Hydrogels For Bone Tissue Engineering Research Articles

Fabrication and characterization of hydroxypropyl guar-poly (vinyl alcohol)-nano hydroxyapatite composite hydrogels for bone tissue engineering

Biocompatible bone implants composed of natural materials are highly desirable in orthopedic reconstruction procedures. In this study, novel and ecofriendly bionanocomposite hydrogels were synthesized using a blend of hydroxypropyl guar (HPG), poly vinyl alcohol (PVA), and nano-hydroxyapatite (n-HA) under freeze-thaw and mild reaction conditions. The hydrogel materials were characterized using various techniques. TGA studies indicate that both composites, HPG/PVA and HPG/PVA/n-HA, have higher thermal stability compared to HPG alone whereas HPG/PVA/n-HA shows higher stability compared to PVA alone. The HPG/PVA hydrogel shows porous morphology as revealed by the SEM, which is suitable for bone tissue regeneration. Additionally, the hydrogels were found to be transparent and flexible in nature. In vitro biomineralization study performed in simulated body fluid shows HPG/PVA/n-HA has an apatite like structure. The hydrogel materials were employed as extracellular matrices for biocompatibility studies. In vitro cell viability studies using mouse osteoblast MC3T3 cells were performed by MTT, Trypan blue exclusion, and ethidium bromide/acridine orange staining methods. The cell viability studies reveal that composite materials support cell growth and do not show any signs of cytotoxicity compared to pristine PVA. Osteoblastic activity was confirmed by an increased alkaline phosphatase enzyme activity in MC3T3 bone cells grown on composite hydrogel matrices.

Incorporation of rosuvastatin-loaded chitosan/chondroitin sulfate nanoparticles into a thermosensitive hydrogel for bone tissue engineering: preparation, characterization, and cellular behavior

Rosuvastatin (RSV) has been shown to have significant impact on the simulation of bone regeneration after local injection. The current study aimed to develop a localized controlled delivery system from RSV by incorporating RSV-loaded chitosan/chondroitin sulfate (CTS/CS) nanoparticles into thermosensitive Pluronic F127/hyaluronic acid (PF127/HA) hydrogel. RSV-loaded CTS/CS nanoparticles were prepared by ionic gelation, and the impact of various formulation variables was assessed using the Box–Behnken design. Consequently, optimized RSV-loaded nanoparticles were incorporated into the PF127/HA hydrogel. Rheological properties, degradation rates of hydrogels, and the release rate of RSV from hydrogel were examined. Mean particle size, zeta potential, entrapment efficiency, and mean release time of the optimized RSV-loaded nanoparticles were confirmed as 283.2 ± 16 nm, –31.2 ± 6.8 mV, 63.1 ± 4.2%, and 6.14 ± 0.3 h, respectively. The hydrogel containing 3% w/v CTS/CS nanoparticles existed as a solution with low viscosity at room temperature converted to a semisolid upon increasing the temperature to 35 °C. Hydrogel engrafted with CTS/CS showed controlled release of RSV during 48 h with superior in vitro gel stability. As revealed by cytotoxicity and mineralization assays, incorporation of RSV-loaded particles into PF127/HA hydrogel led to improvement in osteoblast viability and proliferation.

Acrylic acid grafted tamarind kernel polysaccharide-based hydrogel for bone tissue engineering in absence of any osteo-inducing factors

ABSTRACTPurpose: With increased life expectancy, disorders in lifestyle and other clinical conditions, and the changes in the connective tissues such as in bone, impose diverse biomedical problems. Cells belong to osteogenic lineages are extremely specific for their surface requirements. Therefore, suitable surfaces are the critical bottle neck for successful bone tissue engineering. This study involves assessment of polysaccharide-based hydrogel which effectively allows growth, differentiation and mineralisation of osteogenic cells even in the absence of osteogenic inducing factors. Materials and methods: Tamarind Kernel Polysaccharide was grafted with acrylic acid at different mole ratio. The critical parameter, surface morphology for bio application was assessed by SEM. MTT assay has been performed with hydrogels on Saos-2 cells. The biocompatibility and adhesion of different cell lines (F-11, Saos-2, Raw 264.7 and MSCs) on hydrogel surface was performed by Phalloidin and DAPI staining. Further the differentiation, mineralization and expression of different osteogenic markers, ALP assay, Alizarin Red staining and q-PCR was performed. Results: The hydrogels show highly porous and interconnected pores. MTT assay demonstrates the hydrogel have no cytotoxicity towards Saos-2 cells and are suitable for proliferation of different lineage of cell lines. ALP, Alizarin red staining and q-PCR assay shows that the hydrogel surface enhances the differentiation, mineralization and expression of different osteogenic genes in Saos-2 cells in the absence of any osteogenic inducing factors. Conclusion Synthesized hydrogel surface triggers signalling events towards osteogenesis even in the absence of added growth factors. We proposed that this material can be used for effective bone tissue engineering in vitro at low cost.

Differentiation of osteoclast precursors on gellan gum-based spongy-like hydrogels for bone tissue engineering

Bone tissue engineering with cell-scaffold constructs has been attracting a lot of attention, in particular as a tool for the efficient guiding of new tissue formation. However, the majority of the current strategies used to evaluate novel biomaterials focus on osteoblasts and bone formation, while osteoclasts are often overlooked. Consequently, there is limited knowledge on the interaction between osteoclasts and biomaterials. In this study, the ability of spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels to support osteoclastogenesis was investigated in vitro. First, the spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels were characterized in terms of microstructure, water uptake and mechanical properties. Then, bone marrow cells isolated from the long bones of mice and cultured in spongy-like hydrogels were treated with 1,25-dihydroxyvitamin D3 to promote osteoclastogenesis. It was shown that the addition of HAp to spongy-like gellan gum hydrogels enables the formation of larger pores and thicker walls, promoting an increase in stiffness. Hydroxyapatite-reinforced spongy-like gellan gum hydrogels support the formation of the aggregates of tartrate-resistant acid phosphatase-stained cells and the expression of genes encoding DC-STAMP and Cathepsin K, suggesting the differentiation of bone marrow cells into pre-osteoclasts. The hydroxyapatite-reinforced spongy-like gellan gum hydrogels developed in this work show promise for future use in bone tissue scaffolding applications.

Oxygen-distribution within 3-D collagen I hydrogels for bone tissue engineering

Tissue engineering (TE) approaches typically envisage the structural and functional reconstitution of previously damaged tissue in situ. An adequate three-dimensional environment is therefore of fundamental importance for the designated cells associated to the scaffold material. The sufficient supply with nutrients and oxygen in vitro and in vivo mark thereby critical challenges of TE. In this study, we intended to analyse the level of locally dissolved oxygen within 3-D cell-loaded collagen I gels in vitro. For the analysis of the oxygen levels in situ, we employed an optical fibre-based micro sensor setup, as well as a camera supported non-invasive optical sensor foil based technique. These complementary analytical tools enable the identification, localization, and temporal follow-up investigation of specified regions of interest within TE constructs. Human adipose-derived mesenchymal stem cells (hAdMSCs) cultured in collagen I gels under normoxic conditions were analysed periodically and kinetically up to 70 days – thereby revealing dynamic changes of the level of dissolved oxygen inside the gel constructs. Dependent on the applied cell concentration, the in vitro oxygen concentration (cO2) within the gels reached physiological ranges (7–9%) after 21 days, or 35 days of culture. The minimal cO2 was measured after 35 days in vitro, featuring an oxygen level of 4.8 ± 1.3%. Upon prolonged culture, a plateau-like status of the cO2 around 8–9% established, indicating a change in the physiological activity of the cells under investigation. The expression patterns of BCL2, CASP3 and MCM5 revealed significant differences among the proliferative and apoptotic stages of the cell-loaded samples at the investigated time points of 7 and 70 days in culture. In summary, these data show the temporary dynamic nature of the oxygen distribution in cell-loaded gel constructs. The applied technique is an ideal tool for the evaluation of multiple parameters affecting the oxygen distribution in vitro. We conclude that it takes 5 weeks for establishing an equilibrium of cO2. Levels reached in a 3-D gel construct are comparable with physiological oxygenation ranges in bone-associated tissues.

Decellularized Hydrogels in Bone Tissue Engineering: A Topical Review.

Nowadays, autograft and allograft techniques represent the main solution to improve bone repair. Unfortunately, autograft technique is expensive, invasive and subject to infections and hematoma, frequently affecting both donor sites and surgical sites. A recent advance in tissue engineering is the fabrication of cell-laden hydrogels with custom-made geometry, depending on the clinical case. The use of ECM (Extra-Cellular Matrix)-derived Hydrogels from bone tissue is the new opportunity to obtain good results in bone regeneration.Several micro-engineering techniques and approaches are available to fabricate different cell gradients and zonal structures in hydrogels design, in combination with the advancement in biomaterials selection. In this review, we analyse the stereolithografy, the Bio-patterning, the 3D bioprinting and 3D assembly, the Laser-Induced Forward Transfer Bioprinting (LIFT), the Micro-extrusion bioprinting, the promising Electrospinning technology, the Microfluidics and the Micromolding. Several mechanical properties are taken into account for bone regeneration scaffolds. However, each typology of scaffold presents some advantages and some concerns. The research on biomaterials is the most promising for bone tissue engineering: the new biomimetic materials will allow us to obtain optimal results in the next clinical application of basic research.

Gellan gum-hydroxyapatite composite spongy-like hydrogels for bone tissue engineering.

Osteoinductive biomaterials represent a promising approach to advance bone grafting. Despite promising, the combination of sustained biodegradability, mechanical strength, and biocompatibility in a unique biomaterial that can also support cell performance and bone formation in vivo is demanding. Herein, we developed gellan gum (GG)-hydroxyapatite (HAp) spongy-like hydrogels to mimic the organic (GG) and inorganic (HAp) phases of the bone. HAp was successfully introduced within the GG polymeric networks, as determined by FTIR and XRD, without compromising the thermostability of the biomaterials, as showed by TGA. The developed biomaterials showed sustained degradation, high swelling, pore sizes between 200 and 300 μm, high porosity (>90%) and interconnectivity (<60%) that was inversely proportional to the total polymeric amount and to CaCl2 crosslinker. CaCl2 and HAp reinforced the mechanical properties of the biomaterials from a storage modulus of 40 KPa to 70-80 KPa. This study also showed that HAp and CaCl2 favored the bioactivity and that cells were able to adhere and spread within the biomaterials up to 21 days of culture. Overall, the possibility to tailor spongy-like hydrogels properties by including calcium as a crosslinker and by varying the amount of HAp will further contribute to understand how these features influence bone cells performance in vitro and bone formation in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 479-490, 2018.

3D- Printed Poly(\u03b5-caprolactone) Scaffold Integrated with Cell-laden Chitosan Hydrogels for Bone Tissue Engineering

Synthetic polymeric scaffolds are commonly used in bone tissue engineering (BTE) due to their biocompatibility and adequate mechanical properties. However, their hydrophobicity and the lack of specific cell recognition sites confined their practical application. In this study, to improve the cell seeding efficiency and osteoinductivity, an injectable thermo-sensitive chitosan hydrogel (CSG) was incorporated into a 3D-printed poly(ε-caprolactone) (PCL) scaffold to form a hybrid scaffold. To demonstrate the feasibility of this hybrid system for BTE application, rabbit bone marrow mesenchymal stem cells (BMMSCs) and bone morphogenetic protein-2 (BMP-2) were encapsulated in CSG. Pure PCL scaffolds were used as controls. Cell proliferation and viability were investigated. Osteogenic gene expressions of BMMSCs in various scaffolds were determined with reverse transcription polymerase chain reaction (RT-PCR). Growth factor releasing profile and mechanical tests were performed. CCK-8 assay confirmed greater cell retention and proliferation in chitosan and hybrid groups. Confocal microscopy showed even distribution of cells in the hybrid system. After 2-week osteogenic culture in vitro, BMMSCs in hybrid and chitosan scaffolds showed stronger osteogenesis and bone-matrix formation. To conclude, chitosan/PCL hybrid scaffolds are a favorable platform for BTE due to its capacity to carry cells and drugs, and excellent mechanical strength.

A bioprintable form of chitosan hydrogel for bone tissue engineering

Bioprinting can be defined as 3D patterning of living cells and other biologics by filling and assembling them using a computer-aided layer-by-layer deposition approach to fabricate living tissue and organ analogs for tissue engineering. The presence of cells within the ink to use a ‘bio-ink’ presents the potential to print 3D structures that can be implanted or printed into damaged/diseased bone tissue to promote highly controlled cell-based regeneration and remineralization of bone. In this study, it was shown for the first time that chitosan solution and its composite with nanostructured bone-like hydroxyapatite (HA) can be mixed with cells and printed successfully. MC3T3-E1 pre-osteoblast cell laden chitosan and chitosan-HA hydrogels, which were printed with the use of an extruder-based bioprinter, were characterized by comparing these hydrogels to alginate and alginate-HA hydrogels. Rheological analysis showed that all groups had viscoelastic properties. It was also shown that under simulated physiological conditions, chitosan and chitosan-HA hydrogels were stable. Also, the viscosity values of the bio-solutions were in an applicable range to be used in 3D bio-printers. Cell viability and proliferation analyses documented that after printing with bio-solutions, cells continued to be viable in all groups. It was observed that cells printed within chitosan-HA composite hydrogel had peak expression levels for early and late stages osteogenic markers. It was concluded that cells within chitosan and chitosan-HA hydrogels had mineralized and differentiated osteogenically after 21 days of culture. It was also discovered that chitosan is superior to alginate, which is the most widely used solution preferred in bioprinting systems, in terms of cell proliferation and differentiation. Thus, applicability and printability of chitosan as a bio-printing solution were clearly demonstrated. Furthermore, it was proven that the presence of bone-like nanostructured HA in alginate and chitosan hydrogels improved cell viability, proliferation and osteogenic differentiation.

Acellular mineral deposition within injectable, dual‐gelling hydrogels for bone tissue engineering

This study sought to characterize the composition and morphology of acellular mineralization occurring in thermally and chemically gelable hydrogels comprising copolymers of hydrophobic N-isopropylacrylamide as a function of hydrogel hydrophobicity and culture medium formulation. The deposition of calcium phosphate (CaP) mineral was hypothesized to occur with increasing hydrogel hydrophobicity and presence of serum proteins in the culture medium. Two hydrogel compositions with a solid content of 15 and 20 wt % were examined in serum-containing and nonserum-containing media for 0, 14, 28, and 56 days. Using biochemical assays, calcium, but not phosphate content, was found to significantly increase over time in hydrophobic hydrogels soaked in cell culture medium with fetal bovine serum. Significant increases in the calcium to phosphate ratio were observed within these hydrogels from day 0 to 56, with mineralization indicated by von Kossa histological staining. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) were used to analyze CaP mineral characteristics. No crystalline apatitic reflection peaks were observed using XRD, which was supported by the lack of observable mineral deposits as observed using SEM/EDX. However, FTIR showed the presence of new absorption peaks in the serum-containing samples at 28 and 56 days which suggested the formation of an immature apatitic-like mineral. The ability to undergo hydrophobicity-dependent and protein-mediated mineralization demonstrates the potential of these dual-gelling hydrogels as acellular self-mineralizing materials for bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 110-117, 2017.

Self-assembled high-strength hydroxyapatite/graphene oxide/chitosan composite hydrogel for bone tissue engineering

Graphene hydrogel has shown greatly potentials in bone tissue engineering recently, but it is relatively weak in the practical use. Here we report a facile method to synthesize high strength composite graphene hydrogel. Graphene oxide (GO), hydroxyapatite (HA) nanoparticles (NPs) and chitosan (CS) self-assemble into a 3-dimensional hydrogel with the assistance of crosslinking agent genipin (GNP) for CS and reducing agent sodium ascorbate (NaVC) for GO simultaneously. The dense and oriented microstructure of the resulted composite gel endows it with high mechanical strength, high fixing capacity of HA and high porosity. These properties together with the good biocompatibility make the ternary composite gel a promising material for bone tissue engineering. Such a simultaneous crosslinking and reduction strategy can also be applied to produce a variety of 3D graphene-polymer based nanocomposites for biomaterials, energy storage materials and adsorbent materials.

Macroporous poly(lactic acid) construct supporting the osteoinductive porous chitosan-based hydrogel for bone tissue engineering

Abstract Poor mechanical performance of porous chitosan-hydroxyapatite systems is the main limitation in bone tissue engineering. If we merge good mechanical performance of poly(lactic acid) construct with osteoinductive and bioresorbable properties of chitosan-hydroxyapatite porous hydrogel, we can obtain a material that meets necessary requirement for bone tissue substituent. With this in mind, we propose the combination of 3D printing technique and the thermally-induced phase separation method for simultaneous modification of biological properties of poly(lactic acid) and load-bearing properties of chitosan-hydroxyapatite porous hydrogel. 3D printed poly(lactic acid), PLA, construct has been used as a mechanical support with very large pore diameter of 960 ± 50 μm allowing enough free space (∼60% of porosity) to form porous composite hydrogel by freeze gelation. In situ formation of hydroxyapatite within chitosan hydrogel has ensured higher human mesenchymal stem cell osteogenesis during 21 days of culture. Positive modification of poly(lactic acid) has been simultaneously utilized to improve the compressive strength of composite hydrogel which has been confirmed by Young’s modulus ranging from lower values reported for cancellous bone in dry state. Considering positive osteogenic signal accompanied with suitable mechanical properties, our scaffolds have shown good potential as bone tissue substituent.

Biomineralised interpenetrating network hydrogels for bone tissue engineering

Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but their use is limited by inadequate mechanical strength and poor biocompatibility. In this study, to better mimic the mineral component and the mechanical strength of natural bone, two biocompatible materials, 2-hydroxyethyl agarose and poly(ethylene glycol) diacrylate, were combined with nanocrystalline hydroxyapatite (nHAp)-coated poly(lactic-co-glycolic acid) (PLGA) microspheres. A novel composite interpenetrating network (IPN) hydrogel scaffold was created to investigate its mechanical and osteoconductive performance for bone tissue engineering-related applications. The inclusion of nHAp-coated PLGA microspheres in an IPN hydrogel led to an increase in compressive modulus. In the absence of nHAp-coated microspheres, cell viability dropped to 59·1% at 3 weeks post-encapsulation. However, by incorporating nHAp-coated microspheres, cell viability improved to 80·6%. The capacity of composite IPN hydrogels to promote bone formation in cell culture was assessed. In the presence of mineralised microspheres, a composite IPN gel showed a significant increase in alkaline phosphatase activity and calcium (Ca) deposition following 3 weeks of incubation when compared with plain IPNs. This technology may be also applied to other cell-based applications where the improved mechanical integrity and osteoconductivity of cell-containing IPN hydrogels may be used to mimic bone tissue replacement.

Biocompatible and highly homogenous graphene-hydroxyapatite hydrogel for bone tissue engineering

Event Abstract Back to Event Biocompatible and highly homogenous graphene-hydroxyapatite hydrogel for bone tissue engineering Xingyi Xie1, 2, Kaiwen Hu1, Dongdong Fang3, Lihong Shang1, Simon Tran3 and Marta Cerruti1 1 McGill University, Department of Mining and Materials Engineering, Canada 2 Sichuan University, College of Polymer Science and Engineering, China 3 McGill University, Craniofacial Tissue Engineering, Canada Introduction: Recently, graphene (G) has shown its potential for applications in tissue engineering[1]-[3]. While most of the studies were conducted on two dimensional membranes of G or graphene oxide (GO), fewer investigations were made on three dimensional G or GO structures which could be readily applied as tissue regeneration scaffolds. The promise of such 3D graphene structures was proved in a recent study[4], where 3D graphene foams prepared by chemical vapor deposition (CVD) promoted osteogenic differentiation. However, the cost of CVD may limit the use of such method. In our study, we prepare a GO/hydroxyapatite (HA) composite homogeneous hydrogel via a simple and low cost hydrothermal treatment of colloidal HA and GO. Materials and Methods: We start from commercial GO aqueous suspensions (lateral dimensions 0.5–5 µm) and citrate-stabilized HA hydrocolloids, which we prepare according to[5]. GO and HA are mixed to generate homogenous suspensions with a final GO concentration of 4 mg mL−1 and variable mass ratios of HA to GO. The samples are named based on this ratio: for example, G/HA-40 is a gel prepared with an HA/GO feed ratio of 40/60 (wt/wt). The control sample rGO is a reduced GO without HA added. Results and Discussion: During the hydrothermal treatment, GO is partially deoxygenated and reduced to graphene (rGO), and encapsulates HA nanoparticles inside the hydrogel[6],[7]. SEM image in Figure 1 shows a homogenous distribution of HA on hydrogels with as much as 80% HA. The resulting hydrogel is highly conductive, mechanically robust and can be used as cell culture scaffold for MSCs. Cell culture results in Figure 2 a-c on mouse multipotent MSCs show high cell viability on both rGO and G/HA hydrogels. The cell densities on the two are similar and roughly correspond to 1/2 of that of the polystyrene control (Figure 2d), higher than what observed on CVD graphene foam (4). A closer SEM examination (Figure 2 e and f) shows that cells are well spread and firmly attach to the both rGO and G/HA hydrogels. Cells on g/HA show filamentous extensions which directly interact with the material. Conclusion: We have successfully developed a strategy to prepare highly homogenous G/HA composite hydrogel materials based on colloidal chemistry and hydrothermal treatment of GO. The strong conductive G/HA hydrogel shows excellent biocompatibility and improved cell affinity compared to previous studies, demonstrating its potential for applications in tissue regeneration. We are currently investigating the osteogenic differentiation of MSCs cultured on these nanocomposites. Figure 1. SEM images of rGO and G/HA composites at different scales. Uniform distribution of HA NPs is observed in the porous composites Figure 2. Mouse multipotent mesenchymal stromal cell (MSC) viability and morphology on tissue culture polystyrene (TCP), rGO and G/HA-40 following 2 days of culture. a–c, Live/dead stain shows that the majority of the attached cells are viable (green), and only few cells have died (red). Insets show a higher magnification of live/dead cells. d, The percentage of live cells is as high as ~93% without statistical differences across the three materials (P>0.05), as measured by averaging at least 8 images at 50× magnification from at least 3 specimens. The live cell densities on rGO and G/HA-40 are statistically the same (P>0.05), but significantly lower than on TCP (*, P<0.05). e,f, Cells are well spread on rGO, but more elongated on G/HA-40, with filamentous extensions (* labels) directly interacting with the material. Canada Research Chair foundation; Natural Sciences and Engineering Research Council (NSERC) of Canada; the Fonds de recherche du Québec Nature et Technologie (FQRNT); Center for Self-Assembled Chemical Structures; McGill Engineering Doctoral Award; Sichuan University Scholarship Fund for; Prof. Thomas Skopek; Dr Mohamed Nur Abdallah; Dr Younan Liu; Prof. Faleh Tamimi Marino; Prof. Bernard Drouin

Porous hydrogel of wool hair keratin as a substrate for cell culture

Event Abstract Back to Event Porous hydrogel of wool hair keratin as a substrate for cell culture Ozaki Yuki1*, Yusuke Takagi1*, Hideki Mori1* and Masayuki Hara1* 1 Osaka Prefecture Univertisy, Department of Biological Science, Graduate School of Science,, Japan Introduction: Keratin is contained rich in animal epidermal tissues including hair, nail, horn, and also common proteins such as wool fibers. Keratins have not been so much utilized as Type I collagen as biomaterials although much amount is available as a bio-resource. In this study, we show a novel simple method to prepare a sponge-like porous keratin hydrogel. Wool keratin was extracted for 18 h at 60 oC in a solution containing 8 M guanidine hydrochloride and 2-mercaptoethanol, followed by dialysis against water. Specific water content (%) was calculated as an index for swelling of the gel according to the following equation. Specific water content = (Ww-Wd)/Wd×100 (%) Wd: dry weight of the gel after lyophilization; Ww: wet weight of the gel after swelling in either PBS(-) or DMEM. We tried cell culture on the gel and evaluated the cell growth by WST assay. All animal experiments were conducted in accordance with institutional guidelines and national standards with approval from the Animal Experiment Committee of Osaka Prefecture University. Results and Discussion: Extracted keratin was aggregated and recrosslinked to form a porous hydrogel like a sponge. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) proved that Type I and Type II keratin subunits were extracted by that procedure without degradation by hydrolysis. The porous keratin gel had a highly porous structure and a fast-swelling property in rehydration after freeze-drying. The gel could be autoclaved without significant change of mechanical strength and shapes. It had also high mechanical strength both in the tensile test and the measurement of dynamic viscoelasticity. The gel is flexible but mechanically durable especially for tensile strain. Those property was different from that of the chemically crosslinked type I collagen gel. Three types of animal cells, PC12 cells, HOS cells and murine embryonic fibroblasts, well attached and grew on the surface of the porous hydrogel. We observed those cells by fluorescent microscopy after live/dead staining and also by a scanning electron microscope. The keratin hydrogel has properties suitable for scaffold in tissue engineering. We tried some preliminary studies on application of the keratin hydrogel for bone tissue engineering by culturing osteoblasts. Reference: Ozaki, Y; Takagi, Y; Mori, H; Hara, M. Mater. Sci. Eng. C 2014, 42, 146–154. Keywords: Tissue Engineering, stem cell, 3D scaffold, Biodegradable material Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Regenerative medicine: biomaterials for control of tissue induction Citation: Yuki O, Takagi Y, Mori H and Hara M (2016). Porous hydrogel of wool hair keratin as a substrate for cell culture. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01232 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Ozaki Yuki, Osaka Prefecture Univertisy, Department of Biological Science, Graduate School of Science,, Sakai-city, Osaka Prefecture, Japan, yuki.ozaki@cira.kyoto-u.ac.jp Dr. Yusuke Takagi, Osaka Prefecture Univertisy, Department of Biological Science, Graduate School of Science,, Sakai-city, Osaka Prefecture, Japan, st304016@edu.osakafu-u.ac.jp Dr. Hideki Mori, Osaka Prefecture Univertisy, Department of Biological Science, Graduate School of Science,, Sakai-city, Osaka Prefecture, Japan, morihide@b.s.osakafu-u.ac.jp Dr. Masayuki Hara, Osaka Prefecture Univertisy, Department of Biological Science, Graduate School of Science,, Sakai-city, Osaka Prefecture, Japan, hara@b.s.osakafu-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ozaki Yuki Yusuke Takagi Hideki Mori Masayuki Hara Google Ozaki Yuki Yusuke Takagi Hideki Mori Masayuki Hara Google Scholar Ozaki Yuki Yusuke Takagi Hideki Mori Masayuki Hara PubMed Ozaki Yuki Yusuke Takagi Hideki Mori Masayuki Hara Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Injectable dual-gelling cell-laden composite hydrogels for bone tissue engineering

The present work investigated the osteogenic potential of injectable, dual thermally and chemically gelable composite hydrogels for mesenchymal stem cell (MSC) delivery in vitro and in vivo. Composite hydrogels comprising copolymer macromers of N-isopropylacrylamide were fabricated through the incorporation of gelatin microparticles (GMPs) as enzymatically digestible porogens and sites for cellular attachment. High and low polymer content hydrogels with and without GMP loading were shown to successfully encapsulate viable MSCs and maintain their survival over 28 days in vitro. GMP incorporation was also shown to modulate alkaline phosphatase production, but enhanced hydrogel mineralization along with higher polymer content even in the absence of cells. Moreover, the regenerative capacity of 2 mm thick hydrogels with GMPs only, MSCs only, or GMPs and MSCs was evaluated in vivo in an 8 mm rat critical size cranial defect for 4 and 12 weeks. GMP incorporation led to enhanced bony bridging and mineralization within the defect at each timepoint, and direct bone-implant contact as determined by microcomputed tomography and histological scoring, respectively. Encapsulation of both GMPs and MSCs enabled hydrogel degradation leading to significant tissue infiltration and osteoid formation. The results suggest that these injectable, dual-gelling cell-laden composite hydrogels can facilitate bone ingrowth and integration, warranting further investigation for bone tissue engineering.

Bio-inspired mineralization of hydroxyapatite in 3D silk fibroin hydrogel for bone tissue engineering

To fabricate hard tissue implants with bone-like structure using a biomimetic mineralization method is drawing much more attentions in bone tissue engineering. The present work focuses in designing 3D silk fibroin hydrogel to modulate the nucleation and growth of hydroxyapatite crystals via a simple ion diffusion method. The study indicates that Ca2+ incorporation within the hydrogel provides the nucleation sites for hydroxyapatite crystals and subsequently regulates their oriented growth. The mineralization process is regulated in a Ca2+ concentration- and minerlization time-dependent way. Further, the compressive strength of the mineralized hydrogels is directly proportional with the mineral content in hydrogel. The orchestrated organic/inorganic composite supports well the viability and proliferation of human osteoblast cells; improved cyto-compatibility with increased mineral content. Together, the present investigation reports a simple and biomimetic process to fabricate 3D bone-like biomaterial with desired efficacy to repair bone defects.

Development of silk fibroin/nanohydroxyapatite composite hydrogels for bone tissue engineering

This work presents a novel composite hydrogel consisting of silk fibroin (SF) and nanohydroxyapatite (nanoHA) prepared by a new and innovative method using ethanol as gelling agent capable of forming hydrogels in few minutes. The properties of the composite material, such as the microstructure as well as the chemical and physical properties were studied. Moreover in␣vitro studies of osteoblastic citocompatibility were performed. The microporosity and macroporosity obtained combined with interconnected porous structure and a uniform dispersion of nanoHA particles throughout the fibroin matrix makes composite hydrogel suitable for bone regeneration. The compression modulus of composite hydrogels was increased as the nanoHA concentration increased from 10 to 15wt.% and the water uptake ability of these materials decreased with the incorporation of nanoHA. The metabolic and alkaline phosphatase activities of osteoblastic cells were improved with the incorporation of nanoHA in the SF matrix providing a more promising material for bone tissue engineering.

Bioactive nanoengineered hydrogels for bone tissue engineering: a growth-factor-free approach.

Despite bone's impressive ability to heal after traumatic injuries and fractures, a significant need still exists for developing strategies to promote healing of nonunion defects. To address this issue, we developed collagen-based hydrogels containing two-dimensional nanosilicates. Nanosilicates are ultrathin nanomaterials with a high degree of anisotropy and functionality that results in enhanced surface interactions with biological entities compared to their respective three-dimensional counterparts. The addition of nanosilicates resulted in a 4-fold increase in compressive modulus along with an increase in pore size compared to collagen-based hydrogels. In vitro evaluation indicated that the nanocomposite hydrogels are capable of promoting osteogenesis in the absence of any osteoinductive factors. A 3-fold increase in alkaline phosphatase activity and a 4-fold increase in the formation of a mineralized matrix were observed with the addition of the nanosilicates to the collagen-based hydrogels. Overall, these results demonstrate the multiple functions of nanosilicates conducive to the regeneration of bone in nonunion defects, including increased network stiffness and porosity, injectability, and enhanced mineralized matrix formation in a growth-factor-free microenvironment.

3D polysaccharide based hydrogel for bone tissue engineering

Hydrogels have attracted considerable attention in biomedical engineering applications due to their many favorable biomimetic properties. Hydrogels based on proteins or carbohydrates can also function as effective extracellular matrices to direct cellular behavior. Recently, polysaccharide based hydrogels have become particularly interesting as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the hydrogel with potential applications for bone tissue engineering. The aim of this study was to verify the biological potential of a new carboxymethyl cellulose—hydroxyapatite hybrid hydrogel in bone tissue regeneration. Mesenchymal stem cells were seeded on hydrogel scaffold, in presence and absence of hydroxyapatite, for 7, 14 and 21 days. Cell viability assay and morphological analysis were carried out to evaluate biocompatibility and cell adhesion of the materials. Real Time PCR for genes involved in tissue regeneration was carried out to assay the influence of the scaffold in cell differentiation. Results showed a high cell viability and biocompatibility of the tested material, confirmed by morphological analysis. The evaluation of osteoblast markers demonstrated the osteogenic induction of the 3D material enriched with hydroxyapatite in the production of mineralized extracellular matrix compared to the carboxymethyl cellulose based material. In conclusion, our data show that carboxymethyl cellulose—hydroxyapatite hybrid hydrogel may have great potential in bone tissue engineering applications.