Leukocyte-platelet-rich Plasma Research Articles

Leukocyte Platelet-Rich Plasma-Derived Exosomes Restrained Macrophages Viability and Induced Apoptosis, NO Generation, and M1 Polarization.

Chronic refractory wounds refer to wounds that cannot be repaired timely. Platelet-rich plasma (PRP) has significant potential in chronic wound healing therapy. The exosomes isolated from PRP were proved to exhibit more effectiveness than PRP. However, the therapeutic potential of exosomes from PRP on chronic refractory wounds remained elusive. Hence, this study aimed to clarify the action of exosomes from PRP on chronic refractory wounds by evaluating the response of macrophages to exosomes. Pure platelet-rich plasma (P-PRP) and leukocyte platelet-rich plasma (L-PRP) were prepared from the fasting venous blood of healthy volunteers. Exosomes were extracted from P-PRP and L-PRP using ultracentrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. Macrophages were obtained by inducing THP-1 cells with phorbol-12-myristate-13 acetate (PMA). The internalization of exosomes into macrophages was observed utilizing confocal laser scanning microscopy after being labeled with PKH67. Cell viability was determined by CCK-8 assay. Cell apoptosis was measured utilizing a flow cytometer. The polarization status of M1 and M2 macrophages were evaluated by detecting their markers. Nitric oxide (NO) detection was conducted using the commercial kit. Exosomes from P-PRP and L-PRP were absorbed by macrophages. Exosomes from L-PRP restrained viability and induced apoptosis of macrophages. Besides, exosomes from P-PRP promoted M2 polarization, and exosomes from L-PRP promoted M1 polarization. Furthermore, exosomes from L-PRP promoted NO generation of macrophages. Exosomes from L-PRP restrained viability, induced apoptosis and NO generation of macrophages, and promoted M1 polarization, while exosomes from P-PRP increased M2 polarization. The exosomes from L-PRP presented a more effective effect on macrophages than that from P-PRP, making it a promising strategy for chronic refractory wound management.

Pure platelet-rich plasma promotes semaphorin-3A expression: a novel insight to ameliorate intervertebral disk degeneration in vitro

IntroductionIntervertebral disk degeneration (IVDD) can be effectively treated using platelet-rich plasma (PRP). While the exact process is fully understood, it is believed that using pure PRP (P-PRP) without leukocytes is a better option for preventing IVDD. Semaphorin-3A (Sema3A), an inhibitor of angiogenesis and innervation, is essential for preserving IVDD’s homeostasis. Whether PRP prevents IVDD by modifying Sema3A has yet to receive much research. This work aims to clarify how P-PRP affects Sema3A when IVDD develops in vitro.MethodsNucleus pulposus cells (NPCs) isolated from 8-week-old male Sprague-Dawley rats were exposed to 10 ng/ml IL-1β and then treated with P-PRP or leukocyte platelet-rich plasma (L-PRP) in vitro, followed by measuring cell proliferation, apoptosis and microstructures, inflammatory gene and Sema3A expression, as well as anabolic and catabolic protein expression by immunostaining, quantitative real-time polymerase chain reaction (qPCR), western blot, and enzyme-linked immunosorbent assay (ELISA).ResultsIn comparison with L-PRP, P-PRP had a higher concentration of growth factors but a lower concentration of inflammatory substances. P-PRP increased the proliferation of NPCs, while IL-1 relieved the amount of apoptosis due to its intervention. Anabolic genes, aggrecan, and collagen II had higher expression levels. MMP-3 and ADAMTS-4, two catabolic or inflammatory genes, showed lower expression levels. Sema3A activity was enhanced after P-PRP injection, whereas CD31 and NF200 expression levels were suppressed.ConclusionsP-PRP enhanced the performance of NPCs in IVDD by modifying the NF-κB signaling pathway and encouraging Sema3A expression, which may offer new therapy options for IVDD.The translational potential of this articleThe findings provide a new therapeutic target for the treatment of IVDD and show a novel light on the probable mechanism of PRP and the function of Sema3A in the progression of IVDD.

The wound healing effect of local leukocyte platelet-rich plasma after total hip arthroplasty: A randomized controlled trial.

Rapid wound closure is important after arthroplasty procedures to prevent postoperative complications. Platelets are rich in growth factors and leukocytes contribute to innate immunity. We hypothesized that topical leukocyte platelet-rich plasma (L-PRP) derived from the blood of patients would be beneficial to wound healing. In this randomized controlled trial, patients subjected to elective total hip arthroplasty (THA) were assigned by concealed allocation either L-PRP application onto the sutured fascia or no application (control) after the THA intervention. In addition, all patients received 1.5g protein/kg, 5 g L-arginine, 500 mg vitamin C and 44 mg zinc daily over the 4-week postoperative period to obtain optimal nutrition. The primary endpoint was complete healing of the skin incision. The secondary endpoints were blood transfusions, length of hospital stay, pain and wound infections. Sixteen patients in the L-PRP group and 17 patients in the control group completed the trial. L-PRP treatment accelerated complete wound healing after 3 weeks (seven in the L-PRP group vs. zero in the control group, p=0.003) and after 4 weeks (12 in the L-PRP group vs. six in the control group, p=0.037). No postoperative superficial wound infections occurred within 4 weeks, and there were no significant differences in the other secondary outcomes. L-PRP generated in 10 sex-matched healthy volunteers revealed increased concentrations of platelets (5.8-fold) and leukocytes (2.3-fold) compared with those in whole blood. Furthermore, the concentration of keratinocyte mitogen epidermal growth factor in L-PRP (380 ± 130 pg/ml, mean ± SD) was higher (p < 0.001) than that in serum (130 ± 26 pg/ml). In conclusion, a single intraoperative local application of L-PRP promoted wound healing after THA, possibly mediated by EGF receptor agonists.

Growth Factor Delivery to a Cartilage-Cartilage Interface Using Platelet-Rich Concentrates on a Hyaluronic Acid Scaffold

To determine whether (1) human leukocyte-platelet-rich plasma (L-PRP) or (2) leukocyte-platelet-rich fibrin (L-PRF) delivered on a hyaluronic acid (HA) scaffold at a bovine chondral defect, a simulated cartilage tear interface, invitro would improve tissue formation based on biomechanical, histologic, and biochemical measures. L-PRF and L-PRP were prepared from 3 healthy volunteer donors and delivered in conjunction with HA scaffolds to defects created in full-thickness bovine cartilage plugs harvested from bovine femoral condyle and trochlea. Specimens were cultured invitro for up to 42 days. Treatment groups included an HA scaffold alone and scaffolds containing L-PRF or L-PRP. Cartilage repair was assessed using biomechanical testing, histology, DNA quantification, and measurement of sulfated glycosaminoglycan and collagen content at 28 and 42 days. L-PRF elicited the greatest degree of defect filling and improvement in other histologic measures. L-PRF-treated specimens also had the greatest cellularity when compared with L-PRP and control at day 28 (560.4 μg vs 191.4 μg vs 124.2 μg, P= .15); at day 48, there remained a difference, although not significant, between L-PRF versus L-PRP (761.1 μg vs 589.3 μg, P= .219) . L-PRF had greater collagen deposition when compared with L-PRP at day 42 (40.1 μg vs 16.3 μg, P < .0001). L-PRF had significantly greater maximum interfacial strength compared with the control at day 42 (10.92 N vs 0.66 N, P= .015) but had no significant difference compared with L-PRP (10.92 N vs 6.58 N, P= .536). L-PRP facilitated a greater amount of sulfated glycosaminoglycan production at day 42 when compared with L-PRF (15.9 μg vs 4.3 μg, P= .009). Delivery of leukocyte-rich platelet concentrates in conjunction with a HA scaffold may allow for improvements in cartilage healing through different pathways. L-PRF was not superior to L-PRP in its biomechanical strength, suggesting that both treatments may be effective in improving biomechanical strength of healing cartilage through different pathways. The delivery of platelet-rich concentrates in conjunction HA scaffolds may augment healing cartilaginous injuries.

Effects of Leukocyte-Platelet-Rich Plasma on the Alveolar Bone Changes During Orthodontic Tooth Movement in Rabbits: A Micro-CT Study

Objective: To investigate the effects of leukocyte-platelet-rich plasma (L-PRP) on the alveolar bone changes at the compression and tension sides during orthodontic tooth movement. Materials and Methods: Around 20 New Zealand white rabbits were used in a split-mouth design. The maxillary first premolar was moved mesially with a nickel-titanium closed-coil spring. One side of the maxilla was randomly injected with L-PRP, while the contralateral side served as the control which received normal saline. The alveolar bone adjacent to the maxillary first premolar was scanned using microcomputed tomography at days 0, 7, 14, and 28. Microstructural parameters including bone volume fraction, trabecular thickness, and trabecular separation of alveolar bone were assessed on the compression and tension sides of the maxillary first premolar. Results: Compared between the groups, the L-PPR group showed a significantly decreased bone volume fraction on the compression side on days 7 and 14 but significantly increased bone volume fraction on the tension side on day 14. However, there were no statistically significant differences in the parameters of trabecular thickness and trabecular separation. Conclusion: Local administration of L-PRP may promote bone resorption on the compression side and bone formation on the tension side at the initial stage of orthodontic tooth movement.

The influence of leukocyte-platelet-rich plasma on accelerated orthodontic tooth movement in rabbits

ObjectiveTo determine the effects of a local injection of leukocyte-platelet-rich plasma (L-PRP) on orthodontic tooth movement in rabbits.MethodsTwenty-three male New Zealand white rabbits were included in a split-mouth design. Tooth movement with a 100-g nickel-titanium closed-coil spring was performed on the maxillary first premolars. L-PRP was injected submucosally at the buccal and lingual areas of the first premolar in one random side of the maxilla and the other side served as the control and received normal saline. The amount of tooth movement was assessed on three-dimensional digital models on days 0, 3, 7, 14, 21, and 28. Histological findings and osteoclast numbers were examined on day 0 as the baseline and on days 7, 14, and 28.ResultsThe L-PRP group showed significantly greater cumulative tooth movement at all observed periods. However, a significantly higher rate of tooth movement was observed only on days 0–7 and 7–14. The osteoclast numbers were significantly increased in the L-PRP group on days 7 and 14.ConclusionsLocal injection of L-PRP resulted in a transient increase in the rate of tooth movement and higher osteoclast numbers.

Leukocyte inclusion within a platelet rich plasma-derived fibrin scaffold stimulates a more pro-inflammatory environment and alters fibrin properties.

One of the main differences among platelet-rich plasma (PRP) products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF) and leukocyte-platelet rich plasma (L-PRP) scaffolds was determined by enzyme-linked immunosorbent assay (ELISA) and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions.

Platelet-rich plasma for androgenetic alopecia: a pilot study.

Androgenetic alopecia is a common condition, with severe attendant psychosocial implications, and for which it is difficult to obtain a satisfactory degree of clinical improvement. To explore the possible clinical benefit of injecting platelet-derived growth factors into the scalp of patients using a specific autologous blood concentrate. Two injections of a leukocyte platelet-rich plasma (L-PRP) with the addition of concentrated plasmatic proteins were administered at baseline and after 3 months (single spin at baseline and double-spin centrifugation at 3 months). Macrophotographs were taken at baseline and after 6 months, and 2 independent evaluators rated them using Jaeschke rating of clinical change. Sixty-four consecutive patients were enrolled. Some improvement was seen in all patients by 1 evaluator and in 62 by the other. The mean change in clinical rating was 3.2 (95% confidence interval [CI], 2.9-3.5) and 3.9 (95% CI, 3.5-4.3), and the proportion of patients reaching a clinically important difference was 40.6% and 54.7%, according to the 2 evaluators, respectively. Our pilot study may provide preliminary evidence that this treatment may induce some degree of clinical advantage for male- and female-pattern baldness. This may warrant the design of randomized controlled clinical trials to formally test this procedure.

Morphogen and proinflammatory cytokine release kinetics from PRGF-Endoret fibrin scaffolds: evaluation of the effect of leukocyte inclusion.

The potential influence of leukocyte incorporation in the kinetic release of growth factors from platelet-rich plasma (PRP) may explain the conflicting efficiency of leukocyte platelet-rich plasma (L-PRP) scaffolds in tissue regeneration. To assess this hypothesis, leukocyte-free (PRGF-Endoret) and L-PRP fibrin scaffolds were prepared, and both morphogen and proinflammatory cytokine release kinetics were analyzed. Clots were incubated with culture medium to monitor protein release over 8 days. Furthermore, the different fibrin scaffolds were morphologically characterized. Results show that leukocyte-free fibrin matrices were homogenous while leukocyte-containing ones were heterogeneous, loose and cellular. Leukocyte incorporation produced a significant increase in the contents of proinflammatory cytokines interleukin (IL)-1β and IL-16 but not in the platelet-derived growth factors release (<1.5-fold). Surprisingly, the availability of vascular endothelial growth factor suffered an important decrease after 3 days of incubation in the case of L-PRP matrices. While the release of proinflammatory cytokines was almost absent or very low from PRGF-Endoret, the inclusion of leukocytes induced a major increase in these cytokines, which was characterized by the presence of a latent period. The PRGF-Endoret matrices were stable during the 8 days of incubation. The inclusion of leukocytes alters the growth factors release profile and also increased the dose of proinflammatory cytokines.

Does Autologous Leukocyte-Platelet–Rich Plasma Improve Tendon Healing in Arthroscopic Repair of Large or Massive Rotator Cuff Tears?

To evaluate the clinical and magnetic resonance imaging (MRI) outcome of arthroscopic rotator cuff repair with the use of leukocyte-platelet-rich plasma (L-PRP) in patients with large or massive rotator cuff tears. A comparative cohort of patients with large or massive rotator cuff tears undergoing arthroscopic repair was studied. Two consecutive groups of patients were included: rotator cuff repairs with L-PRP injection (group 1, n= 35) and rotator cuff repairs without L-PRP injection (group 2, n= 35). A double-row cross-suture cuff repair was performed by a single surgeon with the same rehabilitation protocol. Patients were clinically evaluated with the Constant score; Simple Shoulder Test score; University of California, Los Angeles (UCLA) score; and strength measurements by use of a handheld dynamometer. Rotator cuff healing was evaluated by postoperative MRI using the Sugaya classification (type 1 to type 5). We prospectively evaluated the 2 groups at a minimum 2-year follow-up. The results did not show differences in cuff healing between the 2 groups (P= .16). The size of recurrent tears (type 4 v type 5), however, was significantly smaller in group 1 (P= .008). There was no statistically significant difference in the recurrent tear rate (types 4 and 5) between the 2 groups (P= .65). There was no significant difference between group 1 and group 2 in terms of University of California, Los Angeles score (29.1 and 30.3, respectively; P= .90); Simple Shoulder Test score (9.9 and 10.2, respectively; P= .94); Constant score (77.3 and 78.1, respectively; P= .82); and strength (7.5 and 7.0, respectively; P= .51). In our study the use of autologous L-PRP did not improve the quality of tendon healing in patients undergoing arthroscopic repair of large or massive rotator cuff tears based on postoperative MRI evaluation. The only significant advantage was that the L-PRP patients had smaller iterative tears. However, the functional outcome was similar in the 2 groups of patients. Level III, case-control study.

Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors

Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma.

Leukocyte-platelet-rich plasma (L-PRP) impairs the osteoconductive capacity of the autograft associated to changes in the immunolocalization of TGF-β1 and its co-expression with Wnt10b and CD34 cells

BackgroundThis study evaluated the osteoconductive effect of an autograft, in the presence or absence of the L-PRP, using histomorphometric analysis of the bone formed, and we compared the results in the presence of TGF-β1, Wnt10b and CD34 detected by immunohistochemistry. Materials and methodsTwo bone defects were produced in the calvaria of 20 rabbits. The defects were treated with autograft and autograft combined with L-PRP. The animals were euthanized at 15 and 40 days post-surgery. Data were analyzed by Student-Newman–Keuls (p ≤ 0.05) test for histomorphometric and immunohistochemical interpretation. ResultsThe results revealed that the presence of bone matrix was significantly less in the defects treated with L-PRP. These results coincided with changes of the immunolocalization of the TGF-β1. In the L-PRP-free groups the TGF-β1 was restricted to bone matrix while the CD34 was scarce and the Wnt10b occurred in peritrabecular cells. In contrast, in defects that received L-PRP the presence of TGF-β1 occurred in cells, which occupied whole area of defect. These TGF-β1+ cells also were co-expressed to Wnt10b and CD34. ConclusionThese results suggest that L-PRP induces a cross-reaction between TGF-β1 and Wnt10b, which stimulates the self-renewal and maintenance of CD34+ stem cells immunophenotype, impairing the osteoconductivity properties of the autograft.

Leukocyte‐Platelet‐Rich Plasma (L‐PRP) Induces an Abnormal Histophenotype in Craniofacial Bone Repair Associated with Changes in the Immunopositivity of the Hematopoietic Clusters of Differentiation, Osteoproteins, and TGF‐β1

Leukocyte-platelet-rich plasma (L-PRP) is considered an important source of growth factors, especially Transforming growth factor β 1 (TGF-β1), which modulates the proliferation and regulation of mesenchymal cells, and also exerts an influence on the hematopoiesis, osteogenesis, and adipogenesis in bone microenvironment. Thus, the aim of this study was to evaluate the effect of L-PRP on the calvarial bone repair and compare its results on the presence of TGF-β1, CD34, CD45, bone morphogenetic protein 2 (BMP2), BMPR1B, and Runx2 proteins detected by immunohistochemistry. Four bone defects were created on the calvaria of 23 rabbits. The defects were treated with autograft, L-PRP alone, and L-PRP mixed with autograft. The animals were euthanized at 2, 4, and 6 weeks post-surgery. Unlike autograft and sham groups, the defects treated with L-PRP demonstrated significant positivity to TGF-β1, while the BMP2 was scarce. These results coincided with the lower bone matrix deposited and larger medullary area, which were composed of fibrosis, when treated with only L-PRP, or intense adiposity on defects filled with L-PRP mixed with autograft. The fibrosis that occurred was associated with a minor percentage of osteoproteins, intense presence of CD34(+) CD45(-) cells, and significant expression of TGF-β1 in all time periods analyzed. The adiposity occurred from the major presence of osteoprogenitor BMPR1B (+) Runx2(+) cells simultaneously to BMP2(-) TGF-β1(+) and CD34(+) CD45(+/-) expressions predominantly on the earlier period. From this study, it can be concluded that the L-PRP used alone or mixed to autograft hindered the osteoneogenesis due to suppression of immunoexpression of BMP2, while the immunopositivity of TGF-β1 was intense. When used alone, the L-PRP induced a fibrotic condition associated with TGF-β1 presence and lack of osteoproteins, but when L-PRP was mixed to autograft, it induced the presence of the osteolineage cells (BMPR1B (+) Runx2(+) ), but also inhibited the terminal osteoblastic maturation associated with the lack of BMP2 and the presence of TGF-β1(+) , a fact that contributed to cellular transdifferentiation into fat cells.

Succinylated gelatine: an alternative to hydroxyethyl starch for labelling leukocytes with 99mTc-HMPAO

Hydroxyethyl starch (HES) is the most used plasma expander in the sedimentation of the erythrocytes during the radiolabelling procedure for leukocytes in vitro. To evaluate the usefulness of succinylated gelatine (GEL), another colloidal plasma expander, as an alternative to HES in this process. Two identical blood samples were obtained from 30 patients referred to white blood cell scintigraphy. The first sample was used to label leukocytes with Tc-HMPAO using the routine procedure, with HES. The other sample was used to label leukocytes with Tc-HMPAO using the same procedure, with GEL. The cell concentration of the leukocyte-platelet-rich plasma (LPRP) achieved after blood sedimentation was analysed. Labelling efficiency was calculated and the eosin Y viability and red cell/leukocyte ratio were evaluated from the final labelled cell suspension. Leukocytes and platelets recovered in LPRP were not statistically different between both HES and GEL samples (leukocytes: 8.10x10/microl+/-3.82 and 7.80x10/microl+/-3.47; platelets: 411x10/microl+/-182 and 406x10/microl+/-172, respectively). There were no significant differences between both agents on the labelling efficiency (HES: 80.3%+/-6.6%; GEL: 80.1%+/-6.3%), the eosin Y viability (HES: 99.2%+/-1.3%; GEL: 99.3%+/-1.1%) and the red cell/leukocyte ratio (HES: 1.21+/-0.7; GEL: 0.9+/-0.5). These results show that succinylated gelatine can be used instead of hydroxyethyl starch in the labelling of leukocytes with Tc-HMPAO.