Equine Veterinary Journal ISSN 0425-1644 DOI: 10.1111/evj.12321
Short Communications
The embryogenesis of the equine femorotibial joint: The equine interzone F. JENNER*, G. J. V. M. VAN OSCH†, W. WENINGER‡, S. GEYER‡, T. STOUT§, R. VAN WEEREN§ and P. BRAMA¶ Equine Hospital, University of Veterinary Medicine Vienna, Austria † Department of Orthopaedics and Department of Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam, The Netherlands ‡ Center for Anatomy and Cell Biology, Medical University of Vienna, Austria § Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands ¶ Section Veterinary Clinical Sciences, School of Veterinary Medicine, University College Dublin, Ireland. *Correspondence email: fl[email protected]; Received: 27.05.14; Accepted: 10.07.14
Summary Reasons for performing study: Articular cartilage regeneration is the focus and goal of considerable research effort. Since articular chondrocytes descend from a distinct cohort of progenitor cells located in embryonic nascent joints (interzones), establishing the timing of equine interzone formation is an essential first step towards understanding equine joint and articular cartilage development. Objectives: To establish the time frame during which the equine femorotibial interzone forms. Study design: Descriptive anatomical study. Methods: Equine embryos were harvested at 37 (E37), 40, 42, 45, 50 and 65 days’ gestation. The femorotibial interzone was examined using high-resolution episcopic microscopy of E37, E42, E45, E50 and E65. Additional histology and collagen-II-immunohistochemistry were performed on E42. Results: At E37, the femorotibial interzone is first visible as a uniform layer, while at E42 the interzone is fully formed and consists of 3 morphologically distinct layers. The first evidence of cavitation was seen at E45. At E50, the cruciate ligaments were well formed and by E65, joint formation appeared complete. Conclusions: The embryogenesis of the equine femorotibial joint is similar to the developmental timeline of stage-matched human and murine embryos. Further studies looking at interzone formation on a cellular and molecular level may further our understanding of the intricate developmental patterns and pathways of articular cartilage development. Keywords: horse; joint development; embryo; articular cartilage; embryogenesis; interzone
Introduction The interzone has a pivotal role in joint formation demonstrated by joint ablation when surgically removed in embryos [1,2]. Lineage tracing experiments have demonstrated that interzone cells form all articular structures such as articular cartilage, ligaments and synovial lining [2–5]. Despite the high incidence of cartilage and joint disease in horses and the popularity of horses as research animals for osteoarthritis and cartilage tissue engineering, there are no studies of the equine interzone and the timing of interzone formation is not known. Using magnetic resonance microscopy in a companion study, we narrowed down the time frame during which interzone formation probably occurs to gestational Days 35–50 [6]. In the current study, we performed high-resolution episcopic microscopy (HREM) [7], histology and immunohistochemistry (IHC) to determine when the femorotibial interzone forms in the horse. While this study is descriptive, it provides a first insight into the development of equine joints and the related development of articular cartilage. It also creates a basis for additional studies aiming at biomimicry to provide solutions for regeneration of articular cartilage.
Materials and methods Conceptuses were endoscopically recovered from pregnant mares on Days 37 (E37), 42 (E42), 45 (E45) and 65 (E65) after ovulation and then fixed in 4% paraformaldehyde. All embryos were staged according to the Carnegie staging criteria for human embryos, modified by O’Rahilly and Müller in 1987 [8] and adapted for the horse by Acker et al. [9]. The Carnegie staging system classifies embryos into 23 stages based on a combination of their gestational age (postovulatory days), size (crown–rump length), external morphology and organ system development [8].
620
High-resolution episcopic microscopy Equine embryos E37, E42, E45 and E65 were dissected to create limb segments of the area of interest
Short Communications
The embryogenesis of the equine femorotibial joint: The equine interzone F. JENNER*, G. J. V. M. VAN OSCH†, W. WENINGER‡, S. GEYER‡, T. STOUT§, R. VAN WEEREN§ and P. BRAMA¶ Equine Hospital, University of Veterinary Medicine Vienna, Austria † Department of Orthopaedics and Department of Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam, The Netherlands ‡ Center for Anatomy and Cell Biology, Medical University of Vienna, Austria § Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands ¶ Section Veterinary Clinical Sciences, School of Veterinary Medicine, University College Dublin, Ireland. *Correspondence email: fl[email protected]; Received: 27.05.14; Accepted: 10.07.14
Summary Reasons for performing study: Articular cartilage regeneration is the focus and goal of considerable research effort. Since articular chondrocytes descend from a distinct cohort of progenitor cells located in embryonic nascent joints (interzones), establishing the timing of equine interzone formation is an essential first step towards understanding equine joint and articular cartilage development. Objectives: To establish the time frame during which the equine femorotibial interzone forms. Study design: Descriptive anatomical study. Methods: Equine embryos were harvested at 37 (E37), 40, 42, 45, 50 and 65 days’ gestation. The femorotibial interzone was examined using high-resolution episcopic microscopy of E37, E42, E45, E50 and E65. Additional histology and collagen-II-immunohistochemistry were performed on E42. Results: At E37, the femorotibial interzone is first visible as a uniform layer, while at E42 the interzone is fully formed and consists of 3 morphologically distinct layers. The first evidence of cavitation was seen at E45. At E50, the cruciate ligaments were well formed and by E65, joint formation appeared complete. Conclusions: The embryogenesis of the equine femorotibial joint is similar to the developmental timeline of stage-matched human and murine embryos. Further studies looking at interzone formation on a cellular and molecular level may further our understanding of the intricate developmental patterns and pathways of articular cartilage development. Keywords: horse; joint development; embryo; articular cartilage; embryogenesis; interzone
Introduction The interzone has a pivotal role in joint formation demonstrated by joint ablation when surgically removed in embryos [1,2]. Lineage tracing experiments have demonstrated that interzone cells form all articular structures such as articular cartilage, ligaments and synovial lining [2–5]. Despite the high incidence of cartilage and joint disease in horses and the popularity of horses as research animals for osteoarthritis and cartilage tissue engineering, there are no studies of the equine interzone and the timing of interzone formation is not known. Using magnetic resonance microscopy in a companion study, we narrowed down the time frame during which interzone formation probably occurs to gestational Days 35–50 [6]. In the current study, we performed high-resolution episcopic microscopy (HREM) [7], histology and immunohistochemistry (IHC) to determine when the femorotibial interzone forms in the horse. While this study is descriptive, it provides a first insight into the development of equine joints and the related development of articular cartilage. It also creates a basis for additional studies aiming at biomimicry to provide solutions for regeneration of articular cartilage.
Materials and methods Conceptuses were endoscopically recovered from pregnant mares on Days 37 (E37), 42 (E42), 45 (E45) and 65 (E65) after ovulation and then fixed in 4% paraformaldehyde. All embryos were staged according to the Carnegie staging criteria for human embryos, modified by O’Rahilly and Müller in 1987 [8] and adapted for the horse by Acker et al. [9]. The Carnegie staging system classifies embryos into 23 stages based on a combination of their gestational age (postovulatory days), size (crown–rump length), external morphology and organ system development [8].
620
High-resolution episcopic microscopy Equine embryos E37, E42, E45 and E65 were dissected to create limb segments of the area of interest
