4. Characterization of the Stemness Features of CGF Cells and Osteogenic Potential
In recent years CGF was widely studied as an autologous blood derivative able to promote tissue repair, vascularization, cell migration, and differentiation
[11][18][19][20][21]. Tissue repair is a complex mechanism that takes place over four phases: inflammatory process, cell proliferation, differentiation, and ECM remodeling. The process involves cytokines, growth factors, and MMPs
[15]. Despite a large literature on CGF use and applications in the regenerative medicine field
[20][22], up to the present, no data are provided on the metabolomic profile of CGF, and very few studies investigated the kinetic release of CGF growth factors and MMPs over a long time and analyzed the CGF cellular component. The aim of this work was to characterize the CGF metabolites composition, the amount of growth factors and MMPs released by CGF over a period of 28 days, and to study in detail the CGF cellular components.
GC/MS metabolomics analysis highlighted the high concentration of L-glutamic acid and taurine in CGF and the statistically different amount of the two analytes between the CGF and PPP fractions. These results are quite interesting considering the CGF application in the field of regenerative medicine. Indeed, it was demonstrated that ECM proteins and biomaterials, functionalized with amino acid sequences rich in glutamic acid, induced osteogenic differentiation, and mineralization of marrow stromal cells
[23]. In fact, glutamic acid residues are known to act as a nucleation point for calcium phosphate mineralization
[24]. Furthermore, taurine, a non-essential amino acid, has been shown to have positive effects on bone mass and influence bone metabolism
[25]. Taurine was also shown to promote the differentiation of human MSC into osteoblasts and to upregulate the expression of osteoblast markers as osterix, Runx2, osteopontin, and alkaline phosphatase via ERK1/2 signaling
[26]. In a recent study, it was reported that the ability of CGF to promote the osteoblast differentiation of BMSC
[11]. This capacity could be due to the high levels of L-glutamic acid and taurine and to prolong release from CGF of some growth factors, as reported in the present study. In fact, the initial amount of some bioactive molecules extracted from CGF was analyzed soon after preparation, then their release from CGF was quantified over time. It was found that CGF extract contained growth factors such as VEGF, TGF-β1 and BMP-2, and MMPs (such as MMP-2 and MMP-9), confirming previous studies
[27][28][29]. Moreover, to mimic the natural release of soluble factors, we cultured CGF, without any manipulation, in cell culture medium, at different times, until 28 days. We found that growth factors and MMPs were gradually released over time up to 28 days from CGF preparation, following specific release kinetics. In particular, VEGF was released slowly up to 14 days, when it reached its maximum value and gradually decreased over time. Similar to VEGF, TGF-β1 and BMP-2 were also released slowly. They peaked at 21 days, and their values remained high up to 28 days. The matrix-degrading enzymes MMP-9 and MMP-2 were released faster than the growth factors and peaked after seven days, with MMP-9 more abundant than MMP-2, then gradually decreased over time. The present findings reported, for the first time, a continuous and prolonged release of multiple bioactive factors over time, suggesting that CGF is suitable in promoting the complex and long process of tissue regeneration. To the best of our knowledge, 28 days is the longest time analyzed for the release of factors from the CGF. Indeed, previous studies analyzed CGF growth factors release within shorter time intervals
[9][30][31][32].
Two phases in the release of growth factors by CGF have been reported
[33]: an immediate phase, which could be attributed to the instantaneous release from activated platelets during centrifugation or to simple diffusion; a late phase with accumulation peaks at 14 days, which could be explained by the release of growth factors after degradation of the fibrin structure and by the production of growth factors from the CGF resident cells
[20][34][35].
Concordantly, it was found that the growth factors and MMPs released in the conditioned medium from the CGF reached higher amounts than the initial ones extracted from the CGF, suggesting a role of CGF-resident cells in the synthesis and secretion of these factors. In particular, the amount of VEGF in the CGF-conditioned medium after a 14-day incubation was even greater than the amount of VEGF extracted from the CGF. These results agree with our previous study showing that CGF-derived cells expressed and released angiogenic factors, including VEGF
[21].
Growth factors are considered essential elements in tissue regeneration; they play a critical role in regulating processes involved in wound healing and tissue repair, so their amounts and release kinetics, as it found, could be important to better assess the efficacy of CGF.
Among the multiple growth factors released by CGF, VEGF is a crucial molecule in tissue repair and regeneration since it is implicated in angiogenesis, blood vessel growth from pre-existing vasculature and vasculogenesis, and de novo formation of blood vessels
[36]. VEGF has been demonstrated to stimulate endothelial cell proliferation and promote angiogenesis by binding to a high-affinity receptor, and its signaling is considered a rate-limiting step in the initiation of angiogenesis
[37]. However, due to the very short half-life of VEGF
[38][39], low efficacy is achieved when administered as free proteins because high doses have a prohibitive cost and often cause undesirable effects
[40]. Therefore, a sustained release system of VEGF is required to provide ideal therapeutic effects, which could be achieved by CGF application.
In our experimental conditions, TGF-β1 was the most abundant growth factor contained in and released by CGF over time. TGF-β1 is a secreted protein that regulates many cellular functions, including the control of immune and stem cell growth, proliferation, differentiation, apoptosis, development, and tissue remodeling following injury
[41][42]. Thus, the release of TGF-β1 is desirable in wound healing sites and particularly in the oral cavity, where several types of cells, like fibroblasts and osteoblasts, must be stimulated to proliferate. Temporal and spatial activation of TGF-β is involved in the recruitment of stem/progenitor cells and participation in the tissue regeneration/remodeling process. BMP-2, another important member of the TGF-β superfamily, plays a key role in the development of bone and cartilage. It is a highly potent growth factor able to promote the differentiation and maturation of osteoblasts
[43]. It was found that BMP-2 was the growth factor released by CGF in the lowest amounts. However, BMP-2 has been shown to be released from platelets, mainly at low pH
[44], which is the common environment of wound healing sites
[45]. Therefore, the use of CGF could improve the repair processes by locally stimulating the release of BMP-2 at the injury site. Moreover, it was also found that CGF released the MMP-2 and MMP-9. MMPs are matrix-degrading enzymes implicated in many biological processes, including inflammation and cell migration during wound healing and tissue repair in coordination with several growth factors and cytokines
[46].
The importance of the resident and circulating cells in the processes of tissue regeneration is well established
[14][15]; therefore, besides growth factors and molecules contained in and released by CGF, which focused on the characterization of its cellular components.
SEM observation did not reveal the presence of cells on the surface of CGF but showed a fibrin framework denser than inside of CGF, where large populations of activated platelets and cells were present. Immunohistochemistry analysis of CGF showed a very uniform distribution of nucleated cells entrapped in the fibrin network. The sections reacted positively to CD34, CD45, and CD105 immunolabelling. Indeed, the presence of different cell populations is known: hematopoietic stem cells, lymphocytes, monocytes, and fibroblast-like cells
[1]. Our recent findings showed that when CGF, without manipulation, is released into the culture medium, cells are able to adhere to the plate and proliferate
[21]. Here it was shown that the release of cells from CGF seemed to be rather slow, and most of the cells were found in the plate only after cutting CGF on the 14th day. This aspect could be correlated with hematoxylin-eosin staining data and with CGF fibrin network structure observed by SEM analysis: indeed, while at the initial stage CGF cell distribution was homogeneous all over the section, after two and four weeks, cells seemed to migrate from the center where fibrin network was less dense to the peripherical area of the sections, where fibrin appeared to be more densely intertwined. This scenario might explain either why cells were retained into CGF so long (up to 28 days) and the sustained release kinetics of CGF growth factors and MMPs.
Di Liddo et al. recently reported that the leukocyte- and platelet-rich fibrin product called CPL-MB acts as an artificial stem niche containing autologous multipotent cells with defined stemness properties
[47]. In our work, CGF primary cells showed fibroblast-like and spherical morphology; however, after few passages, cell populations appeared to be enriched in spindle-shaped cells and showed different surface markers with respect to cells resident in the CGF. Indeed, adherent cells expressed a high level of CD105 and CD45 surface markers; whereas, CD34 was scarcely detectable. Since it was found that CGF primary cells exhibited monocyte markers, such as CD31, CD45, CD14, and CD36,
[48][49] which was assumed that they might be monocyte-derived cells. The primary CGF cells did not appear as mesenchymal stem cells derived from peripheral blood since they did not express CD73 and CD90 mesenchymal markers; however, they showed mesenchymal, hematopoietic, and endothelial stem cell features. Indeed, it has been demonstrated that monocyte-derived cells expressing CD105, CD45, and CD14 exhibit mesenchymal cell features and are able to differentiate into different cell lines
[48].
In addition, CGF primary cells express genes that denote the molecular signature of stem cell pluripotency, including Oct3/4 and Nanog. The transcription factor Oct3/4 is thought to be indispensable for pluripotency in stem cells and is expressed in multipotent progenitor cells isolated from peripheral blood
[17]. Nanog is a key factor in the self-renewing of embryonic stem cells, which remained pluripotent after multiple passages, but it has a heterogeneous expression mode; indeed, Nanog-negative cells show a higher propensity for differentiation
[50]. The results show low Nanog mRNA levels. It also reported high STAT4 mRNA levels. STAT4 is a key transcription factor involved in promoting cell-mediated immunity, but its expression is not restricted to lymphoid cells. Activated monocytes expressed STAT4 in response to Interferon-alfa
[51], a cytokine that downregulates osteoblastogenesis
[52], though increases the formation of calcific nodules under osteogenic conditions in human aortic valve interstitial cells
[53].
Finally, to better characterize the use of CGF in the field of regenerative medicine, since CGF primary cells seem to display several pluripotency markers, the ability of these cells to differentiate into osteoblasts was tested. Interestingly, we found that CGF primary cells, kept three weeks in osteogenic medium, were able to differentiate into osteoblasts as demonstrated by the formation of mineralized nodules, the expression of the osteogenic markers RUNX2, COL1a1, and OCN, and the loss of stem cell markers
[11]. These results suggest that CGF could also represent a source of cells with stem features, thus expanding its potential applications.