A Cycle of Freezing and Thawing as a Modified Method for Activating Platelets in Platelet-rich Plasma to Use in Regenerative Medicine

INTRODUCTION

Platelet-rich plasma (PRP) preparations are safe, reliable, and cost-effective products that are used for accelerating healing after a surgical procedure or injury. Platelet-rich plasma comprises an increased concentration of platelets compared to whole blood. As a growth factor pool for improving tissue regeneration, PRP has been widely used in different clinical scenarios, such as in orthopedics and ophthalmology. Depending on the purpose, PRP can be used with or without previous platelet activation. Upon activation under physiological and pathological circumstances, platelets release a large amount of bioactive molecules that possess regenerative features.^1–4 Platelet-rich plasma and other platelet-derived products seem to be promising alternative tools besides the treatment options for infected wounds.^5,6 Platelets can be activated by adding collagen, thrombin, and/or calcium in contact with glass. Cryopreservation process has also been recently shown to activate platelets and activation associated markers such as P-selectins.⁷ The purpose of this study is to evaluate a cycle of freezing/thawing and its effect on the amount of growth factor in PRP.

MATERIALS AND METHODS

RESULTS

Twenty donors were included in the study, from whom 5 mL of whole blood was obtained. Their mean age was 36.3 ± 8.4 years, and the mean weight was 83 ± 10 kg. The mean platelet count and the autologous PRP were 238.5 ± 44.7 × 10³/μL and 544.7 ± 161.5 × 10³/μL, respectively. Overall, there was an increase in the concentration of the three growth factors (Table 1), but only PDGF was statistically significant. There were not any significant differences between the growth factor levels of freeze/thawed and calcium-activated PRP. A cycle of freezing/thawing was the only independent factor associated with growth factor yield in the multivariate model.

DISCUSSION

The rationale behind the widespread use of platelet-derived products is the abundance of growth factors and other signal transmitting molecules along with their ease to acquire them.⁸ Under normal circumstances, growth factors and bioactive peptides are secreted in response to injury, acting in a well-organized and controlled medium for contributing to the healing process. Their contribution in the healing process occurs sequentially in the inflammation, repair, and remodeling phases. In general, platelets remain in the inactivated, quiescent state and require a trigger signal to actively participate in hemostasis and wound healing.⁹ During the latter, activated platelets secrete both dense and alfa granules. Dense granules contain ADP, ATP, serotonin and calcium, and alfa granules contain clotting factors and platelet-derived growth factors. The contents of these granules play a critical role in normal platelet activity.¹⁰ Platelet-derived growth factors have specific cellular target receptors. Upon binding their targets, they show morphogenic and mitogenic effects, induce cell migration, and support cell proliferation, differentiation and chemotaxis. Additionally, they support mesenchymal cells in bone marrow, adipose tissue and cord blood, increase the effective proliferating number of cells, and cell differentiation, thus speeding up the wound healing process. The secreted platelet-derived growth factors bind to the tyrosine kinase receptors and induce the propagation of signal transduction toward the nucleus by the explicit pathways and signaling cascades and activate target expression.¹¹

Platelet-rich plasma is obtained by transferring the blood sample into the acid/citrate/dextrose tubes and centrifuged them. In order to gain the supernatant, consisting of PRP, platelet pellet, and PPP, a high-speed centrifugation is required. Besides the protocol for obtaining PRP, platelet activation is required for the secretion and enmeshment of growth factors. However, the preferred method of activation may exert a negative impact on matrix development, growth factor content, and wound healing.¹² The most preferred method of activating the platelets in PRP is the addition of pig thrombin and calcium, which results in the formation of platelet rich gel. When these reagents are added, gel formation occurs quickly, being these reagents frequently applied to the wound site. Most protocols only use calcium to activate platelets due to the risk of developing antibodies against pig thrombin, and clotting factors V and IX, with the resultant development of severe coagulopathy. Currently, a standard method to obtain platelet gel does not exist.

As mentioned earlier, addition of pig thrombin and calcium for activating the platelets in PRP results in the formation of platelet-rich gel. Under some circumstances, it may be preferred that the activated and degranulated platelets remains in liquid form, such as PRP drops used for the treatment of dry eye syndrome and corneal ulcers.

Use of PRP gel form have been previously described. Cetinkaya et al.⁵ reported that antimicrobial properties of PRP have led to the suggestion that PRP may be a viable treatment option for the treatment of surgical site infections. In vitro studies on the antimicrobial efficacy of PRP have shown that PRP inhibits the growth of methicillin-sensitive and -resistant Staphylococcus aureus (MRSA), Group A Streptococci, E. coli, and N. gonorrhoeae, while other authors have shown that PRP used in conjunction with vancomycin is effective against MRSA growth. The conclusion of this animal study was that PRP gel seems to enhance MRSA-infected surgical wound healing, by decreasing chronic suppurative inflammation and MRSA cell counts in wounds.

Later, Çetinkaya and associates⁶ revealed in their in vitro study that PRP and PPP had antimicrobial effect on three resistant microorganisms such as MRSA, ESBL-positive K. pneumoniae, and carbapenem-resistant P. aeruginosa, while they had no significant effect on VRE. When these results were considered as a whole, antimicrobial effect of PRP was more effective than PPP; however, it was statistically significant only in MRSA and P. aeruginosa group and only in the first hours of the study. This group stated that considering the trend on diabetes and antibiotic resistance rates, treating chronic infected wounds is going to continue to be one of the most important and the most difficult fields in medicine in the future. The conclusion of that study was that emerging PRP and other platelet-derived products seem to be a promising alternative tool besides antibiotic treatment, debridement, negative pressure wound therapy, hyperbaric oxygen therapy, and other treatment options for treating such infections.

Other authors describe the implications of the cycle of freezing and thawing effects over platelet-derived growth factor functions. Sekido et al.¹³ studied cell cultures and showed that activities of growth factors released from cryopreserved platelets were similarly effective and adequate as the growth factors released from fresh platelets. Similarly, Roffi et al.¹⁴ demonstrated that a cycle of freezing and thawing did not change the secretion of platelet-derived growth factors and their effects on chondrocytes and sinoviocytes. Ronci et al.¹⁵ showed that the use of PRP activated after a cycle of freezing and thawing, efficiently treated persistent ocular epithelial defects.

The concept of lyses of platelets by freezing was also addressed in a publication by Weed et al.¹⁶ There are differences between their study and our own regarding the method of PRP product preparation. In their study, an average of 240 mL of leukocyte-reduced platelets and a similar amount of plasma were collected using the Spectra blood cell separator. Aliquots of platelets and plasma were placed into 12 vials and frozen at less than or equal to −18°C within 12 hours of collection. In our study, samples of whole blood were obtained from voluntary donors by venipuncture and collected into citrated tubes, and PRP was obtained by using centrifuge as detailed in the methods section, not by apheresis as described in the Weed’ study. While the PDGF increase obtained by our method was approximately three times higher than the basal levels, in their study, they obtained approximately forty times more compared to the basal levels. But as their method needs, apheresis is neither practical nor easy to apply. Also, our samples were cryopreserved and ready immediately at −80°C for 24 hours, not at −18°C within 12 hours of collection. Lastly, their PRP product did not lead to statistically significant accelerated wound healing. They stated that there could be many reasons for this, but did not discuss timing and degree of freezing.

The concept of lyses of platelets by freezing was addressed by Sonker et al.¹⁷ In their study they aimed to compare different preparation methods, concentration techniques, and storage durations in order to optimize the yield of growth factors. Concentration of growth factors was performed by using double freeze thaw technique or CaCl₂-induced degranulation technique. Double freeze thaw is a different concentration technique compared to the single freeze thaw concentration technique we used in our study. With theirs the PRP sample is frozen at −80°C, it is thawed at room temperature after 48 hours, and refrozen immediately thereafter. Just before analyzing it was again thawed and centrifuged for 10 minutes at 10,000 rpm in a microcentrifuge. They did not explain or give any reference about why they used this method, but they probably aimed to lead a complete release of all growth factors from intracellular granules into the surrounding liquid. Further studies are needed to compare the levels of growth factors in the lysate and the lysate activity between both single and double freeze thaw concentration techniques.

Our study showed that growth factors were released at higher concentrations from the platelets after activation with a cycle of freezing/thawing compared to the basal values in the autologous PRP without formation of gel. Although it is thought that the platelet levels present in the blood-derived products play a key role in the regenerative process rather than the fibrin matrix or the methods chosen for platelets activation, our study established that a cycle of freezing/thawing was the only independent factor affecting the growth factors content of the PRP. In the freezing/thawing activated PRP, platelets activation has been obtained without inducing fibrin matrix or coagulation, but there was different amounts of fibrin matrix or coagulation in the calcium activated PRP.

It is important to recognize that standardization of PRP preparations is one of the major concerns in the field of regenerative medicine, and although biological studies like ours give important indications for the development of treatments, the results do not always directly translate into clinical findings.

CONCLUSION

With its features of being simple, inexpensive and easy for standardization, a cycle of freezing/thawing may be the method of choice for PRP activation procedure, especially for obtaining PRP rich in PDGF, without inducing fibrin matrix, like the eye drops rich in GF used for the treatment of persistent ocular epithelial defects. Further clinical studies are recommended to obtain a better understanding of the effects of a cycle of freezing/thawing for activation of platelets in PRP on patients’ symptoms and functional improvement.

Informed Consent

The study was approved by the Institutional Ethics Committee of Gulhane Military Medical Academy. Informed consent was obtained from all study participants.

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