Water-Insoluble, Thermostable, Crosslinked Gelatin Matrix for Soft Tissue Implant Development.

In this present study, the material science background of crosslinked gelatin (GEL) was investigated. The aim was to assess the optimal reaction parameters for the production of a water-insoluble crosslinked gelatin matrix suitable for heat sterilization. Matrices were subjected to enzymatic degradation assessments, and their ability to withstand heat sterilization was evaluated. The impact of different crosslinkers on matrix properties was analyzed. It was found that matrices crosslinked with butanediol diglycidyl ether (BDDE) and poly(ethylene glycol) diglycidyl ether (PEGDE) were resistant to enzymatic degradation and heat sterilization. Additionally, at 1 v/v % crosslinker concentration, the crosslinked weight was lower than the starting weight, suggesting simultaneous degradation and crosslinking. The crosslinked weight and swelling ratio were optimal in the case of the matrices that were crosslinked with 3% and 5% v/v BDDE and PEGDE. FTIR analysis confirmed crosslinking, and the reduction of free primary amino groups indicated effective crosslinking even at a 1% v/v crosslinker concentration. Moreover, stress-strain and compression characteristics of the 5% v/v BDDE crosslinked matrix were comparable to native gelatin. Based on material science measurements, the crosslinked matrices may be promising candidates for scaffold development, including properties such as resistance to enzymatic degradation and heat sterilization.

Professional Athletes Maintain High TNF-Alpha or IFN-Gamma Related Inflammatory Status after Recovering from COVID-19 Infection without Developing a Neutralizing Antibody Response.

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 as a Biomaterial and Therapeutic Agent in Regenerative Medicine.

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.

Application of Injectable, Crosslinked, Fibrin-Containing Hyaluronic Acid Scaffolds for In Vivo Remodeling.

The present research aimed to characterize soft tissue implants that were prepared with the use of crosslinked hyaluronic acid (HA) using two different crosslinkers and multiple reagent concentrations, alone or in combination with fibrin. The effect of the implants was evaluated in an in vivo mouse model, after 4 weeks in one group and after 12 weeks in the other. The explants were compared using analytical methods, evaluating microscopic images, and a histology analysis. The kinetics of the degradation and remodeling of explants were found to be greatly dependent on the concentration and type of crosslinker; generally, divinyl sulfone (DVS) resists degradation more effectively compared to butanediol diglycidyl ether (BDDE). The presence of fibrin enhances the formation of blood vessels, and the infiltration of cells and extracellular matrix. In summary, if the aim is to create a soft tissue implant with easier degradation of the HA content, then the use of 2-5% BDDE is found to be optimal. For a longer degradation time, 5% DVS is the more suitable crosslinker. The use of fibrin was found to support the biological process of remodeling, while keeping the advances of HA in void filling, enabling the parallel degradation and remodeling processes.

Overview of Tissue Engineering Patent Strategies and Patents from 2010 to 2020, Including Outcomes.

In the regenerative medicine and tissue engineering (TE) field, it is often an overlooked and challenging aspect to fathom how TE-related basic research could be translated to applied and translational research, ultimately aiming to develop and market a product or service. The aim of the present article is to investigate the patents in the field of TE and to look up relevant patents, type of applicants, and outcomes of relevant patents over the last 10 years. Besides referencing these patents, it was also the aim to collect data on companies related to the relevant patents to investigate the current commercial status of the product or service that the patent relates to.

Early Transfusion of Convalescent Plasma Improves the Clinical Outcome in Severe SARS-CoV2 Infection.

INTRODUCTION: Plasma harvested from convalescent COVID-19 patients (CCP) has been applied as first-line therapy in the early phase of the SARS-CoV2 pandemic through clinical studies using various protocols. METHODS: We present data from a cohort of 267 hospitalized severe COVID-19 patients who received CCP. No transfusion-related complications were reported, indicating the overall safety of CCP therapy. RESULTS: Patients who eventually died from COVID-19 received CCP significantly later (3.95 versus 5.22 days after hospital admission) and had higher interleukin 6 (IL-6) levels (28.9 pg/ml versus 102.5 pg/ml) than those who survived. In addition, CCP transfusion caused a significant reduction in the overall inflammatory status of the patients regardless of the severity of disease or outcome, as evidenced by decreasing C-reactive protein, IL6 and ferritin levels. CONCLUSION: We conclude that CCP transfusion is a safe and effective supplementary treatment modality for hospitalized COVID-19 patients characterized by better expected outcome if applied as early as possible. We also observed that IL-6 may be a suitable laboratory parameter for patient selection and monitoring of CCP therapy effectiveness.

Cell Attachment Capacity and Compounds of Fibrin Membranes Isolated from Fresh Frozen Plasma and Cryoprecipitate.

Fibrin membranes are widely used in regenerative medicine because they are biocompatible, biodegradable, contain growth factors, and support cell attachment. Most commonly they are produced from serum, but they can also be isolated from activated plasma. To increase the fibrinogen concentration of plasma, cryoprecipitate isolation is a possible solution. In this work, cryoprecipitate was prepared from fresh frozen plasma, isolated by plasmapheresis. The concentration of cellular elements, fibrinogen, total protein, and immunoglobulins among others was measured in different concentrations of cryoprecipitates. After activation with Ca-gluconate, fibrin membranes were produced in different thicknesses, and human mesenchymal stem cells were seeded onto the membranes. They were visualized by live-dead staining and their viability was determined by XTT. The platelet-derived growth factor AB content was quantified by ELISA. Our results showed that fibrinogen and platelet concentration can be multiplied in plasma by cryoprecipitate isolation, which affects the thickness and slightly the growth factor content of the membranes. According to live-dead staining, the thickness of the membranes does not influence cell attachment, and XTT measurement did not reveal a significant difference in cell attachment capacity either; however, a growing trend could be observed in the case of some membranes.

A Pilot Clinical Study of Hyperacute Serum Treatment in Osteoarthritic Knee Joint: Cytokine Changes and Clinical Effects.

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.

Crosslinked Hyaluronic Acid Gels with Blood-Derived Protein Components for Soft Tissue Regeneration.

Hyaluronic acid (HA) is an ideal initial material for preparing hydrogels, which may be used as scaffolds in soft tissue engineering based on their advantageous physical and biological properties. In this study, two crosslinking agents, divinyl sulfone (DVS) and butanediol diglycidyl ether, were used to investigate their effect on the properties of HA hydrogels. As HA hydrogels alone do not promote cell adhesion on the scaffold, fibrin and serum from platelet-rich fibrin (SPRF) were combined with the scaffold; the aim was to create a material intended to be used as soft tissue implant that facilitates new tissue formation, and degrades over time. The chemical changes were characterized and cell attachment capacity of the protein-containing gels was examined using human mesenchymal stem cells, and viability was assessed using live-dead staining. Fourier-transform infrared measurements revealed that linking fibrin into the gel was more effective than linking SPRF. The scaffolds were found to be able to support cell adherence onto the hydrogels, and the best result was achieved when HA was crosslinked with DVS and contained fibrin. The most promising derivative, 5% DVS-crosslinked fibrin-containing hydrogel, was injected subcutaneously into C57BL/6 mice for 12 weeks. The scaffold was proven to be biocompatible, remodeling, and vascularization occurred, while shape and integrity were maintained. Impact statement Fibrin was combined with crosslinked hyaluronic acid (HA) for regenerative application, the structure of the combination of crosslinked HA with blood-derived protein was analyzed and effective coating was proven. It was observed that the fibrin content led to better mesenchymal stem cell attachment in vitro. The compositions showed biocompatibility, connective tissue and vascularization took place when implanted in vivo. Thus, a biocompatible, injectable gel was produced, which is a potential candidate for soft tissue implantation.

Ketamine decreases cell viability of bone explants and impairs bone healing in rats.

BACKGROUND: Ketamine is a widely used anesthetic in experimental medicine. We have also used ketamine for surgical interventions and imaging in rats and found significantly impaired ossification between identically performed experiments, which only differed in the number of anesthetic events. In order to investigate this phenomenon, we estimated the absorbed ionizing radiation and also studied whether ketamine administration has disadvantageous effect on bone cell viability. METHODS: Spongious bone chips and parietal bone disks were harvested from rats. Explants were incubated in stem cell media containing 0.02, 0.2 and 2 mM ketamine. After 3 days of incubation, tetrazolium-based spectrophotometric assay was performed to measure cell viability. Size-specific dose estimation was used to calculate ionizing radiation of computed tomography imaging. RESULTS: We found that ketamine supplementation with 0.2 mM slightly decreased cell viability, while 2 mM caused significant reduction both in the spongious and cortical explants. The cumulative ionizing radiation was found to be negligible compared to irradiation dosages used to impair ossification. CONCLUSIONS: We conclude that multiple ketamine administration was responsible for the diminished regenerative potential of bone tissue in the present experimental setup. For this reason, we suggest that ketamine anesthesia should be avoided in studies investigating bone regeneration.

Investigation of Cytokine Changes in Osteoarthritic Knee Joint Tissues in Response to Hyperacute Serum Treatment.

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-1ß) and the concentration of 39 biomarkers was measured in the co-culture supernatant after hyperacute serum treatment. The IL-1ß 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-1ß, 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.

Serum albumin-coated bone allograft (BoneAlbumin) results in faster bone formation and mechanically stronger bone in aging rats.

Serum albumin-coated bone allografts (BoneAlbumin) have successfully supported bone regeneration in various experimental models by activating endogenous progenitors. However, the effect of tissue aging, linked to declining stem cell function, has yet to be explicitly examined within the context of BoneAlbumin's regenerative capacity. Stem cell function was tested with an in vitro attachment assay, which showed that albumin coating increases stem cell attachment on demineralized bone surfaces in an aging cell population. Bone regeneration was investigated in vivo by creating critical size bone defects on the parietal bones of aging female rats. Demineralized bone matrices with and without serum albumin coating were used to fill the defects. Bone regeneration was determined by measuring the density and the size of the remaining bone defect with computed tomography (CT). Microcomputed tomography (MicroCT) and mechanical testing were performed on the parietal bone explants. In vivo CT and ex vivo microCT measurements showed better regeneration with albumin-coated grafts. Additionally, the albumin-coated group showed a twofold increase in peak fracture force compared with uncoated allografts. In the present study, serum albumin-coated demineralized bone matrices successfully supported faster and functionally superior bone regeneration in aging rats. Because stem cell function, a key contributor of bone remodelling, decreases with age and serum albumin is an effective activator of endogenous progenitor cells, this method could be an effective and safe adjuvant in bone regeneration of aging adult and osteo-compromised populations.

The Composition of Hyperacute Serum and Platelet-Rich Plasma Is Markedly Different despite the Similar Production Method.

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.

Biological and Mechanical Properties of Platelet-Rich Fibrin Membranes after Thermal Manipulation and Preparation in a Single-Syringe Closed System.

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.

Hyperacute serum has markedly better regenerative efficacy than platelet-rich plasma in a human bone oxygen-glucose deprivation model.

AIM: Platelet-rich plasma (PRP) and hyperacute serum (HAS) were compared in a novel human model of ex vivo bone damage induced by oxygen-glucose deprivation (OGD). MATERIALS & METHODS: Osteoarthritic subchondral bone pieces were harvested from discarded femoral heads during hip replacement surgery and subjected to transient OGD. RESULTS: Proteome profiling revealed that PRP is more angiopoietic, whereas HAS is more antiangiopoietic in composition. However, treatment of OGD-exposed bone with multiple PRP preparations had no effect on cell counts, whereas HAS restored cell proliferation capacity and rescued viable cell number following OGD. CONCLUSION: A similar pro-proliferation effect was observed with recombinant growth factors, indicating that HAS may be an alternative agent for enhancing the regeneration of damaged bone cells.

The Effects of Hyperacute Serum on the Elements of the Human Subchondral Bone Marrow Niche.

Mesenchymal stem cells (MSCs) are widely used in laboratory experiments as well as in human cell therapy. Their culture requires animal sera like fetal calf serum (FCS) as essential supplementation; however, animal sera pose a risk for clinical applications. Human blood derivatives, for example, platelet-rich plasma (PRP) releasates, are potential replacements of FCS; however, it is unclear which serum variant has the best effect on the given cell or tissue type. Additionally, blood derivatives are commonly used in musculoskeletal diseases like osteoarthritis (OA) or osteonecrosis as "proliferative agents" for the topical MSC pool. Hyperacute serum (HAS), a new serum derivative, has been designed to approximate the natural coagulation cascade with a single-step, additive-free preparation method. We investigated the effects of HAS on monolayer MSC cultures and in their natural niche, in 3D subchondral bone and marrow explants. Viability measurements, RT-qPCR evaluation for gene expression and flow cytometry for cell surface marker analysis were performed to compare the effects of FCS-, PRP-, or HAS-supplemented culture media. Monolayer MSCs showed significantly higher metabolic activity following 5 days' incubation in HAS, and osteoblast-specific mRNA expression was markedly increased, while cells also retained their MSC-specific cell surface markers. A similar effect was observed on bone and marrow explants, which was further confirmed with confocal microscopy analysis. Moreover, markedly higher bone marrow preservation was observed with histology in case of HAS supplementation compared to FCS. These findings indicate possible application of HAS in regenerative solutions of skeletal diseases like OA or osteonecrosis.

The Effects of Hyperacute Serum on Adipogenesis and Cell Proliferation of Mesenchymal Stromal Cells.

Fat tissue, due to its high concentration of stem cells, has a role in aesthetic medicine and reconstructive surgery. However, poor survival of the transplanted cells still limits the usefulness of this material in regenerative medicine. Several studies indicated that platelet-rich plasma (PRP) may improve adipose tissue viability due to its growth factor content. This study aimed at investigating the effects of PRP and hyperacute serum (HAS) on the adipogenic lineage in vitro. PRP was prepared by using two centrifugation steps in the presence of anticoagulants, and HAS was isolated from activated platelet-rich fibrin within 10 min of blood drawing to prevent the propagation of inflammatory cascades. Metabolic activity and proliferation rate of human bone marrow-derived mesenchymal stem cells (hMSCs) cultivated in media supplemented with three types of serum additives (fetal calf serum [FCS], human PRP, or HAS) was determined by using a tetrazolium assay. Adipogenesis was evaluated in standard and pro-adipogenic media and tested by oil red staining, triglyceride content, and expression of specific genes. Adipogenic regulators in the sera were measured by multiplex ELISA assays. We observed that proliferation of hMSCs was supported by both FCS and HAS in a time-dependent manner, but surprisingly, PRP had a much weaker effect (change in proliferation rate after 5 days relative to metabolic activity on day 0-FCS: 5.4-fold change, HAS: 5.8-fold change, serum free 1.9-fold change, PRP: 3.0-fold change, p < 0.05). Lipogenesis was only observed in groups with adipogenic differentiation medium, with HAS showing a significantly stronger effect than PRP. This was confirmed by intensive accumulation of lysochrome dye in lipid droplets, higher triglyceride concentration, and elevated expression of specific adipogenic genes. Measurement of lipogenic proteins in the sera revealed that both PRP and HAS are abundant in them; however, PRP also contains anti-adipogenic factors, which explains its weaker and less reliable effect. The results of this study suggest that HAS provides more robust support than PRP in hMSCs proliferation as well as lipogenic differentiation, indicating that it may be a better adjuvant in fat grafting procedures.

Serum albumin as a local therapeutic agent in cell therapy and tissue engineering.

Albumin is a major plasma protein that has become ubiquitous in regenerative medicine research. As such, many studies have examined its structure and advantageous properties. However, a systematic and comprehensive understanding of albumin's role, capabilities and therapeutic potential still eludes the field. In the present work, we review how albumin is applied in tissue engineering, including cell culture and storage, in vitro fertilization and transplantation. Furthermore, we discuss how albumin's physiological role extends beyond a carrier for metal ions, fatty acids, pharmacons and growth factors. Albumin acts as a bacteriostatic coating that simultaneously promotes attachment and proliferation of eukaryotic cells. These properties with the combination of free radical scavenging, neutrophil activation and as a buffer molecule already make the albumin protein beneficial in healing processes supporting functional tissue remodeling. Nevertheless, recent data revealed that albumin can be synthesized by osteoblasts and its local concentration is raised after bone trauma. Interestingly, by increasing the local albumin concentration in vivo, faster bone healing is achieved, possibly because albumin recruits endogenous stem cells and promotes the growth of new bone. These data also suggest an active role of albumin, even though a specific receptor has not yet been identified. Together, this discussion sheds light on why the extravascular use of the albumin molecule is in the scope of scientific investigations and why it should be considered as a local therapeutic agent in regenerative medicine. © 2016 BioFactors, 43(3):315-330, 2017.