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：

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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, 鄧力, 羅靜聰, 等. 硬組織切片技術在組織工程骨研究中的應用. 中國修復重建外科雜志, 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, 李曉琴, 張輝, 等. 塑料包埋結合四環素熒光標記制作不脫鈣骨組織切片的實驗研究. 臨床與實驗病理學雜志, 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.	Hammarstr?m 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.	楊戈爾. 低溫冷凍過程中胞內冰晶生長機制與應用研究. 上海:上海交通大學, 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, 周瑩, 龍飛. 不同組織冰凍切片的制作方法. 江西醫學院學報, 2006, 46(4):13.

1. 李秀群, 鄧力, 羅靜聰, 等. 硬組織切片技術在組織工程骨研究中的應用. 中國修復重建外科雜志, 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, 李曉琴, 張輝, 等. 塑料包埋結合四環素熒光標記制作不脫鈣骨組織切片的實驗研究. 臨床與實驗病理學雜志, 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. Hammarstr?m 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. 楊戈爾. 低溫冷凍過程中胞內冰晶生長機制與應用研究. 上海:上海交通大學, 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, 周瑩, 龍飛. 不同組織冰凍切片的制作方法. 江西醫學院學報, 2006, 46(4):13.

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Chinese Journal of Reparative and Reconstructive Surgery

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

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