LIGAMENT CELLS ARE ORGANIZED IN A 3-D NETWORK THAT IS DISRUPTED DURING HEALING
*Lo, I (A-Arthritis Society); *Marchuk, L; *Sutherland, C; *Barclay, L; *Timmerman, S; *Hart, D (A-MRC, Arthritis Society, Canadian Arthritis Network); *Frank, C
(A-MRC, Arthritis Society, Canadian Arthritis Network); *Rattner, J; +*Rattner, J (A-Arthritis Society)
+*McCaig Centre For Joint Injury And Arthritis Research, University of Calgary, Calgary, Alberta, Canada. Department of Anatomy and Cell Biology, 3330 Hospital
Drive, N.W., Calgary, AB, Canada, T2N 4N1, (403)-220-4478, Fax: (403)-283-8737, [email protected]
Introduction: Ligaments are dense connective tissues which attach bones
across joints. Because ligaments are relatively hypocellular, much of the
interest in ligament and ligament healing has focused on the study of the
extracellullar matrix (ECM) and its mechanical properties [1]. Ligament cells
have largely been ignored and their cytoarchitecture and organization have not
been described in detail either in normal or injured ligament.
Purpose: The purpose of this study was to characterize the cellular
architecture and organization of the normal medial collateral ligament (MCL)
and anterior cruciate ligament (ACL) in the ovine model and to compare this
organization to that found in ligaments undergoing healing.
Histological section of normal
MCL (figure 1a), normal
ACL (figure 1b), 14 week
MCL scar (figure 2a) and 14
week ACL scar (figure 2b).
Histologic sections stained
with DAPI (left column) for
cell nuclei and
immunofluorescence labelling
for vimentin (right column).
Hypothesis: We hypothesized that ligament cells would have a distinctive 3dimensional (3-D) organization within ligaments, which would be disrupted
during healing. We also hypothesized that ACL cells would be more
disorganized than MCL cells during healing.
Methods: Skeletally mature (3 year old), suffix/suffix-cross female sheep
were used in this study. In six animals, the sheep underwent a unilateral MCL
and posterolateral band of the ACL (i.e. partial ACL) transection. This model
has previously been used to study both extraarticular and intraarticular
ligament healing producing relevant scar tissue in both ligament injuries [2].
All animals were sacrificed by Euthanyl overdose (MTC Pharmaceuticals:
Cambridge, Ontario) and the mid-substance of the MCL or posterolateral band
of the ACL were harvested at 0 weeks, 2 weeks, 6 weeks and 3 months postinjury. Tissue samples were quick frozen in Tissue Tek OCT compound
(Sakura Finetek; Torrance, CA) immediately following harvesting and 6 µm
sections were fixed in 100% methanol at –20oC. Tissue was then washed in
PBS and incubated at 37oC with primary antibody for 30 minutes. Following
three washes, the sections were incubated for an additional 30 minutes with an
appropriate secondary antibody. After three final washes, the sections were
mounted and counterstained with DAPI (4’6-diaminidino-2-phenyl-indole).
Antibodies to the cytoskeletal proteins vimentin (Boehringer Mannheim,
Laval, Quebec) and β-tubulin (Sigma Chemicals Co, Saint Louis, MO), Ki-67,
a marker for proliferating cells (Novoastra Laboratories, UK), and connexin43, a gap junction protein were used. All specimens were examined with a
Zeiss Axiophot fluorescence microscope.
Results: Results demonstrated that in normal ligaments, cells were organized
in an elaborate fashion. In both MCLs and ACLs cut along the length of the
ligament the majority of cells are arranged in longitudinal rows. Each cell
possessed long cytoplasmic projections that connect adjacent cells within a
row (Figure 1a). When cut in cross section, ligament cells appeared stellate
with cytoplasmic projections extending into the extracellular matrix (Figure
1b). Where the cellular projections came in contact with each other, connexin
43 was present.
Cells in both MCL and ACL scars were more abundant and lacked the
directional alignment seen in normal ligament. As demonstrated by
immunolabelling with Ki67, dividing ligament cells appeared as clusters
within the ECM which decreased with increasing time from injury. MCLs
contained a higher number of these clusters then ACLs (not shown). The most
striking finding in both MCL and ACL scar tissue appeared at 14 weeks,
where large tissues gaps were detected (figure 2a,b). In MCLs (figure 2a) the
majority of these areas contained cellular projections that were connected by
gap junctions (as demonstrated by connexin 43). In ACLs, these areas
remained devoid of both cells and cytoplasmic projections (figure 2b).
Discussion: In contrast to spherical cells seen in cartilage, ligament cells
possess cellular projections that extend for some distance into the ECM and
connect cells via gap junctions. This 3-D architecture may be critical in
forming an important load-sensing system allowing ligaments to modulate the
composition of its ECM in response to changes in loading. Indeed, recent
evidence in tendons has demonstrated that a similar architecture exists in this
type of connective tissue and that, in vitro, tensile load-induced changes in
DNA and collagen synthesis can be inhibited by gap junction blockers [3].
Thus, ligaments may also respond in a similar manner.
Interestingly, MCLs and ACLs also demonstrated differences in their
cellular organization during healing. The MCLs contained a higher number of
dividing cells, more organization and an intact 3-D architecture. In contrast,
ACL scars contained a large number of flaws devoid of cells, cytoplasmic
projections and gap junctions. Assuming that gap junctions are important in
cellular communication, these flaws may compromise ligament integrity by
preventing a coordinated response to changes in its biomechanical
environment. This may help explain some of the differences in the healing
capacity of the ACL and MCL.
Conclusion: Both MCL and ACL cells contained long cytoplasmic
projections which extend into the ECM and appear to be arranged in a
complex 3-D network. Following injury this network is disrupted and
disorganized. ACL scars contained flaws, which may disrupted this loadsensing system and compromise ligament healing.
References:
[1] Jackson DW et al.The Anterior Cruciate Ligament., 1993.
[2] Timmerman S et al. Trans Orthop Res Soc 25(2), 788.
[3] Banes AJ et al. CORR 367S:S356, 1999.
Poster Session - Ligament and Tendon Biology - Hall E
47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California
0701