Page 268 - IJB-8-3

Bone Sialoprotein enhances Bone Regeneration
RUNX2 gene expression after 3 and 5 days compared of 200 nM approximately corresponding to the used
with those in untreated osteoblast cultures . In addition, concentration of 5 μg in this study.
[4]
enhanced relative RUNX2 and SP7 mRNA expression was
detected in human MSCs seeded onto titanium surfaces 3.4. Calvaria defect model
functionalized with BSP by Im et al. [11,41] . In monoculture, To test the effect of immobilized BSP in collagen
expression of OPN was decreased after addition of BSP. type I in vivo, we performed an in vivo model to analyze
As these two molecules have been described as molecules fracture healing in a critical size defect model in the
that can overtake the function of each other and influence calvaria of rats. Various small animal models exist to
their reciprocal expression, this is not surprising . characterize bone regeneration. We chose rats as model
[6]
Moreover, Gordon et al. as well as Im et al. as they are the standard model to test new biomaterials
boosted cell differentiation by adding supplements for studies on bone regeneration and physiology as their
such as ascorbic acid, β-glycerol phosphate, and bone biology is similar to humans. For the first study, a
dexamethasone [4,41] . Effects, depending on different critical size defect in the calvaria was employed, which
concentrations, were observed for physisorbed BSP on is a standard model for testing new materials. We chose
titanium only on day 4 . Coating with a 280 μg/mL the standard size for critical size defects in calvarial rat
[11]
BSP solution displayed higher gene expression rates of models of 5 mm . BMP-7 was used as a positive control
[45]
ALP, Col1, SPARC, RUNX2, and SP7 compared with as its effects on bone formation have been described
those of the lower concentration of 50 μg BSP/mL. before. Nevertheless, as BMPs demonstrate negative
However, no tendencies were observed after 7 days. side effects, alternatives, for example, BSP, are urgently
Modification through covalent coupling showed on day required.
4 a tendency for increased expression by the lower BSP We used three groups (empty defect, collagen gel
concentration (particularly for ALP, RUNX2, and SP7 alone [CG], and collagen gel + BMP-7 [CG + BMP-7])
mRNA expression). This is in contrast with the findings as controls and two test groups (CG + 0.5 μg BSP and
on day 7, which indicate a higher effect by coating with CG + 5 μg BSP) with two BSP concentrations. Figure 2
280 μg/mL BSP (RUNX2, SP7, and SPARC). On CPC demonstrates that the created defect represents a critical
scaffolds, BSP concentrations of 50 μg/mL decreased size defect as even after 8 weeks. No bone regeneration
gene expression of ALP and SPARC, whereas 200 μg/ was observed in the group without any implanted material.
mL did not change marker expression . Coating of In the BMP-7 group, the defect is already closed after
[12]
BSP on ceramic and synthetic polymer materials with 3 weeks and the bone thickness increases after 8 weeks. In
concentrations of 1 μg/mL and 10 μg/mL BSP showed the CG group, a slight bone formation could be observed
no influence on hMSC differentiation in vitro as well as after 3 weeks, which has further grown after 8 weeks;
in vivo . a small positive effect of collagen type I alone on bone
[42]
Taking into account, the described effects in regeneration has already been described by others . The
[46]
literature and comparing them with our results, one can group with immobilized 0.5 μg BSP showed a similar
conclude that rather low concentrations of BSP influence bone growth like the CG-group, whereas the higher BSP
osteogenic marker gene expression. However, it seems concentration showed an increased bone formation with
that the effect varies depending on the analyzed cell an almost closed defect after 8 weeks (Figure 3A and B).
type and depending on the material employed as carrier To further analyze these results, the ratio of BV/
material. We hypothesized that the best impact is obtained TV was calculated using ImageJ. As expected, all groups
when BSP is immobilized in collagen, a component of the demonstrated significantly enhanced BV/TV ratios
ECM. This hypothesis is supported by the fact that BSP compared to the empty defect group. From the collagen
contains a highly conserved collagen-binding domain, groups, only the positive control with immobilized BMP-
which explains why a binding and thereby immobilization 7 showed significant differences compared to the CG
of the molecule in a collagen type I gel is possible . group. BSP at both concentrations showed no significant
[19]
The characterization of the interaction between type I differences compared to CG alone although a positive
collagen and BSP has been described before. Very tendency could be observed in the CG group with the
early works from Fujisawa et al. showed that BSP was higher BSP concentration (Figure 3C).
absorbed to the collagen fibrils and preferentially bound To confirm the results, HE and Masson-Goldner
to the α 2 chain . Tye et al. showed that the binding of trichrome histological stainings were performed. In the
[43]
BSP to collagen type I is hydrophobic and Baht et al. empty defect (Figure 4), the two holes are clearly visible,
[19]
demonstrated higher affinities for helical domains , only filled with connective tissue (light rose) and residual
[44]
which are also present in the collagen used in this study. bone in the middle (dark pink). Staining of the other
They also demonstrated a concentration dependent groups confirmed the radiological and quantitative results
binding curve, with a saturation at a concentration with the highest bone formation seen in the area of the set
260 International Journal of Bioprinting (2022)–Volume 8, Issue 3