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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.
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Plasma Rico en Plaquetas , Suero , Proteínas Sanguíneas/química , Fraccionamiento Químico , Humanos , Fibrina Rica en Plaquetas , Plasma Rico en Plaquetas/química , Suero/químicaRESUMEN
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.
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Cabeza Femoral/metabolismo , Plasma Rico en Plaquetas , Proteoma/metabolismo , Suero , Cabeza Femoral/citología , Glucosa/metabolismo , Humanos , Técnicas de Cultivo de Órganos , Oxígeno/metabolismoRESUMEN
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.
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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.
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Regeneración Ósea/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Criopreservación/métodos , Fertilización In Vitro/efectos de los fármacos , Trasplante de Órganos/métodos , Albúmina Sérica/farmacología , Regeneración Ósea/fisiología , Técnicas de Cultivo de Célula , Citocinas/química , Citocinas/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metales Pesados/química , Metales Pesados/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Ingeniería de TejidosRESUMEN
PURPOSE: Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. RESULTS: Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs' transplanted vessels. CONCLUSION: We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration.
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Blood serum fractions are hotly debated adjuvants in bone replacement therapies. In the present experiment, we coated demineralized bone matrices (DBM) with serum albumin and investigated stem cell attachment in vitro and bone formation in a rat calvaria defect model. In the in vitro experiments, we observed that significantly more cells adhere to the serum albumin coated DBMs at every time point. In vivo bone formation with albumin coated and uncoated DBM was monitored biweekly by computed tomography until 11 weeks postoperatively while empty defects served as controls. By the seventh week, the bone defect in the albumin group was almost completely closed (remaining defect 3.0 ± 2.3%), while uncoated DBM and unfilled control groups still had significant defects (uncoated: 40.2 ± 9.1%, control: 52.4 ± 8.9%). Higher density values were also observed in the albumin coated DBM group. In addition, the serum albumin enhanced group showed significantly higher volume of newly formed bone in the microCT analysis and produced significantly higher breaking force and stiffness compared to the uncoated grafts (peak breaking force: uncoated: 15.7 ± 4 N, albumin 46.1 ± 11 N). In conclusion, this investigation shows that implanting serum albumin coated DBM significantly reduces healing period in nonhealing defects and results in mechanically stronger bone. These results also support the idea that serum albumin coating provides a convenient milieu for stem cell function, and a much improved bone grafting success can be achieved without the use of exogenous stem cells.
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Materiales Biocompatibles Revestidos , Matriz Extracelular/química , Osteogénesis/efectos de los fármacos , Cráneo/lesiones , Células Madre/metabolismo , Animales , Técnica de Desmineralización de Huesos , Adhesión Celular , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Masculino , Ratas , Ratas Wistar , Albúmina Sérica , Cráneo/metabolismo , Cráneo/patologíaRESUMEN
The key drawback of using demineralized bone matrix (DBM) is its low initial mechanical stability due to the severe depletion of mineral content. In the present study, we investigated the long-term regeneration of DBM in a critical size bone defect model and investigated the remineralization after 6 months. Bone defects were created in the cranium of male Wistar rats which were filled with DBM or left empty as negative control. In vivo bone formation was monitored with computed tomography after 11, 19, and 26 weeks postoperatively. After 6 months, parietal bones were subjected to micro-CT. Mineral content was determined with spectrophotometric analysis. After 11 weeks the DBM-filled bone defects were completely closed, while empty defects were still open. Density of the DBM-treated group increased significantly while the controls remained unchanged. Quantitative analysis by micro-CT confirmed the in vivo results, bone volume/tissue volume was significantly lower in the controls than in the DBM group. The demineralization procedure depleted the key minerals of the bone to a very low level. Six months after implantation Ca, P, Na, Mg, Zn, and Cr contents were completely restored to the normal level, while K, Sr, and Mn were only partially restored. The remineralization process of DBM is largely complete by the 6th month after implantation in terms of bone density, structure, and key mineral levels. Although DBM does not provide sufficient sources for any of these minerals, it induces a faster and more complete regeneration process. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1336-1342, 2016.
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Calcificación Fisiológica , Matriz Extracelular/trasplante , Osteogénesis , Cráneo , Microtomografía por Rayos X , Animales , Estudios de Seguimiento , Masculino , Ratas , Ratas Wistar , Cráneo/diagnóstico por imagen , Cráneo/lesiones , Cráneo/metabolismo , Cráneo/patologíaRESUMEN
Exercise capacity and dietary restriction (DR) are linked to improved quality of life, including enhanced brain function and neuro-protection. Brain derived neurotrophic factor (BDNF) is one of the key proteins involved in the beneficial effects of exercise on brain. Low capacity runner (LCR) and high capacity runner (HCR) rats were subjected to DR in order to investigate the regulation of BDNF. HCR-DR rats out-performed other groups in a passive avoidance test. BDNF content increased significantly in the hippocampus of HCR-DR groups compared to control groups (p<0.05). The acetylation of H3 increased significantly only in the LCR-DR group. However, chip-assay revealed that the specific binding between acetylated histone H3 and BNDF promoter was increased in both LCR-DR and HCR-DR groups. In spite of these increases in binding, at the transcriptional level only, the LCR-DR group showed an increase in BDNF mRNA content. Additionally, DR also induced the activity of cAMP response element-binding protein (CREB), while the content of SIRT1 was not altered. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was elevated in HCR-DR groups. But, based on the levels of nuclear respiratory factor-1 and cytocrome c oxidase, it appears that DR did not cause mitochondrial biogenesis. The data suggest that DR-mediated induction of BDNF levels includes chromatin remodeling. Moreover, DR does not induce mitochondrial biogenesis in the hippocampus of LCR/HCR rats. DR results in different responses to a passive avoidance test, and BDNF regulation in LCR and HCR rats.
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Factor Neurotrófico Derivado del Encéfalo/genética , Epigénesis Genética , Conducta Alimentaria/fisiología , Hipocampo/metabolismo , Memoria a Corto Plazo/fisiología , Carrera/fisiología , Animales , Proteína de Unión a CREB/metabolismo , Histonas/metabolismo , Masculino , Factores Nucleares de Respiración/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Ratas , Sirtuina 1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The use of bone allografts is contraindicated in septic revision surgery due to the high risk of graft reinfection. Antibiotic release from the graft may solve the problem and these combinations can theoretically be used for prevention or even therapy of infection. The present study investigated whether amoxicillin, ciprofloxacin, and vancomycin alone or in combination with chitosan or alginate are suitable for short-term or long-term bone coating. Human bone allografts were prepared from femoral head and lyophilized. Antibiotic coating was achieved by incubating the grafts in antibiotic solution and freeze-drying again. Two biopolymers chitosan and alginate were used for creating sustained-release implantable coatings and the drug release profile was characterized in vitro by spectrophotometry. Using lyophilization with or without chitosan only resulted in short-term release that lasted up to 48 hours. Alginate coating enabled a sustained release that lasted for 8 days with amoxicillin, 28 days with ciprofloxacin coating, and 50 days with vancomycin coating. Using only implantable biodegradable allograft and polymers, a sustained release of antibiotics was achieved with ciprofloxacin and vancomycin for several weeks. Since the calculated daily release of the antibiotic was lower than the recommended IV dose, the calcium alginate coated bone graft can support endoprosthesis revision surgery.
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Aloinjertos/efectos de los fármacos , Materiales Biocompatibles , Trasplante Óseo/efectos adversos , Infecciones/tratamiento farmacológico , Amoxicilina/administración & dosificación , Quitosano/administración & dosificación , Quitosano/química , Ciprofloxacina/administración & dosificación , Humanos , Infecciones/patología , Polímeros/administración & dosificación , Polímeros/química , Vancomicina/administración & dosificaciónRESUMEN
We examined the susceptibility of fast and slow twitch muscle fibers in the quadriceps muscle to eccentric exercise-induced muscle damage. Nine healthy men (age: 22.5 ± 1.6 years) performed maximal eccentric quadriceps contractions at 120°·s-1 over a 120° of knee joint range of motion for 6 consecutive days. Biopsies were taken from the vastus lateralis muscle before repeated bouts of eccentric exercise on the third and seventh day. Immunohistochemical procedures were used to determine fiber composition and fibronectin activity. Creatine kinase (CK) and lactate dehydrogenase (LDH) were determined in serum. Average torque was calculated in each day for each subject. Relative to baseline, average torque decreased 37.4% till day 3 and increased 43.0% from the day 3 to day 6 (p < 0.001). Creatine kinase and LDH were 70.6 and 1.5 times higher on day 3 and 75.5 and 1.4 times higher on day 6. Fibronectin was found in fast fibers in subjects with high CK level on day 3 and 7 after exercise, but on day 7, fibronectin seemed in both slow and fast fibers except in muscles of 2 subjects with high fast fiber percentage. Peak torque and muscle fiber-type composition measured at baseline showed a strong positive association on day 3 (r = 0.76, p < 0.02) and strong negative association during recovery between day 3 and day 6 (r = -0.76, p < 0.02), and day 1 and day 6 (r = 0.84, p < 0.001). We conclude that the damage of fast fibers preceded the damage of slow fibers, and muscles with slow fiber dominance were more susceptible to repeated bouts of eccentric exercise than fast fiber dominance muscles. The data suggest that the responses to repeated bouts of eccentric exercise are fiber-type-dependent in the quadriceps muscle, which can be the basis for the design of individualized strength training protocols.
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Ejercicio Físico/fisiología , Fibras Musculares de Contracción Rápida/química , Fibras Musculares de Contracción Lenta/química , Músculo Cuádriceps/química , Músculo Cuádriceps/fisiopatología , Torque , Creatina Quinasa/sangre , Fibronectinas/análisis , Humanos , L-Lactato Deshidrogenasa/sangre , Masculino , Contracción Muscular , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Mialgia/fisiopatología , Músculo Cuádriceps/patología , Factores de Tiempo , Adulto JovenRESUMEN
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.
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Cell therapy holds the promise for a novel modality in the surgical toolkit; however, delivery of cells into damaged soft tissues constitutes a challenge. The authors hypothesized that growing stem cells on the surface of absorbable sutures in vitro and then implanting them via stitching would be a suitable delivery route for cell therapy. Fibronectin, poly-L-lysine, and albumin coatings were used to increase attachment of human and rat bone-marrow-derived mesenchymal stem cells (BMSC) to polyfilament absorbable sutures in vitro. Fluorescence microscopy was performed to localize the cells on the suture. After 48 hours of incubation, the albumin-coated sutures had the highest cell number, and after 168 hours cell number reached confluency. In the in vivo experiments, a 10-mm incision was made on the triceps surae muscle of male Wistar rats and rat BMSC coated sutures were placed into the muscle. Two days after the implantation, cells were seen on the surface of the sutures as well as in the surrounding muscle tissue. Long-term results at 5 weeks showed that transplanted cells survived and the sutures were partly absorbed. In conclusion, coating absorbable sutures with proteins, especially serum albumin, improves attachment and proliferation of cells, and only 48 hours in culture is enough to cover the sutures sufficiently. Using these stitches in vivo resulted in short-term and long-term survival of cells. As a result, albumin-coated suture can be a vehicle for stem cell therapy in soft tissues such as muscle, tendon, or peripheral nerves.
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Albúminas/química , Materiales Biocompatibles Revestidos/química , Trasplante de Células Madre/métodos , Células Madre/citología , Suturas , Albúminas/farmacología , Análisis de Varianza , Animales , Núcleo Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Masculino , Ratas , Ratas WistarRESUMEN
We have investigated the effects of 2 weeks of insulin-like growth factor-1 (IGF-1) supplementation (5 µg/kg per day) and 6 weeks of exercise training (60% of the maximal oxygen consumption [VO2 max]) on neurogenesis, DNA damage/repair, and sirtuin content in the hippocampus of young (3 months old) and old (26 months old) rats. Exercise improved the spatial memory of the old group, but IGF-1 supplementation eliminated this effect. An age-associated decrease in neurogenesis was attenuated by exercise and IGF-1 treatment. Aging increased the levels of 8-oxo-7,8-dihydroguanine (8-oxoG) and the protein Ku70, indicating the role of DNA damage in age-related neuropathology. Acetylation of 8-oxoguanine DNA glycosylase (OGG1) was detected in vivo, and this decreased with aging. However, in young animals, exercise and IGF-1 treatment increased acetylated (ac) OGG1 levels. Sirtuin 1 (SIRT1) and SIRT3, as DNA damage-associated lysine deacetylases, were measured, and SIRT1 decreased with aging, resulting in a large increase in acetylated lysine residues in the hippocampus. On the other hand, SIRT3 increased with aging. Exercise-induced neurogenesis might not be a causative factor of increased spatial memory, because IGF-1 plus exercise can induce neurogenesis in the hippocampus of older rats. Data revealed that the age-associated increase in 8-oxoG levels is due to decreased acetylation of OGG1. Age-associated decreases in SIRT1 and the associated increase in lysine acetylation, in the hippocampus, could have significant impact on function and thus, could suggest a therapeutic target.