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Blood-derived products, such as citrate platelet-rich plasma (CPRP) and hyperacute serum (HAS), are recognized for their rich growth factor content. When human dermal fibroblast (HDF) cells are exposed to combined mitogenic and DNA-damaging stimuli, it can lead to an increased burden of senescent cells and a modified senescence-associated secretory phenotype. In this study, the senescent state was comprehensively assessed through various methods, including phosphorylated histone H2AX (γH2AX) staining, p21 and p16 q-PCR, p21-western blot, growth curves, and senescence-associated ß-galactosidase staining. Two primary treatments with blood products were administered, one early (immediately after etoposide) and the other late (11 days after etoposide treatment). The effects of the blood product treatment were evaluated by measuring interleukin 6 and 8 (IL-6 and IL-8) levels, as well as collagen 1 (COL1) and p21 mRNA expression. Additionally, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays, cell size measurements, viability assays, and cell number calculations were conducted. The results revealed that cells treated with hyperacute serum in the early treatment phase exhibited the lowest observed IL-6 and IL-8 levels. In contrast, a clear inflammatory response for IL-8 was observed in cells treated with hyperacute serum and citrate platelet-rich plasma during the late treatment. Furthermore, an upregulation of COL1 expression was observed in the early treatment, while cells in the late treatment group remained unaffected. Notably, citrate platelet-rich plasma-treated cells showed a decrease in COL1 expression. Overall, the treatment with blood products appears to have slightly positive effects on skin rejuvenation.

We have previously reported that serum albumin-coated bone allograft (BoneAlbumin, BA) is an effective bone substitute. It improves bone regeneration at the patellar and tibial donor sites six months after harvesting bone-patellar tendon-bone (BPTB) autografts for primary anterior cruciate ligament reconstruction (ACLR). In the present study, we examined these donor sites seven years after implantation. The study group (N = 10) received BA-enhanced autologous cancellous bone at the tibial and BA alone at the patellar site. The control group (N = 16) received autologous cancellous bone at the tibial and blood clot at the patellar site. We evaluated subcortical density, cortical thickness, and bone defect volume via CT scans. At the patellar site, subcortical density was significantly higher in the BA group at both time points. There was no significant difference in cortical thickness between the two groups at either donor site. The control group's bone defect significantly improved and reached the BA group's values at both sites by year seven. Meanwhile, the bone defects in the BA group did not change significantly and were comparable to the six-month measurements. No complications were observed. There are two limitations in this study: The number of patients recruited is small, and the randomization of the patients could have improved the quality of the study as the control group patients were older compared to the study group patients. Our 7-year results seem to demonstrate that BA is a safe and effective bone substitute that supports faster regeneration of donor sites and results in good-quality bone tissue at the time of ACLR with BPTB autografts. However, studies with a larger number of patients are required to definitively confirm the preliminary results of our study.

Introduction: Professional athletes are endangered by COVID-19 and belong to the high-risk population due to their lifestyle. To obtain information on the behavior of COVID-19 in professional athletes, serological, cytokine, and virus neutralization capacities were analyzed., Materials and Methods: Hungarian national teams participated in international sports events during the early phases of the COVID-19 epidemic in 2020. Altogether, 29 professional athletes volunteered to donate plasma. Their serological status was evaluated by IgA, IgM, and IgG ELISAs and the highest virus neutralization titer in an in vitro live tissue assay. Plasma cytokine patterns were analyzed with a Bioplex multiplex ELISA system., Results: Surprisingly, only one athlete (3%) had anti-SARS-CoV-2 IgG, while IgA was more common (31%). Neither plasma showed direct virus neutralization in a titer over 1:10; hence, they were not suitable for reconvalescent treatment. The 'cytokine storm' markers IL-6 and IL-8 were at baseline levels. In contrast, either the TNF-alpha-related cytokines or the IFN-gamma-associated cytokines were elevated. There was a strong negative correlation between the TNF-alpha- or IFN-gamma-related cytokines., Conclusions: Professional athletes are susceptible to the SARS-CoV-2 infection without developing long-term immunity through neutralizing immunoglobulins. Elevated secretory and cellular immunity markers indicate that these systems are probably responsible for virus elimination in this subpopulation.

Albumin is a constitutional plasma protein, with well-known biological functions, e.g., a nutrient for stem cells in culture. However, albumin is underutilized as a biomaterial in regenerative medicine. This review summarizes the advanced therapeutic uses of albumin, focusing on novel compositions that take advantage of the excellent regenerative potential of this protein. Albumin coating can be used for enhancing the biocompatibility of various types of implants, such as bone grafts or sutures. Albumin is mainly known as an anti-attachment protein; however, using it on implantable surfaces is just the opposite: it enhances stem cell adhesion and proliferation. The anticoagulant, antimicrobial and anti-inflammatory properties of albumin allow fine-tuning of the biological reaction to implantable tissue-engineering constructs. Another potential use is combining albumin with natural or synthetic materials that results in novel composites suitable for cardiac, neural, hard and soft tissue engineering. Recent advances in materials have made it possible to electrospin the globular albumin protein, opening up new possibilities for albumin-based scaffolds for cell therapy. Several described technologies have already entered the clinical phase, making good use of the excellent biological, but also regulatory, manufacturing and clinical features of serum albumin.

Hyperacute serum (HAS) is a blood derivative product that promotes the proliferation of various cell types and controls inflammation in vitro. The aim of this study is to investigate the regenerative potential of different formulations of HAS, including lyophilized and hyaluronic acid combined versions, to obtain a stable and standardized therapeutic in osteoarthritis (OA), which may be able to overcome the variability limitations of platelet-rich plasma (PRP). Primary human osteoarthritic chondrocytes were used for testing cellular viability and gene expression of OA-related genes. Moreover, a co-culture of human explants of cartilage, bone and synovium under inflammatory conditions was used for investigating the inflammatory control capacities of the different therapeutics. In this study, one formulation of lyophilized HAS achieved the high cell viability rates of liquid HAS and PRP. Gene expression analysis showed that HAS induced higher Col1a1 expression than PRP. Cytokine quantification from supernatant fluids revealed that HAS treatment of inflamed co-cultures significantly reduced levels of IL-5, IL-15, IL-2, TNFα, IL-7 and IL-12. To conclude, lyophilized HAS is a stable and standardized therapeutic with high potential in joint regeneration.

Intra-articular injection of different types of blood-derived products is gaining popularity and clinical importance in the treatment of degenerative cartilage disorders such as osteoarthritis. The regenerative potential of two types of platelet-rich plasma (PRP), prepared in the presence of EDTA (EPRP) and citrate (CPRP) and an alternative blood product-hyperacute serum (hypACT) was evaluated using a 3D osteoarthritic chondrocyte pellet model by assessing the metabolic cell activity, cartilage-related gene expression and extracellular matrix deposition within the pellets. Chondrocyte viability was determined by XTT assay and it revealed no significant difference in metabolic activity of OA chondrocyte pellets after supplementation with different blood products. Nevertheless, the selection of blood products influenced the cartilage-related genes expression, ECM morphology and the tissue quality of pellets. Both PRP types had a different biological effect depending upon concentration and even though CPRP is widely used in clinics our assessment did not reveal good results in gene expression either tissue quality. HypACT supplementation resulted in superior cartilage-related genes expression together with tissue quality and seemed to be the most stable product since no remarkable changes were observed between the two different concentrations. All in all, for successful regenerative therapy, possible molecular mechanisms induced by blood-derived products should be always carefully investigated and adapted to the specific medical indications.

The serum fraction of platelet-rich fibrin (hyperacute serum) has been shown to improve cartilage cell proliferation in in vitro osteoarthritic knee joint models. We hypothesize that hyperacute serum may be a potential regenerative therapeutic for osteoarthritic knees. In this study, the cytokine milieu at the synovial fluid of osteoarthritic knee joints exposed to hyperacute serum intraarticular injections was investigated. Patients with knee osteoarthritis received three injections of autologous hyperacute serum; synovial fluid was harvested before each injection and clinical monitoring was followed-up for 6 months. Forty osteoarthritic-related cytokines, growth factors and structural proteins from synovial fluid were quantified and analysed by Multivariate Factor Analysis. Hyperacute serum provided symptomatic relief regarding pain and joint stability for OA patients. Both patients "with" and "without effusion knees" had improved VAS, KOOS and Lysholm-Tegner scores 6 months after of hyperacute serum treatment. Synovial fluid analysis revealed two main clusters of proteins reacting together as a group, showing strong and significant correlations with their fluctuation patterns after hyperacute serum treatment. In conclusion, hyperacute serum has a positive effect in alleviating symptoms of osteoarthritic knees. Moreover, identified protein clusters may allow the prediction of protein expression, reducing the number of investigated proteins in future studies.

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. One major driver of OA is inflammation, in which cytokines such as IL-6, TNF-α and IL-1β are secreted by activated chondrocytes, as well as synovial cells-including macrophages. Intra-articular injection of blood products-such as citrate-anticoagulated plasma (CPRP), hyperacute serum (hypACT), and extracellular vesicles (EVs) isolated from blood products-is gaining increasing importance in regenerative medicine for the treatment of OA. A co-culture system of primary OA chondrocytes and activated M1 macrophages was developed to model an OA joint in order to observe the effects of EVs in modulating the inflammatory environment. Primary OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from voluntary healthy donors and the monocytic cell line THP-1 were differentiated and activated into proinflammatory M1 macrophages. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot. Gene expression analysis of chondrocytes by RT-qPCR revealed increased type II collagen expression, while cytokine profiling via ELISA showed lower TNF-α and IL-1β levels associated with EV treatment. In conclusion, the inflammation model provides an accessible tool to investigate the effects of blood products and EVs in the inflammatory context of OA.

One option to fight joint degradation and inflammation in osteoarthritis is the injection of activated blood products into the synovial space. It has been demonstrated that hyperacute serum is the most proliferative among plasma products, so we investigated how the cytokine milieu of osteoarthritic knee joint reacts to hyperacute serum treatment in vitro. Cartilage, subchondral bone, and synovial membrane explanted from osteoarthritic knees were stimulated by interleukin-1 beta (IL-1β) and the concentration of 39 biomarkers was measured in the co-culture supernatant after hyperacute serum treatment. The IL-1β stimulation triggered a strong inflammatory response and enhanced the concentrations of matrix metalloproteinase 3 and 13 (MMP-3 and MMP-13), while hyperacute serum treatment reduced inflammation by decreasing the concentrations of IL-1β, tumor necrosis factor alpha (TNF-α), interleukin-6 receptor alpha (IL-6Rα), and by increasing the level of interleukin-1 antagonist (IL-1RA) Cell viability increased by day 5 in the presence of hyperacute serum. The level of MMPs-1, 2, and 9 were higher on day 3, but did not increase further until day 5. The concentrations of collagen 1 alpha 1 (COL1A1) and osteonectin were increased and receptor activator of nuclear factor kappa-B ligand (RANKL) was reduced in response to hyperacute serum. We concluded that hyperacute serum treatment induces cell proliferation of osteoarthritic joint tissues and affects the cytokine milieu towards a less inflamed state.

Autologous blood derived products, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are widely applied in regenerative therapies, in contrast to the drawbacks in their application, mainly deriving from the preparation methods used. Eliminating the disadvantages of both PRP and PRF, hyperacute serum (HAS) opens a new path in autologous serum therapy showing similar or even improved regenerative potential at the same time. Despite the frequent experimental and clinical use of PRP and HAS, their protein composition has not been examined thoroughly yet. Thus, we investigated and compared the composition of HAS, serum, PRP and plasma products using citrate and EDTA by simple laboratory tests, and we compared the composition of HAS, serum, EDTA PRP and plasma by Proteome Profiler and ELISA assays. According to our results the natural ionic balance was upset in both EDTA and citrate PRP as well as in plasma. EDTA PRP contained significantly higher level of growth factors and cytokines, especially platelet derived angiogenic and inflammatory proteins, that can be explained by the significantly higher number of platelets in EDTA PRP. The composition analysis of blood derivatives revealed that although the preparation method of PRP and HAS were similar, the ionic and protein composition of HAS could be advantageous for cell function.

Matrix-assisted autologous chondrocyte transplantation (MACT) for focal articular cartilage defects often fails to produce adequate cartilage-specific extracellular matrix in vitro and upon transplantation results in fibrocartilage due to dedifferentiation during cell expansion. This study aimed to redifferentiate the chondrocytes through supplementation of blood-products, such as hyperacute serum (HAS) and platelet-rich plasma (PRP) in vitro. Dedifferentiated monolayer chondrocytes embedded onto collagen type I hydrogels were redifferentiated through supplementation of 10% HAS or 10% PRP for 14 days in vitro under normoxia (20% O₂) and hypoxia (4% O₂). Cell proliferation was increased by supplementing HAS for 14 days ( p < 0.05) or by interchanging from HAS to PRP during Days 7⁻14 ( p < 0.05). Sulfated glycosaminoglycan (sGAG) content was deposited under both HAS, and PRP for 14 days and an interchange during Days 7⁻14 depleted the sGAG content to a certain extent. PRP enhanced the gene expression of anabolic markers COL2A1 and SOX9 ( p < 0.05), whereas HAS enhanced COL1A1 production. An interchange led to reduction of COL1A1 and COL2A1 expression marked by increased MMP13 expression ( p < 0.05). Chondrocytes secreted less IL-6 and more PDGF-BB under PRP for 14 days ( p < 0.0.5). Hypoxia enhanced TGF-β1 and BMP-2 release in both HAS and PRP. Our study demonstrates a new approach for chondrocyte redifferentiation.

Platelet-rich fibrin (PRF) membrane is a three-dimensional biodegradable biopolymer, which consists of platelet derived growth factors enhancing cell adhesion and proliferation. It is widely used in soft and hard tissue regeneration, however, there are unresolved problems with its clinical application. Its preparation needs open handling of the membranes, it degrades easily, and it has a low tensile strength which does not hold a suture blocking wider clinical applications of PRF. Our aim was to produce a sterile, suturable, reproducible PRF membrane suitable for surgical intervention. We compared the biological and mechanical properties of PRF membranes created by the classical glass-tube and those that were created in a single-syringe closed system (hypACT Inject), which allowed aseptic preparation. HypACT Inject device produces a PRF membrane with better handling characteristics without compromising biological properties. Freeze-thawing resulted in significantly higher tensile strength and higher cell adhesion at a lower degradation rate of the membranes. Mesenchymal stem cells seeded onto PRF membranes readily proliferated on the surface of fresh, but even better on freeze/thawed or freeze-dried membranes. These data show that PRF membranes can be made sterile, more uniform and significantly stronger which makes it possible to use them as suturable surgical membranes.

Biodegradable scaffolds are widely used to transplant stem cells into various tissues. Recent studies showed that living stem cells can be attached to the surface of absorbable sutures in vitro . Soaking the absorbable material polyglactin in a cell culture medium and thereby creating a stem cell biofilm on its surface may initiate the absorption process even before implantation; therefore, the physicochemical properties of the suture may be compromised in vivo . We found that pre-incubation of sutures in cell culture media in vitro results in tensile strength reduction and faster suture absorption in a rat model of muscle injury. Shorter incubation times of up to 48 h do not influence absorption or tensility; therefore, it is advisable to limit incubation times to two days for polyglactin-based cell delivery protocols.