Kenneth E. Gonsalves, Craig R. Halberstadt, Cato T. Laurencin, Lakshmi S. Nair. Biomedical Nanostructures
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Recently, using a triphasic scaffold predesigned for interface tissue
engineering, Spalazzi et al. evaluated the effects of osteoblast and fibroblast
interactions in 3D coculture. It was found that phase-specific cell distribution
and coculture resulted in the formation and maintenance of distinct yet
continuous ligament, interface, and bone-like matrix regions on the scaffold
[54]. In contrast to the 2D study, cell proliferation was not reduced in 3D
coculture and consequently, the observed cell-matrix production was
significantly more extensive. It is possible that the 3D coculture response is
more representative of systemic effects of cellular interactions as opposed to
the local effects seen in 2D coculture models. These observations highlight the
importance of considering multiscale and multidimensional effects on
cellcell interactions and demonstrate the utility of in vitro coculture and
triculture model systems for investigating the mechanisms of interface
regeneration.
14.7 MECHANISM OF CELLULAR INTERACTIONS DURING
COCULTURE
It has been well established that cellcell physical contact and communica-
tions are regulated by a variety of cell-adhesion molecules and cellcell
junctions. Of these, cadherins, which represent a large family of calcium-
dependent, transme mbrane adhesion molecules, are particularly impor tant
in the dynamic regulation of cellcell adhesion and communications.
Cadherins are powerful modulators of tissue mor phogenesis during
embryonic development, as they direct cell s orting [55, 56], cell migration
[57, 58] as well as cell adhesion [59, 60]. Cadherins are structurally classified
into type I and type II subgroups, and are named in accordance to the tissue
type from which they were first isolated. Type I cadherins consist of cadherin
E (epithelial), N (neural), P (placental), and R (retinal) [61] while type II
cadherins consist of cadherin-5, -6, -8, -11, and -12 [61]. During developm ent,
cell sorting is regulated by differential expression of cadherins, which in turn
promotes tissue separation. For example, the selective expression of
E-cadherin in the ectoderm versus N-cadherin in the developing neural
epithelium allows the two regions to split and form separately [55, 56] .
Compartmentalization of tissues and formation of tissue boundaries are also
regulated by cadherins [62, 63]. In the adul t tissue, cadhe rins are essential for
tissue homeostasis in actively growing tissues such as the epidermis [64–66]
and bone [67]. They also help to maintain tissue organization by promoting
contact inhibition and preventing the invasion of tumor cells [60, 68]. The
extracellular domains of cadherins interact with those of adjacent cells in
order to mediate cell adhesion. The intracellular cadherin domain also binds
to cytoplasmic proteins such as catenins (a,b) and p120, which in turn
interacts with the cytoskeleton and facilitate cell migration and shape
changes [69].
366 BIOMEDICAL NANOSTRUCTURES
The mechanism of the various osteoblast, fibroblast, and chondrocyte
interactions discussed in this chapter, in particular the mechanism related to
heterotypic cellular interactions, is not known. In addition to above described
cellular communication mediated by paracrine and autocrine factors during
coculture, cellcell interactions through cadherins will likely play a significant
role, as post ligament reconstruction surgery, fibroblasts, and osteoblasts will
come into physical contact with each other at the soft tissue-to-bone junction.
Full elaboration of the osteoblast phenotype is directly regulated by
N-cadherins [70–72], which is present or upregula ted during osteogenic
differentiation [67, 70, 73]. Cadherins are also believed to be involved in
osteoinduction of mesenchymal stem cells by modulating b-catenin signalling
[74]. Aside from cadherins, connexins which represent a family of gap junction
proteins, are also important for directing adjacent osteoblast, osteocyte, and
periosteal fibroblast communications. Interestingly, cadherins have also been
reported to directly modulate the formation of tight junctions [75, 76].
Cellcell adhesion is also essential for chondrogenesis [77–79], as N-
cadherin expression has been reported in the developing chick limb
mesenchyme, and its expression increases as condensation progresses [77,
78]. When the function of N-cadherin was neutralized through antibody
binding, inhibition of condensation and chondrogenesis was reported [77].
Condensation is further mediated by parallel expression of Ca-independent
membrane glycoproteins such as cell adhesion molecules (CAMs), in particular
N-CAM, which has been reported to play a role in chondrogenesis [79–81].
Cadherin-11 (OB-Ca dherin) is a marker of the loosely connected and
migratory cellular elements of the mesenchyme, and it is consistently expressed
in osteoblastic cells, directly affecting cell sorting, alignment, and separation
through differentiation [82]. It has been reported to regulate osteogenesis and
chondrogenesis of mesenchymal stem cells in the presence of N-cadherin and is
functionally distinct from N-cadherin during endochondral ossification and
chondrogenesis [82]. Gap junctions (connexins 32 and 43) have been identified
in rat digital flexor tendons, suggesting the presence of a sophisticated cell
communication network within the tissue, which may be critical for
mechanotransduction [83]. Moreover, in vitro loading of tendon fibroblasts
regulated N-cadherin and vinculin expression [84]. While the expression of
adhesion molecules in ligament fibroblasts has not been examined, cadherin-11
expression has been identified in synoviocytes and are believed to be significant
for syn ovial tissue organization [69]. Therefore, in the formation of the
multitissue, multicell interface betw een ligament or tendon and bone, it is
anticipated that cellcell adhesions via cadherin complexes or gap junctions
will facilitate critical cellular communications, which will control cell migration
or dictate tissue organization at the interface.
The current understanding of cadherin function is deeply rooted in
developmental biology, which may or may not be transferable to the case of
tissue injury. While the injury model is a physiologically more relevant model
for the soft tissue graft-to-bone junction, the expression and distribution of
MULTISCALE COCULTURE MODELS FOR ORTHOPEDIC INTERFACE TISSUE ENGINEERING 367
cadherins and adherens junctions during tendon-to-bone healing are poorly
understood. Nevertheless, the dynamic modulation of cadherin expression and
associated changes in cellcell communications will be essential in the
regulation of multitissue organization formed via tissue engineering methods,
and should be a critical focus in elucidating the mechanism for interface
regeneration and homeostasis.
14.8 CONCLUSIONS
Multiscale and multidimensional cellular interactions are essential for organ
homeostasis, repair, and regeneration. Biomimetic coculture models are
powerful tools for deciphering the relative contributions of cellcell contact,
soluble factors, substrate geometry, as well as interaction scale. Studies
utilizing coculture models have provided new insights into the role of cellular
interactions in the regeneration of the multitissue interface between bone and
soft tissue. As such, the precise mechanism of cellular interactions that drives
tissue organization and homeostasis at the interface remains elusive. Findings
from these and future studies in orthopedic interface tissue engineering will
promote the design of a new generation of integrative fixation devices, as well
as facilitating the development of modern tissue engineering strategies for the
formation of complex tissues and functional organ systems.
14.9 ACKNOWLEDGMENTS
The authors would like to gratefully acknowledge funding support from the
Wallace H. Coulter Foundation and the National Institutes of Health.
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PART III
Clinical Applications
of Nanostructures