Multiplexed tape-stabilized cryohistology of mineralized large animal specimens

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Hannah M. Zlotnick
Xi Jiang
Robert L. Mauck
Nathaniel A. Dyment


mineralization, bone, cryohistology, large animal, tissue interface


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.


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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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. PMID:7654463