2.1. Cell Viability and Cellular Immunophenotype
In a standard culture medium without osteogenic differentiation additives, the viability of adherent fibroblast-like cells of human adipose tissue was greater than 92% by day 14 (
Table 1). More than 95% of the adherent cells expressed the markers CD73, CD90, and CD105 and exhibited weak (less than 1%) expression of the hematopoietic cell markers CD45, CD34, CD20, and CD14 (
Table 1). In a previous study, after 21 days of cultivation in specialized StemPro® Differentiation Kit medium (Thermo Fisher Scientific, Waltham, MA, USA), the test culture of human adipose-derived MSCs (hAMSCs) showed positive staining for alizarin red (osteoblasts), alcian blue (chondroblasts) and oil red O (adipocytes) [
26]. The minimal morphological criteria for cultured MSCs are (1) a viability of greater than 90% [
27]; (2) positivity for the markers CD73, CD90, and CD105 and negativity for the blood cell markers CD45, CD34, CD20, and CD14 [
19,
20]; and (3) the ability to adhere to plastic and differentiate in vitro toward osteogenesis, chondrogenesis and adipogenesis [
28]. Thus, the culture of hAMSCs used in this experiment satisfied the minimal morphological criteria for MSCs.
The physicochemical properties of materials affect the functional activity and differentiation of MSCs [
29]. The surface roughness of the microarc CaP coating can control the osteogenic differentiation of adherent MSCs upon direct contact, as reflected in the correlations of the Ra index with cell markers of osteoblasts (ALP and OCN). In this case, the average roughness index (Ra) is closely related to the thickness and mass of the CaP coating [
21], which, when degraded, releases calcium and phosphorus ions. These ions can regulate the expression of MSC markers [
30]. Indeed, contact of hAMSCs with CaP-coated samples for 21 days was found to suppress the immunophenotypic traits of hAMSCs adhered to plastic as the relief of the CaP coating increased, while the population of hematopoietic cells (CD45
+CD34
+CD14
+CD20
+) increased [
22].
The results shown in
Table 1 indicate that a statistically significant increase (almost 2-fold) in the presentation of hematopoietic cell antigens due to the contact of the cell culture with CaP-coated titanium samples occurred by the 14th day of observation and slightly preceded the decreases in the expression of CD73, CD90, and CD105 markers.
2.2. In Vitro Osteogenic Differentiation
The main part of the plastic surface was occupied by fibroblast-like cells weakly stained by alizarin red S when hAMSCs were cultured for 14 or 21 days in a standard nutrient medium without the osteogenic supplements. Single small foci of calcification appeared in the ECM, which showed the osteogenic differentiation of individual stem cells (
Figure 1a,c) including hAMSC culture after 14-day contact with the microarc CaP coating (
Figure 1b). In turn, significant increase in the number and an area of mineralized nodules (
Figure 1d,
Table 2) indicated an initiated differentiation of hAMSCs into osteoblasts around the CaP-coated Ti substrates.
Enhanced mineralization (calcification) of the ECM in the MSCs cultured on plastic, caused by the microarc CaP coating, was identified previously by day 21 via alizarin red staining [
21,
22,
26], indicating in vitro the appearance of MSCs in osteoblastic hematopoietic niches [
31]. Apparently, the establishment of such microterritories begins at 14 days under conditions of autocrine and paracrine secretion of chemokines as signaling molecules in the hematopoietic niches induced by the CaP coating [
5].
The thickness of one side (~46 µm,
Table 1 and
Table 2) of the soluble microarc CaP coating allows us to consider it to be scaffold-like, and to be able to determine the behavior of hAMSCs by direct contact with the microrelief, as well as by indirect action through Ca
2+ ions and inorganic phosphate (Pi). A wavelike dissolution/precipitation of a microarc CaP coating with a loss of more than 9% of the initial thickness was shown to occur after 8 weeks of in vitro degradation in a model biological fluid [
32]. For a calcium-deficient (Ca/P ratio < 1) microarc CaP coating, the Ca
2+ and to a greater extent Pi concentrations in model biological fluids are usually 0.5 mM per week [
33] due to the balance of ion release and reverse precipitation. Both extracellular calcium and phosphorus contribute to osteogenic differentiation and mineralization of the ECM over a wide range of concentrations (0.5–16 mM for Ca
2+; 0.09–8 mM for Pi) [
8,
34].
A gradient yield of Ca
2+ and Pi was also noted for other types of CaP-containing scaffolds [
35], accompanied by the formation of an alkaline microenvironment that determines the realization of the osteogenic potential of MSCs [
36]. This potential can be realized through various mechanisms, including CaR activity, the activation of osteoblastic differentiation and the formation of mineralization nodules in the ECM [
6]. Ca
2+ and Pi upregulate the expression of OCN, Runx2 and BMP-2 [
30]. Pi can initiate signaling via extracellular signal-regulated kinases (ERK) and cAMP/protein kinase pathways, and activation of these signaling pathways increases
BMP-2 expression [
37].
2.3. Expression of Osteogenic, Cytokine, and Chemokine Genes
Numerous number of genes and transcription factors are necessary for the proliferation and differentiation of MSCs. Moreover, the individual knockout of each of these genes only partially changes the behavior of MSCs [
9]. Therefore, for the development of regenerative medicine, the molecular basis (primarily genes and transcription factors) of the response of MSCs [
10] to various stimuli must be identified.
Correlation analysis showed that in hAMSC cultured for 14 days around samples with a microarc CaP coating,
RUNX2-
BMP2- and
BMP6-
ALPL expressions were correlated with high coefficients (
r = 0.96;
p < 0.05;
n = 7) (
Figure 2). However, as shown in
Table 3, statistically significant upregulation of osteogenic genes (1.4-fold that in hAMSCs cultured on plastic) was observed only for
RUNX2,
BMP6, and
ALPL (
Table 3).
Notably, as indicated in
Table 3, the expression of osteogenic genes began along with 7-fold increase in the transcription of
hTERT, which regulates cell proliferation. Day 14 corresponds to the completion of the formation of three-dimensional MSC/osteoblast cell culture conditions and the beginning of the formation of mineralization nodules.
The transcription factor Runx2 (core-binding factor subunit alpha-1=cbfa1) is considered a main determinant of osteoblast genesis from MSCs [
38]. It regulates the expression of many osteoblast genes (ALP, OPN, OCN, and matrix metalloproteinase 13). Runx-driven osteogenesis is characterized by sequential expression of marker molecules (BMPs, ALP, and OCN), which alone do not always lead to mineralization of the bone matrix [
12].
ALPL gene expression occurs during the early stages of osteogenesis [
12]. ALP activity is necessary for subsequent mineralization of the ECM in the alkaline microenvironment [
36] and appears within 7–14 days after contact with osteogenic scaffolds [
35]. Ca
2+ ions formed during degradation of biomaterials are inducers of ALP expression [
35] and the differentiation of MSCs into osteoblasts is triggered through the expression of calcium-binding proteins [
39]. In turn, an increase in the level of ALPL gene mRNA expression ensures the presence of free Pi in the cellular microenvironment, and this Pi binds with calcium to form CaP minerals in the bone [
37].
In turn, the BGLAP gene encodes OCN, a protein that is expressed during the late stages of osteogenesis, is a marker of the terminal differentiation of MSCs into secreting osteoblasts and is produced during ECM mineralization [
40]. The promoter of this gene is regulated by the transcription factor Runx2 and is activated by the BMP-2 [
41].
Thus, the increased expression of the hTERT, RUNX2, BMP6, and ALPL genes but not
BGLAP on day 14 of in vitro culture reflects a chain of events associated with the ongoing proliferation and differentiation of hAMSCs into osteoblasts as well as with the early stages of ECM mineralization. Indeed, a mineralized ECM with intense alizarin red staining was observed only by day 21 in a coculture system containing hAMSCs and a micro-arc CaP coating [
5].
Most researchers agree that osteoblasts attach more easily to surfaces with a coarse microtopography [
42,
43], which may enhance osteogenic cell differentiation [
44] than to those with a smooth microtopography. Independent groups of scientists revealed that increased relative mRNA expression levels of OPN, OCN [
45],
RUNX2 and
ALPL [
17] are associated with osteogenic activity, and found that bone marrow MSCs cultured in vitro on rough surfaces demonstrate more intense alizarin red staining than MSCs under similar conditions on flat [
17,
46] and plastic [
22] surfaces. These results may indicate that surface topography modulates the osteogenic differentiation of MSCs and the ECM mineralization.
The results obtained by Sutherland et al. (2005) also indicate that cell attachment and proliferation are dependent on surface topography and that the cytoskeleton exhibits higher stress levels on coarser surfaces [
47]. According to McCafferty et al. (2014), stiffness, topography, and surface chemistry can cause cytoskeletal remodeling and focal adhesion formation prior to MSC differentiation via integrin-mediated signaling pathways [
48]. Cytoskeletal alterations can affect the organization and distribution of organelles and DNA, which regulate the functioning and biological activity of cells [
49]. Shafrir et al. (2002) showed that microfilaments cross the nuclear pores and connect to the nuclear membrane, thereby providing a path for the transduction of signals induced by mechanical stimuli [
50].
Research by McBeath and Prowse (2013) confirmed that the topography of the artificial matrix can regulate the osteogenic differentiation of MMSCs by altering the cytoskeleton [
51,
52]. In addition, research has suggested that the distribution of the actin cytoskeleton, in particular filamentous actin (F-actin), varies on rough surfaces [
53]. The actin cytoskeleton plays an important role in the osteogenic differentiation of MSCs [
54]; it is modified and changed as MSCs differentiate into osteoblasts instead of the large number of thin, parallel microfilament bundles that propagate throughout the cytoplasm in undifferentiated MSCs, thick bundles of actin filaments are located on the periphery of differentiated cells [
54].
Structural alterations in the cytoskeleton lead to signal transduction to the nucleus and are associated with the activation of the nuclear transcription factors YAP (yes-associated protein 1) and TAZ (WW domain-containing transcription regulator protein 1, WWTR1) which are regulated by the actin cytoskeleton; this link explains the participation of mechanical stimuli in the osteogenic differentiation of MSCs [
55,
56]. Yang et al. (2016) convincingly demonstrated that different surface topographies differentially affected the activation of the transcription factors YAP/TAZ, leading to changes in the relative expression levels of osteogenic genes [
17]. In turn, the transcription factors YAP/TAZ mediate the differentiation of MSCs by inducing the Runx2 coactivator, an osteoblast-specific transcription factor that affects the expression of osteogenic genes [
57,
58].
However, we did not find that the expression of the tested osteogenic genes in a 14-day in vitro culture of hAMSCs on plastic around samples coated by soluble microarc CaP coating was dependent on its physical parameters (roughness, weight, and thickness). The correlation coefficients of physical and biological factors varied between −0.43 and 0.52. Apparently, there are other pathways leading to activated expression of osteogenic differentiation-related genes that are not associated with CaP surface features and poor concentrations of Ca2+ and Pi in an intercellular medium.
Considering this possibility, the negative correlation between
RUNX2 mRNA expression and the production of the osteoclastogenic macrophage colony stimulating factor (M-CSF) (
r = −0.79;
p < 0.05) is interesting (
Figure 2). Lienau et al. (2010) showed that hematopoietin gene expression is suppressed during chondrogenesis and endochondral ossification but not during bone remodeling [
59].
In other words, in the context of the actively developing field of osteoimmunology [
60], the osteogenic activity of numerous cytokines and chemokines [
14] is noteworthy as a variation on the cytokine/chemokine-mediated initiation of osteogenesis, constituting an alternative to the known signaling pathways.
2.4. Cytokine and Chemokine Secretion
In our study, by the 14th day of contact with CaP-coated Ti substrates the relative mRNA expression levels of the IL-18 gene and genes encoding some chemokines (CXCL1, CCL27) decreased significantly. However,
CCL7 activity remained elevated relative to that in hAMSCs cultured on plastic without CaP-coated samples (
Table 3).
Secretion by hAMSCs cultured for 14 days on plastic (2D control) (
Table 4; abbreviations of cytokines/chemokines are enclosed) was classified according to [
61]:
High (more than 1 ng/mL) concentration of SCGF-b;
Average (0.1–1 ng/mL) concentrations of HGF and MIF;
Low (1–100 pg/mL) concentrations of IL-2Ra; IL-3; IL-12 (p40); IL-16; IL-18; IFNα2; M-CSF; β-NGF; LIF; MCP-3 (CCL7); MIG (CXCL9); GROα (CXCL1); SCF; SDF-1α (CXCL12); TRAIL CTACK;
Minimal (<1 pg/mL) concentrations of IL-1α and TNFβ mediators;
The inflammatory biomolecules are able to regulate osteogenesis [
2,
14]. In the presence of samples with a microarc CaP coating, a change in the secretory profile of hAMSCs was observed (
Table 4). Specifically, the concentrations of IL-18, GROα (CXCL1) and SCF increased significantly (
p < 0.05) (by 26%, 15% and 267%, respectively). In contrast, the levels of HGF (by 44%) and LIF (by 29%) decreased.
During the formation of a microarc CaP coating, its thickness, controlled by the technological parameters of the microarc device, determines the mass of CaP in the coating (
r = 0.96;
p <0.001;
n = 7) and the average surface roughness index (Ra) (
r = 0.96;
p < 0.001;
n = 7). The tested physical properties of the microarc CaP coating (thickness, mass, and roughness) in the 14-day hAMSC culture affected the secretion of IL-18 (
r = 0.77–0.79;
p < 0.01;
n = 10) and SDF- 1α (CXCL12) (
r = 0.69–0.73;
p < 0.02;
n = 10) (
Figure 2).
Notably, the secretion of inflammatory/migratory cytokines/chemokines was in antiphase (according to the feedback principle) with the expression of the mRNA of their genes, with the mRNA expression of the corresponding genes, with the exception of the CLEC11A gene/SCGFb pair (
Table 3 and
Table 4). In turn, strong inverse correlations were found between the mRNA expression level of
IL-18 with that of
ALPL (−0.79;
p < 0.03;
n = 7) and
BMP6 (−0.82;
p < 0.02;
n = 7) and of
BMP6 with those of the chemotaxis-related genes CXCL1 (−0.79;
p < 0.04;
n = 7) and CCL27 (−0.82;
p < 0.02;
n = 7) (
Figure 2). According to Cornish et al. (2003), IL-18 plays a role as an autocrine/paracrine mitogen in both osteogenic and chondrogenic cells [
62] and may be a trigger of osteogenic differentiation [
63].
The chemokine GROα (CXCL1), a mediator of MSC chemotaxis [
64], apparently contributes to the formation of a network of inflammatory cytokines/chemokines and one of the hematopoietic stem cell (HSCs) growth factors (SCGF-b) (
Figure 2). CXCL1, on the other hand, can induce osteoclastogenic activity [
65]. Therefore, the molecular (gene/secretory) activity of hAMSCs in a 14-day in vitro culture (
Table 3 and
Table 4) may indicate the end of the proinflammatory phase and a switch to regenerative (osteogenic) processes [
14], induced by the physical-chemical properties of the microarc CaP coating.
Interestingly, increasing level of IL-18 is directly correlated with an increasing concentration of SCF (
r = 0.82;
p < 0.03;
n = 7), a key signaling niche molecule in HSCs [
66]. Moreover, the increase in the percentage of cells expressing hematopoietic markers CD45
+34
+14
+20
+ was correlated with an increase in the SCF concentration in the hAMSC culture (
r = 0.77;
p < 0.05;
n = 10) (
Figure 2).
Osteoblasts form niches for HSCs including lymphoid stem cells [
31]. Proinflammatory IL-18 is likely involved in the initiation of both the osteogenic differentiation of hAMSCs and the formation of osteoblastic hematopoietic niches. The low concentrations of potential inducer molecules in the total volume (1.5 mL) of the culture medium should not be confusing. The local concentrations of these factors can be extremely high near individual cells and the forming hematopoietic microterritories. Indeed, we previously noted the association of osteogenic/hematopoietic processes in cultured hAMSCs in contact with a microarc CaP coating for 14 days [
5].
In mice, acute bone inflammation occurring within 7–14 days after injury triggers the subsequent phase of skeletal repair, leading to fracture healing [
4]. If the timely switching of the phases of inflammation/regeneration is disrupted, including disruptions provoked by the implant, complications develop (e.g., infection, osteonecrosis, osteoporosis, pseudoarthrosis, and bone nonunion), making the prognosis significantly less favorable. Curing large bone tissue defects is a major clinical problem worldwide. Therefore, the expression of inflammatory cytokines and their receptors is of functional importance to bone remodeling, and signaling pathway modulation is a promising strategy for controlling bone regeneration [
14]. However, the timing and mechanisms required for the induction of the inflammatory and regenerative reactions remain unclear.