Journal of Functional Biomaterials — Open Access Journal

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

Tobramycin is a potent antimicrobial aminoglycoside and its effective delivery by encapsulation within nanoparticle carriers could increase its activity against infections through a combination of sustained release and enhanced uptake. Effective antimicrobial therapy against a clinically relevant model bacteria (Pseudomonas aeruginosa) requires sufficient levels of therapeutic drug to maintain a drug concentration above the microbial inhibitory concentration (MIC) of the bacteria. Previous studies have shown that loading of aminoglycoside drugs in poly(lactic-co-glycolic) acid (PLGA)-based delivery systems is generally poor due to weak interactions between the drug and the polymer. The formation of complexes of tobramycin with dioctylsulfosuccinate (AOT) allows the effective loading of the drug in PLGA-nanoparticles and such nanoparticles can effectively deliver the antimicrobial aminoglycoside with retention of tobramycin antibacterial function. Full article

Biodentine is one of the most successful and widely studied among the second generation of calcium silicate-based endodontic cements. Despite its popularity, the setting reactions of this cement system are not currently well understood. In particular, very little is known about the formation and structure of the major calcium silicate hydrate (C-S-H) gel phase, as it is difficult to obtain information on this poorly crystalline material by the traditional techniques of powder X-ray diffraction analysis (XRD) and Fourier transform infrared spectroscopy (FTIR). In this study, the hydration reactions of Biodentine are monitored by XRD, FTIR, isothermal conduction calorimetry and, for the first time, ²⁹Si magic angle spinning nuclear magnetic resonance spectroscopy (²⁹Si MAS NMR) is used to investigate the structures of the anhydrous calcium silicate phases and the early C-S-H gel product. XRD analysis indicated that the anhydrous powder comprises 73.8 wt% triclinic tricalcium silicate, 4.45 wt% monoclinic β-dicalcium silicate, 16.6 wt% calcite and 5.15 wt% zirconium oxide. Calorimetry confirmed that the induction period for hydration is short, and that the setting reactions are rapid with a maximum heat evolution of 28.4 mW g⁻¹ at 42 min. A progressive shift in the FTIR peak maximum from 905 to 995 cm⁻¹ for the O-Si-O stretching vibrations accompanies the formation of the C-S-H gel during 1 week. The extent of hydration was determined by ²⁹Si MAS NMR to be 87.0%, 88.8% and 93.7% at 6 h, 1 day and 1 week, respectively, which is significantly higher than that of MTA. The mean silicate chain length (MCL) of the C-S-H gel was also estimated by this technique to be 3.7 at 6 h and 1 day, and to have increased to 4.1 after 1 week. The rapid hydration kinetics of Biodentine, arising from the predominance of the tricalcium silicate phase, small particle size, and ‘filler effect’ of calcite and zirconium oxide, is a favorable characteristic of an endodontic cement, and the high values of MCL are thought to promote the durability of the cement matrix. Full article

Nerve injuries requiring surgery are a significant problem without good clinical alternatives to the autograft. Tissue engineering strategies are critically needed to provide an alternative. In this study, we utilized aligned nanofibers that were click-modified with the bioactive peptide RGD for rat sciatic nerve repair. Empty conduits or conduits filled with either non-functionalized aligned nanofibers or RGD-functionalized aligned nanofibers were used to repair a 13 mm gap in the rat sciatic nerve of animals for six weeks. The aligned nanofibers encouraged cell infiltration and nerve repair as shown by histological analysis. RGD-functionalized nanofibers reduced muscle atrophy. During the six weeks of recovery, the animals were subjected to motor and sensory tests. Sensory recovery was improved in the RGD-functionalized nanofiber group by week 4, while other groups needed six weeks to show improvement after injury. Thus, the use of functionalized nanofibers provides cues that aid in in vivo nerve repair and should be considered as a future repair strategy. Full article

Nanofibrous-based pH sensors have shown promise in a wide range of industrial and medical applications due to their fast response time and good mechanical properties. In the present study, we fabricated pH-sensitive sensors of nanofibrous membranes by electrospinning polyurethane (PU)/poly 2-acrylamido-2-methylpropanesulfonic acid (PAMPS)/graphene oxide (GO) with indicator dyes. The morphology of the electrospun nanofibers was examined using scanning electron microscopy (SEM). The effect of hydrophilic polymer ratio and concentration of GO on the sensing response time was investigated. The sensitivity of the membranes was studied over a wide pH range (1–8) in solution tests, with color change measured by calculating total color difference using UV-vis spectroscopy. The membranes were also subjected to vapor tests at three different pH values (1, 4, 8). SEM results show the successful fabrication of bimodal fiber diameter distributions of PU (mean fiber diameter 519 nm) and PAMPS (mean fiber diameter 78 nm). Sensing response time decreased dramatically with increasing concentrations of PAMPS and GO. The hybrid hydrophobic/hydrophilic/GO nanofibrous membranes are capable of instantly responding to changes in solution pH as well as detecting pH changes in chemical vapor solution in as little as 7 s. Full article

Blood-derived growth factor preparations have long been employed to improve perfusion and aid tissue repair. Among these, platelet-rich plasma (PRP)-based therapies have seen the widest application, albeit with mixed clinical results to date. Hypoxia-preconditioned blood products present an alternative to PRP, by comprising the complete wound healing factor-cascade, i.e., hypoxia-induced peripheral blood cell signaling, in addition to platelet-derived factors. This study set out to characterize the preparation of hypoxia preconditioned serum (HPS), and assess the utility of HPS–fibrin hydrogels as vehicles for controlled factor delivery. Our findings demonstrate the positive influence of hypoxic incubation on HPS angiogenic potential, and the individual variability of HPS angiogenic factor concentration. HPS–fibrin hydrogels can rapidly retain HPS factor proteins and gradually release them over time, while both functions appear to depend on the fibrin matrix mass. This offers a means of controlling factor retention/release, through adjustment of HPS fibrinogen concentration, thus allowing modulation of cellular angiogenic responses in a growth factor dose-dependent manner. This study provides the first evidence that HPS–fibrin hydrogels could constitute a new generation of autologous/bioactive injectable compositions that provide biochemical and biomaterial signals analogous to those mediating physiological wound healing. This therefore establishes a rational foundation for their application towards biomimetic tissue regeneration. Full article

(1) Background: The tissue engineering field has been working to find biomaterials that mimic the biological properties of autogenous bone grafts. (2) Aim: To evaluate the osteoconduction potential of injectable calcium phosphate cement implanted in critical defects in rat calvaria. (3) Methods: In the calvarial bone of 36 rats, 7-mm diameter critical size defects were performed. Afterwards, the animals were randomly divided into three groups according to filler material: a blood clot group (BC), blood clot membrane group (BCM), and an injectable β-tricalcium phosphate group (HBS) cement group. After periods of 30 and 60 days, the animals were euthanized, the calvaria was isolated, and submitted to a decalcification process for later blades confection. Qualitative and quantitative analysis of the neoformed bone tissue were conducted, and histometric data were statistically analyzed. (4) Results: Sixty days post-surgery, the percentages of neoformed bone were 10.67 ± 5.57 in group BC, 16.71 ± 5.0 in group BCM, and 55.11 ± 13.20 in group HBS. The bone formation values in group HBS were significantly higher (p < 0.05) than in groups BC and BCM. (5) Conclusions: Based on these results, it can be concluded that injectable calcium phosphate cement is an osteoconductive material that can be used to fill bone cavities. Full article

β-tricalcium phosphate (β-TCP) is one of the most common bioceramics, widely applied in bone cements and implants. Herein we synthesized β-TCP by solid state reaction in the presence of increasing amounts of two biologically active ions, namely strontium and zinc, in order to clarify the structural modifications induced by ionic substitution. The results of X-ray diffraction analysis indicate that zinc can substitute for calcium into a β-TCP structure up to about 10 at% inducing a reduction of the cell parameters, whereas the substitution occurs up to about 80 at% in the case of strontium, which provokes a linear increase of the lattice constants, and a slight modification into a more symmetric structure. Rietveld refinements and solid-state ³¹P NMR spectra demonstrate that the octahedral Ca(5) is the site of β-TCP preferred by the small zinc ion. ATR-FTIR results indicate that zinc substitution provokes a disorder of β-TCP structure. At variance with the behavior of zinc, strontium completely avoids Ca(5) site even at high concentration, whereas it exhibits a clear preference for Ca(4) site. The infrared absorption bands of β-TCP show a general shift towards lower wavenumbers on increasing strontium content. Particularly significant is the shift of the infrared symmetric stretching band at 943 cm⁻¹ due to P(1), that is the phosphate more involved in Ca(4) coordination, which further supports the occupancy preference of strontium. Full article

Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and homeostasis. The aim of the present study was to investigate an accurate, simple and sensitive alcian blue (AB) method for quantifying heparin in biological samples. A method for analyzing heparin was developed and parameters such as volume, precipitation time, solvent component, and solubility time were evaluated. The AB dye and heparin samples were allowed to react at 4 ℃ for 24 h. The heparin-AB complex was dissolved in 25 N NaOH and 2-Aminoethanol (1:24 v/v). The optical density of the solution was analyzed by UV-Vis spectrometry at 620 nm. The modified AB method was validated in accordance with U.S. Food and Drug Administration guidelines. The limit of detection was found to be 2.95 µg/mL. Intraday and interday precision ranged between 2.14–4.83% and 3.16–7.02% (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5%, confirming good accuracy. In addition, this study has discovered the interdisciplinary nature of protein detection using the AB method. The basis for this investigation was that the fibrous protein inhibits heparin-AB complex whereas globular protein does not. Further, we measured the content of sulfated GAGs (sGAGs; expressed as heparin equivalent) in the ECM of decellularized porcine liver. In conclusion, the AB method may be used for the quantitative analysis of heparin in ECM scaffolds for tissue engineering applications. Full article

The biological effects and cellular activations triggered by monosodium urate (MSU) and calcium pyrophosphate dihydrate (monoclinic: m-CPPD) crystals might be modulated by protein coating on the crystal surface. This study is aimed at: (i) Identifying proteins adsorbed on m-CPPD crystals, and the underlying mechanisms of protein adsorption, and (ii) to understand how protein coating did modulate the inflammatory properties of m-CPPD crystals. The effects of protein coating were assessed in vitro using primary macrophages and THP1 monocytes. Physico-chemical studies on the adsorption of bovine serum albumin (BSA) upon m-CPPD crystals were performed. Adsorption of serum proteins, and BSA on MSU, as well as upon m-CPPD crystals, inhibited their capacity to induce interleukin-1-β secretions, along with a decreased ATP secretion, and a disturbance of mitochondrial membrane depolarization, suggesting an alteration of NLRP3 inflammasome activation. Proteomic analysis identified numerous m-CPPD-associated proteins including hemoglobin, complement, albumin, apolipoproteins and coagulation factors. BSA adsorption on m-CPPD crystals followed a Langmuir-Freundlich isotherm, suggesting that it could modulate m-CPPD crystal-induced cell responses through crystal/cell-membrane interaction. BSA is adsorbed on m-CPPD crystals with weak interactions, confirmed by the preliminary AFM study, but strong interactions of BSA molecules with each other occurred favoring crystal agglomeration, which might contribute to a decrease in the inflammatory properties of m-CPPD crystals. These findings give new insights into the pathogenesis of crystal-related rheumatic diseases and subsequently may open the way for new therapeutic approaches. Full article

Additive manufacturing has great potential for personalized medicine in osseous fixation surgery, including maxillofacial and orthopedic applications. The purpose of this study was to demonstrate 3D printing methods for the fabrication of patient-specific fixation implants that allow for localized drug delivery. 3D printing was used to fabricate gentamicin (GS) and methotrexate (MTX)-loaded fixation devices, including screws, pins, and bone plates. Scaffolds with different infill ratios of polylactic acid (PLA), both without drugs and impregnated with GS and MTX, were printed into cylindrical and rectangular-shaped constructs for compressive and flexural strength mechanical testing, respectively. Bland PLA constructs showed significantly higher flexural strength when printed in a Y axis at 100% infill compared to other axes and infill ratios; however, there was no significant difference in flexural strength between other axes and infill ratios. GS and MTX-impregnated constructs had significantly lower flexural and compressive strength as compared to the bland PLA constructs. GS-impregnated implants demonstrated bacterial inhibition in plate cultures. Similarly, MTX-impregnated implants demonstrated a cytotoxic effect in osteosarcoma assays. This proof of concept work shows the potential of developing 3D printed screws and plating materials with the requisite mechanical properties and orientations. Drug-impregnated implants were technically successful and had an anti-bacterial and chemotherapeutic effect, but drug addition significantly decreased the flexural and compressive strengths of the custom implants. Full article

The conventional applicability of biomaterials in the field of bone tissue engineering takes into consideration several key parameters to achieve desired results for prospective translational use. Hence, several engineering strategies have been developed to model in the regenerative parameters of different forms of biomaterials, including bioactive glass and β-tricalcium phosphate. This review examines the different ways these two materials are transformed and assembled with other regenerative factors to improve their application for bone tissue engineering. We discuss the role of the engineering strategy used and the regenerative responses and mechanisms associated with them. Full article

Porous composite scaffold using an alginate and bioactive glass ICIE16M was synthesized by a simple freeze-drying technique. The scaffold was characterized using compression testing, Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), X-ray microtomography (XMT) and scanning electron microscopy (SEM). The bioactivity of the scaffold was evaluated by its ability to form apatite on its surface in simulated body fluid (SBF). The data collected showed evidence that the novel material produced had an appropriate pore size for osteoconduction, with an average pore size of 110 µm and maximum pore size of 309 µm. Statistical analysis confirmed that the glass filler significantly (P < 0.05) increased the collapse yield of the scaffolds compared with pure alginate scaffolds. The ICIE16M glass had an amorphous structure, favorable for bioactivity. Full article

Aim: The purpose of the present study is to assess the color stability of two calcium silicate-based cements (CSCs) used in regenerative endodontic procedures (REPs). Methods: A total of 40 acrylic single-rooted transparent teeth, with immature apex, were used. Root canals were filled up to 3 mm below the level of the cementoenamel junction, with either saline solution (Mineral Trioxide Aggregate (MTA)/saline and Biodentine/saline) or blood (MTA/blood and Biodentine/blood). Subsequently, ProRoot MTA^® or Biodentine^TM was placed in the root canal to create a cervical barrier. Color measurement was carried out at four different evaluation periods (3 h, 72 h, 7 days, and 6 months). Shade analysis within the L* a* b* color space was performed and color variation (∆E) calculated. The significance level for statistical analysis was set at p < 0.05. Results: The four groups showed a significant decrease in L* values over time. The ΔE value increased over time for all groups but was not statistically significant for the Biodentine/blood group. Two-way ANOVA showed no interaction between the CSC and treatment (contact with saline solution or blood). CSC used was the factor responsible for ΔE over time, inducing statistically significant color variations from T_3H to T_7D (p = 0.04) and T_3H to T_6M (p < 0.01). After 6 months, MTA/saline had 5.08 (p = 0.001) higher ΔE than Biodentine/Saline and the MTA/blood had 3.65 (p = 0.009) higher than Biodentine/blood. Conclusions: After 6 months, regardless of blood exposure, Biodentine exhibits superior color stability compared to MTA. Biodentine might be a suitable alternative to MTA as a cervical barrier material in REPs. Full article

This study investigated the impact of different calcium reagents on the morphology, composition, bioactivity and biocompatibility of two-component (CaO-SiO₂) glasses produced by the Stöber process with respect to their potential application in guided tissue regeneration (GTR) membranes for periodontal repair. The properties of the binary glasses were compared with those of pure silica Stöber particles. The direct addition of calcium chloride (CC), calcium nitrate (CN), calcium methoxide (CM) or calcium ethoxide (CE) at 5 mol % with respect to tetraethyl orthosilicate in the reagent mixture gave rise to textured, micron-sized aggregates rather than monodispersed ~500 nm spheres obtained from the pure silica Stöber synthesis. The broadening of the Si-O-Si band at ~1100 cm⁻¹ in the infrared spectra of the calcium-doped glasses indicated that the silicate network was depolymerised by the incorporation of Ca²⁺ ions and energy dispersive X-ray analysis revealed that, in all cases, the Ca:Si ratios were significantly lower than the nominal value of 0.05. The distribution of Ca²⁺ ions was also found to be highly inhomogeneous in the methoxide-derived glass. All samples released soluble silica species on exposure to simulated body fluid, although only calcium-doped glasses exhibited in vitro bioactivity via the formation of hydroxyapatite. The biocompatibilities of model chitosan-glass GTR membranes were assessed using human MG63 osteosarcoma cells and were found to be of the order: CN < pure silica ≈ CC << CM ≈ CE. Calcium nitrate is the most commonly reported precursor for the sol-gel synthesis of bioactive glasses; however, the incomplete removal of nitrate ions during washing compromised the cytocompatibility of the resulting glass. The superior bioactivity and biocompatibility of the alkoxide-derived glasses is attributed to their ease of dissolution and lack of residual toxic anions. Overall, calcium ethoxide was found to be the preferred precursor with respect to extent of calcium-incorporation, homogeneity, bioactivity and biocompatibility. Full article