## 4 Discussion  
According to the results of our study, histological and histomorphometric analysis demonstrated that the seeding of PDLSCs onto \(\beta\)-TCP, both untreated and pretreated with CAP, led to improved osteogenesiseration. Our results also showed that after two weeks, most of the sites that were treated with PDLSCs had a minor or no inflammatory reaction compared to the groups without the cells, in which the inflammation was moderate up to the fourth week. In addition to histological examination of the tissue samples, we assessed the expression of several osteogenic markers, namely BMP-2, BMP-4, collagen-1, Runx-2, and osteonectin, via immunohistochemical staining. Bone morphogenetic proteins belong to the TGF-\(\beta\) superfamily and are involved in both initiation and terminal osteogenic differentiation [30]. Among other family members, BMP-2, -4, -6, -7, and -9 are especially important for bone formation, as they induce a signaling cascade involving the Runx-2 transcription  
Figure 7: Immunohistochemical analysis of the osteonectin-positive cells in the defects after 2 (**a–d**) and 4 (**e–h**) weeks postoperatively: (**a,e**)–group I—\(\beta\)-TCP; (**b,f**)–group II—plasma-treated \(\beta\)-TCP; (**c,g**)–group III—\(\beta\)-TCP and PDLSCs; (**d,h**)–group IV—plasma-treated \(\beta\)-TCP and PDLSCs. (i) Median values of immunostaining calculated by determining the osteonectin-positive areas (brown-colored cells) are presented on the graph. The magnification in all the micrographs is \(40\times\). **\(p<0.01\); ***\(p<0.001\)—compared with group I; \(p<0.05\)—compared with group III. factor to increase osteeoprogenitor cell proliferation, differentiation, and synthesis of extracellular matrix (ECM) components [31]. Collagen-1 is the main element of the bone ECM and is also expressed at the earliest stages of osteogenesis [32], while osteonectin is a glycoprotein expressed in more mature osteoblasts that binds Ca and initiates mineralization and cell-ECM interaction [33]. In general, we observed the highest percentages of immunostained cells for all the tested markers in the defects filled with CAP-treated \(\beta\)-TCP and PDLSCs, which additionally supports the beneficial therapeutic effects of such a combined approach on osteeoregeneration. BMP-2, BMP-4, collagen-1, and Runx-2 were increased already two weeks after the implantation of the plasma-treated material seeded with the cells. This increase in the early markers of osteogenesis implies an accelerated initial phase of bone healing. Although both groups with the cells showed increased production of BMPs, the defects filled with CAP-treated \(\beta\)-TCP and PDLSCs showed significantly more BMP expression than with the cells seeded on untreated material. The increased presence of BMPs in lesions treated with PDLSCs may be due to the increased production of these factors by the implanted MSCs, which stay longer at the implantation site due to plasma-induced alterations in the grafting material. On the other hand, CAP-treated \(\beta\)-TCP alone stimulated collagen-1 production after two weeks and osteonectin production after four weeks, which is in line with their respective roles in the dynamics of bone formation. The same trend was demonstrated in the group with CAP-treated \(\beta\)-TCP with the cells. It is interesting that four weeks after the implantation, collagen-1 and osteonectin were expressed more in the defects filled with PDLSCs and CAP-treated \(\beta\)-TCP than in those filled with untreated \(\beta\)-TCP and cells. This could be the effect of the alteration in the hydrophilicity of the plasma-treated surface. Even though a few studies have investigated the influence of plasma-treated biomaterial surfaces on pro-osteogenic cell activities in vitro [15; 34; 35], to the best of our knowledge, there is no research that has explored the in vivo effects of surface-modified calcium phosphate biomaterials in combination with MSCs on bone regeneration.