scholarly journals Evaluation of allogeneic freeze-dried platelet lysate in cartilage exposed to interleukin 1-β in vitro

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Livia Camargo Garbin ◽  
C. Wayne McIlwraith ◽  
David D. Frisbie
Keyword(s):  
Musculoskeletal Diseases ◽  
Platelet Rich Plasma ◽  
Autologous Blood ◽  
Interleukin 1 ◽  
Platelet Lysate ◽  
Freeze Dried ◽  
Platelet Concentration ◽  
High Concentrations ◽  
Disease Modifying Treatment

Abstract Background Platelet-rich plasma (PRP) as well as other platelet-derived products have been used as a potential disease-modifying treatment for musculoskeletal diseases, such as osteoarthritis (OA). The restorative properties of such products rely mainly on the high concentrations of growth factors, demonstrating encouraging results experimentally and clinically. Yet, the autologous blood-derived nature of the PRP product lead to limitations that precludes it’s widespread use. The main limitations for PRP use are; product variability, the need for minimum laboratory settings in most cases, and the need for storage at low temperatures to preserve its properties. Based on these limitations, the objective of this study was to investigate an allogeneic off-the-shelf platelet lysate (PL) in cartilage exposed to interleukin 1β (IL-1β). For this purpose, blood and cartilage were harvested from eight skeletally mature and healthy horses. Blood was processed into PL aliquots and divided into three groups (Frozen, Freeze-dried and Filtered freeze-dried), used in autologous and allogeneic conditions and in three different concentrations (1.5, 3 and 6-fold). Different PL preparations were then applied in cartilage culture with interleukin-1 beta and cultured for 10 days. Cartilage and media samples were collected and analyzed for total GAG and 35SO4-labeled GAG content. Results No significant differences between the controls and PL groups in cartilage and media were demonstrated. The effects of PL on cartilage matrix were concentration dependent and intermediate concentrations (3-fold) in PL showed increased 35SO4-labelled GAG in cartilage. Conclusion In conclusion, the allogeneic freeze-dried PL presented equivalent effects compared to frozen autologous PL. Intermediate platelet concentration on average demonstrated improved results, demonstrating less GAG loss compared to other concentrations.

scholarly journals The development of a novel autologous blood glue aiming to improve osseointegration in the bone-implant interface

2020 ◽  
Vol 9 (7) ◽  
pp. 402-411
Author(s):  
Anita Sanghani-Kerai ◽  
Melanie Coathup ◽  
Robyn Brown ◽  
George Lodge ◽  
Liza Osagie-Clouard ◽  
...  
Keyword(s):  
Fibrin Glue ◽  
Platelet Rich Plasma ◽  
Autologous Blood ◽  
Autologous Bone Marrow ◽  
Cost Effective ◽  
Maximum Growth ◽  
Implant Surface ◽  
Bioactive Substances ◽  
High Concentrations

Aims For cementless implants, stability is initially attained by an interference fit into the bone and osteo-integration may be encouraged by coating the implant with bioactive substances. Blood based autologous glue provides an easy, cost-effective way of obtaining high concentrations of growth factors for tissue healing and regeneration with the intention of spraying it onto the implant surface during surgery. The aim of this study was to incorporate nucleated cells from autologous bone marrow (BM) aspirate into gels made from the patient’s own blood, and to investigate the effects of incorporating three different concentrations of platelet rich plasma (PRP) on the proliferation and viability of the cells in the gel. Methods The autologous blood glue (ABG) that constituted 1.25, 2.5, and 5 times concentration PRP were made with and without equal volumes of BM nucleated cells. Proliferation, morphology, and viability of the cells in the glue was measured at days 7 and 14 and compared to cells seeded in fibrin glue. Results Overall, 2.5 times concentration of PRP in ABG was capable of supporting the maximum growth of cells isolated from the BM aspirate and maintain their characteristics. Irrespective of PRP concentration, cells in ABG had statistically significantly higher viability compared to cells in fibrin glue. Conclusion In vitro this novel autologous gel is more capable of supporting the growth of cells in its structure for up to 14 days, compared to commercially available fibrin-based sealants, and this difference was statistically significant. Cite this article: Bone Joint Res 2020;9(7):402–411.

scholarly journals Comparative Evaluation of the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes and Platelet-Rich Plasma: An In Vitro Analysis

Biomedicines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 16 ◽  
Author(s):  
Philipp Moog ◽  
Katharina Kirchhoff ◽  
Sanjar Bekeran ◽  
Anna-Theresa Bauer ◽  
Sarah von Isenburg ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Autologous Blood ◽  
Aortic Ring ◽  
Peripheral Blood Cell ◽  
Angiogenic Factor ◽  
Angiogenic Potential ◽  
Tissue Repair And Regeneration ◽  
Platelet Concentration ◽  
Vitro Analysis

Blood-derived factor preparations are being clinically employed as tools for promoting tissue repair and regeneration. Here we set out to characterize the in vitro angiogenic potential of two types of frequently used autologous blood-derived secretomes: platelet-rich plasma (PRP) and hypoxia preconditioned plasma (HPP)/serum (HPS). The concentration of key pro-angiogenic (VEGF) and anti-angiogenic (TSP-1, PF-4) protein factors in these secretomes was analyzed via ELISA, while their ability to induce microvessel formation and sprouting was examined in endothelial cell and aortic ring cultures, respectively. We found higher concentrations of VEGF in PRP and HPP/HPS compared to normal plasma and serum. This correlated with improved induction of microvessel formation by PRP and HPP/HPS. HPP had a significantly lower TSP-1 and PF-4 concentration than PRP and HPS. PRP and HPP/HPS appeared to induce similar levels of microvessel sprouting; however, the length of these sprouts was greater in HPP/HPS than in PRP cultures. A bell-shaped angiogenic response profile was observed with increasing HPP/HPS dilutions, with peak values significantly exceeding the PRP response. Our findings demonstrate that optimization of peripheral blood cell-derived angiogenic factor signalling through hypoxic preconditioning offers an improved alternative to simple platelet concentration and release of growth factors pre-stored in platelets.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals In vitro responses to platelet-rich-plasma are associated with variable clinical outcomes in patients with knee osteoarthritis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Habib Zahir ◽  
Bijan Dehghani ◽  
Xiaoning Yuan ◽  
Yurii Chinenov ◽  
Christine Kim ◽  
...  
Keyword(s):  
Knee Osteoarthritis ◽  
Clinical Outcomes ◽  
Inflammatory Responses ◽  
Platelet Rich Plasma ◽  
Autologous Blood ◽  
Limited Information ◽  
Knee Oa ◽  
Patient Reported ◽  
Treatment Comparisons

AbstractAutologous blood-derived products such as platelet-rich plasma (PRP) are widely used to treat musculoskeletal conditions, including knee osteoarthritis (OA). However, the clinical outcomes after PRP administration are often variable, and there is limited information about the specific characteristics of PRP that impact bioactivity and clinical responses. In this study, we aimed to develop an integrative workflow to evaluate responses to PRP in vitro, and to assess if the in vitro responses to PRP are associated with the PRP composition and clinical outcomes in patients with knee OA. To do this, we used a coculture system of macrophages and fibroblasts paired with transcriptomic analyses to comprehensively characterize the modulation of inflammatory responses by PRP in vitro. Relying on patient-reported outcomes and achievement of minimal clinically important differences in OA patients receiving PRP injections, we identified responders and non-responders to the treatment. Comparisons of PRP from these patient groups allowed us to identify differences in the composition and in vitro activity of PRP. We believe that our integrative workflow may enable the development of targeted approaches that rely on PRP and other orthobiologics to treat musculoskeletal pathologies.

scholarly journals Autophagy Is Independent of the Chondroprotection Induced by Platelet-Rich Plasma Releasate

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Fan Yang ◽  
Haoran Hu ◽  
Wenjing Yin ◽  
Guangyi Li ◽  
Ting Yuan ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Interleukin 1 ◽  
Cartilage Degeneration ◽  
Inflammatory Stress ◽  
Catabolic Genes ◽  
Transmission Electron ◽  
Similar Expression Pattern ◽  
Vehicle Treatment

Background. Platelet-rich plasma (PRP) has been shown to be a promising therapeutic agent against osteoarthritis (OA), whereas its chondroprotection mechanism is not fully elucidated. Autophagy is considered an important biological process throughout the development of OA. Therefore, the objective of the present study is to investigate the role of autophagy in the chondroprotection and compare the effects of releasate between L-PRP and P-PRP. Methods. PRP were prepared from rat blood. Rat chondrocytes pretreated in the presence or absence of interleukin-1 beta (IL-1β) were incubated with PRP releasate. The expressions of OA-related genes and autophagy-related genes were determined by RT-PCR and western blot, respectively. Autophagic bodies were assessed by transmission electron microscopy and the autophagy flux was monitored under the confocal microscopy. The effect of PRP on autophagy was further investigated in the milieu of autophagy activator, rapamycin, or autophagy inhibition by downregulation of Atg5. The effect of PRP on cartilage repair and autophagy was also evaluated in an OA rat model. Results. In vitro, PRP releasate increased the expression of the anabolic genes, COL2 and Aggrecan, and decreased the expression of the catabolic genes, whereas the expression of autophage markers, Atg5 and Beclin-1, as well as the ratio of LC3 II/LC3 I, was not significantly altered in normal or IL-1β-treated chondrocytes. Similar expression pattern was found following the activation (rapamycin) or inhibition (Atg5 silencing) of autophagy. In vivo, PRP releasate ameliorated posttraumatic cartilage degeneration while the expression of LC3 was comparable to that in the vehicle treatment group. Conclusions. PRP releasate promoted the anabolic gene expression, relieved inflammatory stress in chondrocytes, and ameliorated cartilage degeneration, but autophagy was independent of these processes.

scholarly journals Effect of Combined Leukocyte-Poor Platelet-Rich Plasma and Hyaluronic Acid on Bone Marrow–Derived Mesenchymal Stem Cell and Chondrocyte Metabolism

Cartilage ◽  
2019 ◽  
pp. 194760351985873 ◽  
Author(s):  
Alexander M. Satin ◽  
Jolanta B. Norelli ◽  
Nicholas A. Sgaglione ◽  
Daniel A. Grande
Keyword(s):  
Bone Marrow ◽  
Hyaluronic Acid ◽  
Cellular Proliferation ◽  
Culture Media ◽  
Platelet Rich Plasma ◽  
Interleukin 1 ◽  
Cellular Growth ◽  
Sprague Dawley ◽  
Vitro Effect

ObjectiveGiven the potential applications of combined biologics, the authors sought to evaluate the in vitro effect of combined platelet-rich plasma (PRP) and hyaluronic acid (HA) on cellular metabolism.DesignBone marrow–derived mesenchymal stem cells (BMSCs) and chondrocytes were obtained from the femurs of Sprague-Dawley rats. An inflammatory model was created by adding 10 ng/mL interleukin-1-beta to culture media. Non-crosslinked high-molecular-weight HA, activated-PRP (aPRP), and unactivated-PRP (uPRP) were tested. Cellular proliferation and gene expression were measured at 1 week. Genes of interest included aggrecan, matrix metalloproteinase (MMP)-9, and MMP-13.ResultsCombined uPRP-HA was associated with a significant increase in chondrocyte and BMSC proliferation at numerous preparations. There was a trend of increased chondrocyte aggrecan expression with combined PRP-HA. The greatest and only significant decrease in BMSC MMP-9 expression was observed with combined PRP-HA. While a significant reduction of BMSC MMP-13 expression was seen with PRP and HA-alone, a greater reduction was observed with PRP-HA. MMP-9 chondrocyte expression was significantly reduced in cells treated with PRP-HA. PRP-alone and HA-alone at identical concentrations did not result in a significant reduction. The greatest reduction of MMP-13 chondrocyte expression was observed in chondrocytes plus combined PRP-HA.ConclusionsWe demonstrated a statistically significant increase in BMSC and chondrocyte proliferation and decreased expression of catabolic enzymes with combined PRP-HA. These results demonstrate the additive in vitro effect of combined PRP-HA to stimulate cellular growth, restore components of the articular extracellular matrix, and reduce inflammation.

scholarly journals Micro-Clotting of Platelet-Rich Plasma Upon Loading in Hydrogel Microspheres Leads to Prolonged Protein Release and Slower Microsphere Degradation

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1712
Author(s):  
Miran Hannah Choi ◽  
Alexandra Blanco ◽  
Samuel Stealey ◽  
Xin Duan ◽  
Natasha Case ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Intervertebral Disc Degeneration ◽  
Therapeutic Potential ◽  
Platelet Rich Plasma ◽  
Autologous Blood ◽  
The Body ◽  
Protein Release ◽  
Tissue Degeneration

Platelet-rich plasma (PRP) is an autologous blood product that contains a variety of growth factors (GFs) that are released upon platelet activation. Despite some therapeutic potential of PRP in vitro, in vivo data are not convincing. Bolus injection of PRP is cleared rapidly from the body diminishing its therapeutic efficacy. This highlights a need for a delivery vehicle for a sustained release of PRP to improve its therapeutic effect. In this study, we used microfluidics to fabricate biodegradable PRP-loaded polyethylene glycol (PEG) microspheres. PRP was incorporated into the microspheres as a lyophilized PRP powder either as is (powder PRP) or first solubilized and pre-clotted to remove clots (liquid PRP). A high PRP loading of 10% w/v was achieved for both PRP preparations. We characterized the properties of the resulting PRP-loaded PEG microspheres including swelling, modulus, degradation, and protein release as a function of PRP loading and preparation. Overall, loading powder PRP into the PEG microspheres significantly affected the properties of microspheres, with the most pronounced effect noted in degradation. We further determined that microsphere degradation in the presence of powder PRP was affected by platelet aggregation and clotting. Platelet aggregation did not prevent but prolonged sustained PRP release from the microspheres. The delivery system developed and characterized herein could be useful for the loading and releasing of PRP to promote tissue regeneration and wound healing or to suppress tissue degeneration in osteoarthritis, and intervertebral disc degeneration.

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.

Low concentrations of interleukin-1 stimulate and high concentrations inhibit insulin release from isolated rat islets of Langerhans

1986 ◽  
Vol 113 (4) ◽  
pp. 551-558 ◽  
Author(s):  
Giatgen A. Spinas ◽  
Thomas Mandrup-Poulsen ◽  
Jens Mølvig ◽  
Leif Bæk ◽  
Klaus Bendtzen ◽  
...  
Keyword(s):  
Insulin Release ◽  
Interleukin 1 ◽  
Insulin Biosynthesis ◽  
Rat Islets ◽  
Low Concentrations ◽  
Isolated Rat Islets ◽  
High Concentrations ◽  
Rat Islets Of Langerhans ◽  
Isolated Rat

Abstract. Isolated rat islets were incubated either with crude, affinity-purified or recombinant human interleukin-1 for 1 to 6 days. A significant (20–60%) increase of insulin release was observed at low concentrations of all three interleukin-1-containing preparations. In contrast, higher concentrations dose-dependently inhibited the insulin release. The increased insulin secretion occurred at concentrations below those necessary to augment the mitogen response to phytohaemagglutinin of murine thymocytes in vitro. These doses (0.05-0.5 U/ml) correspond to 0.2-2 ng of recombinant interleukin-1 per ml, equal to approximately 0.01-0.1 pmol/ml. In doses of 0.6-1.8 U/ml affinitypurified interleukin-1 significantly increased the islet insulin content per ng of DNA, indicating a stimulation of insulin-biosynthesis. The data support the concept that low concentrations of interleukin-1 may play a role in priming the physiological secretion of insulin.

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