Hydrophilic poly (ethylene glycol) coating on PDLLA/BCP bone scaffold for drug delivery and cell culture

A well developed porous poly-D-L-lactide (PDLLA)/biphasic calcium phosphate (BCP) scaffold was coated with a hydrophilic poly (ethylene glycol) (PEG)/vancomycin composite for drug delivery and surface modification. The PDLLA/BCP scaffold, obtained by a salt-leaching method, possessed highly inter-connected pores (250–350 μm) and a high porosity (83.8%). The hydrophilic PEG was used to effectively entrap the drug inside the scaffold and to enhance the wettability of the hydrophobic surface of the PDLLA/BCP matrix. The scaffold with PEG/vancomycin coatings was fabricated by injecting the PEG/vancomycin composite solution into the pre-vacuumized scaffold. A standardized bacterial assay showed that the drug was still active after association with the bone scaffold. The in-vitro drug release study of vancomycin showed an initial burst release followed by a slower sustained release. The drug release behavior in vitro was investigated in detail by controlling the composite solution parameters: PEG molecular weight and PEG concentration. The release profiles showed that an increase in the PEG molecular weight and concentration resulted in a slower drug release rate. The water contact angles of the scaffold surface decreased after being coated with PEG. The in-vitro osteoblast culture experiment confirmed the biocompatibility of the scaffold for the growth of osteoblasts.     

Development of poly (lactic-co-glycolic acid)-collagen scaffolds for tissue engineering

Collagen as an important extra-cellular matrix (ECM) in many tissues is weakly antigenic and the structure of collagen sponges is highly porous with interconnected pores effective for cell infiltration and mass transfer of oxygen and nutrients. Its application as a scaffold is limited by poor mechanical strength and rapid biodegradation. In this paper, we attempt to graft hydrolyzed PLGA fiber surfaces with collagen by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), and then embed these collagen-grafted PLGA fibers in collagen sponge to form a hybrid PLGA-collagen scaffold. For further stability, we cross-linked the collagen in the scaffold and used it in rat liver cell cultivation. The scaffold was characterized by mechanical micro-tester, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Results showed that (1) the scaffolds exhibited isotropic and interconnected porous structure; (2) the compression modulus of this scaffold was enhanced 50 fold compared to the collagen scaffolds. The cell attachment and cytotoxicity of this scaffold were studied. Cell attachment was improved remarkably and the cytotoxicity of the hybrid PLGA-collagen scaffold was lower than that of the un-grafted PLGA-collagen scaffolds using alamarBlue™ assay normalized to the DNA content in each scaffold. This new hybrid scaffold has potential applications for tissue engineering.     

Controlled release of BSA by microsphere-incorporated PLGA scaffolds under cyclic loading

Localized delivery of bioactive molecules from porous biodegradable scaffolds is very important in advanced tissue engineering strategies, and it is necessary to study the delivery under dynamic loading which mimics the in vivo biomechanical environments. In this study, bovine serum albumin (BSA), a model of bioactive proteins, was incorporated into porous poly(l-lactide-co-glycolide) (PLGA) scaffolds by seeding BSA-loaded microspheres onto the scaffold pore wall, where the microspheres of poly(ethylene glycol)-b-poly(l-lactide) (PELA) were prepared by double emulsion technique. The in vitro release behavior of BSA from the scaffold under dynamic cyclic loading was studied in comparison with that under a static condition as well as from PELA microspheres. It was observed that the microsphere-incorporated scaffold prolonged BSA release with respect to the microspheres. The cyclic loading accelerated the release of BSA from the scaffold and the cumulative release on day 10 reached 85% of the totally encapsulated BSA. The delivery under a dynamic condition would be an initial study of in vivo localized delivery of growth factors.     

Biotemplating of Luffa cylindrica sponges to self-supporting hierarchical zeolite macrostructures for bio-inspired structured catalytic reactors

Biomorphic self-supporting MFI-type zeolite frameworks with hierarchical porosity and complex architecture were prepared using a 2-step (in-situ seeding and secondary crystal growth) hydrothermal synthesis in the presence of a biological template (Luffa sponge), employed as a macroscale sacrificial structure builder. The bio-inspired zeolitic replica inherited the complex spongy morphology and the intricate open-porous architecture of the biotemplate. Moreover, it exhibited reasonable mechanical stability in order to study the applicability of the biomorphic catalyst in a technical catalytic process. A bio-inspired catalytic reactor utilising the self-supporting ZSM-5 scaffold in monolithic configuration was developed in order to test the catalytic performance of the material.     

The growth of stem cells within β-TCP scaffolds in a fluid-dynamic environment

A three-dimensional dynamic perfusion system was developed to provide mass transport and nutrient supply to permit the cell proliferation during the long-term culture inside a β-tricalcium phosphate (β-TCP) scaffold. Also the flow field throughout the scaffold was studied. The porous cylindrical scaffold with a central channel was seeded with the sheep mesenchymal stem cells (MSCs). Then the cell-seeded scaffolds were continuously perfused with the complete α-MEM medium by a peristaltic pump for 7, 14 and 28 days, respectively. Histological study showed that the cell proliferation rates were different throughout the whole scaffolds and the different cell coverage was shown in different positions of the scaffold. Unoccupied spaces were found in many macropores. A computational fluid dynamics (CFD) modeling was used to simulate the flow conditions within perfused cell-seeded scaffolds to give an insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.52 mm/s) and shear stress (approximately 0.0055 Pa) were found to correspond to increased cell proliferation within the cell–scaffold constructs. Flow speeds were between 0.25 and 0.75 mm/s and shear stresses were between 0.003 and 0.008 Pa in approximately 75% of the regions. This method exhibits novel capabilities to compare the results obtained for different perfusion rates or different scaffold microarchitectures. It may allow specific fluid velocities and shear stresses to be determined to optimize the perfusion flow rate, porous scaffold architecture and distribution of in vitro tissue growth.     

Fabrication of a composite vascular scaffold using electrospinning technology

Intermolecular forces and morphology demonstrated that there was an excellent compatibility between silk fibroin and gelatin. The silk fibroin/gelatin composite vascular scaffold (inner diameter 4.5 mm) was prepared successfully by electrospinning. The scaffold was treated with ethanol to enhance the water-resistant ability and biomechanical properties. After ethanol treatment, the scaffold could hardly dissolve in the water, and FTIR showed that the conformation of the treated silk fibroin/gelatin composite vascular scaffold was mainly β-sheets. The electrospun silk fibroin/gelatin vascular scaffold possessed outstanding biomechanical properties. In vitro cell culture and in vivo subcutaneous implantation demonstrated that the electrospun silk fibroin/gelatin vascular scaffold had an appropriate biocompatibility. The results indicated that the electrospun silk fibroin/gelatin composite vascular scaffold could be considered as an ideal candidate for tissue-engineered blood vessel.     

Osteogenic differentiation and mineralization in fibre-reinforced tubular scaffolds: theoretical study and experimental evidences

The development of composite scaffolds with well-organized architecture and multi-scale properties (i.e. porosity, degradation) represents a valid approach for achieving a tissue-engineered construct capable of reproducing the medium- and long-term in vitro behaviour of hierarchically complex tissues such as spongy bone. To date, the implementation of scaffold design strategies able to summarize optimal scaffold architecture as well as intrinsic mechanical, chemical and fluid transport properties still remains a challenging issue. In this study, poly ɛ-caprolactone/polylactid acid (PCL/PLA) tubular devices (fibres of PLA in a PCL matrix) obtained by phase inversion/salt leaching and filament winding techniques were proposed as cell instructive scaffold for bone osteogenesis. Continuous fibres embedded in the polymeric matrix drastically improved the mechanical response as confirmed by compression elastic moduli, which vary from 0.214 ± 0.065 to 1.174 ± 0.143 MPa depending on the relative fibre/matrix and polymer/solvent ratios. Moreover, computational fluid dynamic simulations demonstrated the ability of composite structure to transfer hydrodynamic forces during in vitro culture, thus indicating the optimal flow rate conditions that, case by case, enables specific cellular events—i.e. osteoblast differentiation from human mesenchymal stem cells (hMSCs), mineralization, etc. Hence, we demonstrate that the hMSC differentiation preferentially occurs in the case of higher perfusion rates—over 0.05 ml min^–1—as confirmed by the expression of alkaline phosphate and osteocalcin markers. In particular, the highest osteopontin values and a massive mineral phase precipitation of bone-like phases detected in the case of intermediate flow rates (i.e. 0.05 ml min^–1) allows us to identify the best condition to stimulate the bone extracellular matrix in-growth, in agreement with the hydrodynamic model prediction. All these results concur to prove the succesful use of tubular composite as temporary device for long bone treatment.     

Bioactive glass nanotube scaffold with well-ordered mesoporous structure for improved bioactivity and controlled drug delivery

In this study, a novel mesoporous bioactive glass nanotube (MBGN) scaffold has been fabricated via template-assisted sol-gel method using bacterial cellulose (BC) as template and nonionic block copolymer (P123) as pore-directing agent. The scaffold was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, and N₂ adsorption-desorption analysis. Furthermore, simvastatin was used to evaluate the loading efficiency and release kinetics of the scaffold. The obtained scaffold displays nanofiber-like morphology, ordered mesopores on the tube walls, and interconnected three-dimensional (3D) network structure that completely replicates the BC template. In addition, it shows dual pore sizes (16.2 and 3.3 nm), large specific surface area (537.2 m² g⁻¹) and pore volume (1.429 cm³ g⁻¹). More importantly, the scaffold possesses excellent apatite-forming ability and sustainable drug release as compared to the counterpart scaffold without mesopores. This unique scaffold can be considered a promising candidate for drug delivery and bone tissue regeneration.     

Antheraea assama Silk Fibroin‐Based Functional Scaffold with Enhanced Blood Compatibility for Tissue Engineering Applications

The architecture and surface chemistry of a scaffold determine its utility in tissue engineering (TE). Conventional techniques have limitations in fabricating a scaffold with control over both architecture and surface chemistry. To ameliorate this, in this report, we demonstrate the fabrication of an Antheraea assama silk fibroin (AASF)‐based functional scaffold. AASF is a non‐mulberry variety having superior qualities to mulberry SF and is largely unexplored in the context of TE. First, a 3D scaffold with biomimetic architecture is fabricated. The scaffold is subsequently made blood compatible by modifying the surface chemistry through a simple sulfation reaction. EDX and FTIR analysis demonstrate the successful sulfation of the scaffold. SEM observations reveal that sulfation has no any effect on the scaffold architecture. TGA reveals that it has increased thermal stability. The sulfation reaction significantly improves the overall hydrophilicity of the scaffold, as is evident from the increase in water holding capacity; this possibly enhances the blood compatibility. The enhancement in blood compatibility of the sulfated scaffold is determined from in vitro haemolysis, protein adsorption and platelet adhesion studies. The sulfated scaffold is non‐toxic and supports cell adhesion and growth, as revealed by indirect and direct contact‐based in vitro cytotoxicity assays. This study reveals that the AASF‐based functional scaffold, which has biomimetic architecture and blood‐compatible surface chemistry, could be suitable for TE applications.     

Multiphasic construct studied in an ectopic osteochondral defect model

In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials.     

Osteogenic effect of hyaluronic acid sodium salt in the pores of a hydroxyapatite scaffold

According to previous reports, a large volume of bone marrow cells (1 × 10⁷ cells/ml) is required for bone regeneration in the pores of a scaffold in vivo. We theorized that immersion of a porous hydroxyapatite (HA) scaffold in hyaluronic acid solution would facilitate bone formation in the scaffold at 1 × 10⁶ cells/ml density of bone marrow cells. The cells were respectively seeded into pores of the cylindrical HA scaffolds with a hollow center after immersion in hyaluronic acid solution or in culture medium. The scaffolds were implanted in the dorsal subcutis of rats for 4 weeks. Thereafter, serially sectioned paraffin specimens were made and observed histologically. Bone formation was observed in many pores of HA scaffold by immersion in hyaluronic acid solution. However, there were no or less pores with new bone formation in the scaffold by immersion in culture medium. The cells were cultured with and without hyaluronic acid in vitro. There was no significant difference in bone formation in vitro with and without hyaluronic acid. The results of this study suggest that hyaluronic acid binds to the cells on the wall of three-dimensional structure and effectively promotes new bone formation.     

Fabrication and characterization of bioactive silk fibroin/wollastonite composite scaffolds

Composite scaffolds of silk fibroin (SF) with bioactive wollastonite were prepared by freeze-drying. X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy analysis showed that random coil and β-sheet structure co-existed in the SF scaffold. The mechanical performance, surface hydrophilicity and water-uptake capacity of the composite scaffolds were improved compared with those of pure SF scaffold. The bioactivity of the composite scaffold was evaluated by soaking in a simulated body fluid (SBF), and formation of a hydroxycarbonate apatite (HCA) layer was determined by FT-IR and XRD. The results showed that the SF/wollastonite composite scaffold was bioactive as it induced the formation of HCA on the surface of the composite scaffold after soaking in SBF for 5 days. In vitro cell attachment and proliferation tests showed that the composite scaffold was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, the incorporation of wollastonite into the SF scaffold can enhance both the mechanical strength and bioactivity of the scaffold, which suggests that the SF/wollastonite composite scaffold may be a potential biomaterial for tissue engineering.     

Label-free magnetic resonance imaging to locate live cells in three-dimensional porous scaffolds

Porous scaffolds are widely tested materials used for various purposes in tissue engineering. A critical feature of a porous scaffold is its ability to allow cell migration and growth on its inner surface. Up to now, there has not been a method to locate live cells deep inside a material, or in an entire structure, using real-time imaging and a non-destructive technique. Herein, we seek to demonstrate the feasibility of the magnetic resonance imaging (MRI) technique as a method to detect and locate in vitro non-labelled live cells in an entire porous material. Our results show that the use of optimized MRI parameters (4.7 T; repetition time = 3000 ms; echo time = 20 ms; resolution 39 × 39 µm) makes it possible to obtain images of the scaffold structure and to locate live non-labelled cells in the entire material, with a signal intensity higher than that obtained in the culture medium. In the current study, cells are visualized and located in different kinds of porous scaffolds. Moreover, further development of this MRI method might be useful in several three-dimensional biomaterial tests such as cell distribution studies, routine qualitative testing methods and in situ monitoring of cells inside scaffolds.     

Effects of extrusion and heat treatment on the microstructure and tensile properties of in situ TiBw/Ti6Al4V composite with a network architecture

In situ TiB whisker reinforced Ti6Al4V (TiBw/Ti64) composites with a network architecture were extruded and heat treated in order to further improve their mechanical properties. The microstructure results show that the equiaxed network architecture was extruded to column network architecture and TiB whisker to alignment distribution. The transformed β phase is formed and the residual stress generated during extrusion obviously decreases after water quenching and aging processes. The tensile test results show that the strength, elastic modulus and ductility of the composites can be significantly improved by the subsequent extrusion, and then, the strength can be further improved by water quenching and aging processes after hot extrusion deformation. The elastic modulus of the as-sintered composites with a novel network microstructure follows the upper bound of Hashin-Shtrikman (H-S) theory before extrusion, while that of the as-extruded composites with a column network microstructure agrees well with the prediction from Halpin-Tsai equation.     

Morphology and degradation properties of PCL/HYAFF11® composite scaffolds with multi-scale degradation rate

The analysis of scaffold degradation is a promising strategy for understanding the dynamic changes in texture and pore morphology which accompany polymer resorption, and for collecting same fundamental indicators regarding the potential fate of the scaffold in the biological environment. In this study, we investigate the morphology and degradation properties of three composite scaffolds based on poly(ε-caprolactone) (PCL) embedded with benzyl ester of hyaluronic acid (HYAFF11®) phases, and, in turn, different reinforcement systems – i.e., calcium phosphate particles or continuous poly(lactic acid) (PLA) fibres. Scanning electron microscopy (SEM) and μ-tomography supported by digital image analysis enabled a not invasive investigation of the scaffold morphology, providing a quantitative assessment of porosity (which ranged from 63.1 to 82.8), pore sizes (which varied from 170.5 to 230.4 μm) and pore interconnectivity. Thermal analyses (DSC and TGA) and Raman spectroscopy demonstrated the multi-scale degradation of the composite with highly tailoring degradation kinetics depending on the component material phases and scaffold architecture changes, due to their conditioning in simulated in vivo environment (i.e., SBF solution). These results demonstrate that the judicious mixing of materials with faster (i.e., HYAFF11) and slower (i.e., PLA and PCL) degradation kinetics, different size and shape (i.e., domains, particles or long fibres), certainly concurs to design a smart composite scaffold with time-controlled degradation which can support the regeneration of a large variety of tissues, from the cartilage to the bone.     

Rapid adhesion and proliferation of keratinocytes on the gold colloid/chitosan film scaffold

The gold colloid/chitosan film scaffold, which could enhance the attached ratio and accelerate proliferation of newborn mice keratinocytes, was fabricated by nanotechnology and self-assembly technology. This nanometer scaffold was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The keratinocytes were cultured and observed on three different extracellular matrices (ECM): gold colloid/chitosan film scaffold, chitosan film and cell culture plastic (control groups). 6 h, 12 h, 24 h after inoculation, the cell attached ratios were calculated respectively. In comparison to control groups, this scaffold could significantly (P < 0.01) increase the attached ratio of keratinocytes and promote their growth. Meanwhile, there were not any fusiform fibroblasts growing on this scaffold. The rapidly proliferating keratinocytes were indentified and characterized by immunohistochemistry and transmissive electron microscope (TEM), which showed the cells maintain their biological activity well. The results indicated that gold colloid/chitosan film scaffold was nontoxic to keratinocytes, and was a good candidate for wound dressing in skin tissue engineering.     

Engineered synovial joint condyle using demineralized bone matrix

In an effort to develop tissue-engineered bio-joints, a novel demineralized joint scaffold was achieved by peeling off the cartilage layer of a distal femur joint condyle. Primary chondrocytes were then seeded onto the demineralized joint condyle scaffolds and cultured in vitro for 6 weeks. Histological staining and biochemical assays of the engineered joints showed that after 6 weeks in vitro culture, a cartilaginous layer had formed on the demineralized joint scaffold that was similar to native synovial articular cartilage with respect to palpation and texture. Meanwhile, the engineered joint condyle cartilage demonstrated rudimentary morphological and structural resemblance to native cartilage. Intense and uniform safranin-O red staining was found in engineered joint condyle cartilage. Furthermore, glycosaminoglycan (GAG) assays confirmed that there were no statistical differences in the GAG/DNA ratio between the engineered joint cartilage and native cartilage (p > 0.05). In conclusion, a novel scaffold and a practical method have therefore been developed for total joint tissue engineering based on demineralized bone scaffold. The morphological appearance of the engineered joint and the rudimentary biochemical quantification resemble that of a native articular condyle.     

Stimulation of healing within a rabbit calvarial defect by a PCL/PLGA scaffold blended with TCP using solid freeform fabrication technology

The purpose of this study was to investigate the healing capacity within an 8-mm rabbit calvarial defect using a polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffold blended with tri-calcium phosphate (TCP) that was constructed using solid freeform fabrication (SFF) technology. The PCL/PLGA/TCP scaffold showed a 37?% higher compressive strength and rougher surface than the PCL/PLGA scaffold. In animal experiments, new bone formation was analyzed using microcomputed tomography (micro-CT) and histological and histometric analyses. The PCL/PLGA/TCP groups had significantly greater neo-tissue areas as compared with the control groups at 4 and 8 weeks (P?<?0.05). The PCL/PLGA/TCP group had significantly greater bone density as compared with the control and PCL/PLGA groups at 4 and 8 weeks (P?<?0.005). The results of this study suggest that the PCL/PLGA/TCP scaffold fabricated using SFF technology is useful for recovering and enhancing new bone formation in bony defects in rabbits.     

The microscopic network structure of mussel (Mytilus) adhesive plaques

Marine mussels of the genus Mytilus live in the hostile intertidal zone, attached to rocks, bio-fouled surfaces and each other via collagen-rich threads ending in adhesive pads, the plaques. Plaques adhere in salty, alkaline seawater, withstanding waves and tidal currents. Each plaque requires a force of several newtons to detach. Although the molecular composition of the plaques has been well studied, a complete understanding of supra-molecular plaque architecture and its role in maintaining adhesive strength remains elusive. Here, electron microscopy and neutron scattering studies of plaques harvested from Mytilus californianus and Mytilus galloprovincialis reveal a complex network structure reminiscent of structural foams. Two characteristic length scales are observed characterizing a dense meshwork (approx. 100 nm) with large interpenetrating pores (approx. 1 µm). The network withstands chemical denaturation, indicating significant cross-linking. Plaques formed at lower temperatures have finer network struts, from which we hypothesize a kinetically controlled formation mechanism. When mussels are induced to create plaques, the resulting structure lacks a well-defined network architecture, showcasing the importance of processing over self-assembly. Together, these new data provide essential insight into plaque structure and formation and set the foundation to understand the role of plaque structure in stress distribution and toughening in natural and biomimetic materials.     

Design of tunable protein-releasing nanoapatite/hydrogel scaffolds for hard tissue engineering

Hierarchically structured porous scaffolds based on nanocrystalline carbonated hydroxyapatite reinforced hydrogels (Gellan or Agarose) have been tested as protein release matrices while evaluation their in vitro biocompatibility. The shaping method used develops under mild conditions thus allowing the incorporation of labile substances. The Bovine Serum Albumin (BSA), employed as a model protein, has been included by using two drug-inclusion strategies: during the scaffolds preparation (in situ process) or by injection of an aqueous protein solution within (ex situ process). The release studies showed a more controlled BSA delivery when the protein was incorporated during the scaffold preparation when compared to that where the protein has been loaded in a second step (ex situ process). The release patterns can also be tailored as a function of the scaffold composition (ceramic/polysaccharide ratio and nature) as well as the drying technology employed. Biocompatibility studies demonstrated that these scaffolds, regardless of the composition, allow the culture of osteoblasts on and around the material, thus supporting the potential use of these biomaterials for bone tissue engineering.