Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Léčba diabetických ran je problematická, časově i finančně náročná a omezující pro pacienty. Krevní destičky jsou fyziologicky přítomné v procesu hojení akutní rány, kde produkují růstové a angiogenní faktory, cytokiny, chemokiny, molekuly zprostředkující adhesi buněk a další bioaktivní látky napomáhající hojení. Lyzát připravený z plazmy obohacené o krevní destičky bude zabudován do degradovatelných i nedegradovatelných nanovlákenných nosičů připravených ze syntetických i přirozených polymerů dále modifikovaných fibrinem, a to s cílem postupného uvolňování růstových faktorů a dalších biologicky aktivních látek. Bioaktivita těchto nanovlákenných krytů bude hodnocena in vitro v kulturách lidských keratinocytů, dermálních fibroblastů, cévních endotelových buněk a kmenových buněk tukové tkáně. Nanovlákenné kryty s nejlepšími vlastnostmi budou aplikovány in vivo na rány u diabetických potkanů, a následně bude hodnocena jejich schopnost stimulace hojení ran.; Treatment of diabetic wounds is problematic, long-lasting, expensive and restrictive for patients. Platelets are physiologically present in the process of healing of acute wounds, where they secret growth and angiogenic factors, cytokines, chemokines, cell adhesion-mediating molecules and other bioactive substances that support healing. Lysate prepared from platelet-rich plasma will be incorporated into degradable and non-degradable nanofibrous dressings, prepared from synthetic and natural polymers further modified with fibrin, in order to assure controlled release of growth factors and other bioactive substances. Bioactivity of these dressings will be evaluated in vitro in cultures of human keratinocytes, dermal fibroblasts, vascular endothelial cells and adipose tissue-derived stem cells. Nanofibrous dressings with the best properties will be applied in vivo on wounds of diabetic rats, and their ability to stimulate wound healing will be evaluated.
- Keywords
- řízené uvolňování, diabetické rány, lyzát z trombocytů, diabetický potkan, hojení ran, wound healing, diabetic wounds, platelet lysate, control release, diabetic rat, plasma obohacená destičkami, epidermální keratinocyty, dermální fibroblasty, kmenové buňky tukové tkáně, platelet-rich plasma, epidermal keratinocytes, dermal fibroblasts, adipose-tissue stem cells,
- NML Publication type
- závěrečné zprávy o řešení grantu AZV MZ ČR
New studies have shown the great potential of the combination of in situ enzymatically cross-linked hydrogels based on tyramine derivative of hyaluronic acid (HA-TA) with platelet-rich plasma (PRP) and platelet lysate in regenerative medicine. This study describes how the presence of PRP and platelet lysate affects the kinetics of gelation, viscoelastic properties, swelling ratio, and the network structure of HA-TA hydrogels and how the encapsulation of PRP in hydrogels affects the bioactivity of released PRP determined as the ability to induce cell proliferation. The properties of hydrogels were tuned by a degree of substitution and concentration of HA-TA derivatives. The addition of platelet derivatives to the reaction mixture slowed down the cross-linking reaction and reduced elastic modulus (G') and thus cross-linking efficiency. However, low-swellable hydrogels (7-190%) suitable for soft tissue engineering with G' 200-1800 Pa were prepared with a gelation time within 1 min. It was confirmed that tested cross-linking reaction conditions are suitable for PRP incorporation because the total bioactivity level of PRP released from HA-TA hydrogels was ≥87% and HA-TA content in the hydrogels and thus mesh size (285-482 nm) has no significant effect on the bioactivity level of released PRP.
Osteoarthritisis a highly prevalent musculoskeletal disorder characterized by degradation of cartilage and synovial fluid (SF). Platelet derivatives as platelet-rich plasma (PRP) and platelet lysate have great potential in the treatment of osteoarthritis because they contain biologically active substances including growth factors (GFs). Rapid release of GFs and their short biological half-life are factors that can limit the therapeutic impact of PRP therapy. Herein, the first work that describes hydrogels based on polyaldehyde derivative of hyaluronic acid (HA-OX) as carriers of platelet derivatives for in situ applications is presented, which can be a possible solution to the problem. HA-OX hydrogels containing 50% (w/w) of PRP or platelet lysate can be injected using a syringe due to low viscosity(<10 Pa s) and injection force (<20 N), and reach elastic modulus up to 2000 Pa. Insulin-like GF-1 and Platelet-derived GF-AB release from HA-OX hydrogels (mesh size 297-406 nm) by Fickian and non-Fickian diffusion respectively. The released PRP GFs maintain their ability to induce cell proliferation (87%-92%). Based on the obtained results, the unique concept of a new material that can restore viscoelastic properties of SF and at the same time gradually deliver GFs from platelet derivatives is designed.
INTRODUCTION: The formation of diabetic ulcers (DU) is a common complication for diabetic patients resulting in serious chronic wounds. There is therefore, an urgent need for complex treatment of this problem. This study examines a bioactive wound dressing of a biodegradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) covered by a thin fibrin layer for sustained delivery of bioactive molecules. METHODS: Electrospun PLCL/PCL nanofibers were coated with fibrin-based coating prepared by a controlled technique and enriched with human platelet lysate (hPL), fibroblast growth factor 2 (FGF), and vascular endothelial growth factor (VEGF). The coating was characterized by scanning electron microscopy and fluorescent microscopy. Protein content and its release rate and the effect on human saphenous vein endothelial cells (HSVEC) were evaluated. RESULTS: The highest protein amount is achieved by the coating of PLCL/PCL with a fibrin mesh containing 20% v/v hPL (NF20). The fibrin coating serves as an excellent scaffold to accumulate bioactive molecules from hPL such as PDGF-BB, fibronectin (Fn), and α-2 antiplasmin. The NF20 coating shows both fast and a sustained release of the attached bioactive molecules (Fn, VEGF, FGF). The dressing significantly increases the viability of human saphenous vein endothelial cells (HSVECs) cultivated on a collagen-based wound model. The exogenous addition of FGF and VEGF during the coating procedure further increases the HSVECs viability. In addition, the presence of α-2 antiplasmin significantly stabilizes the fibrin mesh and prevents its cleavage by plasmin. DISCUSSION: The NF20 coating supplemented with FGF and VEGF provides a promising wound dressing for the complex treatment of DU. The incorporation of various bioactive molecules from hPL and growth factors has great potential to support the healing processes by providing appropriate stimuli in the chronic wound.
- MeSH
- alpha-2-Antiplasmin MeSH
- Endothelial Cells MeSH
- Wound Healing MeSH
- Humans MeSH
- Nanofibers * MeSH
- Bandages MeSH
- Polyesters pharmacology MeSH
- Vascular Endothelial Growth Factor A * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Despite several scientific or ethical issues, fetal bovine serum (FBS) remains the standard nutrient supplement in the mesenchymal stem cell cultivation medium. Cell amplification plays an important role in human stem cell therapies. Increasing interest in this field has supported attempts to find suitable human alternatives to FBS for in vitro cell propagation. Human platelet lysate (hPL) has recently been determined as one of them. Our study aimed to evaluate the influence of 2% hPL in the growth medium for in vitro expansion of human natal dental pulp stem cells (hNDP-SCs). The effect was determined on proliferation rate, viability, phenotype profile, expression of several markers, relative telomere length change, and differentiation potential of four lineages of hNDP-SCs. As a control, hNDP-SCs were simultaneously cultivated in 2% FBS. hNDP-SCs cultivated in hPL showed a statistically significantly higher proliferation rate in initial passages. We did not observe a statistically significant effect on mesenchymal stem cell marker (CD29, CD44, CD73, CD90) or stromal-associated marker (CD13, CD166) expression. The cell viability, relative telomere length, or multipotency remained unaffected in hNDP-SCs cultivated in hPL-medium. In conclusion, hPL produced under controlled and standardized conditions is an efficient serum supplement for in vitro expansion of hNDP-SCs.
- MeSH
- Cell Differentiation MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mesenchymal Stem Cells * MeSH
- Cell Proliferation MeSH
- Nutrients MeSH
- Dental Pulp * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Platelet concentrates and especially their further product platelet lysate, are widely used as a replacement for cell culturing. Platelets contain a broad spectrum of growth factors and bioactive molecules that affect cellular fate. However, the cellular response to individual components of the human platelet concentrate is still unclear. The aim of this study was to observe cellular behavior according to the individual components of platelet concentrates. The bioactive molecule content was determined. The cells were supplemented with a medium containing 8% (v/v) of platelet proteins in plasma, pure platelet proteins in deionized water, and pure plasma. The results showed a higher concentration of fibrinogen, albumin, insulin growth factor I (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), in the groups containing plasma. On the other hand, chemokine RANTES and platelet-derived growth factor bb (PDGF-bb), were higher in the groups containing platelet proteins. The groups containing both plasma and plasma proteins showed the most pronounced proliferation and viability of mesenchymal stem cells and fibroblasts. The platelet proteins alone were not sufficient to provide optimal cell growth and viability. A synergic effect of platelet proteins and plasma was observed. The data indicated the importance of plasma in platelet lysate for cell growth.
- MeSH
- Albumins MeSH
- Becaplermin metabolism MeSH
- Cell Culture Techniques methods MeSH
- Chemokines metabolism MeSH
- Fibrinogen metabolism MeSH
- Fibroblast Growth Factor 7 MeSH
- Fibroblasts metabolism MeSH
- Hepatocyte Growth Factor MeSH
- Insulin-Like Growth Factor I MeSH
- Plasma chemistry MeSH
- Culture Media chemistry MeSH
- Humans MeSH
- Mesenchymal Stem Cells metabolism MeSH
- Platelet-Rich Plasma metabolism MeSH
- Cell Proliferation drug effects MeSH
- Proto-Oncogene Proteins c-sis metabolism MeSH
- Blood Platelets chemistry metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.
- MeSH
- Adipose Tissue, Brown cytology metabolism MeSH
- Adipocytes, Brown cytology metabolism MeSH
- Scapula cytology metabolism MeSH
- Mice MeSH
- Proteins genetics metabolism MeSH
- Gene Expression Regulation * MeSH
- Cell Separation methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Video-Audio Media MeSH
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
In vitro and in vivo analyses are closely connected, and the reciprocal relationship between the two comprises a key assumption with concern to the conducting of meaningful research. The primary purpose of in vitro analysis is to provide a solid background for in vivo and clinical study purposes. The fields of cell therapy, tissue engineering, and regenerative medicine depend upon the high quality and appropriate degree of the expansion of mesenchymal stromal cells (MSCs) under low-risk and well-defined conditions. Hence, it is necessary to determine suitable alternatives to fetal bovine serum (FBS-the laboratory gold standard) that comply with all the relevant clinical requirements and that provide the appropriate quantity of high-quality cells while preserving the required properties. Human serum (autologous and allogeneic) and blood platelet lysates and releasates are currently considered to offer promising and relatively well-accessible MSC cultivation alternatives. Our study compared the effect of heat-inactivated FBS on MSC metabolism as compared to its native form (both are used as the standard in laboratory practice) and to potential alternatives with concern to clinical application-human serum (allogeneic and autologous) or platelet releasate (PR-SRGF). The influence of the origin of the serum (fetal versus adult) was also determined. The results revealed the key impact of the heat inactivation of FBS on MSCs and the effectiveness of human sera and platelet releasates with respect to MSC behaviour (metabolic activity, proliferation, morphology, and cytokine production).
- Publication type
- Journal Article MeSH
The essential historical knowledge and expertise developed over the past 5-6 decades on the safety / efficacy of conventional blood components therapy by blood transfusion establishments have guided the development of validated methods which have ensure optimal safety margins for frozen blood and its bioproducts with or even without pathogen reduction. Newer generations of pathogen reduced frozen red blood cell, plasma and platelet products and the standardised and safer pooling of human platelet lysate are now become available for potential clinical use. These types of whole blood-derived bioproducts not only reduce the risk of transmission of range of pathogenic blood-borne pathogen. As cryopreservation can be combined with PRT without significantly compromising in vitro quality characteristics or physiological capabilities, it allows us to maximize the available inventory of these blood products in both civil and military trauma settings. The main objective of this overview is to update readers and scientific / medical communities of the various building blocks needed to optimally grantee the pathogen safety of whole blood-derived bioproducts, with minimal untoward events to the recipients. While this is an emerging area, we are seeing the numerous potential opportunities that cryopreservation and pathogen inactivation can have on the transfused patient outcomes. This manuscript is informed by recent publications on this topic.
- MeSH
- Blood Transfusion methods MeSH
- Cryopreservation methods MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin-chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin-chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin-chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%-90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin-chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin-chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.
- Publication type
- Journal Article MeSH
