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Monoaldehydes, due to natural origin and therapeutic activity, have attracted great attention for their ability to crosslink chitosan hydrogels for biomedical applications. However, most studies have focused on single-component hydrogels. In this work, chitosan-based hydrogels, crosslinked for the first time with 2,3,4-trihydroxybenzaldehyde (THBA), were modified with pectin (PC), bioactive glass (BG), and rosmarinic acid (RA). All of these were not only involved in the crosslinking, but also modulated properties or imparted completely new ones. THBA functioned as a crosslinker, resulting in improved mechanical properties, high swelling capacity and delayed degradation and also imparted high antioxidant activity and antiproliferative effect on cancer cells without cytotoxicity for normal cells. Hydrogels containing PC showed enhanced mechanical strength, while the combination with BG gave improved stability in PBS. All hydrogels modified with BG exhibited the ability to mineralise in SBF. The addition of RA enhanced antioxidant and anticancer activities and promoting the mineralisation process.

Biological hydrogels are highly promising materials for bone tissue engineering (BTE) due to their high biocompatibility and biomimetic characteristics. However, for advanced and customized BTE, precise tools for material stabilization and tuning material properties are desired while optimal mineralisation must be ensured. Therefore, reagent-free crosslinking techniques such as high energy electron beam treatment promise effective material modifications without formation of cytotoxic by-products. In the case of the hydrogel gelatin, electron beam crosslinking further induces thermal stability enabling biomedical application at physiological temperatures. In the case of enzymatic mineralisation, induced by Alkaline Phosphatase (ALP) and mediated by Calcium Glycerophosphate (CaGP), it is necessary to investigate if electron beam treatment before mineralisation has an influence on the enzymatic activity and thus affects the mineralisation process. The presented study investigates electron beam-treated gelatin hydrogels with previously incorporated ALP and successive mineralisation via incubation in a medium containing CaGP. It could be shown that electron beam treatment optimally maintains enzymatic activity of ALP which allows mineralisation. Furthermore, the precise tuning of material properties such as increasing compressive modulus is possible. This study characterizes the mineralised hydrogels in terms of mineral formation and demonstrates the formation of CaP in dependence of ALP concentration and electron dose. Furthermore, investigations of uniaxial compression stability indicate increased compression moduli for mineralised electron beam-treated gelatin hydrogels. In summary, electron beam-treated mineralized gelatin hydrogels reveal good cytocompatibility for MG-63 osteoblast like cells indicating a high potential for BTE applications.

Biomaterial surface modification represents an important approach to obtain a better integration of the material in surrounding tissues. Different techniques are focused on improving cell support as well as avoiding efficiently the development of infections, such as by modifying the biomaterial surface with amine groups (–NH2). Previous studies showed that –NH2 groups could promote cell adhesion and proliferation. Moreover, these chemical functionalities may be used to facilitate the attachment of molecules such as proteins or to endow antimicrobial properties. This mini-review gives an overview of different techniques which have been used to obtain amine-rich coatings such as plasma methods and adsorption of biomolecules. In fact, different plasma treatment methods are commonly used with ammonia gas or by polymerization of precursors such as allylamine, as well as coatings of proteins (for example, collagen) or polymers containing –NH2 groups (for example, polyethyleneimine). Moreover, this mini-review will present the methods used to characterize such coatings and, in particular, quantify the –NH2 groups present on the surface by using dyes or chemical derivatization methods.

In the paper, synthesis of chitosan-based hydrogels modified with Aloe vera juice is presented. The novelty of the research was a combination of hydrogel materials with properties beneficial in viewpoint of their use as modern wound dressings and Aloe vera juice supporting the wound healing process. Hydrogels have been obtained via UV radiation. The impact of the amount of the crosslinking agent as well as the introduction of the Aloe vera juice into the hydrogel matrix has been determined. Performed measurements involved analysis of the swelling ability, characteristics of the surface roughness, determining the release profile of Aloe vera and the contact angles of hydrogels. Furthermore, the analysis of the dehydration process of the polymer membrane, investigations on the cytotoxicity of hydrogels via MTT reduction assay and the neutral red uptake assay as well as the studies on the pro-inflammatory activity have also been performed. It was proved that the addition of Aloe vera juice improves the hydrophilic properties of the materials (e.g. contact angle changed from 82.5° to 73.0°). Next, the use of 25% more of the crosslinker resulted even in the increase of the contact angle by 86%. Modified hydrogels showed higher swelling properties even by 15% than unmodified materials. Furthermore, obtained hydrogels show an ability to release Aloe vera – after 5 h approx. 80% of this additive has been released in an acidic environment. Tested materials do not exhibit cytotoxic properties, the addition of Aloe vera results in an improvement of the viability of L929 murine fibroblasts and, importantly, these materials show lower pro-inflammatory activity than the positive control. Performed investigations allow to state that obtained materials show a great application potential.

The “Multifunctional biologically active composites for applications in bone regenerative medicine” project is carried out within the TEAM-NET program of the Foundation for Polish Science financed by the European Union under the European Regional Development Fund. The authors gratefully acknowledge the financial support. T.E.L.D. thanks N8 Agrifood for its financial support in the framework of the pump priming grant “Food2Bone”. Hydroxyapatite (HAp) is a bioactive ceramic with great potential for the regeneration of the skeletal system. However, its mechanical properties, especially its brittleness, limit its application. Therefore, in order to increase its ability to transmit stresses, it can be combined with a polymer phase, which increases its strength without eliminating the important aspect of bioactivity. The presented work focuses on obtaining organic–inorganic hydrogel materials based on whey protein isolate (WPI) reinforced with nano-HAp powder. The proportion of the ceramic phase was in the range of 0–15%. Firstly, a physicochemical analysis of the materials was performed using XRD, FT-IR and SEM. The hydrogel composites were subjected to swelling capacity measurements, potentiometric and conductivity analysis, and in vitro tests in four liquids: distilled water, Ringer’s fluid, artificial saliva, and simulated body fluid (SBF). The incubation results demonstrated the successful formation of new layers of apatite as a result of the interaction with the fluids. Additionally, the influence of the materials on the metabolic activity according to ISO 10993-5:2009 was evaluated by identifying direct contact cytotoxicity towards L-929 mouse fibroblasts, which served as a reference. Moreover, the stimulation of monocytes by hydrogels via the induction of nuclear factor (NF)-κB was investigated. The WPI/HAp composite hydrogels presented in this study therefore show great potential for use as novel bone substitutes.

This study involves the synthesis of hydroxyapatite and describes the preparation and characterization of polymer coatings based on poly(ethylene glycol) diacrylate and poly(ethylene glycol) and modified with bovine serum albumin and hydroxyapatite. Hydroxyapatite was obtained by wet chemical synthesis and characterized by X-ray diffraction and FTIR spectroscopy, and its Ca/P molar ratio was determined (1.69 ± 0.08). The ceramic and bovine serum albumin were used in the preparation of composite materials with the polymeric matrix. The chemical composition of coatings was characterized with FTIR spectroscopy, and their morphology was recorded with SEM imaging. Moreover, the measurements of surface roughness parameters and stereometric research were performed. The prepared coatings were subjected to in vitro studies in simulated body fluid and artificial saliva. Changes in chemical composition and morphology after immersion were examined with FTIR spectroscopy and SEM imaging. Based on the conducted research, it can be stated that applied modifiers promote the biomineralization process. The roughness analysis confirmed prepared materials were characterized by the micrometer-scale topography. The materials morphology and roughness, and the morphology of the newly formed apatite deposit, were dependent on the type of the used modifier, and the artificial fluid used in in vitro studies.

A novel versatile biocompatible hydrogel of whey protein isolate (WPI) and two types of tannic acid (TAs) was prepared by crosslinking of WPI with TAs in a one-step method at high temperature for 30 min. WPI is one common protein-based preparation which is used for hydrogel formation. The obtained WPI-TA hydrogels were in disc form and retained their integrity after sterilization by autoclaving. Two TA preparations of differing molecular weight and chemical structure were compared, namely a polygalloyl glucose-rich extract-ALSOK 02-and a polygalloyl quinic acid-rich extract-ALSOK 04. Hydrogel formation was observed for WPI solutions containing both preparations. The swelling characteristics of hydrogels were investigated at room temperature at different pH values, namely 5, 7, and 9. The swelling ability of hydrogels was independent of the chemical structure of the added TAs. A trend of decrease of mass increase (MI) in hydrogels was observed with an increase in the TA/WPI ratio compared to the control WPI hydrogel without TA. This dependence (a MI decrease-TA/WPI ratio) was observed for hydrogels with different types of TA both in neutral and acidic conditions (pH 5.7). Under alkaline conditions (pH 9), negative values of swelling were observed for all hydrogels with a high content of TAs and were accompanied by a significant release of TAs from the hydrogel network. Our studies have shown that the release of TA from hydrogels containing ALSOK04 is higher than from hydrogels containing ALSOK 02. Moreover, the addition of TAs, which display a strong anti-cancer effect, increases the cytotoxicity of WPI-TAs hydrogels against the Hep-2 human laryngeal squamous carcinoma (Hep-2 cells) cell line. Thus, WPI-TA hydrogels with prolonged drug release properties and cytotoxicity effect can be used as anti-cancer scaffolds.

To improve the integration of a biomaterial with surrounding tissue, its surface properties may be modified by adsorption of biomacromolecules, e.g., fibrils. Whey protein isolate (WPI), a dairy industry by-product, supports osteoblastic cell growth. WPI&rsquo, s main component, &beta, lactoglobulin, forms fibrils in acidic solutions. In this study, aiming to develop coatings for biomaterials for bone contact, substrates were coated with WPI fibrils obtained at pH 2 or 3.5. Importantly, WPI fibrils coatings withstood autoclave sterilization and appeared to promote spreading and differentiation of human bone marrow stromal cells (hBMSC). In the future, WPI fibrils coatings could facilitate immobilization of biomolecules with growth stimulating or antimicrobial properties.

The adsorption of biomolecules on biomaterial surfaces can promote their integration with surrounding tissue without changing their bulk properties. For biomaterials in bone reconstruction, the promotion of osteogenic differentiation and reduction of inflammation are desirable. Fibrillar coatings are interesting because of fibrils&rsquo, high surface area-volume ratio, aiding adsorption and adhesion. Fibrils also serve as a matrix for the immobilization of biomolecules with biological activity, such as the phenolic compound phloroglucinol (PG), the subunit of marine polyphenols. The aim of this work was to investigate the influence of PG coatings on fibroblast- and osteoblast-like cells to increase the osseointegration of titanium implants. Collagen fibril coatings, containing PG at low and high concentrations, were produced on titanium alloy (Ti6Al4V) scaffolds generated by additive manufacturing (AM). These coatings, especially PG-enriched coatings, reduced hydrophobicity and modulated the behavior of human osteosarcoma SaOS-2 and mouse embryonic fibroblast 3T3 cell lines. Both osteoblastic and fibroblastic cells spread and adhered well on PG-enriched coatings. Coatings significantly reduced the inflammatory response. Moreover, osteogenic differentiation was promoted by collagen coatings with a high PG concentration. Thus, the enrichment of collagen fibril coatings with PG is a promising strategy to improve Ti6Al4V implants for bone contact in orthopedics and dentistry and is worthy of further investigation.

Whey protein isolate (WPI) is a by-product from the production of cheese and Greek yoghurt comprising &beta, lactoglobulin (&beta, lg) (75%). Hydrogels can be produced from WPI solutions through heating, hydrogels can be sterilized by autoclaving. WPI hydrogels have shown cytocompatibility and ability to enhance proliferation and osteogenic differentiation of bone-forming cells. Hence, they have promise in the area of bone tissue regeneration. In contrast to commonly used ceramic minerals for bone regeneration, a major advantage of hydrogels is the ease of their modification by incorporating biologically active substances such as enzymes. Calcium carbonate (CaCO3) is the main inorganic component of the exoskeletons of marine invertebrates. Two polymorphs of CaCO3, calcite and aragonite, have shown the ability to promote bone regeneration. Other authors have reported that the addition of magnesium to inorganic phases has a beneficial effect on bone-forming cell growth. In this study, we employed a biomimetic, marine-inspired approach to mineralize WPI hydrogels with an inorganic phase consisting of CaCO3 (mainly calcite) and CaCO3 enriched with magnesium using the calcifying enzyme urease. The novelty of this study lies in both the enzymatic mineralization of WPI hydrogels and enrichment of the mineral with magnesium. Calcium was incorporated into the mineral formed to a greater extent than magnesium. Increasing the concentration of magnesium in the mineralization medium led to a reduction in the amount and crystallinity of the mineral formed. Biological studies revealed that mineralized and unmineralized hydrogels were not cytotoxic and promoted cell viability to comparable extents (approximately 74% of standard tissue culture polystyrene). The presence of magnesium in the mineral formed had no adverse effect on cell viability. In short, WPI hydrogels, both unmineralized and mineralized with CaCO3 and magnesium-enriched CaCO3, show potential as biomaterials for bone regeneration.

Metallic implants have dominated the biomedical implant industries for the past century for load-bearing applications, while the polymeric implants have shown great promise for tissue engineering applications. The surface properties of such implants are critical as the interaction of implant surfaces, and the body tissues may lead to unfavourable reactions. Desired implant properties are biocompatibility, corrosion resistance, and antibacterial activity. A polymer coating is an efficient and economical way to produce such surfaces. A lot of research has been carried out on chitosan (CS)-modified metallic and polymer scaffolds in the last decade. Different methods such as electrophoretic deposition, sol-gel methods, dip coating and spin coating, electrospinning, etc. have been utilized to produce CS coatings. However, a systematic review of chitosan coatings on scaffolds focussing on widely employed techniques is lacking. This review surveys literature concerning the current status of orthopaedic applications of CS for the purpose of coatings. In this review, the various preparation methods of coating, and the role of the surface functionalities in determining the efficiency of coatings are discussed. Effect of nanoparticle additions on the polymeric interfaces and in regulating the properties of surface coatings are also investigated in detail.

This work explores novel biocomposite hydrogels fabricated using 40% (wt/vol) solution of whey protein isolate (WPI, from the food industry) mixed with increasing concentrations of synthetic aragonite rod-like powder of 0, 100, 200 and 300 mg/ml (named WPI0, WPI100, WPI200 and WPI300). FTIR results showed that aragonite was successfully incorporated into the WPI hydrogel network. SEM and micro-CT investigations revealed that aragonite was mainly concentrated near the edges of the composite samples, except in WPI300, which had homogeneous aragonite distribution. The pore diameters ranged from 18 to 778 μm while averaged pore size was the lowest for WPI0 at 30 μm and highest for WPI200 at 103 μm. The mean compression modulus was highest for WPI300 at 3.16 MPa. After 28 days in physiological conditions WPI300 had maximum mean swelling of 4.3% and there was the highest degradation rate for WPI200 and WPI300 and lowest for WPI100 and WPI0. The osteoblast-like MG63 cell metabolic and alkaline phosphatase activities in direct contact experiments with composites increased with increasing aragonite content over 3 weeks. Moreover, the degradation products of these composites were non-cytotoxic and led to mineral-like deposits in extracellular matrix. These WPI-aragonite biocomposite hydrogels are potent candidates for bone repair applications. Keywords: Whey protein isolate, Aragonite, Bone graft, Inexpensive, Degradation, Cytocompatible

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.

Osteochondral defects remain a huge problem in medicine today. Biomimetic bi- or multi-phasic scaffolds constitute a very promising alternative to osteochondral autografts and allografts. In this study, a new curdlan-based scaffold was designed for osteochondral tissue engineering applications. To achieve biomimetic properties, it was enriched with a protein component - whey protein isolate as well as a ceramic ingredient - hydroxyapatite granules. The scaffold was fabricated via a simple and cost-efficient method, which represents a significant advantage. Importantly, this technique allowed generation of a scaffold with two distinct, but integrated phases. Scanning electron microcopy and optical profilometry observations demonstrated that phases of biomaterial possessed different structural properties. The top layer of the biomaterial (mimicking the cartilage) was smoother than the bottom one (mimicking the subchondral bone), which is beneficial from a biological point of view because unlike bone, cartilage is a smooth tissue. Moreover, mechanical testing showed that the top layer of the biomaterial had mechanical properties close to those of natural cartilage. Although the mechanical properties of the bottom layer of scaffold were lower than those of the subchondral bone, it was still higher than in many analogous systems. Most importantly, cell culture experiments indicated that the biomaterial possessed high cytocompatibility towards adipose tissue-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells in vitro. Both phases of the scaffold enhanced cell adhesion, proliferation, and chondrogenic differentiation of stem cells (revealing its chondroinductive properties in vitro) as well as osteogenic differentiation of these cells (revealing its osteoinductive properties in vitro). Given all features of the novel curdlan-based scaffold, it is worth noting that it may be considered as promising candidate for osteochondral tissue engineering applications.

Hydrogels mineralized with calcium phosphate (CaP) are increasingly popular bone regeneration biomaterials. Mineralization can be achieved by phosphatase enzyme incorporation and incubation in calcium glycerophosphate (CaGP). Gellan gum (GG) hydrogels containing the enzyme phytase and chitosan oligomer were mineralized in CaGP solution and characterized with human osteoblast-like MG63 cells and adipose tissue-derived stem cells (ADSC). Phytase induced CaP formation. Chitosan concentration determined mineralization extent and hydrogel mechanical reinforcement. Phytase-induced mineralization promoted MG63 adhesion and proliferation, especially in the presence of chitosan, and was non-toxic to MG63 cells (with and without chitosan). ADSC adhesion and proliferation were poor without mineralization. Chitosan did not affect ADSC osteogenic differentiation.

Hydrogels, which are versatile three-dimensional structures containing polymers and water, are very attractive for use in biomedical fields, but they suffer from rather weak mechanical properties. In this regard, biocompatible particles can be used to enhance their mechanical properties. The possibility of loading such particles with drugs (e.g. enzymes) makes them a particularly useful component in hydrogels. In this study, micro/nanoparticles containing various ratios of Ca

Porous titanium alloy Ti6Al4V scaffolds manufactured via electron beam melting (EBM®) reveal broad prospects for applications in bone tissue engineering. However, local inflammation and even implant failure may occur while placing an implant into the body. Thus, the application of drug carriers to the surface of a metallic implant can provide treatment at the inflammation site. In this study, we propose to use polyelectrolyte (PE) microcapsules formed by layer-by-layer (LbL) synthesis loaded with both porous calcium carbonate (CaCO3) microparticles and the anti-inflammatory drug dexamethasone (DEX) to functionalize implant surfaces and achieve controlled drug release. Scanning electron microscopy indicated that the CaCO3 microparticles coated with PE bilayers loaded with DEX had a spherical shape with a diameter of 2.3 ± 0.2 μm and that the entire scaffold surface was evenly coated with the microcapsules. UV spectroscopy showed that LbL synthesis allows the manufacturing of microcapsules with 40% DEX. According to high performance liquid chromatography (HPLC) analysis, 80% of the drug was released within 24 h from the capsules consisting of three bilayers of polystyrene sulfonate (PSS) and poly(allylamine)hydrochloride (PAH). The prepared scaffolds functionalized with CaCO3 microparticles loaded with DEX and coated with PE bilayers showed hydrophilic surface properties with a water contact angle below 5°. Mouse embryonic fibroblast cells were seeded on Ti6Al4V scaffolds with and without LbL surface modification. The surface modification with LbL PE microcapsules with CaCO3 core affected cell morphology in vitro. The results confirmed that DEX had no toxic effect and did not prevent cell adhesion and spreading, thus no cytotoxic effect was observed, which will be further studied in vivo.

Coating biomedical implant surfaces with biopolymers is an easy and inexpensive way to impart functionalities. Numerous biocompatible biopolymers, including cationic crustacean-derived chitosans, have been used. Here, substrates of Titanium (Ti), commonly used for bone contact applications, were coated with chitosan and one of three polyphenol-rich plant extracts (PPrPE) and characterized physicochemically. Hela cell adhesion and growth of methicillin-resistant Staphylococcus aureus (MRSA) were studied. Chitosan and PPrPE on surfaces were detected by FTIR and XPS. Chitosan coatings, both with and without PPrPE functionalization, did not inhibit MRSA growth and promoted Hela cell adhesion. The effect of PPrPE functionalization remained unclear.

Hydrogels are popular materials for tissue regeneration. Incorporation of biologically active substances, e.g. enzymes, is straightforward. Hydrogel mineralization is desirable for bone regeneration. Here, hydrogels of Gellan Gum (GG), a biocompatible polysaccharide, were mineralized biomimetically with CaCO 3 using a double enzymatic approach. The enzymes urease (U) and carbonic anhydrase (CA) were incorporated in GG hydrogels. Hydrogels were incubated in a mineralization solution containing U substrate (urea) and calcium ions. U converts urea to ammonia (which raises pH) and CO 2 . CA catalyses the reaction of CO 2 with water to form HCO 3 − , which undergoes deprotonation to form CO 3 2− , which react with Ca 2+ to form insoluble CaCO 3 . All hydrogels containing U+CA were mineralized more with calcite and stiffer than hydrogels containing U. Mineralization with calcite promoted proliferation and spreading of osteoblast-like cells.

In this work, for the first time, a material derived from food industry waste – whey protein isolate – and a material commonly used in bone regeneration – bioactive glasses – were combined to obtain novel composite biomaterials with potential applications in bone tissue engineering (BTE). Additionally, to obtain pro-angiogenic properties, sol–gel-derived BGs doped with Cu2+ and Co2+ ions were used. Using a simple gas foaming method, ready-to-use (sterile), bioactive scaffolds with high porosity (above 70%), fully connected pore networks, and pore size suitable for BTE applications (80–350 μm) were obtained. Furthermore, scaffolds showed additional functionalities – calcium phosphate-forming ability and gradual release of therapeutic ions. Porous WPI/BG composites showed great potential for use as novel bone substitutes.

Introducing collagen the basic ingredient of bone tissue into the structure of chitosan gels that are formed at physiological body temperature, aims to create so-called biomimetic structures, which are close in composition to the natural composition of bone tissue. The aim of this study was to determine the influence of mineralization on the structural properties of thermosensitive chitosan-collagen gels containing alkaline phosphatase (ALP) by SEM, XRD FTIR and XPS analyses, compared to the previously presented structure of chitosan-collagen gels before mineralization.

Polyphenols are known for their antimicrobial activity, whilst both polyphenols and the globular protein β-lactoglobulin (bLG) are suggested to have antioxidant properties and promote cell proliferation. These are potentially useful properties for a tissue-engineered construct, though it is unknown if they are retained when both compounds are used in combination. In this study, a range of different microbes and an osteoblast-like cell line (human fetal osteoblast, hFOB) were used to assess the combined effect of: (1) green tea extract (GTE), rich in the polyphenol epigallocatechin gallate (EGCG), and (2) whey protein isolate (WPI), rich in bLG. It was shown that approximately 20–48% of the EGCG in GTE reacted with WPI. GTE inhibited the growth of Gram-positive bacteria, an effect which was potentiated by the addition of WPI. GTE alone also significantly inhibited the growth of hFOB cells after 1, 4, and 7 days of culture. Alternatively, WPI significantly promoted hFOB cell growth in the absence of GTE and attenuated the effect of GTE at low concentrations (64 µg/mL) after 4 and 7 days. Low concentrations of WPI (50 µg/mL) also promoted the expression of the early osteogenic marker alkaline phosphatase (ALP) by hFOB cells, whereas GTE inhibited ALP activity. Therefore, the antioxidant effects of GTE can be boosted by WPI, but GTE is not suitable to be used as part of a tissue-engineered construct due to its cytotoxic effects which negate any positive effect WPI has on cell proliferation.

Hybrid materials, or hybrids incorporating both organic and inorganic constituents, are emerging as a very potent and promising class of materials due to the diverse, but complementary nature of the properties inherent of these different classes of materials. The complementarity leads to a perfect synergy of properties of desired material and eventually an end-product. The diversity of resultant properties and materials used in the construction of hybrids, leads to a very broad range of application areas generated by engaging very different research communities. We provide here a general classification of hybrid materials, wherein organics–in-inorganics (inorganic materials modified by organic moieties) are distinguished from inorganics–in–organics (organic materials or matrices modified by inorganic constituents). In the former area, the surface functionalization of colloids is distinguished as a stand-alone sub-area. The latter area—functionalization of organic materials by inorganic additives—is the focus of the current review. Inorganic constituents, often in the form of small particles or structures, are made of minerals, clays, semiconductors, metals, carbons, and ceramics. They are shown to be incorporated into organic matrices, which can be distinguished as two classes: chemical and biological. Chemical organic matrices include coatings, vehicles and capsules assembled into: hydrogels, layer-by-layer assembly, polymer brushes, block co-polymers and other assemblies. Biological organic matrices encompass bio-molecules (lipids, polysaccharides, proteins and enzymes, and nucleic acids) as well as higher level organisms: cells, bacteria, and microorganisms. In addition to providing details of the above classification and analysis of the composition of hybrids, we also highlight some antagonistic yin-&-yang properties of organic and inorganic materials, review applications and provide an outlook to emerging trends.

The process of porous calcium carbonate (CaCO3 ) covering on electrospun poly(e-caprolactone) (PCL) fibers is described in this study. Uniform CaCO3 coatings, composed of vaterite microparticles and its aggregates, were formed on PCL fibers by mineral precipitation from solution under ultrasonic treatment. The porous structure of CaCO3 in vaterite polymorphic form is useful for loading of various substances (drugs and nanoparticles), and this property makes vaterite an appropriate material for design of drug delivery systems. Such mineralization was implemented to attain therapeutic and/or biological activity of tissue engineering scaffolds based on electrospun PCL, by means of CaCO3 coatings. Various structures and polymorphs of CaCO3 coatings were obtained by variation of growth conditions (time of fiber incubation in work solution, ultrasonic treatment of this system). Coating homogeneity, CaCO3 polymorphic form, morphology, and CaCO3 mass can be controlled by number of successive stages of fibrous material treatment. Cytotoxicity tests showed that PCL fibers mineralized with CaCO3 did not release substances toxic for cells. SEM images of PCL/CaCO3 scaffolds cultured with cells demonstrate that scaffolds supported cell adhesion and spreading. The presented results show the new technique of controlled PCL scaffold mineralization with vaterite, and an opportunity of using PCL/CaCO3 as scaffolds for tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 94-103, 2017.

In this work, composite membranes were investigated as future components of a layered implant for the reconstruction of nasal septum. Incorporation of zinc ions into nasal implants could potentially provide antibacterial properties to decrease or eliminate bacterial infections and subsequent surgical complications. Two types of membranes were prepared using an electrospinning method: PCL with bioglass and PCL with bioglass doped with Zn. The aim of this work was to investigate the influence of bioglass addition on the morphology, fiber diameter and composition of the membranes. The apatite-forming ability was examined in Simulated Body Fluid (SBF). The cytotoxicity of the membranes, ALP activity and in vitro mineralization were evaluated in cell culture. The mineralization and ALP activity was higher for polycaprolactone membranes modified with Zn doped bioglass than compared to pure PCL membranes or control material. The results proved that the presence of $Zn^{2+}$ in the electrospun membranes = influence the osteogenic differentiation of cells.

Polysaccharides of marine origin are gaining interest as biomaterial components. Bacteria derived from deep-sea hydrothermal vents can produce sulfated exopolysaccharides (EPS), which can influence cell behavior. The use of such polysaccharides as components of organic, collagen fibril-based coatings on biomaterial surfaces remains unexplored. In this study, collagen fibril coatings enriched with HE800 and GY785 EPS derivatives were deposited on titanium alloy (Ti6Al4V) scaffolds produced by rapid prototyping and subjected to physicochemical and cell biological characterization. Coatings were formed by a self-assembly process whereby polysaccharides were added to acidic collagen molecule solution, followed by neutralization to induced self-assembly of collagen fibrils. Fibril formation resulted in collagen hydrogel formation. Hydrogels formed directly on Ti6Al4V surfaces, and fibrils adsorbed onto the surface. Scanning electron microscopy (SEM) analysis of collagen fibril coatings revealed association of polysaccharides with fibrils. Cell biological characterization revealed good cell adhesion and growth on bare Ti6Al4V surfaces, as well as coatings of collagen fibrils only and collagen fibrils enhanced with HE800 and GY785 EPS derivatives. Hence, the use of both EPS derivatives as coating components is feasible. Further work should focus on cell differentiation.

The present study focuses on biopolymer surface modification of cp-Titanium with Chitosan, Gelatin, and Sodium Alginate. The biopolymers were spin coated onto a cp-Titanium substrate and further subjected to Electrochemical Impedance Spectroscopic (EIS) characterization. Artificial Neural Network (ANN) was developed to predict the Open Circuit Potential (OCP) values and Nyquist plot for bare and biopolymer coated cp-Titanium substrate. The experimental data obtained was utilized for ANN training. Two input parameters, i.e., substrate condition (coated or uncoated) and time period were considered to predict the OCP values. Backpropagation Levenberg-Marquardt training algorithm was utilized in order to train ANN and to fit the model. For Nyquist plot, the network was trained to predict the imaginary impedance based on real impedance as a function of immersion periods using the Back Propagation Bayesian algorithm. The biopolymer coated cp-Titanium substrate shows the enhanced corrosion resistance compared to uncoated substrates. The ANN model exhibits excellent comparison with the experimental results in both the cases indicating that the developed model is very accurate and efficiently predicts the OCP values and Nyquist plot. Keywords: Cp-Ti, Chitosan, Gelatin, Sodium alginate, Electrochemical impedance spectroscopy (EIS), Artificial neural network (ANN)

Paclitaxel is a natural, highly lipophilic anti proliferative drug widely used in medicine. We have studied the release of tritium-labeled paclitaxel (3H-PTX) from matrices destined for the coating of vascular stents and produced by the electrospinning method from the solutions of polycaprolactone (PCL) with paclitaxel (PTX) in hexafluoisoropropanol (HFIP) and/or solutions of PCL with PTX and human serum albumin (HSA) in HFIP or HIFP-dimethyl sulphoxide (DMSO) blend. The release of PTX has been shown to depend on the solvent and the composition of electrospinning solution, as well as the composition of the surrounding medium, particularly the concentration of free PTX and PTX-binding biomolecules present in human serum. It was shown that 3D matrices can completely release PTX without weight loss. Two-phase PTX release from optimized 3D matrices was obtained: ~27% of PTX was released in the first day, another 8% were released over the next 26 days. Wherein ~2.8%, ~2.3%, and ~0.25% of PTX was released on day 3, 9, and 27, respectively. Considering PTX toxicity, the rate of its diffusion through the arterial wall, and the data obtained the minimum cytostatic dose of the drug in the arterial wall will be maintained for at least three months.

Ti6Al4V is a popular biomaterial for load-bearing implants for bone contact, which can be fabricated by additive manufacturing technologies. Their long-term success depends on their stable anchoring in surrounding bone, which in turn depends on formation of new bone tissue on the implant surface, for which adhesion and proliferation of bone-forming cells is a pre-requisite. Hence, surface coatings which promote cell adhesion and proliferation are desirable. Here, Ti6Al4V discs prepared by additive manufacturing (EBM) were coated with layers of pectins, calcium-binding polysaccharides derived from citrus (C) and apple (A), which also contained alkaline phosphatase (ALP), the enzyme responsible for mineralization of bone tissue. Adhesion and proliferation of human bone marrow stromal cells (hBMSC) were assessed. Proliferation after 7 days was increased by A-ALP coatings and, in particular, by C-ALP coatings. Cell morphology was similar on coated and uncoated samples. In conclusion, ALP-loaded pectin coatings promote hBMSC adhesion and proliferation.

This study aimed to develop hybrid electrospun chitosan&ndash, phospholipid nanofibers and investigate the effect of phospholipid (P) content and chitosans (Ch) molecular weights (Mw) and degree of acetylation (DA), on the morphological, mechanical and mucoadhesive properties of the nanofibers. Electrospun Ch/P nanofibers exhibited a smooth and uniform surface with average diameters ranging from 300 to 1000 nm, as observed by scanning electron microscopy (SEM). The average diameter of the nanofibers was observed to increase with the increase of the Mw and degree of deacetylation of Ch, and phospholipid content. The elastic and adhesive properties of the nanofibers were determined by atomic force microscopy, and displayed higher values for higher Mw and lower DA Ch used. The elastic modulus of electrospun Ch/P hybrid fibers determined for the different conditions tested was found to be in the range of 500 and 1400 MPa. Furthermore, electrospun Ch/P nanofibers displayed mucoadhesive properties expressed by the work of adhesion calculated after the compression of the nanofibers against a section of pig small intestine. Our results showed that the increase in phospholipid content and DA of Ch decrease the work of adhesion, while the increase of Mw resulted in slightly higher work of adhesion of the nanofibers.

The present work focuses on the development of novel injectable, self-gelling composite hydrogels based on two types of low esterified amidated pectins from citrus peels and apple pomace. Sol-gelderived, calcium-rich bioactive glass (BG) fillers in a particle form are applied as delivery vehicles for the release of Ca2+ ions to induce internal gelation of pectins. Composites were prepared by a relatively simple mixing technique, using 20% w/v BG particles of two different sizes (2.5 and

Enrichment of hydrogels with inorganic particles improves their suitability for bone regeneration by enhancing their mechanical properties, mineralizability, and bioactivity as well as adhesion, proliferation, and differentiation of bone-forming cells, while maintaining injectability. Low aggregation and homogeneous distribution maximize particle surface area, promoting mineralization, cell-particle interactions, and homogenous tissue regeneration. Hence, determination of the size and distribution of particles/particle agglomerates in the hydrogel is desirable. Commonly used techniques have drawbacks. High-resolution techniques (e.g., SEM) require drying. Distribution in the dry state is not representative of the wet state. Techniques in the wet state (histology, µCT) are of lower resolution. Here, self-gelling, injectable composites of Gellan Gum (GG) hydrogel and two different types of sol-gel-derived bioactive glass (bioglass) particles were analyzed in the wet state using Synchrotron X-ray radiation, enabling high-resolution determination of particle size and spatial distribution. The lower detection limit volume was 9 × 10(-5) mm(3) . Bioglass particle suspensions were also studied using zeta potential measurements and Coulter analysis. Aggregation of bioglass particles in the GG hydrogels occurred and aggregate distribution was inhomogeneous. Bioglass promoted attachment of rat mesenchymal stem cells (rMSC) and mineralization.

The paper presents the structural studies of mineralized chitosan hydrogels. Hydrogels produced by using sodium beta-glycerophosphate (Na-β-GP) as a neutralizing agent. Mineralization was performed method “post loading”, which consisted in sorption to the gels structure Ca ions. In order to obtain – in the structure of gels – compounds similar to the hydroxyapatites present naturally in bone tissue, gels after sorption were modified in: pH 7 buffer and sodium hydrogen phosphate. In order to determine the structural properties of the gels, the following methods were used: infrared spectroscopy with Fourier transformation, FTIR, X-ray diffractometry, XRD, scanning electron microscopy, SEM.

Gellan gum hydrogels functionalized with alkaline phosphatase were enzymatically mineralized with phosphates in mineralization medium containing calcium (Ca) and zinc (Zn) to improve their suitability as biomaterials for bone regeneration. The aims of the study were to endow mineralized hydrogels with antibacterial activity by incorporation of Zn in the inorganic phase, and to investigate the effect of Zn incorporation on the amount and type of mineral formed, the compressive modulus of the mineralized hydrogels and on their ability to support adhesion and growth of MC3T3-E1 osteoblast-like cells. Mineralization medium contained glycerophosphate (0.05 m) and three different molar Ca:Zn ratios, 0.05:0, 0.04:0.01 and 0.025:0.025 (all mol/dm3 ), hereafter referred to as A, B and C, respectively. FTIR, SAED and TEM analysis revealed that incubation for 14 days caused the formation of predominantly amorphous mineral phases in sample groups A, B and C. The presence of Zn in sample groups B and C was associated with a drop in the amount of mineral formed and a smaller mineral deposit morphology, as observed by SEM. ICP-OES revealed that Zn was preferentially incorporated into mineral compared to Ca. Mechanical testing revealed a decrease in compressive modulus in sample group C. Sample groups B and C, but not A, showed antibacterial activity against biofilm-forming, methicillin-resistant Staphylococcus aureus. All sample groups supported cell growth. Zn incorporation increased the viable cell number. The highest values were seen on sample group C. In conclusion, the sample group containing the most Zn, i.e. group C, appears to be the most promising. Copyright © 2015 John Wiley & Sons, Ltd.

Polycaprolactone (PCL) is a biodegradable and biocompatible polyester whose low melting point facilitates production of 3D porous scaffolds with precisely defined dimensions and internal architecture by rapid prototyping techniques. To improve the suitability of such PCL scaffolds for bone regeneration applications, they were coated with inorganic layers of calcium phosphate (CaP) and CaP doped with 0.6% w/v magnesium (CaP+Mg) using pulsed laser deposition (PLD) and characterized in vitro using osteoblast-like Saos-2 cells. Saos-2 cells were able to adhere to all scaffolds. CaP+Mg coatings significantly increased activity of alkaline phosphatase (ALP), an early differentiation marker, after 7 days. However, gene expression of ALP after 7 days was markedly lower on the same scaffolds. These data show the feasibility of coating PCL with CaP layers by PLD and the possibility of influencing osteoblastic differentiation by magnesium doping of the CaP coating.

Polyelectrolyte complexes (PEC) of chitosan and ulvan were fabricated to study alkaline phosphatase (ALP) mediated formation of apatitic minerals. Scaffolds of the PEC were subjected to ALP and successful mineral formation was studied using SEM, Raman and XRD techniques. Investigation of the morphology via SEM shows globular structures of the deposited minerals, which promoted cell attachment, proliferation and extracellular matrix formation. The PEC and their successful calcium phosphate based mineralization offers a greener route of scaffold fabrication towards developing resorbable materials for tissue engineering.

This article reports on a study of the mineralisation behaviour of CaCO3 deposited on electrospun poly(ϵ-caprolactone) (PCL) scaffolds preliminarily treated with low-temperature plasma. This work was aimed at developing an approach that improves the wettability and permeability of PCL scaffolds in order to obtain a superior composite coated with highly porous CaCO3, which is a prerequisite for biomedical scaffolds used for drug delivery. Since PCL is a synthetic polymer that lacks functional groups, plasma processing of PCL scaffolds in O2, NH3, and Ar atmospheres enables introduction of highly reactive chemical groups, which influence the interaction between organic and inorganic phases and govern the nucleation, crystal growth, particle morphology, and phase composition of the CaCO3 coating. Our studies showed that the plasma treatment induced the formation of O- and N-containing polar functional groups on the scaffold surface, which caused an increase in the PCL surface hydrophilicity. Mineralisation of the PCL scaffolds was performed by inducing precipitation of CaCO3 particles on the surface of polymer fibres from a mixture of CaCl2- and Na2CO3-saturated solutions. The presence of highly porous vaterite and nonporous calcite crystal phases in the obtained coating was established. Our findings confirmed that preferential growth of the vaterite phase occurred in the O2-plasma-treated PCL scaffold and that the coating formed on this scaffold was smoother and more homogenous than those formed on the untreated PCL scaffold and the Ar- and NH3-plasma-treated PCL scaffolds. A more detailed three-dimensional assessment of the penetration depth of CaCO3 into the PCL scaffold was performed by high-resolution micro-computed tomography. The assessment revealed that O2-plasma treatment of the PCL scaffold caused CaCO3 to nucleate and precipitate much deeper inside the porous structure. From our findings, we conclude that O2-plasma treatment is preferable for PCL scaffold surface modification from the viewpoint of use of the PCL/CaCO3 composite as a drug delivery platform for tissue engineering. © 2018 The Royal Society of Chemistry.

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component β-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI.

There are relatively few nanotechnologies that can produce nanocomposite scaffolds for cell growth. Electrospinning has emerged as the foremost method of producing nanofibrous biomimetic scaffolds for tissue engineering applications. In this study diamond nanoparticles were integrated into a polymer solution to develop a nanocomposite scaffold containing poly(lactide-co-glycolide) (PLGA) loaded with diamond nanoparticles. To investigate the effect of adding diamond nanoparticles to PLGA scaffolds, primary human mesenchymal stem cells (hMSCs) were seeded on the scaffolds. The cytocompatibility results showed that addition of diamond nanoparticles did not impinge upon cell proliferation, nor was there a cytotoxic cellular response after 9 days in culture. Scanning electron microscopy, transmission electron microscopy, atomic force microscopy and confocal microscopy enabled qualitative characterization of the fibres and revealed cell morphology and number. Furthermore, surface roughness was measured to evaluate diamond nanoparticle modifications, and no significant difference was found between the diamond nanocomposite and pure polymer scaffolds. On the other hand, bright spots on phase images performed by atomic force microscopy suggested a higher hardness at certain points on fibers of the PLGA-nanodiamond composites, which was supported by nanoindentation measurements. This study shows that PLGA nanofibers can be reinforced with nanodiamond without adversely affecting cell behaviour, and thus it sets the foundation for future application of these scaffolds in bone tissue engineering.

In the current study, the effect of post-plasma grafting of 2-aminoethyl methacrylate (AEMA) and the subsequent covalent immobilization of gelatin (GelB) on poly( l -lactide-co-glycolide) (PLGA) thin films were investigated. The applied modification resulted in surface chemistry changes of PLGA. More specifically, an increase of nitrogen from 0 at% to 14 at% with a concomitant decrease in carbon and oxygen concentration was observed. The samples were more hydrophilic after the treatment as reflected by a decrease of the water contact angle from 72° to 33° and more rough at the nanoscale as shown by atomic force microscopy (increase of R a roughness from 0.7 nm to 10 nm). The growth of osteoblast-like MG-63 cells was enhanced on biofunctionalised PLGA-AEMA-GelB surfaces and the cells were more homogenously distributed than on non-modified PLGA. Our findings are especially important for tissue engineering applications, where substrates supporting homogenous cell cultures are particularly promising.

Hydrogel biomaterials can be easily enriched with bioactive substances such as the mineralization-promoting enzyme alkaline phosphatase (ALP). In this study, poly(vinyl alcohol) (PVA) hydrogels designed for osteochondral regeneration containing incorporated ALP were mineralized with calcium phosphate (CaP) and magnesium phosphate (MgP) by incubation in solutions of 0.1 M calcium or magnesium glycerophosphate (CaGP, MgGP). Hydrogels incubated in water served as controls. More mineral was formed in hydrogels incubated in CaGP than in MgGP. Rheometry revealed that mechanical strength (storage modulus) decreased in the order: CaGP>MgGP>water. Physicochemical charaterization showed that hydrogels incubated in CaGP appeared to be mineralized with apatite and amorphous CaP, while hydrogels incubated in MgGP appeared to be mineralized with plate-like MgP crystals and amorphous MgP. Hydrogels incubated in water were devoid of mineralization. Cell viability testing showed that proliferation on hydrogels incubated in MgGP was comparable to that on non-mineralized samples and superior to that on hydrogels incubated in CaGP. The results prove the principle of enzymatic mineralization of PVA hydrogels with CaP and MgP. Further work may concentrate on in vivo evaluation of the suitability of these mineralized hydrogels for bone or osteochondral regeneration applications.

In this paper the properties of thermosensitive chitosan hydrogels prepared with the use of chitosan glutamate and β-glycerophosphate are presented. The study is focused on the determination of changes in the hydrogel structure in different environments: during conditioning in water and buffer at pH 7 and pH 2 respectively. The structure of gels was observed under the Scanning Electron Microscopy (SEM) and was investigated by infrared (IR) spectroscopy. The crystallinity of gel structure was determined by X-ray diffraction analysis (XRD). On the basis of structural changes during the conditioning in water a mechanism of their formation was proposed.

Thermosensitive injectable hydrogels for bone regeneration consisting of chitosan, sodium beta-glycerophosphate (Na-β-GP) and alkaline phosphatase (ALP) were enriched with oxygenated perfluorodecalin (PFD), a liquid hydrophobic perfluorochemical with high oxygen affinity, in order to improve cell growth on the hydrogels. Furthermore, influence of PFD concentration on hydrogel physicochemical properties relevant for bone regeneration, namely gelation speed, radiopacity and homogenicity, was investigated. Addtionally, ALP-mediated and non-ALP-mediated mineralization were evaluated by incubation in 0.1 M calcium glycerophosphate and simulated body fluid. 2% (w/v) chitosan hydrogels containing 2.5 mg/ml ALP were enriched with PFD at five concentrations, namely 0 (control), 0.069, 0.138, 0.207 and 0.276 ml/ml hydrogel, denoted A, B, C, D and E, respectively. Rheometrical investigations revealed that gelation speed increased with increasing PFD concentration. Micro-CT analysis revealed homogenicity of all sample groups except E and that radiopacity increased in the order B>C>A>D>E. ALP-mediated and non-ALP-mediated mineralization were not affected adversely by PFD. Growth of human adipose tissue-derived mesenchymal stem cells (ADSC) encapsulated in hydrogels was markedly higher in sample groups containing PFD, i.e. B–E. Hence, incorporation of oxygenated PFD can improve the suitability of hydrogels as bone regeneration materials.

The use of porous titanium-based implant materials for bone contact has been gaining ground in recent years. Selective laser melting (SLM) is a rapid prototyping method by which porous implants with highly defined external dimensions and internal architecture can be produced. The coating of porous implants produced by SLM with ceramic layers based on calcium phosphate (CaP) remains relatively unexplored, as does the doping of such coatings with magnesium (Mg) to promote bone formation. In this study, Mg-doped coatings of the CaP types octacalcium phosphate and hydroxyapatite (HA) were deposited on such porous implants using the pulsed laser deposition method. The coated implants were subsequently implanted in a rabbit femoral defect model for 6 months. Uncoated implants served as a reference material. Bone-implant contact and bone volume in the region of interest were evaluated by histopathological techniques using a tri-chromatographic Masson-Goldner staining method and by microcomputed tomography (µCT) analysis of the volume of interest in the vicinity of implants. Histopathological analysis revealed that all implant types integrated directly with surrounding bone with ingrowth of newly formed bone into the pores of the implants. Biocompatibility of all implant types was demonstrated by the absence of inflammatory infiltration by mononuclear cells (lymphocytes), neutrophils, and eosinophils. No osteoclastic or foreign body reaction was observed in the vicinity of the implants. µCT analysis revealed a significant increase in bone volume for implants coated with Mg-doped HA compared to uncoated implants.