YBX1 is required for maintaining myeloid leukemia cell survival by regulating BCL2 stability in an m6A-dependent manner.

RNA-binding proteins (RBPs) are critical regulators of transcription and translation that are often dysregulated in cancer. Although RBPs are increasingly recognized as being important for normal hematopoiesis and for hematologic malignancies as oncogenes or tumor suppressors, RBPs that are essential for the maintenance and survival of leukemia remain elusive. Here we show that YBX1 is specifically required for maintaining myeloid leukemia cell survival in an N6-methyladenosine (m6A)-dependent manner. We found that expression of YBX1 is significantly upregulated in myeloid leukemia cells, and deletion of YBX1 dramatically induces apoptosis and promotes differentiation coupled with reduced proliferation and impaired leukemic capacity of primary human and mouse acute myeloid leukemia cells in vitro and in vivo. Loss of YBX1 has no obvious effect on normal hematopoiesis. Mechanistically, YBX1 interacts with insulin-like growth factor 2 messenger RNA (mRNA)-binding proteins (IGF2BPs) and stabilizes m6A-tagged RNA. Moreover, YBX1 deficiency dysregulates the expression of apoptosis-related genes and promotes mRNA decay of MYC and BCL2 in an m6A-dependent manner, which contributes to the defective survival that results from deletion of YBX1. Thus, our findings have uncovered a selective and critical role of YBX1 in maintaining myeloid leukemia survival, which might provide a rationale for the therapeutic targeting of YBX1 in myeloid leukemia.

Liquid platelet-rich fibrin promotes the regenerative potential of human periodontal ligament cells.

OBJECTIVE: To compare the biological effect of PRP and liquid-PRF on human periodontal ligament cells (hPDLCs) in vitro. METHODS: The liquid-PRF was processed with centrifugation at 700 g for 3 min, and PRP was processed according to Curasan's protocol. Migration and proliferation assay were performed by a scratch/Transwell assay and a CCK-8 assay, respectively. To investigate hPDLC differentiation, alkaline phosphatase (ALP) assay, Alizarin Red S staining, and gene expression level detection of Runx2, Col1a1, and OCN were conducted. Furthermore, cells cultured with lipopolysaccharide (LPS) to induce an inflammation condition were utilized to investigate the impact of liquid-PRF on inflammatory resolution. RESULTS: Either PRP or liquid-PRF can promote proliferation, migration of hPDLCs, and osteogenic differentiation of hPDLCs. It was noteworthy that liquid-PRF demonstrated a significantly higher ability to promote the biological differentiation and mineralization of hPDLCs compared with PRP. Lastly, when hPDLCs were incubated with LPS, cells cultured with liquid-PRF showed significantly lower mRNA expression levels of inflammatory genes. CONCLUSIONS: Liquid-PRF notably promoted hPDLC activity and attenuated the inflammatory state induced by LPS.

Comparison of platelet-rich fibrin (PRF) produced using 3 commercially available centrifuges at both high (~ 700 g) and low (~ 200 g) relative centrifugation forces.

OBJECTIVES: Platelet-rich fibrin (PRF) has gained tremendous momentum in recent years as a natural autologous growth factor derived from blood capable of stimulating tissue regeneration. Owing to its widespread use, many companies have commercialized various centrifugation devices with various proposed protocols. The aim of the present study was to compare 3 different commercially available centrifuges at both high and low g-force protocols. MATERIALS AND METHODS: PRF was produced on three commercially available centrifuges including the IntraSpin Device (IntraLock), the Duo Quattro (Process for PRF), and Salvin (Salvin Dental). Two separate protocols were tested on each machine including the original leukocyte and platelet-rich fibrin (L-PRF) protocol (~ 700 RCF max (~ 400 RCF clot) for 12 min) as well as the advanced platelet-rich fibrin (A-PRF+) protocol (~ 200 g RCF max (~ 130 g RCF clot) for 8 min). Each of the tested groups was compared for cell numbers, growth factor release, scanning electron microscopy (SEM) for morphological differences, and clot size (both weight and length/width). RESULTS: The present study found that PRF clots produced utilizing the low-speed centrifugation speeds (~ 200 g for 8 min) produce clots that (1) contained a higher concentration of evenly distributed platelets, (2) secreted higher concentrations of growth factors over a 10 day period, and (3) were smaller in size. This was irrespective of the centrifugation device utilized and consistently observed on all 3 devices. The greatest impact was found between the protocols utilized (up to a 200%). Interestingly, it was further revealed that the centrifugation tubes used had a much greater impact on the final size outcome of PRF clots when compared to centrifugation devices. It was found that, in general, the Process for PRF tubes produced significantly greater-sized clots when compared to other commercially available tubes. The Salvin Dental tubes also produced significantly greater PRF clots when compared to the IntraLock tubes on each of the tested centrifugation devices. CONCLUSIONS: The present study demonstrated the reproducibility of a scientific concept (reduction in RCF produces PRF clots with more evenly distributed cells and growth factors) utilizing different devices. Furthermore, (and until now overlooked), it was revealed for the first time that the centrifugation tubes are central to the quality production of PRF. Future research investigating tube characteristics thus becomes critically important for the future optimization of PRF. CLINICAL RELEVANCE: This is the first study to reveal the marked impact of centrifugation tubes on the final production of PRF. Future study thus becomes markedly important to further optimize the quality of PRF-based matrices. It was further found that little variability existed between the centrifugation devices if optimized centrifugation protocols (lower centrifugation speeds) were utilized.

A novel method for harvesting concentrated platelet-rich fibrin (C-PRF) with a 10-fold increase in platelet and leukocyte yields.

BACKGROUND AND OBJECTIVES: Liquid platelet rich fibrin (PRF; often referred to as injectable PRF) has been utilized as an injectable formulation of PRF that is capable of stimulating tissue regeneration. Our research group recently found that following standard L-PRF protocols (2700 RPM for 12 min), a massive increase in platelets and leukocytes was observed directly within the buffy-coat layer directly above the red blood cell layer. The purpose of this study was to develop a novel harvesting technique to isolate liquid PRF directly from this buffy coat layer and to compare this technique to standard i-PRF. MATERIALS AND METHODS: Standard high g-force L-PRF and low g-force i-PRF protocols were utilized to separate blood layers. Above the red blood corpuscle layer, sequential 100-µL layers of plasma were harvested (12 layers total; i.e., 1.2 mL, which represents the total i-PRF volume), and 3 layers (3 × 100 µL) were harvested from the red blood cell layer to quantify blood cells. Each layer was then sent for complete blood count (CBC) analysis, and the cell numbers were quantified including red blood cells, leukocytes, neutrophils, lymphocytes, monocytes, and platelets. The liquid PRF that was directly collected from the buffy-coat layer following L-PRF protocols was referred to as concentrated PRF (C-PRF). RESULTS: The i-PRF protocol typically yielded a 2- to 3-fold increase in platelets and a l.5-fold increase in leukocyte concentration from the 1- to 1.2-mL plasma layer compared to baseline concentrations in whole blood. While almost no cells were found in the first 4-mL layer of L-PRF, a massive accumulation of platelets and leukocytes was found directly within the buffy coat layer demonstrating extremely high concentrations of cells in this 0.3-0.5-mL layer (~ 20-fold increases). We therefore proposed harvesting this 0.3- to 0.5-mL layer directly above the red blood cell corpuscle layer as liquid C-PRF. In general, i-PRF was able to increase platelet numbers by ~ 250%, whereas a 1200-1700% increase in platelet numbers could easily be achieved by harvesting this 0.3-0.5 mL of C-PRF (total platelet concentrations of > 2000-3000 × 109 cells/L). CONCLUSION: While conventional i-PRF protocols increase platelet yield by 2-3-fold and leukocyte yield by 50%, we convincingly demonstrated the ability to concentrate platelets and leukocytes over 10-fold by harvesting the 0.3-0.5 mL of C-PRF within the buffy coat following L-PRF protocols. CLINICAL RELEVANCE: Previous studies have demonstrated only a slight increase in platelet and leukocyte concentrations in i-PRF. The present study described a novel harvesting technique with over a 10-fold increase in platelets and leukocytes that can be further utilized for tissue regeneration.

Evaluation of 24 protocols for the production of platelet-rich fibrin.

BACKGROUND: The aim of this study was to evaluate 24 protocols for the production of platelet rich fibrin (PRF) produced via horizontal centrifugation to better understand cell separation following protocols at various times and speeds. METHODS: All protocols were compared utilizing a recent method to quantify cells in PRF in 1 mL sequential layers pipetted from the upper layer downwards until all 10 mL were harvested. In total, 960 complete blood counts (CBCs) were investigated. Both solid and liquid-based PRF protocols were investigated following 24 protocols involving 6 relative centrifugal force (RCF) values (100, 200, 400, 700, 1000 and 1200g) at 4 centrifugation times (3, 5, 8 and 12 min). RESULTS: In general, platelets could more easily accumulate in the upper 4 layers when compared to leukocytes owing to their lower cellular density. Protocol time seemed to have a greater impact on the final cell layer separation when compared to the effect of speed. Protocols of greater than 8 min at 400g led to no leukocyte accumulation in the upper PRF layers (found specifically within the buffy coat). Protocols at or below 200g were unable to effectively accumulate platelets/leukocytes. The optimal centrifugation speed and time for solid-PRF ranged between 400 and 700g for 8 min. It was noted that variability in patient baseline platelet/leukocyte/erythrocyte counts (hematocrit) significantly affected cell layer separation. This finding was more pronounced at lower centrifugation speeds. CONCLUSIONS: Within the investigated ranges, a protocol of 700g for 8 min presented the highest yield of platelets/leukocytes evenly distributed throughout the upper PRF layers.

Injectable-platelet rich fibrin using the low speed centrifugation concept improves cartilage regeneration when compared to platelet-rich plasma.

The aim of the present study was to evaluate the effect of injectable platelet-rich fibrin (i-PRF) on cultivated chondrocytes and osteochondral regeneration in critical-sized osteochondral defect of the rabbit's knee in comparison to autologous platelet-rich plasma (PRP). Chondrocytes were first investigated for their ability to proliferate and differentiate in response to PRP and i-PRF. Thereafter, full-thickness critical-sized osteochondral defects 5 mm in diameter and 5 mm in depth were created in the knee joint of 12 adult female New Zealand White rabbits. Defects were regenerated with either PRP or i-PRF and compared to control. Animals were sacrificed at 4 and 12 weeks postoperatively and evaluated histologically by macroscopic and microscopic examination for cartilage regeneration. i-PRF significantly promoted chondrocyte proliferation and mRNA levels of Sox9, collagen type II, and aggrecan when compared to PRP and control. Histological analysis revealed that at 4 weeks, macroscopic ICRS scores from the i-PRF group were significantly enhanced when compared to the PRP and control groups. At 12 weeks post surgery, the microscopic ICRS scores demonstrated that the i-PRF group significantly improved cartilage regeneration when compared to PRP. In conclusion, the use of i-PRF using the low speed centrifugation concept significantly promoted chondrocyte activity and further improved cartilage regeneration when compared to PRP. The histological results revealed early and better cartilage regeneration within 4 weeks postoperatively when i-PRF was utilized and the results were maintained at 12 weeks. Future clinical studies are now needed investigating the regenerative potential of i-PRF in comparison to PRP for knee regeneration.

Ptip safeguards the epigenetic control of skeletal stem cell quiescence and potency in skeletogenesis.

Stem cells remain in a quiescent state for long-term maintenance and preservation of potency; this process requires fine-tuning regulatory mechanisms. In this study, we identified the epigenetic landscape along the developmental trajectory of skeletal stem cells (SSCs) in skeletogenesis governed by a key regulator, Ptip (also known as Paxip1, Pax interaction with transcription-activation domain protein-1). Our results showed that Ptip is required for maintaining the quiescence and potency of SSCs, and loss of Ptip in type II collagen (Col2)+ progenitors causes abnormal activation and differentiation of SSCs, impaired growth plate morphogenesis, and long bone dysplasia. We also found that Ptip suppressed the glycolysis of SSCs through downregulation of phosphoglycerate kinase 1 (Pgk1) by repressing histone H3 lysine 27 acetylation (H3K27ac) at the promoter region. Notably, inhibition of glycolysis improved the function of SSCs despite Ptip deficiency. To the best of our knowledge, this is the first study to establish an epigenetic framework based on Ptip, which safeguards skeletal stem cell quiescence and potency through metabolic control. This framework is expected to improve SSC-based treatments of bone developmental disorders.

YBX1 regulates the survival of chronic myeloid leukemia stem cells by modulating m6A-mediated YWHAZ stability.

PURPOSE: Chronic myeloid leukemia (CML) is a myeloproliferative disease derived from hematopoietic stem cells (HSCs) that harbor Philadelphia chromosome (Ph chromosome). In clinic, leukemia stem cells (LSCs) in CML are insensitive to the treatment with tyrosine kinase inhibitors, and are responsible for disease relapse. However, the molecular mechanisms for maintaining LSCs survival remain elusive. METHODS: CML patient-derived cell lines and BCR-ABL-induced CML mouse model were used to explore the role of YBX1 in regulating the survival of CML LSCs. Bone marrow transduction and transplantation, and colony-forming unit assay were used to investigate LSC function. The underlying mechanism of how YBX1 regulates LSCs survival were assessed using flow cytometry, RNA sequencing, western blot, RNA decay assay, co-immunoprecipitation and RNA immunoprecipitation. RESULTS: Here we show that RNA-binding protein YBX1 plays an important role in regulating survival of CML LSCs. We find that YBX1 expression is significantly increased in CML cells, and confirm that YBX1 is required for maintaining survival of LSCs. Deletion of YBX1 impairs the propagation of CML through blocking cell proliferation and inducing apoptosis of LSCs. Mechanistically, we find that YBX1 regulates expression of apoptotic associated genes. YBX1 cooperates with RNA m6A reader IGF2BPs to stabilize YWHAZ transcript in an m6A-dependent manner, and loss of YBX1 decreases YWHAZ expression by accelerating mRNA decay. Restoration of YWHAZ efficiently rescues the defects of YBX1-deficient CML cells. CONCLUSION: Our findings reveal a critical role of YBX1 in maintaining survival of CML LSCs, which provides a rationale for targeting YBX1 in CML treatment.

Decoding m6A RNA methylome identifies PRMT6-regulated lipid transport promoting AML stem cell maintenance.

N6-methyladenosine (m6A) is a common chemical modification for mammalian mRNA and exhibits high dynamics in various biological processes. However, dynamics of m6A RNA methylome during leukemogenesis remains unknown. Here, we delineate a comprehensive m6A landscape during acute myeloid leukemia (AML) development and identify PRMT6 as a key for maintaining AML stem cells. We observe an obvious change in m6A methylome during leukemogenesis and find that protein arginine methyltransferase PRMT6 and m6A reader IGF2BP2 maintain the function of human and murine leukemia stem cells (LSCs). Genetic deletion or pharmacological inhibition of PRMT6 damages AML development and LSC function. Mechanistically, IGF2BP2 stabilizes PRMT6 mRNA via m6A-mediated manner, which catalyzes H3R2me2a and suppresses lipid transporter MFSD2A expression. PRMT6 loss upregulates MFSD2A expression that increases docosahexaenoic acid levels and impairs LSC maintenance. Collectively, our findings reveal a critical role of PRMT6-MFSD2A signaling axis in AML development and provide a therapeutic strategy for targeting LSCs.

Role of the Demethylase AlkB Homolog H5 in the Promotion of Dentinogenesis.

Dentinogenesis is a key process in tooth formation and is regulated by a series of pre- and post-transcriptional regulations. N6-methyl-adenosine (m6A), which is the most prevalent internal chemical modification that can be removed by the RNA demethylase AlkB homolog H5 (ALKBH5), has recently been reported to be involved in several biological processes. However, the exact function of ALKBH5-mediated m6A modification in tooth development remains unclear. Here, we showed that Alkbh5 was expressed in pre-odontoblasts, polarizing odontoblasts, and secretory odontoblasts. Alkbh5 overexpression in the mouse dental papilla cell line mDPC6T promoted odontoblastic differentiation. Conditional knockout of Alkbh5 in Dmp1-expressing odontoblasts led to a decrease in number of odontoblasts and increased pre-dentin formation. Mechanistically, RNA sequencing and m6A sequencing of Alkbh5-overexpressing mDPC6T cells revealed that Alkbh5 promoted odontoblast differentiation by prolonging the half-life of Runx2 transcripts in an m6A-dependent manner and by activating the phosphatidylinositol 3-kinase/protein kinase B pathway. Notably, the loss of Alkbh5 expression in odontoblasts impaired tertiary dentin formation in vivo. These results suggested that the RNA demethylase ALKBH5 plays a role in dentinogenesis.

PTIP governs NAD+ metabolism by regulating CD38 expression to drive macrophage inflammation.

NAD+ metabolism is involved in many biological processes. However, the underlying mechanism of how NAD+ metabolism is regulated remains elusive. Here, we find that PTIP governs NAD+ metabolism in macrophages by regulating CD38 expression and is required for macrophage inflammation. Through integrating histone modifications with NAD+ metabolic gene expression profiling, we identify PTIP as a key factor in regulating CD38 expression, the primary NAD+-consuming enzyme in macrophages. Interestingly, we find that PTIP deletion impairs the proinflammatory response of primary murine and human macrophages, promotes their metabolic switch from glycolysis to oxidative phosphorylation, and alters NAD+ metabolism via downregulating CD38 expression. Mechanistically, an intronic enhancer of CD38 is identified. PTIP regulates CD38 expression by cooperating with acetyltransferase p300 in establishing the CD38 active enhancer with enriched H3K27ac. Overall, our findings reveal a critical role for PTIP in fine-tuning the inflammatory responses of macrophages via regulating NAD+ metabolism.

Differential m6A RNA landscapes across hematopoiesis reveal a role for IGF2BP2 in preserving hematopoietic stem cell function.

N6-methyladenosine (m6A) on mRNA plays critical roles in various cellular processes. However, the landscape and dynamics of m6A modification in hematopoietic system remain unknown. Here, we delineate a comprehensive m6A landscape across hematopoietic hierarchy and uncover that IGF2BP2 is required for preserving the function of hematopoietic stem cells (HSCs). Our data reveal a cell-type-specific m6A landscape in hematopoiesis. m6A modifications arise mostly in the early stage of hematopoiesis and prefer to play distinct roles for determining mRNA fates in HSCs and committed progenitors. Mechanistically, increased m6A-IGF2BP2 expression controls transcriptional state and maintenance of HSCs. IGF2BP2 deficiency induces quiescence loss and impairs HSC function. Moreover, IGF2BP2 loss increases mitochondrial activity of HSCs by accelerating Bmi1 mRNA decay, leading to de-repression of mitochondria-related genes. Collectively, our results present a fascinating portrait of m6A modification of hematopoietic hierarchy and reveal a key role of IGF2BP2 in maintaining HSC function by restraining mitochondrial activity.

Leukemogenic Chromatin Alterations Promote AML Leukemia Stem Cells via a KDM4C-ALKBH5-AXL Signaling Axis.

N6-methyladenosine (m6A) is a commonly present modification of mammalian mRNAs and plays key roles in various cellular processes. m6A modifiers catalyze this reversible modification. However, the underlying mechanisms by which these m6A modifiers are regulated remain elusive. Here we show that expression of m6A demethylase ALKBH5 is regulated by chromatin state alteration during leukemogenesis of human acute myeloid leukemia (AML), and ALKBH5 is required for maintaining leukemia stem cell (LSC) function but is dispensable for normal hematopoiesis. Mechanistically, KDM4C regulates ALKBH5 expression via increasing chromatin accessibility of ALKBH5 locus, by reducing H3K9me3 levels and promoting recruitment of MYB and Pol II. Moreover, ALKBH5 affects mRNA stability of receptor tyrosine kinase AXL in an m6A-dependent way. Thus, our findings link chromatin state dynamics with expression regulation of m6A modifiers and uncover a selective and critical role of ALKBH5 in AML that might act as a therapeutic target of specific targeting LSCs.

Effect of Liquid Platelet-rich Fibrin and Platelet-rich Plasma on the Regenerative Potential of Dental Pulp Cells Cultured under Inflammatory Conditions: A Comparative Analysis.

INTRODUCTION: Platelet-rich plasma (PRP) has been widely used in regenerative dentistry for over 2 decades. Nevertheless, previous studies have shown that its growth factor content is released over a short time period, and the application of anticoagulants limits its regenerative potential. Therefore, a second-generation platelet concentrate (liquid platelet-rich fibrin [PRF]) was developed without the use of anticoagulants and with shorter centrifugation times. The purpose of the present study was to compare the cellular regenerative activity of human dental pulp cells (hDPCs) when cultured with either liquid PRF or traditional PRP. METHODS: The regenerative potential of hDPCs isolated from healthy human third molars (18-22 years, n = 5) was investigated in both normal and inflammatorylike conditions (lipopolysaccharide [LPS]) and assessed for their potential for dentin repair. The effects of liquid PRF and PRP were assessed for cellular migration, proliferation, and odontoblastic differentiation using a transwell assay, scratch assay, proliferation assay, alkaline phosphatase assay, alizarin red staining, and real-time polymerase chain reaction for genes encoding collagen type 1 alpha 1, dentin sialophosphoprotein, and dentin matrix protein 1, respectively. The effects of both platelet concentrates were also assessed for their ability to influence nuclear translocation of nuclear factor kappa B (p65) by immunofluorescence, and reverse-transcription polymerase chain reaction for genes encoding interleukin-1ß, tumor necrosis factor alpha, and nuclear factor kappa B (p65) during an inflammatory condition. RESULTS: Both PRP and liquid PRF increased the migration and proliferation of hDPCs when compared with the control group, whereas liquid PRF showed a notable significant increase in migration when compared with PRP. Furthermore, liquid PRF induced significantly greater alkaline phosphatase activity, alizarin red staining, and a messenger RNA expression of genes encoding collagen type 1 alpha 1, dentin sialophosphoprotein, and dentin matrix protein 1 when compared with PRP. When hDPCs were cultured with LPS to stimulate an inflammatory environment, a marked decrease in dentin-related repair was observed. When liquid PRF was cultured within this inflammatory environment, the reduced regenerative potential in this LPS-produced environment was significantly and markedly improved, facilitating hDPC regeneration. The messenger RNA expression of inflammatory markers including tumor necrosis factor alpha, interleukin-1ß, and p65 were all significantly decreased in the presence of liquid PRF, and, furthermore, liquid PRF also inhibited the transport of p65 to the nucleus in hDPCs (suggesting a reduced inflammatory condition). CONCLUSIONS: The findings from the present study suggest that liquid PRF promoted greater regeneration potential of hDPCs when compared with traditional PRP. Furthermore, liquid PRF also attenuated the inflammatory condition created by LPS and maintained a supportive regenerative ability for the stimulation of odontoblastic differentiation and reparative dentin in hDPCs.

A novel method for evaluating and quantifying cell types in platelet rich fibrin and an introduction to horizontal centrifugation.

Platelet rich fibrin (PRF) has been utilized clinically as a platelet concentrate capable of stimulating tissue regeneration. Interestingly, several protocols have been proposed with little data obtained regarding the final cell counts following centrifugation. The aim of the present study was to compare different commercially available centrifuges and their respective protocols utilizing a novel method to quantify cells. One millimeter blood layers following centrifugation were sequentially pipetted from the upper layer downward until all 10 mL were harvested in sequential samples. Thereafter, each sample was sent for CBC analysis to accurately quantify precisely cell numbers within each separate blood layer following centrifugation. The results from this study revealed that L-PRF protocols (2700 rpm × 12 min) produced a clot with the majority of platelets and leukocytes concentrated within the buffy coat with relatively no cells found within the first 4 mL of L-PRF. Slower centrifugation protocols produced using the A-PRF protocols (1300 rpm × 8 min) produced a more evenly distributed number of platelets throughout PRF. Injectable-PRF (i-PRF) protocols produced the highest concentration of leukocytes/platelets, however, the total number of leukocytes and platelets were significantly lower owing to the decreased total volume collected. Horizontal centrifugation produced a significant increase in both the number and concentration of platelets and leukocytes (up to 3.5× higher for either solid/liquid PRF). When compared to either fixed or angled centrifuge (InstraSpin, Process for PRF). In conclusion, the present study revealed a novel/accurate method to quantify cells following PRF protocols. Furthermore, PRF produced via horizontal centrifugation accumulated a higher number and concentration of platelets/leukocytes when compared to either fixed-angle centrifugation.

Effects of concentrated growth factor on proliferation, migration, and differentiation of human dental pulp stem cells in vitro.

Concentrated growth factor, a novel autologous plasma extract, contained various growth factors which promoted tissue regeneration. In this study, we aimed to investigate the biological effects of concentrated growth factor on human dental pulp stem cells. The microstructure and biocompatibility of concentrated growth factor scaffolds were evaluated by scanning electron microscopy. Cell proliferation and migration, odontoblastic and endothelial cell differentiation potential were assessed after exposing dental pulp stem cells to different concentrations (5%, 10%, 20%, 50%, or 80%) of concentrated growth factor extracts. The results revealed that concentrated growth factor scaffolds possessed porous fibrin network with platelets and leukocytes, and showed great biocompatibility with dental pulp stem cells. Higher cell proliferation rates were detected in the concentrated growth factor-treated groups in a dose-dependent manner. Interestingly, in comparison to the controls, the low doses (<50%) of concentrated growth factor increased cell migration, alkaline phosphatase activity, and mineralized tissue deposition, while the cells treated in high doses (50% or 80%) showed no significant difference. After stimulating cell differentiation, the expression levels of dentin matrix protein-1, dentin sialophosphoprotein, vascular endothelial growth factor receptor-2 and cluster of differentiation 31 were significantly upregulated in concentrated growth factor-supplemented groups than those of the controls. Furthermore, the dental pulp stem cell-derived endothelial cells co-induced by 5% concentrated growth factor and vascular endothelial growth factor formed the most amount of mature tube-like structures on Matrigel among all groups, but the high-dosage concentrated growth factor exhibited no or inhibitory effect on cell differentiation. In general, our findings confirmed that concentrated growth factor promoted cell proliferation, migration, and the dental pulp stem cell-mediated dentinogenesis and angiogenesis process, by which it might act as a growth factor-loaded scaffold to facilitate dentin-pulp complex healing.