Comparative Evaluation of Growth Factor Concentrations Between PRF Membranes Amongst Type-2 Diabetics - A Cross-Sectional Study

Introduction

Regeneration of lost tissue necessitates the use of growth factors, of which denovo synthesized Platelet Rich Fibrin (PRF) has been used extensively.¹ This PRF processed from blood finds its application in the regenerative areas of the dental and medical fields.² A pioneering work by Choukroun in 2001, this supraphysiological growth factor concentration in PRF allows its release from a 3-Dimensional matrix capable of sustaining tissue growth which is required for improved tissue healing.³ Leukocyte and Platelet Rich Fibrin (L-PRF) originated first finds its immeasurable medical and dental applications in healing of tissues and regenerative areas.⁴ One of the developments in PRF is the introduction of a low-speed centrifugation concept with Advanced- Platelet Rich Fibrin (A-PRF) and A-PRF+ preparations. These provide a slower release of growth factors over time due to the ability of the fibrin matrix to hold proteins within its fibrin network, as well as cells capable of extending the release of growth factors into their surrounding microenvironment.⁵ Growth factors regulate cell proliferation, cell activity, chemotaxis, cell differentiation, stimulate and attract stem cells to the site of injury, promoting cell mitosis and inducing angiogenesis and osteogenesis.⁶ The newer formulation A-PRF+ has been shown to increase growth factor release of Transforming Growth Factor Beta-1 (TGF-β1), Platelet Derived Growth Factor (PDGF-AA, PDGFAB, PDGF-BB), Vascular Endothelial Growth Factor (VEGF), Insulin Growth Factor (IGF) and Fibroblast Growth Factor (FGF). These PRF preparations enhance fibroblast migration, proliferation, and collagen mRNA levels.^5,7 Periodontal regeneration is the regeneration of the tooth supporting tissues⁸ and use of this A-PRF as a regenerative and barrier membrane has shown promising results.⁹

Diabetes mellitus (DM) is a highly prevalent metabolic disease characterized by hyperglycaemia. Current global estimates indicate that approximately 537 million adults are living with diabetes as of 2021. This figure is predicted to escalate to 643 million by 2030 and 783 million by 2045.¹⁰ DM is associated with impairments in the wound healing process and reduced growth factor release in the local wound area.^11-13 Thus, it is imperative to employ a substance that not only hastens wound healing but also has regenerative potential, and PRF is one such material. Till date, there are no studies to show PRF membrane growth factor release in diabetic individuals. Hence, this novel study compared growth factor release from L-PRF, A-PRF, and A-PRF+ in diabetic individuals.

Materials and Methods

This was a cross sectional experimental study. An informed written consent from all the subjects and approval by the Ethical Committee from DAPM RV Dental College in accordance with Helsinki Declaration of 1975, as revised in 2013 were obtained before start of the study. G* power software was used to estimate the sample size. A total of selected 90 samples yielded 95% power to detect significant difference with effect size of 0.40 and significance level at 0.05. Samples were divided into groups A, B, C, comprising of 30 samples in each group (i.e., from healthy, well-controlled, and poorly-controlled diabetic individuals, respectively) based on their Glycated Haemoglobin (HbA1c) levels. Group-A included samples with HbA1c <6.5, groups-B with HbA1c in the range of 6.5-7.5, and groups-C samples with HbA1c >7.5%. Individuals within the age group of 35-55 years were selected. Diabetic individuals under medication (oral hypoglycaemic drugs) were included. Patients with any other systemic disease such as hypertension, bleeding disorders, obesity, any diagnosed malignancies, history of other medications such as oral anticoagulants, anti-platelet drugs, or immunosuppressive drugs, pregnant or lactating females, individuals with history of smoking, alcohol consumption were excluded from the study.

The blood samples were obtained from the individuals reporting to the Department of Periodontology, R.V. Dental College, Bangalore, Karnataka. A total of 17 mL of blood was withdrawn from each patient, of which 2 mL of blood was collected into an EDTA coated vacutainer for analysis of HbA1c, CBC and platelet parameters analysis (platelet count (PC), Plateletcrit (PCT), Mean Platelet Volume (MPV), Platelet to Large Cell Ratio (PLCR), Platelet Distribution Width (PDW)). The remaining blood was collected into uncoated vacutainers (BD glass vacutainers, Franklin Lakes, USA) for preparation of the three PRF membranes (paired sample) following the protocols using the Remi8c centrifuge machine: centrifugation at 2,700 rpm for 12 minutes for L-PRF; centrifugation at 1,300 rpm for 14 minutes for A-PRF; and centrifugation at 1,300 rpm for 8 minutes for A-PRF+. The clots thus obtained after separation from platelet poor plasma were formed into the membrane by gentle compression on the PRF box with drainage of the excess fluid (Figure 1).

The centrifugation process separates the blood into different layers based on the density of cells. The obtained buffy coat from these protocols were separated from red blood cells with scissor, after gently pulling out from the vacutainer. Then fibrin clots were weighed and placed on the PRF Xpression box for gentle compression by slightly compressing until they closed completely the metal cover as per the recommendation of the manufacturer. Platelet poor plasma will be squeezed out. Membranes were gently lifted and were ready for use. Finally, a total of 90 membranes from the groups were ready for analysis. The membranes were stored in sterile Dulbecco’s Modified Eagle’s Medium at -200°C before quantification. At desired time points, the following growth factors: VEGF, IGF-1, and FGF-21 were quantified using ELISA assays [Everon Lifesciences, Shanghai Korain Biotech Co., Ltd. (BT Bioassay)] according to the manufacturer’s protocol. Kinesis kits with catalogue no. E-0080Hu for VEGF, E-0103Hu for IGF-1, and E-1983Hu for FGF-21 were used for Vascular Endothelial Growth Factor, Insulin-Like Growth Factor-1, and Fibroblast Growth Factor-21, respectively.

Data and statistical analysis

All the data obtained from the sample analysis and lab reports was transferred to an excel sheet and subjected to statistical analysis. The data showed a non-normal distribution. Hence, non-parametric tests (Mannwhitney) were applied. The p value was found to be equal or within 0.05 (p <0.05) and was considered statistically significant. All the recorded parameters were statistically analyzed using SPSS (Statistical Package for Social Sciences) version 20 [IBM SPASS statistics, IBM Corp. released 2011]. Comparison of blood parameters between the three groups (Groups A, B, and C) was performed using one-way ANOVA test followed by a Tukey post hoc to determine which specific groups differed from each other. Comparison between L-PRF, A-PRF, and A-PRF+ among the individual groups for growth factor release was done by using the Independent T test. Comparison between the three groups for growth factor release (VEGF, IGF-1, FGF-21) was analyzed using one way-ANOVA, followed by Tukey’s post hoc test.

Results

In the present study, a total of 90 samples were divided into groups A, B, C, comprising of 30 samples in each group based on their HbA1c levels. Comparison of demographic data and blood parameters is shown in Table 1, Table 2 and Table 3. 

A statistically significant difference (Table 3) in mean platelet count (F=32.39; p=0.001), WBC (/cumm) (F=13.59; p=0.001), Plateletcrit (PCT) (F=35.41; p=0.001), Mean Platelet Volume (MPV) (F=11.51; p=0.001), Platelet to Large Cell Ratio (PLCR) (F=15.76; p=0.001), Platelet Distribution Width (PDW) (F=3.6; p=0.041) between healthy, well controlled diabetes and poorly controlled diabetes were seen. Poorly controlled diabetes group displayed a statistically significant higher platelet count on comparison (Chart 1). A statistically significant higher level of VEGF, IGF-1, and FGF-21 release (Chart 2) was observed among A-PRF+ when compared to L-PRF and A-PRF at days 1, 3, and 7 in healthy individuals. 

Similarly, statistics show a statistically significant higher level of FGF-21 among A-PRF + when compared to L-PRF and A-PRF at 1 day and day 3. However, there were no statistically significant differences in IGF-1 levels between A-PRF and A-PRF+. A statistically significant higher level of VEGF and IGF-1 release was noted among A-PRF + when compared to L-PRF and A-PRF at 3 days and 7 days. Also, a statistically significant higher level of FGF-21 was noted among A-PRF + when compared to A-PRF and L-PRF at 1 day.

Pearson’s correlation showed a statistically significant positive correlation between HbA1c (%) and WBC count (r=0.649; p=0.042) in Group A. FGF-21 growth factor had a strong positive correlation with HbA1c levels (r=0.722; p=0.018) in Group B. In Group C, a statistically significant strong positive correlation between HbA1c (%) and PLCR (r=0.792; p=0.006) and PDW (r=0.827; p=0.003) was noted. Pearson correlation of HbA1c levels and FGF-21 levels (f=165.122) showed a very strong correlation between HbA1c and FGF-21.

Discussion

The present study assessed and compared the changes in growth factor release (VEGF, IGF-1, and FGF-21) from three different platelet rich fibrin membranes that were prepared at different centrifugation times amongst healthy, well controlled diabetics, and poorly controlled diabetics. The present study showed statistically significant higher values of platelet indices like MPV, PDW, PCT, PLCR and higher mean white blood cell count and mean platelet count in diabetic individuals, especially in poorly controlled diabetics when compared to well-controlled diabetics and healthy individuals. A statistically significant and strong positive correlation between HbA1c levels and PLCR and PDW was also noted. Comparison of mean growth factor levels revealed that poorly controlled diabetics displayed statistically significant higher VEGF, IGF-1, and FGF-21 levels at days 1, 3, and 7 when compared to healthy and well controlled diabetic individuals. Diabetes is a chronic, metabolic disease and is an established risk factor for periodontitis. The morbidity and severity of periodontitis positively correlate with poorly controlled or long-standing diabetes.¹⁴ Diabetes mellitus is associated with impairments in the wound healing process and tissue regeneration. Results of studies on regenerative therapies in diabetics have shown that autologous biomaterials can be used effectively as a regenerative material.^15,16

Platelet hyperactivity in patients with diabetes is multifactorial and is associated with hyperglycemia, insulin resistance, inflammatory and oxidative states. Alterations in several platelet functional parameters have been reported in diabetes. Statistically significant higher values of platelet indices in diabetic individuals in the present study are in accordance with the findings of Ulutas K et al., in which higher HbA1c levels were associated with higher levels of MPV, PCT, and PDW.¹⁷ Hekimsoy et al.,¹⁸ elucidated that higher MPV corresponds to larger platelets which are younger, more reactive and agreeable. Hence, they contain denser granules which secrete higher amounts of biologically active growth factors and cytokines than smaller platelets. One of the plausible mechanisms of increased MPV in DM is the osmotic swelling due to hyperglycemia and perhaps due to a shorter life span of platelets in diabetic patients.¹⁹

This increased mean platelet count in diabetics is in consonance with the findings of Thomas et al., Zuberi et al.²⁰ It is, however, in contrast with the findings of a study conducted by Hekimsoy et al. ²¹ Elevated WBC count in poorly controlled diabetics is associated with insulin resistance which plays a role in the pathogenesis of diabetes and is also associated with macro and micro vascular complications in diabetes.²² Activities like sustained production of pro-inflammatory cytokines, impaired angiogenic response, microvascular complications, impaired macrophage and neutrophil function, impaired keratinocytes and fibroblast migration and proliferation, and impaired production of healing-associated factors like impaired growth factor production in the local wound area have been reported by Patel et al.¹³ Glycation of haemoglobin causes a deficient supply of nutrients and oxygen to tissues, which further delays the wound healing process and may lead to local ischaemia.³ Hence, it can be speculated that PRF would aid in wound healing in diabetics as it acts as a reservoir of cells, growth factors, and cytokines that are released in supraphysiological concentrations.

The present study demonstrated that there was a significantly higher release of growth factors (VEGF, IGF-1, and FGF-21) from A-PRF+ when compared to A-PRF in all the groups. These findings corroborate a previous study by El Baghdadi et al., ²³ who found that A-PRF+ is linked to increased growth factor release. Also, as A-PRF+ shows a more porous structure, allowing more space for trapped platelets and immune cells and therefore a higher and more sustained release of growth factors, as demonstrated by El Bagdadi et al., and Pitzurra et al. ²⁴ Higher VEGF, IGF-1, and FGF-21 levels at days 1, 3, and 7 were observed in poorly controlled diabetics.

Chronic hyperglycaemia has been reported to stimulate the synthesis and secretion of VEGF. Although VEGF levels are higher in the serum of diabetics, they seem to be present in a lower concentration in the local wound area. This can be justified by the fact that there are decreased VEGF mRNA levels, abnormal patterns of VEGF receptors, increased VEGFR-1 (related to inflammation) level and decreased VEGFR-2 (responsible for angiogenesis) level in diabetic wounds.²⁵ Also, accumulation of advanced glycation end-products (AGEs) in diabetics can cause interference with VEGF and its receptors and signaling pathways.²⁶ A significant increase in IGF-1 levels from A-PRF+ could be with an increase in HbA1c levels. Compensatory up-regulation of hepatic IGF-1 synthesis occurs, resulting in an increase in free IGF-I levels. Also, there are impairments in its interaction with the IGF1 receptor in diabetics, leading to impaired wound healing. This is however, in contrast with the findings by Aleidi et al., who showed low serum IGF-1 in diabetics. IGF-1 is considered one of the key mediators in wound healing and mesenchymal cell proliferation. IGF-1 released from PRF has been shown to stimulate growth, proliferation, and differentiation of human PDL stem cells by increasing RUNX2, SP7, and OCN.²⁷ PRF and IGF-1 bind to receptors on the cell surface to mediate their effects. This, therefore, induces a signalling cascade and ultimately affects gene expression and protein synthesis. So IGF-1 through PRF in diabetics may provide anabolic effects on the bone. Significant higher release of FGF-21 at day 1, 3, and 7 from A-PRF+ prepared from poorly controlled diabetic individuals and also a positive correlation between HbA1c levels and FGF-21 in diabetics were seen in this study. This could be due to an increased hepatic production of FGF-21 as a result of the metabolic changes.²⁸ However, hyperglycemia may cause resistance to the actions of FGF-21, thereby increasing its levels in the serum of diabetic individuals. A recent study has demonstrated that FGF-21 enhances the osteogenic activity of BMP-2 by up regulating the BMP-2 mediated Smad pathway.²⁹ The limitation of the present study could be conducting the study in multi-centric places with the effect of particular oral anti-diabetic medication the patient is taking. Hence, future direction of study should be aimed at conducting the study in a multicentric area and standardizing the patient’s oral hypoglycemic medication. Also, the clinical application in various medical and dental fields must be planned in well-controlled diabetic individuals.

Conclusion

Thus, from the present study, it could be concluded that diabetic A-PRF+ has the highest concentration of growth factors and could be used in the healing of tissues and regenerative procedures. The abundance of growth factors in A-PRF+ in diabetic individuals can be harnessed and utilized in soft and hard tissue regeneration. As such, diabetic individuals need some regenerative material so as to replace lost tissue and bring about faster wound healing so that their life expectancy can be improved. Hence, A-PRF+ could be planned in clinical applications for diabetic individuals in regenerative therapies.

Acknowledgment

The present study is self-funded

Conflict of interest statement

This study is self-funded and hence there is no conflict of interest among the authors.