Three-Dimensional Imaging and Quantitative Analysis of Blood Vessel Distribution in The Meniscus of Transgenic Mouse after Tissue Clearing

Document Type : Original Article


1 Sports Medicine Institute of Fudan University, Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai, China

2 Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Institutes of Brain Science, Brain Science Collaborative Innovation Centre, State Key Laboratory of Medical Neurobiology, Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine, Fudan University, Shanghai, China

3 China Shanghai Key Laboratory of Acupuncture Mechanism and Acupoint Function, Shanghai, China


Objective: Blood supply to the meniscus determines its recovery and is a reference for treatment planning. This study
aimed to apply tissue clearing and three-dimensional (3D) imaging in exploring the quantitative distribution of blood
vessels in the mouse meniscus.
Materials and Methods: In this experimental study, tissue clearing was performed to treat the bilateral knee joints of
transgenic mice with fluorescent vascular endothelial cells. Images were acquired using a light sheet microscope and
the vascular endothelial cells in the meniscus was analysed using 3D imaging. Quantitative methods were employed
to further analyse the blood vessel distribution in the mouse meniscus.
Results: The traditional three-equal-width division of the meniscus is as follows: the outer one-third is the red-red zone
(RR), the inner one-third is the white-white zone (WW), and the transition area is the red-white zone (RW). The division
revealed significant signal differences between the RW and WW (P<0.05) zones, but no significant differences between
the RR and RW zones, which indicated that the division might not accurately reflect the blood supply of the meniscus.
According to the modified division (4:2:1) in which significant differences were ensured between the adjacent zones,
we observed that the width ratio of each zone was 38 ± 1% (RR), 24 ± 1% (RW), and 38 ± 2% (WW). Furthermore, the
blood supply to each region was verified. The anterior region had the most abundant blood supply. The fluorescence
count in the anterior region was significantly higher than in the central and posterior regions (P<0.05). The blood supply
of the medial meniscus was superior to the lateral meniscus (P<0.05).
Conclusion: Analysis of the blood supply to the mouse meniscus under tissue clearing and 3D imaging reflect
quantitative blood vessel distribution, which would facilitate future evaluations of the human meniscus and provide
more anatomical references for clinicians.


Main Subjects

  1. Hutchinson ID, Moran CJ, Potter HG, Warren RF, Rodeo SA. Restoration of the meniscus: form and function. Am J Sports Med. 2014; 42(4): 987-998.
  2. Tanaka T, Fujii K, Kumagae Y. Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus. Knee Surg Sports Traumatol Arthrosc. 1999; 7(2): 75-80.
  3. Chambers HG, Chambers RC. The natural history of meniscus tears. J Pediatr Orthop. 2019; 39 (6 Suppl 1): S53-S55.
  4. Hidaka C, Ibarra C, Hannafin JA, Torzilli PA, Quitoriano M, Jen SS, et al. Formation of vascularized meniscal tissue by combining gene therapy with tissue engineering. Tissue Eng. 2002; 8(1): 93-105.
  5. Kobayashi K, Fujimoto E, Deie M, Sumen Y, Ikuta Y, Ochi M. Regional differences in the healing potential of the meniscus-an organ culture model to eliminate the influence of microvasculature and the synovium. Knee. 2004; 11(4): 271-278.
  6. Uchida R, Horibe S, Shiozaki Y, Shino K. All-inside suture repair for isolated radial tears at the midbody of the lateral meniscus. Arthrosc Tech. 2019; 8(12): e1451-e1456.
  7. Fox A J, Bedi A, Rodeo SA. The basic science of human knee menisci: structure, composition, and function. Sports Health. 2012; 4(4): 340-351.
  8. Fox AJ, Wanivenhaus F, Burge AJ, Warren RF, Rodeo SA. The human meniscus: a review of anatomy, function, injury, and advances in treatment. Clin Anat. 2015; 28(2): 269-287.
  9. Day B, Mackenzie WG, Shim SS, Leung G. The vascular and nerve supply of the human meniscus. Arthroscopy. 1985; 1(1): 58-62.
  10. Longo UG, Campi S, Romeo G, Spiezia F, Maffulli N, Denaro V.Biological strategies to enhance healing of the avascular area of the meniscus. Stem Cells Int. 2012; 2012: 528359.
  11. Michel PA, Domnick CJ, Raschke MJ, Hoffmann A, Kittl C, Herbst E, et al. Age-related changes in the microvascular density of the human meniscus. Am J Sports Med. 2021; 49(13): 3544-3550.
  12. Beaufils P, Pujol N. Management of traumatic meniscal tear and degenerative meniscal lesions. Save the meniscus. Orthop Traumatol Surg Res. 2017; 103(8S): S237-S244.
  13. Aman ZS, Dickens JF, Dekker TJ. Meniscal repair techniques for middle- and posterior-third tears. Arthroscopy. 2021; 37(3): 792- 794.
  14. Lubowitz JH, Brand JC, Rossi MJ. Nonoperative management of degenerative meniscus tears is worth a try. Arthroscopy. 2020; 36(2): 327-328.
  15. Stärke C, Kopf S, Petersen W, Becker R. Meniscal repair. Arthroscopy. 2009; 25(9): 1033-1044.
  16. Hevesi M, Krych AJ, Kurzweil PR. Meniscus tear management: indications, technique, and outcomes. Arthroscopy. 2019; 35(9): 2542-2544.
  17. Kopf S, Beaufils P, Hirschmann MT, Rotigliano N, Ollivier M, Pereira H, et al. Management of traumatic meniscus tears: the 2019 ESSKA meniscus consensus. Knee Surg Sports Traumatol Arthrosc. 2020; 28(4): 1177-1194.
  18. Takata Y, Nakase J, Shimozaki K, Asai K, Tsuchiya H. Autologous adipose-derived stem cell sheet has meniscus regeneration-promoting effects in a rabbit model. Arthroscopy. 2020; 36(10): 2698- 2707.
  19. Moatti A, Cai Y, Li C, Sattler T, Edwards L, Piedrahita J, et al. Threedimensional imaging of intact porcine cochlea using tissue clearing and custom-built light-sheet microscopy. Biomed Opt Express. 2020; 11(11): 6181-6196.
  20. Arnoczky SP, Warren RF. Microvasculature of the human meniscus. Am J Sports Med. 1982; 10(2): 90-95.
  21. Arnoczky SP, Warren RF. The microvasculature of the meniscus and its response to injury. An experimental study in the dog. Am J Sports Med. 1983; 11(3): 131-141.
  22. Crawford MD, Hellwinkel JE, Aman Z, Akamefula R, Singleton JT, Bahney C, et al. Microvascular anatomy and intrinsic gene expression of menisci from young adults. Am J Sports Med. 2020; 48(13): 3147-3153.
  23. Luo W, Yi Y, Jing D, Zhang S, Men Y, Ge WP, et al. Investigation of postnatal craniofacial bone development with tissue clearingbased three-dimensional imaging. Stem Cells Dev. 2019; 28(19): 1310-1321.
  24. Broothaerts W, Cordeiro F, Corbisier P, Robouch P, Emons H. Log transformation of proficiency testing data on the content of genetically modified organisms in food and feed samples: is it justified? Anal Bioanal Chem. 2020; 412(5): 1129-1136.
  25. Feng C, Wang H, Lu N, Tu XM. Log transformation: application and interpretation in biomedical research. Stat Med. 2013; 32(2): 230-239.
  26. Beaufils P, Becker R, Kopf S, Englund M, Verdonk R, Ollivier M, et al. Surgical management of degenerative meniscus lesions: the 2016 ESSKA meniscus consensus. Knee Surg Sports Traumatol Arthrosc. 2017; 25(2): 335-346.
  27. Englund M, Roemer FW, Hayashi D, Crema MD, Guermazi A. Meniscus pathology, osteoarthritis, and the treatment controversy. Nat Rev Rheumatol. 2012; 8(7): 412-419.
  28. Krych AJ, Hevesi M, Leland DP, Stuart MJ. Meniscal root injuries. J Am Acad Orthop Surg. 2020; 28(12): 491-499.
  29. Cinque ME, DePhillipo NN, Moatshe G, Chahla J, Kennedy MI, Dornan GJ, et al. Clinical outcomes of inside-out meniscal repair according to anatomic zone of the meniscal tear. Orthop J Sports Med. 2019; 7(7): 2325967119860806.
  30. Jacob G, Shimomura K, Krych AJ, Nakamura N. The meniscus tear: a review of stem cell therapies. Cells. 2019; 9(1): 92.
  31. Barber-Westin SD, Noyes FR. Clinical healing rates of meniscus repairs of tears in the central-third (red-white) zone. Arthroscopy. 2014; 30(1): 134-146.
  32. Noyes FR, Barber-Westin SD. Repair of complex and avascular meniscal tears and meniscal transplantation. J Bone Joint Surg Am. 2010; 92(4): 1012-1029.
  33. Hennerbichler A, Moutos FT, Hennerbichler D, Weinberg JB, Guilak F. Repair response of the inner and outer regions of the porcine meniscus in vitro. Am J Sports Med. 2007; 35(5): 754-762.
  34. Clark CR, Ogden JA. Development of the menisci of the human knee joint. Morphological changes and their potential role in childhood meniscal injury. J Bone Joint Surg Am. 1983; 65(4): 538-547.
  35. Petersen W, Tillmann B. Age-related blood and lymph supply of the knee menisci. A cadaver study. Acta Orthop Scand. 1995; 66(4): 308-312.
  36. Williams LB, Adesida AB. Angiogenic approaches to meniscal healing. Injury. 2018; 49(3): 467-472.
  37. Ribera J, Pauta M, Melgar-Lesmes P, Córdoba B, Bosch A, Calvo M, et al. A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury. Am J Physiol Gastrointest Liver Physiol. 2017; 313(5): G492-G504.
  38. Chahla J, Papalamprou A, Chan V, Arabi Y, Salehi K, Nelson TJ, et al. Assessing the resident progenitor cell population and the vascularity of the adult human meniscus. Arthroscopy. 2021; 37(1): 252-265.
  39. Krych AJ, Bernard CD, Kennedy NI, Tagliero AJ, Camp CL, Levy BA, et al. Medial versus lateral meniscus root tears: is there a difference in injury presentation, treatment decisions, and surgical repair outcomes? Arthroscopy. 2020; 36(4): 1135-1141.
  40. Hohmann E. Editorial commentary: discovery: progenitor cells and endothelial cells are found in the white-white zone of the meniscus, but this does not mean that these tears heal or should be repaired. Arthroscopy. 2021; 37(1): 266-267.