Dynamic viscoelastic properties of collagen gels in the presence and absence of collagen fibrils

We measured the dynamic viscoelasticities of collagen gels prepared and modified by four different methods: i) collagen gels cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) after their preparation, ii) collagen gels cross-linked simultaneously with their preparation, iii) collagen gels irradiated with gamma rays after their preparation, and iv) collagen gels directly formed from an acidic collagen solution by gamma-cross-linking. Dynamic viscoelasticities of all samples were measured using a rheometer before and after heating for 30 min at 80 °C. The collagen gels sequentially cross-linked by 125 mM EDC after preparation and then heated exhibited mechanically strong properties (storage modulus G′, 7010 Pa; loss modulus G″, 288 Pa; Young's modulus E, 0.012 in the rapidly-increasing phase and 0.095 in the moderately-increasing phase; tensile strain, 5.29; tensile stress σ, 0.053). We generally conclude that the G′ value decreases when gels without fibrils are heated. On the other hand, well cross-linked collagen gels with thick fibrils, such as gels sequentially cross-linked with 125 mM EDC after preparation or gamma-cross-linked conventional gels irradiated at 40 kGy, exhibit a distinct increase in G′ value after heating. Those gels also have thick, twisted, or fused fibrils of collagen.     

Effect of accelerated aging on the viscoelastic properties of Elast-Eon™: A polyurethane with soft poly(dimethylsiloxane) and poly(hexamethylene oxide) segments

The viscoelastic properties of cylinders (diameter 5 mm, height 2.2 ± 0.2 mm) of Elast-Eon? 3, (a polyurethane with poly(dimethylsiloxane) and poly(hexamethylamine oxide) segments) were investigated before and after the specimens had undergone accelerated aging in saline solution at 70 °C for 38, 76 and 114 days (to simulate aging at 37 °C, for 1, 2 and 3 years, respectively). All sets of specimens were immersed in physiological saline solution at 37 °C during testing and the properties were measured using dynamic mechanical analysis (DMA). A sinusoidal cyclic compression of 40 N ± 5 N was applied over a frequency range, f, of 0.02–100 Hz. Values of the storage, E′, and loss, E″, moduli were found to depend on f; the dependence of E′ or E″ on the logarithm (base 10) of f was represented by a second-order polynomial. After accelerated aging, the E′ and E″ increased significantly (p < 0.05) and the specimens became darker and more opaque. SEM images showed that accelerated aging affected the surface morphology but ATR-FTIR spectra did not show any appreciable change in surface chemistry. DSC thermograms showed some slight changes in thermal properties following accelerated aging.     

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.     

Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering

Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p > 0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP–G–CA–ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP–G–CA–ET). DBP–G–CA–ET exhibited a significant (p > 0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (p < 0.05). Lyophilized acellular detoxified GLUT cross-linked bovine pericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions.     

In situ synthesis and luminescence characteristics of complexes of europium(III) with 4,6-diacetylresorcinol

The complexes of europium(III) with 4,6-diacetylresorcinol (H₂DAR) and a co-ligand (phen, bpy or 2,2′-bipyridine N,N′-dioxide (2,2′-bpyO₂)) were in situ synthesized in silica matrix via a two-step gel process. The formation of complexes in silica gel was confirmed by the luminescence excitation spectra. The silica gels that contain in situ synthesized europium complexes exhibit the characteristic emission bands of the Eu(III). The results show that there are two ways to enhance the emission intensity of the Eu(III): (i) synthesize the complex in silica matrix and (ii) synthesize the complex with a co-ligand, which coordinates with Eu(III) in the composite system and can efficiently transfer the energy from 4,6-diacetylresorcinol to the Eu(III). The order of the luminescence intensities of the complexes is: Eu₂(DAR)₃(phen)₂-(sol–gel) > Eu₂(DAR)₃(2,2′-bpyO₂)₂-(sol–gel) > Eu₂(DAR)₃ (bpy)₂-(sol–gel) > Eu₂(DAR)₃-(sol–gel) > pure Eu₂(DAR)₃·4H₂O.     

Cross-linking of collagen with laccases and tyrosinases

Oxidation of acid soluble collagen (ASC), collagen suspension and BrCN-peptides (BrCN-P) with tyrosinases from B. obtusa (BoT1, BoT2) and A. bisporus (AbT) and laccases from T. versicolor (TvL) and T. hirsuta (ThL) resulted in UV/VIS peaks at 475 nm and 305 nm indicating formation of reactive o-quinones and cross-linked components. Concomitant oxygen consumption was higher for the low molecular weight enzymes (TvL and BoT2) indicating limited accessibility. SDS-PAGE and SEC bands at higher MW demonstrated the formation of cross-linked material. LC-MS/MS analysis suggested the involvement of tyrosine residues in cross-linking without major changes of sequence similarities to untreated collagen. However, an increase of the SEC α-peak together with a decrease of β-peak and the 1235/1450 cm^? 1 ratio (FTIR) indicated partial degradation. Crosslinking was enhanced by phenolic molecules such as catechine which lead to increased denaturation temperature and reduced degradation by microbial collagenase. The tensile strength was increased whereas resistance to compressive forces was not influenced.     

Bone induction by biomimetic PLGA copolymer loaded with a novel synthetic RADA16-P24 peptide in vivo

Bone morphogenetic protein-2 (BMP-2) is a key bone morphogenetic protein, and poly(lactic-co-glycolic acid) (PLGA) has been widely used as scaffold for clinical use to carry treatment protein. In the previous studies, we have synthesized BMP-2-related peptide (P24) and found its capacity of inducing bone regeneration. In this research, we have synthesized a new amphiphilic peptide Ac-RADA RADA RADA RADA S[PO4]KIPKASSVPTELSAISTLYLDDD-CONH2 (RADA16-P24) with an assembly peptide RADA16-Ion the P24 item of BMP2 to form divalent ion-induced gelatin. Two methods of physisorption and chemical cross-linking were used to bind RADA16-P24 onto the surface of the copolymer PLGA to synthesize RADA16-P24–PLGA, and its capacity of attaching bone marrow stromal cells (BMSCs) was evaluated in vitro and inducing ectopic bone formation was examined in vivo. In vitro our results demonstrated that RADA16-P24–PLGA copolymer prepared by physisorbing or prepared by chemical cross-linking had a peptide binding rate of (2.0180 ± 0.5296)% or (10.0820 ± 0.8405)% respectively (P < 0.05). In addition the BMSCs proliferated vigorously in the RADA16-P24–PLGA biomaterials. Significantly the percentage of BMSCs attached to RADA16-P24–PLGA composite prepared by chemical cross-linking and physisorbing were (71.4 ± 7.5) % or (46.7 ± 5.8) % (P < 0.05). The in vivo study showed that RADA16-P24–PLGA chemical cross-linking could better induce ectopic bone formation compared with RADA16-P24–PLGA physisorbing and PLGA. It is concluded that the PLGA copolymer is a good RADA16-P24 carrier. This novel RADA16-P24–PLGA composite has strong osteogenic capability.     

Preparation of YAG gel coated ZrB2–SiC composite prepared by gelcasting and pressureless sintering

In this paper, gelcasting and pressureless sintering of YAG gel coated ZrB₂–SiC (YZS) composite were conducted. YAG gel coated ZrB₂–SiC (YZS) suspension was firstly prepared through sol–gel route. Poly (acrylic acid) was used as dispersant. YZS suspension had the lowest viscosity when using 0.6 wt.% PAA as dispersant. Gelcasting was conducted based on AM–MBAM system. The gelcast YZS sample was then pressureless sintered to about 97% density. During sintering, YAG promoted the densification process from solid state sintering to liquid phase sintering. The average grain sizes of ZrB₂ and SiC in the YZS composite were 3.8 and 1.3 μm, respectively. The flexural strength, fracture toughness and microhardness were 375 ± 37 MPa, 4.13 ± 0.45 MPa m^1/2 and 14.1 ± 0.5 GPa, respectively.     

In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Studies on optical,chemical, and electrical properties of rapid SiO2 atomic layer deposition using tris(tert-butoxy)silanol and trimethyl-aluminum

Rapid SiO₂ atomic layer deposition (ALD) was used to deposit amorphous, transparent, and conformal SiO₂ films using tris(tert-butoxy)silanol (TBS) and trimethyl-aluminum (TMA) as silicon oxide source and catalytic agent, respectively. The growth rate of the SiO₂ films drastically increased to a maximum value (2.3 nm/cycle) at 200 °C and slightly decreased to 1.6 nm/cycle at 275 °C. The SiO₂ thin films have C–H species and hydrogen content (～8 at%) at 150 °C because the cross-linking rates of SiO₂ polymerization may reduce below 200 °C. There were no significant changes in the ratio of O/Si (～2.1) according to the growth temperatures. On the other hand, the film density slightly increased from 2.0 to 2.2 although the growth rate slightly decreased after 200 °C. The breakdown strength of SiO₂ also increases from 6.20 ± 0.82 to 7.42 ± 0.81 MV/cm. These values suggest that high cross-linking rate and film density may enhance the electrical property of rapid SiO₂ ALD films at higher growth temperature.     

Crosslinked collagen–gelatin–hyaluronic acid biomimetic film for cornea tissue engineering applications

Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents the crosslinked collagen (Col)–gelatin (Gel)–hyaluronic acid (HA) films developed by making use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the crosslinker. The test results on the physical and biological properties indicate that the CGH631 film (the mass ratio of Col:Gel:HA = 6:3:1) has appropriate optical performance, hydrophilicity and mechanical properties. The diffusion properties of the CGH631 film to NaCl and tryptophan are also satisfactory and the measured data are 2.43 × 10^? 6 cm²/s and 7.97 × 10^? 7 cm²/s, respectively. In addition, cell viability studies demonstrate that the CGH631 film has good biocompatibility, on which human corneal epithelial cells attached and proliferated well. This biocompatible film may have potential use in cornea tissue engineering.     

Gelation of photopolymerized hyaluronic acid grafted with glycidyl methacrylate

Experiments have tracked the ambient gelation of a series of hydrophilic hyaluronic acid (HA) resins grafted with glycidyl methacrylate (GM) and photopolymerized as a function of dose. The resin mixtures range in GMHA concentration between 0.5 and 1.5% w/w in phosphate buffered saline (PBS). Illuminated at 20 mW/cm², the dynamic viscosity (η(t)) has been tracked and characterized using the Boltzmann log-sigmoidal model. A gelled viscosity of ~ 10 Pa s was determined at 0.5% w/w which rose to ~ 50 Pa s at or above 1% w/w. More curing agent marginally increased the gel viscosity at each concentration. Time constants associated with viscosity advancement were shortest at [GMHA] = 1.0%; higher concentrations are attributed with lower quantum efficiency when illuminated. Subsequent frequency sweeps replicated already published work using similar GHMA concentrations in PBS. G′ values ranged from 100 to 500 Pa over the formulation range with expected sensitivity to GMHA and curing agent concentration. Overall, the sigmoidal model represented this advancing viscosity data well, and further analysis of the physical significance of these model parameters may help in understanding photopolymerization of this complicated formulation more broadly.     

Production and in vitro characterization of 3D porous scaffolds made of magnesium carbonate apatite (MCA)/anionic collagen using a biomimetic approach

3D porous scaffolds are relevant biomaterials to bone engineering as they can be used as templates to tissue reconstruction. The aim of the present study was to produce and characterize in vitro 3D magnesium-carbonate apatite/collagen (MCA/col) scaffolds. They were prepared by using biomimetic approach, followed by cross-linking with 0.25% glutaraldehyde solution (GA) and liofilization. Results obtained with Fourier-transform infrared spectroscopy (FT-IR) confirmed the type-B carbonate substitution, while by X-ray diffraction (XRD), a crystallite size of ~ 10 nm was obtained. Optical and electron microscopy showed that the cylindrical samples exhibited an open-porous morphology, with apatite nanocrystals precipitated on collagen fibrils. The cross-linked 3D scaffolds showed integrity when immersed in culture medium up to 14 days. Also, the immersion of such samples into an acid buffer solution, to mimic the osteoclastic resorption environment, promotes the release of important ions for bone repair, such as calcium, phosphorus and magnesium. Bone cells (SaOs2) adhered, and proliferated on the 3D composite scaffolds, showing that synthesis and the cross-linking processes did not induce cytotoxicity.     

Development,characterization, and in vitro bioactivity studies of sol–gel bioactive glass/poly(l-lactide) nanocomposite scaffolds

Developing materials combining the advantages of synthetic polymers and bioactive glass nanoparticles can provide an efficient bone engineering scaffold. In this study, sol–gel bioactive glass (SG) nanoparticles were synthesized by quick alkali-mediation; sol–gel derived bioactive glass/poly(l-lactide) nanocomposite scaffolds were then developed. The influence of the glass content on the porosity of nanocomposite scaffolds was evaluated by SEM. The results showed that the neat polymer scaffold (PLA) has a highly interconnected porous structure with a maximum pore size of about 250 μm. For the composite scaffold containing 25 wt.% glass (SGP25), the decrease in the maximum pore size, (to about 200 μm) was not significant while for the SGP50 composite scaffold containing 50 wt.% glass it was a significant decrease (to about 100 μm). The apparent porosity of the scaffolds was 56.56% ± 7.15, 54.14% ± 3.84, and 53.11% ± 3.99 for PLA, SGP25, and, SGP50 respectively. FT-IR, TGA, and XRD results revealed some interaction of the glass filler with the polymeric matrix in the scaffolds. The degradation study showed that, by increasing the glass content in the scaffolds, the water absorption decreased, the weight loss increased, and the cumulative ion concentrations released from them also increased. This indicates the possibility of modulating the degradation rate by varying the glass/polymer ratio. At the end of the incubation period, the weight losses were around 5.44% ± 0.96, 32.50% ± 2.73, and 41.47% ± 3.02 for the PLA, SGP25, and SGP50, respectively. Moreover, the water uptake reached 119.65% ± 18.88 and 93.39% ± 13.01 for SGP25 and SGP50, respectively. The addition of the SG to the scaffolds was found to enhance their in vitro bioactivity. Therefore, these nanocomposite scaffolds have a potential to be applied in bone engineering. All data are expressed as mean ± standard deviation (n = 3).     

Effect of processing methods on physicochemical properties of titania nanoparticles produced from natural rutile sand

Titania (TiO₂) nanoparticles were produced from natural rutile sand using different approaches such as sol–gel, sonication and spray pyrolysis. The inexpensive titanium sulphate precursor was extracted from rutile sand by employing simple chemical method and used for the production of TiO₂ nanoparticles. Particle size, crystalline structure, surface area, morphology and band gap of the produced nanoparticles are discussed and compared with the different production methods such as sol–gel, sonication and spray pyrolysis. Mean size distribution (d₅₀) of obtained particles is 76 ± 3, 68 ± 3 and 38 ± 3 nm, respectively, for sol–gel, sonication and spray pyrolysis techniques. The band gap (3.168 < 3.215 < 3.240 eV) and surface area (36 < 60 < 103 m² g^?1) of particles are increased with decreasing particle size (76 > 68 > 38 nm), when the process methodology is changed from sol–gel to sonication and sonication to the spray pyrolysis. Among the three methods, spray pyrolysis yields high-surface particles with active semiconductor bandgap energy. The effects of concentration of the precursor, pressure and working temperature are less significant for large-scale production of TiO₂ nanoparticles from natural minerals.     

Spontaneous patterning and nanoparticle encapsulation in carboxymethylcellulose/alginate/dextran hydrogels and sponges

Novel polysaccharide sponges containing a network of capillaries and pore structures have been prepared by freeze drying of Ca²⁺ ion cross-linked sodium carboxymethylcellulose/sodium alginate hydrogels with or without addition of dextran. The iontropic gels consisted of capillaries, 5 to 40 µm in width, which comprised small pores, 1–25 µm in size. Gold, Fe₃O₄ or TiO₂ nanoparticles were encapsulated in the patterned gels and the mechanical strength of the resulting sponges investigated.     

Physico-chemical/biological properties of tripolyphosphate cross-linked chitosan based nanofibers

In this study, chitosan-PEO blend, prepared in a 15 M acetic acid, was electrospun into nanofibers (~ 78 nm diameter) with bead free morphology. While investigating physico-chemical parameters of blend solutions, effect of yield stress on chitosan based nanofiber fabrication was clearly evidenced. Architectural stability of nanofiber mat in aqueous medium was achieved by ionotropic cross-linking of chitosan by tripolyphosphate (TPP) ions. The TPP cross-linked nanofiber mat showed swelling up to ~ 300% in 1 h and ~ 40% degradation during 30 day study period. 3T3 fibroblast cells showed good attachment, proliferation and viability on TPP treated chitosan based nanofiber mats. The results indicate non-toxic nature of TPP cross-linked chitosan based nanofibers and their potential to be explored as a tissue engineering matrix.     

Preparation of organic and carbon xerogels using high-temperature–pressure sol–gel polymerization

To prepare organic gels at temperatures higher than normal boiling point of solvent, a method was developed using sol–gel polymerization in atmosphere saturated by vapor of solvent. To illustrate the advantages of proposed method, two series of gels were prepared using the conventional (T_curing = 70 °C) and the high temperature (T_curing = 140–170 °C) sol–gel polymerization. While no drying shrinkage was observed in our proposed method, 5–18% linear shrinkage occurred in conventional method depending on resin concentration in sol. Moreover, rising of curing temperature reduced the required time for preparation of organic gels from 5 days to lower than 5 h. The effects of processing parameters were investigated on physical and mechanical properties of organic xerogels. The results revealed that resin concentration significantly affects both density and compressive strength of final xerogels. While the curing temperature had no obvious effect on density, the raising of curing temperature significantly enhance the strength of organic xerogels. Carbon xerogels prepared by pyrolysis of novolac aerogels in inert atmosphere. The textures of the carbon xerogels were denser than corresponding organic xerogels, as evidenced by scanning electron microscopy (SEM) images. N₂ adsorption tests indicated that carbon aerogels were mainly meso or macroporous depending on resin concentration in initial sol.     

Studies on fish scale collagen of Pacific saury (Cololabis saira)

We purified and characterized Type I collagen from the scales of the Pacific saury (Cololabis saira) and compared it with collagen from other organisms. Subunit composition of C. saira collagen (2α1 + α2) was similar to that of red sea bream (Pagrus major) and porcine collagen. C. saira collagen did not form a firm gel after neutralization of pH in solution. The temperature of denaturation (24–25 °C) of C. saira collagen was slightly lower than that of P. major collagen (26–27 °C). The contents of proline and hydroxyproline were lower in red sea bream and Pacific saury collagen than in porcine collagen. Circular dichroism spectra and Fourier-transformed infrared spectra showed that heat denaturation caused unfolding of the triple helices in all three collagens.     

Comparison of electrical conductivities of various brain phantom gels: Developing a ‘brain gel model’

The use of conducting gels to mimic brain and other tissues is of increasing interest in the development of new medical devices. Currently, there are few such models that can be utilized at physiologic temperatures. In this work, the conductivities of agar, agarose and gelatin gels were manipulated by varying NaCl concentration from 0–1 mg/ml. The AC conductivity was measured at room and physiological temperatures (37 °C) in the 100–500 Hz frequency range. Conductivity (σ) was nearly independent of frequency but increased linearly with NaCl concentration and was higher at physiological temperatures in these gels. A formula for predicting conductivity as a function of NaCl concentration was derived for each gel type. The overall goal is to develop a ‘brain gel model’, for studying low frequency electrical properties of the brain and other tissues at physiological temperatures.