Antibody desolvation with sodium chloride and acetonitrile generates bioactive protein nanoparticles.

About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2). Their size could be controlled by the addition of low concentrations of sodium chloride between 0.5 and 2 mM. Protein particles could be redissolved in aqueous solutions and redissolved antibodies retained their binding activity as evaluated in cell binding assays and exemplified for trastuzumab in an ELISA.

The Effect of Alendronate on Osteoclastogenesis in Different Combinations of M-CSF and RANKL Growth Factors.

Bisphosphonates (BPs) are compounds resembling the pyrophosphate structure. BPs bind the mineral component of bones. During the bone resorption by osteoclasts, nitrogen-containing BPs are released and internalized, causing an inhibition of the mevalonate pathway. As a consequence, osteoclasts are unable to execute their function. Alendronate (ALN) is a bisphosphonate used to treat osteoporosis. Its administration could be associated with adverse effects. The purpose of this study is to evaluate four different ALN concentrations, ranging from 10-6 to 10-10 M, in the presence of different combinations of M-CSF and RANKL, to find out the effect of low ALN concentrations on osteoclastogenesis using rat and human peripheral blood mononuclear cells. The cytotoxic effect of ALN was evaluated based on metabolic activity and DNA concentration measurement. The alteration in osteoclastogenesis was assessed by the activity of carbonic anhydrase II (CA II), tartrate-resistant acid phosphatase staining, and actin ring formation. The ALN concentration of 10-6 M was cytotoxic. Low ALN concentrations of 10-8 and 10-10 M promoted proliferation, osteoclast-like cell formation, and CA II activity. The results indicated the induction of osteoclastogenesis with low ALN concentrations. However, when high doses of ALN were administered, their cytotoxic effect was demonstrated.

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.

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.

Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer.

BACKGROUND: The primary objective of Tissue engineering is a regeneration or replacement of tissues or organs damaged by disease, injury, or congenital anomalies. At present, Tissue engineering repairs damaged tissues and organs with artificial supporting structures called scaffolds. These are used for attachment and subsequent growth of appropriate cells. During the cell growth gradual biodegradation of the scaffold occurs and the final product is a new tissue with the desired shape and properties. In recent years, research workplaces are focused on developing scaffold by bio-fabrication techniques to achieve fast, precise and cheap automatic manufacturing of these structures. Most promising techniques seem to be Rapid prototyping due to its high level of precision and controlling. However, this technique is still to solve various issues before it is easily used for scaffold fabrication. In this article we tested printing of clinically applicable scaffolds with use of commercially available devices and materials. Research presented in this article is in general focused on "scaffolding" on a field of bone tissue replacement. RESULTS: Commercially available 3D printer and Polylactic acid were used to create originally designed and possibly suitable scaffold structures for bone tissue engineering. We tested printing of scaffolds with different geometrical structures. Based on the osteosarcoma cells proliferation experiment and mechanical testing of designed scaffold samples, it will be stated that it is likely not necessary to keep the recommended porosity of the scaffold for bone tissue replacement at about 90%, and it will also be clarified why this fact eliminates mechanical properties issue. Moreover, it is demonstrated that the size of an individual pore could be double the size of the recommended range between 0.2-0.35 mm without affecting the cell proliferation. CONCLUSION: Rapid prototyping technique based on Fused deposition modelling was used for the fabrication of designed scaffold structures. All the experiments were performed in order to show how to possibly solve certain limitations and issues that are currently reported by research workplaces on the field of scaffold bio-fabrication. These results should provide new valuable knowledge for further research.

Osteogenic differentiation of 3D cultured mesenchymal stem cells induced by bioactive peptides.

OBJECTIVES: Bioactive peptides derived from receptor binding motifs of native proteins are a potent source of bioactive molecules that can induce signalling pathways. These peptides could substitute for osteogenesis promoting supplements. The work presented here compares three kinds of bioactive peptides derived from collagen III, bone morphogenetic protein 7 (BMP-7) and BMP-2 with their potential osteogenic activity on the model of porcine mesenchymal stem cells (pMSCs). MATERIALS AND METHODS: pMSCs were cultured on electrospun polycaprolactone nanofibrous scaffolds with different concentrations of the bioactive peptides without addition of any osteogenic supplement. Analysis of pMSCs cultures included measurement of the metabolic activity and proliferation, immunofluorescence staining and also qPCR. RESULTS: Results showed no detrimental effect of the bioactive peptides to cultured pMSCs. Based on qPCR analysis, the bioactive peptides are specific for osteogenic differentiation with no detectable expression of collagen II. Our results further indicate that peptide derived from BMP-2 protein promoted the expression of mRNA for osteocalcin (OCN) and collagen I significantly compared to control groups and also supported deposition of OCN as observed by immunostaining method. CONCLUSION: The data suggest that bioactive peptide with an amino acid sequence of KIPKASSVPTELSAISTLYL derived from BMP-2 protein was the most potent for triggering osteogenic differentiation of pMSCs.

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.

Highly efficient mesenchymal stem cell proliferation on poly-ε-caprolactone nanofibers with embedded magnetic nanoparticles.

In this study, we have developed a combined approach to accelerate the proliferation of mesenchymal stem cells (MSCs) in vitro, using a new nanofibrous scaffold made by needleless electrospinning from a mixture of poly-ε-caprolactone and magnetic particles. The biological characteristics of porcine MSCs were investigated while cultured in vitro on composite scaffold enriched with magnetic nanoparticles. Our data indicate that due to the synergic effect of the poly-ε-caprolactone nanofibers and magnetic particles, cellular adhesion and proliferation of MSCs is enhanced and osteogenic differentiation is supported. The cellular and physical attributes make this new scaffold very promising for the acceleration of efficient MSC proliferation and regeneration of hard tissues.

Significant improvement of biocompatibility of polypropylene mesh for incisional hernia repair by using poly-ε-caprolactone nanofibers functionalized with thrombocyte-rich solution.

Incisional hernia is the most common postoperative complication, affecting up to 20% of patients after abdominal surgery. Insertion of a synthetic surgical mesh has become the standard of care in ventral hernia repair. However, the implementation of a mesh does not reduce the risk of recurrence and the onset of hernia recurrence is only delayed by 2-3 years. Nowadays, more than 100 surgical meshes are available on the market, with polypropylene the most widely used for ventral hernia repair. Nonetheless, the ideal mesh does not exist yet; it still needs to be developed. Polycaprolactone nanofibers appear to be a suitable material for different kinds of cells, including fibroblasts, chondrocytes, and mesenchymal stem cells. The aim of the study reported here was to develop a functionalized scaffold for ventral hernia regeneration. We prepared a novel composite scaffold based on a polypropylene surgical mesh functionalized with poly-ε-caprolactone (PCL) nanofibers and adhered thrombocytes as a natural source of growth factors. In extensive in vitro tests, we proved the biocompatibility of PCL nanofibers with adhered thrombocytes deposited on a polypropylene mesh. Compared with polypropylene mesh alone, this composite scaffold provided better adhesion, growth, metabolic activity, proliferation, and viability of mouse fibroblasts in all tests and was even better than a polypropylene mesh functionalized with PCL nanofibers. The gradual release of growth factors from biocompatible nanofiber-modified scaffolds seems to be a promising approach in tissue engineering and regenerative medicine.

Abdominal closure reinforcement by using polypropylene mesh functionalized with poly-ε-caprolactone nanofibers and growth factors for prevention of incisional hernia formation.

Incisional hernia affects up to 20% of patients after abdominal surgery. Unlike other types of hernia, its prognosis is poor, and patients suffer from recurrence within 10 years of the operation. Currently used hernia-repair meshes do not guarantee success, but only extend the recurrence-free period by about 5 years. Most of them are nonresorbable, and these implants can lead to many complications that are in some cases life-threatening. Electrospun nanofibers of various polymers have been used as tissue scaffolds and have been explored extensively in the last decade, due to their low cost and good biocompatibility. Their architecture mimics the natural extracellular matrix. We tested a biodegradable polyester poly-ε-caprolactone in the form of nanofibers as a scaffold for fascia healing in an abdominal closure-reinforcement model for prevention of incisional hernia formation. Both in vitro tests and an experiment on a rabbit model showed promising results.

A cell-free nanofiber composite scaffold regenerated osteochondral defects in miniature pigs.

The aim of the study was to evaluate the effect of a cell-free hyaluronate/type I collagen/fibrin composite scaffold containing polyvinyl alcohol (PVA) nanofibers enriched with liposomes, basic fibroblast growth factor (bFGF) and insulin on the regeneration of osteochondral defects. A novel drug delivery system was developed on the basis of the intake effect of liposomes encapsulated in PVA nanofibers. Time-controlled release of insulin and bFGF improved MSC viability in vitro. Nanofibers functionalized with liposomes also improved the mechanical characteristics of the composite gel scaffold. In addition, time-controlled release of insulin and bFGF stimulated MSC recruitment from bone marrow in vivo. Cell-free composite scaffolds containing PVA nanofibers enriched with liposomes, bFGF, and insulin were implanted into seven osteochondral defects of miniature pigs. Control defects were left untreated. After 12 weeks, the composite scaffold had enhanced osteochondral regeneration towards hyaline cartilage and/or fibrocartilage compared with untreated defects that were filled predominantly with fibrous tissue. The cell-free composite scaffold containing PVA nanofibers, liposomes and growth factors enhanced migration of the cells into the defect, and their differentiation into chondrocytes; the scaffold was able to enhance the regeneration of osteochondral defects in minipigs.

Time-regulated drug delivery system based on coaxially incorporated platelet α-granules for biomedical use.

AIM: Platelet derivatives serve as an efficient source of natural growth factors. In the current study, α-granules were incorporated into coaxial nanofibers. MATERIALS & METHODS: A nanofiber scaffold containing α-granules was prepared by coaxial electrospinning. The biological potential of the nanofiber scaffold was evaluated in chondrocyte and mesenchymal stem cell cultivation studies. Additionally, the concentration of TGF-ß1 was determined. RESULTS: Microscopy studies showed that intact α-granules were incorporated into the coaxial nanofibers. The cultivation tests showed that the novel scaffold stimulated viability and extracellular matrix production of chondrocytes and mesenchymal stem cells. In addition, the concentration of growth factors necessary for the induction of cell proliferation significantly decreased. CONCLUSION: The system preserved α-granule bioactivity and stimulated cell viability and chondrogenic differentiation of mesenchymal stem cells. Core/shell nanofibers incorporating α-granules are a promising system for tissue engineering, particularly cartilage engineering.

Core/shell nanofibers with embedded liposomes as a drug delivery system.

The broader application of liposomes in regenerative medicine is hampered by their short half-life and inefficient retention at the site of application. These disadvantages could be significantly reduced by their combination with nanofibers. We produced 2 different nanofiber-liposome systems in the present study, that is, liposomes blended within nanofibers and core/shell nanofibers with embedded liposomes. Herein, we demonstrate that blend electrospinning does not conserve intact liposomes. In contrast, coaxial electrospinning enables the incorporation of liposomes into nanofibers. We report polyvinyl alcohol-core/poly-ε-caprolactone-shell nanofibers with embedded liposomes and show that they preserve the enzymatic activity of encapsulated horseradish peroxidase. The potential of this system was also demonstrated by the enhancement of mesenchymal stem cell proliferation. In conclusion, intact liposomes incorporated into nanofibers by coaxial electrospinning are very promising as a drug delivery system.

Thin-layer hydroxyapatite deposition on a nanofiber surface stimulates mesenchymal stem cell proliferation and their differentiation into osteoblasts.

Pulsed laser deposition was proved as a suitable method for hydroxyapatite (HA) coating of coaxial poly-ɛ-caprolactone/polyvinylalcohol (PCL/PVA) nanofibers. The fibrous morphology of PCL/PVA nanofibers was preserved, if the nanofiber scaffold was coated with thin layers of HA (200 nm and 400 nm). Increasing thickness of HA, however, resulted in a gradual loss of fibrous character. In addition, biomechanical properties were improved after HA deposition on PCL/PVA nanofibers as the value of Young's moduli of elasticity significantly increased. Clearly, thin-layer hydroxyapatite deposition on a nanofiber surface stimulated mesenchymal stem cell viability and their differentiation into osteoblasts. The optimal depth of HA was 800 nm.