Journal of Functional Biomaterials — Open Access Journal

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

A synthetic technique inspired by the biomineralisation process in nacre has been previously reported to be effective in replicating the nanostructural elements of nacre in 2D chitosan hydrogel films. Here we evaluate the applicability of this synthetic biomineralisation technique, herein called the McGrath method, in replicating the flat tabular morphology of calcium carbonate and other nanostructural elements obtained when 2D chitosan hydrogel films were used, on a 3D porous chitosan hydrogel-based scaffold, hence developing 3D chitosan-calcium carbonate composites. Nozzle extrusion-based 3D printing technology was used to develop 3D porous scaffolds using chitosan hydrogel as the printing ink in a custom-designed 3D printer. The rheology of the printing ink and print parameters were optimised in order to fabricate 3D cylindrical structures with a cubic lattice-based internal structure. The effects of various dehydration techniques, including air-drying, critical point-drying and freeze-drying, on the structural integrity of the as-printed scaffolds from the nano to macroscale, were evaluated. The final 3D composite materials were characterised using scanning electron microscopy, X-ray diffraction and energy dispersive X-ray spectroscopy. The study has shown that McGrath method can be used to develop chitosan-calcium carbonate composites wherein the mineral and matrix are in intimate association with each other at the nanoscale. This process can be successfully integrated with 3D printing technology to develop 3D compartmentalised polymer-mineral composites. Full article

Dicalcium phosphate dihydrate (DCPD) is one of the mineral phases indicated as possible precursors of biological apatites and it is widely employed in the preparation of calcium phosphate bone cements. Herein, we investigated the possibility to functionalize DCPD with aspartic acid (ASP) and poly-aspartic acid (PASP), as models of the acidic macromolecules of biomineralized tissues, and studied their influence on DCPD hydrolysis. To this aim, the synthesis of DCPD was performed in aqueous solution in the presence of increasing concentrations of PASP and ASP, whereas the hydrolysis reaction was carried out in physiological solution up to three days. The results indicate that it is possible to prepare DCPD functionalized with PASP up to a polyelectrolyte content of about 2.3 wt%. The increase of PASP content induces crystal aggregation, reduction of the yield of the reaction and of the thermal stability of the synthesized DCPD. Moreover, DCPD samples functionalized with PASP display a slower hydrolysis than pure DCPD. On the other hand, in the explored range of concentrations (up to 10 mM) ASP is not incorporated into DCPD and does not influence its crystallization nor its hydrolysis. At variance, when present in the hydrolysis solution, ASP, and even more PASP, delays the conversion into the more stable phases, octacalcium phosphate and/or hydroxyapatite. The greater influence of PASP on the synthesis and hydrolysis of DCPD can be ascribed to the cooperative action of the carboxylate groups and to its good fit with DCPD structure. Full article

Biomaterial enhanced regeneration (BER) falls mostly under the broad heading of Tissue Engineering: the use of materials (synthetic and natural) usually in conjunction with cells (both native and genetically modified as well as stem cells) and/or biological response modifiers (growth factors and cytokines as well as other stimuli, which alter cellular activity). Although the emphasis is on the biomaterial as a scaffold it is also the use of additive bioactivity to enhance the healing and regenerative properties of the scaffold. Enhancing regeneration is both moving more toward regeneration but also speeding up the process. The review covers principles of design for BER as well as strategies to select the best designs. This is first general design principles, followed by types of design options, and then specific strategies for applications in skin and load bearing applications. The last section, surveys current clinical practice (for skin and load bearing applications) including limitations of these approaches. This is followed by future directions with an attempt to prioritize strategies. Although the review is geared toward design optimization, prioritization also includes the commercializability of the devices. This means a device must meet both the clinical performance design constraints as well as the commercializability design constraints. Full article

3D printing of polymers can now be considered as a common processing technology for the development of biomaterials. These can be constituted out of polymeric abiotic material alone or can be co-printed with living cells. However, the adaptive and shape-morphing characteristics cannot be developed with the rigid, pre-determined structures obtained by 3D printing. In order to produce functional engineered biomaterials, the dynamic properties/characteristics of the living cells must be attained. 4D printing can be envisaged as a route to achieve these goals. This paper intends to give a brief review of the pioneer 4D printing research that has been developed and to present an insight into future research in this field. Full article

(1) Background: Evaluate the osteoconduction capability of a biphasic calcium phosphate (BCP) ceramic composed of hydroxyapatite and β-tricalcium phosphate 60%/40% in a rat model. (2) Methods: In the calvarial bone of 54 adult male rats, 7-mm diameter critical size defects were performed. The animals were randomly allocated to three experimental groups according to the type of material: blood clot (BCG), blood clot covered with a bovine-derived collagen membrane (MBCG), and BCP ceramic covered with a bovine-derived collagen membrane (BCPG). In each group, 6 animals were euthanatized at post-operative days 7, 30, and 60 for histological and histometric analysis. (3) Results: The qualitative analysis revealed the persistence of the collagen membrane at seven days, with no relevant newly bone formation in all groups. At 30 days, centripetal bone formation was observed residual particles of the biomaterial surrounded by fibroblasts noted in the BCPG. At 60 days, while BCG and MBCG showed a partial maturation with the central part of the defect populated by a fibrous connective tissue, in the BCPG the critical area was entirely occupied by newly formed bone. In the intra groups analysis was noted a significant increase in new bone formation during the experimental period (p < 0.05). At 60 days, BCPG showed a higher percentage area of new bone formation (p < 0.05). (4) Conclusion: BCP promoted a new bone formation by osteoconduction and might be considered a valid alternative in bone regeneration procedures. Full article

Prescription of implant treatments is very widespread at present, mainly due to the low rate of annual loss and, to date, few studies have assessed their survival in the routine clinical practice of dentistry. The purpose of this observational study was to evaluate the effectiveness of dental implants with a calcium-phosphate surface in the daily practice of dental clinics. A multicenter, prospective, non-interventional, observational study was performed, in which three experienced practitioners (one maxillofacial and two oral surgeons) inserted implants using standard external and internal hexagon connections in adult patients requiring ≥1 osseointegrated implants to replace missing teeth. Follow-up was performed for 24 months after implant loading. Two hundred and twelve subjects were included (51.5% men), with a mean age of 51.2 ± 11.90 years, in whom 544 implants were inserted. 87.2% of the patients received 1–4 implants. The preferred connection system was internal hexagon (73.5%). There were nine failures, with an interval survival rate (ISR) at 24 months of 100% and a cumulative survival rate (CSR) of 98.3%. In conclusion, implants with a modified calcium-phosphate surface are associated with a high rate of survival and may be considered a method of choice in clinical practice. Full article

Polymer nanoparticles (NPs) represent one of the most innovative non-invasive approaches for drug delivery applications. NPs main objective is to convey the therapeutic molecule be they drugs, proteins, or nucleic acids directly into the target organ or tissue. Many polymers are used for the synthesis of NPs and among the currently most employed materials several biocompatible synthetic polymers, namely polylactic acid (PLA), poly lactic-co-glycolic acid (PLGA), and polyethylene glycol (PEG), can be cited. These molecules are made of simple monomers which are naturally present in the body and therefore easily excreted without being toxic. The present review addresses the different approaches that are most commonly adopted to synthetize biocompatible NPs to date, as well as the experimental strategies designed to load them with therapeutic agents. In fact, drugs may be internalized in the NPs or physically dispersed therein. In this paper the various types of biodegradable polymer NPs will be discussed with emphasis on their applications in drug delivery. Close attention will be devoted to the treatment of cancer, where both active and passive targeting is used to enhance efficacy and reduce systemic toxicity, and to diseases affecting the central nervous system, inasmuch as NPs can be modified to target specific cells or cross membrane barriers. Full article