- Email: [email protected]
A New Method to Develop Human Dental Pulp Cells and Platelet-rich Fibrin Complex
Regenerative Endodontics
A New Method to Develop Human Dental Pulp Cells and Platelet-rich Fibrin Complex Xuan He, MD, DDS, Wen-Xia Chen, PhD, DDS, Guifei Ban, MD, DDS, Wei Wei, MD, DDS, Jun Zhou, MD, DDS, Wen-Jin Chen, MD, DDS, and Xian-Yu Li, PhD, DDS Abstract Introduction: Platelet-rich fibrin (PRF) has been used as a scaffold material in various tissue regeneration studies. In the previous methods to combine seed cells with PRF, the structure of PRF was damaged, and the manipulation time in vitro was also increased. The objective of this in vitro study was to explore an appropriate method to develop a PRF–human dental pulp cell (hDPC) complex to maintain PRF structure integrity and to find out the most efficient part of PRF. Methods: The PRF-hDPC complex was developed at 3 different time points during PRF preparation: (1) the before centrifugation (BC) group, the hDPC suspension was added to the venous blood before blood centrifugation; (2) the immediately after centrifugation (IAC) group, the hDPC suspension was added immediately after blood centrifugation; (3) the after centrifugation (AC) group, the hDPC suspension was added 10 minutes after blood centrifugation; and (4) the control group, PRF without hDPC suspension. The prepared PRF-hDPC complexes were cultured for 7 days. The samples were fixed for histologic, immunohistochemistry, and scanning electron microscopic evaluation. Real-time polymerase chain reaction was performed to evaluate messenger RNA expression of alkaline phosphatase and dentin sialophosphoprotein. Enzyme-linked immunosorbent assay quantification for growth factors was performed within the different parts of the PRF. Results: Histologic, immunohistochemistry, and scanning electron microscopic results revealed that hDPCs were only found in the BC group and exhibited favorable proliferation. Real-time polymerase chain reaction revealed that alkaline phosphatase and dentin sialophosphoprotein expression increased in the cultured PRF-hDPC complex. The lower part of the PRF released the maximum quantity of growth factors. Conclusions: Our new method to develop a PRF-hDPCs complex maintained PRF structure integrity. The hDPCs were distributed in the buffy coat, which might be the most efficient part of PRF. (J Endod 2016;42:1633–1640)
Key Words Human dental pulp cells, platelet-rich fibrin, platelet-rich fibrin–human dental pulp cell complex, scaffold
D
ental pulp regeneraSignificance tion, which is considOur study showed that a PRF-hDPC complex can ered one of the most be developed when hDPC suspension is added promising therapeutic apbefore blood centrifugation during PRF preparaproaches for irreversible tion, presenting a suitable method to combine pulpitis and periapical disPRF with seed cells to maintain PRF structure ease, consists of 3 key integrity and biological activity. components: seed cells, biomaterial scaffolds, and growth factors (1, 2). The scaffold is an essential element of tissue engineering and regeneration. Ideal scaffolds for tooth or dental pulp regeneration should be biocompatible, nontoxic, absorbable, microporous, and easy to handle, and they should promote efficient cell proliferation and migration and provide support for angiogenesis and new tissue outgrowth (3). Platelet-rich fibrin (PRF), a second generation of platelet concentrates adapted to simplified preparation with no biochemical blood handling procedure (4, 5), was first produced for specialized use in oral and maxillofacial surgery in 2001 by Choukroun et al (6). Because of the solid consistency of fibrin, PRF is slowly destroyed by remodeling, similar to a natural blood clot (7). Depending on the local and continuous delivery of a wide range of growth factors, PRF promotes the processes of wound healing and tissue repair (8). Recently, PRF has been used as a scaffold material for tissue engineering and regeneration of various tissues, such as periosteal tissue (9), periodontal ligament (10), maxillofacial soft tissue (11), dental pulp (12), and myocardium tissue (13). Although these studies have achieved favorable results, it is difficult to combine PRF evenly with seed cells because PRF is a solid biomaterial. Two methods were used in previous studies: the seed cells were either seeded on the surface of the PRF membrane or mixed with minced PRF fragments (9, 11–13). However, the fibrin network of PRF was destroyed partially or even totally. Nevertheless, the fibrin architecture directly influences the biology of all fibrin-based biomaterials (14–16). To remedy this defect, it is necessary to search for a new method to combine PRF with seed cells without any damage to the fibrin structure. Human dental pulp cells (hDPCs), a heterogeneous population that contains progenitor/stem cells of odontoblast lineage (17, 18), were the main cellular component found in the human dental pulp. The isolated dental pulp cells expressed mesenchymal stem cell markers and could differentiate in vitro into odontogenic, adipogenic, and chondrogenic lineages (19). In our study, hDPCs were used as seed cells to explore a suitable method to prepare a PRF-hDPC complex. Growth factors are crucial signal molecules that could instruct stem cells to accomplish tissue regeneration. Research has shown that PRF releases high quantities of 3 main growth factors (transforming growth factor beta 1 [TGF-b1], platelet-derived
From the Department of Operative Dentistry and Endodontology, College and Hospital of Stomatology, Guangxi Medical University, Nanning, Guangxi, China. Address requests for reprints to Dr Wen-Xia Chen, Department of Operative Dentistry and Endodontology, College and Hospital of Stomatology, Guangxi Medical University, No. 10 Shuangyong Road, Nanning, Guangxi, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.08.011
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Regenerative Endodontics growth factor [PDGF]-AB, and vascular endothelial growth factor [VEGF]) and an important coagulation matricellular glycoprotein (thrombospondon-1 [TSP-1]) for at least 7 days (20). PRF accumulates approximately 97% of the platelets and >50% of the leukocytes of the blood harvest and develops a fibrin network similar to a natural blood clot although platelet and leukocyte degranulation implies a large release of growth factors that promote cell proliferation, matrix remodeling, and the protection of healing cells. As a scaffold biomaterial, PRF gel consists of 3 main visible parts: a fibrin yellow portion termed the fibrin matrix composing the main body; a red portion located at the end of the PRF gel (full of red blood cells [RBCs]); and between these 2 areas, a whitish layer called the buffy coat (21). Histologic analysis has revealed that platelets and leukocytes are concentrated in the buffy coat (22). Considering the heterogeneous structure of PRF, it is necessary to find out the most efficient part of this biomaterial. This study explored a suitable method to prepare a PRF-hDPC complex and investigated the growth forms of hDPCs in PRF. The secondary objective of this work was to find out the most efficient part of PRF through growth factor release analysis.
Materials and Methods All the experiments were performed with the approval of the Institutional Review Board of the College and Hospital of Stomatology, Guangxi Medical University, Guangxi, China (approval number 2014005). Written informed consent from the donors was obtained for the use of all human tissues involved in our research (including dental pulp tissue and blood).
Primary Culture and Characterization of hDPCs Dental pulps were obtained from sound human third molars (age range 18–25 years) extracted for therapeutic reasons. The derived tissues were washed with Dulbecco modified Eagle medium (DMEM; Hyclone, Logan, UT) and minced into 0.5- to 1-mm pieces using microscissors. The tissue blocks were cultured in DMEM supplemented with 20% fetal bovine serum (Hyclone) and a complex of 100 U/mL penicillin G and 100 mg/mL streptomycin. The medium was replaced every 3 days, and cells were subcultured at 70% confluence. Cell cultures between the third and eighth passages were used. To characterize the cell lineage of hDPCs, morphologic analysis was performed by immunocytochemical staining for vimentin and keratin (Zhongshan Jinqiao Co, Beijing, China). The immunoreaction was conducted according to the instructions of the SP kit (Zhongshan Jinqiao Co). PRF Preparation Venous blood from 2 healthy donors was collected from the cubital vein for PRF preparation using a previously reported protocol (4). Briefly, blood samples were taken without anticoagulant in 5-mL vacuum blood collection tubes and immediately centrifuged at 400g for 10 minutes. The PRF was obtained in the middle of the tube, between the acellular plasma at the top and the red corpuscles at the bottom. The PRF gel was used right after obtaining. Effect of PRF Extract on hDPC Proliferation PRF gels were soaked in standard culture mediums (2.5 mL standard culture mediums for every 1.0 g PRF gel) to make the PRF extract. The hDPCs were seeded in 96-well plates at an initial density of 2000 cells per well and cultured in PRF extract for 7 days. The control was standard culture (DMEM supplemented with 10% fetal bovine serum and a mixture of 100 U/mL penicillin G and 100 mg/mL streptomycin) only. At each experimental time (ie, on days 3, 5, and 7), 10 mL 1634
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Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each well. After incubation for 4 hours, the plate absorbance was measured at 450 nm using a microplate reader. The dehydrogenase activity of hDPCs with the PRF extract at each time point was calculated from the absorbance values compared with that of each control.
Preparation of PRF-hDPC Complex hDPCs in the logarithmic growth phase were selected to perform the experiment. The cells were resuspended in phosphate-buffered saline at a density of 105 cells/mL. According to the preparation protocol of PRF, 0.5 mL hDPC suspension was added to the venous blood at 3 different time points: 1. BC group: The hDPC suspension was added before blood centrifugation. 2. IAC group: The hDPC suspension was added immediately after blood centrifugation. 3. AC group: The hDPC suspension was added 10 minutes after blood centrifugation. 4. Control group: The negative control was PRF without hDPC suspension. The prepared PRF-hDPC complexes were cultured in a 6-well plate filled with 1.5 mL standard culture medium for 7 days. All the samples were fixed for histologic, immunohistochemistry, and scanning electron microscopic (SEM) evaluation.
Histologic Analysis After fixation in 4% paraformaldehyde for 24 hours, all the PRFhDPC complex samples were embedded in paraffin wax, sectioned longitudinally along the long axis of the PRF gel, and stained with hematoxylin-eosin. Each individual PRF gel was analyzed histologically under a light microscope to determine the tissue structure. Immunohistochemistry To visualize the localization of leukocytes and hDPCs in plateletrich fibrin gel sections, immunohistochemistry staining for vimentin, CD45 (Zhongshan Jinqiao Co), and dentin sialophosphoprotein (DSPP) (BIOSS, Beijing, China) was performed. The immunoreaction was conducted according to the instructions of the streptavidin/peroxidase kit (Zhongshan Jinqiao Co). SEM Evaluation A morphologic evaluation of the hDPC-PRF complex was performed with a scanning electron microscope. The hDPC-PRF complex was fixed in 2.5% glutaraldehyde for 1 hour and treated for dessication. Specimens were sputter coated with 20 nm gold and subsequently examined in a scanning electron microscope. Photographs were taken using 1000 to 10,000 magnifications. Real-time Polymerase Chain Reaction Analysis For real-time polymerase chain reaction analysis, the total RNA of the PRF-hDPC complex was isolated using RNAiso Plus (Takara, Otsu, Japan) according to the manufacturer’s instructions. The total extracted RNA was applied toward complementary DNA generation with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). The relative gene expression levels of alkaline phosphatase (ALP) and DSPP were evaluated. Real-time polymerase chain reaction was performed using SYBR Premix Ex Taq (Takara) on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) with amplification conditions as follows: 95 C/30 seconds for denaturation followed by 95 C/5 JOE — Volume 42, Number 11, November 2016
Regenerative Endodontics seconds and 64 C/30 seconds for 40 cycles. The relative gene expression was calculated by the 2 DDCt method and normalized to GAPDH. The primer sequences and product sizes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ALP, and DSPP are listed in Supplemental Table S1 (Supplemental Table S1 is available online at www.jendodon.com).
Sample Preparation and Enzyme-linked Immunosorbent Assay Quantification The PRF gel was cut along the midline of the long axis into 2 segments. The lower part contained the buffy coat, and the upper part was without the buffy coat (Fig. 1B). Twelve freshly prepared PRF gels were divided into 3 groups, named the WP group (whole PRF), the LP group (the lower part of PRF containing the buffy coat), and the UP group (the upper part of PRF without the buffy coat). The 3 groups of PRF gels were
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soaked separately in sterile DMEM (2.5 mL DMEM for every 1.0 g PRF gel). The extracts of PRF gel were collected at 24, 72, 120, 168, and 216 hours. At each experimental time, the PRF gel was transferred to new sterile DMEM, and the previous DMEM was stored at 80 C until enzyme-linked immunosorbent assay quantification. When all the samples were collected, quantifications of 4 growth factors were performed using enzyme-linked immunosorbent assay kits (CUSABIO, Hubei, China): human PDGF-BB, human TGF-b1, human insulin-like growth factor 1 (IGF-1), and human basic fibroblast growth factor 3 (FGF2). The optical density was read using a microplate reader set to 450 nm, and the concentrations were calculated.
Statistical Analysis Triplicate experiments were conducted throughout this study to guarantee reproducibility of the results. Statistical analysis was
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Figure 1. (A) Messenger RNA expression of ALP and DSPP in the PRF-hDPC complex. Messenger RNA expression of ALP and DSPP was evaluated by real-time reverse-transcription polymerase chain reaction. Values are present as mean standard deviation (n = 3) normalized to GAPDH. *P < .05 between the 0-day PRF-hDPC complex and the 7-day PRF-hDPC complex. (B–F) Growth factor release in different parts of PRF. (B) After blood centrifugation, the whole PRF gel (WP) was cut along the midline of the long axis into 2 segments. The lower part contained the buffy coat (LP), and the upper part was without the buffy coat (UP). (C–F) Quantification of PDGF-BB, TGF-b1, IGF1, and FGF2 released by different parts of PRF was evaluated by enzyme-linked immunosorbent assay. Values are present as mean standard deviation (n = 3). *P < .05 between the WP group and the LP group. JOE — Volume 42, Number 11, November 2016
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mesenchymal origin (Supplemental Figure S1B and C is available online at www.jendodon.com).
Results
Effect of PRF Extract on hDPC Proliferation After incubation, the PRF extract was found to increase hDPC proliferation. From the Cell Counting Kit-8 assay, the absorbance values of hDPCs with the PRF extract increased about 1.7-fold on days 5 and 7 compared with the control group (P < .05) (Supplemental Figure S2 is available online at www.jendodon.com).
Identification of hDPCs Fibroblastlike morphology with a spindle shape and strong proliferation was visualized in primary hDPCs (Supplemental Figure S1A is available online at www.jendodon.com). The hDPCs showed a positive expression of vimentin but were negative for keratin, indicating their
Figure 2. (A) Prepared PRF-hDPC complex. A fibrin clot (2) was obtained in the middle of the tube, between the acellular plasma (1) at the top and the red corpuscles (3) at the bottom. (B) The PRF-hDPC complex was composed of 3 main parts visible from top to bottom with the naked eyes: fibrin matrix, buffy coat, and red blood cells. FM, fibrin matrix; BC, buffy coat; R, red blood cells. (C–E) Control group (PRF). The fibrin matrix appeared homogeneous in light pink and showed a sparse network structure, and the area of the buffy coat was dark pink and showed a dense network structure. A large number of leukocytes were deposited on the bottom of the buffy coat. (F–H) The BC group. Leukocytes and seeded cells (arrows) with irregular morphology were distributed in the buffy coat. (I–K) The IAC group. Only leukocytes were found in the buffy coat. (L–N) The AC group. Only leukocytes were found in the buffy coat. Sections were stained with hematoxylin-eosin.
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Regenerative Endodontics Construction of the PRF-hDPC Complex A fibrin clot was obtained in the middle of the tube, between the acellular plasma at the top and the red corpuscles at the bottom (Fig. 2A). The PRF-hDPC complex was composed of 3 main parts visible from top to bottom with the naked eye: fibrin matrix, buffy coat, and RBCs (Fig. 2B). In the control group, with hematoxylin-eosin staining, the fibrin matrix appeared homogeneous in light pink and showed a sparse network structure, and the area of the buffy coat was dark pink and showed a dense network structure. A large number of leukocytes were deposited on the bottom of the buffy coat. The leukocytes were stained in dark blue with hematoxylin. The RBCs were stained in red with eosin (Fig. 2C–E). In the BC group, leukocytes and seeded cells with irregular morphology were distributed in the buffy coat of the PRF gel. The seeded cells were larger than leukocytes, and the nuclei were oval
and stained in dark blue with hematoxylin, whereas the cytoplasm was stained red (Fig. 2F–H). In the IAC group and AC group, only leukocytes were found in the buffy coat of the PRF gel similarly to the control group (Fig. 2I–N).
PRF-hDPC Complex in 7 days During the 7 days of incubation, the PRF-hDPC complex was fixed for hematoxylin-eosin staining. In the control group, Leukocytes were dispersed among the buffy coat of PRF gel (Fig. 3A–C). In the BC group, morphology of the seeded cells changed from irregular to spindle shaped. Some spindle cell colonies were found in the buffy coat of the PRF-hDPC complex; the cells gathered into a spiral. A layer of spindle cells was observed along the edge of the buffy coat (Fig. 3D–H). In the IAC and AC groups, only Leukocytes were dispersed among the buffy coat of the PRF gel similarly to the control group (Fig. 3I–N).
Figure 3. The PRF-hDPC complex in 7 days. (A–C) The control group (PRF). Leukocytes were dispersed among the buffy coat. FM, fibrin matrix; BC, buffy coat; R, red blood cells. (D–H) The BC group. (E and F) Spindle cell colonies (arrows) were found in the buffy coat. (G and H) A layer of spindle cells (arrows) was distributed along the edge of the buffy coat. (I–K) The IAC group. Leukocytes were dispersed among the buffy coat. (L–N) The AC group. Leukocytes were dispersed among the buffy coat. Sections were stained with hematoxylin-eosin.
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Figure 4. Immunohistochemical staining of (B, F, and J) vimentin, (C, F, and K) CD45, and (D, H, and I) DSPP for the PRF-hDPC complex in the BC group. (A–D) The 0-day PRF-hDPC complex. Seeded cells (arrows) were distributed in the buffy coat and stained positive for vimentin, negative for CD45, and weakly positive for DSPP. (E–L) The 7-day PRF-hDPC complex. (E–H) Spindle cell colonies and (I–L) a layer of spindle cells along the edge of the buffy coat stained positive for vimentin, negative for CD45, and weakly positive for DSPP. (M–X) The ultrastructure of the PRF-hDPC complex. Scanning electron microscopy. (M–R) The 0-day PRF-hDPC complex. (M–O) Leukocytes were enmeshed in the fibrin network. (P–R) The seeded cells (arrows) were inserted in the interspaces between the fibrin. (S–X) The 7-day PRF-hDPC complex. hDPCs (arrows) adhered firmly in the (S–U) spindle cell colonies and distributed along the edge of (V–X) the buffy coat.
Immunohistochemical Staining for the PRF-hDPC Complex Immunohistochemical staining of vimentin, CD45, and DSPP for the PRF-hDPC complex was performed in the BC group. In the 0-day PRF-hDPC complex, seeded cells were distributed in the buffy coat 1638
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and stained positive for vimentin, negative for CD45, and weakly positive for DSPP (Fig. 4A–D). In the 7-day PRF-hDPC complex, spindle cell colonies and a layer of spindle cells along the edge of the buffy coat stained positive for vimentin, negative for CD45, and weakly positive for DSPP (Fig. 4E–L).
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Regenerative Endodontics Scanning Electron Microscopy of the PRF-hDPC Complex The result of the SEM evaluation was consistent with the light microscopic findings. In the 0-day PRF-hDPC complex, the seeded cells were found only in the BC group. Leukocytes were enmeshed in the fibrin network, and the seeded cells were inserted in the interspaces between the fibrin and had no connection with the fibrin network (Fig. 4M–R). In the 7-day PRF-hDPC complex, hDPCs adhered firmly in the spindle cell colonies and distributed along the edge of the buffy coat (Fig. 4S–X). Messenger RNA Expression of ALP and DSPP in the PRF-hDPC Complex The profile of ALP and DSPP messenger RNA expression was assessed by real-time polymerase chain reaction. During the 7 days of incubation, ALP and DSPP expression was up-regulated in PRF-hDPC complex. ALP expression was increased 5.7-fold compared with the PRF-hDPC complex just prepared, whereas the DSPP expression increased 2.3-fold (P < .05) (Fig. 1A). Growth Factor Released in Different Parts of PRF Certain amounts of PDGF-BB, TGF-b1, and FGF2 were found at each experimental time, even 9 days after the production of the PRF gel. IGF-1 was detected in the first 120 hours. The release peak for PDGF-BB was at 72 hours, and the other 3 growth factors were released at 120 hours. The quantity of growth factors released by the lower part of the PRF (the LP group) was higher than the whole PRF (the WP group) at most times, and the upper part of the PRF (the UP group) hardly released any growth factors (Fig. 1B–F).
Discussion Without any gelling agent such as anticoagulant or bovine thrombin, the slow polymerization during PRF preparation seems to develop a fibrin network similar to the natural one, resulting in more efficient cell migration and proliferation and thus cicatrization (4). In our study, the extract of PRF was found to encourage the gradual proliferation of hDPCs, and similar results were reported by Tsai et al (23), Dohan Ehrenfest et al (24), and Huang et al (25). The researchers showed that PRF stimulated the cell proliferation of gingival fibroblasts, periodontal ligament cells, osteoblasts, and hDPCs. These studies confirm the biocompatibility and lack of cytotoxicity of PRF as a biomaterial. An ideal scaffold should be suitable for the seeding of stem/progenitor cells (26). Unlike hydrogels or platelet-rich plasma (27), PRF cannot be injected with a suspension as a solid material (28). It is difficult to mix the seed cells with PRF by simply adding the cell suspension after centrifugation. In previous studies, the seed cells were either seeded on the surface of the PRF membrane or mixed with minced PRF fragments (9, 11). However, the structure of PRF was damaged, and the manipulation time in vitro was increased. Research has shown that the final architecture of the fibrin matrix influences the strength and the growth factor release potential of PRF (28). In our research, when hDPC suspension was added before blood centrifugation during the preparation protocol of PRF, the histologic results showed that hDPCs were well embedded in the buffy coat of PRF by this method. The immunohistochemistry results revealed that the seeded cells exhibiting irregular morphology and larger size showed positive expression for vimentin, negative for CD45, and weakly positive for DSPP. Furthermore, the morphology of the seeded cells changed from irregular to spindle shape during 7 days of incubation. These results indicated that the seeded cells were indeed hDPCs. In the groups in which hDPCs were added after centrifugation, no cells with definite hDPC features were observed in addition to the blood cells. These results have revealed that a PRF-hDPC complex can be developed by this new method and that the complex maintains the structural integrity of the PRF. JOE — Volume 42, Number 11, November 2016
PRF is derived from the blood and prepared by centrifugation (8, 29). The added hDPCs were treated as a normal cellular ingredient, such as leukocytes, which may explain why leukocytes and hDPCs were distributed in the same position in the PRF. The histologic and SEM analyses indicated that hDPCs seemed to have a separate space from the leukocytes, which were enmeshed in the fibrin network. This phenomenon provided a vacuum for hDPC growth. In brief, the hDPCs were cultured in a 3-dimensional culture condition within PRF gel. Our histologic analysis has revealed that the hDPCs experienced a favorable proliferation in the fibrin matrix of PRF gel 7 days after inoculation. Two forms of hDPC growth were observed in PRF-hDPCs. One is spindle cell colonies; the hDPCs gathered into a spiral mainly distributed in the buffy coat. We speculate that this may because of a wealth of growth factors within the buffy coat, which could promote cell adhesion and proliferation. The other form for hDPC growth is more interesting; a layer of spindle cells was distributed along the edge of the buffy coat and presented positive expression for vimentin, negative for CD45, and weakly positive for DSPP. Cellular migration is one of the crucial parameters for tissue repair and regeneration. TGF-b1, which was rich in PRF, was found to be important promoters of DPSC migration (30). Fibroblasts develop a significant proteolytic activity to move with the fibrin clot through the expression of 2 plasminogen activators (31). However, whether the hDPCs were located at the edge of the buffy coat at the very beginning when the PRF-hDPCs complex was prepared or they migrated to the spot later during incubation needs to be further confirmed. For pulp regeneration, the formation of a layer of odontoblastlike cells lining against the existing dentin surface and producing new dentin is considered to be necessary for regenerated pulp identification (32). Several research groups had observed a layer of odontoblastlike cells lining against the existing dentin surface in regenerated pulplike tissue (12, 33–35). We used to attribute this to the cytokine released by the treated dentin matrix. In the present study, a layer of hDPCs was observed along the edge of the buffy coat in PRF, indicating that PRF may play a synergistic effect with dentin matrix in the formation of odontoblast cells when used as a scaffold for pulp regeneration. ALP is considered as one of the markers indicating odontoblast differentiation and mineralization of dental pulp cells (36). DSPP is the initial translational product of DSPP messenger RNA and then cleaved to dentin phosphoprotein and dentin sialoprotein (37). DSPP plays a crucial role in dentinogenesis (38). ALP and DSPP expression was generally regarded as a marker of odontoblastic differentiation. Chen et al (12) showed that PRF induced the differentiation of DPSCs to odonto-/osteoblastic fates by increasing the expression of the Alp, Dspp, Dmp1, and Bsp genes. In the present study, the ALP and DSPP expression was up-regulated in the PRF-hDPC complex during the 7day incubation, indicating that PRF might maintain its property to promote odontoblastic differentiation in the complex. This effect will be further explored in our animal studies. In an ideal scaffold, it is important to have supplementation with growth factors (26). The PRF membrane exhibits a very significant slow sustained release of many key growth factors for at least 1 week (20) and up to 28 days (39). Considering the heterogeneous structure of PRF, it is necessary to find out the most efficient part of this biomaterial. Here, we showed that PRF gel slowly released certain amounts of PDGF-BB, TGF-b1, IGF-1, and FGF2, which were essential for hDPC proliferation and differentiation (40). The result indicated that most growth factors released by PRF gel might be derived from the buffy coat. This result was expected because platelets and leukocytes were concentrated in the buffy coat of PRF (22). Coincidentally, our study indicated that hDPCs were mainly inserted in the buffy coat area, underscoring the
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Acknowledgments Supported by the National Natural Science Foundation of China (grant no. 81160133). The authors deny any conflicts of interest related to this study.
Supplementary Material Supplementary material associated with this article can be found in the online version at www.jendodon.com (http://dx.doi. org/10.1016/j.joen.2016.08.011).
References 1. Hargreaves KM, Diogenes A, Teixeira FB. Treatment options: biological basis of regenerative endodontic procedures. J Endod 2013;39(Suppl):S30–43. 2. Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–6. 3. Yildirim S, Fu SY, Kim K, et al. Tooth regeneration: a revolution in stomatology and evolution in regenerative medicine. Int J Oral Sci 2011;3:107–16. 4. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): a secondgeneration platelet concentrate. Part I: technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e37–44. 5. Sunitha Raja V, Munirathnam Naidu E. Platelet-rich fibrin: evolution of a secondgeneration platelet concentrate. Indian J Dent Res 2008;19:42–6. 6. Choukroun J, Adda F, Schoeffler C, et al. Une opportunite en paro-implantologie: le PRF. Implantodontie 2001;42:e62. 7. Clark RA. Fibrin and wound healing. Ann N Y Acad Sci 2001;936:355–67. 8. Borie E, Olivi DG, Orsi IA, et al. Platelet-rich fibrin application in dentistry: a literature review. Int J Clin Exp Med 2015;8:7922–9. 9. Gassling V, Douglas T, Warnke PH, et al. Platelet-rich fibrin membranes as scaffolds for periosteal tissue engineering. Clin Oral Implants Res 2010;21:543–9. 10. Ji B, Sheng L, Chen G, et al. The combination use of platelet-rich fibrin and treated dentin matrix for tooth root regeneration by cell homing. Tissue Eng Part A 2015;21: 26–34. 11. Chen Y, Niu Z, Xue Y, et al. Improvement in the repair of defects in maxillofacial soft tissue in irradiated minipigs by a mixture of adipose-derived stem cells and plateletrich fibrin. Br J Oral Maxillofac Surg 2014;52:740–5. 12. Chen YJ, Zhao YH, Zhao YJ, et al. Potential dental pulp revascularization and odonto-/osteogenic capacity of a novel transplant combined with dental pulp stem cells and platelet-rich fibrin. Cell Tissue Res 2015;361:439–55. 13. Sun CK, Zhen YY, Leu S, et al. Direct implantation versus platelet-rich fibrinembedded adipose-derived mesenchymal stem cells in treating rat acute myocardial infarction. Int J Cardiol 2014;173:410–23. 14. Collen A, Koolwijk P, Kroon M, et al. Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells. Angiogenesis 1998;2:153–65. 15. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci 2001;936:11–30.
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16. Buchta C, Hedrich HC, Macher M, et al. Biochemical characterization of autologous fibrin sealants produced by CryoSeal and Vivostat in comparison to the homologous fibrin sealant product Tissucol/Tisseel. Biomaterials 2005;26:6233–41. 17. Alliot-Licht B, Bluteau G, Magne D, et al. Dexamethasone stimulates differentiation of odontoblast-like cells in human dental pulp cultures. Cell Tissue Res 2005;321: 391–400. 18. Huang GT, Shagramanova K, Chan SW. Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro. J Endod 2006;32:1066–73. 19. Wei X, Ling J, Wu L, et al. Expression of mineralization markers in dental pulp cells. J Endod 2007;33:703–8. 20. Dohan Ehrenfest DM, de Peppo GM, Doglioli P, et al. Slow release of growth factors and thrombospondin-1 in Choukroun’s platelet-rich fibrin (PRF): a gold standard to achieve for all surgical platelet concentrates technologies. Growth Factors 2009; 27:63–9. 21. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): a secondgeneration platelet concentrate. Part II: platelet-related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e45–50. 22. Dohan Ehrenfest DM, Del Corso M, Diss A, et al. Three-dimensional architecture and cell composition of a Choukroun’s platelet-rich fibrin clot and membrane. J Periodontol 2010;81:546–55. 23. Tsai C-H, Shen S-Y, Zhao J-H, et al. Platelet-rich fibrin modulates cell proliferation of human periodontally related cells in vitro. J Dent Sci 2009;4:130–5. 24. Dohan Ehrenfest DM, Doglioli P, de Peppo GM, et al. Choukroun’s platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way. Arch Oral Biol 2010;55: 185–94. 25. Huang F-M, Yang S-F, Zhao J-H, et al. Platelet-rich fibrin increases proliferation and differentiation of human dental pulp cells. J Endod 2010;36:1628–32. 26. Nosrat A, Ryul Kim J, Verma P, et al. Tissue engineering considerations in dental pulp regeneration. Iran Endod J 2014;9:30–9. 27. Zhu X, Zhang C, Huang GT, et al. Transplantation of dental pulp stem cells and platelet-rich plasma for pulp regeneration. J Endod 2012;38:1604–9. 28. Dohan Ehrenfest DM, Bielecki T, Jimbo R, et al. Do the fibrin architecture and leukocyte content influence the growth factor release of platelet concentrates? An evidence-based answer comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte- and platelet-rich fibrin (L-PRF). Curr Pharm Biotechnol 2012;13: 1145–52. 29. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin (PRF): a secondgeneration platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e56–60. 30. Howard C, Murray PE, Namerow KN. Dental pulp stem cell migration. J Endod 2010; 36:1963–6. 31. Brown LF, Lanir N, McDonagh J, et al. Fibroblast migration in fibrin gel matrices. Am J Pathol 1993;142:273–83. 32. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 2010;16:605–15. 33. Yang JW, Zhang YF, Wan CY, et al. Autophagy in SDF-1alpha-mediated DPSC migration and pulp regeneration. Biomaterials 2015;44:11–23. 34. Nakashima M, Iohara K. Mobilized dental pulp stem cells for pulp regeneration: initiation of clinical trial. J Endod 2014;40(Suppl):S26–32. 35. Iohara K, Murakami M, Takeuchi N, et al. A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration. Stem Cells Transl Med 2013;2:521–33. 36. Yokose S, Kadokura H, Tajima Y, et al. Establishment and characterization of a culture system for enzymatically released rat dental pulp cells. Calcif Tissue Int 2000; 66:139–44. 37. Butler WT, Brunn JC, Qin C, et al. Extracellular matrix proteins and the dynamics of dentin formation. Connect Tissue Res 2002;43:301–7. 38. Sreenath T, Thyagarajan T, Hall B, et al. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III. J Biol Chem 2003;278: 24874–80. 39. He L, Lin Y, Hu X, et al. A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:707–13. 40. Kim SG, Zhou J, Solomon C, et al. Effects of growth factors on dental stem/progenitor cells. Dent Clin North Am 2012;56:563–75.
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A New Method to Develop Human Dental Pulp Cells and Platelet-rich Fibrin Complex Xuan He, MD, DDS, Wen-Xia Chen, PhD, DDS, Guifei Ban, MD, DDS, Wei Wei, MD, DDS, Jun Zhou, MD, DDS, Wen-Jin Chen, MD, DDS, and Xian-Yu Li, PhD, DDS Abstract Introduction: Platelet-rich fibrin (PRF) has been used as a scaffold material in various tissue regeneration studies. In the previous methods to combine seed cells with PRF, the structure of PRF was damaged, and the manipulation time in vitro was also increased. The objective of this in vitro study was to explore an appropriate method to develop a PRF–human dental pulp cell (hDPC) complex to maintain PRF structure integrity and to find out the most efficient part of PRF. Methods: The PRF-hDPC complex was developed at 3 different time points during PRF preparation: (1) the before centrifugation (BC) group, the hDPC suspension was added to the venous blood before blood centrifugation; (2) the immediately after centrifugation (IAC) group, the hDPC suspension was added immediately after blood centrifugation; (3) the after centrifugation (AC) group, the hDPC suspension was added 10 minutes after blood centrifugation; and (4) the control group, PRF without hDPC suspension. The prepared PRF-hDPC complexes were cultured for 7 days. The samples were fixed for histologic, immunohistochemistry, and scanning electron microscopic evaluation. Real-time polymerase chain reaction was performed to evaluate messenger RNA expression of alkaline phosphatase and dentin sialophosphoprotein. Enzyme-linked immunosorbent assay quantification for growth factors was performed within the different parts of the PRF. Results: Histologic, immunohistochemistry, and scanning electron microscopic results revealed that hDPCs were only found in the BC group and exhibited favorable proliferation. Real-time polymerase chain reaction revealed that alkaline phosphatase and dentin sialophosphoprotein expression increased in the cultured PRF-hDPC complex. The lower part of the PRF released the maximum quantity of growth factors. Conclusions: Our new method to develop a PRF-hDPCs complex maintained PRF structure integrity. The hDPCs were distributed in the buffy coat, which might be the most efficient part of PRF. (J Endod 2016;42:1633–1640)
Key Words Human dental pulp cells, platelet-rich fibrin, platelet-rich fibrin–human dental pulp cell complex, scaffold
D
ental pulp regeneraSignificance tion, which is considOur study showed that a PRF-hDPC complex can ered one of the most be developed when hDPC suspension is added promising therapeutic apbefore blood centrifugation during PRF preparaproaches for irreversible tion, presenting a suitable method to combine pulpitis and periapical disPRF with seed cells to maintain PRF structure ease, consists of 3 key integrity and biological activity. components: seed cells, biomaterial scaffolds, and growth factors (1, 2). The scaffold is an essential element of tissue engineering and regeneration. Ideal scaffolds for tooth or dental pulp regeneration should be biocompatible, nontoxic, absorbable, microporous, and easy to handle, and they should promote efficient cell proliferation and migration and provide support for angiogenesis and new tissue outgrowth (3). Platelet-rich fibrin (PRF), a second generation of platelet concentrates adapted to simplified preparation with no biochemical blood handling procedure (4, 5), was first produced for specialized use in oral and maxillofacial surgery in 2001 by Choukroun et al (6). Because of the solid consistency of fibrin, PRF is slowly destroyed by remodeling, similar to a natural blood clot (7). Depending on the local and continuous delivery of a wide range of growth factors, PRF promotes the processes of wound healing and tissue repair (8). Recently, PRF has been used as a scaffold material for tissue engineering and regeneration of various tissues, such as periosteal tissue (9), periodontal ligament (10), maxillofacial soft tissue (11), dental pulp (12), and myocardium tissue (13). Although these studies have achieved favorable results, it is difficult to combine PRF evenly with seed cells because PRF is a solid biomaterial. Two methods were used in previous studies: the seed cells were either seeded on the surface of the PRF membrane or mixed with minced PRF fragments (9, 11–13). However, the fibrin network of PRF was destroyed partially or even totally. Nevertheless, the fibrin architecture directly influences the biology of all fibrin-based biomaterials (14–16). To remedy this defect, it is necessary to search for a new method to combine PRF with seed cells without any damage to the fibrin structure. Human dental pulp cells (hDPCs), a heterogeneous population that contains progenitor/stem cells of odontoblast lineage (17, 18), were the main cellular component found in the human dental pulp. The isolated dental pulp cells expressed mesenchymal stem cell markers and could differentiate in vitro into odontogenic, adipogenic, and chondrogenic lineages (19). In our study, hDPCs were used as seed cells to explore a suitable method to prepare a PRF-hDPC complex. Growth factors are crucial signal molecules that could instruct stem cells to accomplish tissue regeneration. Research has shown that PRF releases high quantities of 3 main growth factors (transforming growth factor beta 1 [TGF-b1], platelet-derived
From the Department of Operative Dentistry and Endodontology, College and Hospital of Stomatology, Guangxi Medical University, Nanning, Guangxi, China. Address requests for reprints to Dr Wen-Xia Chen, Department of Operative Dentistry and Endodontology, College and Hospital of Stomatology, Guangxi Medical University, No. 10 Shuangyong Road, Nanning, Guangxi, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.08.011
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Regenerative Endodontics growth factor [PDGF]-AB, and vascular endothelial growth factor [VEGF]) and an important coagulation matricellular glycoprotein (thrombospondon-1 [TSP-1]) for at least 7 days (20). PRF accumulates approximately 97% of the platelets and >50% of the leukocytes of the blood harvest and develops a fibrin network similar to a natural blood clot although platelet and leukocyte degranulation implies a large release of growth factors that promote cell proliferation, matrix remodeling, and the protection of healing cells. As a scaffold biomaterial, PRF gel consists of 3 main visible parts: a fibrin yellow portion termed the fibrin matrix composing the main body; a red portion located at the end of the PRF gel (full of red blood cells [RBCs]); and between these 2 areas, a whitish layer called the buffy coat (21). Histologic analysis has revealed that platelets and leukocytes are concentrated in the buffy coat (22). Considering the heterogeneous structure of PRF, it is necessary to find out the most efficient part of this biomaterial. This study explored a suitable method to prepare a PRF-hDPC complex and investigated the growth forms of hDPCs in PRF. The secondary objective of this work was to find out the most efficient part of PRF through growth factor release analysis.
Materials and Methods All the experiments were performed with the approval of the Institutional Review Board of the College and Hospital of Stomatology, Guangxi Medical University, Guangxi, China (approval number 2014005). Written informed consent from the donors was obtained for the use of all human tissues involved in our research (including dental pulp tissue and blood).
Primary Culture and Characterization of hDPCs Dental pulps were obtained from sound human third molars (age range 18–25 years) extracted for therapeutic reasons. The derived tissues were washed with Dulbecco modified Eagle medium (DMEM; Hyclone, Logan, UT) and minced into 0.5- to 1-mm pieces using microscissors. The tissue blocks were cultured in DMEM supplemented with 20% fetal bovine serum (Hyclone) and a complex of 100 U/mL penicillin G and 100 mg/mL streptomycin. The medium was replaced every 3 days, and cells were subcultured at 70% confluence. Cell cultures between the third and eighth passages were used. To characterize the cell lineage of hDPCs, morphologic analysis was performed by immunocytochemical staining for vimentin and keratin (Zhongshan Jinqiao Co, Beijing, China). The immunoreaction was conducted according to the instructions of the SP kit (Zhongshan Jinqiao Co). PRF Preparation Venous blood from 2 healthy donors was collected from the cubital vein for PRF preparation using a previously reported protocol (4). Briefly, blood samples were taken without anticoagulant in 5-mL vacuum blood collection tubes and immediately centrifuged at 400g for 10 minutes. The PRF was obtained in the middle of the tube, between the acellular plasma at the top and the red corpuscles at the bottom. The PRF gel was used right after obtaining. Effect of PRF Extract on hDPC Proliferation PRF gels were soaked in standard culture mediums (2.5 mL standard culture mediums for every 1.0 g PRF gel) to make the PRF extract. The hDPCs were seeded in 96-well plates at an initial density of 2000 cells per well and cultured in PRF extract for 7 days. The control was standard culture (DMEM supplemented with 10% fetal bovine serum and a mixture of 100 U/mL penicillin G and 100 mg/mL streptomycin) only. At each experimental time (ie, on days 3, 5, and 7), 10 mL 1634
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Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each well. After incubation for 4 hours, the plate absorbance was measured at 450 nm using a microplate reader. The dehydrogenase activity of hDPCs with the PRF extract at each time point was calculated from the absorbance values compared with that of each control.
Preparation of PRF-hDPC Complex hDPCs in the logarithmic growth phase were selected to perform the experiment. The cells were resuspended in phosphate-buffered saline at a density of 105 cells/mL. According to the preparation protocol of PRF, 0.5 mL hDPC suspension was added to the venous blood at 3 different time points: 1. BC group: The hDPC suspension was added before blood centrifugation. 2. IAC group: The hDPC suspension was added immediately after blood centrifugation. 3. AC group: The hDPC suspension was added 10 minutes after blood centrifugation. 4. Control group: The negative control was PRF without hDPC suspension. The prepared PRF-hDPC complexes were cultured in a 6-well plate filled with 1.5 mL standard culture medium for 7 days. All the samples were fixed for histologic, immunohistochemistry, and scanning electron microscopic (SEM) evaluation.
Histologic Analysis After fixation in 4% paraformaldehyde for 24 hours, all the PRFhDPC complex samples were embedded in paraffin wax, sectioned longitudinally along the long axis of the PRF gel, and stained with hematoxylin-eosin. Each individual PRF gel was analyzed histologically under a light microscope to determine the tissue structure. Immunohistochemistry To visualize the localization of leukocytes and hDPCs in plateletrich fibrin gel sections, immunohistochemistry staining for vimentin, CD45 (Zhongshan Jinqiao Co), and dentin sialophosphoprotein (DSPP) (BIOSS, Beijing, China) was performed. The immunoreaction was conducted according to the instructions of the streptavidin/peroxidase kit (Zhongshan Jinqiao Co). SEM Evaluation A morphologic evaluation of the hDPC-PRF complex was performed with a scanning electron microscope. The hDPC-PRF complex was fixed in 2.5% glutaraldehyde for 1 hour and treated for dessication. Specimens were sputter coated with 20 nm gold and subsequently examined in a scanning electron microscope. Photographs were taken using 1000 to 10,000 magnifications. Real-time Polymerase Chain Reaction Analysis For real-time polymerase chain reaction analysis, the total RNA of the PRF-hDPC complex was isolated using RNAiso Plus (Takara, Otsu, Japan) according to the manufacturer’s instructions. The total extracted RNA was applied toward complementary DNA generation with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). The relative gene expression levels of alkaline phosphatase (ALP) and DSPP were evaluated. Real-time polymerase chain reaction was performed using SYBR Premix Ex Taq (Takara) on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) with amplification conditions as follows: 95 C/30 seconds for denaturation followed by 95 C/5 JOE — Volume 42, Number 11, November 2016
Regenerative Endodontics seconds and 64 C/30 seconds for 40 cycles. The relative gene expression was calculated by the 2 DDCt method and normalized to GAPDH. The primer sequences and product sizes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ALP, and DSPP are listed in Supplemental Table S1 (Supplemental Table S1 is available online at www.jendodon.com).
Sample Preparation and Enzyme-linked Immunosorbent Assay Quantification The PRF gel was cut along the midline of the long axis into 2 segments. The lower part contained the buffy coat, and the upper part was without the buffy coat (Fig. 1B). Twelve freshly prepared PRF gels were divided into 3 groups, named the WP group (whole PRF), the LP group (the lower part of PRF containing the buffy coat), and the UP group (the upper part of PRF without the buffy coat). The 3 groups of PRF gels were
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soaked separately in sterile DMEM (2.5 mL DMEM for every 1.0 g PRF gel). The extracts of PRF gel were collected at 24, 72, 120, 168, and 216 hours. At each experimental time, the PRF gel was transferred to new sterile DMEM, and the previous DMEM was stored at 80 C until enzyme-linked immunosorbent assay quantification. When all the samples were collected, quantifications of 4 growth factors were performed using enzyme-linked immunosorbent assay kits (CUSABIO, Hubei, China): human PDGF-BB, human TGF-b1, human insulin-like growth factor 1 (IGF-1), and human basic fibroblast growth factor 3 (FGF2). The optical density was read using a microplate reader set to 450 nm, and the concentrations were calculated.
Statistical Analysis Triplicate experiments were conducted throughout this study to guarantee reproducibility of the results. Statistical analysis was
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Figure 1. (A) Messenger RNA expression of ALP and DSPP in the PRF-hDPC complex. Messenger RNA expression of ALP and DSPP was evaluated by real-time reverse-transcription polymerase chain reaction. Values are present as mean standard deviation (n = 3) normalized to GAPDH. *P < .05 between the 0-day PRF-hDPC complex and the 7-day PRF-hDPC complex. (B–F) Growth factor release in different parts of PRF. (B) After blood centrifugation, the whole PRF gel (WP) was cut along the midline of the long axis into 2 segments. The lower part contained the buffy coat (LP), and the upper part was without the buffy coat (UP). (C–F) Quantification of PDGF-BB, TGF-b1, IGF1, and FGF2 released by different parts of PRF was evaluated by enzyme-linked immunosorbent assay. Values are present as mean standard deviation (n = 3). *P < .05 between the WP group and the LP group. JOE — Volume 42, Number 11, November 2016
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mesenchymal origin (Supplemental Figure S1B and C is available online at www.jendodon.com).
Results
Effect of PRF Extract on hDPC Proliferation After incubation, the PRF extract was found to increase hDPC proliferation. From the Cell Counting Kit-8 assay, the absorbance values of hDPCs with the PRF extract increased about 1.7-fold on days 5 and 7 compared with the control group (P < .05) (Supplemental Figure S2 is available online at www.jendodon.com).
Identification of hDPCs Fibroblastlike morphology with a spindle shape and strong proliferation was visualized in primary hDPCs (Supplemental Figure S1A is available online at www.jendodon.com). The hDPCs showed a positive expression of vimentin but were negative for keratin, indicating their
Figure 2. (A) Prepared PRF-hDPC complex. A fibrin clot (2) was obtained in the middle of the tube, between the acellular plasma (1) at the top and the red corpuscles (3) at the bottom. (B) The PRF-hDPC complex was composed of 3 main parts visible from top to bottom with the naked eyes: fibrin matrix, buffy coat, and red blood cells. FM, fibrin matrix; BC, buffy coat; R, red blood cells. (C–E) Control group (PRF). The fibrin matrix appeared homogeneous in light pink and showed a sparse network structure, and the area of the buffy coat was dark pink and showed a dense network structure. A large number of leukocytes were deposited on the bottom of the buffy coat. (F–H) The BC group. Leukocytes and seeded cells (arrows) with irregular morphology were distributed in the buffy coat. (I–K) The IAC group. Only leukocytes were found in the buffy coat. (L–N) The AC group. Only leukocytes were found in the buffy coat. Sections were stained with hematoxylin-eosin.
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Regenerative Endodontics Construction of the PRF-hDPC Complex A fibrin clot was obtained in the middle of the tube, between the acellular plasma at the top and the red corpuscles at the bottom (Fig. 2A). The PRF-hDPC complex was composed of 3 main parts visible from top to bottom with the naked eye: fibrin matrix, buffy coat, and RBCs (Fig. 2B). In the control group, with hematoxylin-eosin staining, the fibrin matrix appeared homogeneous in light pink and showed a sparse network structure, and the area of the buffy coat was dark pink and showed a dense network structure. A large number of leukocytes were deposited on the bottom of the buffy coat. The leukocytes were stained in dark blue with hematoxylin. The RBCs were stained in red with eosin (Fig. 2C–E). In the BC group, leukocytes and seeded cells with irregular morphology were distributed in the buffy coat of the PRF gel. The seeded cells were larger than leukocytes, and the nuclei were oval
and stained in dark blue with hematoxylin, whereas the cytoplasm was stained red (Fig. 2F–H). In the IAC group and AC group, only leukocytes were found in the buffy coat of the PRF gel similarly to the control group (Fig. 2I–N).
PRF-hDPC Complex in 7 days During the 7 days of incubation, the PRF-hDPC complex was fixed for hematoxylin-eosin staining. In the control group, Leukocytes were dispersed among the buffy coat of PRF gel (Fig. 3A–C). In the BC group, morphology of the seeded cells changed from irregular to spindle shaped. Some spindle cell colonies were found in the buffy coat of the PRF-hDPC complex; the cells gathered into a spiral. A layer of spindle cells was observed along the edge of the buffy coat (Fig. 3D–H). In the IAC and AC groups, only Leukocytes were dispersed among the buffy coat of the PRF gel similarly to the control group (Fig. 3I–N).
Figure 3. The PRF-hDPC complex in 7 days. (A–C) The control group (PRF). Leukocytes were dispersed among the buffy coat. FM, fibrin matrix; BC, buffy coat; R, red blood cells. (D–H) The BC group. (E and F) Spindle cell colonies (arrows) were found in the buffy coat. (G and H) A layer of spindle cells (arrows) was distributed along the edge of the buffy coat. (I–K) The IAC group. Leukocytes were dispersed among the buffy coat. (L–N) The AC group. Leukocytes were dispersed among the buffy coat. Sections were stained with hematoxylin-eosin.
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Figure 4. Immunohistochemical staining of (B, F, and J) vimentin, (C, F, and K) CD45, and (D, H, and I) DSPP for the PRF-hDPC complex in the BC group. (A–D) The 0-day PRF-hDPC complex. Seeded cells (arrows) were distributed in the buffy coat and stained positive for vimentin, negative for CD45, and weakly positive for DSPP. (E–L) The 7-day PRF-hDPC complex. (E–H) Spindle cell colonies and (I–L) a layer of spindle cells along the edge of the buffy coat stained positive for vimentin, negative for CD45, and weakly positive for DSPP. (M–X) The ultrastructure of the PRF-hDPC complex. Scanning electron microscopy. (M–R) The 0-day PRF-hDPC complex. (M–O) Leukocytes were enmeshed in the fibrin network. (P–R) The seeded cells (arrows) were inserted in the interspaces between the fibrin. (S–X) The 7-day PRF-hDPC complex. hDPCs (arrows) adhered firmly in the (S–U) spindle cell colonies and distributed along the edge of (V–X) the buffy coat.
Immunohistochemical Staining for the PRF-hDPC Complex Immunohistochemical staining of vimentin, CD45, and DSPP for the PRF-hDPC complex was performed in the BC group. In the 0-day PRF-hDPC complex, seeded cells were distributed in the buffy coat 1638
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and stained positive for vimentin, negative for CD45, and weakly positive for DSPP (Fig. 4A–D). In the 7-day PRF-hDPC complex, spindle cell colonies and a layer of spindle cells along the edge of the buffy coat stained positive for vimentin, negative for CD45, and weakly positive for DSPP (Fig. 4E–L).
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Regenerative Endodontics Scanning Electron Microscopy of the PRF-hDPC Complex The result of the SEM evaluation was consistent with the light microscopic findings. In the 0-day PRF-hDPC complex, the seeded cells were found only in the BC group. Leukocytes were enmeshed in the fibrin network, and the seeded cells were inserted in the interspaces between the fibrin and had no connection with the fibrin network (Fig. 4M–R). In the 7-day PRF-hDPC complex, hDPCs adhered firmly in the spindle cell colonies and distributed along the edge of the buffy coat (Fig. 4S–X). Messenger RNA Expression of ALP and DSPP in the PRF-hDPC Complex The profile of ALP and DSPP messenger RNA expression was assessed by real-time polymerase chain reaction. During the 7 days of incubation, ALP and DSPP expression was up-regulated in PRF-hDPC complex. ALP expression was increased 5.7-fold compared with the PRF-hDPC complex just prepared, whereas the DSPP expression increased 2.3-fold (P < .05) (Fig. 1A). Growth Factor Released in Different Parts of PRF Certain amounts of PDGF-BB, TGF-b1, and FGF2 were found at each experimental time, even 9 days after the production of the PRF gel. IGF-1 was detected in the first 120 hours. The release peak for PDGF-BB was at 72 hours, and the other 3 growth factors were released at 120 hours. The quantity of growth factors released by the lower part of the PRF (the LP group) was higher than the whole PRF (the WP group) at most times, and the upper part of the PRF (the UP group) hardly released any growth factors (Fig. 1B–F).
Discussion Without any gelling agent such as anticoagulant or bovine thrombin, the slow polymerization during PRF preparation seems to develop a fibrin network similar to the natural one, resulting in more efficient cell migration and proliferation and thus cicatrization (4). In our study, the extract of PRF was found to encourage the gradual proliferation of hDPCs, and similar results were reported by Tsai et al (23), Dohan Ehrenfest et al (24), and Huang et al (25). The researchers showed that PRF stimulated the cell proliferation of gingival fibroblasts, periodontal ligament cells, osteoblasts, and hDPCs. These studies confirm the biocompatibility and lack of cytotoxicity of PRF as a biomaterial. An ideal scaffold should be suitable for the seeding of stem/progenitor cells (26). Unlike hydrogels or platelet-rich plasma (27), PRF cannot be injected with a suspension as a solid material (28). It is difficult to mix the seed cells with PRF by simply adding the cell suspension after centrifugation. In previous studies, the seed cells were either seeded on the surface of the PRF membrane or mixed with minced PRF fragments (9, 11). However, the structure of PRF was damaged, and the manipulation time in vitro was increased. Research has shown that the final architecture of the fibrin matrix influences the strength and the growth factor release potential of PRF (28). In our research, when hDPC suspension was added before blood centrifugation during the preparation protocol of PRF, the histologic results showed that hDPCs were well embedded in the buffy coat of PRF by this method. The immunohistochemistry results revealed that the seeded cells exhibiting irregular morphology and larger size showed positive expression for vimentin, negative for CD45, and weakly positive for DSPP. Furthermore, the morphology of the seeded cells changed from irregular to spindle shape during 7 days of incubation. These results indicated that the seeded cells were indeed hDPCs. In the groups in which hDPCs were added after centrifugation, no cells with definite hDPC features were observed in addition to the blood cells. These results have revealed that a PRF-hDPC complex can be developed by this new method and that the complex maintains the structural integrity of the PRF. JOE — Volume 42, Number 11, November 2016
PRF is derived from the blood and prepared by centrifugation (8, 29). The added hDPCs were treated as a normal cellular ingredient, such as leukocytes, which may explain why leukocytes and hDPCs were distributed in the same position in the PRF. The histologic and SEM analyses indicated that hDPCs seemed to have a separate space from the leukocytes, which were enmeshed in the fibrin network. This phenomenon provided a vacuum for hDPC growth. In brief, the hDPCs were cultured in a 3-dimensional culture condition within PRF gel. Our histologic analysis has revealed that the hDPCs experienced a favorable proliferation in the fibrin matrix of PRF gel 7 days after inoculation. Two forms of hDPC growth were observed in PRF-hDPCs. One is spindle cell colonies; the hDPCs gathered into a spiral mainly distributed in the buffy coat. We speculate that this may because of a wealth of growth factors within the buffy coat, which could promote cell adhesion and proliferation. The other form for hDPC growth is more interesting; a layer of spindle cells was distributed along the edge of the buffy coat and presented positive expression for vimentin, negative for CD45, and weakly positive for DSPP. Cellular migration is one of the crucial parameters for tissue repair and regeneration. TGF-b1, which was rich in PRF, was found to be important promoters of DPSC migration (30). Fibroblasts develop a significant proteolytic activity to move with the fibrin clot through the expression of 2 plasminogen activators (31). However, whether the hDPCs were located at the edge of the buffy coat at the very beginning when the PRF-hDPCs complex was prepared or they migrated to the spot later during incubation needs to be further confirmed. For pulp regeneration, the formation of a layer of odontoblastlike cells lining against the existing dentin surface and producing new dentin is considered to be necessary for regenerated pulp identification (32). Several research groups had observed a layer of odontoblastlike cells lining against the existing dentin surface in regenerated pulplike tissue (12, 33–35). We used to attribute this to the cytokine released by the treated dentin matrix. In the present study, a layer of hDPCs was observed along the edge of the buffy coat in PRF, indicating that PRF may play a synergistic effect with dentin matrix in the formation of odontoblast cells when used as a scaffold for pulp regeneration. ALP is considered as one of the markers indicating odontoblast differentiation and mineralization of dental pulp cells (36). DSPP is the initial translational product of DSPP messenger RNA and then cleaved to dentin phosphoprotein and dentin sialoprotein (37). DSPP plays a crucial role in dentinogenesis (38). ALP and DSPP expression was generally regarded as a marker of odontoblastic differentiation. Chen et al (12) showed that PRF induced the differentiation of DPSCs to odonto-/osteoblastic fates by increasing the expression of the Alp, Dspp, Dmp1, and Bsp genes. In the present study, the ALP and DSPP expression was up-regulated in the PRF-hDPC complex during the 7day incubation, indicating that PRF might maintain its property to promote odontoblastic differentiation in the complex. This effect will be further explored in our animal studies. In an ideal scaffold, it is important to have supplementation with growth factors (26). The PRF membrane exhibits a very significant slow sustained release of many key growth factors for at least 1 week (20) and up to 28 days (39). Considering the heterogeneous structure of PRF, it is necessary to find out the most efficient part of this biomaterial. Here, we showed that PRF gel slowly released certain amounts of PDGF-BB, TGF-b1, IGF-1, and FGF2, which were essential for hDPC proliferation and differentiation (40). The result indicated that most growth factors released by PRF gel might be derived from the buffy coat. This result was expected because platelets and leukocytes were concentrated in the buffy coat of PRF (22). Coincidentally, our study indicated that hDPCs were mainly inserted in the buffy coat area, underscoring the
New Method to Develop a PRF-hDPC Complex
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Regenerative Endodontics idea that the buffy coat would be of interest for clinical use and even more effective than the higher part of the fibrin gel (21). Therefore, when harvesting PRF as a scaffold material, practitioners should collect the buffy coat carefully; sometimes it is even necessary to preserve a small RBC layer at the PRF gel end. Our study has shown that a PRF-hDPC complex can be developed when hDPC suspension is added before blood centrifugation during the preparation protocol of PRF, presenting a suitable method to combine PRF with seed cells to maintain PRF structure integrity and the biological activity of seed cells. The hDPCs in the complex were distributed in the buffy coat, which was the most efficient part of this biomaterial in releasing growth factors. However, the poor plasticity of PRF might limit its application in the field of pulp regeneration. We still believe this new method might expand to other fields in which PRF is used as a scaffold for specific tissue engineering. The feasibility of this method must be proven through in vivo studies.
Acknowledgments Supported by the National Natural Science Foundation of China (grant no. 81160133). The authors deny any conflicts of interest related to this study.
Supplementary Material Supplementary material associated with this article can be found in the online version at www.jendodon.com (http://dx.doi. org/10.1016/j.joen.2016.08.011).
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JOE — Volume 42, Number 11, November 2016