The effect of platelet derived growth factor-AB on periodontal ligament fibroblasts: An in vitro study.

BACKGROUND AND OBJECTIVES: Traditional methods of periodontal therapy produce results in healing of tissues by repair; however, what we require is regeneration of the lost tissues. The periodontal ligament (PDL) cells appear to be important in periodontal wound healing. Platelet derived growth factor (PDGF), a potent mitogen and useful mediator for wound healing, has been extensively studied in periodontal regeneration. This in vitro study was designed to evaluate the effect of PDGF-AB on human PDL fibroblasts (hPDLF) at 50, 100 and 150 ng/ml dosages at 24, 48 and 72 hours time duration.
MATERIALS AND METHODS: Tissue explants from three different patients were harvested from the roots of freshly extracted, uninfected and impacted third molars. The cells cultured from all samples were divided into 4 groups: Group-1 was the control group, and the experimental groups were designated as Group-2, Group-3 and Group-4, to test the effect of PDGF-AB at 50, 100 and 150 ng/ml by proliferation assay carried out at 24, 48 and 72 hours.
RESULTS: The results revealed maximum mitogenic response of PDL cells at 100 ng/ml and at 48 hours, suggesting that the mitogenic response of PDGF-AB is both, time and dose dependent.
CONCLUSIONS: The results of this in vitro study suggest that PDGF has maximum mitogenic response on hPDLF at 48 hours and for 100 ng dose. However for clinical application, randomized controlled clinical trials are required to substantiate the results of this in vitro study.

INTRODUCTION

Periodontal disease is a destructive inflammatory disease of the supporting tooth structures′, caused by a complex interaction between the plaque bacteria and host immune system, and is one of the main causes for tooth loss. The presence of appropriate cell types, matrix and signaling molecules are the key factors for the repair and regeneration of tissues. The periodontal ligament (PDL) cells appear to be critical in periodontal wound healing, and the current periodontal regeneration methods are based on the concepts of promoting repopulation of the wound area adjacent to the root surface with cells from the PDL.[1-4] Polypeptide growth factors are a class of natural biological mediators which regulate key cellular events in tissue repair and regeneration including cell proliferation, chemotaxis, differentiation, and matrix synthesis by binding to specific cell surface receptors.[5-9]

Platelet derived growth factor (PDGF) is one such growth factor found to be a major mitogen for mesenchymal cells, chemoattractant for fibroblasts and stimulant for the production of matrix components. Due to the pleotrophic biological activities stimulated by the PDGF, it has been implicated as a major component regulating the events of wound healing. PDGF has been widely investigated as a potentially useful mediator of periodontal regeneration due to its ability to stimulate proliferation and migration of a variety of cells, including PDL cells.[9-14] PDGF is a dimeric molecule and occurs as a combination of two polypeptide subunits, designated as A and B. PDGF acts on the target cells by binding to α and β receptors on their cell surfaces and in turn stimulates them. PDGF has 5 isoforms, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. Among these, 3 isoforms PDGF-AA, PDGF-AB and PDGF-BB have been extensively studied.[715-20] It has been reported that PDGF-AA and PDGF-AB are more rapidly secreted from the producer cell compared to PDGF-BB,[18] and also PDGF-AB is found to have strong mitogenic effect on cells expressing both α and β receptors.[21] Hence, PDGF-AB was selected in our study and mitogenic effects on cultured human PDL fibroblasts (hPDLF) were evaluated.

The effect of PDGF-AB has been studied by many investigators on various cell types. There is lack of unanimity among investigators about effective concentrations and duration of exposure. Oates et al.,[11] reported that the mitogenic response of PDL fibroblasts to PDGF-AB is highest at 50 ng/ml at 24 hours, whereas Dennison et al.,[14] observed significant mitogenic response of PDGF on hPDLF at 20 ng/ml at 48 hours duration. Boyan et al. observed highest mitogenic response of PDGF-AB on hPDLF at a concentration 100 ng/ml and at day five.[22] The present in vitro study was designed to see the proliferative activity of PDGF-AB on PDL fibroblast at different dosages and duration.

MATERIALS AND METHODS

This study was carried out at M. R. Ambedkar Dental College and Hospital, Bangalore, in collaboration with Department of Neuro-Virology National Institute of Mental Health and Neuro Sciences (NIMHANS), Research Institute Bangalore.

Tissue sample collection

All the tissue samples were obtained from medically healthy patients after obtaining ethical clearance by the institutional review board. Informed consent was obtained from all the patients in the prescribed proforma. Patients selected for the study included 2 males and 2 females, aged between 20-30 years. All the tissue samples collected were handled using aseptic conditions, and directly placed in vials containing media for transport to the laboratory for explanting. Transport media consisted of minimal essential media (MEM) containing 1000 units/ml penicillin, 100 μg/ml streptomycin and fungizone 250 μg/100 ml medium. Remaining procedures were conducted in a sterile environment under a laminar flow hood.

Human periodontal ligament cell isolation and culture

Human PDL fibroblasts used in the present study were obtained from PDL tissues harvested from the roots of freshly extracted, uninfected, impacted third molar teeth. Immediately after extraction and under sterile conditions, the periodontal ligament tissue was gently scraped from the middle one-third of the root using a sterile sharp # 15 blade. The tissue specimens were placed in sterile Petri dishes containing a thin layer of MEM containing 10% fetal calf serum (FCS) and teased into small bits of tissue using a sterile sharp # 15 blade. The tissue explants were then placed in 25 cm2 culture flask containing culture media composed of: Dulbecco's Modified Eagles Medium (DMEM), 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37°C in humidified air with 5% CO2 for 2-4 weeks. Out of the 4 samples, one sample showed contamination with bacteria, and thus was discarded while the other 3 samples were monitored and continued for the study. The medium was replaced after 1 week and every 3 days until sufficient cell proliferation was evident. Growth was permitted to continue until the cells attained a confluent monolayer, at which time they were trypsinized (trypsin 0.05%, Ethylenediaminetetraacetic acid,EDTA, 0.53 mm) and the cells were collected by centrifugation at 1000 revolutions per minute (rpm) for 5 minutes. The cells were then transferred to new 25 cm2 culture flasks for continued growth. All cells in this study were utilized between 3rd-4th passage. The cell counts were done using a hemocytometer.

Growth factor reconstitution

Platelet Derived Growth Factor-AB used in this study was obtained from Sigma Aldrich Company. 10 μg of PDGF-AB was reconstituted to a 1 ml solution as per the manufacturer's instructions. Thus, 100 μl containing 1000 ng of PDGF-AB in 1 μl 10 ng of the growth factor was present. Based on these calculations PDGF-AB was used in different dosages as required in this study.

Experimental design

Fibroblasts from all the 3 samples were plated in 96 well culture plate at a density of 1,000 cells per well, in a 200 μl culture medium. 3 rows of the 96 well, plate were utilized for the study. Each row of the 96 well culture plate consisting of 12 wells, was meant for one sample, and 200 μl of sample I was plated onto the first row of the culture plate. These 12 wells were then divided into 4 groups consisting of 3 wells each. The first group, i.e., Group-1 was used as contros and the next three were used as test groups. The test groups were labeled based on the dosages of the PDGF-AB added. Group-2 was meant for 50 ng/ml dose, Group-3 for 100 ng/ml dose and Group-4 for 150 ng/ml dose of PDGF-AB. Similarly, 200 μl of Sample II and Sample III were plated onto the next two rows of the 96 well culture plates and divided into four groups. The reconstituted growth factor was added to the test groups as per the dosages mentioned in our experimental design and Group-1 received no growth factor. Once the addition of growth factor was completed, the 96 well plate was sealed and transferred to the incubator. The proliferation assays were carried out at 24, 48 and 72 hours for each of the samples at dosages 50, 100 ng/ ml and 150 ng/ml, and also the control group by using a hemocytometer.

Statistical methods

Factorial model of the Analysis of variance was used to model the effect of dose and duration. Factorial experiment also studied the interaction between dose and duration. Partial eta square was calculated to evaluate the effect size which would give the strength of association. Uni-variate Analysis of variance was used based on General Linear Model with dosages and duration as an interaction effect to evaluate the significance of differences in the mean cell count at different time duration and dosages.

RESULTS

Table 1 shows mean cell count of all the four groups at different time duration. Table 2 shows the P value for significance in the differences and partial eta squared value for the interactions. The mitogenic response of the PDL cells was both dose and time dependent. Maximum mitogenic response was seen at 48 hours and for Group-3 (100 ng/ml). Figure 1 depicts the means of cell counts at different dosage and duration.

Table 1

Effect of dose and hour on growth on cells in our study

Table 2

Tests of between-subjects effects dependent variable: Growth

Figure 1

Mean cell count at different time duration and dosage of PDGF-AB in our study

At 24 hours the mean cell count was 2333.33±577.35 in Group-1, 6333.33±1154.70 in group-2, 7666.76±1524.53 in group-3 and 6000.00±0.00 in Group-4. The cell count is highest in group-3 and the difference in means between all the test groups is not statistically significant. At 48 hours mean cell count for Group-3 was maximum, 123333.33±20816.66 cells; while the count was 5333.33±1527.53 for group-1, 70000.00±10000.0 cells for Group-2 and 50000.00±10000.0 cells for Group-4. The difference in mean cell count between all the groups was highly significant (P<0.001). At 72 hours there was no statistical significant difference between the mean cell count of the control group (9833.33±1258.31) and mean cell count at Group-2, Group-3 and Group-4, respectively (10666.67±2081.67, 12666.67±20814.67, 11667.67±1527.53). The mean cell count in the control group gradually increased from baseline up to 72 hours. In Group-2, the mean cell count dropped from 70000.00±10000.0 at 48 hours to 10666.67±2081.67 at 72 hours; and, in Group-3 the mean cell count dropped from 123333.33±20816.66 at 48 hours to 12666.67±20814.67 at 72 hours. Similar observations were also seen in Group-4 where the mean cell count dropped from 50000.00±10000.0 at 48 hours to 11667.67±1527.53 at 72 hours and the difference was statistically significant at P<0.001. Table 2 shows the partial eta square value for interaction between cell count and the dose-duration factor. The interaction is highest for duration when considered alone and when the dose and duration is combined the value is higher than dose alone.

DISCUSSION

The process of restoring structure and function of the lost periodontium continues to be a major goal of periodontal treatment. Currently available methods aimed at enhancing regeneration, lack the predictability and efficacy to make them universally accepted by the clinicians. The mainstay of regeneration is stimulating series of events and cascades at a point, which can result in the co-ordination, and completion of integrated tissue formation.[23] The healing of the periodontal wound must be modified if regeneration of lost periodontal tissues with new attachment is to be established. The healing of the periodontal wound involves various cell types, the epithelial cells, the gingival fibroblasts and PDL fibroblasts. Fibroblasts in the PDL are the key cells involved in the regenerative process. If regeneration of lost periodontal tissues is to be established, the PDL fibroblasts have to be recruited and allowed to repopulate onto the root surface. The PDL contains stable cells and these cells normally remain quiescent in tissues in Go – G1 phase, waiting for an appropriate signal to divide. In the presence of mitogenic factors, these cells are activated and thus induce cell division. Extensive research has been carried out in identifying the mitogens and also in understanding the role played by these molecules during regeneration. PDGF is one of the potent mitogens present in the serum, expressed mainly by the platelets, and influences periodontal cell migration, proliferation and synthetic activity. Platelets synthesize a mixture of the three possible PDGF isoforms (70% AB, 20% BB, 10% AA).[13] They differ in their functional properties as well as in their secretory behaviors. PDGF-AA and PDGF-AB are rapidly secreted from the producer cell, whereas PDGF-BB remains to a large extent associated with the producer cell and relatively small amounts are secreted.[18] Many in vitro and in vivo dental wound-healing models have focused on PDGF, alone or in combination with other growth factors and have shown that the wound-healing responses have been enhanced.

Reports have suggested that PDGF-AB has similar mitogenic potential to that of PDGF-BB,[811161819] and is equally potent in stimulating deoxyribonucleic acid synthesis in human fibroblasts.[8] Oates et al. reported that hPDLF has equal number of α and β receptors[24] and PDGF-AB has been found to exhibit stronger mitogenic effect on cells expressing both α and β receptors.[21] Hence, PDGF-AB was selected in our study to evaluate its mitogenic effects on cultured hPDLF. The results of our study showed that PDGF-AB had significant mitogenic effect on hPDLF at all dosages compared to the controls. However, the maximum mitogenic response was observed at 100 ng/ml. The mean cell count at 48 hours with 100 ng was 123333.33±20816.66 and at 50 ng was 70000.00±10000.0. Further, at 150 ng the mean cell count was 50000.00±10000.0. These results indicate that at high dosages of 150 ng, PDGF-AB has an inhibitory effect on cell proliferation. The results of our study are in agreement with those of Boyan et al.,[24] who reported that the hPDLF in response to PDGF-AB is dose dependent and maximum effect was evident at 100 ng/ml.

Our results are contrary to those of Oates et al. who have reported highest mitogenic response at a concentration of 50 ng/ml. The difference may be due to the fact that the highest concentration used in their study was 50 ng/ml.[11] In recent years, platelet rich plasma (PRP) has been used for periodontal regeneration on the premise that higher concentration of PDGF-AB levels at the site of wound healing will have enhanced mitogenic activity and also help in regeneration. Okuda et al.,[25] have estimated the PDGF-AB levels in PRP and reported it to be 182.0±75.5 ng/ml. whereas in human serum the concentration of PDGF-AB was estimated to be at 51.8±33.4 ng/ml. The results of our study have clearly indicated that dosages of 150 ng/ml PDGF-AB will have less mitogenic effect. Hence our observation questions the utility of using PRP, which contains very high level of PDGF-AB, in periodontal regeneration.

The other parameter evaluated in our study was the time duration and the results indicated that maximum mitogenic response of PDGF on hPDLF was observed at 48 hours. The results of our study are in agreement with those of Dennison et al. who reported that maximum mitogenic response of PDGF on hPDLF was observed at 48 hours, and the response was 215% at 48 hours and 114% at 72 hours when compared to the control.[14] Our results are contrary to those of Boyan et al. who reported maximum mitogenic response of PDGF-AA and PDGF-BB on hPDLF at day 7, whereas in our study PDGF-AB showed maximum response at day five.[22] Our results are also contrary to those of Oates et al. who observed significantly higher mitogenic response of PDGF on hPDLF at 24 hours than at 48 hours.[11]

Discrepancies between the results of various published studies may come from several variables, including species differences, cell heterogeneity, differences in culture regimen and differences in the method used to quantitate the biological response. The limitation of our study is that we have evaluated the effect of a single growth factor on PDL cells and in an in vitro condition. Ideally, in an in vivo situation there are multiple growth factors which act together in the repair and regeneration of hard and soft tissues. These molecules are known to have redundant and pleotrophic effect on cells. Hence, ideal combination of growth factors and optimal doses required for regeneration is to be determined to maximize their benefits in clinical applications.[26] The results of the present investigation clearly indicated that PDGF-AB has potent mitogenic effect on hPDLF at dosages of 100 ng/ml at 48 hours. These findings may go a long way to gain maximum clinical advantage of mitogenic potential of PDGF, which holds a great promise to the periodontist for stimulating periodontal regeneration. In vivo studies are required to determine the role of PDGF in wound healing. From the clinical applicability to the utilization of the mitogenic potential of PDGF on hPDLF in achieving periodontal regeneration, further clinical studies with a larger sample size are required to determine at what dosage PDGF exhibits its maximum regenerative potential.