J. Funct. Biomater., Volume 10, Issue 3 (September 2019) – 17 articles

Compared with platelet-rich plasma, the preparation of platelet-rich fibrin (PRF) is simple and has not been overly modified. However, it was recently demonstrated that centrifugation conditions influence the composition of PRF and that silica microparticles from silica-coated plastic tubes can enter the PRF matrix. These factors may also modify platelet distribution. To examine these possibilities, we prepared PRF matrices using various types of blood-collection tubes (plain glass tubes and silica-containing plastic tubes) and different centrifugation speeds. The protocols of concentrated growth factors and advanced-PRF represented high- and low-speed centrifugation, respectively. Platelet distribution in the PRF matrix was examined immunohistochemically. Using low-speed centrifugation, platelets were distributed homogeneously within the PRF matrix regardless of tube types. In high-speed centrifugation, platelets were distributed mainly on one surface region of the PRF matrix in glass tubes, whereas in silica-coated tubes, platelet distribution was commonly more diffusive than in glass tubes. Therefore, both blood-collection tube types and centrifugal conditions appeared to influence platelet distribution in the PRF matrix. Platelets distributed in the deep regions of the PRF matrix may contribute to better growth factor retention and release. However, clinicians should be careful in using silica-coated tubes because their silica microparticles may be a health hazard. Full article

The controlled release or delivery of proteins encapsulated in micro/nanospheres is an emerging strategy in regenerative medicine. For this, micro/nanospheres made from alginate have drawn considerable attention for the use as a protein delivery device because of their mild fabrication process, inert nature, non-toxicity and biocompatibility. Though promising, one key issue associated with using alginate micro/nanospheres is the burst release of encapsulated protein at the beginning of the release, which may be responsible for exerting toxic side effects and poor efficiency of the delivery device. To address this issue, this study aimed to investigate the effect of process parameters of fabricating protein-loaded alginate nanospheres on the initial burst release. The alginate nanospheres were prepared via a combination of water-in-oil emulsification and the external gelation method and loaded with bovine serum albumin (BSA) as a model protein. The examined process parameters included alginate concentration, ionic cross-linking time and drying time. Once fabricated, the nanospheres were then subjected to the examination of BSA release, as well as the characterization of their morphology, size, and encapsulation efficiency. Our results revealed that by properly adjusting the process parameters, the initial burst release can be reduced by 13%. Taken together, our study demonstrates that regulating process parameters of fabricating alginate nanospheres is a possible means to reduce the initial burst release. Full article

Pseudomonas aeruginosa (P. aeruginosa) infections may lead to severe damage of the cornea, mucosa, and skin. The highly aggressive nature of P. aeruginosa and the rise in multi-drug resistance, particularly in nosocomial settings, lead to an increased risk for permanent tissue damage and potentially death. Thus, a growing need exists to develop alternative treatments to reduce both the occurrence of bacterial infection and biofilm development, as well as pathological progression post-infection. Silk derived from Bombyx mori silkworms serves as a unique biomaterial that is biocompatible with low immunogenicity and high versatility, and thereby ideal for stabilizing therapeutics. In this study, we assessed the cytotoxicity of P. aeruginosa on human corneal stromal stem cells and two mucosal cell lines (Caco-2 and HT29-MTX). To determine whether antibiotic-immobilized scaffolds can serve as alternative therapeutics to free, diffuse forms, we developed novel gentamicin-conjugated silk films as functional scaffolds and compared antimicrobial effects and free gentamicin. The advantages of generating a surface coating with a covalently-bound antibiotic may reduce potential side-effects associated with free gentamicin, as well as limit the diffusion of the drug. Our results suggest that gentamicin conjugated to native silk and carboxyl-enriched silk inhibits P. aeruginosa growth. Development of stabilized antibiotic treatments with surface toxicity selective against bacteria may serve as an alternative approach to treat active infections, as well as potential prophylactic use as coatings in high-risk cases, such as post-surgical complications or prolonged hospitalization. Full article

Failure of the cement mantle in total hip arthroplasty is not a simple phenomenon. Cracking, which can be caused by crack initiation and repeated loading, can cause loosening of the acetabular liner component. A previous study showed that addition of a metal layer between the liner and acetabular could reduce the stress at the contact surface of the cement mantle. This study elaborates on the performance of the additional layer. Several material properties of the layer were simulated using finite element analysis for maximum performance. A static contact analysis was used to simulate the stresses at the contact surface of the cement mantle. The results show that an additional layer of cobalt chrome produced the best performance. Full article

Proteins derived from the natural extracellular matrix like collagen or gelatin are common in clinical research, where they are prized for their biocompatibility and bioactivity. Cells are able to adhere, grow and remodel scaffolds based on these materials. Usually, collagen and gelatin are sourced from animal material, risking pathogenic transmission and inconsistent batch-to-batch product quality. A recombinant production in yeast circumvents these disadvantages by ensuring production with a reproducible quality in animal-component-free media. A gelatin mimetic protein, based on the alpha chain of human collagen I, was cloned in Pichia pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. A producing clone was selected and cultivated at the 30 L scale. The protein was secreted into the cultivation medium and the final yield was 3.4 g·L⁻¹. Purification of the target was performed directly from the cell-free medium by size exclusion chromatography. The gelatin mimetic protein was tested in cell culture for biocompatibility and for promoting cell adhesion. It supported cell growth and its performance was indistinguishable from animal-derived gelatin. The gelatin-mimetic protein represents a swift strategy to produce recombinant and human-based extracellular matrix proteins for various biomedical applications. Full article

This study reports on the processing of three-dimensional (3D) chitosan/bioactive glass composite scaffolds. On the one hand, chitosan, as a natural polymer, has suitable properties for tissue engineering applications but lacks bioactivity. On the other hand, bioactive glasses are known to be bioactive and to promote a higher level of bone formation than any other biomaterial type. However, bioactive glasses are hard, brittle, and cannot be shaped easily. Therefore, in the past years, researchers have focused on the processing of new composites. Difficulties in reaching composite materials made of polymer (synthetic or natural) and bioactive glass include: (i) The high glass density, often resulting in glass segregation, and (ii) the fast bioactive glass reaction when exposed to moisture, leading to changes in the glass reactivity and/or change in the polymeric matrix. Samples were prepared with 5, 15, and 30 wt% of bioactive glass S53P4 (BonAlive ^®), as confirmed using thermogravimetric analysis. MicrO–Computed tomography and optical microscopy revealed a flaky structure with porosity over 80%. The pore size decreased when increasing the glass content up to 15 wt%, but increased back when the glass content was 30 wt%. Similarly, the mechanical properties (in compression) of the scaffolds increased for glass content up to 15%, but decreased at higher loading. Ions released from the scaffolds were found to lead to precipitation of a calcium phosphate reactive layer at the scaffold surface. This is a first indication of the potential bioactivity of these materials. Overall, chitosan/bioactive glass composite scaffolds were successfully produced with pore size, machinability, and ability to promote a calcium phosphate layer, showing promise for bone tissue engineering and the mechanical properties can justify their use in non-load bearing applications. Full article

Stainless steel wires are the standard method for sternal closure because of their strength and rigidity, the simplicity of the process, and the short healing time that results from their application. Despite this, problems still exist with sternal stability due to micromotion between the two halves of the dissected and wired sternum. Recently, a novel glass-based adhesive was developed which, in cadaveric trials and in conjunction with wiring, was shown to restrict this micromotion. However, in order to avoid complications during resternotomy, the adhesive should adhere only to the bone and not the sternal wire. In this study, sternal wires were embedded in 8 mm discs manufactured from the novel glass-based adhesive and the constructs were then incubated at 37 °C for one, seven, and 30 days. The discs were manufactured in two different thicknesses: 2 and 3 mm. Wire pull-out tests were then performed on the constructs at three different strain rates (1, 0.1, and 0.01 mm/min). No statistically significant difference in pull-out force was found regardless of incubation time, loading rate, or construct thickness. The pull-out forces recorded were consistent with static friction between the wire and adhesive, rather than the adhesion between them. Scanning electron micrographs provided further proof of this. These results indicate that the novel adhesive may be suitable for sternal fixation without complicating a potential resternotomy. Full article

The synthesis and characterization of an ABA triblock copolymer based on hydrophilic poly(2-methyl-2-oxazoline) (pMeOx) blocks A and a modestly hydrophobic poly(2-iso-butyl-2-oxazoline) (piBuOx) block B is described. Aqueous polymer solutions were prepared at different concentrations (1–20 wt %) and their thermogelling capability using visual observation was investigated at different temperatures ranging from 5 to 80 °C. As only a 20 wt % solution was found to undergo thermogelation, this concentration was investigated in more detail regarding its temperature-dependent viscoelastic profile utilizing various modes (strain or temperature sweep). The prepared hydrogels from this particular ABA triblock copolymer have interesting rheological and viscoelastic properties, such as reversible thermogelling and shear thinning, and may be used as bioink, which was supported by its very low cytotoxicity and initial printing experiments using the hydrogels. However, the soft character and low yield stress of the gels do not allow real 3D printing at this point. Full article

Transparent composite hydrogel in the form of a contact lens made from poly(vinyl alcohol) (PVA) and cellulose nanocrystals (CNCs) was subjected to in vitro biocompatibility evaluation with human corneal epithelial cells (HCE-2 cells). The cell response to direct contact with the hydrogels was investigated by placing the samples on top of confluent cell layers and evaluating cell viability, morphology, and cell layer integrity subsequent to 24 h culture and removal of the hydrogels. To further characterize the lens–cell interactions, HCE-2 cells were seeded on the hydrogels, with and without simulated tear fluid (STF) pre-conditioning, and cell viability and morphology were evaluated. Furthermore, protein adsorption on the hydrogel surface was investigated by incubating the materials with STF, followed by protein elution and quantification. The hydrogel material was found to have affinity towards protein adsorption, most probably due to the interactions between the positively charged lysozyme and the negatively charged CNCs embedded in the PVA matrix. The direct contact experiment demonstrated that the physical presence of the lenses did not affect corneal epithelial cell monolayers in terms of integrity nor cell metabolic activity. Moreover, it was found that viable corneal cells adhered to the hydrogel, showing the typical morphology of epithelial cells and that such response was not influenced by the STF pre-conditioning of the hydrogel surface. The results of the study confirm that PVA-CNC hydrogel is a promising ophthalmic biomaterial, motivating future in vitro and in vivo biocompatibility studies. Full article

Over the past 10 years, stimuli-responsive polymeric biomaterials have emerged as effective systems for the delivery of therapeutics. Persistent with ongoing efforts to minimize adverse effects, stimuli-responsive biomaterials are designed to release in response to either chemical, physical, or biological triggers. The stimuli-responsiveness of smart biomaterials may improve spatiotemporal specificity of release. The material design may be used to tailor smart polymers to release a drug when particular stimuli are present. Smart biomaterials may use internal or external stimuli as triggering mechanisms. Internal stimuli-responsive smart biomaterials include those that respond to specific enzymes or changes in microenvironment pH; external stimuli can consist of electromagnetic, light, or acoustic energy; with some smart biomaterials responding to multiple stimuli. This review looks at current and evolving stimuli-responsive polymeric biomaterials in their proposed applications. Full article

Poly(d,l–lactide–co–glycolide) (PLGA) has been extensively explored for bone regeneration applications; however, its clinical use is limited by low osteointegration. Therefore, approaches that incorporate osteoconductive molecules are of great interest. Graphene oxide (GO) is gaining popularity for biomedical applications due to its ability to bind biological molecules and present them for enhanced bioactivity. This study reports the preparation of PLGA microparticles via Pickering emulsification using GO as the sole surfactant, which resulted in hybrid microparticles in the size range of 1.1 to 2.4 µm based on the ratio of GO to PLGA in the reaction. Furthermore, this study demonstrated that the hybrid GO-PLGA microparticles were not cytotoxic to either primary human fetal cartilage rudiment cells or the human osteoblast-like cell line, Saos-2. Additionally, the GO-PLGA microparticles promoted the osteogenic differentiation of the human fetal cartilage rudiment cells in the absence of exogenous growth factors to a greater extent than PLGA alone. These findings demonstrate that GO-PLGA microparticles are cytocompatible, osteoinductive and have potential as substrates for bone tissue engineering. Full article

Parkinson’s disease (PD) is a neurodegenerative disease associated with loss or dysfunction of dopaminergic neurons located in the substantia nigra (SN), and there is no cure available. An emerging new approach for treatment is to transplant human induced dopaminergic neurons directly into the denervated striatal brain target region. Unfortunately, neurons grafted into the substantia nigra are unable to grow axons into the striatum and thus do not allow recovery of the original connectivity. Towards overcoming this general limitation in guided neuronal regeneration, we develop here magnetic nanoparticles functionalized with proteins involved in the regulation of axonal growth. We show covalent binding of constitutive active human rat sarcoma (RAS) proteins or RAS guanine nucleotide exchange factor catalytic domain of son of sevenless (SOS) by fluorescence correlation spectroscopy and multiangle light scattering as well as the characterization of exchange factor activity. Human dopaminergic neurons were differentiated from neural precursor cells and characterized by electrophysiological and immune histochemical methods. Furthermore, we demonstrate magnetic translocation of cytoplasmic γ-Fe₂O₃@SiO₂ core-shell nanoparticles into the neurite extensions of induced human neurons. Altogether, we developed tools towards remote control of directed neurite growth in human dopaminergic neurons. These results may have relevance for future therapeutic approaches of cell replacement therapy in Parkinson’s disease. Full article

Grafts are required to restore tissue integrity and function. However, current gold standard autografting techniques yield limited harvest, with high rates of complication. In the search for viable substitutes, the number of biomaterials being developed and studied has increased rapidly. To date, low clinical uptake has accompanied inherently high failure rates, with immune rejection a specific and common end result. The objective of this review article was to evaluate published immune assays evaluating biomaterials, and to stress the value that incorporating immune assessment into evaluations carries. Immunogenicity assays have had three areas of focus: cell viability, maturation and activation, with the latter being the focus in the majority of the literature due to its relevance to functional outcomes. With recent studies suggesting poor correlation between current in vitro and in vivo testing of biomaterials, in vitro immune response assays may be more relevant and enhance ability in predicting acceptance prior to in vivo application. Uptake of in vitro immune response assessment will allow for substantial reductions in experimental time and resources, including unnecessary and unethical animal use, with a simultaneous decrease in inappropriate biomaterials reaching clinic. This improvement in bench to bedside safety is paramount to reduce patient harm. Full article

Tissue engineering aims to develop artificial human tissues by culturing cells on a scaffold in the presence of biochemical cues. Properties of scaffold such as architecture and composition highly influence the overall cell response. Electrospinning has emerged as one of the most affordable, versatile, and successful approaches to develop nonwoven nano/microscale fibrous scaffolds whose structural features resemble that of the native extracellular matrix. However, dense packing of the fibers leads to small-sized pores which obstruct cell infiltration and therefore is a major limitation for their use in tissue engineering applications. To this end, a variety of approaches have been investigated to enhance the pore properties of the electrospun scaffolds. In this review, we collect state-of-the-art modification methods and summarize them into six classes as follows: approaches focused on optimization of packing density by (a) conventional setup, (b) sequential or co-electrospinning setups, (c) involving sacrificial elements, (d) using special collectors, (e) post-production processing, and (f) other specialized methods. Overall, this review covers historical as well as latest methodologies in the field and therefore acts as a quick reference for those interested in electrospinning matrices for tissue engineering and beyond. Full article

Carbon fibre reinforced polyether ether ketone (CFR-PEEK) is a suitable material to replace metal implants in orthopaedic surgery. The radiolucency of CFR-PEEK allows an optimal visualisation of the bone and soft tissue structures. We aimed to assess the performance and radiological and clinical outcomes of anterior cervical discectomy and fusion (ACDF) with CFR-PEEK anterior cervical plating (ACP) under first use clinical conditions. We retrospectively studied the prospectively-collected data of 42 patients who underwent ACDF with CFR-PEEK ACP between 2011 and 2016. We assessed clinical outcome (Odom’s criteria, complications) and radiological parameters (global and segmental cervical lordosis, Bridwell score for fusion, adjacent segment degeneration) preoperatively, immediately post-operatively, and after a 12-month follow-up period. Patients’ satisfaction was excellent, good, fair, and poor in 12, 19, 3, and 1 patients, respectively. Two patients developed dysphagia. No hardware failure occurred. Compared with preoperative radiographs, we observed a gain of global cervical lordosis and segmental lordosis (7.4 ± 10.1 and 5.6 ± 7.1 degrees, respectively) at the 12-month follow-up. Bridwell IF grades I, II, and III were observed in 22, 6, and 7 patients, respectively. The 12-month adjacent segment degeneration-free and adjacent segment disease-free survival rates were 93.1% and 96.3%, respectively. We observed a dysphagia rate of 5.7% and a reoperation rate of 4.8%. In conclusion, CFR-PEEK ACP shows positive outcomes in terms of implant safety, restoration of cervical lordosis, and functional recovery, and is suitable for ACDF. Full article

Cell scaffolds are often used in cell transplantation as they provide a solid structural support to implanted cells and can be bioengineered to mimic the native extracellular matrix. Gadolinium fluoride nanoparticles (Gd-NPs) as a contrast agent for Magnetic Resonance Imaging (MRI) were incorporated into poly(lactide-co-glycolide)/chitosan scaffolds to obtain Imaging Labelled Cell Scaffolds (ILCSs), having the shape of hollow spherical/ellipsoidal particles (200–600 μm diameter and 50–80 μm shell thickness). While Gd-NPs incorporated into microparticles do not provide any contrast enhancement in T₁-weighted (T₁w) MR images, ILCSs can release Gd-NPs in a controlled manner, thus activating MRI contrast. ILCSs seeded with human mesenchymal stromal cells (hMSCs) were xenografted subcutaneously into either immunocompromised and immunocompetent mice without any immunosuppressant treatments, and the transplants were followed-up in vivo by MRI for 18 days. Immunocompromised mice showed a progressive activation of MRI contrast within the implants due to the release of Gd-NPs in the extracellular matrix. Instead, immunocompetent mice showed poor activation of MRI contrast due to the encapsulation of ILCSs within fibrotic capsules and to the scavenging of released Gd-NPs by phagocytic cells. In conclusion, the MRI follow-up of cell xenografts can report the host cell response to the xenograft. However, it does not strictly report on the viability of transplanted hMSCs. Full article

Repairs of orbital post-traumatic and extensive malignant defects remain a major surgical challenge, in view of follow-up outcomes. Incorrect surgical management of injured facial structures results in cosmetic, ophthalmic, and social aftereffects. A custom-made knitted TiNi-based mesh (KTNM) endograft was employed to overcome post-surgical complications and post-resected lesions of the orbital area. Preoperative high-resolution computed tomography (CT) imaging and CAD modelling were used to design the customized KTNM in each case. Twenty-five patients underwent surgery utilizing the suggested technique, from 2014 to 2019. In all documented cases, resolution of the ophthalmic malfunction was noted in the early period. Follow-up observation evidenced no relapsed enophthalmos, hypoglobus, or diplopia as late complications. The findings emanating from our clinical observations allow us to claim that the KTNM indicated a high level of biocompatibility. It is simply modified intraoperatively to attach any desired shape/size for implantation and can also be screw-fixed, providing a good supporting ability. The KTNM precisely renders orbitozygomatic outlines and orbital floor, thus recovering the anatomical structure, and is regarded as an attractive alternative to Ti-based meshes and plates. Additionally, we report one of the studied cases, where good functional and cosmetic outcomes have been achieved. Full article