Several members of the bone morphogenetic protein/osteogenic protein (BMP/OP)
and transforming growth factor beta (TGF-B) families are molecular regulators o f
cartilage and bone regeneration. However, their precise mode o f signal transduction
and combined interactions are poorly understood. The presence of several molecular
forms o f these growth factors suggests multiple functions in vivo as well as
synergistic interactions during both embryonic bone development and regeneration
o f cartilage and bone in postnatal life. A functional bioassay for identification of
osteogenic proteins within the bone matrix has been established. The heterotopic
bioassay in rats, together with the improved purification methods, has led to the
purification o f native BMPs. In rats, heterotopic and orthotopic implantation o f TGF-
13 singly fails to initiate new bone formation whereas implantation o f BMPs/OPs
elicit the local differentiation o f new bone, at both sites.
This study presents data which shows that co-administration of TGF-B1 with OP-1
delivered by a collagenous carrier, and implanted in the subcutaneous site o f rats
results in synergistic induction o f bone formation. Changes in hist-logical,
biochemical and molecular response o f the effects o f the morphogens were vu bed
over 7, 12 and 21 days post implantation. Induced cartilage and bone were analyzed
by alkaline phosphatase activity, calcium content, Northern blots and histological
examination of subcutaneous implants in the rats.
Single applications o f TGF-B1 (0.01pg, 0.03pg and O.lpg) on days 7, 12 and 21 gave
rise to negligible alkaline phosphatase activity (0.03-0.09 U/ mg protein) and
calcium content (0.05-0.6 pg/mg tissue). Histological examination showed that all
TGF-B1 implants did not exhibit any sign o f bone formation.
On day 7, OP-1 implants (O .lpg and 0.3 pg) elicited negligible alkaline phosphatase
activity and calcium content. However, higher doses o f OP-1 (1 pg and 3 pg) elicited
alkaline phosphatase activity o f 0.1 U/mg protein and 0.2 U/mg protein.
Combination TGF-Bl (O.Olpg, 0.0.3pg and O.lpg) with OP-1 (O.lpg, 0.3pg, Ip g and
3pg) slightly increased the activity o f alkaline phosphatase activity (0.2 U/mg
protein-0.4 U/mg protein). OP-1 singly gave rise to very low calcium levels o f 0.4
pg/mg tissue to 0.5 pg/mg tissue. Addition o f TGF-Bl to OP-1 resulted in calcium
content rising from 0.2 to 1 pg/mg tissue. Histologically, the specimens o f single
OP-1 applications did not show any sign o f bone formation. On addition o f TGF-Bl
to OP-1 the specimens showed signs o f the beginning o f chondroblastic
differentiation.
On day 12 alkaline phosphatase activity elicited by single applications o f OP-1
ranged from 0.1 U/mg protein to 1 U/mg protein. Addition of l i , : 7- Bl increased the
alkaline phosphatase from 0.8 U/mg protein to 7 U/mg protein. Calcium levels
resulting from single applications of OP-1 ranged from 0.1 to 15 pg/mg tissue Auer
addition of TGF-Bl to OP-1 calcium levels rose from 5 to 20 pg/mg tissue.
Histological analysis showed formation o f cartilage in specimens both of OP-1 solo
and OP-1 in combination with TGF-B1.
On day 21 alkaline phosphatase activity was reduced to a range o f 0.1-0.5 U/mg
protein upon single applications o f OP-1. Addition TGF-131 resulted in a further
decrease in alkaline phosphatase activity. Calcium levels were 10-68 jag/mg tissue on
single applications o f OP-1. Addition o f TGF-131 to OP-1 increased the calcium
levels in the range o f 2-70 gg/mg tissue. Histological examination o f the 3 jig OP-1
solo specimens showed complete chondrolysis whereas the OP-1 (3|ig)/ TGF-B1
(0.03 and 0.1 gg) specimens showed the differentiation o f bone marrow.
Tissues generated in the rat subcutaneous space at 7, 12 and 21 days post
implantation elicited mKNA expression o f OP-1, BMP-3 and TGF-B1. These results
indicate that at least in part, the matrix-induced endochondral bone formation
involves the expression o f some members o f the TGF-B superfamily. Type II
collagen (chondrogenesis marker) and type IV collagen (angiogenesis marker)
mRNAs were also detected on days 12 and 21, respectively.
The present data suggests that TGF-B up regulates the temporal activity o f OP-1 to
induce bone formation. Co-administiation of TGF-B 1 to OP-1 caused an increase in
the alkaline phosphatase activity and calcium content, markers o f bone formation,
which implies that when TGF-B is mixed with OP-1, the cascade o f bone formation
is accelerated. These results may have important therapeutic implications. The
rapidity o f tissue morphogenesis with bone marrow formation is important for
regeneration o f bone in older patients where repair phenomena are temporally
delayed and the healing process is slower than in younger patients. The expression of
multiple members o f the TGF-13 superfamily indicates that the cascade of bone
formation incorporates some members o f the TGF-B superfamily and this may form
the basis for synergistic molecular therapeutics for cartilage and bone regeneration in
clinical contexts.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/14701 |
| Date | 22 May 2014 |
| Creators | Matsaba, Thato Nelly |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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