Scaffold Microarchitecture Research Articles

Decellularization of kidney tissue: comparison of sodium lauryl ether sulfate and sodium dodecyl sulfate for allotransplantation in rat.

An automatic decellularization device was developed to perfuse and decellularize male rats' kidneys using both sodium lauryl ether sulfate (SLES) and sodium dodecyl sulfate (SDS) and to compare their efficacy in kidney decellularization and post-transplantation angiogenesis. Kidneys were perfused with either 1% SDS solution for 4h or 1% SLES solution for 6h. The decellularized scaffolds were stained with hematoxylin and eosin, periodic acid Schiff, Masson's trichrome, and Alcian blue to determine cell removal and glycogen, collagen, and glycosaminoglycan contents, respectively. Moreover, scanning electron microscopy was performed to evaluate the cell removal and preservation of microarchitecture of both SDS and SLES scaffolds. Additionally, DNA quantification assay was applied for all groups in order to measure residual DNA in the scaffolds and normal kidney. In order to demonstrate biocompatibility of the decellularized scaffolds, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were seeded on the scaffolds. In addition, the allotransplantation was performed in back muscle and angiogenesis was evaluated. Complete cell removal in both SLES and SDS groups was observed in scanning electron microscopy and DNA quantification assays. Moreover, the extracellular matrix (ECM) architecture of rat kidney in the SLES group was significantly preserved better than the SDS group. The hUC-MSCs were successfully migrated from the cell culture plate surface into the SDS and SLES decellularized scaffolds. The formation of blood vessels was observed in the kidney in both SLES and SDS decellularized kidneys. The better preservation of ECM than SDS introduces SLES as the solvent of choice for kidney decellularization.

Characterization of Indonesia Decellularized Liver Cubes Scaffold using Scanning Electron Microscopy

Liver biological scaffold was developed in order to resemble native liver tissue environment. It can be achieved by decellularizing native liver tissue that will remove cells and preserve extracellular matrix (ECM). Furthermore, ECM fibers are arranged in a special pattern, which affect liver cell polarity and topography that are important for cells’ implantation, proliferation and differentiation. Therefore, the aim of this study was to evaluate liver cube scaffold topography that was decellularized with fixed multiple sites syringe injection (Indonesia patent number: S00201907930).Rat liver cubes (n=3) underwent decellularization with Ethylene Glycol Tetraacetic Acid (EGTA) immersion and increased Sodium Dodecyl Sulfate (SDS) concentrations using previous multiple sites syringe injection protocol study. Deoxyribonucleic Acid (DNA) concentrations were measured to confirm less DNA materials remaining in scaffolds. Scanning Electron Microscope (SEM) analysis of scaffolds were conducted for topographic characterization compared to undecellularized liver control. Molecular analysis of DNA concentration showed complete removal of DNA material. SEM analysis gave appearance of intact liver cube scaffold microarchitecture. Liver cubes decellularization using multiple sites syringe injection showed good topographic liver scaffold characterization.

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering.

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.

Topographic Guidance in Melt-Electrowritten Tubular Scaffolds Enhances Engineered Kidney Tubule Performance

To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties. A custom-built MEW device was used to fabricate tubular fibrous scaffolds with small diameter sizes (O = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality. Tubular scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 0.5 mm and pore sizes of 0.2 mm were achieved. CiPTEC formed tight monolayers in all scaffold microarchitectures tested, but well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp). To conclude, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

Topographic Guidance in Melt-Electrowritten Tubular Scaffolds Enhances Engineered Kidney Tubule Performance.

Introduction: To date, tubular tissue engineering relies on large, non-porous tubular scaffolds (Ø > 2 mm) for mechanical self-support, or smaller (Ø 150–500 μm) tubes within bulk hydrogels for studying renal transport phenomena. To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties.Materials and Methods: A custom-built melt-electrowriting (MEW) device was used to fabricate tubular fibrous scaffolds with small diameter sizes (Ø = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the tubular scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality.Results: Tubular fibrous scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 1 mm and pore sizes of 0.2 mm were achieved and used for subsequent cell experiments. While HUVEC were unable to bridge the pores, ciPTEC formed tight monolayers in all scaffold microarchitectures tested. Well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp).Discussion and Conclusion: Here, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

The development of natural polymer scaffold-based therapeutics for osteochondral repair.

Due to the limited regenerative capacity of cartilage, untreated joint defects can advance to more extensive degenerative conditions such as osteoarthritis. While some biomaterial-based tissue-engineered scaffolds have shown promise in treating such defects, no scaffold has been widely accepted by clinicians to date. Multi-layered natural polymer scaffolds that mimic native osteochondral tissue and facilitate the regeneration of both articular cartilage (AC) and subchondral bone (SCB) in spatially distinct regions have recently entered clinical use, while the transient localized delivery of growth factors and even therapeutic genes has also been proposed to better regulate and promote new tissue formation. Furthermore, new manufacturing methods such as 3D bioprinting have made it possible to precisely tailor scaffold micro-architectures and/or to control the spatial deposition of cells in requisite layers of an implant. In this way, natural and synthetic polymers can be combined to yield bioactive, yet mechanically robust, cell-laden scaffolds suitable for the osteochondral environment. This mini-review discusses recent advances in scaffolds for osteochondral repair, with particular focus on the role of natural polymers in providing regenerative templates for treatment of both AC and SCB in articular joint defects.

Acute In Vivo Functional Assessment of a Biodegradable Stentless Elastomeric Tricuspid Valve.

Degradable heart valves based on in situ tissue regeneration have been proposed as potentially durable and non-thrombogenic prosthetic alternatives. We evaluated the acute in vivo function, microstructure, mechanics, and thromboresistance of a stentless biodegradable tissue-engineered heart valve (TEHV) in the tricuspid position. Biomimetic stentless tricuspid valves were fabricated with poly(carbonate urethane)urea (PCUU) by double-component deposition (DCD) processing to mimic native valve mechanics and geometry. Five swine then underwent 24-h TEHV implantation in the tricuspid position. Echocardiography demonstrated good leaflet motion and no prolapse and trace to mild regurgitation in all but one animal. Histology revealed patches of proteinaceous deposits with no cellular uptake. SEM demonstrated retained scaffold microarchitecture with proteinaceous deposits but no platelet aggregation or thrombosis. Explanted PCUU leaflet thickness and mechanical anisotropy were comparable with native tricuspid leaflets. Bioinspired, elastomeric, stentless TEHVs fabricated by DCD were readily implantable and demonstrated good acute function in the tricuspid position.

Mechanical properties of tissue formed in vivo are affected by 3D-bioplotted scaffold microarchitecture and correlate with ECM collagen fiber alignment

ABSTRACTPurpose: Musculoskeletal soft tissues possess highly aligned extracellular collagenous networks that provide structure and strength. Such an organization dictates tissue-specific mechanical properties but can be difficult to replicate by engineered biological substitutes. Nanofibrous electrospun scaffolds have demonstrated the ability to control cell-secreted collagen alignment, but concerns exist regarding their scalability for larger and anatomically relevant applications. Additive manufacturing processes, such as melt extrusion-based 3D-Bioplotting, allow fabrication of structurally relevant scaffolds featuring highly controllable porous microarchitectures.Materials and Methods: In this study, we investigate the effects of 3D-bioplotted scaffold design on the compressive elastic modulus of neotissue formed in vivo in a subcutaneous rat model and its correlation with the alignment of ECM collagen fibers. Polycaprolactone scaffolds featuring either 100 or 400 µm interstrand spacing were implanted for 4 or 12 weeks, harvested, cryosectioned, and characterized using atomic-force-microscopy-based force mapping.Results: The compressive elastic modulus of the neotissue formed within the 100 µm design was significantly higher at 4 weeks (p < 0.05), but no differences were observed at 12 weeks. In general, the tissue stiffness was within the same order of magnitude and range of values measured in native musculoskeletal soft tissues including the porcine meniscus and anterior cruciate ligament. Finally, a significant positive correlation was noted between tissue stiffness and the degree of ECM collagen fiber alignment (p < 0.05) resulting from contact guidance provided by scaffold strands.Conclusion: These findings demonstrate the significant effects of 3D-bioplotted scaffold microarchitectures in the organization and sub-tissue-level mechanical properties of ECM in vivo.

Macrophage-Driven Biomaterial Degradation Depends on Scaffold Microarchitecture.

In situ tissue engineering is a technology in which non-cellular biomaterial scaffolds are implanted in order to induce local regeneration of replaced or damaged tissues. Degradable synthetic electrospun scaffolds are a versatile and promising class of biomaterials for various in situ tissue engineering applications, such as cardiovascular replacements. Functional in situ tissue regeneration depends on the balance between endogenous neo-tissue formation and scaffold degradation. Both these processes are driven by macrophages. Upon invasion into a scaffold, macrophages secrete reactive oxygen species (ROS) and hydrolytic enzymes, contributing to oxidative and enzymatic biomaterial degradation, respectively. This study aims to elucidate the effect of scaffold microarchitecture, i.e., μm-range fiber diameter and fiber alignment, on early macrophage-driven scaffold degradation. Electrospun poly-ε-caprolactone-bisurea (PCL-BU) scaffolds with either 2 or 6 μm (Ø) isotropic or anisotropic fibers were seeded with THP-1 derived human macrophages and cultured in vitro for 4 or 8 days. Our results revealed that macroph age-induced oxidative degradation in particular was dependent on scaffold microarchitecture, with the highest level of ROS-induced lipid peroxidation, NADPH oxidase gene expression and degradation in the 6 μm Ø anisotropic group. Whereas, biochemically polarized macrophages demonstrated a phenotype-specific degradative potential, the observed differences in macrophage degradative potential instigated by the scaffold microarchitecture could not be attributed to either distinct M1 or M2 polarization. This suggests that the scaffold microarchitecture uniquely affects macrophage-driven degradation. These findings emphasize the importance of considering the scaffold microarchitecture in the design of scaffolds for in situ tissue engineering applications and the tailoring of degradation kinetics thereof.

Preparation of novel porous calcium silicate scaffold loaded by celecoxib drug using freeze drying technique: Fabrication, characterization and simulation

Abstract In the current investigation, the microarchitecture of bio-nanocomposite scaffold, which is fabricated by natural synthetic diopside and composed of magnetite nanoparticles (MNPs), is considered. The MNPs are tested with various weight fractions (0, 5, 10, and 15 wt%) and are manufactured by the freeze-drying technique using sodium alginate as base matrix for the first time. Due to the limitation of the mechanical properties of calcium phosphates (CaPs) and bioactive glasses (BG), clinical usage of calcium silicate ceramics (CSC) are greatly affected. Therefore, CSCs are produced with the incorporation of metal oxides into the base binary xCaO-ySiO2, as well as the substitution of calcium ions. Furthermore, mechanical and biological properties of CSCs are enhanced, which are a result of the ability to give out bioactive ions and their distinct compositions. After that, the porous bio-nanocomposite scaffolds are investigated for biological and mechanical properties corresponding to hardness versus elastic modulus, apatite formation versus biodegradation rate, wetting properties versus roughness and electrical conductivity of the sample. Then, the composition, microstructure, and physical characteristics are also examined using different techniques such as X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) which is equipped with energy-dispersive X-ray spectroscopy (EDX). The obtained outcomes show that addition of diopside bioceramic enhances the mechanical and physical properties of the samples. It is shown that the prepared porous bio-nanocomposite scaffolds, containing 10 wt% MNPs, represents a better agreement in serving as a bone graft for the cancer disease treatment and hyperthermia term. The results indicate that the specimen with 10 wt% MNPs in the bio-nanocomposite release the celecoxib drug easier, however, its has better porosity and mechanical behavior that make it suitable candidate for bone implantations.

The odontogenic performance of human dental pulp stem cell in 3-dimensional chitosan and nano-bioactive glass-based scaffold material with different pores size

Background This study focused on the development of biocompatible and biodegradable homoporous chitosan scaffolds containing nano-bioactive glass 58S for dental tissue regeneration. For this aim, we developed a new chemical route to obtain homoporous scaffolds that mimic cancellous bone by utilizing H2O2. Materials and methods Different properties of the prepared powder of 58S were analyzed by transmission electron microscopy, thermogravimetric analysis (TGA)/differential scanning colometric (DSC), X-ray diffraction, and Fourier transform infrared spectroscopy. The developed scaffolds were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscope in comparison with heteroporous scaffolds prepared in the absence of H2O2. Stem cells from dental pulp were seeded onto the prepared scaffolds for different periods of time. The ability to differentiate toward an odontogenic lineage was studied for the loaded cells with the prepared scaffolds. Results The size of the prepared nano-bioglass 58S nanoparticles was in the range of 23.9–98.18 nm. The scaffolds prepared using H2O2 exhibited homogenous porous scaffolds in the range of 250–450 μm size when compared with scaffold synthesized in the absence of H2O2, which demonstrated heterogeneous scaffolds in the range of 350–700 μm. The cell studies showed better cell growth and biomineralization with the heterogeneous scaffolds compared with homogenous microarchitecture. Conclusion The microarchitecture of heterogeneous scaffolds plays a significant role in dental pulp stem cell differentiation and calcified tissue development as well as provides new possibilities for dental tissue engineering.

Pore architecture effects on chondrogenic potential of patient-specific 3-dimensionally printed porous tissue bioscaffolds for auricular tissue engineering

ObjectiveThis study aims to determine the effect of auricular scaffold microarchitecture on chondrogenic potential in an in vivo animal model. MethodsDICOM computed tomography (CT) images of a human auricle were segmented to create an external anatomic envelope. Image-based design was used to generate 1) orthogonally interconnected spherical pores and 2) randomly interspersed pores, and each were repeated in three dimensions to fill the external auricular envelope. These auricular scaffolds were then 3D printed by laser sintering poly-l-caprolactone, seeded with primary porcine auricular chondrocytes in a hyaluronic acid/collagen hydrogel and cultured in a pro-chondrogenic medium. The auricular scaffolds were then implanted subcutaneously in rats and explanted after 4 weeks for analysis with Safranin O and Hematoxylin and Eosin staining. ResultsAuricular constructs with two micropore architectures were rapidly manufactured with high fidelity anatomic appearance. Subcutaneous implantation of the scaffolds resulted in excellent external appearance of both anterior and posterior auricular surfaces. Analysis on explantation showed that the defined, spherical micropore architecture yielded histologic evidence of more robust chondrogenic tissue formation as demonstrated by Safranin O and Hematoxylin and Eosin staining. ConclusionsImage-based computer-aided design and 3D printing offers an exciting new avenue for the tissue-engineered auricle. In early pilot work, creation of spherical micropores within the scaffold architecture appears to impart greater chondrogenicity of the bioscaffold. This advantage could be related to differences in permeability allowing greater cell migration and nutrient flow, differences in surface area allowing different cell aggregation, or a combination of both factors. The ability to design an anatomically correct scaffold that maintains its structural integrity while also promoting auricular cartilage growth represents an important step towards clinical applicability of this new technology.

Manufacture and characterisation of EmDerm\u2014novel hierarchically structured bio-active scaffolds for tissue regeneration

There are significant challenges for using emulsion templating as a method of manufacturing macro-porous protein scaffolds. Issues include protein denaturation by adsorption at hydrophobic interfaces, emulsion instability, oil droplet and surfactant removal after protein gelation, and compatible cross-linking methods. We investigated an oil-in-water macro-emulsion stabilised with a surfactant blend, as a template for manufacturing protein-based nano-structured bio-intelligent scaffolds (EmDerm) with tuneable micro-scale porosity for tissue regeneration. Prototype EmDerm scaffolds were made using either collagen, through thermal gelation, fibrin, through enzymatic coagulation or collagen-fibrin composite. Pore size was controlled via surfactant-to-oil phase ratio. Scaffolds were crosslink-stabilised with EDC/NHS for varying durations. Scaffold micro-architecture and porosity were characterised with SEM, and mechanical properties by tensiometry. Hydrolytic and proteolytic degradation profiles were quantified by mass decrease over time. Human dermal fibroblasts, endothelial cells and bone marrow derived mesenchymal stem cells were used to investigate cytotoxicity and cell proliferation within each scaffold. EmDerm scaffolds showed nano-scale based hierarchical structures, with mean pore diameters ranging from 40–100 microns. The Young’s modulus range was 1.1–2.9 MPa, and ultimate tensile strength was 4–16 MPa. Degradation rate was related to cross-linking duration. Each EmDerm scaffold supported excellent cell ingress and proliferation compared to the reference materials Integra™ and Matriderm™. Emulsion templating is a novel rapid method of fabricating nano-structured fibrous protein scaffolds with micro-scale pore dimensions. These scaffolds hold promising clinical potential for regeneration of the dermis and other soft tissues, e.g., for burns or chronic wound therapies.

Effects of microarchitecture and mechanical properties of 3D microporous PLLA-PLGA scaffolds on fibrochondrocyte and L929 fibroblast behavior

There are several reports studying cell behavior on surfaces in 2D or in hydrogels in 3D. However, cell behavior in 3D microporous scaffolds has not been investigated extensively. In this study, poly(L-lactic acid)/poly(lactic acid-co-glycolic acid) (PLLA/PLGA)-based microporous scaffolds were used to study the effects of scaffold microarchitecture and mechanical properties on the behavior of two different cell types, human meniscal fibrochondrocytes and L929 mouse fibroblasts. In general, cell attachment, spreading and proliferation rate were mainly regulated by the strut (pore wall) stiffness. Increasing strut stiffness resulted in an increase in L929 fibroblast attachment and a decrease in fibrochondrocyte attachment. L929 fibroblasts tended to get more round as the strut stiffness increased, while fibrochondrocytes tended to get more elongated. Cell migration increased for both cell types with the increasing pore size. Migrating L929 fibroblasts tended to get more round on the stiff scaffolds, while fibrochondrocytes tended to get more round on the soft scaffolds. This study shows that the behavior of cells on 3D microporous scaffolds is mainly regulated by pore size and strut stiffness, and the response of a cell depends on the stiffness of both cells and materials. This study could be useful in designing better scaffolds for tissue engineering applications.

Design and Structure-Function Characterization of 3D Printed Synthetic Porous Biomaterials for Tissue Engineering.

3D printing is now adopted for use in a variety of industries and functions. In biomedical engineering, 3D printing has prevailed over more traditional manufacturing methods in tissue engineering due to its high degree of control over both macro- and microarchitecture of porous tissue scaffolds. However, with the improved flexibility in design come new challenges in characterizing the structure-function relationships between various architectures and both mechanical and biological properties in an assortment of clinical applications. Presently, the field of tissue engineering lacks a comprehensive body of literature that is capable of drawing meaningful relationships between the designed structure and resulting function of 3D printed porous biomaterial scaffolds. This work first discusses the role of design on 3D printed porous scaffold function and then reviews characterization of these structure-function relationships for 3D printed synthetic metallic, polymeric, and ceramic biomaterials.

Substrate Microarchitecture Shapes the Paracrine Crosstalk of Stem Cells with Endothelial Cells and Osteoblasts

We examined the hypothesis that substrate microarchitecture regulates the crosstalk between human mesenchymal stem cells (hMSC) and cell types involved in bone regeneration. Compared with polyester flat substrates having uniformly distributed homogenous pores (2D), three-dimensional polystyrene substrates with randomly oriented and interconnected pores of heterogeneous size (3D) stimulated the stromal secretion of IGF-1 while lessened the production of VEGFR-1, MCP-1 and IL-6. The medium conditioned by hMSC cultured in 3D substrates stimulated tube formation by human endothelial cells (hEC) to a higher extent than medium from 2D cultures. 3D co-cultures of hMSC and hEC contained higher secreted levels of IGF-1, EGF and FGF-2 than 2D co-cultures, resulting in increased hEC proliferation and migration. Substrate microarchitecture influenced the secretion of factors related to bone remodeling as the ratio RANKL to OPG, and the levels of M-CSF and IL-6 were higher in 3D co-cultures of hMSC and human osteoblasts (hOB) than in 2D co-cultures. Cytokine microenvironment in 3D co-cultures stimulated osteoblast matrix reorganization while demoted the late steps of osteoblastic maturation. Altogether, data in this study may unveil a new role of scaffold microarchitecture during bone regeneration, as modulator of the paracrine relationships that hMSC establish with hEC and hOB.

Alginate Bead Based Hexagonal Close Packed 3D Implant for Bone Tissue Engineering.

Success of bone tissue engineering (BTE) relies on the osteogenic microarchitecture of the biopolymeric scaffold and appropriate spatiotemporal distribution of therapeutic molecules (growth factors and drugs) inside it. However, the existing technologies have failed to address both the issues together. Keeping this perspective in mind, we have developed a novel three-dimensional (3D) implant prototype by stacking hexagonal close packed (HCP) layers of calcium alginate beads. The HCP arrangement of the beads lead to a patterned array of interconnected tetrahedral and octahedral pores of average diameter of 142.9 and 262.9 μm, respectively, inside the implant. The swelling pattern of the implants changed from isotropic to anisotropic in the z-direction in the absence of bivalent calcium ions (Ca2+) in the swelling buffer. Incubation of the implant in simulated body fluid (SBF) resulted in a 2.7-fold increase in the compressive modulus. The variation in the relaxation times as derived from the Weichert viscoelasticity model predicted a gradual increase in the interactions among the alginate molecules in the matrix. We demonstrated the tunability of the spatiotemporal drug release from the implant in a tissue mimicking porous semisolid matrix as well as in conventional drug release set up by changing the spatial coordinates of the "drug loaded depot layer" inside the implant. The therapeutic potential of the implant was confirmed against Escherichia coli using metronidazole as the model drug. Detailed analysis of cell viability, cell cycle progression, and cytoskeletal reorganization using osteoblast cells (MG-63) proved the osteoconductive nature of the implant. Expression of differentiation markers such as alkaline phosphatase, runx2, and collagen type 1 in human mesenchymal stem cell in vitro confirmed the osteogenic nature of the implant. When tested in vivo, VEGF loaded implant was found capable of inducing angiogenesis in a mice model. In conclusion, the bead based implant may find its utility in non-load-bearing BTE.

Photolithography of 3D Scaffolds for Artificial Tissue

The present study is focused on the design and fabrication of novel functional 3D-hydrogel scaffolds for regenerative medicine. In order to critically analyzing the effect of the microarchitecture of 3D scaffolds for driving the cellular fate and diffusion of progenitor stem cells we have fabricate a number of scaffolds with different geometry, stiffness and composition. The physical characteristics of the scaffold determine indeed, as well the biochemical factors, the fate of the cells. We use an innovative composite material consisting of hydrogel with different molecular weight and with suitable accordion-like and woodpile structures in order to tailor stiffness and elasticity conferred to the final structure.These novel 3D bioinspired scaffolds were obtained by both single- (1PP) and two-photon polymerization (2PP) processing. In particular, 2PP scaffolds represent a great advantage with respect to previous achievements based on traditional methods. 3D-structures were fabricated with lateral resolution of some microns, allowing an advanced control of pore microarchitecture of defined tensile strength, and the inclusion of albumin microspheres with various functionalities. The morphological, biochemical and functional characteristics are discussed. Moreover, the effects of the structured hydrogel scaffolds on the proliferation and differentiation of adult stem cells is analyzed in view of the fabrication of portion of contractile cardiac muscle to be obtained In Vitro.

PEG-fibrinogen hydrogels for three-dimensional breast cancer cell culture.

Tissue-engineered three-dimensional (3D) cancer models employing biomimetic hydrogels as cellular scaffolds provide contextual in vitro recapitulation of the native tumor microenvironment, thereby improving their relevance for use in cancer research. This study reports the use of poly(ethylene glycol)-fibrinogen (PF) as a suitable biosynthetic hydrogel for the 3D culture of three breast cancer cell lines: MCF7, SK-BR-3, and MDA-MB-231. Modification of the matrix characteristics of PF hydrogels was achieved by addition of excess poly(ethylene glycol) diacrylate, which resulted in differences in Young's moduli, degradation behavior, release kinetics, and ultrastructural variations in scaffold microarchitecture. Cancer cells were maintained in 3D culture with high viability within these hydrogels and resulted in cell-type dependent morphological changes over time. Cell proliferation and 3D morphology within the hydrogels were visualized through immunofluorescence staining. Finally, spatial heterogeneity of colony area within the hydrogels was quantified, with peripheral cells forming colonies of higher area compared to those in the interior regions. Overall, PF-based hydrogels facilitate 3D culture of breast cancer cells and investigation of cellular behavior in response to varying matrix characteristics. PF-based cancer models could be potentially used in future investigations of cancer biology and in anti-cancer drug-testing applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 236-252, 2017.

Endothelial pattern formation in hybrid constructs of additive manufactured porous rigid scaffolds and cell-laden hydrogels for orthopedic applications

Vascularization of tissue engineering constructs (TECs) in vitro is of critical importance for ensuring effective and satisfactory clinical outcomes upon implantation of TECs. Biomechanical properties of TECs have remarkable influence on the in vitro vascularization of TECs. This work utilized in vitro experiments and finite element analysis to investigate endothelial patterns in hybrid constructs of soft collagen gels and rigid macroporous poly(ε-caprolactone)-β-tricalcium phosphate (PCL-β-TCP) scaffold seeded/embedded with human umbilical vein endothelial cells (HUVECs) for bone tissue engineering applications. We first fabricated and characterized well-defined porous PCL-β-TCP scaffolds with identical pore size (500µm) but different strut sizes (200 and 400µm) using additive manufacturing (AM) technology, and then assessed the HUVEC׳s proliferation and morphogenesis within collagen, PCL-β-TCP scaffold, and the collagen-scaffold hybrid construct. Results showed that, in the hybrid construct, the cell population in the collagen component dropped by day 7 but then increased by day 14. Also, cells migrated onto the struts of the scaffold component, proliferated over time, and formed networks on the thinner struts (i.e., 200µm). Also, the thinner struts resulted in formation of long linear cellular cords structures within the pores. Finite element simulation demonstrated principal stress patterns similar to the observed cell-network pattern. It is probable that the scaffold component modulated patterns of principal stresses in the collagen component as biomechanical cues for reorganization of cell network patterns. Also, the scaffold component significantly improved the mechanical integrity of hydrogel component in the hybrid construct for weight-bearing applications. These results have collectively indicated that the manipulation of micro-architecture of scaffold could be an effective means to further regulate and guide desired cellular response in hybrid constructs.