A possible injectable tissue engineered nucleus pulposus constructed with platelet-rich plasma and ADSCs in vitro

Background. Platelet-rich plasma (PRP) is a promising strategy for intervertebral disc degeneration. However, the potential harmful effects of leukocytes in PRP on nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have seldom been studied. This study aimed at comparatively evaluating effects of pure platelet-rich plasma (P-PRP) and leukocyte-containing platelet-rich plasma (L-PRP) on rabbit NPMSCs in vitro. Methods. NPMSCs isolated from rabbit NP tissues were treated with L-PRP or P-PRP in vitro, and then cell proliferation and expression of stem cell markers, proinflammatory cytokines (TNF-α, IL-1β), production of ECM (extracellular matrix-related protein), and NF-κB p65 protein were validated by CCK-8 assay, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and western blot respectively. Results. NPMSCs differentiate into nucleus pulposus-like cells after treatment of PRPs (P-PRP and L-PRP), and NPMSCs exhibited maximum proliferation at a 10% PRP dose. L-PRP had observably higher concentration of leukocytes, TNF-α, and IL-1β than P-PRP. Furthermore, compared to P-PRP, L-PRP induced the differentiated NPMSCs to upregulate the expression of TNF-α and IL-1β, enhanced activation of the NF-κB pathway, increased the expression of MMP-1 and MMP-13, and produced less ECM in differentiated NPMSCs. Conclusions. Both P-PRP and L-PRP can induce the proliferation and NP-differentiation of NPMSCs. Compared to L-PRP, P-PRP can avoid the activation of the NF-κB pathway, thus reducing the inflammatory and catabolic responses.

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A possible Injectable Tissue Engineered Nucleus Pulposus constructed with platelet-rich plasma and ADSCs in Vitro

10.21203/rs.3.rs-20257/v1 ◽

2020 ◽

Author(s):

Zhicheng Zhang ◽

Jian Ma ◽

Dajiang Ren ◽

Fang Li

Keyword(s):

Nucleus Pulposus ◽

Platelet Rich Plasma ◽

Positive Staining ◽

Adipose Derived Stromal Cells ◽

Genes Expression ◽

Significant Difference ◽

Safranin O ◽

Collagen Genes ◽

Prp Gel

Abstract Background: Injectable tissue engineered nucleus pulposus is a new idea for minimally invasive repair for degenerative intervertebral disc. The platelet-rich plasma (PRP) and adipose derived stromal cells(ADSCs) could be harvested from autologous tissue easily. PRP has mixed autologous growth factors and fibrous reticulate structure which may have the potential to make ADSCs differentiated into nucleus pulposus-like cells. The goal of this study was to explore the feasibility of constructing a possible injectable tissue engineered nucleus pulposus with PRP gel scaffold and ADSCs. Methods: After the rabbit ADSCs were identified with flow cytometry, the ADSCs were seeded into PRP gel and cultured in vitro. At the 2nd, 4th and 8th week, the PRP gel/ADSCs complex was observed by macroscopy, histological staining, BrdU immunofluorescence, and scanning electron microscopy. The glycosaminoglycans (GAG) in the PRP gel/ADSCs complex were measured by safranin O staining with spectrophotometry. PRP gel/ADSCs complex genes expression of HIF-1α, Aggrecan, Type Ⅱ collagen were detected by RT-PCR. The injectability of this complex was tested. Results: Macroscopically, the complex was solidified into gel with smooth surface and good elasticity. The safranin O staining confirmed almost no positive staining at 2nd week, however, the positive staining of extracellular matrix was enhanced obviously at 4th, 8th week. The HE staining and SEM demonstrated that the cells were well-distributed in the reticulate scaffold. BrdU immunofluorescence showed that ADSCs can survive and proliferate in PRP gel at each time points. The level of GAG at 4th week was higher than those at 2nd week (P<0.05), and significant difference was also noted between 4th and 8th week (P<0.05). HIF-1α, Aggrecan, Type Ⅱ collagen genes expression at 4th week were much more than those at 2nd week (P<0.05), and significant differences were also noted between 4th and 8th week (P<0.05). The flow rate of complex was 0.287 mL/min when passed through the 19-gauge needle with the 100mmHg pressure. Conclusions: The PRP gel made it possible for rabbit ADSCs differentiated into nucleus pulposus-like cells after mixed culture in vitro. Maybe, it is a better feasible method for construction of autologous injectable tissue engineered nucleus pulposus.

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The Effect of Platelet-Rich Plasma on Morphogenesis and Expression of Chondrogenic Marker Genes by Chondrocyte-Like Cells of the Rat Nucleus Pulposus in Vitro

Cytology and Genetics ◽

10.3103/s0095452719020087 ◽

2019 ◽

Vol 53 (2) ◽

pp. 124-131

Author(s):

E. G. Pedachenko ◽

I. G. Vasilyeva ◽

M. V. Khizniak ◽

N. G. Chopyck ◽

N. P. Oleksenko ◽

...

Keyword(s):

Nucleus Pulposus ◽

Platelet Rich Plasma ◽

Marker Genes ◽

Chondrogenic Marker

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647788 ◽

1973 ◽

Vol 29 (02) ◽

pp. 490-498 ◽

Cited By ~ 6

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.

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Storage of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647709 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 405-416 ◽

Cited By ~ 3

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.

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The Effect of Lysed Platelets on Neutralization of Heparin In Vitro with Protamine as Measured by the Activated Coagulation Time (ACT)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646392 ◽

1991 ◽

Vol 66 (02) ◽

pp. 213-217 ◽

Cited By ~ 7

Author(s):

Arthur P Bode ◽

William J Castellani ◽

Edna D Hodges ◽

Susan Yelverton

Keyword(s):

Platelet Rich Plasma ◽

Coagulation Time ◽

Platelet Factor ◽

Platelet Suspension ◽

Antiheparin Activity ◽

Unit Increase ◽

Heparin Anticoagulation ◽

Heparin Activity ◽

Activated Coagulation Time

SummaryThe effect of lysed platelets on the activated coagulation time (ACT) was studied in heparinized whole blood during titration with protamine. Frozen-thawed washed platelet suspension, or a chromatography fraction thereof, or autologous frozen-thawed platelet-rich plasma was added in various dilutions to freshly drawn blood anticoagulated with 3,000 USP units/1 heparin. After a 10 min incubation, the amount of protamine needed to restore the ACT to baseline ("protamine titration dose") was determined. We found that the protamine titration dose decreased in proportion to the amount of lysed platelet material added; expressed as a percentage of the total number of platelets present, each unit increase in lysed platelets produced a 1.7% ±0.8 (SD) reduction in the protamine dose needed to normalize the ACT. A heparin activity assay showed that this effect was not due to antiheparin activity of lysed platelets such as platelet factor 4 (PF4). Our data indicate that the procoagulant activity of platelet membranes reduced the sensitivity of the ACT to heparin. These findings suggest that membranous platelet microparticles may cause an inaccurate calculation, based on the ACT, of a protamine dose to reverse heparin anticoagulation in cardiopulmonary bypass procedures.

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Haemostatic Platelet Plug Formation in the Isolated Rabbit Mesenteric Preparation - An Analysis of Red Blood Cell Participation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650069 ◽

1980 ◽

Vol 44 (01) ◽

pp. 006-008 ◽

Cited By ~ 12

Author(s):

D Bergqvist ◽

K-E Arfors

Keyword(s):

Whole Blood ◽

Platelet Rich Plasma ◽

Adenosine Diphosphate ◽

In Vitro Test ◽

Pharmacological Agents ◽

Vitro Test ◽

Releasing Effect ◽

Plug Formation

SummaryIn a model using an isolated rabbit mesenteric preparation microvessels were transected and the time until haemostatic plugs formed was registered. Perfusion of platelet rich plasma gave no haemostasis whereas whole blood did. Addition of chlorpromazine or adenosine to the whole blood significantly prolonged the time for haemostasis, and addition of ADP to the platelet rich plasma significantly shortened it. It is concluded that red cells are necessary for a normal haemostasis in this model, probably by a combination of a haemodynamic and ADP releasing effect.The fundamental role of platelets in haemostatic plug formation is unquestionable but there are still problems concerning the stimulus for this process to start. Three platelet aggregating substances have been discussed – thrombin, adenosine diphosphate (ADP) and collagen. Evidence speaking in favour of thrombin is, however, very minimal, and the discussion has to be focused on collagen and ADP. In an in vitro system using polyethylene tubings we have shown that "haemostasis" can be obtained without the presence of collagen but against these results can be argued that it is only another in vitro test for platelet aggregation (1).To be able to induce haemostasis in this model, however, the presence of red blood cells is necessary. To further study this problem we have developed a model where haemostatic plug formation can be studied in the isolated rabbit mesentery and we have briefly reported on this (2).Thus, it is possible to perfuse the vessels with whole blood as well as with platelet rich plasma (PRP) and different pharmacological agents of importance.

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Effect of Aspirin on the Thrombelastogram of Human Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649127 ◽

1973 ◽

Vol 30 (03) ◽

pp. 494-498 ◽

Cited By ~ 1

Author(s):

G de Gaetano ◽

J Vermylen

Keyword(s):

Oral Dose ◽

Single Oral Dose ◽

Platelet Function ◽

Human Blood ◽

Platelet Rich Plasma ◽

Plasma Samples ◽

Release Reaction ◽

Platelet Release

SummaryThrombelastograms of both native blood and re-calcified platelet-rich plasma samples taken from subjects given a single oral dose of aspirin (1 gram) were not significantly different from the pretreatment recordings. Aspirin also did not modify the thrombelastogram when preincubated in vitro with platelet-rich plasma at concentrations inhibiting the platelet “release reaction” by collagen. Thrombelastography therefore cannot evaluate the effect of aspirin on platelet function.

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Reaction of Acetaldehyde with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648393 ◽

1992 ◽

Vol 67 (01) ◽

pp. 126-130 ◽

Cited By ~ 8

Author(s):

Olivier Spertini ◽

Jacques Hauert ◽

Fedor Bachmann

Keyword(s):

Platelet Aggregation ◽

Inhibitory Action ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Shape Change ◽

Reaction Medium ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650089 ◽

1980 ◽

Vol 44 (02) ◽

pp. 081-086 ◽

Cited By ~ 8

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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