Multiplexed tape-stabilized cryohistology of mineralized large animal specimens

² McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

³ Translational Musculoskeletal Research Center, Corporal Michael J Crescenz VA Medical Center, Philadelphia, PA 19104, USA

JBM 2022 , 9(4), 1;

Published: 17 November 2022

© 2022 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )

Download PDF

Cite

XML

Abstract

Tape-stabilized cryohistology is a powerful histological method to reinforce tissue samples during and after sectioning, enhancing the overall image quality. This technique has widely been applied to section mineralized small animal (i.e., mice, rat, rabbit) specimens, but has only been sparsely implemented for large animal samples that have a greater tendency to tear due to their increased surface area. Here, we present an optimized protocol for tape-stabilized cryohistology of undecalcified minipig vertebral body, femoral head, and temporomandibular joint samples. This protocol further develops a pipeline for sequential staining and imaging of the tape-stabilized cryosections. Images from multiple rounds of staining (endogenous bone mineral labels, aligned collagen (polarized light), tartrate resistant phosphatase (TRAP), alkaline phosphatase (AP), and toluidine blue) are overlaid to provide insight into dynamic bone remodeling. Overall, the established multiplexed tape-stabilized cryohistology protocol provides step-by-step instructions and guidance to cryosection large, mineralized tissues, and maximize data output from a single histological section.

Keywords

mineralization

bone

cryohistology

large animal

tissue interface

References

1. Jiang X, Kalajzic Z, Maye P, Braut A, Bellizzi J, Mina M, et al. Histological analysis of GFP expression in murine bone. J Histochem Cytochem. 2005 May;53(5):593–602. https://doi.org/10.1369/jhc.4A6401.2005 PMID:15872052
2. Kawamoto T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol. 2003 May;66(2):123–43. https://doi.org/10.1679/aohc.66.123 PMID:12846553
3. Dyment NA, Jiang X, Chen L, Hong SH, Adams DJ, Ackert-Bicknell C, et al. High-throughput, multi-image cryohistology of mineralized tissues. J Vis Exp. 2016 Sep;2016(115):1–11. https://doi.org/10.3791/54468 PMID:27684089
4. Hong SH, Jiang X, Chen L, Josh P, Shin DG, Rowe D. Computer-Automated Static, Dynamic and Cellular Bone Histomorphometry. J Tissue Sci Eng. 2012 Dec;Suppl 1:004. https://doi.org/10.4172/2157-7552.S1-004 PMID: 25019033
5. Xin X, Jiang X, Wang L, Mikael P, McCarthy MB, Chen L, et al. Histological Criteria that Distinguish Human and Mouse Bone Formed Within a Mouse Skeletal Repair Defect. J Histochem Cytochem. 2019 Jun;67(6):401–17. https://doi.org/10.1369/0022155419836436 PMID:30848692
6. Kamalitdinov TB, Fujino K, Shetye SS, Jiang X, Ye Y, Rodriguez AB, et al. Amplifying Bone Marrow Progenitors Expressing α-Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon-to-Bone Repair. J Orthop Res. 2020 Jan;38(1):105–16. https://doi.org/10.1002/jor.24395 PMID:31228280
7. Hagiwara Y, Dyrna F, Kuntz AF, Adams DJ, Dyment NA. Cells from a GDF5 origin produce zonal tendon-to-bone attachments following anterior cruciate ligament reconstruction. Ann N Y Acad Sci. 2020 Jan;1460(1):57–67. https://doi.org/10.1111/nyas.14250 PMID:31596513
8. Tsinman TK, Jiang X, Han L, Koyama E, Mauck RL, Dyment NA. Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly. FASEB J. 2021 Aug;35(8):e21779. https://doi.org/10.1096/fj.202100499R PMID:34314047
9. Wei Y, Sun H, Gui T, Yao L, Zhong L, Yu W, et al. The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair. Elife. 2021 Jun;10:e62917. https://doi.org/10.7554/eLife.62917 PMID:34085927
10. Killian ML, Cavinatto L, Shah SA, Sato EJ, Ward SR, Havlioglu N, et al. The effects of chronic unloading and gap formation on tendon-to-bone healing in a rat model of massive rotator cuff tears. J Orthop Res. 2014 Mar;32(3):439–47. https://doi.org/10.1002/jor.22519 PMID:24243733
11. Fisher MB, Belkin NS, Milby AH, Henning EA, Söegaard N, Kim M, et al. Effects of Mesenchymal Stem Cell and Growth Factor Delivery on Cartilage Repair in a Mini-Pig Model. Cartilage. 2016 Apr;7(2):174–84. https://doi.org/10.1177/1947603515623030 PMID:27047640
12. Fortier LA, Chapman HS, Pownder SL, Roller BL, Cross JA, Cook JL, et al. BioCartilage improves cartilage repair compared with microfracture alone in an equine model of full-thickness cartilage loss. Am J Sports Med. 2016 Sep;44(9):2366–74. https://doi.org/10.1177/0363546516648644 PMID:27298478
13. Ashinsky BG, Gullbrand SE, Wang C, Bonnevie ED, Han L, Mauck RL, et al. Degeneration alters structure-function relationships at multiple length-scales and across interfaces in human intervertebral discs. J Anat. 2021 Apr;238(4):986–98. https://doi.org/10.1111/joa.13349 PMID:33205444
14. Zlotnick HM, Locke RC, Stoeckl BD, Patel JM, Gupta S, Browne KD, et al. Marked differences in local bone remodelling in response to different marrow stimulation techniques in a large animal. Eur Cell Mater. 2021 May;41:546–57. https://doi.org/10.22203/eCM.v041a35 PMID:34008855
15. Zlotnick HM, Locke RC, Hemdev S, Stoeckl BD, Gupta S, Peredo AP, et al. Gravity-based patterning of osteogenic factors to preserve bone structure after osteochondral injury in a large animal model. Biofabrication. 2022 Jul;14(4):044101. https://doi.org/10.1088/1758-5090/ac79cd PMID:35714576
16. Pfeifer CG, Fisher MB, Carey JL, Mauck RL. Impact of guidance documents on translational large animal studies of cartilage repair. Sci Transl Med. 2015 Oct;7(310):310re9. https://doi.org/10.1126/scitranslmed.aac7019 PMID:26491080
17. van Gaalen SM, Kruyt MC, Geuze RE, de Bruijn JD, Alblas J, Dhert WJ. Use of fluorochrome labels in in vivo bone tissue engineering research. Tissue Eng Part B Rev. 2010 Apr;16(2):209–17. https://doi.org/10.1089/ten.teb.2009.0503 PMID:19857045
18. Huja SS, Fernandez SA, Hill KJ, Li Y. Remodeling dynamics in the alveolar process in skeletally mature dogs. Anat Rec A Discov Mol Cell Evol Biol. 2006 Dec;288(12):1243–9. https://doi.org/10.1002/ar.a.20396 PMID:17075846
19. Bradbeer JN, Zanelli JM, Lindsay PC, Pearson J, Reeve J. Relationship between the location of osteoblastic alkaline phosphatase activity and bone formation in human iliac crest bone. J Bone Miner Res. 1992 Aug;7(8):905–12. https://doi.org/10.1002/jbmr.5650070807 PMID:1442204
20. Ballanti P, Coen G, Taggi F, Mazzaferro S, Perruzza I, Bonucci E. Extent of alkaline phosphatase cytochemistry vs. extent of tetracycline fluorescence in the evaluation of histodynamic variables of bone formation. Bone. 1995 May;16(5):493–8. https://doi.org/10.1016/8756-3282(95)00079-S PMID:7654463

Previous article in this issue

Next article in this issue

Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TAB, Published by POL Scientific

Publisher's Core Philosophy

We are committed to support the scientific community by publishing impactful research and enhancing communication among scientists. At POL Scientific, we are continuously looking for ways to accelerate scientific progress and to strive for transparency and open communication, making knowledge freely accessible without barrier.

171 Skyview St. San Francisco, CA 94131 U.S.A.

info@polscientific.com