Corrigendum to "The Effect of Intravenous Autologous Activated Platelet-Rich Plasma Therapy on "Profibrotic Cytokine" IL-1ß Levels in Severe and Critical COVID-19 Patients: A Preliminary Study".

[This corrects the article DOI: 10.1155/2021/9427978.].

Phase I/II Clinical Trial of Autologous Activated Platelet-Rich Plasma (aaPRP) in the Treatment of Severe Coronavirus Disease 2019 (COVID-19) Patients.

BACKGROUND: The outbreak of Coronavirus Disease 2019 (COVID-19) has been increasing rapidly. This disease causes an increase in proinflammatory cytokine production that leads to cytokine storm or cytokine release syndrome (CRS). Autologous activated platelet-rich plasma (aaPRP) contains various types of growth factors and anti-inflammatory cytokines that may have the potential to suppress CRS. This study of phase I/II trial was aimed to evaluate the safety and efficacy of aaPRP to treat severe COVID-19 patients. METHODS: A total of 10 severe COVID-19 patients from Koja Regional Public Hospital (Koja RPH) were admitted to the intensive care unit (ICU). All patients received aaPRP administration three times. Primary outcomes involving the duration of hospitalization, oxygen needs, time of recovery, and mortality were observed. Secondary outcomes involving C-reactive protein (CRP), neutrophil, lymphocyte, and lymphocyte-to-CRP (LCR) and neutrophil-lymphocyte ratio (NLR) were analyzed. RESULTS: All patients were transferred to the ICU with a median duration of 9 days. All patients received oxygen at enrollment and nine of ten patients recovered from the ICU and transferred to the ward room. There was one patient who passed away in the ICU due to heart failure. The results of secondary outcomes showed that CRP value and lymphocytes counts were significantly decreased while neutrophils, LCR, and NLR were slightly increased after aaPRP administration. CONCLUSIONS: Our results of the phase I/II trial demonstrated that the use of aaPRP in severe COVID-19 patients was safe and not associated with serious adverse events, which showed that aaPRP was a promising adjunctive therapy for severe COVID-19 patients.

The Effect of Intravenous Autologous Activated Platelet-Rich Plasma Therapy on "Profibrotic Cytokine" IL-1ß Levels in Severe and Critical COVID-19 Patients: A Preliminary Study.

INTRODUCTION: Elevated concentration of proinflammatory cytokines followed by hyperinflammation is one of the hallmarks of severe and critical COVID-19. In the short term, this may result in ARDS and lung injury; subsequently, this may cause pulmonary fibrosis-a disease with poor prognosis-in the long run. Among the cytokines, interleukin-1ß (IL-1ß) is one of the most overexpressed in COVID-19. We speculate that administration of intravenous activated autologous platelet-rich plasma (aaPRP), which contains interleukin-1 receptor antagonist (IL-1RA), would lower IL-1ß levels and benefit the severe and critical COVID-19 patients. METHODS: After acquiring ethical clearance, we recruited 12 adult COVID-19 patients of both sexes from the Koja Regional Hospital (Jakarta, Indonesia) ICU. After selection, seven patients were included and divided into two groups, severe and critical. In addition to three doses of aaPRP, both groups received the same treatment of antiviral, steroid, and antibiotics. Quantification of plasma IL-1ß levels was performed by beads multiplex assay a day before the first aaPRP administration and a day after the second and third aaPRP administration. PaO2/FiO2 ratio and lung injury scores were evaluated a day before and a day after each aaPRP administration. RESULTS: Severe and critical patients' initial plasma IL-1ß concentration was 4.71 pg/mL and 3.095 pg/mL, respectively. After 2 treatments with aaPRP, severe patients' plasma IL-1ß concentration decreased 12.48 pg/mL, while critical patients' plasma IL-1ß concentration increased to 18.77 pg/mL. Furthermore, after 3 aaPRP treatments, significant amelioration of patients' PaO2/FiO2 ratio from 71.33 mmHg at baseline to 144.97 mmHg was observed (p < 0.05). However, no significant improvement in lung injury score was observed in severe and critical groups. All severe patients and one critical patient recovered. CONCLUSION: The use of aaPRP may prevent pulmonary fibrosis in severe COVID-19 patients through the reduction of patients' plasma IL-1ß concentration and the amelioration of PaO2/FiO2 ratio.

Effect of ascorbic acid on morphology of post-thawed human adipose-derived stem cells.

BACKGROUND: Ascorbic acid-2-phosphate has been reported to play a role in cell division and to suppress aging of cell. However, post-thawed cell morphology on various concentration of ascorbic acid is still unclear. In this study, we aimed to observe the morphology of post-thawed adipose-derived stem cells (ADSCs) in medium containing L-ascorbic acid-2-phosphate (LAA2P) (50 and 100 µg/mL). METHODS: The cells were isolated from adipose tissue. Isolated cells then cultured and cryopreserved in liquid nitrogen. We detected mRNA expression of type 1 collagen on day 5. Cell seeded in T25 flask using basal medium [Dulbecco's modified Eagle's medium (DMEM) only] as a control group, DMEM with 10% fetal bovine serum (FBS) and antibiotics as DMFA group, while DMFA with ascorbic acid (50 and 100 µg/mL) as ascorbic acid treatment group. RESULTS: The results showed that the cells cultured in DMEM only attached until 96 hours of observation while serum groups with or without ascorbic acid supplementation showed the proliferation until 240 hours of observation. The highest spread size of cell was in a serum group without ascorbic acid supplementation and the highest yield of cells showed in a group with 50 µg/mL of ascorbic acid supplementation. Reduced mRNA expression of type 1 collagen which related to aging was showed in cells cultured without ascorbic acid supplementation. CONCLUSIONS: These results showed that ascorbic acid increased the cell division and suppressed the aging processes indicated by normal spread cell in size compared to cell cultured in DMFA without ascorbic acid supplementation.

Safety of Technique and Procedure of Stromal Vascular Fraction Therapy: From Liposuction to Cell Administration.

BACKGROUND: Stromal vascular fraction (SVF) therapy has been performed over the past six years to treat 421 patients by our group in five clinical centers. Autologous SVF, which is a substance containing stem cells, was isolated from lipoaspirate, mixed with platelet-rich plasma (PRP), and administered to patients with degenerative diseases, autoimmune diseases, trauma, aging, and other diseases with unknown etiology. This study aimed to determine the safety of SVF and PRP that were given through infusion, spinal, and intra-articular injection. METHODS: The lipoaspirate was treated with a tissue-dissociating enzyme, and then, through centrifugation, SVF was isolated. In addition, blood was drawn from each patient, and PRP was isolated. Autologous PRP and SVF were administered to all subjects by intravenous (IV) injection. A minority group within the population received an additional spinal or intra-articular injection. The type of intervention was determined by each disease evaluation. The cell doses and adverse events for each patient were documented and analyzed. RESULTS: Cell dose that was considered to be safe was less than 10 billion SVF cells in 250 cc of normal saline, for IV injection, and less than 1 billion SVF, for intra-articular and spinal injection. Adverse events were not severe and were treated successfully. Any observed adverse events were identified as a result of spinal or intra-articular injections and were not related to SVF or PRP. CONCLUSIONS: Our results showed that administration of high dose of SVF until 10 billion cells in a majority of 421 patients through infusion, spinal, and intra-articular injection was feasible without causing major adverse events and should be further investigated in well-designed phase I-II clinical trial to address the safety and efficacy of therapy.

In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold.

BACKGROUND: Cartilage tissue engineering is a promising technique for repairing cartilage defect. Due to the limitation of cell number and proliferation, mesenchymal stem cells (MSCs) have been developed as a substitute to chondrocytes as a cartilage cell-source. This study aimed to develop cartilage tissue from human adipose-derived stem cells (ADSCs) cultured on a Bombyx mori silk fibroin scaffold and supplemented with 10% platelet-rich plasma (PRP). METHODS: Human ADSCs and PRP were characterized. A silk fibroin scaffold with 500 µm pore size was fabricated through salt leaching. ADSCs were then cultured on the scaffold (ADSC-SS) and supplemented with 10% PRP for 21 days to examine cell proliferation, chondrogenesis, osteogenesis, and surface marker expression. The messenger ribonucleic acid (mRNA) expression of type 2 collagen, aggrecan, and type 1 collagen was analysed. The presence of type 2 collagen confirming chondrogenesis was validated using immunocytochemistry. The negative and positive controls were ADSC-SS supplemented with 10% foetal bovine serum (FBS) and ADSC-SS supplemented with commercial chondrogenesis medium, respectively. RESULTS: Cells isolated from adipose tissue were characterized as ADSCs. Proliferation of the ADSC-SS PRP was significantly increased (p < 0.05) compared to that of controls. Chondrogenesis was observed in ADSC-SS PRP and was confirmed through the increase in glycosaminoglycans (GAG) and transforming growth factor-ß1 (TGF-ß1) secretion, the absence of mineral deposition, and increased surface marker proteins on chondrogenic progenitors. The mRNA expression of type 2 collagen in ADSC-SS PRP was significantly increased (p < 0.05) compared to that in the negative control on days 7 and 21; however, aggrecan was significantly increased on day 14 compared to the controls. ADSC-SS PRP showed stable mRNA expression of type 1 collagen up to 14 days and it was significantly decreased on day 21. Confocal analysis showed the presence of type 2 collagen in the ADSC-SS PRP and positive control groups, with high distribution outside the cells forming the extracellular matrix (ECM) on day 21. CONCLUSION: Our study showed that ADSC-SS with supplemented 10% PRP medium can effectively support chondrogenesis of ADSCs in vitro and promising for further development as an alternative for cartilage tissue engineering in vivo.

Combination of the stromal vascular fraction and platelet-rich plasma accelerates the wound healing process: pre-clinical study in a Sprague-Dawley rat model.

BACKGROUND: Adipose-derived stem cells (ADSCs) improve wound healing owing to their angiogenic potency. However, the production of large quantities of ADSCs for clinical applications is laborious. In this study, the efficacy of the stromal vascular fraction (SVF; non-expanded ADSCs) combined with platelet-rich plasma (PRP) which contains abundant growth factors, for wound healing was evaluated using an animal model. METHODS: PRP from venous blood and SVF from lipoaspirates were harvested from six donors. PRP, SVF, SVF + PRP, and saline solution as a negative control were injected to second degree burn wounds in the backs of 24 male Sprague-Dawley rats. On the seventh day after injection, rats were euthanized and wounds were analyzed microscopically and macroscopically. RESULTS: Wounds closed faster in the SVF + PRP group than in the control group or PRP or SVF alone groups, with less inflammation, prominent signs of re-epithelization, more skin appendages and blood vessels, and a higher rate of hair growth. No infection or rat death occurred during the trial. CONCLUSIONS: The combination of SVF and PRP may provide an additive stimulatory effect to support angiogenesis and accelerate the wound healing process; accordingly, this combination is a potential alternative to ADSC treatment.

Evaluation of platelet-rich plasma from diabetic donors shows increased platelet vascular endothelial growth factor release.

BACKGROUND: Platelet-rich plasma (PRP) contains pro-angiogenic growth factors including vascular endothelial growth factor (VEGF). Angiogenesis is a necessary component of wound healing in instances of diabetic foot ulcers (DFU). PRP composition varies depending on methods and donor health status. Our group has developed an improved PRP protocol for diabetes treatment. The aims of this study were to examine the levels of the pro-angiogenic factor VEGF in these patient populations with and without diabetes. METHODS: PRP was prepared using 24 mL of whole blood from 13 diabetic and 10 non-diabetic patients registered at Klinik Hayandra. Whole blood in sodium citrate tubes were centrifuged at 1,000 rpm for 5 minutes followed by plasma separation. Plasma samples were centrifuged at 3,000 rpm for 5 minutes. Upper platelet-poor plasma layers were discarded, leaving 5 mL of concentrated platelet containing plasma (PRP). Concentrated plasma samples were mixed, aliquoted, stored at -86 °C, and pooled for platelet count, VEGF, and total protein analyses. Platelet counting was also performed using fresh whole blood and PRP to measure changes following PRP preparation. RESULTS: Diabetic donors had higher whole blood platelet counts than non-diabetic donors, but this difference was not statistically significant. An average increase of more than 250% in platelet number after PRP preparation using our method was noted in both groups. Freezing-thawing samples at -86 °C lysed more than 90% of PRP platelets regardless of diabetes status. Diabetic PRP had lower mean total protein and higher VEGF concentrations. Lysed platelets from diabetic donors released more VEGF than those from non-diabetic donors. CONCLUSIONS: PRP from diabetic donors had higher VEGF content making autologous PRP application a promising treatment for DFU. However, this should be investigated another appropriate clinical trial.

Apoptosis of Adipose-Derived Stem Cells Induced by Liposomal Soybean Phosphatidylcholine Extract.

BACKGROUND: Recently, Phosphatidylcholine (PC) has been used as an off-label treatment for lipolysis injection, which is associated with inflammatory reaction due to sodium deoxycholate, an emulsifier, so that inflammation as side effect occurs in those patients. Liposome formulation from soybean lipid was thought to be a better and safer alternative. This study aimed to analyze the mechanism of Liposomal Soybean Phosphatidylcholine (LSPC) extract from Indonesian soybeans (containing 26% PC) to induce Adipose-derived Stem Cells (ASCs) death in vitro. METHODS: Liposomes were prepared using thin film hydration method followed by a stepwise extrusion process to produce a small amount of 41.0-71.3 nm. Liposomal soybean phosphatidylcholine extract (LSPCE), liposomal purified PC (LPCC), and solution of PC+SD were used for comparison. Annexin V fluorescein Isothiocyanate/Propidium Iodide (FITC/PI) double staining by flow cytometry and also measurement of caspase-3 activity using ELISA were used to quantify the rate of apoptosis. ASCs viability was measured using MTT assay after induction with liposomes. Morphological changes were shown using a phase-contrast, inverted microscope and Transmission-Electron Microscope (TEM). RESULTS: The flow cytometry results showed that cells treated with both LSPCE and LPCC showed increase in early apoptosis beginning at 6 hr after incubation, which was confirmed by caspase 3 measurement. MTT assay showed that both LSPCE and LPCC could decrease viability of cells. Cells treated with LSPCE and LPCC showed some rounded cells, which was an early sign of cell death. Cells treated with SD showed extensive membrane damage with necrosis features using TEM. CONCLUSION: The results above demonstrated that LSPCE induced apoptosis of ASCs.