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Bone Sialoprotein enhances Bone Regeneration
by a Tukey-HSD or Games-Howell post hoc test. In lower BSP concentration demonstrated a better effect on
contrast, non-normally distributed data were evaluated proliferation than the 5 times higher concentration speaking
with the Kruskal–Wallis test. For pairwise comparisons, for a concentration dependency, also demonstrated in
the Mann–Whitney U-test was used. P < 0.05 was another study with BSP on titanium . It might be that
[11]
considered statistically significant (*P < 0.05, **P < 0.01, BSP in higher concentration effects other cells engaged
and ***P < 0.001). Due to multiple testing, the P-values in bone remodeling, for example, osteoclasts , however,
[8]
were adjusted through Bonferroni-Holm method. this has to be addressed in follow-up experiments using
other cells as well as wider concentrations ranges.
3. Results and discussion In summary, BSP was able to induce proliferation

3.1. BSP immobilized in collagen type I enhances of hOB in mono- as well as in co-culture speaking for a
proliferation of hOB in mono and in coculture positive effect, when BSP is applied in combination with
collagen type I.
The first step to characterize the effect of BSP immobilized
in collagen type I were in vitro assays. hOBs were seeded 3.2. Effect of BSP encapsulation in collagen gels
in collagen type I gels with and without BSP (Figure S1) on gene expression in hOB monoculture
and proliferation was analyzed using the alamarBlue To further understand the effect of BSP encapsulated
assay (Figure S2A). Next proliferation in a coculture in collagen type I, gene expression analyses were
model with endothelial cells was analyzed as it has been performed. The ALP expression of hOBs was significantly
demonstrated that this system is effective in regulating downregulated in collagen gels containing 5 μg/mL BSP
cell proliferation and osteoblastic differentiation after 1 day as well as in gels with 1 μg/mL BSP after
(Figure S2B). Figure S2 demonstrates that BSP applied 4 days. However, expression increased after 7 days with
in low concentrations enhanced proliferation of primary the lower BSP concentration of 1 μg/mL (Figure 2A).
hOBs in mono- and in co-culture with endothelial cells. Gene expression analyses of hOB monocultures
The most significant effect was observed after 4 days. revealed a decreased expression of OPN after BSP
Regarding morphology of hOBs, no differences addition (Figure 2B). After 4 days, two key factors of
were observed whether they were seeded in mono- or osteoblastic differentiation were upregulated (SP7 in all
in co-culture with HUVECs (Figure S1). One varying BSP groups and RUNX2 in high concentrated BSP gels
aspect in co-culture is the ratio of hOB and HUVECs. In (Figure 2C and D).
our experiments, we used a ratio of 1:1; ratios ranging up
to 1:10 have been described depending on the endothelial 3.3. Effect of BSP encapsulation in collagen
cell type used [35,36] . However, in our setting, the ratio of gels on gene expression in hOB and HUVEC
1:1 has shown to be best . coculture
[30]
Comparing the effects of BSP coated materials on
different cells, varying effects were observed. Moreover, To imitate the physiological surroundings, cocultures of
the materials used as well as the conditions employed hOB and HUVEC were analyzed to detect intercellular
varied making comparisons difficult. When human influences. In cocultures, the trend in gene expression
primary osteoblasts were seeded on titanium implants, of osteogenic markers was similar to those of hOB
a rather suppressive effect of BSP on proliferation was monocultures. Significant changes occurred merely after
detected . Regarding cell proliferation on calcium 4 days of culture. OPN (1 and 5 μg/mL BSP; Figure 2F)
[11]
phosphate scaffolds, a low concentration of BSP could as well as RUNX2 (5 μg/mL BSP, Figure 2G) expression
enhance proliferation of human primary osteoblasts increased. In coculture examinations, the SP7 expression
to a small extent 3 days after seeding . Other studies was not affected by BSP supplementation (Figure 2H)
[12]
demonstrated that BSP coating showed no positive in contrast to a significant upregulation by both BSP
influence on cell proliferation . However, this effect concentrations in hOB monocultures.
[37]
could also be an indication for increased osteogenicity Enhancement of the transcription factors RUNX2
as several studies have demonstrated that an increase and SP7 gene expression caused by BSP employing
in osteogenic activity is correlated to a decrease in cell various coating or expression methods as well as different
proliferation [6,38,39] . materials (titanium, calcium-phosphate-cements, cell
An increase in proliferation can also be observed culture materials, etc.) has already been demonstrated
when cells are cultured in coculture. As described in various studies [4,11,12,41] . The observation of higher
by several authors, endothelial cells and osteoblasts RUNX2 and SP7 levels on day 4 compared with those on
influence proliferation and differentiation of each other day 7 is consistent with the findings of Gordon et al. who
due to growth factor release . Another remarkable point showed that supplementation of 2 μg/mL BSP to primary
[40]
is the BSP concentration applied. In our experiments, the rat calvaria osteoblasts significantly increased SP7 and
258 International Journal of Bioprinting (2022)–Volume 8, Issue 3