 1:100 in PBS containing 3% bovine serum albumin for 60 minutes at room temperature. After washing in PBS, slides were incubated for 5 minutes at room temperature in a solution containing 0.05% 3,3' diaminobenzidine tetrahydrochloride, 0.01% hydrogen peroxide and 0.05 M TrisHCl (pH 7.6) to visualise the immune complexes. The sections were counter-stained with methyl green or haematoxylin. As a positive control, rabbit knee joints were used to confirm immunoreaction. For a negative control, some slides were incubated with 3% BSA in PBS instead of the primary antibody.
Results Histological Observations In the immediately grafted tissue on day 0 after grafting, periosteal cells showed a fibroblastic appearance with surrounding connective tissue. Periosteal cells were scarce. At this stage, periosteal cells did not express any osteo/chondrogenic marker (Figure 1). By day 7 after grafting, the number of periosteal cells increased remarkably with CD44 expression. Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
Ueno, Kagawa, Kanou, et al
a
b
c
Figure 1. Histological findings of immediately harvested periosteum. (a) Fibroblastic cells (arrows) in the harvested periosteum (haematoxylin and eosin; original magnification, x 60); (b) fibroblastic cells show no immunoreaction to CD44 (counter-stained with haematoxylin, original magnification, x 60); and (c) fibroblastic cells show no immunoreaction to Sox9 (counter-stained with haematoxylin, original magnification, x 60).
Most of these cells started to express Sox9. The Sox9positive cells became larger in size and were of chondroblastic appearance. However, some cells expressed no Sox9 (Figure 2). Fourteen days after grafting, grafted periosteum formed cartilage with type 2 collagen production. Periosteal cells with Sox9 immunoreaction showed chondrogenic differentiation. At this stage, some Sox9-negative cells showed osteogenic cell differentiation producing woven bone with vascular invasion from the surrounding connective tissue (Figure 3). Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
By day 21 after grafting, cartilage in the periosteum developed and partial endochondral ossification with blood invasion was observed. Sox9 was not detected in hypertrophic chondrocytes (data not shown).
Discussion Although periosteal grafts show chondrogenic potential and have been used to repair defects in articular cartilage, the immunolocalisation of Sox9 in the endochondral ossification from grafted periosteum has not been well documented. In the rat periosteal graft model described here, periosteal cells 37
Immunoexpression of Sox9 in the Periosteum
a
b
c
d
Figure 2. Histological findings of grafted periosteum 7 days after grafting. (a) The number of periosteal cells is remarkably increased in the grafted tissue and proliferating cells become round in shape (haematoxylin and eosin; original magnification, x 60). (b) Most proliferating periosteal cells show CD44 immunoreaction (arrows) [counter-stained with haematoxylin; original magnification, x 60]; and (c) Sox9 immunoreaction (arrows) [counter-stained with haematoxylin; original magnification, x 60]. (d) Some periosteal cells present at the periphery of the graft show no immunoreaction to Sox9 (counter-stained with haematoxylin; original magnification, x 60).
that differentiated to chondrocytes showed Sox9 immunoexpression. This finding suggested that Sox9 was related to the chondrogenic differentiation of periosteal cells in the grafted condition.
results of these studies agree with the results of this study and suggest the relationship of Sox9 with chondrogenic differentiation of osteo/chondrogenic precurser cells in the periosteum.
Although some reports suggested that Sox9 was related to chondrogenic differentiation,12 the exact role of Sox9 has not been clarified. Bi et al suggested that the major function of Sox9 in foetal growth cartilage was the maintenance of the chondrocytic phenotype as well as inhibition of hypertrophic chondrocyte differentiation.9 Kulyk et al used micromass culture of an embryonic cartilage differentiation model to examine the temporal pattern of Sox9 and demonstrated activation of Sox9 RNA expression in the prechondrogenic phase of culture. 13 The
In the present study, Sox9 immunoreaction was detected in the rat periosteal graft model. We used CD44 as a marker of chondrogenic cells at the proliferating stage in the grafted periosteum in accordance with Gilson et al’s work.14 CD44 is a cell surface hyaluronan binding protein. CD44 is commonly found on the surface of several different cell types, including lymphocytes, epithelial cells, and melanoma cells. Recently, CD44 has been identified on the surface of cells to have a capability to differentiate osteocytes, osteoclasts, and chondrogenic periosteal cells.
38
Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
Ueno, Kagawa, Kanou, et al
a
b
c
d
Figure 3. Histological findings of grafted periosteum 14 days after grafting. (a) Periosteal graft forms cartilage (thick arrows) and there is little bone formation (thin arrows) in the peripheral area (haematoxylin and eosin; original magnification, x 40). Most periosteal cells have differentiated into chondrocytes and show (b) type 2 collagen (counter-stained with haematoxylin; original magnification, x 100); and (c) Sox9 (counter-stained with haematoxylin; original magnification, x 100). (d) Osteoblasts differentiated from periosteal cells show no Sox9 immunoreaction (counter-stained with haematoxylin; original magnification, x 100). Abbreviations: Car = cartilage induced from grafted periosteum; Bo = directly formed new bone.
Periosteal cell in grafted periosteum Sox9-positive
Sox9-negative
Direct bone formation Chondrogenic differentiation
Endochondral ossification
Figure 4. The role of Sox9 in the process of cartilage formation in the grafted periosteum. Sox9-positive cells differentiate into chondrocytes in the grafted periosteum. Sox9-negative cells directly differentiate into osteoblasts. Asian J Oral Maxillofac Surg Vol 18, No 1, 2006
In the present study, CD44-positive chondrogenic periosteal cells showed expression of Sox9 prior to cartilage formation, as shown in Figure 2. However, some periosteal cells without Sox9 expression in grafted tissue directly differentiated into osteoblasts. These results suggest that Sox9 is related to the regulation of chondrogenic differentiation of grafted periosteal cells (Figure 4). Recent research has suggested the efficacy of the periosteum graft for the repair of articular cartilage. O’Driscoll and Recklies reported cartilage formation from cultured rabbit tibial periosteum with transforming growth factor-β and emphasised the efficacy of the periosteal graft for the repair of the damaged surface of articular cartilage.1 The authors of this study have also reported the efficacy of the periosteum graft for the repair of damaged mandibular head in the rabbit and demonstrated 39
Immunoexpression of Sox9 in the Periosteum
histologically the regeneration of the articular cartilage surface.3 However, wide cartilage defects cannot be fully repaired by these conventional periosteal grafts. Recently, gene inductive therapy using bone growth factor and transcription factor has been attempted in the field of hard tissue engineering. Kishimoto et al enhanced bone formation by BMP-4 gene transfer using in vivo electroporation.6 Sox9 is possibly an important transcription factor of chondrogenic cell differentiation, and these results may contribute to the development of gene inductive therapy for the repair of bone and cartilage defects. The findings of this study suggest the relation of Sox9 to chondrogenic differentiation of periosteal cells. However, further studies using additional techniques such as double staining with both type 2 collagen and Sox9 are required in order to confirm the exact role of Sox9.
References 1. O’Driscoll SW, Recklies AD. Chondrogenesis in periosteal explants. J Bone Joint Surg Am 1994;76:1042-1051. 2. Honda MJ, Yada T, Ueda M, Kimata K. Cartilage formation by serial passaged cultured chondrocytes in a new scaffold: hybrid 75:25 poly (L-Lactide-e-Caprolactone) sponge. J Oral Maxillofac Surg 2004;62:1510-1516. 3. Ueno T, Kagawa T, Fujii T, Kanou M. Regeneration of mandibular head from grafted periosteum. Ann Plast Surg 2003;51:77-83. 4. Hanada K, Solchaga LA, Caplan AI. BMP-2 induction and TGF-β1 modulation of rat periosteal cell chondrogenesis. J Cell Biochem 2001;81:284-294.
40
5. Ueno T, Mizukawa N, Sugahara T. Experimental study of bone formation from autogenous periosteal graft following insulin-like growth factor-one administration. J Craniomaxillofac Surg 1999;27:308-313. 6. Kishimoto K, Watanabe Y, Nakamura H, Kokubun S. Ectopic bone formation by electroporat transfer of bone morphogenetic protein-4 gene. Bone 2002;31:340-347. 7. Cheung M, Briscoe J. Neural crest development is regulated by the transcription factor Sox9. Development 2003;130: 5681-5693. 8. Spokony RF, Aoki Y, Saint-Germain N, Magner-Fink E, Saint-Jeannet JP. The transcription factor Sox9 is required for cranial neutral crest. Development 2001;129:421-432. 9. Bi W, Huang W, Whitworth DJ, Min DJ, Zhang Z, Behringer RR. Haploinsufficiency of Sox9 results in defective cartilage primordial and premature skeletal mineralization. Proc Natl Acad Sci USA 2001;98:66986703. 10. Bell DM, Leung KK, Wheatley SC, Ng LJ, Zhou S, Ling K, Sham W, Koopman P, Tam PP, Cheah KS. Sox9 directly regulate the type two collagen gene. Nat Genet 1997;16: 174-178. 11. Aigner T, Gebhard PM, Schmid E, Bau B, Harley V, Poschl E. Sox9 expression does not correlate with type two collagen expression in adult articular chondrocytes. Matrix Biol 2003;22:363-372. 12. Ikeda T, Kawaguchi S, Kamekura S. Distinct roles of Sox5. Sox6, and Sox9 in differentiation of chondrogenic differentiation. J Bone Miner Metab 2005;23:337-340. 13. Kulyk WM, Franklin JL, Hoffman LM. Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme. Exp Cell Res 2000;255:327-332. 14. Gilson R, Mcculloch CA, Zohar R. Stromal mesenchymal progenitor cells. Leuko Lymphoma 1999;32:211-221.
Asian J Oral Maxillofac Surg Vol 18, No 1, 2006