Injectable platelet-rich fibrin positively regulates osteogenic differentiation of stem cells from implant hole via the ERK1/2 pathway

Abstract Bone regeneration in dentistry is a dynamic approach for treating critical size bone defects that are unlikely to self-heal. Human bone marrow stem cell (hBMSCs) therapies are being tested clinically for various disorders and have remarkable clinical advancements in bone regeneration. Injectable platelet-rich fibrin (i-PRF), which is obtained from autologous blood centrifuged at 700 rpm (60 G) for 3 min can promote osteogenic differentiation of this cell, but the mechanism remains unclear. The objectives of this study were to explore the contents of i-PRF further and investigate its effect on the cell behavior of hBMSCs and the underlying molecular mechanisms. The results showed that i-PRF contained 41 cytokines, including macrophage colony-stimulating factor (M-CSF) and β-nerve growth factor (β-NGF), which had not been reported before. The Cell Counting Kit-8 and wound healing assay showed that 10% and 20% i-PRF improved the proliferation rate and the migration capacity of hBMSCs without toxicity to cells. Besides, the expression of osteogenic markers and the capacity to form mineralized nodules of hBMSCs were promoted by 20% i-PRF. Furthermore, i-PRF activated the ERK pathway, and the ERK inhibitor attenuated its effects. In summary, i-PRF promotes hBMSCs proliferation and migration and facilitates cell osteogenesis through the ERK pathway, which has promising potential in bone regeneration. Plain Language Summary What is the context? Bone defects caused by trauma or tumor is a great challenge in clinical practice. However, there is the good news that the bone defect in the oral can self-regenerate, the bone remodeling may take several months to several years and shows apparent individual differences. Different strategies, surgical techniques, and materials have been employed to induce an optimal outcome in guided bone regeneration. Blood products have been widely used in dentistry due to their excellent biocompatibility, growth factor content, ease of collection, and ability to be produced by the human body. Limited data suggest that Injectable platelet-rich fibrin positively regulates osteogenic differentiation of stem cells, but further evidence is needed to quantify this effect. What is new? It is unclear how many growth factors i-PRF contains in previous studies, so we detected 41 kinds of growth factors, more than has been previously appreciated, and found that all growth factors were measured in the samples, and the difference was in the amount of expression. In our research, we explored the role of i-PRF in the osteogenesis of hBMSCs through the effects of different concentrations of i-PRF on the proliferation, migration, and osteogenic differentiation of hBMSCs. Currently, most current research focuses on observing phenomena, and we wondered by what mechanism the i-PRF regulates stem cell function. We found that i-PRF can regulate the molecular mechanism of the osteogenic differentiation of hBMSCs in vitro by activating the MAPK/ERK signaling pathway. What is the impact? I-PRF promotes hBMSCs proliferation and migration and facilitates cell osteogenesis through the ERK pathway. The favorable cytobiological effects of i-PRF on hBMSCs might be the basis for i-PRF applications in bone regenerative.


Plain Language Summary
What is the context?
• Bone defects caused by trauma or tumor is a great challenge in clinical practice.However, there is the good news that the bone defect in the oral can self-regenerate, the bone remodeling may take several months to several years and shows apparent individual differences.• Different strategies, surgical techniques, and materials have been employed to induce an optimal outcome in guided bone regeneration.• Blood products have been widely used in dentistry due to their excellent biocompatibility, growth factor content, ease of collection, and ability to be produced by the human body.• Limited data suggest that Injectable platelet-rich fibrin positively regulates osteogenic differentiation of stem cells, but further evidence is needed to quantify this effect.
What is new?
• It is unclear how many growth factors i-PRF contains in previous studies, so we detected 41 kinds of growth factors, more than has been previously appreciated, and found that all growth factors were measured in the samples, and the difference was in the amount of expression.
• In our research, we explored the role of i-PRF in the osteogenesis of hBMSCs through the effects of different concentrations of i-PRF on the proliferation, migration, and osteogenic differentiation of hBMSCs.• Currently, most current research focuses on observing phenomena, and we wondered by what mechanism the i-PRF regulates stem cell function.We found that i-PRF can regulate the molecular mechanism of the osteogenic differentiation of hBMSCs in vitro by activating the MAPK/ERK signaling pathway.

Introduction
Patients with tooth loss can present a significant challenge for implantology regarding anatomical limitations and technical difficulties.The presence of maxillary sinus and mandibular nerve canal limits proper implant placement.Although there is the good news that the bone defect in the oral can self-regenerate, bone remodeling may take several months to several years and shows apparent individual differences.Different strategies, surgical techniques, and materials have been employed to induce an optimal outcome in guided bone regeneration [1].The bone gain was significantly reduced when healing complications occurred [2].
To reduce the incidence of postoperative complications, plateletrich plasma (PRP) was introduced into the research of stomatology by Marx in 1998 and is considered the first generation of blood products for oral treatment.Over the decades, blood products evolved the concept of platelet-rich fibrin (PRF), advanced PRF(A-PRF), and injectable PRF (i-PRF).These are becoming an attractive and widely-used approach in regenerative dentistry [3][4][5][6][7].Among them, 7 ml of elbow forearm whole blood was centrifuged at 700 rpm for 3 min, and the yellow upper liquid obtained was i-PRF [8].It is simple to prepare and use, and there is no biological modification.Perhaps most crucially, it is a straightforward and affordable procedure regardless of one's financial situation [9].Besides the results from a study favored the use of the naturally-formulated i-PRF when compared to traditional PRP with anti-coagulants [10].
Recent experiments in this area suggested that i-PRF contains more abundant cytokines, including transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), interleukin, IL, IL-1β, IL-6, and other proinflammatory cytokines [6,11,12], but how many types of growth factors i-PRF contains have not reported.Of these known factors, VEGF could regulate sprouting angiogenesis by activating the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway [13].The blood product PRF increased ERK phosphorylation in U2OS cells [14].Notably, combining i-PRF and bone substitutes may provide biologically active molecules and a scaffold for osteogenesis [15], but the underlying molecular mechanism has not yet been clarified.
Therefore, the present study was designed to investigate the cytobiological effects of i-PRF on human bone marrow mesenchymal stem cells (hBMSCs) in vitro and to further explore the molecular mechanisms in i-PRF-induced osteogenesis differentiation of hBMSCs.

Ethics
This study was approved by the local ethics committee (Ethics Committee of the Department of Stomatology of Lanzhou University, Project number LZUKQ-2020-030).Human bone marrow mesenchymal stem cells were obtained from the patients' implant holes.The use of anonymous bone marrow samples and for research purposes informed consent was acquired from all donors.Sampling of peripheral blood from healthy volunteers to generate i-PRF and all healthy volunteers (n = 6) signed informed consent.

I-PRF preparation and analysis
Six volunteers aged 20-40 years were recruited to produce i-PRF according to an existing protocol [8,16].Briefly, samples were drawn in sterile plastic tubes (VACUETTE, Austria) and centrifuged at 700 rpm for 3 min (60×g) at room temperature in a centrifuge (TR-18, Trausim, China).After the centrifugation process, the upper liquid layer of about 1 ml was collected as i-PRF.After solidification, the freshly obtained i-PRF were placed in a six-well plate by adding 2.5% glutaraldehyde and placed in a 4°C refrigerator overnight.The fixing solution was discarded, washed with PBS 1× three times, and then dehydrated with gradient ethanol of 50%, 60%, 70%, 80%, 90%, and 100% for 10 min each time.The critical point was dried, sprayed with gold, and observed under a scanning electron microscope.According to the instructions, growth factor expression levels were detected by the human growth factor antibody array-membrane (ab134002, Abcam, Cambridge, UK).

I-PRF treatment
1 ml i-PRF from 7 ml blood was put into a 15 mL centrifuge tube, and 9 ml α-MEM was added to make the conditioned medium containing 10% of i-PRF at constant volume, 20%, and 40% of i-PRF according to this method.HBMSCs were divided into 4 experimental groups, each of which was treated with different concentrations of i-PRF.Without i-PRF served as the control.

Cell proliferation assay
CCK-8 assay was performed according to the manufacturer's instructions to assess cell proliferation with i-PRF treatment.Briefly, hBMSCs (4 × 103cells/well) were seeded in 96-well plates containing 10% FBS complete medium and incubated for 2 h.Next, 100 µl medium supplemented with 10, 20, or 40% concentration of i-PRF was changed to each well and incubated for 1, 3 5,7 days.Then, at the corresponding time point, 10 μl of Cell Counting Kit-8 reagent (Beyotime, China) was added to each plate for 2 h at 37°C.The absorbance at a wavelength of 450 nm was detected using an automatic microplate reader (Tecan, Swiss) to be detected as an index of cell proliferation and viability.

Live/Dead cell staining
Live/dead cell staining (AAT, American) was performed according to the manufacturer's instructions to assess cell viability with different concentrations of i-PRF treatment [8].HBMSCs (1 × 104 cells/well) were seeded in a 6-well plate incubated for 2 h.Then 3 ml medium supplemented with 10, 20, and 40% concentration of i-PRF were changed to each well and incubated for 24 h, next washed with PBS 1× two times.Incubated, the cells were in the staining working solution at room temperature for 4 h, and the cell state and viability were observed under a fluorescence microscope (Zeiss, American).

Scratch test
The cells (1 × 104 cells/well) were seeded in 12-well plates, reached 80% confluence, and then made about 5 mm scratch.After washing and removing the floating cells and debris with sterile PBS, the culture medium was changed to a 1 ml conditioned medium without serum.At 0 h, 12 h, and 48 h, the migration effect was observed under an inverted microscope, and wound width was quantified using ImageJ software.

I-PRF osteogenic differentiation induction
Ten percent fetal bovine, serum high sugar DMEM (containing 100 U/ml of penicillin, 100 μg/ml of streptomycin, 2 mM L-glutamine), which contains 50 μM of ascorbic acid, 10 mM of β-glycerol phosphate and 100 nM of ascorbic acid to obtain osteogenic induction medium (OIM).HBMSCs were divided into 6 experimental groups, three groups were cultured in OIM (two groups were treated with 10% and 20% i-PRF, respectively, without i-PRF as the control group), and the remaining three groups were cultured in essential culture (two groups were treated with 10% and 20% i-PRF respectively, without i-PRF as the control group).In 7 days, total protein was extracted to assess osteogenesis marker expression; in 3 weeks, samples were detected via Alizarin Red S staining.

Alizarin red S staining
All specimens were fixed with 4% paraformaldehyde for 30 minutes at room temperature.Alizarin red S solution was added to each well for 30 minutes to stain.Nonspecific staining was removed by repeated washing with distilled water.The staining of calcium mineral deposits was recorded under bright-field microscopy.The calcium concentration was quantitated by the cetylpyridinium chloride (Sigma, American).Absorbances were read at 570 nm automatic microplate reader (Tecan, Swiss).

Real-time quantitative PCR (RT-qPCR)
After 7 days of culture, the cells were harvested in Trizol and processed for total ribonucleic acid (RNA) extraction using the classical method with phenol-chloroform.The amount and purity of isolated RNA were quantified using the NanoDrop spectrophotometer by calculating the 260/280 ratio.A total of RNA was reverse-transcribed into 500 ng/μL cDNA using a PrimeScript RT Master Mix system (AgBio, China), and the final volume was 45 μL.Real-time PCR was carried out using 20 μL final reaction volumes of iTaq Universal SYBR Green Supermix (AgBio, China) in a StepOne Plus real-time PCR system (QuantStudio, American).The following primers were used: collagen type I (COL1), osteocalcin (OCN), osteopontin (OPN), and runtrelated transcription factor 2(Runx2).The sequences of the primers are listed below.Relative levels of the mentioned genes were normalized to β-actin.

Western blotting
To further identify whether the MAPK/ERK pathway was involved in the osteogenic differentiation of hBMSCs stimulated by 20% i-PRF.HBMSCs were divided into 4 experimental groups, and two groups were treated with 2 μM U0126 (CST, USA).Cells were harvested and lysed in RIPA buffer (Beyotime, China) supplemented with protease inhibitors.Following concentration determination, protein samples (25 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane by western blotting.Membranes were blocked in 5% skim milk for 2 h and incubated with antibodies extracellular-signal-regulated kinases ERK (1:1000; Immunoway), phosphorylated-ERK (1:1000; Immunoway), and βactin (1:1000, Immunoway) respectively, at 4°C overnight.After washing within Tris-buffered saline including Tween 20 (TBST) three times (10 minutes each), the membranes were incubated by using horseradish-peroxidase-conjugated goat anti-rabbit (1:5000, Immunoway) as a secondary antibody at the average temperature for two hours, the immunoreactive bands had been noticed adopting a developed chemiluminescent detection reagent and further visualized through the explosion of the blot to X-ray film (FUSION SOLO6S.EDGE), and grayscale analysis was performed with ImageJ software.

Statistical analysis
The number of samples in each group was three, and each experiment was repeated once.The statistical significance was analyzed by ANOVA tests and t-test using GraphPad Prism.The value of p < .05 was considered statistically significant.

i-PRF preparation and analysis
The samples were observed under a scanning electron microscope (SEM) as a high-density three-dimensional network structure (Figure 1Aa)., and a large number of white blood cells and platelets were embedded in its complex fibrin structure (Figure 1Ac).The types of growth factors in i-PRF were detected semi-quantitatively by human growth factor array membrane detection, which not only contained known factors such as EGF, IGFBP-2, PDGF, but also M-CSF-R (macrophage colonystimulating factor receptor) and β-nerve growth factor (β-NGF), which had not been reported (Figure 1B).But how many kinds of soluble factors is still a mystery.

i-PRF improved the cell proliferation and no cytotoxic effects
To identify the effects of i-PRF on hBMSCs cell viability, the proliferation rate and cytotoxic effects were measured by CCK-8 assay and live/dead cell staining.All experimental groups had no i-PRF promotes osteogenic differentiation of hBMSCs 3 cytotoxic effects on cells, and the tests groups improved the proliferation rate at 3, 5 days compared control group (3d: 10% i-PRF: P < .001,20% i-PRF: P < .01,40% i-PRF: P < .05;5d: 10% i-PRF: P < .001,20% i-PRF: P < .0001,40% i-PRF: P < .01), on the 7th day, there was no significant difference between the 40% i-PRF and the control group.(10%i-PRF: P < .01,20% i-PRF: P < .01)(Figure 2A).And no dead cells were found in all experimental groups, indicating that i-PRF has good biocompatibility and no cell toxicity (Figure 2B).

I-PRF improved hBmscs osteogenesis differentiation
According to the previous experiment, we decided to evaluate the effects of 10% and 20% of i-PRF on hBMSCs differentiation; Alizarin S Red staining and RT-qPCR were applied to the next experiment.It was found that the group of 20% i-PRF with OIM provoked expression of Runx2, COL1A a slight significant increase compared with the OIM group (P < .01)(Figure 4A).There was a slight increase in the expression of OPN and OCN within the 20% group than 10% i-PRF, but this was not statistically significant (Figure 4A).Furthermore, both 10% and 20% i-PRF in OIM promoted mineralized nodule formation compared to the control group, with the staining intensity in the 20% i-PRF group being most noticeable (P < .0001)(Figure 4Bf).These results show that i-PRF can promote hBMSCs migration, proliferation, and osteogenic differentiation, especially at 20%.

Involvement of ERK1/2 in regulating osteogenic differentiation of hBmscs induced by i-PRF
Complete the above experiment and select 20% i-PRF for further study.To identify whether hBMSCs osteogenic differentiation was induced via the ERK pathway, an ERK-specific inhibitor U0126 was used to reduce ERK expression.Western blot analysis showed that U0126 attenuated i-PRF-induced p-ERK and Runx2 expression (Figure 5A).RT-qPCR analysis also had corresponding results.The expression of genes ERK1/2 and Runx2 decreased after U0126 was used (Figure 5B).Alizarin red S staining revealed that ERK inhibitor attenuated the increased mineralized nodule formation of hBMSCs induced by i-PRF (Figure 5C)

Discussion
Blood products have been widely used in dentistry due to their excellent biocompatibility, growth factor content, ease of collection, and ability to be produced by the human body [17].They also have been widely used in implantology for stimulating new bone formation and might help to improve the regeneration of nerve fibers in peri-implant bone [18,19].The clinical use of such preparations is primarily based on the rationale that activated platelets release high levels of growth factors and cytokines [20,21], which are used to regulate the wound healing process and influence the cells involved in tissue reconstruction, and vascular and bone remodeling [22].Such as fibroblast growth factor (FGF), IGF-1, TGF-β1, and PDGF, are essential for development, maturation, and repair in craniofacial tissues as they create an appropriate microenvironment for tissue development that promotes cell and tissue growth [23].They also promote periodontal tissue regeneration and treat bone defects associated with post-extraction or periodontitis [24][25][26].Growth factors released from i-PRF showed significantly higher levels of total long-term, like PDGF-AA, PDGF-AB, EGF, and IGF-1 [8,27], and the role of the soluble factors in tissue generation is indisputable.In our study, the fibrin structure observed under SEM was the same as in previous reports [28], and the fibrin of i-PRF was closely linked with a small network septum and could accommodate platelets, white blood cells, and red blood cells in it.This structure was conducive to the storage of platelets and other cells in the septum, and at the same time, it was conducive to the integration with patient tissues when transferred to the human body [29].Blood centrifuged at a lower centrifugation rate in a non-glass tube produces movable platelet-rich fibrin called i-PRF, clinicians have used this technique to facilitate the aggregation or capping of biomaterials wound healing [30], and i-PRF could release more and longer growth factors [8,10].However, it is unclear how many growth factors i-PRF contains, so we detected 41 kinds of growth factors, more than has been previously appreciated, and found that all growth factors were measured in the samples, and the difference was in the amount of expression.Among them, M-CSF-R and β-NGF have not been reported, and this is the first main finding of the present study.NGF is a polypeptide protein belonging to the neurotrophic factor family, and its receptor is expressed in both normal tissues and fracture callus [31,32].This factor promotes the differentiation of rat bone marrow stromal cells into osteoblasts and enhances their mineralization ability [33].Furthermore, the other factor CSF-1 plays an essential role in regulating the proliferation and differentiation of monocytes, plays a crucial role in bone remodeling and the development of tissue-specific macrophages, and treatment with CSF-1 has been shown to accelerate bone healing in a model of tibia injury [34].However, these two factors' roles in i-PRF are unclear and need further study.Blood product conditioned medium can promote cell proliferation, migration, and osteogenic differentiation, attributing to the presence of the abundance of growth factors in the blood products [35].And a recent study reported that i-PRF significantly increased the migration of cells when compared to PRP, and i-PRF promoted more osteogenic differentiation [10].In our research, we explored the role of i-PRF in the osteogenesis of hBMSCs through the effects of different concentrations of i-PRF on the proliferation, migration, and osteogenic differentiation of hBMSCs.It was chosen because higher concentrations of growth factors and blood products have been reported to potentiate several vital properties in MSCs [36,37].In addition, the i-PRF acquisition process is simple and does not contain anticoagulants and calcium chloride to activate the polymerization of platelets and fibrin during the preparation process [8].Some authors suggest that when the concentration of PRF is too high, cell proliferation is inhibited and reported that 10% PRF is a suitable concentration [38].However, some scholars have reported that PRF can promote the proliferation of human jaw mesenchymal stem cells in a dose-dependent manner [39].The optimal concentration of growth factors plays a crucial role during tissue regeneration.As described above, i-PRF can release more growth factors for a longer time.Through this experiment, we got that 20% of i-PRF created the appropriate microenvironment for cell proliferation, migration, and osteogenic differentiation of stem cells from implant holes in vitro; whether this concentration could be the optimal choice in clinical treatment needs further study.
Since i-PRF has been recently introduced, it has not been thoroughly investigated for its potential.It is now being studied in various fields, studies on bone regeneration in oral and maxillofacial reconstruction, guided bone regeneration, sinus lifting, and dental implant placement, as well as cleft palate repair [7,15,[40][41][42].To further assess the efficacy of this material, it can even be applied in various surgical procedures such as orthognathic surgeries, osteogenesis and so on [7].But, most of the current research focuses on observing phenomena, and we wondered by what mechanism the i-PRF regulates stem cell function.Osteogenesis is a series of operations rather than direct formation.It involves different stages and these differentiation stages are controlled by the interaction of many signaling pathways and transcription factors.These transcription factors are critical to stimulating cell differentiation by regulating the expression of downstream osteogenic genes.Moreover, the transcription factor Runx2 regulates the differentiation of mesenchymal stem cells into osteoblasts and further maturation into osteoblasts.Runx2 can stimulate or inhibit osteoblast differentiation and acts as a modulator [43].Some studies have reported that ERK phosphorylates Runx2 in osteoblasts, suggesting that ERK can induce osteogenic gene expression programs by activating Runx2 [44].In the present study, Alizarin S Red staining assay showed that pretreatment with a suitable concentration of U0126 abolished the i-PRF-induced hBMSCs osteogenic capacity.Furthermore, we found that i-PRF can regulate the molecular mechanism of the osteogenic differentiation of hBMSCs in vitro by activating the MAPK/ERK signaling pathway.Western blotting and RT-qPCR analysis showed that ERK 1/2 to phosphorylate (p-ERK 1/2), an effector protein in the MAPK/ERK signaling pathway, is significantly expressed after hBMSCs stimulated by i-PRF, the protein expression of Runx2 same as p-ERK.We further speculated that i-PRF induced osteogenesis through MAPK/ERK pathway.Further study will focus on the exact relationship between the gene Runx2 and the MAPK/ERK signaling pathway in the microenvironment created by soluble factors in i-PRF.
Based on these observations, we concluded that i-PRF activates the MAPK/ERK pathway to promote the osteogenic differentiation of hBMSCs.At the same time, i-PRF (containing more than 41 growth factors) significantly promoted the proliferation and migration of hBMSCs.Further studies will explore the molecular effects within i-PRF, not only in osteogenesis.Animal models and human trials are necessary to elucidate the regenerative potential of i-PRF and its response in clinical situations.

Figure 1 .Figure 2 .
Figure 1.I-PRF analysis.(A).SEM micrographs show the pore morphology of i-PRF.(B) the human growth factor array membrane shows 41 growth factors in i-PRF, (a) the exposed array membrane.(b) the expression of each factor.

Figure 3 .
Figure 3.The migration capacity of the hBmscs treated with different concentrations of i-PRF conditioned medium.(A).The cell migration processes were observed under a microscope at 12 and 48 hours.(B) the wound closure percentages of hBmscs cultured in 10% and 20% i-PRF were significantly increased at 48 hours compared with the control group.Mean ± SD (****p<.0001).

Figure 4 .
Figure 4. Role of i-PRF in hBmscs osteogenesis differentiation.(A).RT-qPCR showed that 20% of i-PRF can promote the expression of gene Runx2 and COL1 compared with the control group, but only in the OIM-containing groups with statistical significance.The expression of OPN and OCN within the 20% group than 10% i-PRF, but this was not statistically significant.(B) Alizarin red staining and quantitative analysis showed that the number of calcified nodules in 10% and 20% i-PRF with OIM was significantly increased compared with the OIM group.Without OIM, 20% of i-PRF promoted the formation of calcified nodules.N=3, mean ± SD (****p<.0001,**p<.01,*p<.05).The error bars represent the standard deviation.