Comparison of bioactive substances in novel-developed freeze-dried platelet-rich plasma (PRP) and activated normal PRP, and investigation of bioactive substance levels after long-term storage.

Introduction: In recent years, biotherapy in orthopedics has become widespread, and platelet-rich plasma (PRP) has been readily used to treat sports injuries and osteoarthritis. Production of freeze-dried PRP (PRP-FD) results in PRP that is in powder form, allowing it to be stored for long periods at room temperature. Using this technology, we have developed Valuable Platelet-Derived Factor Concentrate Freeze Dry (VFD). However, whether VFD contains sufficient levels of bioactive substances (BS) remains unknown and retains the same levels of BS during long-term storage. In this study, we examined whether VFD contains sufficient amounts of BS and whether they retain these BS levels during long-term storage. Methods: Peripheral blood was collected from 10 healthy men (mean ± SD: 46.5 ± 15 years old) and various BS, including transforming growth factor ß (TGF-ß), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), tissue inhibitors of metalloproteinases-1 (TIMP-1), interleukin-1 receptor antagonist (IL-1ra), matrix metallopeptidase-9 (MMP-9), and interleukin-6 (IL-6), were compared between VFD and normal PRP samples, including both leukocyte-rich PRP (LR-PRP) and leukocyte-poor PRP (LP-PRP). VFD was prepared using two rounds of centrifugation. LP-PRP and LR-PRP were activated by freezing and thawing before measurement. To evaluate the effects of long-term storage, the BS of VFD purified from five professional football players was compared between baseline and 1 year after storage. Results: In terms of the growth factors, the TGF-ß and EGF levels were higher in LR-PRP than in VFD and LP-PRP (p < 0.05), while the bFGF levels were higher in VFD than in the LR-PRP and LP-PRP groups (p < 0.01). In terms of anti-inflammatory cytokines, the TIMP-1 level was lower in VFD than that in the other groups (p < 0.01), whereas the IL-1ra levels were higher in VFD than those in LP-PRP (p < 0.05) and lower than those in LR-PRP (p < 0.01). In terms of inflammatory enzymes and cytokines, the IL-1ra level was higher in VFD than that in LP-PRP (p < 0.05) and lower than that in LR-PRP (p < 0.01), whereas the IL-6 levels did not differ among the groups. Furthermore, the TGF-ß, bFGF, TIMP-1, and IL-1ra levels were 5.61 â†’ 3.38 (x103 pg/µL), 61.0 â†’ 63.0 (pg/µL), 3.4 â†’ 2.7 (x105 pg/µL), and 14.9 â†’ 14.5 (x103 pg/µL) at baseline and 1 year later, respectively. No significant differences in the BS levels were observed between baseline and 1 year after storage. Conclusions: The VFD samples prepared in this study exhibited higher levels of anti-inflammatory cytokines than LP-PRP and contained growth factor levels similar to LP-PRP and LR-PRP. In addition, the BS levels in VFD samples were maintained after one year of storage. These results suggest that VFD can be prepared and stored and may serve as a novel treatment strategy for sports injuries in high-risk groups, such as athletes.

Study protocol for a randomised placebo controlled trial of platelet-rich plasma injection to prevent post-traumatic knee osteoarthritis after anterior cruciate ligament reconstruction.

INTRODUCTION: The elevated cytokine levels in patients suffering from anterior cruciate ligament (ACL) rupture may lead to acute post-traumatic arthritis (APTA) and post-traumatic osteoarthritis (PTOA). Due to its chondrogenerative and anti-inflammatory effect, platelet-rich plasma (PRP) therapy is expected to show a positive outcome in APTA and PTOA. The proposed trial aims to quantitatively measure the efficacy of PRP injection in arresting post-traumatic cartilage degeneration among patients after ACL reconstruction. METHODS AND ANALYSIS: This will be a single-blind, randomised, prospective, controlled clinical trial designed following the Consolidated Standards of Reporting Trials guidelines. After ACL reconstruction, 80 patients will be randomised to receive either leucocyte-poor PRP injection after joint aspiration or a placebo control group receiving only joint aspiration. Participants (age 20-49 years) will be those who have undergone ACL reconstruction within the past 2 weeks with a body mass index<35 and Kellgren Lawrence osteoarthritis grade<2. The primary outcome will include MRI-T2 values of knee cartilage at 6 months. The secondary outcomes will include pain assessment by Visual Analogue Scale, Knee injury and Osteoarthritis Outcome Score, blood and urine test, physical findings, measurements for muscle strength and joint stability. ETHICS AND DISSEMINATION: The study was approved by The Independent Ethics Committee for Clinical Trials of the Japanese Association for the Promotion of State-of-the-Art Medicine. Results of the trial and each of the outcomes will be shared via conferences and publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: jRCTb030200391.

The immature platelet fraction affects the efficacy of platelet rich plasma therapy for knee osteoarthritis.

INTRODUCTION: Platelet-rich plasma (PRP) therapy is used to treat pathological conditions such as degenerative inflammatory diseases including osteoarthritis (OA) by enhancing tissue repair and promoting anti-inflammatory effects. Although PRP therapy for patients with knee OA improved pain and functional scores, the association of clinical outcomes and quality of PRP including cell composition and concentration is unclear. METHODS: Therefore, this study analyzed blood cell counts, including the immature platelet fraction (IPF), in peripheral blood and PRP of 144 patients with knee OA who underwent PRP therapy. The mean leukocyte and platelet concentrations in whole blood and PRP were analyzed using an XN-1000 automated hematology analyzer. Visual analogue scale (VAS) scores and knee injury and osteoarthritis outcome scores (KOOS) before and 1 month after a single PRP injection were also determined. RESULTS: Higher platelet and lower leukocyte concentration rates were observed in PRP compared with whole blood. The platelet concentration in whole blood was negatively correlated with VAS improvement. The percentage of IPF (IPF%) in whole blood was positively correlated with VAS improvement and KOOS (pain) improvement, whereas the IPF% in PRP tended to correlate with VAS improvement. Furthermore, multivariate logistic regression demonstrated the high IPF% in whole blood was significantly associated with VAS improvement. The low percentage of neutrophil (neutrophil%) in PRP was significantly associated with the VAS improvement and KOOS (ADL) improvement. CONCLUSIONS: Therefore, PRP efficacy for OA might depend on the patient's biological status.

Optical Trapping-Polarized Raman Microspectroscopy of Single Ethanol Aerosol Microdroplets: Droplet Size Effects on Rotational Relaxation Time and Viscosity.

Optical trapping-polarized Raman microspectroscopy of single ethanol (EtOH) microdroplets with a diameter (d) of 6.1-16.5 µm levitated in an EtOH vapor-saturated air/N2 gas atmosphere has been explored to elucidate the vibrational and rotational motions of EtOH in the droplets at 22.0 °C. The Raman spectral bandwidth of the C-C stretching vibrational mode observed for an aerosol EtOH microdroplet was narrower than that of bulk EtOH, suggesting that the vibrational/rotational motions of EtOH in the aerosol system were restricted compared to those in the bulk system. In practice, polarized Raman microspectroscopy demonstrated that the rotational relaxation time (τrot) of EtOH in an aerosol microdroplet with d = 16. 5 µm was slower (2.33 ps) than that in a bulk EtOH (1.65 ps), while the vibrational relaxation times (τvib) in the aerosol and bulk EtOH systems were almost comparable with one another: 0.86-0.98 ps. Furthermore, although the τvib value of an aerosol EtOH microdroplet was almost unchanged irrespective of d as described above, the τrot value increased from 2.33 to 3.57 ps with a decrease in d from 16.5 to 6.1 µm, which corresponded to the increase in EtOH viscosity (η) from 1.33 to 2.04 cP with the decrease in d. The droplet size dependences of τrot and η in an aerosol EtOH microdroplet were discussed in terms of the gas/droplet interfacial molecular arrangements of EtOH and Laplace pressure experienced by a spherical EtOH microdroplet in the gas phase.

Optical Trapping-Microspectroscopy of Single Aerosol Microdroplets in Air: Supercooling of Dimethylsulfoxide Microdroplets.

We report temperature (T = +22.5 ∼ -57.0 °C)-controlled optical trapping of single dimethylsulfoxide (DMSO) droplets with the diameter (d) of 7-15 µm in air. Optically levitated DMSO microdroplets containing 0.1 mol/dm3 (=M) potassium iodide (KI) as an additive for reducing the vapor pressure of DMSO in air have been suggested to take supercooled liquid states even below the freezing temperature (fp) of the bulk DMSO liquid (fp = +18.4 °C in the presence of 0.1 M KI) as seen in bright-field microscopic observations of the droplet. Clear evidence for supercooling of an aerosol DMSO microdroplet below fp has been obtained by in situ optical trapping-polarized Raman microspectroscopy of the droplet down to -14.9 °C. Analysis of the polarized Raman spectral data of an aerosol DMSO droplet (d = ∼10 µm) has demonstrated that the droplet at +22.5, +0.2, or -14.9 °C is characterized by the rotational relaxation time (τrot) of a DMSO molecule in the droplet being 1.95, 2.58, or 3.90 ps, respectively. On the basis of the τrot values and the Stokes-Einstein equation (τrot = 8πa3η/kBT where a, η, kB are the radius (1.883 Å) of a DMSO molecule, the viscosity in DMSO, and the Boltzmann constant, respectively), the η values in the DMSO microdroplet in air at +22.5, +0.2, or -14.9 °C have been estimated to be 2.39, 2.94, or 4.20 cP, respectively, while that of bulk DMSO liquid at +20.5 °C is 1.98 cP. We also report the T-dependence (+22.5 > T > -14.9 °C) of the viscosity in a single aerosol DMSO microdroplet (d = ∼10 µm) and the effects of aerosolization in air on the viscosity in DMSO.

Acute application of cisplatin affects methylation status in neuroblastoma cells.

The pharmacological mechanism of the anti-cancer effect of cisplatin is well known to be DNA intercalation, but the direct or indirect effects of cisplatin on protein expression in cancer cells remain to be explained. In this study, we used a proteomic approach to clarify the early impact of cisplatin on protein expression. In a 2-dimensional gel electrophoresis proteomic experiment, the application of cisplatin for 24 h increased the expression of four proteins and decreased the levels of one protein in neuroblastoma IMR-32 cells. Levels of S-adenosyl-L-homocysteine hydrolase, a key enzyme in methylation metabolism, were increased the most. Therefore, we examined the methylation status of histone proteins. Histone H3K9 methylation was reduced by the application of cisplatin for 24 h. These results suggest that acute cisplatin treatment alters methylation status. Thus, these data can help clarify the unknown pharmacological mechanisms of cisplatin, including the anticancer effect, adverse effects and/or the mechanism of drug resistance.