scholarly journals The optimal platelet concentration in platelet-rich plasma for proliferation of human cells in vitro—diversity, biases, and possible basic experimental principles for further research in the field: A review

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10303
Author(s):  
Olav K. Straum
Keyword(s):  
Human Cell ◽  
Platelet Rich Plasma ◽  
Human Cells ◽  
Proliferation Rate ◽  
In Vitro Studies ◽  
General Tendency ◽  
Platelet Concentration ◽  
Volume Group

Background In the last decades, several in vitro studies have tested the effect of plate-rich plasma (PRP) on the proliferation of human cells in search of a wizard for the use of PRP in a clinical setting. However, the literature displays striking differences regarding this question despite the relatively similar experimental design. The aim of this review is twofold: describe and explain this diversity and suggest basic principles for further in vitro studies in the field. The optimal platelet concentration in vivo will also be discussed. Methods A search in mainly EMBASE and PubMed was performed to identify in vitro studies that investigate the effect of different PRP concentrations on human cell proliferation. The assessment of bias was based on the principles of “Good Cell Culture Practice” and adapted. Results In total, 965 in vitro studies were detected. After the initial screening, 31 studies remained for full-text screening. A total of 16 studies met the criteria of final inclusion and appeared relatively sound. In general, the studies state consistently that PRP stimulates the proliferation of the human cell. Two main types of experimental techniques were detected: 1. The Fixed PRP Concentration Group using a fixed PRP concentration throughout the experiment, which leads to a substantial decrease in nutrition available at higher concentrations. 2. The Fixed PRP Volume Group using a fixed PRP-to-media ratio (Vol/Vol) throughout the experiment. A general tendency was observed in both groups: when the PRP to media ratio increased (Vol/Vol), the proliferation rate decreased. Further, The Low Leukocyte group observed a substantial higher optimal PRP concentration than The High leukocyte group. No prominent tendencies was seen regarding anticoagulants, activation methods, and blood donor (age or sex). Discussion Two major biases regarding optimal proliferation in vitro is pointed out: 1. Too high PRP volume. It is speculated that the techniques used by some studies led to an adverse growth condition and even cell starvation at higher concentrations. 2. High leukocyte levels. Reduced proliferation rate due to proinflammatory substances released during degranulation of leukocytes. Conclusions The two main biases may explain the bell-shaped effect of PRP and the detrimental effects at higher platelet concentrations observed in several studies. These biases may also explain the low optimal PRP concentration observed in some studies. Even if one universal optimal PRP concentration does not exist, the review indicates that PRP concentrations in the upper parts of the scale is optimal or at least beneficial. Finally, following basic experimental principles are suggested. 1: The PRP/media ratio (Vol/Vol) should be kept as constant. 2: The PRP/media ratio should provide a sufficient nutrition supply, that is, PRP ≤ 10% (Vol/Vol). 3: The cell density per well (cells/mL) should be defined. 4: Leukocyte level should be kept low, preferable depleted (< 0.1 PLT/µL).

scholarly journals Systematic Review—The Potential Implications of Different Platelet-Rich Plasma (PRP) Concentrations in Regenerative Medicine for Tissue Repair

2020 ◽  
Vol 21 (16) ◽  
pp. 5702 ◽  
Author(s):  
Pietro Gentile ◽  
Simone Garcovich
Keyword(s):  
Systematic Review ◽  
Tissue Regeneration ◽  
Tissue Repair ◽  
Platelet Rich Plasma ◽  
Platelet Concentration ◽  
Volume Group ◽  
Scopus Database ◽  
Meta Analyses

The number of studies evaluating platelet-rich plasma (PRP) concentration has substantially grown in the last fifteen years. A systematic review on this field has been realized by evaluating in the identified studies the in vitro PRP concentration—also analyzing the platelet amount—and the in vivo PRP effects in tissue regeneration compared to any control. The protocol has been developed in agreement with the Preferred Reporting for Items for Systematic Reviews and Meta-Analyses-Protocols (PRISMA-P) guidelines. Multistep research of the PubMed, MEDLINE, Embase, PreMEDLINE, Ebase, CINAHL, PsycINFO, Clinicaltrials.gov, Scopus database and Cochrane databases has permitted to identify articles on different concentrations of PRP in vitro and related in vivo impact for tissue repair. Of the 965 articles initially identified, 30 articles focusing on PRP concentration have been selected and, consequently, only 15 articles have been analyzed. In total, 40% (n = 6) of the studies were related to the fixed PRP Concentration Group used a fixed PRP concentration and altered the platelet concentration by adding the different volumes of the PRP (lysate) to the culture. This technique led to a substantial decrease in nutrition available at higher concentrations. Sixty percent (n = 9) of the studies were related to the fixed PRP Volume Group that used a fixed PRP-to-media ratio (Vol/Vol) throughout the experiment and altered the concentration within the PRP volume. For both groups, when the volume of medium (nutrition) decreases, a lower rate of cell proliferation is observed. A PRP concentration of 1.0 × 106 plt/μL, appears to be optimal thanks to the constant and plentiful capillary nutrition supply and rapid diffusion of growth factors that happen in vivo and it also respects the blood decree-law. The PRP/media ratio should provide a sufficient nutrition supply to prevent cellular starvation, that is, PRP ≤ 10% (Vol/Vol) and thus best mimic the conditions in vivo.

Transplantation of Adipose-derived Cells for Periodontal Regeneration: A Systematic Review

2019 ◽  
Vol 14 (6) ◽  
pp. 504-518 ◽  
Author(s):  
Dilcele Silva Moreira Dziedzic ◽  
Bassam Felipe Mogharbel ◽  
Priscila Elias Ferreira ◽  
Ana Carolina Irioda ◽  
Katherine Athayde Teixeira de Carvalho
Keyword(s):  
Systematic Review ◽  
Animal Models ◽  
Platelet Rich Plasma ◽  
Periodontal Regeneration ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Cell Sources ◽  
Study Designs

This systematic review evaluated the transplantation of cells derived from adipose tissue for applications in dentistry. SCOPUS, PUBMED and LILACS databases were searched for in vitro studies and pre-clinical animal model studies using the keywords “ADIPOSE”, “CELLS”, and “PERIODONTAL”, with the Boolean operator “AND”. A total of 160 titles and abstracts were identified, and 29 publications met the inclusion criteria, 14 in vitro and 15 in vivo studies. In vitro studies demonstrated that adipose- derived cells stimulate neovascularization, have osteogenic and odontogenic potential; besides adhesion, proliferation and differentiation on probable cell carriers. Preclinical studies described improvement of bone and periodontal healing with the association of adipose-derived cells and the carrier materials tested: Platelet Rich Plasma, Fibrin, Collagen and Synthetic polymer. There is evidence from the current in vitro and in vivo data indicating that adipose-derived cells may contribute to bone and periodontal regeneration. The small quantity of studies and the large variation on study designs, from animal models, cell sources and defect morphology, did not favor a meta-analysis. Additional studies need to be conducted to investigate the regeneration variability and the mechanisms of cell participation in the processes. An overview of animal models, cell sources, and scaffolds, as well as new perspectives are provided for future bone and periodontal regeneration study designs.

scholarly journals Centrifugation Conditions in the L-PRP Preparation Affect Soluble Factors Release and Mesenchymal Stem Cell Proliferation in Fibrin Nanofibers

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2729 ◽  
Author(s):  
Melo ◽  
Luzo ◽  
Lana ◽  
Santana
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
Clinical Practices ◽  
Soluble Factors ◽  
Tnf Α ◽  
Platelet Concentration ◽  
Tgf Β1

Leukocyte and platelet-rich plasma (L-PRP) is an autologous product that when activated forms fibrin nanofibers, which are useful in regenerative medicine. As an important part of the preparation of L-PRP, the centrifugation parameters may affect the release of soluble factors that modulate the behavior of the cells in the nanofibers. In this study, we evaluated the influences of four different centrifugation conditions on the concentration of platelets and leukocytes in L-PRP and on the anabolic/catabolic balance of the nanofiber microenvironment. Human adipose-derived mesenchymal stem cells (h-AdMSCs) were seeded in the nanofibers, and their viability and growth were evaluated. L-PRPs prepared at 100× g and 100 + 400× g released higher levels of transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-BB due to the increased platelet concentration, while inflammatory cytokines interleukin (IL)-8 and tumor necrosis factor (TNF)-α were more significantly released from L-PRPs prepared via two centrifugation steps (100 + 400× g and 800 + 400× g) due to the increased concentration of leukocytes. Our results showed that with the exception of nanofibers formed from L-PRP prepared at 800 + 400× g, all other microenvironments were favorable for h-AdMSC proliferation. Here, we present a reproducible protocol for the standardization of L-PRP and fibrin nanofibers useful in clinical practices with known platelet/leukocyte ratios and in vitro evaluations that may predict in vivo results.

Comparison Of The Effects Of Aspirin In Humans Ex Vivo In Platelet-Rich Plasma And Whole Blood

1981 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Trisodium Citrate ◽  
Blood Platelet ◽  
Maximal Response ◽  
Blood Samples ◽  
Platelet Concentration

The effect of aspirin on human platelet function is usually assessed using platelet-rich plasma (PRP). Some preliminary results in vitro suggested that the effect of aspirin appears to be greater in PRP than whole blood. To explore this possibility further, a comparison of the effect of aspirin in humans ex vivo has been made taking measurements simultaneously in whole blood and PRP at 2 platelet concentrations. Blood samples (36ml) were drawn from 7 male volunteers after a light breakfast. Each took 300mg soluble aspirin and blood samples were drawn again 2 hours later. Blood was mixed with 0.1 volumes 129nM trisodium citrate. Some (30ml) was then centrifuged to prepare PRP and platelet -poor plasma (PPP) by standard techniques. Platelet concentration of some PRP was adjusted with PPP to equal that of the corresponding blood sample; the rest was adjusted to 350,000 per μl. Aggregation in response to collagen (Horm, Munich) was measured photometrically at 37°. Aggregation in 0.5ml aliquots of whole blood was measured after 4 min stirring with 154mM NaCl (control) or collagen at 37° as the fall in single platelet count determined using an Ultraflo- 100 whole blood platelet counter (Clay Adams). The concentrations of collagen producing a 50% maximal response (EC50) in PRP and blood were determined. Dose-ratios for each volunteer were calculated by dividing the EC50 obtained after aspirin by the corresponding value obtained before aspirin.The effect of aspirin was significantly (p<0.001) less in blood than PRP. Whether or not the results in whole blood more closely reflect the effect of aspirin in vivo remains to be determined.

Recovery Of Platelet Function After Aspirin Ingestion: In Vivo And In Vitro Studies

1981 ◽  
Author(s):  
B A Bradlow ◽  
N Chetty ◽  
M Birnbaum
Keyword(s):  
Platelet Function ◽  
Recovery Of Function ◽  
Platelet Rich Plasma ◽  
Recovery Period ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Partial Recovery ◽  
Mean Values

Blood was taken from normal volunteers before and two hours after a 600mg oral dose of aspirin (ASA). Platelet rich plasma (PRP) was prepared from both samples. Mixtures of normal and ASA treated PRP were tested for aggregation responses and for production of thromboxane B2 (TXB2) by RIA (New England Nuclear). Normal aggregation responses were found when the mixture contained 26% of normal platelets (NP) with ADP, 40% NP with adrenalin, 54% NP with collagen and 52% NP with arachidonic acid (AA)(Mean Values). TXB2 production in the mixtures increased when the proportion of normal platelets was raised above 10 to 30%. These findings indicate partial restoration of platelet function by an admixture of 10-20% NP to ASA treated platelets although 50% NP was necessary to restore all functions tested. In vivo studies on seven volunteers given 300mg ASA showed partial recovery of malondialdehyde production after 72 hours, partial return of aggregation responses to collagen, ADP, adrenalin and AA at 96 hours and partial or full return of aggregation responses in all subjects by 120 hours. Since new platelets enter the circulation at the rate of 10-15% per day the results of our in vitro studies suggest that some recovery of platelet function might occur as soon as 24 hours after a dose of ASA and full recovery within 3-5 days. The longer recovery period indicated by the in vivo studies may be attributable to the effect of ASA on megakaryocyte cyclooxygenase.Conclusion: ASA administered once every 24 hours may be sufficient to suppress platelet function adequately and continuously but partial recovery of function might occur 48-72 hours after ASA ingestion.

scholarly journals Platelet Concentration in Platelet-Rich Plasma Affects Tenocyte BehaviorIn Vitro

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Ilaria Giusti ◽  
Sandra D’Ascenzo ◽  
Annalisa Mancò ◽  
Gabriella Di Stefano ◽  
Marianna Di Francesco ◽  
...  
Keyword(s):  
Mechanical Stability ◽  
Platelet Rich Plasma ◽  
Tendon Healing ◽  
Inhibitory Effects ◽  
Global Effect ◽  
Platelet Concentration ◽  
Normal Human ◽  
New Strategies

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 106 plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 106, 1 × 106 plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, thesein vitrodata strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducingin vivotendon healing.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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