Effects of the local mechanical environment on vertebrate tissue differentiation during repair: does repair recapitulate development?
1 Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Boston
University Medical Center, 715 Albany Street, Housman-205, Boston, MA
02118-2526, USA
2 Department of Restorative Sciences and Biomaterials, Boston University
School of Dental Medicine, Boston, MA 02118, USA
3 Department of Biomedical Engineering, Boston University, Boston, MA 02115,
USA
* Author for correspondence (e-mail: bones{at}bu.edu)
Accepted 10 April 2003
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Summary |
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Key words: local mechanical environment, mechanical loading, mechanobiology, tissue differentiation, tissue architecture, gene expression, finite element model, skeleton, cartilage, bone
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Introduction |
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However, aside from simple changes in tissue architecture, this
mechanosensitivity of the skeleton is also likely to include a direct
influence on gene expression, tissue molecular architecture and tissue type
during the processes of development and healing (Carter et al.,
1998a,b
;
Claes and Heigele, 1999
;
Claes et al., 2002
; Cullinane
et al., 1999
,
2002
; Hartman and Tabin, 2001;
Elder et al., 2001
;
Gardner et al., 2000
;
Loboa et al., 2001
;
Smith-Adaline et al., 2002
;
Waanders et al., 1998
). In
fact, several studies have made direct parallels between joint development and
fracture repair based on this relationship
(Cullinane et al., 2002
;
Ferguson et al., 1999
). Thus,
not only does the mechanical environment initiate tissue formation and
resorption due to exercise and disuse, for example, but it can also regulate
the very type of tissue that will form during development or healing.
Appropriate mechanical stimulation is essential in directing complex tissue
differentiation and architecture during joint development
(Carter et al., 1998b;
Eckstein et al., 2002
;
Heegaard et al., 1999
;
Sarin and Carter, 2000
;
Smith et al., 1992
;
van der Meulen and Carter,
1995
), and the mechanical properties of the resulting tissues can
be correlated to the applied load
(Grodzinsky et al., 2000
).
Thus, cartilage, and specifically articular cartilage, demonstrates direct
dependence on the mechanical environment for normal development and
maintenance (Beaupre et al.,
2000
; Grodzinsky et al.,
2000
; Loboa-Polefka et al.,
2002
). Evidence of this relationship can be found in studies of
joint immobilization in which the absence of mechanical loading significantly
alters tissue developmental pathways even in developmentally predetermined
joint tissues (de Rooji et al.,
2001
; Hall, 1972
;
Smith et al., 1992
). In this
way, the mechanical environment can specifically foster cartilage formation
instead of bone or fibrous tissues, and it can regulate the architecture of
those tissues down to their molecular configuration
(Cullinane et al., 2002
).
The model
This study was designed to empirically test the mechanobiological paradigm
as it applies to gene expression, tissue differentiation and tissue
architecture in a healing skeletal defect. The goal of this experimental
design was to mimic the local mechanical environment during early joint
development (post-segmentation) using a custom-designed external fixation
device capable of inducing bending and shear loads within a healing bone
defect. Finite element models (FEMs) generate estimates of stress and strain
distributions within the defects that are then used to predict tissue type and
distribution based on a mechanobiologically derived tissue differentiation
fate map (MFM) based on Carter et al.
(1988).
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Materials and methods |
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The cortical bone of the femoral diaphysis served as a rigid boundary
because the bone is several orders of magnitude stiffer than the materials
within the early healing defect. The defect is represented by a middle segment
of the tube with different mechanical properties from the cortical bone
portion of the tube and the medullary canal. Values for the mechanical
properties of the defect tissues were taken from the literature for an
equivalent early stage of maturation
(Gardner et al., 2000). It was
expected that the early callus would be representative of a fluid to
semi-solid phase material with hydrostatic forces dominating. The bending and
shear models were used to (1) estimate local mechanical loading conditions
and, using that information, to (2) predict the patterns of tissue
differentiation within the defect.
The FEM is comprised of a number of nodes and brick elements. We estimated
the brick elements to be 0.05 mm in magnitude. The FE analysis was performed
using I-DEAS software (Schroff Development Corp., Mission, KS, USA). Solids
including bone and condensed cell masses were meshed into elements using mesh
generation software. Stress and strain distributions were estimated by the
FEMs, and tissue types were assigned based on a mechanobiologically derived
tissue differentiation fate map (MFM) based on Carter et al.
(1988,
1998a
,1998b
).
Hydrostatic stress and maximum principal tensile strain were calculated for
the different mechanical actions, and spatial tissue predictions were assigned
based upon quantitative, as well as relative, stress and strain levels
according to Giori et al.
(1993
). The tissue types we
predicted based on our mechanical stimulations included cartilage (under
relatively high hydrostatic compressive stress), fibrocartilage (under
relatively high hydrostatic stress and high tensile strain), bone (under
relatively low hydrostatic stress and low hydrostatic strain) and fibrous
tissue (under relatively low hydrostatic stress but high tensile strain).
External fixation
A total of twelve SpragueDawley rats (Rattus norvegicus
Berkkenhaut 1769) weighing 421±34 g were used in this study. Animal
care and experimental protocols were followed in accordance with NIH
guidelines and approved by our institution's Animal Care and Use Committee.
Four animals were used in each of the groups that were subjected to bending,
shear or alternating bending and shear. The external fixator was modified from
a previous model, while the surgical procedure was identical to that used in a
previous report from our laboratory
(Cullinane et al., 2002).
Briefly, the external fixator was surgically applied to the right femur using
four bicortical pins, and an osteotomy was created leaving a 2 mm defect
within the femoral diaphysis. The external fixator, in conjunction with the
linkage system, was capable of imposing either 12° symmetrical bending or
10% symmetrical cortical shear, depending on the actuator pin insertion
configuration (Fig. 2A). The
fixator body was composed of two articulating solid aluminum rectangular
prisms with cortical pin holes and included clamping and locking screws. The
clamping screws fastened the cortical pins into the fixator, and the locking
screws arrested the fixator pivot or shear actions
(Fig. 2B).
|
Mechanical stimulation
The stimulation protocol followed that established for a previous study
from our laboratory (Cullinane et al.,
2002). To perform the mechanical stimulations, an oscillating
linkage system was built that supplied a rotational moment that could be
applied to the fixators as an oscillating vertical displacement
(Fig. 3). The vertical
displacement was translated by the fixator into either 10% shear or 12°
bending. The action was applied using a servomotor (model #2602-010,
QCI-23-5-E-01; Quicksilver Controls Inc., Covina, CA, USA). The speed of the
motor was controlled using PC-based software. The shaft of the servomotor was
coupled to one of two eccentric shafts of a torque transducer (model #1102-50;
Lebow Products, Troy, MI, USA). The other shaft of the transducer was
connected to the linkage system. A torque sensor incorporated within the
system was connected to an external data acquisition board (model #100;
InstruNet Inc., Cambridge, MA, USA) and acted as a bridge voltage sensor,
measuring the torque transduced to the fixator. Based on the manufacturer's
specifications, the torque sensor was mapped to 278.6 mV Nm-1, with
the input torque sampled at 60 Hz for the duration of each experimental
session. The peak torque required to induce the respective motions prior to
animal attachment was recorded as the baseline value. The loading apparatus
was calibrated prior to every application using InstruNet and Quicksilver
PC-based software.
|
There were three mechanical stimulation protocols executed during this study: (1) bending at 12°, (2) 10% shear and (3) alternating 12° bending and 10% shear (percentage of cortex diameter). All mechanical stimulations were symmetrical to the alignment of the cortices and cyclical for the 15-min stimulation period. Starting at post-operative day three and continuing for six weeks, the mechanical stimulations were induced for six consecutive days, with one day of rest each week. The fixator on each animal was attached to the linkage system that instituted the respective bending and shear actions initiated by the motor. The results from these three treatment groups were compared with those of previous control specimens.
During each mechanical session, the treatment animals were anesthetized, the fixators were attached to the linkage, the locking screws were removed, and cyclic stimulations were applied for 15 min at a frequency of 1 Hz. A dedicated computer coordinated the application of the mechanical treatment and data acquisition during calibration and treatment. The locking screws were replaced upon completion of each session. Once recovered from the anesthesia, the animals were returned to the housing room and allowed to ambulate freely in their cages.
Moment analysis
A torque sensor incorporated within the system was connected to an external
data acquisition board (model #100; InstruNet Inc.) and acted as a bridge
voltage sensor, measuring the torque transduced to the fixator and the
resistance to the applied torque. Based on the manufacturer's specifications,
the torque sensor was mapped to 278.6 mV Nm-1, with the input
torque sampled at 60 Hz for the duration of each experimental session. The
loading apparatus was calibrated prior to every application using InstruNet
and Quicksilver PC-based software. The peak torque required to induce the
respective motions prior to animal attachment was recorded as the baseline
value. The torque sensor then constantly monitored the torsional resistance to
the motor via the skeletal defect within the animal. These resistance
data were captured for each animal during every daily stimulation period, the
data were then averaged among all the animals for every day of stimulation,
and the result was a mean daily moment resistance for every day of the
stimulation protocol. The mean relative moment resistance was charted for the
entire 35-day stimulation period.
Histology
Animals were euthanized at the termination of the study and the femora were
excised. Standard histological methods were employed to generate serial 5
µm sagittal sections for standard histology and histomorphometry
(Cullinane et al., 2002). The
sections were mounted on glass slides, and even-numbered slides were stained
using Safranin-O (cartilage) and Fast Green (bone), while odd-numbered slides
were stained with Alcian Blue and counter-stained with eosin (proteoglycans).
The 5 µm decalcified histological specimens were examined under a light
microscope using 1.25x to 40x objectives. Dark-field images were
obtained through the use of a polarizing filter, which highlighted collagen
fibrils for quantification of their orientation and conformity within the
extracellular matrix.
Histomorphometrics
Tissue type composition
Tissue type area composition was quantified using ImagePro ® software
(Atlanta, GA, USA). We quantified the percentage of bone and cartilage for
each of the treatment groups and the control group, as well as rat knee and
lumbar intervertebral joints. The entire defect and joint were quantified for
tissue percentage within a standardized area of interest, including 2.5 mm in
both directions proximal and distal to the defect or joint center. Tissue type
ratios were generated for each treatment group and the controls, as well as
actual native rat joints. Comparisons were made to identify similarities in
tissue composition ratios between the treatment groups and the native rat
joints.
Collagen architecture quantification
In order to characterize the molecular organization of the newly formed
cartilage tissues, collagen fibril orientation and angular agreement were
quantified using polarizing light microscopy and histomorphometric analyses
using Matlab ® and ImagePro ®. Fast Fourier transforms were performed
on digitized images of polarized light micrographs, and the preferred collagen
fibril orientation was determined by the most intense region in their power
spectra (Fig. 4). This
procedure was performed as previously described by Cullinane et al.
(2002). Polarized light
micrographs were taken from predetermined superficial, intermediate and deep
regions of the experimentally derived cartilage tissues in order to highlight
collagen fibrils. These images were then incorporated into a Matlab Fourier
transform analysis to determine mean collagen fibril orientation and fibrillar
agreement (Cullinane et al.,
2002
).
|
Molecular analyses
Molecular analyses of the expression of specific genes or proteins was
carried out by in situ hybridization and immunostaining in order to
confirm tissue types and to identify expression of the growth and
differentiating factor 5 (GDF-5), respectively. In situ hybridization
was carried out for collagen type II using a commercially available probe for
RNA labeling. Linearized plasmids containing this gene were purchased from
Pharmigen Corp. (San Diego, CA, USA). Single stranded 35S-labeled
cRNA probes were generated by in vitro transcription (Pharmigen
Corp.). Linearized plasmids containing each of the selected genes for analysis
were transcribed using [35S]uridine triphosphate
([35S]UTP; NEN Life Science Products, Inc., Boston, MA, USA) and T7
RNA polymerase, then digested with DNase, phenol extracted and ethanol
precipitated. The labeling efficiency for the cRNA products was determined by
scintillation counting and adjusted to a concentration of
3x105 c.p.m µl-1 of probe for each in
situ assay.
Tissue procurement
Tissue samples were fixed overnight in freshly prepared 4% paraformaldehyde
at 0°C, followed by decalcification in 14% EDTA for up to eight weeks.
Decalcified samples were paraffin embedded.
Tissue preparation and sectioning
Fixed and decalcified tissues were dehydrated in graded ethanol up to 100%,
transferred to xylenes, then embedded in paraffin. 5 µm-thin paraffin
sections were placed on poly L-lysine-coated slides, dried
overnight and used immediately or stored at 4°C.
Probe preparation
Sense and antisense 35S-labeled cRNA probes were used for
hybridization. Vectors were appropriately linearized and incubated with either
T7 or SP6 RNA polymerase in the presence of [35S]UTP, unlabeled
nucleotides, 10 mmol l-1 dithiothreitol (DTT) and RNasin RNase
inhibitor (Promega, Madison, WI, USA). Labeled cRNA probes were separated from
free nucleotides using a Mini Quick Spin RNA column (Roche Molecular
Biochemicals, Indianapolis, IN, USA).
Prehybridization
Slides were deparaffinized in xylenes followed by rehydration in graded
ethanol solutions, rinsed in 0.85% NaC1 (5 min) and 1x
phosphate-buffered saline (PBS; 5 min). Sections were treated with proteinase
K (20 µg ml-1) for 8 min at 37°C. Slides were dipped
successively in 1x PBS (5 min), 4% paraformaldehyde (5 min), acetylated
in 0.25% acetic anhydride. GDF-5 expression was examined by
immunohistochemistry. For these studies, an antibody to GDF-5 was obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Briefly,
histochemical staining was carried out using antigen retrieved at 199°F
for 10 min in 10 mmol l-1 sodium citrate. The anti-GDF-5 antibody
(0.5 µg ml-1) was applied to the sections, followed by a
biotinylated secondary antibody and horseradish peroxidase (HRP)-conjugated
streptavidine complex, and visualized with DAB chromogen.
Statistical analysis
Data are presented as means ± S.D. All histomorphometric
results, including collagen preferred fiber angle and fiber angle conformity,
were compared between the control and treatment groups using analysis of
variance (ANOVA) and Tukey's post-hoc test at an level of
0.05, with P values of <0.05 interpreted as significant. All
sample sizes for the specific groups were determined by power statistics
calculations: based on a coefficient of variation of 25% in the data and
accepting
and ß errors of 5.0%.
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Results |
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Peak strain levels in the bending group reached 7.87x10-6,
while peak strain in the shear groups reached only
1.95x10-11, with a more narrow range of strain distribution
in the proximaldistal direction. According to the MFM based on Carter
et al. (1988), fibrous tissues
would form in the bending group within the estimated range of
7.87x10-6 to 6.07x10-6, with cartilage
forming in the range from 5.84x10-6 to
3.37x10-6 and bone within the range from
2.70x10-6 to 2.20x10-6. The shear group
tissue differentiation ranges for strain include 1.95x10-11
to 1.56x10-11 for fibrous tissue, 1.47x10-11
to 8.79x10-12 for cartilage and 7.82x10-12
to 2.93x10-12 for bone.
The stress and strain distributions were then incorporated into graphic models of expected tissue differentiation for each of the mechanical stimulations (Fig. 6). The graphic model predictions were based on the stress and strain results from the FEMs, interpreted by the MFM. The areas of higher compressive stress were predicted to encourage cartilage differentiation whereas the areas of extreme tensile strain were predicted to promote the differentiation of fibrous tissue. The areas within the high compressive stress region but that are shielded by previous cartilage formation are predicted to foster bone. These areas of subchondral bone were predicted to form arch-like structures, peaking at the neutral axis of bending.
|
Moment analysis
Fig. 7 details the results
of the moment analysis for the three mechanical stimulation regimens for the
entire 35-day stimulation period, normalized to the stimulation device without
an animal attached. ANOVA found a significant difference among the groups
(P<0.001, N=4), with the bending group being
significantly different from both the shear and combination groups. The
bending group experienced the greatest moment resistance, followed by the
shear and combination groups. After initial fluctuations, the three groups
appeared to cycle together, with some temporal offset initially in the
combination group, and with a magnitudinal difference in the bending group.
The bending and combination groups experienced an initial peak at
approximately 810 days following the onset of stimulation. This peak
was followed by a day 1517 mutual low for all three groups. A
subsequent mutual peak at days 2325 then followed for all three groups,
followed by a mutual low at days 3032. The day 10 peak coincides with
the maturation and peak of the cartilaginous stage of callus healing.
|
Radiology
The weekly radiographs illustrated the onset of bony bridging across the
defects in the control specimens, while the treatment defects each
demonstrated defect translucency and complete non-union in all specimens
(Fig. 8). Areas of reduced
density represent cartilage or fibrous tissue, while high-density areas
represent mineralized tissues such as bone. A distinctive arch-shaped
structure spanning the defect cortices can be seen in several of the bending
group specimens.
|
General histology
The shear treatment was preceded by an experimental test to determine an
appropriate shear magnitude. This test, using two shear magnitudes,
demonstrated two completely different tissue outcomes. One group experienced
10% shear magnitude while the other experienced 25% shear. The 10% magnitude
shear group developed a cartilage band across the entire defect, while the 25%
shear defect developed only fibrous tissue across the defect
(Fig. 9).
|
The mechanical treatment groups all demonstrated the presence of a cartilage band spanning the entire defect, while the control specimens demonstrated bony bridging of the defect (Fig. 10). The cartilage tissues stained red while the bone and fibrous tissues stained blue-green. The bending specimens acquired an arched appearance to their cartilage and the underlying subchondral bone arch on at least one side of the defect, while the shear and combination groups showed parallel and evenly distributed cartilage bands.
|
Histomorphometrics
Tissue type composition
We found that the ratio of cartilage to bone in our experimental tissues
was very similar to that in articular cartilage, especially in comparison with
control endochondral healing callus (Table
1). The mean control ratio of bone to cartilage was 94:6. In the
experimental results, the mean bending group ratio of bone to cartilage was
78:22 and the shear group was 80:20. The mean combination group ratio was
82:18. None of the treatment groups were significantly different from the knee
joint but all were significantly different from the control
(P<0.05, N=4 per group). Thus, we found consistent ratios
of cartilage to bone in our experimental tissues, and these mirrored the knee
joint mean of roughly 80:20. The intervertebral joint was unique in that its
cartilage to bone ratio was significantly different from all others
(P<0.00062), approximating 50:50.
|
Collagen architecture quantification
The experimentally generated cartilage tissues demonstrated visually
distinct zones of collagen fibril organization with specialized fiber
orientations in each zone (Fig.
11).Obvious were the superficial and deep zones, with a less
obvious transitional intermediate zone. Mean collagen fibril angles were not
significantly different among the different treatment groups for each of the
layers, with the exception of the shear intermediate layer
(Table 2), which was
significantly different from both the bending and combination groups
(P<0.001, N=4 per group). The intervertebral joint tissue
was not used in this analysis due to its specialized structural
configuration.
|
|
Molecular analysis
In situ hybridization confirmed the presence of type II collagen
within the tissues differentiating in the experimental defects
(Fig. 12). Type II collagen is
a marker molecule for all forms of cartilage, and its presence confirms that
the tissue differentiating within the defect is cartilage. We observed
collagen type II expression at constant but relatively low levels throughout
the tissue. However, higher levels of expression were seen in a band of cells
adjacent to the area of fibrous tissue where cartilage cavitation was
initiating (Fig. 12A). We also
saw a weaker band of labeled cells adjacent to the subchondral bone formed
under the cartilage band. Immunohistology also identified the presence of
growth and differentiating factor 5 (GDF-5) within the cells of the bending
experimental cartilage (Fig.
13). The positive presence of this molecule is indicated by a
brown stain located around the cells differentiating within the defect. Its
presence in the experimental tissues is contrasted by its absence in the
controls.
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Discussion |
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The FEMs accurately predicted the production and persistence of cartilage within the defects and went as far as to predict the presence of fibrous tissues in specific areas of both the bending and shear models. The mechanical stimulations created uniform cartilage bands across the entire defects that persisted well past the timeframe of bony bridging found in the controls. The bending model predicted the presence of stress and strain distributions, peaking in the center of the defect and diminishing toward the cortices. These distributions resulted in an arch-like bony structure forming across the medullary canal in the bending specimens. The results of the shear pilot study are particularly interesting because they underline the extremely divergent tissue types one can expect based solely on differences in the magnitude of the local mechanical environments. These results in particular serve as a potent example of the predictive power of the mechanobiological paradigm.
The moment resistance data is interesting because it appears to coincide with tissue developmental timing events such as the onset of cartilage formation (1520 days post-surgery) and perhaps even the formation of ligament-like connective tissues on the periphery of the defect. Tissue segmentation events (chiefly cartilage) probably resulted in the steep declines in moment resistance following the day 10 and day 25 peaks, while connective tissue formations such as ligament-like tissues probably resulted in increases in moment resistance. The magnitude of moment resistance is probably related to the mechanics of bending versus shear and, specifically, the extreme tension and compression generated via bending. This information goes far in explaining the significant difference in the bending group data. The individual highs and lows are probably related to the tissue types being formed within the defects, and thus are time-dependent as well as mechanobiologically dependent. Thus, the appearance of a tissue is constrained by time and physiological processes as well as by mechanobiological principles, while the architecture and maintenance of a tissue is related more to the mechanical environment.
The histomorphometric results verify that the ratio of cartilage to bone
within the experimentally treated defects is in large part controlled by
mechanics. The collagen fibrils within the experimentally derived tissues
demonstrate organized patterns that resemble those found in articular
cartilage. Finally, specific mechanical stimuli can trigger the expression of
the genes encoding collagen type II (cartilage formation) and GDF-5 (bone and
joint formation). These results further suggest that induced mechanical
stimulation during the process of bone defect repair can cause a
recapitulation of developmental events from joint formation. It is interesting
to note that during stable fracture repair, GDF-5 expression appears
in a tightly defined window during the endochondral phase of fracture and
disappears as soon as bone replacement is initiated
(Cho et al., 2002). Such
results suggest that in the absence of continued mechanical intervention, the
expression of this gene is downregulated and would further suggest that it
plays an important role either in the maintenance of cartilage or in the
retardation of further endochondral maturation. It may reflect the possibility
that the experimentally induced activity of this gene is a vestigial attribute
of the mechanisms of original joint formation.
Our molecular results are encouraging because they demonstrate two
principal findings: first, the presence of collagen type II confirms that our
experimentally derived tissues are true cartilage and, second, a gene
associated with the in utero development of joints (GDF-5)
is upregulated as a result of the bending stimulation
(Storm and Kingsley, 1999).
The comparison between the in situ reactions in normal postnatal long
bones and those obtained from the mechanically induced cartilage was very
informative. High levels of cartilage mRNA expression were not observed in
fully differentiated joint tissues but were observed with very intensely
labeled areas of cartilage formation within the epiphyseal growth plate.
Similarly, in the areas of mechanically induced cartilage formation we
observed collagen type II expression at low levels throughout the tissue.
However, higher levels of expression were seen in a band of cells adjacent to
the area of fibrous tissue where cartilage cavitation was initiating. We also
saw a weaker band of labeled cells adjacent to the subchondral bone that
formed under the cartilage band. These results suggest that the mechanical
environment has a direct and quantifiable effect on gene expression and tissue
differentiation within healing bone defects.
The origin of the cells that populate the defect following the surgical
procedure and during the duration of the experiment is an intriguing question.
Very little is known about the precise origin of the cells invading the callus
during the many stages of defect repair
(Denker et al., 2001;
Hunziker et al., 2001
;
O'Driscoll and Fitzsimmons,
2001
). The hematoma probably arises from vascular cells that
invade the defect and fill the gap, while cells that form the cartilage tissue
originate from the periosteum (Denker et
al., 2001
; Hunziker et al.,
2001
; O'Driscoll and
Fitzsimmons, 2001
). In bone formation, cells must invade from the
marrow as they do during initial endochondral bone formation. The origin of
the cells within the defect may be academic since it seems that the local
mechanical environment can regulate and direct their maturation trajectory to
mature cells.
Finally, the experimentally generated tissues and their molecular architecture took on joint-like characteristics in several aspects of our analyses. This is an intuitive outcome in our estimation, as the mechanical interventions were designed to mimic the actions of a developing joint. These results emphasize, on numerous levels, the importance of the mechanical environment in tissue differentiation during both development and repair. It should also be noted that cases of mechanically unstable fractures will likewise demonstrate the presence of cartilage within a healing bone defect, but the location, amount and architecture of that cartilage differs markedly from our precisely mechanically generated cartilages. A classic pseudoarthrosis or `false joint' is typically a random conglomeration of fibrotic tissue, cartilage and bone. This configuration is directly related to the random instability of the local mechanical environment and its variant magnitudes.
The outcomes of this study confirm that mechanobiological principles can accurately predict gene expression, tissue differentiation and tissue architecture based on manipulations of the local mechanical environment during healing. The results further emphasize the important role the local mechanical environment plays in the everyday development and repair of the vertebrate body. Further studies need to be conducted in order to determine the precise relationships between the physical environment and gene expression, tissue development and tissue repair.
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Acknowledgments |
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References |
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