A favorite patients of mine has been so kind to continue to do research on how to save meibomian glands. We are trying to figure out if we can inject IGF-1 (Insulin Growth Factor 1) into the meibomian glands to make them grow back better than Platelet Rich Plasma.
Some key points:
1. IFG-1 can help meibomian glands cells.
2. There is more IGF-1 in Cord Blood Serum than in peripheral blood (see below).
3. High glucose environments and Diabetes is toxic to meibomian gland cells (see paper below).
4. PRP and Cord Blood Serum both have high levels of IGF-1, but I have not seen a head to head study to see which one has the most in a sample.
REFERENCES:
1.
The Effects of Insulin-like Growth Factor 1 and Growth
Hormone on Human Meibomian Gland Epithelial Cells
Juan Ding, PhD; David A. Sullivan, PhD
2014 AMA
IMPORTANCE
A phase 1 study has reported that dry eye disease is the most common adverse effect of human exposure to the antibody figitumumab, an anticancer drug that prevents insulin-like growth factor 1 (IGF-1) from binding to its receptor. We hypothesized that the mechanism underlying this effect is the inhibition of IGF-1 action in epithelial cells of the meibomian gland.
OBJECTIVES
To test the hypothesis that IGF-1 stimulates meibomian gland function in vitro and to examine whether growth hormone, a closely related hormone of IGF-1, has the same effect.DESIGN,
SETTING, AND MATERIAL
Immortalized human meibomian gland epithelial cells were
cultured in the presence or the absence of IGF-1, growth hormone, and an IGF-1 receptor–blocking antibody. Signaling pathways, cell proliferation, neutral lipid staining, and a key protein involved in lipid biogenesis were evaluated.
INTERVENTION
Application of IGF-1 and growth hormone to human meibomian gland epithelial cells.
MAIN OUTCOMES AND MEASURES
Immunoblotting, cell counting, and neutral lipid staining.
RESULTS
Insulin-like growth factor 1 activated the phosphoinositol 3-kinase/Akt and forkhead box O1 pathways (showing a dose-dependent effect on immunoblotting), stimulated cellular
proliferation (about 1.8-fold increase in cell number), increased sterol regulatory element-binding protein 1 expression (about 3-fold increase on immunoblotting), and promoted lipid accumulation in human meibomian gland epithelial cells (about 2-fold
increase in lipid staining). These IGF-1 actions, which may be blocked by cotreatment with the anti–IGF-1 antibody, were accompanied by inconsistent effects on extracellular
signal-regulated kinase phosphorylation. We were not able to demonstrate activation of Akt, forkhead box O1, extracellular signal-regulated kinase, Janus kinase 2, or signal transducers and activators of transcription 5, induced cell proliferation, or lipid accumulation in these cells by growth hormone application
CONCLUSIONS AND RELEVANCE
Our results support the hypothesis that IGF-1 acts on human
meibomian gland epithelial cells and may explain why treatment with figitumumab, the IGF-1
inhibitor, causes dry eye disease. Ophthalmic care for dry eye disease may be needed when
patients with cancer undergo treatment with drugs that inhibit IGF-1 action.
(5) The Effects of Insulin-like Growth Factor 1 and Growth Hormone on Human Meibomian Gland Epithelial Cells | Request PDF. Available from: https://www.researchgate.net/publication/261757494_The_Effects_of_Insulin-like_Growth_Factor_1_and_Growth_Hormone_on_Human_Meibomian_Gland_Epithelial_Cells [accessed Oct 02 2018].
2.
3.
Transfus Apher Sci. 2018 Aug;57(4):549-555. doi: 10.1016/j.transci.2018.06.001. Epub 2018 Jun 13.
Comparison of growth factor and interleukin content of adult peripheral blood and cord bloodserum eye drops for cornea and ocular surface diseases.
Buzzi M1, Versura P2, Grigolo B3, Cavallo C3, Terzi A1, Pellegrini M4, Giannaccare G4, Randi V1, Campos EC4.
Author information
- 1
- Emilia Romagna Cord Blood Bank-Transfusion Service, S.Orsola-Malpighi Teaching Hospital, Bologna, Italy.
- 2
- Ophthalmology Unit, DIMES, Alma Mater Studiorum University of Bologna and S.Orsola-Malpighi Teaching Hospital, Bologna, Italy. Electronic address: piera.versura@unibo.it.
- 3
- RAMSES Laboratory, Department of Research & Innovation, Istituto Ortopedico Rizzoli, Bologna, Italy.
- 4
- Ophthalmology Unit, DIMES, Alma Mater Studiorum University of Bologna and S.Orsola-Malpighi Teaching Hospital, Bologna, Italy.
Abstract
INTRODUCTION:
Various blood-derived products have been proposed for the topical treatment of ocular surface diseases. The aim of the study was to compare the different content of Growth Factors (GFs) and Interleukins (ILs) in peripheral blood (PB-S) and Cord Blood (CB-S) sera.
MATERIALS AND METHODS:
Sera were obtained from 105 healthy adult donors (PB-S) and 107 umbilical/placental veins at the time of delivery (CB-S). The levels of epithelial-GF (EGF), fibroblast-GF (FGF), platelet-derived-GF (PDGF), insulin-GF (IGF), transforming-GF alpha (TGF-α,) and beta 1-2-3 (TGF-β1-β2-β3), vascular endothelial-GF (VEGF), nerve-GF (NGF), Interleukin (IL)-1β,IL-4,IL-6,IL-10, and IL-13 were assessed by Bio-Plex Protein Array System (Bio-Rad Laboratories, CA, USA). The Mann-Whitney test for unpaired data was applied to compare GFs and ILs levels in the two sources. The associations among each GF/IL level and the obstetric data for CB-S and hematological characteristics for PB-S were also investigated.
RESULTS:
The levels of EGF, TGF-α, TGF-β2, FGF, PDGF, VEGF, NGF, IL-1B, IL-4, IL-6, IL-10, and IL-13 were significantly higher in CB-S compared to PB-S. Conversely, the levels of IGF-1, IGF-2, and TGF-β1 were significantly higher in PB-S. The female sex and the weight of the child showed a significant association in predicting EGF and PDGF levels.
CONCLUSION:
A significantly different content in those GFs and ILs was demonstrated in the two blood sources. Since each GF/IL selectively regulates different cellular processes involved in corneal healing, the use of PB-S or CB-S should be chosen on the basis of the cellular mechanism to be promoted in each clinical case.
4.
Clin Chim Acta. 2018 Aug;483:89-93. doi: 10.1016/j.cca.2018.04.027. Epub 2018 Apr 21.
Sustained or higher levels of growth factors in platelet-rich plasma during 7-day storage.
Abstract
BACKGROUND:
The effectiveness of platelet-rich plasma (PRP) for treating soft tissue injuries is still controversial. Most of PRPs were prepared simply by concentrating in volume and were injected right after preparation in physician offices. Neither platelet count nor growth factors were quantitated in advance. We prepared and stored leukocyte and platelet-rich plasma (L-PRP) by regular separation protocols for blood components in the blood bank. And we investigated the dynamic change of growth factors in the L-PRPs over the period of storage.
METHODS:
The L-PRPs were prepared by 2-step centrifugation and stored agitatedly at 22 °C for 7 days in the platelet incubator of blood bank. Levels of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-basic, hepatocyte growth factor (HGF), insulin-like growth factor (IGF)-1, platelet derived growth factor (PDGF)-AB, endothelial growth factor (EGF), and transforming growth factor (TGF) over the period of storage were evaluated daily after freeze-thawing to release growth factors from platelet.
RESULTS:
Compared to original whole blood, platelet concentration, VEGF, FGF-basic, PDGF-AB, EGF, and TGF-beta1 levels of L-PRPs significantly increased after PRP preparation. Both HGF and IGF-1 in L-PRPs remained the original plasma level. Platelet, FGF, and TGF-beta1 concentrations sustained during storage, and concentrations of VEGF, HGF, IGF-1, PDGF-AB, and EGF in L-PRPs increased over the period of storage.
Systematic review and meta-analysis have showed that single-shot L-PRP treatment for tendinopathy had strongly positive effect on pain control at 3 months follow-up [26], and intra-articular PRP treatment for knee osteoarthritis was more efficacious on pain relief and physical function improvement at 12 months follow-up [27]. The growth factors in PRP could be the reason for the effectiveness, because VEGF, PDGF, and TGF-beta are essential in tissue healing process [1]. Our data showed that VEGF, FGF-basic, PDGF-AB, EGF, and TGF-beta1 are released by platelet, and further investigation should be performed to clarify whether and how these growth factors participate in tissue regeneration after PRP injection.
5. Conclusions
PRP injection is a controversial but widely applied medical procedure for tendon tear injury, osteoarthritis, et al. Multiple injections of PRPs have been implicated for sustaining and achieving better regeneration effect [28]. Most of the current PRP separation devices are designed only for a single injection. Our data demonstrated levels of growth factorsmaintained well during 7-day storage in blood bank. Therefore, multiple injections of stored PRPs could become applicable by our protocol.
CONCLUSIONS:
During the storage in blood bank, platelet counts and 7 growth factors sustained or reached higher level than L-PRP obtained on first day. Multiple injections of stored PRPs could become applicable by our protocol.
Clinica Chimica Acta
Volume 483, August 2018, Pages 89-93
Sustained or higher levels of growth factors in platelet-rich plasma during 7-day storage
Keywords
Platelet-rich plasma
VEGF
PDGF-AB
EGF
FGF-basic
TGF-beta 1
1. Introduction
Platelet-rich plasma (PRP) opens a vision for potentially restoring the normal function of tissue following injury or degeneration, and it is widely used by the specialist in the fields of physical medicine and rehabilitation, orthopedics and rheumatology. PRP contains various growth factors and cytokines, which were considered to improve tissue repair and regeneration when delivered to target tissue [1,2]. However, the effectiveness of PRP for treating soft tissue injuries is still controversial [3].
The underlying reasons attributing to the controversial therapeutic effectiveness are complicated. Firstly, precise injection of PRP is crucial for delivering growth factors-rich plasma to the culprit. Several procedures such as ultrasound-guidance, arthroscopy-guidance or anatomical guidance were used. Accuracy was proven to be improved with the use of ultrasound-guided injection compared with anatomical guidance [4,5]. Secondly, in literature, the patient parameters were diverse. In studies of PRP treatment in knee osteoarthritis, the outcome was better in younger individuals with a low degree of cartilage degeneration [6,7]. Studies of different populations could hardly be combined together for generating recommendations of high quality and sufficient evidence. Thirdly, treatment efficacy among various PRP preparation protocols and kits was unable to be compared. Most of them were prepared simply by concentrating in volume and were injected right after preparation in physician offices. No consensus dosage protocols could therefore be established due to lack of platelet or growth factors quantification in advance [8].
Two types of PRPs, pure PRP (P-PRP, or leukocyte-poor PRP) and leukocyte-and PRP (L-PRP), must be differentiated at least before any comparison [9,10]. PRP method and buffy coat method are two common methods for preparing the platelet concentrate in blood bank. Platelet concentrate belongs to L-PRP. In PRP method, the whole blood is centrifuged by soft spin at first to separate and discard red blood cells, and then the supernatant is centrifuged by high speed to concentrate platelet [8]. Whereas in buffy coat method [8], the whole blood is centrifuged at high speed, and the product is divided into three layers: the bottom layer is red blood cells, the middle part is platelet and white blood cells, and the top layer is platelet poor plasma. The middle-buffy coat layer is retrieved.
Reports on growth factors of PRP mainly focused on vascular endothelial growth factor(VEGF), fibroblast growth factor (FGF)-basic, hepatocyte growth factor (HGF), insulin-like growth factor (IGF)-1, platelet derived growth factor (PDGF), endothelial growth factor (EGF), and transforming growth factor (TGF) [11]. VEGF, FGF-basic, and PDGF are related with proangiogenic activity and chemotaxis [12]. HGF is a mitogen for endothelial cells [13]; IGF-1 is a mediator in growth and repair of skeletal muscle, chemotaxis and enhance bone formation [14]. EGF could induce tubule formation, endothelial cell proliferation and migration [13]; TGF also involves in anti-angiogenic activity and chemotaxis [12].
Herein, we produced L-PRP by the PRP method for preparing platelet concentrate according to American Association of Blood Banks (AABB) technical manual [15], and evaluated dynamic concentration changes of VEGF, FGF basic, HGF, IGF-1, PDGF-AB, EGF, and TGF-beta1 in L-PRPs through the 7-day storage at 22 °C with agitation in blood bank.
2. Subjects and methods
2.1. Study subjects and blood collection
Peripheral bloods were collected from 6 healthy volunteer donors (3 males and 3 females, 40 to 56 years old) using a close system consisting of a primary blood bag containing anticoagulant citrate-phosphate-dextrose-adenine (CPDA) -1 and two satellite bags (JMS, Corp.; Singapore). The study was approved by the Ethics Committee of the CGMH (IRB no. 201600506A3), and all study subjects signed an informed consent.
2.2. Platelet-rich plasma preparation and storage
Freshly collected whole blood in the primary bag was centrifuged at 515 g for 5 min (J6-MI, Beckman Coulter Inc.; California, USA) to separate and discard red blood cells, and the supernatant plasma containing platelets was transferred into the first satellite bag. The supernatant plasma was centrifuged again at 3427 g for 6.5 min to concentrate platelets. Supernatant plasma lacking platelets (known as platelet-poor plasma, PPP) was transferred into the second satellite bag, and 30 ml of PRP remained in the first satellite bag. The final L-PRP was kept in the platelet incubator (Model 628, HOTECH Instruments Corp.; Taiwan) with agitation at 22 °C for 7 days in blood bank.
2.3. Hematological analysis and growth factor quantification
Aliquot of 2.5 mL was drawn out of the L-PRP bag each day during its 7-day storage (Table 1). The plasma of original whole blood specimen (day 0) and all the PRP aliquots (day 1 – day7) were analyzed by a hematological analyzer XE-5000 (Sysmex Corp.; Japan). After hematological analysis, specimens (day 0 – day 7) were frozen and stored at −80 °C for the following growth factors quantification in batch.
Table 1
| Day 1a | Day 2a | Day 3a | Day 4a | Day 5a | Day 6a | Day 7a | |
|---|---|---|---|---|---|---|---|
| L-PRP remained (mL) | 30 | 27.5 | 25 | 22.5 | 20 | 17.5 | 15 |
| Aliquot drawn (mL) | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
- a
-
Aliquot of 2.5 mL was drawn from the blood bag immediately after PRP preparation on day 1 and another aliquot of 2.5 mL was drawn from each blood bag every 24 h during the storage period. L-PRP, leukocyte- and platelet-rich plasma.
Growth factors could be released from platelets by deep freeze-thawing [16]. All frozen stocked specimens were simply thawed in room temperature and then subjected for quantifying growth factors. We used Quantikine ELISA Kits (R&D Diagnostics; Minnesota, USA) to quantify VEGF (Cat. #DVE00), FGF basic (#DFB50), HGF (#DHG00), IGF-1(#DG100), PDGF-AB (#DHD00C), EGF (#DEG00), and TGF-beta1 (#DB100B). Procedures were carried out following the manufacturer’s instructions.
2.4. Statistical analysis
Paired Student’s t-test was used to analyze for significant differences of growth factor levels between the original plasma (day 0) and PRP (day 1). Shapiro-Wilk original test was performed for normality of the data. Univariate approach of a repeated measures analysis of variance (rANOVA) with post-hoc Tukey’s honestly significant difference (HSD) test was used to determine whether platelet counts and growth factor levels changed significantly throughout the storage period (p < 0.05 was considered statistically significant). Mauchly’s sphericity test was used to validate the sphericity (an important assumption of rANOVA). If sphericity is violated, Huynh-Feldt correction (when ε ≥ 0.75) or Greenhouse-Geisser correction (when ε < 0.75) will be applied. Statistical analysis was performed using Microsoft Office Excel 2007 (Microsoft Inc.; Washington, USA) and SPSS Statistics 18.0 (IBM Corp.; New York, USA).
3. Results
Total 153 to 170 mL whole blood were collected and processed from each of the six healthy volunteers (Table 2). Their platelet concentrations were all above 150,000/μL. Thirty milliliters of PRP were prepared from each donation by the two-step centrifugation. Final concentrations of PRPs became 1.6 to 5.7 folds of their original concentration (Table 2). The WBC reduction rates after PRP preparation were all above 83.0%. Platelet counts of PRPs remained unchanged during storage at 22 °C with agitation for 7 days (Table 3); statistically different platelet concentrations were not observed among storage days (rANOVA, p-value = 0.152).
Table 2
| Sample | Whole blood | Platelet-rich plasma | ||||||
|---|---|---|---|---|---|---|---|---|
| Volume (mL) | WBC (1000/uL) | Platelet (1000/uL) | Volume (mL) | WBC (1000/uL) | Platelet (1000/uL) | Yield (folds) | WBC reductiona(%) | |
| 1 | 165 | 3.05 | 218 | 30 | 2.86 | 1232 | 5.7 | 83.0 |
| 2 | 170 | 5.54 | 212 | 30 | 2.92 | 953 | 4.5 | 90.7 |
| 3 | 153 | 6.30 | 221 | 30 | 0.40 | 354 | 1.6 | 98.8 |
| 4 | 163 | 4.42 | 279 | 30 | 0.15 | 829 | 3.0 | 99.4 |
| 5 | 165 | 5.01 | 236 | 30 | 3.86 | 978 | 4.1 | 86.0 |
| 6 | 170 | 6.42 | 278 | 30 | 4.57 | 897 | 3.2 | 87.4 |
- a
-
WBC reduction was calculated as: [(WBC of whole blood x volume of whole blood – WBC of platelet-rich plasma x volume of platelet-rich plasma)/(WBC of whole blood x volume of whole blood)] × 100. WBC, white blood cell.
Table 3
| Platelet count (1000/uL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
|---|---|---|---|---|---|---|---|---|
| Sample 1 | 218 | 1232 | 1203 | 1255 | 1235 | 1277 | 1179 | 1251 |
| Sample 2 | 212 | 953 | 971 | 970 | 1047 | 1004 | 951 | 801 |
| Sample 3 | 221 | 354 | 378 | 396 | 381 | 377 | 405 | 332 |
| Sample 4 | 279 | 829 | 1002 | 927 | 951 | 1011 | 984 | 956 |
| Sample 5 | 236 | 978 | 1006 | 986 | 1000 | 1023 | 1067 | 1028 |
| Sample 6 | 278 | 897 | 966 | 906 | 911 | 976 | 932 | 885 |
Plasma VEGF levels of original whole blood specimens (day 0) ranged from 40.3 to 86.0 pg/mL (Table 4A). After centrifuge preparation and deep freezing procedure, the mean VEGF level of PRPs increased significantly from 300.0 pg/mL to 511.9 pg/mL on day 1 (t-test, p < 0.001). Statistically significant differences (rANOVA, p < 0.023) were observed among storage days, and post-hoc Tukey HSD test (k = 7, n = 6, α = 0.05) showed VEGF level was statistically significant increasing between day 1 and day 2, day 2 and day 3, day 4 and day 5, and between day 6 and day 7.
Table 4
| (A) | ||||||||
|---|---|---|---|---|---|---|---|---|
| VEGF (pg/mL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| Sample 1 | 40.3 | 324.6 | 372.1 | 504.9 | 569.9 | 645.2 | 741.6 | 813.1 |
| Sample 2 | 52.6 | 511.9 | 628.5 | 771.8 | 882.0 | 1012.5 | 1039.1 | 1175.7 |
| Sample 3 | 86.0 | 448.3 | 480.8 | 521.9 | 469.3 | 483.7 | 477.9 | 535.8 |
| Sample 4 | 61.7 | 300.0 | 310.6 | 329.2 | 364.9 | 374.9 | 435.5 | 515.9 |
| Sample 5 | 70.7 | 311.8 | 391.8 | 493.8 | 774.9 | 972.3 | 1246.1 | 1284.2 |
| Sample 6 | 60.8 | 329.9 | 378.8 | 397.6 | 481.8 | 628.2 | 633.5 | 714.7 |
| (B) | ||||||||
|---|---|---|---|---|---|---|---|---|
| FGF-basic (pg/mL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| Sample 1 | 30.4 | 192.8 | 235.8 | 244.0 | 230.7 | 189.9 | 203.3 | 220.6 |
| Sample 2 | 6.0 | 192.3 | 221.1 | 206.3 | 201.7 | 147.7 | 168.5 | 169.0 |
| Sample 3 | 4.8 | 12.3 | 13.9 | 11.7 | 11.9 | 14.3 | 13.0 | 18.1 |
| Sample 4 | 32.8 | 94.3 | 102.2 | 80.4 | 107.8 | 86.3 | 85.8 | 112.6 |
| Sample 5 | 23.8 | 136.0 | 138.6 | 156.7 | 159.3 | 119.2 | 122.4 | 139.2 |
| Sample 6 | 40.3 | 90.3 | 105.9 | 108.2 | 114.9 | 125.9 | 114.6 | 154.7 |
| (C) | ||||||||
|---|---|---|---|---|---|---|---|---|
| HGF (pg/mL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| Sample 1 | 553 | 557 | 556 | 578 | 579 | 622 | 637 | 670 |
| Sample 2 | 632 | 572 | 561 | 579 | 642 | 665 | 671 | 744 |
| Sample 3 | 600 | 511 | 515 | 516 | 526 | 531 | 536 | 525 |
| Sample 4 | 658 | 571 | 603 | 636 | 650 | 645 | 668 | 668 |
| Sample 5 | 576 | 669 | 674 | 690 | 707 | 736 | 860 | 893 |
| Sample 6 | 501 | 629 | 632 | 697 | 749 | 692 | 705 | 787 |
| (D) | ||||||||
|---|---|---|---|---|---|---|---|---|
| IGF-1 (ng/mL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| Sample 1 | 60.4 | 62.6 | 66.7 | 66.4 | 70.9 | 69.5 | 69.9 | 69.9 |
| Sample 2 | 61.4 | 66.4 | 69.3 | 80.1 | 77.0 | 76.3 | 75.4 | 85.4 |
| Sample 3 | 88.0 | 92.9 | 106.0 | 109.9 | 112.8 | 92.1 | 111.6 | 115.3 |
| Sample 4 | 69.5 | 73.7 | 84.4 | 84.2 | 87.8 | 89.7 | 79.1 | 90.0 |
| Sample 5 | 46.5 | 44.7 | 48.6 | 48.3 | 54.5 | 57.0 | 59.3 | 58.2 |
| Sample 6 | 67.7 | 64.0 | 65.4 | 76.9 | 80.8 | 89.7 | 89.4 | 84.4 |
| (E) | ||||||||
|---|---|---|---|---|---|---|---|---|
| PDGF-AB (pg/mL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| Sample 1 | 1763 | 22,398 | 24,599 | 32,405 | 34,421 | 40,580 | 45,288 | 47,866 |
| Sample 2 | 1247 | 25,594 | 29,347 | 35,522 | 41,818 | 47,370 | 45,774 | 54,714 |
| Sample 3 | 3654 | 21,164 | 21,572 | 22,339 | 24,601 | 25,211 | 25,835 | 27,225 |
| Sample 4 | 7939 | 30,016 | 31,595 | 35,640 | 38,289 | 42,281 | 42,543 | 46,047 |
| Sample 5 | 5799 | 39,668 | 38,371 | 42,206 | 49,505 | 47,513 | 60,367 | 70,111 |
| Sample 6 | 5163 | 28,211 | 29,738 | 32,782 | 34,066 | 33,709 | 35,241 | 43,307 |
| (F) | ||||||||
|---|---|---|---|---|---|---|---|---|
| EGF (pg/mL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| Sample 1 | 111 | 1574 | 1797 | 2079 | 2488 | 2845 | 3202 | 3436 |
| Sample 2 | 25 | 1541 | 1844 | 1955 | 2371 | 2752 | 2781 | 3265 |
| Sample 3 | 219 | 1430 | 1561 | 1593 | 1684 | 1630 | 1846 | 1709 |
| Sample 4 | 350 | 2526 | 2642 | 2740 | 2915 | 2992 | 3236 | 3529 |
| Sample 5 | 202 | 1771 | 1634 | 1768 | 1829 | 1720 | 2687 | 3290 |
| Sample 6 | 280 | 1643 | 1818 | 1948 | 1980 | 2394 | 2391 | 2528 |
| (G) | ||||||||
|---|---|---|---|---|---|---|---|---|
| TGF-beta1 (pg/mL) | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
| Sample 1 | 20,570 | 111,476 | 139,439 | 112,362 | 108,320 | 126,698 | 146,100 | 133,768 |
| Sample 2 | 9489 | 152,715 | 151,707 | 197,058 | 159,830 | 144,681 | 153,505 | 169,248 |
| Sample 3 | 31,875 | 59,534 | 69,489 | 80,697 | 75,371 | 59,675 | 64,804 | 66,148 |
| Sample 4 | 80,563 | 131,598 | 135,523 | 118,904 | 134,416 | 145,182 | 138,204 | 151,295 |
| Sample 5 | 43,332 | 176,103 | 138,485 | 160,110 | 138,033 | 169,594 | 147,262 | 151,166 |
| Sample 6 | 52,358 | 107,363 | 140,779 | 105,940 | 147,034 | 138,582 | 102,993 | 100,092 |
Plasma FGF levels of original whole blood specimens (day 0) ranged from 4.8 to 40.3 pg/mL (Table 4B). After centrifuge preparation and deep freezing procedure, the mean FGF basic level of PRPs increased significantly from 12.3 pg/mL to a plateau level of 192.8 pg/mL on day 1 (t-test, p < 0.001). No statistically significant difference (rANOVA, p = 0.146) was observed among storage days.
Plasma HGF levels of original whole blood specimens (day 0) ranged from 501 to 658 pg/mL (Table 4C). After centrifuge preparation and deep freezing procedure, the mean HGF level of PRPs remained as the similar level of original plasma (from 511 to 669 pg/mL on day 1, t-test, p = 0.9609). However, statistically significant differences (rANOVA, p = 0.004) were observed among storage days, and post-hoc Tukey HSD test (k = 7, n = 6, α = 0.05) showed HGF level was statistically significant increasing only between day 2 and day 3.
Plasma IGF-1 levels of original whole blood specimens (day 0) ranged from 46.5 to 88.0 ng/mL (Table 4D). The mean IGF-1 level of PRPs showed the same pattern as HGF after centrifuge preparation and deep freezing procedure. However, statistically significant differences (rANOVA, p = 0.009) were observed among storage days, and post-hoc Tukey HSD test (k = 7, n = 6, α = 0.05) showed IGF-1 level was statistically significant increasing only between day 1 and day 2.
Plasma PDGF-AB levels of original whole blood specimens (day 0) ranged from 1247 to 7939 pg/mL (Table 4E). After centrifuge preparation and deep freezing procedure, the mean PDGF-AB level of PRPs increased significantly from 21,164 pg/mL to 39,668 pg/mL on day 1 (t-test, p<0.001). Statistically significant differences (rANOVA, p = 0.001) were observed among storage days, and post-hoc Tukey HSD test (k = 7, n = 6, α = 0.05) showed PDGF-AB level was statistically significant increasing between day 2 and day 3, day 3 and day 4, and between day 6 and day 7.
Plasma EGF levels of original whole blood specimens (day 0) ranged from 25 to 350 pg/mL (Table 4F). After centrifuge preparation and deep freezing procedure, the mean EGF level of PRPs increased significantly from 1430 pg/mL to 2526 pg/mL on day 1 (t-test, p < 0.001). Statistically significant differences (rANOVA, p = 0.001) were observed among storage days, and post-hoc Tukey HSD test (k = 7, n = 6, α = 0.05) showed EGF level was statistically significant increasing between day 2 and day 3, and between day 3 and day 4.
The mean TGF-beta1 level of PRPs showed the same pattern as FGF basic after centrifuge preparation and deep freezing procedure (Table 4G). Plasma TGF-beta1 levels of original whole blood specimens (day 0) ranged from 9689 to 80,563 pg/mL. After centrifuge preparation and deep freezing procedure, the mean TGF-beta1 level of PRPs increased significantly from 59,534 pg/mL to a plateau level of 176,103 pg/mL on day 1 (t-test, p < 0.001). No statistically significant difference (rANOVA, p = 0.924) was observed among storage days.
4. Discussion
Our observation revealed VEGF, FGF-basic, PDGF-AB, EGF, and TGF-beta1 were most likely platelet-released factors after freeze-thawing. Several studies measured growth factorsof PRP after platelet activation and releasing growth factors. Paola et al. considered FGF basic was a plasmatic factor and VEGF was not detectable in L-PRP [17], and they prepared L-PRP by activation at 37 °C for 1 h and storage at 4 °C for 16 h. Hesham, et al. [18] and Barry, et al. [19] activated PRP shortly for 10 min and then centrifuged 5 min before measuring growth factors. Hesham, et al. presented significantly higher levels of FGF basic after activation. Both studies showed detectable and significantly increased VEGF after activation. Conflict results on VEGF may be resulted from delayed measurement after activation, VEGF is a fragile factor sensitive to temperature [20]. Our data may be more convincible because all growth factors were measured immediately after freeze-thawing procedure.
HGF and IGF-1 were considered as plasma factors because they presented consistent levels throughout the centrifugation preparation and freeze-thaw procedure. Although HGF increased to 2.5-fold of the original whole blood after L-PRP preparation in the previous report, its increment was not significant statistically [17]. IGF-1 was also recognized as a plasmatic factor by Paola, et al. [17] and Barry et al. [19], whereas Hesham, et al. showed L-PRP had significantly higher levels of IGF-1 than the original whole blood [18]. Hesham, et al. proposed that IGF-1 was released by other cell types rather than platelets, because IGF-1 levels of PRP and PPP were not different significantly.
PDGF-AB, TGF beta1, and EGF in PRP have been widely investigated in literature, and they were quantified in terms of the secreted amount per platelet. Our L-PRP contained 18.2 to 59.8 pg PDGF-AB, 90.5 to 180.1 pg TGF beta1, and 1278 to 4039 fg EGF per 106 platelets. Paola, et al. [17] reported that 22.7 to 52.5 pg PDGF-AB were secreted by 106 platelets, and other authors reported similar values: 27.4 to 48.8 pg/106 platelets [21], and 21.9 to 44.1 pg/106 platelets [22]. TGF beta1 secreted by 106 platelets in L-PRP was also similar in literature including 55.4 to 126.7 pg [23], 161.7 to 185.4 pg [21], and 200.2 to 437.0 pg [17]. However, EGF secreted by 106 platelets in L-PRP was greatly variable in past studies: 243.2 to 633.8 fg/106 platelets [17] and 1498.1 to 3020.7 fg/106 platelets [21].
During the storage at 22 °C with agitation for 7 days, platelet counts and 7 growth factors sustained or reached higher level than L-PRP obtained on first day. However, Gary, et al. [24] demonstrated that reduction of platelet counts and PDGF-BB in L-PRP which were stored on shaker at room temperature over an 8-day period. Because viability of platelets remains well at 22 °C with agitation for 7 days [25], their content of growth factors were probably maintained as well based on our observation. Taken together, L-PRP should be preserved no >7 days at 22 °C with agitation for effective usage.
In the results of VEGF, HGF, PDGF-AB, and EGF, their concentrations in sample 5 rapidly increased, when compared to other samples. Because of limited sample size, further investigation should be performed and include more subjects to elucidate underlying causes. In our study, sample size had enough statistical power (>0.8) in paired Student’s t-test to evaluate whether there were significant differences of growth factor levels between the original plasma (day 0) and PRP (day1). However, rANOVA had poor statistical power (<0.8) to support significant differences of platelet counts and growth factor levels throughout the storage period. Therefore, more samples should be included to increase the statistical power in determining whether concentrations of VEGF, HGF, IGF-1, PDGF-AB, and EGF would increase over the period of storage.
Systematic review and meta-analysis have showed that single-shot L-PRP treatment for tendinopathy had strongly positive effect on pain control at 3 months follow-up [26], and intra-articular PRP treatment for knee osteoarthritis was more efficacious on pain relief and physical function improvement at 12 months follow-up [27]. The growth factors in PRP could be the reason for the effectiveness, because VEGF, PDGF, and TGF-beta are essential in tissue healing process [1]. Our data showed that VEGF, FGF-basic, PDGF-AB, EGF, and TGF-beta1 are released by platelet, and further investigation should be performed to clarify whether and how these growth factors participate in tissue regeneration after PRP injection.
5. Conclusions
PRP injection is a controversial but widely applied medical procedure for tendon tear injury, osteoarthritis, et al. Multiple injections of PRPs have been implicated for sustaining and achieving better regeneration effect [28]. Most of the current PRP separation devices are designed only for a single injection. Our data demonstrated levels of growth factorsmaintained well during 7-day storage in blood bank. Therefore, multiple injections of stored PRPs could become applicable by our protocol.
Conflicts of interest
None.
Research funding
None declared.
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Stability of umbilical cord blood-platelet lysate
In order to assay for stability, different batches of UCB-PL were stored at -70˚C for nine months after which we analyzed the concentrations of PDGF- AB, TGF-β1, IGF-I, and bFGF. Because the freeze/ thaw cycles affect the absolute concentrations of cytokines, we processed the samples in such a way that they did not differ in the number of freeze/ thaw cycles. -
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Table 2
Concentration of major growth factors in umbilical cord blood-platelet lysate (UCB-PL) and peripheral blood platelet lysate (PB-PL)
Sample bFGF (ng/ml) TGF-β1(ng/ml) IGF-1(ng/ml) PDGF-AB (ng/ml) Total protein (mg/ml) UCB-PL PB-PL UCB-PL PB-PL UCB-PL PB-PL UCB-PL PB-PL UCB-PL PB-PL
1 0.066 0.072 55.38 29.75 470.96 320.61 675.03 372.8 74.85 42 2 0.054 0.0737 51.36 31.39 436.00 322.36 627.13 463.4 65.33 30 3 0.057 0.040 56.64 38.59 505.93 343.34 638.29 332.6 73.65 55 4 0.063 0.059 58.204 27.98 575.86 327.60 553.81 318.8 62.45 44 5 0.054 0.045 51.63 18.85 558.38 313.62 580.02 392.6 91.225 45 6 0.066 0.044 ND ND 487 ND 490.91 493.4 85.6 82 7 0.054 0.072 ND ND 477.2 ND 480.63 367.8 67.8 46 Mean ± SD 0.059± 0.005 0.058± 0.014 56.04± 3.45 29.31± 7.102 501.62± 49.67 325.50± 11.153 577.97± 74.29 391.62± 64.83 90.46± 6.21 49.14 ± 16.25 P value 0.854 0.0008 0.002 0.004 0.0006
bFGF; Basic fibroblast growth factor, TGF-β1; Transforming growth factor-beta1; IGF-1; Insulin growth factor-I, and PDGF-AB; Plateletderived growth factor-AB. -
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Other notes:
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5. Plasma IGF-1 levels of original whole blood specimens (day 0) ranged from 46.5 to 88.0 ng/mL (Table 4D). The mean IGF-1 level of PRPs showed the same pattern as HGF after centrifuge preparation and deep freezing procedure. However, statistically significant differences (rANOVA, p = 0.009) were observed among storage days, and post-hoc Tukey HSD test (k = 7, n = 6, α = 0.05) showed IGF-1 level was statistically significant increasing only between day 1 and day 2.HGF and IGF-1 were considered as plasma factors because they presented consistent levels throughout the centrifugation preparation and freeze-thaw procedure. Although HGF increased to 2.5-fold of the original whole blood after L-PRP preparation in the previous report, its increment was not significant statistically [17]. IGF-1 was also recognized as a plasmatic factor by Paola, et al. [17] and Barry et al. [19], whereas Hesham, et al. showed L-PRP had significantly higher levels of IGF-1 than the original whole blood [18]. Hesham, et al. proposed that IGF-1 was released by other cell types rather than platelets, because IGF-1 levels of PRP and PPP were not different significantly.Kabiri et al. reported that the PRP has rich concentration of a few GFs like IGF