An Autologous Platelet Rich Plasma Stimulates Periodontal Ligament Regeneration

Journal of Periodontology; Copyright 2013 DOI: 10.1902/jop.2013.120556

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An Autologous Platelet Rich Plasma Stimulates Periodontal Ligament Regeneration

Eduardo Anitua, PhD,*† María Troya, MSc †, and Gorka Orive, PhD†‡

*Private practice in dentistry, Vitoria, Spain.

†Biotechnology Institute, Vitoria, Spain.

‡NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, School of Pharmacy, Vitoria-Gasteiz, Spain. Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Vitoria-Gasteiz,

Spain. Background: Regeneration of periodontal tissues is one of the most important goals for the

treatment of periodontal disease. The technology of plasma rich in growth factors provides a biological approach for the stimulation and acceleration of tissue healing. The purpose of this study was to evaluate the biological effects of this technology on primary human periodontal ligament fibroblasts.

Materials and Methods: We studied the response of periodontal ligament cells to this pool of growth factors on cell proliferation, cell migration, secretion of several biomolecules, cell adhesion and expression of α2 integrin. Cell proliferation and adhesion were evaluated by means of a fluorescence-based method. Cell migration was performed on culture inserts. The release of different biomolecules by periodontal ligament fibroblasts was quantified through enzyme-linked immunosorbent assay. The α2 integrin expression was assessed through Western-blot.

Results: This autologous technology significantly stimulated cell proliferation, migration, adhesion and synthesis of many growth factors from cells including vascular endothelial growth factor (VEGF), thrombospondin 1 (TSP-1), connective tissue growth factor (CTGF), hepatocyte growth factor (HGF) and pro-collagen type I. The α2 integrin expression was lower in plasma rich in growth factors treated cells compared with non-stimulated cells though no statistically significant differences were observed.

Conclusions: This Plasma rich in growth factors exerts positive effects on periodontal ligament fibroblasts, which could be positive for periodontal regeneration.

KEYWORDS Periodontal ligament, platelet rich plasma, growth factors, wound healing.

Periodontal diseases are characterized by affecting the composition and integrity of all the structures involved in the periodontium resulting in the destruction of the connective tissue, the loss of attachment and finally the resorption of alveolar bone. Periodontal therapy is aimed to control periodontal tissue inflammation and to prevent further attachment loss and regenerate periodontal supporting tissues.1

The complex structure of the periodontium, which consists of the gingival soft connective tissue, the periodontal ligament and the mineralized tissues (cementum and bone), makes periodontal wound healing to be a unique process.2 Periodontal ligament play a major role in the periodontal wound healing process. It is complex, composed of several different cell populations and characterized by rapid turnover and high capacity for renewal and remodeling. Among these heterogeneous cells, fibroblasts are predominant and they have been suggested to present cementoblast-like and osteoblasts-like properties, as well as to contain a population of progenitor cells.2-5

During the last few decades a number of different treatments have been described to promote periodontal regeneration. Unfortunately, current therapeutic approaches are


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unable to achieve reliable and predictable regeneration.2, 6 From a biological perspective, periodontal regeneration demands the availability of appropriate cell types, together with a favorable local environment. Furthermore, cell migration, adhesion, proliferation and differentiation need to be precisely coordinated.1, 7, 8

Growth factors are natural proteins that regulate the main cellular events involved in tissue regeneration and its application has become an area of increasing interest in periodontal regenerative medicine.8-11 Platelet rich plasma provides an interesting alternative to deliver concentrated amounts of these growth factors to the wound site. The technology of plasma rich in growth factors represents a biological approach for the stimulation and acceleration of tissue healing. Upon activation with calcium chloride, platelets pour out their growth factor content to the local milieu that mediate and accelerate cell function, driving tissue regeneration process.12-15

The positive effects of this technology on gingival fibroblasts16 and oral osteoblasts17 has been already reported; however, its effect on the periodontal ligament remains to be investigated. Here, we evaluate the regenerative potential of this technology on the periodontal ligament. To address this, plasma rich in growth factors prepared from 3 different donors, was assessed over the main biological events of the wound healing process including cell proliferation, migration, adhesion and secretion of growth factors.

MATERIAL AND METHODS

Isolation of Primary Cultures of Human Periodontal Ligament Fibroblasts Primary cultures of human periodontal ligament fibroblasts (hPDLF) were obtained from three different healthy patients (two females and one male, aged 18-27) undergoing a simple extraction of non-impacted wisdom teeth. No history of inflammation was reported. Written informed consent was obtained from each subject prior to donation of the tissue. The study was performed following the principles of the Declaration of Helsinki. The cells were established by the explant method. Briefly, each tooth sample was rinsed in phosphate-buffered saline containing 50µg/ml gentamicin and 2.5µg/ml amphotericin B.§ Periodontal ligament tissue was mechanically removed from the middle third of the tooth roots using a scalpel. Explants were rinsed again in phosphate-buffered saline containing 50µg/ml gentamicin and 2.5µg/ml amphotericin B,║ minced into smaller portions, placed in 6-well plates and incubated in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (1:1 volume)¶ supplemented with 15% fetal bovine serum (FBS),# 2 mMglutamine,** 50µg /ml gentamicin and 2.5µg/ml amphotericin B†† in a humidified atmosphere at 37ºC with 5% CO2. After reaching approximately confluence, primary fibroblasts were detached with animal origin-free trypsin-like enzyme‡‡ and subcultured. Cell viability was assessed by trypan blue dye exclusion.§§ All experiments were performed using cells from these three donors, between the third and the fifth passages.

Characterization of Human Periodontal Ligament Fibroblasts Cells were characterized by immunofluorescence. The connective tissue markers were fibronectin,║║ collagen type I, ¶¶ and periostin## (a typical marker used for periodontal ligament fibroblasts identification18) while the osteogenic marker was osteopontin.***

Cells were fixed for 10 minutes in 4% formaldehyde for type I collagen and osteopontin antigens and in methanol for fibronectin and periostin antigen. Cells for


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type I collagen and osteopontin staining were permeabilized with 1% Triton X-100, in phosphate buffered saline (PBS) for 10 minutes and for periostin staining with 0.1%Tween 20††† in phosphate buffered saline (PBS) for 1 hour. After that, cells were blocked with fetal bovine serum (10% in PBS) for 30 minutes, and incubated for 1 h with the primary antibodies. Next, cells were incubated with their appropriate secondary antibodies, goat anti-mouse immunoglobulin G conjugated with Alexa Fluor 488 fluorochrome or goat anti-rabbit immunoglobulin G conjugated with Alexa Fluor 594 fluorochrome. ‡‡‡

Finally, the cell nuclei were stained with Hoechst 33342, mounted, and visualized under a fluorescence microscope.

Histological Analysis Parts of the periodontal ligaments were fixed with 4% formaldehyde for 24 hours. The samples were dehydrated in increasing graded alcohols, cleared in xylene substitute║║║

and included in paraffin.

The obtained blocks were cut at 3.5µm thickness. Paraffin was removed with xylene substitute¶¶¶ and the sections were rehydrated in increasing ethanol content. Then, slides were stained with hematoxylin-eosin### and May Grünwald- Giemsa.****

Plasma Rich in Growth Factors Technology Due to the great variety of platelet rich plasma protocols available, which may lead to different biological effects we would like to emphasize that the results obtained here, are only related to an autologous plasma preparation that is rich in growth factors.

The technology used in the current study involved use of the patient's own blood to obtain an autologous cocktail of proteins and growth factors and a fibrin scaffoldBlood from three different donors (donor 1-3) was collected into 9-ml tubes with 3.8% (wt/v) sodium citrate, after written informed consent was provided. Samples were centrifuged at 580 g for 8 min†††† at room temperature.

The 2 ml above the buffy coat (F2), without included it, were separated in each donor.

Platelets and leukocytes counts were performed with a hematology analyzer. ‡‡‡‡ Plasma preparations were incubated with the activator§§§§ at 37ºC in glass tubes for 1 hour. The supernatants were collected by aspiration after centrifugation at 1000 g for 20’ at 4ºC. Finally, the plasma obtained from each donor was aliquoted and stored at -80ºC until use. Different Growth factors were measured in the supernatants using commercially available Quantikine colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) kits║║║║ (Table 1).

Assessment of the Time-Dependent Effect of Plasma Rich in Growth Factors on hPDLF Proliferation Cells were seeded at a density of 5000 cells per cm2 on 96-well optical bottom black plates and maintained for 24h in culture medium containing 15%FBS. The medium was then replaced with serum free medium supplemented with either: 0.2%FBS, as a control of non-stimulation (NS); or 20% (v/v) F2 supernatant from each donor. After 24, 48 and 72 hours of culture, cell proliferation was evaluated by CYQUANT cell proliferation assay.¶¶¶¶ Briefly, treatments were discarded and wells were washed carefully with PBS. Then the microplates were frozen at -80ºC until assayed. After thawing the plates at


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room temperature, wells were incubated with RNase A (1.35 Ku/ml) diluted in cell lysis buffer for 1 hour at room temperature. Then, 2x GR dye/cell-lysis buffer was added to each sample well, mixed gently and incubated for 5 minutes at room temperature, protected from light. Sample fluorescence was measured with a fluorescence microplate reader.####A DNA standard curve was included in each assay for converting sample fluorescence values into DNA concentration.

Migration Assay In order to evaluate the effect of PRGF-Endoret on the migratory potential of hPDLF, they were plated at a density of 25000cells/cm2 in culture inserts*****placed on a 24-well plate with culture medium and 15%FBS. After reaching confluence, the inserts were carefully removed and a cell-free gap was created between two separated cell monolayers. The cells were washed with PBS and incubated for 24 hours with the corresponding treatments: 0.2%FBS or 20% (v/v) F2 supernatant from each donor. After this period, treatments were removed and cells were nuclear stained with Hoechst 33342.

Phase contrast images of the central part of the septum before treatment and phase contrast and fluorescence images after the treatment time were captured with a digital camera coupled to an inverted microscope. ††††† Cell counting was done by mean of the software Image J, Version 1.45. ‡‡‡‡‡ Results were expressed as cell number/mm2.

Synthesis of Biomolecules and Extracellular Matrix (EM) Components In order to examine the effect of this plasma rich in growth factors on the expression of different biomolecules and extracellular matrix components by hPDLF, cells were seeded on 12-well plates at a density of 6000 cells/cm2 with culture medium and 15%FBS until confluence. After that, the medium was changed and the corresponding treatments (0.2%FBS or 20% (v/v) F2 supernatant from each donor) were added for 72 hours. Then, culture medium was collected and centrifuged for 10 min at 460g and stored at -80ºC until use. In order to determine the amount of growth factor added with each treatment, additional wells were kept without cells.

Vascular endothelial growth factor (VEGF), thrombospondin 1 (TSP-1), hepatocyte growth factor (HGF),§§§§§ connective tissue growth factor (CTGF)║║║║║ and procollagen type I¶¶¶¶¶ concentrations were determined in conditioned culture medium using enzyme-linked immunosorbent assay (ELISA) kits.

Cell Adhesion to Collagen Type I hPDLF were plated at a density of 20000 cells/cm2 onto 96-well optical bottom black plates with the appropriates treatments (0.2%FBS or 20% (v/v) F2 supernatant from each donor) for 10, 20 or 30 minutes. The plates were previously coated with 10 μg/cm2 collagen type I##### and blocked by FBS.

After the different times of treatment cell adhesion was stopped by carefully pouring off the medium and attached cells were quantified using CYQUANT cell proliferation assay, ****** as already described above.


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Quantification of α2 Integrin Expression by Western Blot Analysis hPDLF were seeded at a density of 20000 cells/cm2 with 0.2%FBS or 20% (v/v) F2 supernatant from each donor on a surface coated with 10 μg/cm2 collagen type I††††††

and blocked by FBS. After 5 or 24 hours, treatments were removed and cells were washed carefully with PBS and lysed with Mammalian Protein Extraction Reagent (MPER) supplemented with protease and phosphatase inhibitors. ‡‡‡‡‡‡ Lysates were clarified by centrifugation at 14000g for 10 min, and the supernatants were then collected. Proteins in the cell lysates were concentrated using Amicon ultra-0.5 (3k) filters.§§§§§§ Protein concentration from the lysates was determined with the BCA Protein Assay Reagent. ║║║║║║ Equal amounts of protein were resolved through SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes.¶¶¶¶¶¶ The membranes were blocked with 5% non-fat dry milk in Tris buffered saline containing 0.1% Tween 20 (TBST) for 1 hour at room temperature. Then, the membranes were incubated with a mouse antibody against integrin α-2###### and with a rabbit antibody against β-actin, as loading control******* overnight at 4ºC. After several washing with TBST, the blots were treated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse secondary antibodies††††††† for 1 hour. Then, the blots were washed again with TBST and developed by chemiluminescence with Immun-Star HRP substrate using the Chemidoc image analyzer. ‡‡‡‡‡‡‡

Statistical Analysis Univariate analysis of variance was used to assess the potential differences among treatments for each experimental procedure according to the different cell lines used. In order to identify the differences, post-hoc analysis based on Bonferroni tests were performed. Statistical differences between groups were accepted for p-values lower than 0.05. Results are expressed as mean ± standard deviations. All samples were preformed in triplicate.

RESULTS The human periodontal ligament fibroblasts (hPDLF) showed elongated and spindle-shaped appearance in culture.

Periodontal ligament cells represent a heterogeneous population of cells, with fibroblastic characteristics, such as fibroblast morphology and collagen production and osteoblastic characteristics, such as alkaline phosphatase activity and expression of bone-associated proteins.19 So, several markers of both types of cells were used to characterized. Cells were positive for all the analyzed markers. The histological sections showed a characteristic pattern of connective tissue orientated fibers, rich in cells and with a variable number of blood vessels. (Data not shown). Platelet counts and the levels of some of the most important growth factors present in PRGF-Endoret of each donor are shown in table 1.

Effect of Plasma Rich in Growth Factors on hPDLF Proliferation Proliferation of human periodontal ligament fibroblasts increased after treatment with PRGF-Endoret for both 48h and 72h compared to the negative control (p<0.05). The effect of the three donors was similar, increasing cell proliferation approximately 3 and 4-fold after 48h or 72h respectively (fig.1). After 24 hours of treatment however, only


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the donor 1 exerted a statistically significant increase in the proliferation compared with the remaining two donors. Non-stimulated cells did not show any obvious sign of proliferation over time.

Migration Assay Figure 2 shows the migratory capacity of hPDLF after treatment with this pool of growth factors for 24h. In fact, this plasma rich in growth factors prepared from the 3 donors induced a significant stimulatory effect in the migration process. Cell migration was enhanced 33, 44 and 27-fold when the cells were treated with this cocktail of growth factors from donor 1, 2 and 3 respectively. No statistical differences were found among those 3 donors. Images of the figure 2 highlight the potent stimulatory effect of plasma rich in growth factors on hPDLF migration.

Synthesis of Biomolecules and Extracellular Matrix (EM) Components All the proteins measured were significantly increased when the cells were treated with plasma rich in growth factors compared to non-stimulation condition. The synthesis of the angiogenic factor VEGF increased significantly (approximately 5-fold) once the cells were treated with this technology for 72h. Similar results were observed for TSP-1. Regarding the factors HGF and CTGF, this pool of growth factors also exerted an increase of its synthesis with respect to control. In the case of HGF, results obtained with plasma rich in growth factors from the 3 donors were statistically significant compared with the non-stimulated cells, while in the case of CTGF, differences were significant for 1 out of 3 donors. Finally, the expression of the main component of the extracellular matrix, procollagen type I, was also significantly stimulated by this technology. (Fig.3)

Cell Adhesion This plasma rich in growth factors promoted hPDLF attachment to collagen type I more rapidly compared to non-stimulation conditions (fig. 4). Cell attachment was pronounced in the initial follow-up times and decreased over time. In fact, after 30 minutes, only donor 1 showed statistically significant differences with respect to control. (Fig.4).

Integrins are a family of dimeric proteins that mediate cell to cell and extracellular matrix adhesion. Integrin α2 functions as a collagen receptor on platelets and fibroblasts. Not only plays an important role in cellular adhesion, but may function in intracellular signal transmission.20, 21 The expression of α2 integrin was evaluated in this assay. After treatment with this cocktail of growth factors the expression of that integrin was lower than in the control group after both 5 and 24h; however, no statistically significant differences were found (fig. 5).

In addition, α2 integrin expression increased over time until the end of the assay.

DISCUSSION Periodontal regeneration requires the coordinated formation of new alveolar bone, cementum and functional periodontal ligament. However, traditional therapies remain insufficient to stimulate complete and functional periodontal regeneration. The development of novel methods based on the use of growth factors, biomaterials and tissue engineering represents a real challenge for the future. As it has been reported


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appropriate cells, signals and scaffolds are the basic components to reconstruct natural tissues.1, 22-26

Recently, the use of growth factors has emerged as a new strategy to achieve periodontal regeneration.6, 10, 27 The use of platelet-rich plasma is one of the approaches available for modulating and enhancing periodontal healing as a source to release multiple endogenous growth factors to the wound area.

In this study, we have evaluated the effects of an autologous platelet rich plasma, on periodontal ligament cells. This pioneering autologous technology, free of leukocytes, provides a cocktail of proteins and growth factors from the same patient’s blood. This versatile technology permits the elaboration of 4 different formulations with therapeutic potential.12, 28, 29 Effects of this technology on tissue regeneration have been demonstrated in many different medical fields. 29

Results obtained in the present study confirm that both proliferation and migration of periodontal ligament fibroblasts were significantly increased when cells were cultured with this plasma rich in growth factors. Moreover, the effect of the 3 different donors used in this assay was nearly the same, which highlight the homogeneity of the approach. Some of the growth factors present in platelet rich plasma (table 1) may be the responsible for this effect including VEGF, PDGF, IGF, HGF and TGF-β1.7, 9, 30, 31

In addition to increase periodontal ligament cell migration and proliferation, this technology enhances delivery of growth factors to the wound area. The secretion of the proangiogenic factor VEGF stimulated by PRGF-Endoret is of great significance since the periodontal ligament is highly vascular.2 However, there must be a balance in all the biological process. Hence, it is also important that release of TSP-1 was also increased by this biological approach. In addition to its role as an antiangiogenic factor, TSP-1 is a major activator of TGF-β1, which in turn, mediates many processes in wound healing. Moreover, TSP-1 has anti-inflammatory and adhesive effects.32 Plasma rich in growth factors also stimulates periodontal ligament cells to synthesize HGF, which has been reported to exert anti-fibrotic and anti-inflammatory effects. 33 CTGF is another factor stimulated by plasma rich in growth factors in periodontal ligament cells. This factor regulates a variety of cellular functions in periodontal tissue, including extracellular matrix production, angiogenesis, cell migration, proliferation and adhesion.34, 35

Periodontal wound healing requires synthesis and remodeling of the extracellular matrix, which consists mainly of collagen I.22 According with our results, the pool of growth factors is capable of upregulating extracellular matrix production by stimulating the secretion of procollagen type I by periodontal ligament fibroblasts.

Adhesion organizes the signaling networks that regulate migration and other cellular processes including proliferation, gene expression and cell survival.36 Our results show that plasma rich in growth factors favors periodontal ligament cells to adhere to a collagen type I matrix earlier. As cells must remain in a dynamic state during the process of wound healing, cell adhesion must happen as soon as possible to ensure that the rest of processes, such as proliferation or migration also occur actively to maintain this dynamic cellular state.

Integrins are a class of cell adhesion molecules composed of αβ heterodimers that mediate cell to cell and cell to substrate interactions. In addition to provide a physical link between cells and the extracellular matrix, integrins transmit signals across the cytoplasmic membrane and initiate signaling cascades, which regulate many cellular functions, including gene expression, cell proliferation, migration and survival.37, 38


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The effect of plasma rich in growth factors on the α2 integrin expression, which functions as a collagen receptor on fibroblasts among others20, 21 has been also evaluated. Despite the differences are not statistically significant there is less protein when the cells were cultured with the pool of autologus morphogens. As mentioned above, this technology stimulates the cells to adhere faster; although the expression of α2 integrin was reduced. The latter could be explained by the fact that the state of the cells cultured with plasma rich in growth factors is more dynamic, that is, they are in a constant movement to proliferate, migrate and therefore cells have to attach and detach constantly. In fact, integrin-mediated adhesions are dynamic structures that must be created and disassembled during migration and cell division. 36 It has been demonstrated that optimum cell speed occurs at intermediate levels of expression of α5β1 or α2β136 and that cell motility was inversely correlated to the expression of integrin α1 and α2 collagen receptors.39 However, non stimulated cells normally remain static, which could explain the fact that α2 integrin expression was higher.

Nevertheless, since many molecules are involved in cell adhesion20, 38, 40 further research is need to elucidate the exact mechanism and molecules that participate in this rapid stimulation of cell adhesion by plasma rich in growth factors and at the same time mantain the cells in a dynamic condition.

It is important to understand that each of the periodontal components has its very specialized function and structure. Indeed, proper functioning of the periodontium is only achieved through structural integrity and interaction between its components. Therefore, the fact that this technology stimulates gingival fibroblasts16, oral osteoblasts17 and periodontal ligament fibroblasts, makes it ideal for a complete periodontal regeneration.

In summary, this preliminary in vitro study shows how plasma rich in growth factors stimulates the proliferation, migration, adhesion and secretion of some of the main biomolecules of human periodontal ligament cells.

Conflict of Interest EA, MT and GO are scientists at BTI Biotechnology Institute. This biotechnology company has developed the technology of plasma rich in growth factors.

ACKNOWLEDGMENTS AND SOURCE OF FUNDING The study was partially self-funded by the authors and their institution (BTI Biotechnology Institute) and partially funded by the Basque Government from the Basque Country SAIOTEK program (S-PE11BI002; research support program, basque science, technology and innovation).

REFERENCES 1. Benatti BB, Silverio KG, Casati MZ, Sallum EA, Nociti FH, Jr. Physiological features of periodontal

regeneration and approaches for periodontal tissue engineering utilizing periodontal ligament cells. J Biosci Bioeng 2007;103:1-6.

2. Marchesan JT, Scanlon CS, Soehren S, Matsuo M, Kapila YL. Implications of cultured periodontal ligament cells for the clinical and experimental setting: a review. Arch Oral Biol 2011;56:933-943.

3. Volponi AA, Pang Y, Sharpe PT. Stem cell-based biological tooth repair and regeneration. Trends Cell Biol 2010;20:715-722.

4. Nomura Y, Ishikawa M, Yashiro Y, et al. Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells. Histochem Cell Biol 2012;137:719-732.

5. Iwata T, Yamato M, Zhang Z, et al. Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use. J Clin Periodontol 2010;37:1088-1099.


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6. Chen FM, Shelton RM, Jin Y, Chapple IL. Localized delivery of growth factors for periodontal tissue regeneration: role, strategies, and perspectives. Med Res Rev 2009;29:472-513.

7. Ramseier CA, Rasperini G, Batia S, Giannobile WV. Advanced reconstructive technologies for periodontal tissue repair. Periodontol 2000 2012;59:185-202.

8. Chen FM, Zhang J, Zhang M, An Y, Chen F, Wu ZF. A review on endogenous regenerative technology in periodontal regenerative medicine. Biomaterials 2010;31:7892-7927.

9. Javed F, Al-Askar M, Al-Rasheed A, Al-Hezaimi K. Significance of the platelet-derived growth factor in periodontal tissue regeneration. Arch Oral Biol 2011;56:1476-1484.

10. Chen FM, An Y, Zhang R, Zhang M. New insights into and novel applications of release technology for periodontal reconstructive therapies. J Control Release 2011;149:92-110.

11. Lee J, Stavropoulos A, Susin C, Wikesjo UM. Periodontal regeneration: focus on growth and differentiation factors. Dent Clin North Am 2010;54:93-111.

12. Anitua E, Alkhraisat MH, Orive G. Perspectives and challenges in regenerative medicine using plasma rich in growth factors. J Control Release 2012;157:29-38.

13. Anitua E, Sanchez M, Prado R, Orive G. The P makes the difference in plasma rich in growth factors (PRGF) technology. Platelets 2011;22:473-474; author reply 475.

14. Anitua E, Sanchez M, Prado R, Orive G. Plasma rich in growth factors: the pioneering autologous technology for tissue regeneration. J Biomed Mater Res A 2011;97:536.

15. Nurden AT. Platelets, inflammation and tissue regeneration. Thromb Haemost 2011;105 Suppl 1:S13-33.

16. Anitua E, Troya M, Orive G. Plasma rich in growth factors promote gingival tissue regeneration by stimulating fibroblast proliferation and migration and by blocking transforming growth factor-beta1-induced myodifferentiation. J Periodontol 2012;83:1028-1037.

17. Anitua E, Tejero R, Zalduendo MM, Orive G. Plasma Rich in Growth Factors (PRGF-Endoret) Promotes Bone Tissue Regeneration by Stimulating Proliferation, Migration and Autocrine Secretion on Primary Human Osteoblasts. J Periodontol 2012:1-14.

18. Rios HF, Ma D, Xie Y, et al. Periostin is essential for the integrity and function of the periodontal ligament during occlusal loading in mice. J Periodontol 2008;79:1480-1490.

19. Jonsson D, Nebel D, Bratthall G, Nilsson BO. The human periodontal ligament cell: a fibroblast-like cell acting as an immune cell. J Periodontal Res 2011;46:153-157.

20. Ivaska J, Heino J. Interplay between cell adhesion and growth factor receptors: from the plasma membrane to the endosomes. Cell Tissue Res 2010;339:111-120.

21. Wickstrom SA, Fassler R. Regulation of membrane traffic by integrin signaling. Trends Cell Biol 2011;21:266-273.

22. Chen FM, Jin Y. Periodontal tissue engineering and regeneration: current approaches and expanding opportunities. Tissue Eng Part B Rev 2010;16:219-255.

23. Dangaria SJ, Ito Y, Luan X, Diekwisch TG. Successful periodontal ligament regeneration by periodontal progenitor preseeding on natural tooth root surfaces. Stem Cells Dev 2011;20:1659-1668.

24. Hughes FJ, Ghuman M, Talal A. Periodontal regeneration: a challenge for the tissue engineer? Proc Inst Mech Eng H 2010;224:1345-1358.

25. Ishikawa I, Iwata T, Washio K, et al. Cell sheet engineering and other novel cell-based approaches to periodontal regeneration. Periodontol 2000 2009;51:220-238.

26. Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920-926.

27. Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835-870.

28. Anitua E. Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants 1999;14:529-535.


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29. Anitua E, Sanchez M, Orive G. Potential of endogenous regenerative technology for in situ regenerative medicine. Adv Drug Deliv Rev 2010;62:741-752.

30. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen 2008;16:585-601.

31. Raja S, Byakod G, Pudakalkatti P. Growth factors in periodontal regeneration. Int J Dent Hyg 2009;7:82-89.

32. Lopez-Dee Z, Pidcock K, Gutierrez LS. Thrombospondin-1: multiple paths to inflammation. Mediators Inflamm 2011;2011:296069.

33. Bendinelli P, Matteucci E, Dogliotti G, et al. Molecular basis of anti-inflammatory action of platelet-rich plasma on human chondrocytes: mechanisms of NF-kappaB inhibition via HGF. J Cell Physiol 2010;225:757-766.

34. Dangaria SJ, Ito Y, Walker C, Druzinsky R, Luan X, Diekwisch TG. Extracellular matrix-mediated differentiation of periodontal progenitor cells. Differentiation 2009;78:79-90.

35. Takeuchi H, Kubota S, Murakashi E, et al. Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells. J Periodontal Res 2009;44:161-169.

36. Huttenlocher A, Horwitz AR. Integrins in cell migration. Cold Spring Harb Perspect Biol [serial online]. 2011;3(9):a005074. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21885598. Accessed Sep.

37. Geiger B, Bershadsky A, Pankov R, Yamada KM. Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk. Nat Rev Mol Cell Biol 2001;2:793-805.

38. Geiger B, Yamada KM. Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol [serial online]. 2011;3(5). Available from: http://www.ncbi.nlm.nih.gov/pubmed/21441590. Accessed May.

39. Lallier TE, Miner QW, Jr., Sonnier J, Spencer A. A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts. Cell Tissue Res 2007;328:339-354.

40. Legate KR, Wickstrom SA, Fassler R. Genetic and cell biological analysis of integrin outside-in signaling. Genes Dev 2009;23:397-418.

Correspondence: Eduardo Anitua. Instituto Eduardo Anitua; c/ Jose Maria Cagigal 19, 01007 Vitoria (Spain). Phone: +34 945160653 Fax: +34 945160657, E-mail:

[email protected] Submitted September 11, 2012; accepted for publication December 3, 2012.

Figure 1

Plasma rich in growth factors promotes hPDLF proliferation. (A-D) Phase contrast images after 72h illustrating the proliferative effect of plasma rich in growth factors. Scale bar: 300 µm. (E) Evolution of periodontal ligament cell proliferation over time after culturing with 0.2% FBS or 20% F2. N.S.: non-stimulated cells. *Statistically significant differences between treated and non-stimulated cells (p < 0.05). †Statistically significant differences between control and donor 1 (p < 0.05). ‡Statistically significant differences between donor 1 and donor 2 (p < 0.05).


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Figure 2

Plasma rich in growth factors stimulates periodontal ligament cell migration. (A-D) Hoechst staining showed the strong differences on periodontal fibroblast migration after 24 hours with both plasma rich in growth factors treatment and control. Scale bar: 300 µm. (E) Migration cell rates revealed an obvious increase when cells were cultured with any of the three donors of plasma rich in growth factors for 24 hours. N.S.: non-stimulated cells. *Statistically significant differences between treatment and non-stimulation (p < 0.05).

Figure 3

Biomolecules secretion by human periodontal ligament cells. Plasma rich in growth factors significantly stimulated the secretion of VEGF, TSP-1, HGF and procollagen type I. In the case of CTGF the differences were only statistically significant between donor 1 and control. N.S.: non-stimulated cells. *Statistically significant differences between treatment and non-stimulation (p < 0.05). †Statistically significant differences between donor 1 and donor 2 (p < 0.05). ‡Statistically significant differences between donor 3 and the others two donors (p < 0.05). §Statistically significant differences between donor 1 and non-stimulation condition (p < 0.05).

Figure 4

Plasma rich in growth factors promotes periodontal ligament cell adhesion to a collagen type I matrix. Cells treated with plasma rich in growth factors had an earlier adhesion than the non stimulated ones. N.S.: non-stimulated cells. *Statistically significant differences between treatment and non-stimulation (p < 0.05). †Statistically significant differences between donor 3 and the others two donors (p < 0.05). ‡Statistically significant differences between control and donor 1 (p < 0.05). §Statistically significant differences between donor 1 and donor 3 (p < 0.05).

Figure 5

Relative expression of α2 integrin by human periodontal ligament fibroblasts. (A) Western blot analysis of α2 integrin after 5 hours of being cultured under control conditions or with plasma rich in growth factors. (B) Western blot analysis of α2 integrin after 24 hours of being cultured under control conditions or with plasma rich in growth factors. No statistically significant differences were found among treatments.

β-actin was used as a loading control.


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Table 1

Concentration of several growth factors, platelet and leukocyte count in plasma rich in growth factors.

Growth factors concentration

Donors

Leukocyte count (106/ml)

Platelet count (106/ml)

Endostatin (ng/ml) HGF (pg/ml) IGF-I

(ng/ml) PDGF-AB

(pg/ml) TGF-β1 (pg/ml)

TSP-1 (µg/ml)

VEGF (pg/ml)

1 0,1 498 (2,8x) 115 960 96 23515 44050 32 110

2 0,1 442 (3x) 105 840 104 21795 46850 29 380

3 0,1 580 (2,5x) 110 1150 75 33430 54100 36 15

§ Sigma-Aldrich, St Louis, MO, USA

║ Sigma-Aldrich

¶ Gibco-Invitrogen, Grand Island, NY, USA

# Biochrom AG, Leonorenstr, Berlin, Germany

** Sigma-Aldrich

†† Sigma-Aldrich

‡‡ Gibco-Invitrogen

§§ Sigma-Aldrich

║║ Sigma-Aldrich

¶¶ Chemicon-Millipore, Billerica, MA, USA

## Abcam, Cambridge, UK

*** Sigma-Aldrich

††† Sigma Aldrich

‡‡‡ Molecular Probes-Invitrogen, Grand Island, NY, USA

§§§ Leica DM IRB, Leica Microsystems, Wetzlar, Germany

║║║ Sigma- Aldrich

¶¶¶ Sigma- Aldrich

### Sigma- Aldrich

**** Sigma- Aldrich

†††† PRGF-Endoret Technology, BTI Biotechnology Institute, S.L., Miñano, Álava, Spain

‡‡‡‡ Micros 60, Horiba ABX, Montpelier, France

§§§§ PRGF-Endoret Technology

║║║║R&D Systems, Minneapolis, MN, USA

¶¶¶¶ Molecular Probes-Invitrogen

#### Twinkle LB 970, Berthold Technologies, Bad Wildbad, Germany

***** Ibidi GmbH, Martinsried, Germany

††††† Leica DM IRB

‡‡‡‡‡ National Institute of Health, Bethesda, MD, USA

§§§§§ R&D Systems

║║║║║ USCN Life Science Inc., Wuhan, China

¶¶¶¶¶ TaKaRa, Shiga, Japan

##### Sigma-Aldrich

****** Molecular Probes-Invitrogen

†††††† Sigma-Aldrich


13

‡‡‡‡‡‡ Pierce Biotechnology, Bonn, Germany

§§§§§§ Chemicon-Millipore

║║║║║║Pierce Biotechnology

¶¶¶¶¶¶ Bio-Rad Laboratories, Munich, Germany

###### BD Biosciences, San Jose, CA

******* Abcam

††††††† Bio-Rad Laboratories

‡‡‡‡‡‡‡ Bio-Rad Laboratories


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