A Chick Embryo in-Vitro Model of Knee Morphogenesis

Document Type: RESEARCH PAPER

Authors

1 Orthopedic Trauma Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA

2 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, USA

Abstract

 
 Background: In this feasibility study, a mechanically loaded in-vitro tissue culture model of joint morphogenesis using the isolated lower extremity of the 8 day old chick embryo was developed to assess the effects of mechanical loading on joint morphogenesis.
Methods: The developed in-vitro system allows controlled flexion and extension of the chick embryonic knee with a range of motion of 20 degrees from a resting position of 90-100 degrees of flexion. Joint morphogenesis at 2, 3, 4 and 7 days of culture was assessed by histology and micro MRI in 4 specimen types: undisturbed in-ovo control embryos, in-ovo paralyzed embryos, in-vitro unloaded limb cultures, and in-vitro loaded limb cultures. Relative glycosaminoglycan (GAG) concentration across the joint was assessed with an MRI technique referred to as dGEMRIC (delayed gadolinium enhanced MRI of cartilage) where T1 is proportional to glycosaminoglycan concentration.
Results: Average T1 over the entire tissue image for the normal control (IC) knee was 480 msec; for the 4 day loaded specimen average T1 was 354 msec; and for the 7 day loaded specimens T1 was 393 msec. The 4 day unloaded specimen had an average T1 of 279 msec while the 7 day unloaded specimen had an average T1 of 224 msec. The higher T1 values in loaded than unloaded specimens suggest that more glycosaminoglycan is produced in the loaded culture than in the unloaded preparation.
Conclusion: Isolated limb tissue cultures under flexion-extension load can be viable and exhibit more progression of joint differentiation and glycosaminoglycan production than similarly cultured but unloaded specimens. However, when compared with controls consisting of intact undisturbed embryos in-ovo , the isolated loaded limbs in culture do not demonstrate equivalent amounts of absolute growth or joint differentiation.

Keywords


  1. Fell HB. The histogenesis of cartilage and bone in the long bones of the embryonic fowl. J Morphol. 1925; 40(3):417-59.
  2. Fell HB. Experiments in the differentiation in vitro of cartilage and bone. Part 1 Arch Exp Zellforsh. 1928; 18(7):390-412.
  3. Fell HB, Canti RG. Experiments on the development in vitro of the avian knee-joint. Proc R Soc Lond B Biol Sci. 1934; 116(799):316-51.
  4. Ruano-Gil D, Nardi-Vilardaga J, Tejedo-Mateu A. Influence of extrinsic factors on the development of the articular system. Acta Anat. 1978; 101(1):36-44.
  5. Ruano-Gil D, Nardi-Vilardaga J, Teixidor-Johe A. Embryonal hypermobility and articular development. Acta Anat. 1985; 123(2):90-2.
  6. Hosseini A, Hogg DA. The effects of paralysis on skeletal development in the chick embryo. I. General effects. J Anat. 1991; 177(9):159-68.
  7. Drachman DB, Sokoloff L. The role of movement in embryonic joint development. Dev Biol. 1966; 14(3):401-20.
  8. Drachman DB, Weiner LP, Price DL, Chase J. Experimental arthrogryposis caused by viral myopathy. Arch Neurol. 1976; 33(5):362-7.
  9. Mikic B, Johnson TL, Chhabra AB, Schalet BJ, Wong M, Hunsiker EB. Differential effects of embryonic immobilization on the development of fibrocartilagenous skeletal elements. J Rehabil Res Dev. 2000; 37(2):127-33.
  10. Pitsillides AA. Early effects of embryonic movement: ‘a shot out of the dark’. J Anat. 2006; 208(4):417-31.
  11. Roddy KA, Kelly GM, Van Es MH, Murphy P, Prendergast PJ. Dynamic patterns of mechanical stimulation co-localise with growth and cell proliferation during morphogenesis in the avian embryonic knee joint. J Biomech. 2011; 44(1):143-9.
  12. Nowlan NC, Chandaria V, Sharpe J. Immobilized chicks as a model system for early-onset developmental dysplasia of the hip. J Orthop Res. 2014; 32(6):777-85.
  13. Nowlan NC, Sharpe J. Joint shape morphogenesis precedes cavitation of the developing hip joint. J Anat. 2014; 224(4):482-9.
  14. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951; 88(1):49-92.
  15. Hogg DA, Hosseini A. The effects of paralysis on skeletal development in the chick embryo. Comp Biochem Physiol Comp Physiol. 1992; 103(1):25-8.
  16. Llusa-Perez M, Susu-Vergara S, Ruano-Gil D. Recording of chick embryo movements and their correlation with joint development. Acta Anat. 1988; 132(1):55-8.
  17. Allen RG, Burstein D, Gray ML. Monitoring glycosaminoglycan replenishment in cartilage explants with gadolinium-enhanced magnetic resonance imaging. J Orthop Res. 1999; 17(3):430-6.
  18. Murray PD, Drachman DB. The role of movement in the development of joints and related structures: the head and neck in the chick embryo. J Embryol Exp Morph. 1969; 22(3):349-71.
  19. Rajan KT, Merker HJ. Preceedings: Joint formation in culture. Ann Rheum Dis. 1975; 34(2):200.
  20. Mitrovic D. Development of the articular cavity in paralyzed chick embryos and in the chick embryo limb buds cultured on chorioallantoic membranes. Acta Anat. 1982; 113(4):313-24.
  21. Watson SJ, Bekoff A. A kinematic analysis of hindlimb motility in 9- and 10-day old chick embryos. J Neurobiol. 1990; 21(4):651-60.