Bone Healing and Failure - Engineered Bone: Tissue Engineering Intelligence Unit - page 51

Recently Cho et al 10 have characterized the differential temporal expression of members of
the TGF- β superfamily during murine fracture healing. This study also looked at various
cytokines (IL-6, IL-1 β ), cartilaginous matrix proteins, including type II collagen and osseous
matrix proteins, including osteoclacin. The members of the TGF- β superfamily analyzed in-
cluded: BMP-2, -3, -4, -5, -6, -7, -8, GDF-1, -3, -5, -6, -8, -9, -10 and TGF- β 1, - β 2, - β 3. The
findings of this study were as follows; Peak expression of IL-1 β and IL-6 was seen 1 day follow-
ing fracture after which these cytokines showed a rapid decline until day 3 to near undetectable
levels. Peak expression of collagens type II and X occurred on day 7 and characterized the
chondrogenic phase. As endochondral ossification progressed type II collagen expression sub-
sided. Expression of type X collagen was slower to regress resulting in a 4.7 fold difference
between these at day 14. Osteogenesis was observed over the whole period of fracture healing,
reflected by increasing expression of type I collagen and osteoclacin (OC) beginning within 24
hours of injury. Maximal osteogenic differentiation, using these criteria, was seen between days
14 and 21.
The temporal expression of members of the TGF- β superfamily in relation to the phases of
fracture repair were as outline below; BMP-2 was the earliest to be expressed and also showed
a second elevation late during the period of peak osteogenesis. In contrast, BMP-4, -7, -8, and
-3 showed a clear restriction in expression to the osteogenic period between days 14 and 21.
BMP-5 and -6 were induced at the onset of injury and were elevated throughout the healing
period.
The GDFs showed a more restrictive expression pattern with GDF-6 specifically induced
on day 1 with little expression beyond this time point. GDF-5 was restricted to day 7 and day
14 when the chondrogenic phase was most pronounced. GDF-1 was also restricted to the
chondrogenic phase though levels of expression were very low. GDF-3, -6, and -9 were not
detected at any time.
Analysis of TGFs showed that TGF- β 1 was expressed at very high levels in unfractured
bones and showed a sharp rise on day 1 following fracture returning rapidly to baseline levels.
TGF- β 2 and TGF- β 3 were only highly expressed following fracture and were not detected to
any extent in unfractured bones. Both showed peak expression within the chondrogenic period
however TGF- β 3 remained elevated approximately 1 week more than TGF- β 2 (until 21 days).
A summary of the temporal expression of the TGF- β superfamily found in this study is as
follows: TGF- β 1, BMP-2 and GDF-8 during the inflammatory phase, GDF-5, TGF- β 2 and
TGF- β 3 during the chondrogenic phase, and BMP-3a, -4, -7, -8 during the osteogenic phase.
The authors 10 went on to determine the relative importance of each of these growth factors
in terms of total expression over the time course of fracture healing, and the relative basal to
peak levels of expression for the time point having the highest fold induction. In general it was
found that those factors showing the highest relative peak levels of expression also had the
greatest levels of overall expression. Among these, TGF- β 1 was the highest followed by BMP-3,
-5, TGF- β 3 and GDF-10. Both GDF-5 and -8 had undetectable basal levels of expression and
showed the lowest levels of induced expression but because of their near absence of basal ex-
pression in intact bone these factors showed the greatest fold up-regulation.
It is interesting that molecules that are structurally very similar have very different roles in
fracture healing. An example of this is BMP-2 and BMP-4 which share high sequence similar-
ity but are expressed differently during the bone repair process. Early induction of BMP-2
occurs on day 1 following fracture and is sustained until day 21. BMP-4 however reaches its
peak expression during osteogenesis. Given this, it is possible that BMP-2 may serve as a trigger
in the fracture healing cascade and regulates the subsequent expression of other BMPs. It has
been shown that BMP-2 up-regulates the expression of BMP-3 and -4 in fetal rat calvarial
osteoblasts and places the expression of these genes downstream of BMP-2 during osteoblastic
differentiation. 78 It would appear that the coordinated expression of multiple BMPs during
fracture healing is an important concept in bone repair, with BMP-2 being a possible trigger to
the cascade.