Cellular Response to Individual Components of the Platelet Concentrate.

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.

A Simple Drug Delivery System for Platelet-Derived Bioactive Molecules, to Improve Melanocyte Stimulation in Vitiligo Treatment.

Vitiligo is the most common depigmentation disorder of the skin. Currently, its therapy focuses on the halting of the immune response and stimulation of the regenerative processes, leading to the restoration of normal melanocyte function. Platelet-rich plasma (PRP) represents a safe and cheap regenerative therapy option, as it delivers a wide spectrum of native growth factors, cytokines and other bioactive molecules. The aim of this study was to develop a simple delivery system to prolong the effects of the bioactive molecules released from platelets. The surface of electrospun and centrifugally spun poly-ε-caprolactone (PCL) fibrous scaffolds was functionalized with various concentrations of platelets; the influence of the morphology of the scaffolds and the concentration of the released platelet-derived bioactive molecules on melanocytes, was then assessed. An almost two-fold increase in the amount of the released bioactive molecules was detected on the centrifugally spun vs. electrospun scaffolds, and a sustained 14-day release of the bioactive molecules was demonstrated. A strong concentration-dependent response of melanocyte to the bioactive molecules was observed; higher concentrations of bioactive molecules resulted in improved metabolic activity and proliferation of melanocytes. This simple system improves melanocyte viability, offers on-site preparation and is suitable for prolonged topical PRP administration.

Poly-ε-caprolactone and polyvinyl alcohol electrospun wound dressings: adhesion properties and wound management of skin defects in rabbits.

Aim: This study evaluates the effect of electrospun dressings in critical sized full-thickness skin defects in rabbits. Materials & methods: Electrospun poly-ε-caprolactone (PCL) and polyvinyl alcohol (PVA) nanofibers were tested in vitro and in vivo. Results: The PCL scaffold supported the proliferation of mesenchymal stem cells, fibroblasts and keratinocytes. The PVA scaffold showed significant swelling, high elongation capacity, limited protein adsorption and stimulation of cells. Nanofibrous dressings improved wound healing compared with the control group in vivo. A change of the PCL dressing every 7 days resulted in a decreased epithelial thickness and type I collagen level in the adhesive group, indicating peeling off of the newly formed tissue. In the PVA dressings, the exchange did not affect healing. Conclusion: The results demonstrate the importance of proper dressing exchange.

Different diameters of titanium dioxide nanotubes modulate Saos-2 osteoblast-like cell adhesion and osteogenic differentiation and nanomechanical properties of the surface.

The formation of nanostructures on titanium implant surfaces is a promising strategy to modulate cell adhesion and differentiation, which are crucial for future application in bone regeneration. The aim of this study was to investigate how the nanotube diameter and/or nanomechanical properties alter human osteoblast like cell (Saos-2) adhesion, growth and osteogenic differentiation in vitro. Nanotubes, with diameters ranging from 24 to 66 nm, were fabricated on a commercially pure titanium (cpTi) surface using anodic oxidation with selected end potentials of 10 V, 15 V and 20 V. The cell response was studied in vitro on untreated and nanostructured samples using a measurement of metabolic activity, cell proliferation, alkaline phosphatase activity and qRT-PCR, which was used for the evaluation of osteogenic marker expression (collagen type I, osteocalcin, RunX2). Early cell adhesion was investigated using SEM and ELISA. Adhesive molecules (vinculin, talin), collagen and osteocalcin were also visualized using confocal microscopy. Moreover, the reduced elastic modulus and indentation hardness of nanotubes were assessed using a TriboIndenter™. Smooth and nanostructured cpTi both supported cell adhesion, proliferation and bone-specific mRNA expression. The nanotubes enhanced collagen type I and osteocalcin synthesis, compared to untreated cpTi, and the highest synthesis was observed on samples modified with 20 V nanotubes. Significant differences were found in the cell adhesion, where the vinculin and talin showed a dot-like distribution. Both the lowest reduced elastic modulus and indentation hardness were assessed from 20 V samples. The nanotubes of mainly 20 V samples showed a high potential for their use in bone implantation.

A polypropylene mesh modified with poly-ε-caprolactone nanofibers in hernia repair: large animal experiment.

PURPOSE: Incisional hernia repair is an unsuccessful field of surgery, with long-term recurrence rates reaching up to 50% regardless of technique or mesh material used. Various implants and their positioning within the abdominal wall pose numerous long-term complications that are difficult to treat due to their permanent nature and the chronic foreign body reaction they trigger. Materials mimicking the 3D structure of the extracellular matrix promote cell adhesion, proliferation, migration, and differentiation. Some electrospun nanofibrous scaffolds provide a topography of a natural extracellular matrix and are cost effective to manufacture. MATERIALS AND METHODS: A composite scaffold that was assembled out of a standard polypropylene hernia mesh and poly-ε-caprolactone (PCL) nanofibers was tested in a large animal model (minipig), and the final scar tissue was subjected to histological and biomechanical testing to verify our in vitro results published previously. RESULTS: We have demonstrated that a layer of PCL nanofibers leads to tissue overgrowth and the formation of a thick fibrous plate around the implant. Collagen maturation is accelerated, and the final scar is more flexible and elastic than under a standard polypropylene mesh with less pronounced shrinkage observed. However, the samples with the composite scaffold were less resistant to distracting forces than when a standard mesh was used. We believe that the adverse effects could be caused due to the material assembly, as they do not comply with our previous results. CONCLUSION: We believe that PCL nanofibers on their own can cause enough fibroplasia to be used as a separate material without the polypropylene base, thus avoiding potential adverse effects caused by any added substances.

Needleless emulsion electrospinning for the regulated delivery of susceptible proteins.

In the present work, we developed a novel needleless emulsion electrospinning technique that improves the production rate of the core/shell production process. The nanofibres are based on poly-ε-caprolactone (PCL) as a continuous phase combined with a droplet phase based on Pluronic F-68 (PF-68). The PCL-PF-68 nanofibres show a time-regulated release of active molecules. Needleless emulsion electrospinning was used to encapsulate a diverse set of compounds to the core phase [i.e. 5-(4,6-dichlorotriazinyl) aminofluorescein -PF-68, horseradish peroxidase, Tetramethylrhodamine-dextran, insulin growth factor-I, transforming growth factor-ß and basic fibroblast growth factor]. In addition, the PF-68 facilitates the preservation of the bioactivity of delivered proteins. The system's potential was highlighted by an improvement in the metabolic activity and proliferation of mesenchymal stem cells. The developed system has the potential to deliver susceptible molecules in tissue-engineering applications.

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers.

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

Platelet-functionalized three-dimensional poly-ε-caprolactone fibrous scaffold prepared using centrifugal spinning for delivery of growth factors.

Bone and cartilage are tissues of a three-dimensional (3D) nature. Therefore, scaffolds for their regeneration should support cell infiltration and growth in all 3 dimensions. To fulfill such a requirement, the materials should possess large, open pores. Centrifugal spinning is a simple method for producing 3D fibrous scaffolds with large and interconnected pores. However, the process of bone regeneration is rather complex and requires additional stimulation by active molecules. In the current study, we introduced a simple composite scaffold based on platelet adhesion to poly-ε-caprolactone 3D fibers. Platelets were used as a natural source of growth factors and cytokines active in the tissue repair process. By immobilization in the fibrous scaffolds, their bioavailability was prolonged. The biological evaluation of the proposed system in the MG-63 model showed improved metabolic activity, proliferation and alkaline phosphatase activity in comparison to nonfunctionalized fibrous scaffold. In addition, the response of cells was dose dependent with improved biocompatibility with increasing platelet concentration. The results demonstrated the suitability of the system for bone tissue.