The effect of platelet-rich plasma on osteoblast and periodontal ligament cell migration, proliferation and differentiation.
ABSTRACT Platelet-rich plasma is used to deliver growth factors, in a safe and convenient manner, for enhancing bone and periodontal regeneration. However, conflicting reports regarding its effectiveness suggest that further study of the relevant cellular mechanisms is required. The aim of this study was to investigate the in vitro effect of platelet-rich plasma on osteoblasts and periodontal ligament cell function.
Various concentrations of platelet-rich plasma (100, 50, 20 and 10%) and platelet-poor plasma, obtained from human donors, were applied to primary cultures of human osteoblasts and periodontal ligament cells. [(3)H]-Thymidine incorporation, crystal violet staining and MTT assays were utilized to assess DNA synthesis and proliferation. Migration was determined by assessing the cell response to a concentration gradient, while differentiation was assessed using Alazarin Red staining.
Platelet-rich plasma and platelet-poor plasma had stimulatory effects on the migration of both human osteoblasts and periodontal ligament cells. At 24 h, DNA synthesis was suppressed by the application of the various concentrations of platelet-rich plasma, but over a 5-d period, a beneficial effect on proliferation was observed, especially in response to 50% platelet-rich plasma. Platelet-poor plasma resulted in the greatest enhancement of cellular proliferation for both cell types. At a concentration of 50%, platelet-rich plasma and platelet-poor plasma facilitated differentiation of both cell types.
Platelet-rich plasma can exert a positive effect on osteoblast and periodontal ligament cell function, but this effect is concentration specific with maximal concentrations not necessarily resulting in optimal outcomes. Platelet-poor plasma also appears to have the ability to promote wound healing-associated cell function.
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ABSTRACT: Wound healing in an incisional wound is a highly predictable process which has been studied extensively hour-by-hour and day-by-day. Healing in a periodontal defect following gingival flap surgery is, conceptually, a more complex process as one wound margin consists of calcified tissue, including the avascular and rigid root surface. Another complicating factor in this wound healing is the transgingival position of the tooth. Experimental studies, however, have indicated that healing at a dento-gingival interface under optimal conditions occurs at the same rate as in a skin wound. Generally, periodontal healing is characterized by maturation of gingival connective tissue, limited regeneration of alveolar bone and cementum, and the formation of a long junctional epithelium. Such observations have nurtured the hypothesis that the epithelium of the surgical flap needs to be prevented from early access to the root surface during the healing period to achieve connective tissue repair of the root surface-gingival flap interface. Recent experimental findings suggest, however, that connective tissue repair to the root surface following reconstructive periodontal surgery is a function of the establishment and maintenance of a root surface-adhering fibrin clot. Since fibrin adherence to the wound margins is a natural event, it is additionally suggested that apical migration of the gingival epithelium in periodontal surgical wounds may only follow interruption of the adherence of the fibrin clot to the root surface.Journal of Periodontology 04/1992; 63(3):158-65. · 2.40 Impact Factor
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ABSTRACT: The well-orchestrated, complex series of events resulting in the repair of cutaneous wounds are, at least in part, regulated by polypeptide growth factors. This review provides a detailed overview of the known functions, interactions, and mechanisms of action of growth factors in the context of the overall repair process in cutaneous wounds. An overview of the cellular and molecular events involved in soft tissue repair is initially presented, followed by a review of widely studied growth factors and a discussion of commonly utilized preclinical animal models. The article concludes with a summary of the preliminary results from human clinical trials evaluating the effects of growth factors in the healing of chronic skin ulcers. Throughout, the interactions among the growth factors in the wound-healing process are emphasized.Critical Reviews in Oral Biology & Medicine 02/1993; 4(5):729-60.
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ABSTRACT: Guided bone regeneration is an accepted surgical method employed in implant dentistry to increase the quantity and quality of the host bone in areas of localized alveolar defects. The lack of predictability in osseous regenerative procedures with various grafting materials suggests that improvement in the osteoinductive properties of these materials is highly desirable. Platelet-rich plasma (PRP), a modification of fibrin glue made from autologous blood, is being used to deliver growth factors in high concentration to sites requiring osseous grafting. Growth factors released from the platelets include platelet-derived growth factor, transforming growth factor beta, platelet-derived epidermal growth factor, platelet-derived angiogenesis factor, insulin-like growth factor 1, and platelet factor 4. These factors signal the local mesenchymal and epithelial cells to migrate, divide, and increase collagen and matrix synthesis. PRP has been suggested for use to increase the rate of bone deposition and quality of bone regeneration when augmenting sites prior to or in conjunction with dental implant placement Only 6 human studies using PRP have been found in the dental implant literature and 5 were case series or reports. Thus, there is clearly a lack of scientific evidence to support the use of PRP in combination with bone grafts during augmentation procedures. This novel and potentially promising technique requires well-designed, controlled studies to provide evidence of efficacy.The International journal of oral & maxillofacial implants 18(1):93-103. · 1.91 Impact Factor
The Effect of Platelet-Rich Plasma on Osteoblast and Periodontal Ligament Cell
Migration, Proliferation and Differentiation
Creeper F*, Lichanska AM*, Marshall RI*, Seymour GJ#, Ivanovski S‡
* School of Dentistry, The University of Queensland, Australia
#School of Dentistry, University of Otago, New Zealand
‡School of Dentistry and Oral Health, Griffith University, Australia
Effect of PRP on cell function
School of Dentistry and Oral Health
Gold Coast Campus
Griffith University QLD 4222
Platelet-rich plasma, osteoblasts, periodontal ligament cells, growth factors
Background and objective: The use of platelet-rich plasma (PRP) aims to safely and
conviniently deliver growth factors in order to enhance bone and periodontal
regeneration. However, conflicting reports regarding its effectiveness suggest that
further study of the relevant cellular mechanisms is required. The aim of this study
was to investigate the in vitro effect of PRP on osteoblasts and periodontal ligament
Methods: Various concentrations of PRP (100%, 50%, 20% and 10%) and platelet-
poor plasma (PPP) obtained from human donors was applied to primary cultures of
human osteoblast and periodontal ligament cells. 3H-Thymidine incorporation, crystal
violet and MTT assays were utilized to assess DNA synthesis and proliferation.
Migration was determined by assessing cell response to a concentration gradient,
while differentiation was assessed using Alazarin Red staining.
Results: PRP and PPP had stimulatory effects on the migration of both cell types. At
24 hours, DNA synthesis was suppressed by the application of the various
concentrations of PRP, but over a 5 day period, a beneficial effect on proliferation
was observed, especially in response to the intermediate concentrations of 50% PRP.
Platelet-poor plasma (PPP) resulted in the greatest enhancement of cellular
proliferation for both cell types. 50% PRP and PPP facilitated differentiation of both
Conclusion: PRP can exert a positive effect on osteoblast and periodontal ligament
cell function, but this effect is concentration specific with maximal concentrations not
necessarily resulting in optimal outcomes. Furthermore, PPP appears to also have the
ability to promote wound healing associated cell function.
During the early stages of wound healing, platelet released growth factors, including
platelet derived growth factor (PDGF), insulin like growth factor -1 (IGF) and
transforming growth factor β (TGFβ), initiate a cascade of cellular and molecular
events which result in wound healing in a highly regulated and coordinated fashion
(1-3). The understanding that platelet-derived growth factors play an important role in
wound healing has led to the development of recombinant growth factors aimed at
influencing and enhancing repair and regeneration. The application of these growth
factors to bone and periodontal regeneration has been investigated using in vitro and
in vivo models with promising results (4-9) that have provided the basis for
subsequent human clinical studies (10, 11).
In addition to the use of recombinant growth factors, concentrated formulations of
platelets, known as platelet rich plasma (PRP), have also been investigated as a
potential source of autologous growth factors. PRP is a volume of autologous plasma
that has a platelet concentration approximately three to four times above baseline
levels (12). Hence, it can be considered a concentration of all the platelet derived
growth factors and plasma components of the individual patient in their biologically
determined ratios (13,14).
Whitman et al first described the use of PRP in the dental setting and showed that
PRP application to the underlying tissues allowed more predictable flap adaptation
and haemostasis and ensured a more definitive seal than primary closure alone (15).
Subsequently, there has been considerable interest in examining this method of
promoting wound healing and regeneration. In particular, it has been proposed that
PRP may be utilized alone or in conjunction with various graft materials to deliver
growth factors to the wound site, especially in order to enhance bone and/or
Many investigations have been conducted on the clinical effect of PRP on bone
regeneration (for review see (3)) and periodontal regeneration (16-18), in addition to
several in vitro studies (19-26) aimed at establishing the biological rationale for this
treatment. However, there is a lack of consensus regarding the effectiveness of PRP
with early promising clinical reports (16, 27-29) being tempered by subsequent
negative findings (30-32). Hence, the aim of this study was to investigate the in vitro
effect of platelet-rich plasma on the cells that are critical for periodontal and bone
regeneration, namely periodontal ligament cells and osteoblasts, in terms of the key
cellular functions associated with wound healing and regeneration, namely migration,
proliferation and differentiation.
MATERIALS AND METHODS
During surgical extraction of third molars, teeth and bone chips were collected in
explant media containing Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal calf serum (FCS), 100units/ml Penicillin, 100µg/ml
Streptomycin, Fungizone (2.5mg/ml) and non-essential amino acids. The study was
approved by the human ethics committee of the University of Queensland and
informed consent was obtained prior to the collection of the samples.
Periodontal ligament cells and osteoblasts were obtained as previously described (33,
34). Briefly, the bone chips and periodontal ligament fragments obtained from the
middle third of the root surface were minced into smaller tissue portions, transferred
to 25cm2 tissue culture flasks (Coning Incorporated, Corning NY, USA) and
incubated in explant media at 37°C and a humidified atmosphere containing 5% CO2.
One week following establishment of the explants, the explant media was changed to
standard media containing DMEM, 10% FCS, 50units/ml Penicillin, 50µg/ml
Streptomycin and non-essential amino acids.
Following cell growth from the explants, the cells were detached from the plate using
0.2% trypsin and 0.02% EDTA (Sigma Chemical Co, St Louis, USA), and were
subsequently propagated by passaging in a 1:3 split until sufficient numbers were
obtained to carry out the required experiments. Cells from passages 3 to 5 were used
in this study.
Each experiment described in this study was carried out using three individual
primary cell lines from different donors and was repeated in triplicate for each cell
PRP was prepared from healthy patients immediately prior to application on the cells.
Whole venous blood was collected in lithium heparin coated collection tubes (Becton,
Dickinson and Company, NJ, USA) and initially centrifuged at 1500RPM for 10
minutes to separate the red blood cell (RBC) portion from the platelet-rich and
platelet-poor plasma. The upper layer of the RBC portion was included as the platelets
containing the largest amount of growth factors, and hence having the greatest
potential biological activity, are larger and mix with the upper 1mm of the red blood
cells. The inclusion of this small RBC layers imparted a red tinge to the PRP (28, 35) .
The PRP and PPP portions were then extracted and centrifuged again at 2500RPM for
10 minutes to separate the PRP from the PPP.
Dilutions of the PRP and PPP for the various experiments were obtained by mixing
with standard serum-free media (DMEM, non-essential amino acids and
penicillin/streptomysin). Both autogenous and allogenic PRP/PPP preparations were
used with no differences on cell functions being observed (data not shown), and hence
the results are combined.
Using Transwell Permeable (TP) Supports (Corning Incorporated, Corning NY,
USA) (26, 36) with 6.5mm diameter and 8µm pore size, migration from one side of a
membrane to the other was examined after 6 hours in response to five treatments –
100% PRP, 50% PRP, and 10% PRP, as well as 50% PPP and serum free medium.
300µl of treatment media per well was placed into a 24-well plate and allowed to
incubate for 30 minutes. Care was taken to ensure that the treatment media was settled
at the bottom of the well, and then serum free media was gently applied on top until
the layer just contacted the under side of the TP supports. Cells were seeded at a
concentration of 2x103 onto the outer surface of the TP supports and were incubated
for six hours.
After the incubation period, the TP supports was removed and washed with
phosphate-buffered saline solution (PBS) and the outer surface was carefully wiped
dry with a flattened cotton bud and a microbrush to remove non-migrated cells. The
TP supports were then placed into a fresh 24-well plate containing 300μl crystal violet
for 15 minutes, and subsequently removed, washed by flooding with tap water until
free dye was no longer visible and allowed to air dry. This stain was then solubilised
and extracted with 33% glacial acetic acid and absorbance was read in a
spectrophotometer at 570nm. The absorbance reading is directly proportional to the
number of cells that migrated from the outer to the inner surface of the TP supports, in
response to the various treatments placed in the 24 well plates.
Proliferation of the periodontal ligament cells and osteoblasts was assessed using
three methods, which were based on different outcome measures. The treatments
utilized were 0% FCS (negative control), 10% FCS (positive control), 50% PRP, 20%
PRP, 10% PRP and 50% PPP. All treatments were diluted with serum-free media.
100% PRP was not utilized as it resulted in total loss of cell viability (data not
3H Thymidine Incorporation (DNA synthesis) Assay
The 3H-Thymidine incorporation assay is based on the incorporation of radio-labelled
3H-thymidine into the replicating DNA strands during mitosis and therefore measures
DNA synthesis. As previously described (37), the cells were seeded at a concentration
of 2x104 cells per well in 24 well (Nunclon, Denmark) plates in standard cell culture
media. These cells were allowed to attach overnight. Subsequently, the cells were
exposed to media containing 0% FCS for 48 hours in order to synchronize the cells in
the Go phase of the cell cycle. After this time, the treatment media was added and the
cells were allowed to incubate for 24 hours. For the last four hours, the cells were
pulse-labelled with 10µCi 3H-Thymidine per well. Cells were then lysed with 1.5%
SDS for 15 minutes, combined with scintillation fluid and radioactivity was measured
using a liquid scintillation counter (Beckman Instruments, Fullerton, California,
Crystal Violet (Colorimetric) Assay
The crystal violet colorimetric assay directly measures cell numbers that are present.
As previously described (37), cells were seeded into 96 well plates at a concentration
of 2x103 cells per well and allowed to attach overnight in standard media. This media
was then removed and the treatment media were added followed by incubation for 5
days. After this time, the media was removed; the wells were washed with PBS and
stained with crystal violet for 30 minutes. Following the removal of excess stain,
solubilization of the bound crystal violet was carried out with 33% glacial acetic acid
solution and absorbance was measured by a spectrophotometer at 570nm. The relative
spectrophotometer readings are directly proportional to cell numbers.
MTT Proliferation (Cell viability) Assay
The MTTTM (Roche Diagnostics GmbH, Mannheim, Germany) proliferation assay was
also used to assess the proliferative potential of PRP (26). This is a colorimetric assay
where the amount of colour produced is directly proportional to the number of viable
cells. Cells were seeded into 96 well plates at a concentration of 2x103 cells per well
and allowed to attach overnight in standard media containing 10% FCS. Treatment
media was added and the cells were allowed to incubate for 5 days. The MTT
labelling reagent was then added to each well and allowed to incubate for a further 4
hours, after which the cells were washed with PBS and solubilization solution was
added. The plates were then incubated overnight and subsequently read using a
spectrophotometer at 570nm.
Differentiation of the periodontal ligament cells and osteoblasts was assessed by
quantification of Alazarin red staining (38). Cells were plated in 6-well plates at a
concentration of 1 x 105 cells per well and incubated with DMEM containing 10%
FCS (standard media) and allowed to attach overnight. Subsequently, treatment media
of 50% PRP, 20% PRP, 50% PPP, mineralization media (consisting of standard media
with the addition of 50 µg/ml ascorbic acid, 10 mM glycerophosphate and 10-8 M
dexamethasone), standard media and media containing 0% FCS were added and the
cells incubated for 5 days. After this time, the media were removed and cells were
washed and fixed with 95% ethanol for 15 minutes at 4°C. The cells were then stained
with 2% Alizarin Red S (pH 4.1-4.3) for 15 minutes. Calcium forms an alizarin red S-
calcium complex in a chelation process, and red staining is evident in the well. This
stain was then solubilized with 300µl of 33% glacial acetic acid solution and
absorbance was measured by a spectrophotometer at 415nm.
One way ANOVA was used to assess whether there was a statistically significant
effect of the different treatments on cell function. In order to identify statistically
significant differences between the various treatments, post-hoc analysis was carried
out using SPSS software (SPSS Inc. Chicago, Illanois, USA) and the LSD correction.
Statistical differences between groups were accepted for p-values lower than 0.05.
The results of the migration assay indicated that PRP had a stimulatory effect on
cellular migration of periodontal ligament cells (Figure 1). Concentrations of 100%
(p<0.01), 50% (p<0.001) and 10% PRP (p<0.001) significantly enhanced migration
compared to the negative control of 0%FCS. 50% PPP also significantly enhanced
migration of the periodontal ligament cells (p<0.02), but no difference was found
compared to the various PRP concentrations. There were no significant differences
between the various concentrations of PRP.
The migration of osteoblasts was also significantly enhanced by the various
concentrations of PRP with 100% PRP (p<0.001), 50% PRP (p<0.01) and 10% PRP
(p<0.05) all showing significant stimulation compared to 0% FCS (Figure 2). No
statistical differences were seen between the various concentrations of PRP. 50% PPP
also had a significant migratory stimulus compared to 0% FCS (p<0.01), but was not
significantly different compared to the various PRP concentrations.
3H Thymidine Incorporation (DNA synthesis) Assay
The 3H-thymidine proliferation assays showed similar effects of PRP on the DNA
synthesis of both periodontal ligament cells and osteoblasts (Figures 3 and 4
respectively). PRP exerted a statistically significant decrease in cellular DNA
synthesis of periodontal ligament cells at all concentrations (p<0.001) compared to the
positive control. Furthermore, 50% PPP significantly increased DNA synthesis
compared to all PRP concentrations (p<0.001 for all concentrations), as well as the
positive control (p<0.01) of 10% FCS, indicating its mitogenic effect on these cells.
Similar results were obtained using the osteoblasts, whereby all PRP concentrations
significantly inhibited DNA synthesis (p<0.001) compared to the positive control
(10% FCS). PPP had a significant enhancing effect when compared to all
concentrations of PRP (p<0.001), but showed no difference compared to the positive
control of DMEM containing 10%FCS.
Crystal Violet (Colorimetric) Assay
Continuing the trend from the DNA synthesis assay, in the five day colorimetric
proliferation assay, the periodontal ligament cells (figure 5) showed the greatest
proliferative activity in the presence of 50% PPP, which was significantly higher than
the positive control of 10% FCS (p<0.05), 20% PRP (p<0.001), 10% PRP (p<0.001)
and the negative control of 0% FCS (p<0.001). However, in this assay, 50% PRP
induced a significant increase in cell numbers compared to 0% FCS (p<0.01) and the
lower concentrations of PRP, namely 20% and 10% (p<0.001 for both). There was no
significant statistical difference between 50% PRP, 50% PPP and 10% FCS. In
addition, both 20% and 10% PRP showed no significant difference when compared to
the negative control of 0% FCS.
Similarly, with regards to the osteoblasts (figure 6), 50% PPP was the most beneficial
in terms of proliferation compared to all PRP concentrations, as well as media
containing 10% and 0% FCS (p<0.001). 50% PRP was significantly more mitogenic
compared to 0% FCS (p<0.001), 20% PRP (p<0.001) and 10% PRP (p<0.001),
whereas 10% PRP significantly stimulated proliferation compared to 20% PRP
(p<0.05) and 0% FCS (p<0.05). There were no statistical differences in proliferation
between 20%PRP and 0% FCS, and 50% PRP and 10% FCS.
MTT Proliferation (Cell Viability) Assay
The MTT proliferation box-plots for the periodontal ligament cells and osteoblasts are
shown in figures 7 and 8 respectively. Treatment with 50% PRP significantly
stimulated cell proliferation of periodontal ligament cells compared to media
containing 0% FCS (p<0.001), 10%PRP and 20% PRP (all p< 0.001), but there was
no statistical difference compared to 10% FCS and 50%PPP. Similarly, 50% PPP
was shown to significantly increase proliferation compared to 0% FCS, 10% PRP and
20% PRP (all p<0.001), as well as 10% FCS (p<0.05).
With regards to the osteoblasts, 50% PRP (p<0.001) and 20% PRP (p<0.01)
significantly increased proliferation compared to 0% FCS. Furthermore, 50% PPP
significantly increased proliferation compared to 0% FCS (p<0.001) and 10% PRP
(p<0.01). No significant statistical differences were seen between the various PRP
concentrations and 10% FCS, although there appeared to be trend towards reduced
proliferative activity with reduced PRP concentrations.
The results show that PRP and PPP are capable of inducing differentiation of the
periodontal ligament cells and osteoblasts as shown by the photographs of the wells
with the Alizarin Red S staining of Ca2+ deposits (Figure 9). This was reflected in the
results obtained by solubilization of the staining and reading in the spectrophotometer
at 415nm (Figures 10 and 11).
In the periodontal cells (Figure 10), 50% PRP (p<0.001) and 50% PPP (p<0.05)
induced significant increases in differentiation compared to 0% FCS. In addition, 50%
PRP significantly increased Ca2+ deposition compared to 10% FCS, mineralization
media (p<0.01), 20% PRP (p<0.01) and 10% FCS (p<0.01). The lower concentration
of PRP showed no difference when compared to 50% PPP or mineralization media.
The results obtained with the osteoblast cells indicated that mineralization media
(p<0.05), 50% PRP (p<0.01) and 50% PPP (p<0.05) had a significant increase in
differentiation when compared to 0% FCS and only 50% PRP induced significantly
greater Ca2+deposition compared to 10% FCS (p<0.05) (Figure 11). No other
significant differences were noted.