FLUORESCENCE DISTRIBUTION IN BONE AND CARTILAGE TISSUE BY SECTIONING OF FROZEN UNDECALCIFIED BONE_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SUNZhen ¹ , YINHeyong ² , SUIXiang ¹ , YUXiaoming ¹ ,  PENGJiang ¹ , WANGYu ¹ , WANGAiyuan ¹ , GUOQuanyi ¹ , LIUShuyun ¹ , MENGHaoye ¹ , LUShibi ¹

1. Institute of Orthopedics, General Hospital of Chinese PLA, Beijing, 100853, P. R. China;
2. Medical College of Nankai University;

Corresponding author：

PENGJiang, Email: pengjiang301@126.com

Keywords：

Frozen sections; Undecalcified bone tissue; Immunofluorescence; Rat

DOI：

10.7507/1002-1892.20150306

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To introduce a technique of frozen sections for undecalcified bone and discuss its feasibility by observing the fluorescence distribution of the bone and cartilage. Methods The male Sprague Dawley transgenic rats at the age of 8 weeks, which express green fluorescent protein were selected to isolate the whole knee sectioned by the undecalcified bone frozen section technique. Under the fluorescence and light microscopy, the fluorescence and structure were observed within the organization of slice. Immunohistochemical staining (collagen type Ⅰ and Ⅱ), HE staining, toluidine blue staining, and Alizarin red staining were performed to observe the distribution of fluorescent substance and cartilage and bone structure. Results The thickness of sections prepared by this technology was 6 μm. General observation showed that the structure of sectioned joint was complete. Under the light microscope, the morphology of cartilage cells, the arrangement of subchondral bone, and trabecular bone traveling could be clearly distinguished. Under fluorescence microscope, green fluorescence was shown in the joint soft tissue, cartilage tissue, and bone tissue; collagen type Ⅰ expressed in the bone tissue, collagen type Ⅱ in cartilage tissue. HE staining and toluidine blue staining could clearly distinguish the morphology of the cartilage layer. Alizarin red staining showed the structural integrity of subchondral bone plate and the organization within the meniscus, and proximal tibia cortical bone continuity. Conclusion The fluorescence distribution can be directly observe in the bone and cartilage by sectioning of frozen undecalcified bone. This new technology can shorten the cycle of preparing sections.

Citation： SUNZhen, YINHeyong, SUIXiang, YUXiaoming, PENGJiang, WANGYu, WANGAiyuan, GUOQuanyi, LIUShuyun, MENGHaoye, LUShibi. FLUORESCENCE DISTRIBUTION IN BONE AND CARTILAGE TISSUE BY SECTIONING OF FROZEN UNDECALCIFIED BONE. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(11): 1429-1433. doi: 10.7507/1002-1892.20150306 Copy

1.	李秀群, 邓力, 罗静聪, 等. 硬组织切片技术在组织工程骨研究中的应用. 中国修复重建外科杂志, 2004, 18(3):239-240.
2.	Xu QC, Hao PJ, Yu XB, et al. Hyperlipidemia compromises homing efficiency of systemically transplanted BMSCs and inhibits bone regeneration. Int J Clin Exp Pathol, 2014, 7(4):1580-1587.
3.	Eseonu OI, De Bari C. Homing of mesenchymal stem cells:mechanistic or stochastic? Implications for targeted delivery in arthritis. Rheumatology (Oxford), 2015, 54(2):210-218.
4.	Bouvet-Gerbettaz S, Boukhechba F, Balaguer T, et al. Adaptive immune response inhibits ectopic mature bone formation induced by BMSCs/BCP/plasma composite in immune-competent mice. Tissue Eng Part A, 2014, 20(21-22):2950-2962.
5.	王勇平, 李晓琴, 张辉, 等. 塑料包埋结合四环素荧光标记制作不脱钙骨组织切片的实验研究. 临床与实验病理学杂志, 2012, 28(5):543-545.
6.	Van den Munckhof RJ, Vreeling-Sindelárová H, Schellens JP, et al. Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver. J Histochem Cytochem, 1994, 42(2):177-183.
7.	Fukase Y. Immunohistochemical staining of human teeth, and production of monoclonal antibodies against cementum. J Osaka Dent Univ, 1997, 31(1-2):1-9.
8.	Hill EL, Elde R. An improved method for preparing cryostat sections of undecalcified bone for multiple uses. J Histochem Cytochem, 1990, 38(3):443-448.
9.	Nakamura Y, Tanaka T, Wakimoto Y, et al. Preparation of unfixed and undecalcified frozen sections of adult rat periodontal ligament during experimental tooth movement. Biotech Histochem, 1994, 69(4):186-191.
10.	Kawamoto T, Shimizu M. A method for preparing 2-to 50-micronthick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem Cell Biol, 2000, 113(5):331-339.
11.	Itoh M, Kawamoto T, Tatsukawa H, et al. In situ detection of active transglutaminases for keratinocyte type (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides. J Histochem Cytochem, 2011, 59(2):180-187.
12.	Hata M, Kawamoto T, Kawai M, et al. Differential expression patterns of the tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor. Med Mol Morphol, 2010, 43(2):102-106.
13.	Hammarström L. Autoradiography and histochemistry of mineralized tissues by means of the Ullberg freeze-sectioning technique. Ups J Med Sci, 1986, 91(3):239-243.
14.	Kawamoto T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, wholeanimals, insects and plants. Arch Histol Cytol, 2003, 66(2):123-143.
15.	Aaron JE, Carter DH. Rapid preparation of fresh-frozen undecalcified bone for histological and histochemical analysis. J Histochem Cytochem, 1987, 35(3):361-369.
16.	McElroy HH, Shih MS, Parfitt AM. Producing frozen sections of calcified bone. Biotech Histochem, 1993, 68(1):50-55.
17.	Kawamoto T, Kawamoto K. Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamot's film method (2012). Methods Mol Biol, 2014, 1130:149-164.
18.	杨戈尔. 低温冷冻过程中胞内冰晶生长机制与应用研究. 上海:上海交通大学, 2011.
19.	Haines JW. A technique for embedding undecalcified bone samples for detecting alpha-emitters using vacuum impregnation with Spurr's resin. Biotech Histochem, 1992, 67(1):45-49.
20.	章克萍, 周莹, 龙飞. 不同组织冰冻切片的制作方法. 江西医学院学报, 2006, 46(4):13.

1. 李秀群, 邓力, 罗静聪, 等. 硬组织切片技术在组织工程骨研究中的应用. 中国修复重建外科杂志, 2004, 18(3):239-240.
2. Xu QC, Hao PJ, Yu XB, et al. Hyperlipidemia compromises homing efficiency of systemically transplanted BMSCs and inhibits bone regeneration. Int J Clin Exp Pathol, 2014, 7(4):1580-1587.
3. Eseonu OI, De Bari C. Homing of mesenchymal stem cells:mechanistic or stochastic? Implications for targeted delivery in arthritis. Rheumatology (Oxford), 2015, 54(2):210-218.
4. Bouvet-Gerbettaz S, Boukhechba F, Balaguer T, et al. Adaptive immune response inhibits ectopic mature bone formation induced by BMSCs/BCP/plasma composite in immune-competent mice. Tissue Eng Part A, 2014, 20(21-22):2950-2962.
5. 王勇平, 李晓琴, 张辉, 等. 塑料包埋结合四环素荧光标记制作不脱钙骨组织切片的实验研究. 临床与实验病理学杂志, 2012, 28(5):543-545.
6. Van den Munckhof RJ, Vreeling-Sindelárová H, Schellens JP, et al. Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver. J Histochem Cytochem, 1994, 42(2):177-183.
7. Fukase Y. Immunohistochemical staining of human teeth, and production of monoclonal antibodies against cementum. J Osaka Dent Univ, 1997, 31(1-2):1-9.
8. Hill EL, Elde R. An improved method for preparing cryostat sections of undecalcified bone for multiple uses. J Histochem Cytochem, 1990, 38(3):443-448.
9. Nakamura Y, Tanaka T, Wakimoto Y, et al. Preparation of unfixed and undecalcified frozen sections of adult rat periodontal ligament during experimental tooth movement. Biotech Histochem, 1994, 69(4):186-191.
10. Kawamoto T, Shimizu M. A method for preparing 2-to 50-micronthick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem Cell Biol, 2000, 113(5):331-339.
11. Itoh M, Kawamoto T, Tatsukawa H, et al. In situ detection of active transglutaminases for keratinocyte type (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides. J Histochem Cytochem, 2011, 59(2):180-187.
12. Hata M, Kawamoto T, Kawai M, et al. Differential expression patterns of the tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor. Med Mol Morphol, 2010, 43(2):102-106.
13. Hammarström L. Autoradiography and histochemistry of mineralized tissues by means of the Ullberg freeze-sectioning technique. Ups J Med Sci, 1986, 91(3):239-243.
14. Kawamoto T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, wholeanimals, insects and plants. Arch Histol Cytol, 2003, 66(2):123-143.
15. Aaron JE, Carter DH. Rapid preparation of fresh-frozen undecalcified bone for histological and histochemical analysis. J Histochem Cytochem, 1987, 35(3):361-369.
16. McElroy HH, Shih MS, Parfitt AM. Producing frozen sections of calcified bone. Biotech Histochem, 1993, 68(1):50-55.
17. Kawamoto T, Kawamoto K. Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamot's film method (2012). Methods Mol Biol, 2014, 1130:149-164.
18. 杨戈尔. 低温冷冻过程中胞内冰晶生长机制与应用研究. 上海:上海交通大学, 2011.
19. Haines JW. A technique for embedding undecalcified bone samples for detecting alpha-emitters using vacuum impregnation with Spurr's resin. Biotech Histochem, 1992, 67(1):45-49.
20. 章克萍, 周莹, 龙飞. 不同组织冰冻切片的制作方法. 江西医学院学报, 2006, 46(4):13.

Previous Article
EPIDERMAI GROWTH FACTOR SECRETED BY ASTROCYTES STIMULATED WITH TACROLIMUS PROMOTING NEURITE OUTGROWTH
Next Article
RESEARCH PROGRESS OF KLIPPEL-FEIL SYNDROME WITH EAR MALFORMATION

Chinese Journal of Reparative and Reconstructive Surgery

FLUORESCENCE DISTRIBUTION IN BONE AND CARTILAGE TISSUE BY SECTIONING OF FROZEN UNDECALCIFIED BONE

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content