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CGF Membrane Promotes Periodontal Tissue Regeneration Mediated by hUCMSCs through Upregulating TAZ and Osteogenic Differentiation Genes

Concentrated growth factor (CGF) membranes are widely used in basic and clinical research of soft and hard tissue regeneration, but its effect on periodontal tissue regeneration is less studied. This study explored the role of CGF membranes in periodontal tissue regeneration mediated by human umbilical cord mesenchymal stem cells (hUCMSCs). HUCMSCs and human periodontal ligament fibroblasts (HPLFs) were extracted and identified by microscope and flow cytometry. The effects of the extracted CGF membrane on cell viability, osteogenic differentiation ability, osteopontin (OPN) expression, alkaline phosphatase (ALP) content, and osteogenic differentiation-related genes (Runt-related transcription factor 2 (RUNX2); osteocalcin (OCN); ALP), Tafazzin (TAZ) expression, and nuclear transfer were examined by MTT assay, alizarin red staining, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Rescue experiments were performed to examine the effects of TAZ transfection and cell coculture. In the identified hUCMSCs (positive expressions of CD29, CD44, CD146, and CD105), overexpressed TAZ (pc-TAZ) enhanced the promotive effect of CGF membrane on cell viability, cell cycle, mineralization, ALP content and expressions of OPN, TAZ and osteogenic differentiation-related genes, and nuclear transfer. However, silencing TAZ showed opposite effects. The coculture of hUCMSCs and HPLFs further promoted the basic biological functions of HPLFs by upregulating osteogenic differentiation-related genes and COL-1 but downregulated MMP1 expression. Pc-TAZ could enhance the effect of CGF membrane on promoting periodontal tissue regeneration. CGF membrane promoted periodontal tissue regeneration through upregulating TAZ and osteogenic differentiation-related genes.


Introduction
Teeth are the hardest organs in the human body, but periodontal tissues are vulnerable to external stimuli under the influence of various external factors. Lesions in the periodontal tissues could cause periodontitis, leading to the destruction and absorption of periodontal ligaments, cementum, alveolar bone, and other dental support tissues [1,2]. At present, the main periodontal treatment in clinical prac-tice is to remove tartar, plaque, and diseased root surface to eliminate local inflammation of periodontal tissues and prevent further progression [3]. Currently, tissue regeneration technology has become a promising strategy for the treatment of periodontal tissue lesions [4]. Since the guided tissue regeneration (GTR) technology was proposed in 1980, tissue regeneration engineering has attracted much research attention in the past ten years [5,6]. Mesenchymal stem cells play an important role in maintaining tissue self-renewal and self-repair and, therefore, are often used as seed cells for tissue regeneration engineering research [7].
Human umbilical cord mesenchymal stem cells (hUCMSCs) are a type of adult stem cells in the mesenchymal stem cell family, with a high multidirectional differentiation potential and self-renewal ability [8]. Studies confirmed that hUCMSCs could be successfully induced to differentiate into osteoblasts, adipocytes, nerve cells, skin fibroblasts, and other tissue cells in vitro [8,9]. HUCMSCs can express common stem cell surface markers such as CD29, CD44, and CD105 and also markers of embryonic stem cells such as NANOG and DNMT3B [10]. These features of hUCMSCs allow it to become seed cells widely studied in tissue regeneration engineering. Xue et al. [11] used liver homogenate supernatant to simulate the liver tissue microenvironment in vivo and finally induced hUCMSC to differentiate into hepatocytes; Yin et al. [12] found that infusion of hUCMSC caused localized M1 macrophages in the pancreas of type 2 diabetic mice to an anti-inflammatory M2-like state. However, there are few reports studying hUCMSC in the intervention of periodontal disease [13].
Concentrated growth factor (CGF) fibrin membrane is the third generation platelet enrichment [14]. Compared with previous generations of platelet enrichment, the preparation of CGF membranes does not require additional reagents to generate platelet activation or fibrin polymerization and is less likely to develop immune responses or cross infections [15,16]. CGF membrane has been extensively studied in bone tissue engineering, soft tissue injury, cardiovascular disease, and nervous system diseases [17,18] and used in periodontal surgery, extraoral surgery, and implants [19]. However, the roles of CGF fibrin membrane and hUCMSCs in periodontal tissue regeneration have not been reported.
TAZ, also known as the WWTR1 gene, is located on the human chromosome 3q23-q24 [20]. TAZ was originally discovered as a binding protein that can enter the nucleus and bind to transcription factors to activate transcription factor activity and promote gene expression [21]. TAZ functions in the conditional knockout of ephrinB1 in osteogenic progenitors to delay the process of endochondral ossification during fracture repair [22]. Whether the role of CGF fibrin membrane and hUCMSCs in periodontal tissue regeneration was related to TAZ was investigated in our research.
In this study, by preparing CGF fibrin membrane and extracting hUCMSCs and human periodontal ligament fibroblasts (HPLFs), the role and specific mechanisms of CGF fibrin membrane in periodontal tissue regeneration mediated by hUCMSCs were clarified.

Materials and Methods
2.1. Ethics Statement. All the umbilical cord tissues and the venous blood in this experiment were collected from the Department of Oral Medicine of The Second Hospital of Hebei Medical University and conducted under the approval from the Hospital Ethics Committee (No. 201810021FCK). The umbilical cords (n = 3) were obtained from cesarean women without any systemic diseases and used for the pri-mary culture of hUCMSCs. HPLFs were extracted from the premolars (n = 6) obtained from of the periodontal healthy, caries-free young volunteers aged 12-28 years old, together with the venous blood. This study was approved by the Ethics Committee of The Second Hospital of Hebei Medical University (No. 201811013KQK). The sample provider and their guardian signed informed consent for the clinical study.
2.2. Cell Extraction and Culture. The excised umbilical cord tissues were rinsed in normal saline and placed in a DMEM culture medium (C11995500BT, Invitrogen, USA) containing 4% of penicillin and streptomycin (SV30010, Hyclone, USA) at 4°C. After the umbilical cord was fixed, the envelope tissues were separated from the blood vessels of the umbilical cord using a pair of ophthalmic scissors, and the obtained umbilical cord tissues were cut to 1 mm 2 . Type I collagenase (1 mL, C0130, Gibco, USA) and Dispase (1 mL, 17105041, Invitrogen, USA) were used to fully digest the tissues at 37°C. After 16 hours (h), human umbilical cord mesenchymal stem cells were added to a DMEM medium with 20% fetal bovine serum (SH30096.03, Hyclone, USA) to terminate the digestion and resuspend the cells. The finally harvested human umbilical cord mesenchymal stem cells were cultured in a Herocell C1 carbon dioxide incubator (RADOBIO, China) at 37°C with 5% CO 2 . HPLFs were taken from premolars provided by volunteers. Before tooth extraction, volunteers gargled 3% hydrogen peroxide solution (M164, Beijing Land Bridge Technology Co., Ltd., China) for 3 minutes (min), and then the surface of the crown was cleaned with 75% alcohol. The extracted teeth were immediately placed in a prechilled DMEM medium (C11995500BT, Invitrogen, USA) containing penicillin and streptomycin (SV30010, Hyclone, USA) at 4°C. The periodontal ligament tissues were scraped by a sterile blade, cut into 1 mm 3 pieces and then transferred to a DMEM medium containing 20% FBS and cultured in a Herocell C1 carbon dioxide incubator (RADO-BIO, China) at 37°C with 5% CO 2 . After the separation and culture of the two cells, the growth and morphology of the cells were observed under a light microscope (Leica DM4 B & DM6 B., Leica, Germany).

Identification of hUCMSCs.
To confirm that the cells extracted were hUCMSCs, cell surface markers were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, USA). The cell density of hUCMSCs in the third logarithmic growth phase was adjusted to 1 × 10 6 cells/mL and then aliquoted into EP tubes (100 μL). One tube was added with rabbit anti-mouse IgG-PE as a negative control, and the remaining cell suspensions were added with Mesenchymal Stromal Cell Marker (CD44, CD45, CD90 CD29, CD105), Antibody Panel (ab93758, Abcam, UK), Anti-CD34 antibody (ab81289, Abcam, UK), and Anti-CD146 antibody (ab75769, Abcam, UK) and incubated at room temperature in the dark for 60 min. The antibody-incubated cells were resuspended in 500 μL of PBS and then transferred to a flow cytometer to detect the levels of CD29, CD44, CD45, CD34, CD146, and CD105 on the cell surface.

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Preparation of Concentrated Growth Factors (CGF) and
Cell Treatment. The vacuum blood collection tube containing the venous blood of the healthy volunteer was prepared for differential centrifugation as follows for a total of 13 min at 4°C: acceleration for 30 seconds (s); at 2700 rpm for 2 min; at 2400 rpm for 4 min; at 2700 rpm for 4 min; at 3000 rpm for 3 min; deceleration for 36 s and terminated. The blood in the blood collection tube was divided into three layers, the middle of which was the CGF gel layer. By discarding the upper serum and separating the lower red blood cells, the CGF fibrin membrane was removed, cut into 3 × 3 mm 2 , and placed in sterile physiological saline for later use. HUCMSCs were divided into the control group (without CGF fibrin membrane) and CGF1 group (with a piece of CGF fibrin membrane). The prepared CGF was used for cocultivation with hUCMSCs (1 × 10 4 cell/well). When the degree of cell fusion reached 80%, the hUCMSCs were used for subsequent studies.  2.11. Western Blot. By referring to previous research [24], RIPA Lysis Buffer (C05-01001, Bioss, China) was used for total cell protein extraction. In order to detect the distribution of genes in the nucleus and cytoplasm, Cytoplasmic and Nuclear Fractionation kit (SC-003, INVENT, USA) was used to isolate the cytoplasm and nucleus and further extract total proteins. The protein was transferred to the nitrocellulose filter membrane by the method of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%). The process of protein blocking and antibody incubation was completed briefly as follows: the protein on the NC membrane was blocked by the blocking solution for 2 h. The primary antibodies of related genes (TAZ, 1 : 500, ab224239, 44 kDa, Abcam, UK; collagen-1, COL-1, 1 : 500, ab64883, 181 kDa, Abcam, UK; matrix metalloprotein 1, MMP1, 1 : 1000, ab137332, 54 kDa, Abcam, UK; β-actin, 1 : 5000, ab8226, 42 kDa, Abcam, UK; Lamin B1, 1 : 1000, ab220797, 70 kDa, Abcam, UK) were bound to the corresponding antigen in the protein overnight at 4°C. Corresponding secondary antibodies (Goat anti-rabbit IgG, 1 : 5000, ab205718, Abcam, UK; Goat anti-mouse IgG, 1 : 5000, ab205719, Abcam, UK) were bound to the primary antibody for 1.5 h. ECL luminescent fluid (WBK1S0100, Millipore, USA) and gel imaging system (GelDoc XR+, Bio-Rad, USA) were used for the final detection of protein expressions.

Results
3.1. The Identification of hUCMSCs. The extracted hUCMSCs were identified. Observation with a microscope showed that the extracted cells were long spindle-shaped and polarized (Figure 1(a)). The results of flow cytometry analysis (Figure 1(b)) demonstrated that the expression rates of stem cell surface markers CD29, CD44, CD146, and CD105 in the extracted cells were 97.85%, 99.16%, 97.91%, and 97.76%, respectively, and the hematopoietic marker CD34 (1.06%) and CD45 (0.40%) were negative. The above experimental results fully proved the cells extracted were hUCMSCs. By employing MTT assay, alizarin staining, immunofluorescence staining, and ELISA assay, we investigated the effect of CGF fibrin membranes on hUCMSCs. CGF fibrin membrane was found to significantly promote cell viability (Figure 2(a), p < 0:01). As shown in Figure 2(b), after the intervention of CGF fibrin membrane, the red mineralized nodules formed in hUCMSCs on day 21 were obviously more than those in the control group (p < 0:001). The detection results of OPN were shown in Figure 2(c), on day 21, it could be observed that the positive expression of red OPN in the visual field of the CGF group was significantly higher than that of the control group. CGF fibrin membrane also increased ALP content in the cells (Figure 2(d), p < 0:05).

CGF Fibrin Membrane Upregulated the Expression of
Osteogenesis-Related Genes and TAZ and Promoted the Nuclear Transfer of TAZ. CGF fibrin membranes could also regulate the expressions of osteogenic-related genes in cells in addition to its role in affecting basic physiological functions of hUCMSCs. On the 21st day of cell culture, the mRNA expressions of RUNX2, OCN, and ALP were significantly upregulated in the CGF group compared with the control group (Figures 3(a)-3(c), p < 0:001). The detection of TAZ expression in the nucleus and cytoplasm showed that TAZ expression in the cytoplasm and nucleus was

The Effects of Overexpressed or Silencing TAZ on the
Expressions of Genes Related to Osteoblast Differentiation, Osteogenesis, and TAZ. The effects of overexpressed or silencing TAZ on hUCMSCs were investigated using constructed plasmids. The results of Alizarin staining (Figure 4(a)) showed that compared with the vector group, the red mineralized particles in the shTAZ group were decreased (p < 0:001), whereas the red mineralized area in the pc-TAZ group was increased (p < 0:001). CGF fibrin membrane intervention improved the condition of the shTAZ group (Figure 4(a), p < 0:001) and increased the mineralized area of the pc-TAZ group (Figure 4(a), p < 0:01). ShTAZ inhibited the mRNA expressions of RUNX2, OCN, and ALP compared with the vector group (Figures 4(b)-4(d), p < 0:01), whereas pc-TAZ showed the opposite regulatory effect (Figures 4(b)-4(d), p < 0:01). The CGF fibrin membrane also reversed the effect of shTAZ but promoted the effect of pc-TAZ (Figures 4(b)-4(d), p < 0:05). By detecting TAZ protein expression in cytoplasm and nucleus, we found that shTAZ inhibited TAZ protein expression in both cytoplasm and nucleus (Figures 4(e) and 4(f), p < 0:01), whereas pc-TAZ produced the opposite effect (Figures 4(e) and 4(f), p < 0:01). The intervention of CGF fibrin membrane was opposite to shTAZ and showed a promoting effect on pc-TAZ (Figures 4(e) and 4(f), p < 0:05).
3.5. The Identification of HPLFs. We identified the extracted human periodontal fibroblasts. Our observation with a microscope showed that the morphology of the extracted cells was mainly spindle-shaped with varied length of the cell protrusions, and the cell bodies were full, showing a typical fibroblastic morphology (Figure 5(a)). Immunofluorescence staining results demonstrated that Vimentin protein was positive, while Cytokeratin protein was negative ( Figure 5(b)). The above experimental results fully proved that the cells extracted were human periodontal fibroblasts.  We further analyzed cell cycle changes after coculture by performing flow cytometry. Compared with the blank group, each group showed different degrees of promoted cell cycle and reduced G1 phase arrest (Figure 7(a), p < 0:05). pc-TAZ also significantly promoted cell proliferation in S phase (Figure 7(a), p < 0:01), and such an effect was further enhanced by CGF fibrin membrane (Figure 7(a), p < 0:05). Cell mineralization results detected by alizarin were presented in Figure 7(b); after coculture, hUCMSCs promoted the mineralization of HPLFs (p < 0:001). The CGF fibrin

Discussion
Periodontitis was a common infective disease and a main cause of tooth loss. Local periodontal inflammation could destroy the integrity of the epithelium, causing periodontal pathogens to spread to the circulatory system [25]. At the same time, periodontal pockets contained a large amount of inflammatory mediators, especially those related to chronic inflammation, such as tumor necrosis factor α, interleukin, and prostaglandin, which could enter the blood circulation system from periodontal pockets and cause systemic inflammatory reactions [26,27]. Therefore, timely and effective treatment was essential. Traditional periodontitis treatment focuses on inflammation control, aiming at preventing or delaying the disease progression, and it is still difficult to achieve a satisfactory regeneration of periodontal tissues [4]. The clinical effect of periodontal tissue regeneration treatment was greatly improved by the introduction of new technologies and materials such as guided tissue regeneration, bone transplantation, growth factors, and biological materials [3]. HUCMSCs derived from mesoderm are the seed cells of tissue regeneration engineering. Research showed that in a bone healing model based on cell regeneration, hUCMSCs show the same osteogenic characteristics as bone marrow mesenchymal stem cells [28] and could effectively promote fracture healing. In a study comparing the chondrogenic differentiation characteristics of hUCMSCs and bone marrow mesenchymal stem cells, the collagen production capacity of the former was three times higher than the latter [29]. Moreover, the previous study had successfully

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Shang et al. [13] confirmed that hUCMSCs could promote the regeneration of periodontal tissues under inflammatory periodontitis conditions, similar to the role of human   The mRNA expressions of fibrosis-related genes were analyzed by qRT-PCR. β-Actin was employed as an internal control. All experiments were performed in independent triplicate. * p < 0:05, * * p < 0:01, * * * p < 0:001 vs. blank; # p < 0:05, ## p < 0:01, ### p < 0:001 vs. CGF;^p < 0:05,^p < 0:01,^^^p < 0:001 vs. vector; & p < 0:05, && p < 0:01, &&& p < 0:001 vs. CGF + vector; △ p < 0:05, △△ p < 0:01 vs. shTAZ; § p < 0:05, § § p < 0:01 vs. pc-TAZ. 12 Stem Cells International periodontal ligament stem cells. In this study, similarly, coculture of hUCMSCs and HPLFs promoted cell proliferation and differentiation, upregulated the expressions of RUNX2, OCN, ALP, and COL-1, and inhibited MMP1 expression. RUNX2, OCN, and ALP are important regulatory genes of osteogenic differentiation. RUNX2 is a key regulatory factor regulating the differentiation of osteoblasts and osteoclasts to promote bone formation. By regulating the expressions of osteoblast-specific extracellular matrix protein genes and the osteoblast cycle, RUNX2 participates in the differentiation process of osteoblasts, promotes bone form, and inhibits bone resorption [32]. OCN, which is one of the most abundant noncollagenous bone proteins in the human body, is specifically expressed at the end of osteoblast differentiation and could regulate bone mineral deposition and metastasis promote osteoblast differentiation and maturation and bone cell formation [33,34]. Mohamed-Ahmed et al. [35] found that bone marrow mesenchymal stem cells showed higher ALP activity and calcium deposition compared with adipose-derived stem cells. Combined with the findings from previous studies and experimental results of this study, the role of CGF in promoting osteogenic differentiation mediated by hUCMSCs is fully demonstrated.
In this study, CGF could promote cell viability, migration, and osteogenic differentiation; upregulate the expressions of RUNX2, OCN, ALP, and COL-1; and inhibit MMP1 expression, and such effects were enhanced with the intervention of pc-TAZ. Studies indicated that TAZ, which is widely distributed in tissues except thymus and peripheral lymphocytes, is high-expressed in a variety of tumor tissues and, therefore, is considered as a tumor-promoting gene [36]. Park et al. [37] demonstrated that downregulation of the Smad4/TAZ axis leads to delayed osteogenic differentiation of mesenchymal stem cells and increased adipogenesis. These findings further supported the reliability of this study.
CGF fibrin membrane is a blood agglutinate obtained by centrifuging venous blood specimens in a special centrifuge, which can produce fibrin with higher density and richer growth factors and is therefore widely used in many clinical treatments and tissue engineering [38]. Studies found that CGF promotes the proliferation and osteogenic differentiation of gingival mesenchymal stem cells, stimulates the bone differentiation of bone marrow stromal cells, and is an excellent biological material for bone regeneration [17,39]. Our study not only confirmed that CGF fibrin membrane can promote hUCMSCs-mediated periodontal tissue regeneration but also revealed that the promotion is achieved by upregulating the expressions of TAZ and genes related to osteogenic differentiation. The current findings discovered that CGF fibrin membrane has the potential to serve as a bone regeneration biomaterial and provided new understandings to tissue regeneration treatment of periodontitis and other periodontal diseases.

Data Availability
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Conflicts of Interest
The authors declare no conflicts of interest.