PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1
The use of platelet concentrates is becoming very popular in the field of musculoskeletal tissue regeneration. A widespread interest has been shown for platelet-rich plasma (PRP) as an injective treatment or as a surgical augmentation procedure for the repair of tissues with low healing potential, with an increasing number of preclinical and clinical studies over time [
Preclinical evidence supports the use of intra-articular PRP injections for joint degenerative pathology, by targeting not only cartilage but also synovial and meniscal tissues and thus promoting a favorable environment for joint tissue healing [
Platelet count, activation methods, leukocytes/red blood cells content, and number of injections are the most debated aspects that led to a different effect on target tissues. PRP storage is also a key factor: freeze-thawing allows easier patient management, but it is thought to impair platelet function and lifespan, alter the GF release pattern, favor the accumulation of pyrogenic cytokines, and increase the risk of bacterial proliferation [
Whether freeze/thawing PRP might lead to different release kinetics of molecules or different effects on tissue homeostasis with respect to fresh PRP is poorly understood. Perut et al. [
The purpose of this study was to compare fresh and frozen PRPs by analyzing whether the different state might influence the release of bioactive molecules and their effects on chondrocytes and synoviocytes. In particular, since some authors showed that 95% of these molecules are secreted within 1 hour after activation and then platelets release additional proteins in one week [
The study was approved by the Institutional Review Board and the local Ethics Committee, and written informed consent was signed by each donor.
Ten healthy volunteers (Caucasian male, age range: 27–38 years, BMI: normal values) were enrolled to undergo a blood sample collection. Exclusion criteria were systemic disorders, infections, smoking, nonsteroidal anti-inflammatory drug use 5 days before blood donation, haemoglobin values lower than 11 g/dL, and platelet values lower than
To prepare PRP, a 150 mL venous blood sample was collected in a bag containing 21 mL of sodium citrate and centrifuged at 730 g for 15 min. Most of the red blood cells were eliminated and the resulting plasma and buffy-coat were transferred to a separate bag through a closed circuit. After a second centrifugation at 3800 g for 10 min, the supernatant was collected to produce PRP [
All PRP samples (fresh and frozen) were activated with 10% CaCl2 (22.8 mM final concentration) and divided into two aliquots, one incubated for 1 h and the other for 7 days at 37°C in 5%
After centrifugation (for 15 min at 2800 g at 20°C), the released supernatant was collected and stored at −30°C. Interleukin (IL)-1
Chondrocytes (
Synoviocytes were obtained from synovial tissue that was digested with 0.1% Trypsin (Sigma-Aldrich) in PBS at 37°C, 5%
Chondrocytes were plated at a density of
Synoviocytes were plated at a density of
At the end of the incubation time (7 days), culture supernatants were collected and maintained at −80°C until their use in ELISA tests, whereas chondrocytes and synoviocytes were used for proliferation assay and then lysed for RNA extraction.
Chondrocyte and synoviocyte growth in the presence of each PRP formulation was evaluated through the Alamar blue test [
The expression of specific genes by chondrocytes was assayed with real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). IL-1
RNA specific primers for PCR amplification were generated from GeneBank sequences using Primer 3 software (Table
List of primers used in real-time PCR
RNA template | Primer sequences (5′–3′) | Annealing temperature (°C) | References* |
---|---|---|---|
GAPDH | 5′-TGGTATCGTGGAAGGACTCATGAC |
60 | [ |
|
|||
Collagen type II | 5′-GACAATCTGGCTCCCAAC |
60 | PRIMER 3 |
|
|||
Aggrecan | 5′-TCGAGGACAGCGAGGCC |
60 | [ |
|
|||
Sox-9 | 5′-GAG CAG ACG CAC ATC TC |
60 | PRIMER 3 |
|
|||
IL-1 |
5′-GTGGCAATGAGGATGACTTGTT |
60 | PRIMER 3 |
|
|||
IL-6 | 5′-TAGTGAGGAACAAGCCAGAG |
60 | PRIMER 3 |
|
|||
IL-8 | 5′-CCAAACCTTTCCACCC |
60 | PRIMER 3 |
|
|||
IL-10 | 5′-CTTTAAGGGTTACCTGGGTTG |
60 | PRIMER 3 |
|
|||
TNF- |
5′-AGCCCATGTTGTAGCAAACC |
60 | PRIMER 3 |
|
|||
VEGF | 5′-TGATGATTCTGCCCTCCTC |
60 | PRIMER 3 |
|
|||
FGF- |
5′-CGGCTGTACTGCAAAAACGG |
60 | PRIMER 3 |
|
|||
HGF | 5′-ATACTCTTGACCCTCACACC |
60 | PRIMER 3 |
|
|||
TGF- |
5′-CAACAATTCCTGGCGATACCT |
60 | PRIMER 3 |
|
|||
HAS-1 | 5′-TGGTGCTTCTCTCGCTCTACG |
60 | [ |
|
|||
HAS-2 | 5′-AAATGGGATGAATTCTTTGTTTATG |
60 | [ |
|
|||
HAS-3 | 5′-CAGCTGATCCAGGCAATCGT |
60 | [ |
|
|||
TIMP-1 | 5′-CCGACCTCGTCATCAG |
60 | PRIMER 3 |
|
|||
TIMP-3 | 5′-CCTTGGCTCGGGCTCATC |
60 | PRIMER 3 |
|
|||
IL-4 | 5′-CAGTTCCACAGGCACAAG |
60 | PRIMER 3 |
|
|||
IL-13 | 5′-GCACACTTCTTCTTGGTC |
60 | PRIMER 3 |
|
|||
IL-10 | 5′-CTTTAAGGGTTACCTGGGTTG |
60 | PRIMER 3 |
|
|||
MMP-13 | 5′-TCACGATGGCATTGCT |
60 | PRIMER 3 |
*Primer sequences were obtained from published references and were indicated or designed using PRIMER 3.
HA levels were measured in the supernatants of chondrocytes and synoviocytes that had been treated for 7 days with PRP and frozen PRP, using commercial DuoSet ELISA kit (R&D Systems) following the manufacturer’s instructions. Lubricin protein level was measured in the supernatants of chondrocytes using a specific Elisa Kit (PRG4) (Uscn, Life Science Inc., Wuhan, China).
Data concerning the characterization of the PRP and frozen PRP were analyzed by Friedman’s test for multiple comparisons of paired data and, when significant, followed by Bonferroni’s post hoc correction for multiple comparisons (a value of
Statistical analysis was carried out using the Statistica for Windows package release 6.1 (Statsoft Inc., Tulsa, OK) and GraphPad Prism for Windows (CA, USA).
Table
Soluble factor concentrations in fresh (PRP) and frozen PRP 1 h and 7 days after activation. Concentrations are expressed as pg/mL and reported as median values and (interquartile ranges).
Soluble factors | Preparations | Incubation time |
|
|
---|---|---|---|---|
1 hour | 7 days | |||
IL-1 |
PRP | 1.015 (0.83–5.41) | 70.11 (56.81–233.35) |
|
Frozen PRP | 1.22 (0.72–2.32) | 14.43 (1.11–179.20) | NS | |
|
NS | NS | ||
|
||||
TGF- |
PRP | 107861.6 (81652–127793.0) | 103553.4 (64935.69–1341400.0) | NS |
Frozen PRP | 33849.8 (23339.08–54974.0) | 52511.5 (30092.61–201434.0) | NS | |
|
|
|
||
|
||||
PDGF AB/BB | PRP | 27714.68 (18591.50–35850.24) | 31670.63 (18617.58–80462.27) | NS |
Frozen PRP | 17388.90 (8648.29–29500.03) | 6035.78 (4691.41–37053.02) | NS | |
|
|
|
||
|
||||
VEGF | PRP | 157.94 (62.3–238.52) | 226.79 (145.82–743.31) | NS |
Frozen PRP | 147.78 (9.39–209.94) | 204.10 (136.85–632.23) | NS | |
|
NS | NS | ||
|
||||
HGF | PRP | 247.20 (148.75–305.97) | 380.89 (370.58–493.17) |
|
Frozen PRP | 253.68 (109.87–283.97) | 212.77 (149.44–261.94) | NS | |
|
NS |
|
The soluble factors analyzed showed that in frozen PRP both the immediate and delayed releases were similar or slightly lower than those of PRP. In particular, the TGF-
Concerning time-related modifications, in fresh PRP, IL-1
Chondrocytes and synoviocytes grown in the presence of 10% of both PRPs were viable and able to proliferate up to 7 days with no difference between preparations (data not shown). At 7 days, cells are subconfluent.
Collagen type II, aggrecan, and Sox-9 mRNAs were similarly expressed on day 7 in chondrocytes treated with fresh and frozen PRP (Figure
RT-PCR analysis: messenger RNAs expression in chondrocytes grown in presence of 10% fresh or frozen PRP at 7 days. Data were normalized to GAPDH and expressed as a percentage of the reference gene. Boxes indicate the 25% and 75% percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median.
Gene expression analysis on synoviocyte cultures indicated that, among proinflammatory factors, anti-inflammatory factors, and/or anticatabolic factors, IL-1
Expression analysis of factors involved in joint physiopathology: messenger RNAs expression in synoviocytes grown in presence of 10% fresh or frozen PRP at 7 days. Data were expressed as n°mol mRNA ×100000 GAPDH. Hyaluronic acid production was evaluated in culture supernatants and protein production was normalized per number of cells. Boxes indicate the 25% and 75% percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median.
With regard to GFs, cartilage matrix degrading enzymes, and their inhibitors, no difference was found in synoviocyte gene expression levels between fresh PRP and frozen PRP for most of them (VEGF, TGF-
As shown in Figures
Hyaluronan levels in the culture media of human chondrocytes grown in presence of 10% fresh or frozen PRP at 7 days. Mean values are expressed as ng/mL, boxes indicate the 25% and 75% percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median.
The heterogeneous clinical outcome reported in the literature on PRP treatment for joint tissue regeneration reflects the lack of guidelines regarding the use of platelet concentrates, starting from their production up to their clinical application. The increasing awareness on the need for PRP standardization is shown by the numerous biological studies investigating the role of each PRP variable on the healing potential of platelet concentrates [
In this scenario, our aim was to investigate whether PRP freezing/thawing affected the release of GFs from platelets
The results of GF release showed that in frozen PRP the immediate release and the total amount at 7 days were not the same as in fresh PRP. Indeed, it seemed to be similar or slightly lower with respect to the fresh preparation, as reported for TGF-
With regard to proinflammatory cytokines, the immediate release of IL-1
Another key point is the release kinetics of GFs in both PRPs. It has been recently reported that once platelets are activated, an initial burst of GF release is followed by a further sustained release, 3- to 5-fold increase as compared with baseline [
Despite these differences in GF release, fresh and frozen PRPs did not differ in their ability to induce cell proliferation or ECM production and secretion in both chondrocytes and synoviocytes. Concerning gene expression analysis, chondrocytes cultured with both PRPs showed similar results for collagen II, aggrecan, and Sox-9, thus indicating that frozen PRP did not lose or reduce its ability to enhance chondrocyte anabolism. Albeit with no statistical significant difference with respect to frozen PRP, IL-1
Concerning synoviocyte culture, also in this case the two PRP preparations did not induce significant differences in the expression of pro/anti-inflammatory agents and anticatabolic factors, whereas a higher expression of HGF was found in frozen PRP (
Current research aims to optimize PRP production and administration protocols. This study underlines two interesting aspects. The first one is that freeze/thawing affects PRP cell composition and its release of bioactive molecules. The second is that this different release kinetics does not significantly influence the effects on cell cultures. It is important to recognize that biological studies give important indications for the development of treatments, but their results do not always directly translate into clinical findings, as previously shown by the same clinical outcome reported using two biologically completely different procedures [
PRP freezing is a controversial topic. Our results on GF release from platelets
Giuseppe Filardo is consultant and receives institutional support from Finceramica Faenza Spa (Italy), Fidia Farmaceutici Spa (Italy), and CartiHeal (2009) Ltd (Israel). He is a consultant for EON Medica SRL (Italy). He receives institutional support from IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). Elizaveta Kon is a consultant for CartiHeal (2009) Ltd. (Israel) and has stocks of CartiHeal (2009) Ltd (Israel). She is a consultant and receives institutional support from Finceramica Faenza Spa (Italy). She receives institutional support from Fidia Farmaceutici Spa (Italy), IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). Maurilio Marcacci receives royalties and research institutional support from Fin-Ceramica Faenza SpA (Italy). He receives institutional support from Fidia Farmaceutici Spa (Italy), CartiHeal (2009) Ltd (Israel), IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). All the other authors declare that there is no conflict of interests regarding the publication of this paper.
Doctor Roffi Alice participated in writing of the paper. Doctor Filardo Giuseppe participated in writing of the paper and study design. Doctor Assirelli Elisa participated in tests on synoviocytes, data analysis, and writing of the paper. Doctor Cavallo Carola participated in tests on chondrocytes, data analysis, and writing of the paper. Doctor Cenacchi Annarita participated in PRP production and is a hematology consultant. Professor Facchini Andrea is a supervisor and senior consultant and participated in editing. Doctor Grigolo Brunella participated in tests on chondrocytes, data analysis, and writing of the paper and is a consultant. Doctor Kon Elizaveta is a supervisor and participated in editing. Professor Mariani Erminia is a supervisor and senior consultant and participated in editing. Doctor Pratelli Loredana participated in data analysis. Doctor Pulsatelli Lia participated in tests on synoviocytes, data analysis, and writing of the paper and is a consultant. Professor Marcacci Maurilio is a supervisor and senior consultant and participated in editing.
This work was supported by the Italian Ministry of Health (Project “Ricerca Finalizzata”-2009-1498841) and PRRU (Emilia-Romagna Region/University of Bologna Project) 2010–2012.