BackgroundCartilage 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).MethodsHuman 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.ResultsCells 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-β1 (TGF-β1) 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.ConclusionOur 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.

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.

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.

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-1β (IL-1β) 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-1β 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-1β 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-1β concentration was 4.71 pg/mL and 3.095 pg/mL, respectively. After 2 treatments with aaPRP, severe patients’ plasma IL-1β concentration decreased 12.48 pg/mL, while critical patients’ plasma IL-1β 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-1β concentration and the amelioration of PaO2/FiO2 ratio.

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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 ℃, 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 ℃ 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.

Background: Microtia is a congenital malformation in the external ear due to cartilage defect. Adipose-derived stem cells (ADSC) is promising cells to develop cartilage tissue engineering for microtia. In this study, we focused on proliferation and chondrogenesis of ADSC in three different media, which consist of 10% fetal bovine serum (FBS), 10% FBS with L-ascorbic acid, and 10% human platelet rich plasma (PRP). Methods: ADSC were induced to differentiate into adipocytes, chondrocyte and osteocytes. ADSC morphology, proliferation and population doubling time was compared in three different media and analysed. Observation and alcian blue staining were done every 7 days to assess chondrogenic potency of ADSC from each treatment.Results: Isolated ADSC were able to differentiate into adipocytes, osteocytes and chondrocytes. ADSC in all group have fibroblast-like morphology, but cells in 10% FBS and 10% FBS with LAA group were flattened and larger. ADSC in 10% PRP group proliferates faster than 10% FBS with and without LAA. PDT values of ADSC were 34 hours, 44 hours and 48 hours, respectively for 10% PRP, 10% FBS with LAA and 10% FBS group. Alcian blue staining revealed that ADSC in 10% FBS with LAA and 10% PRP were able to proceed to chondrogenesis when cultured time were prolong up to 21 days, but not with ADSC in 10% FBS. Conclusion: We conclude that adding 10% FBS with LAA or 10% PRP into medium culture can support proliferation and chondrogenesis of ADSC. Key words: human ADSC, PRP, L-ascorbic acid, proliferation, chondrogenesis

Introduction: Hyperglycemia in diabetic patients induces elevated pro-inflammatory cytokine production, resulting in cellular damage, which may affect the regenerative function of mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs). Identifying the effect of diabetes on ADSCs and optimization of culture conditions is therefore an important starting point for the application of autologous stem cells to improve clinicial outcomes. The aim of this study was to investigate the effect of diabetes on ADSCs that cultured in low-glucose anti-oxidant-serum supplemented medium. Methods: In this study, freshly isolated stromal vascular fraction (SVF) and expanded ADSCs were compared between diabetic and non-diabetic donors. SVF were isolated from the abdominal fat, and total viable cells and viability were estimated. Fresh SVF were cultured in low-glucose (100 mg/dL) culture medium supplemented with an anti-oxidant and fetal bovine serum (complete culture medium) at a low density for 14 days for the colony formation unit-fibroblast (CFU-F) assay. The remaining SVF were expanded to obtain ADSCs in the complete culture medium, which were evaluated based on MSCs surface marker expression and three lineage differentiation potential. Diabetic and non-diabetic ADSCs were compared with respect to population doubling time and viability after serial passage. Results: Total viable counts (0.97 +/- 0.39 x 109 cells/10 mL of adipose tissue, 0.56 +/- 0.39 x 109 cells/10 mL of adipose tissue, p=0.02, independent t-test), but not viability (98.63 +/- 1.12%, 98.20 +/- 1.21%, p= 0.38, independent t-test), were significantly higher for SVF cells from adipose tissues of non-diabetic donors than diabetic donors. Fewer CFU-F were obtained from cultured diabetic SVF than from non-diabetic SVF. Diabetic and non-diabetic ADSCs had similar differentiation potency and CD73 (99.44 +/- 0.34%, 97.15 +/- 5.37%, p= 0.21, Mann-Whitney U test) and CD90 (97.30 +/- 2.86%, 95.06 +/- 6.32%, p= 0.90, Mann- Whitney U test) expression, but significantly fewer diabetic ADSCs expressed CD105 or endoglin, a marker for angiogenesis (89.91 +/- 7.14%, 57.90 +/- 21.36% for non-diabetic and diabetic groups, p< 0.001, Mann-Whitney U test). Diabetic ADSCs tended to exhibit slower proliferation (4.43 +/- 2.70 days, 3.04 +/- 0.55 days, p= 0.27 in passage 2 (P2); 3.95 +/- 1.55 days, 2.96 +/- 0.91 days, p= 0.21 in P3, independent t-test) and lower viability than those of non-diabetic ADSCs (77.65 +/- 10.61%, 87.13 +/- 10.06%, p= 0.25 in P2; 82.70 +/- 8.07%, 91.15 +/- 3.77%, p= 0.04 in P3, independent t-test). Culture in low-glucose anti-oxidant-serum supplemented medium did not improve CD105 expression (65.14 +/- 5.86%, 71.06 +/- 10.27%, 64.05 +/- 10.04%, p= 0.70, for P1, P2, and P3, respectively, repeated measure ANOVA) and cell proliferation (p= 0.50 for P2 vs. P3, paired t-test) of diabetic ADSCs. Conclusions: Overall, diabetes reduced CD105 expression and ADSCs proliferation, suggesting that the angiogenic potency of diabetic ADSCs is reduced. The diabetic ADSCs in this study were also more prone to cell death caused by handling technique compared to non-diabetic ADSCs. Therefore, more advanced culture techniques should be applied to expand ADSCs from diabetic patients to achieve expected clinical outcomes.