Scaffold Bulk Research Articles

Improved porosity of electrospun poly (Lactic-Co-Glycolic) scaffolds by sacrificial microparticles enhances cellular infiltration compared to sacrificial microfiber.

Electrospinning is a technique used to fabricate nano-/microfiber scaffolds for tissue engineering applications. However, a major limitation of electrospun scaffolds is the high packing density of fibers that leads to poor cellular infiltration. Thus, incorporation of a water soluble sacrificial porogen, polyethylene oxide (PEO), was utilized to fine-tune the porous fraction of the scaffolds and decrease fiber packing density. Poly(lactic-co-glycolic) acid (PLGA) scaffolds were either co-electrospun with sacrificial PEO microfibers or co-electrosprayed with sacrificial PEO microparticles at three different extrusion rates to control the relative morphology and dose of PEO. A dose-dependent response in PLGA scaffold bulk porosity and pore area was noted as PEO content was increased. Notably, PLGA scaffolds after removal of sacrificial PEO microparticles significantly increased the porous fraction and pore area approximately 8, 10, and 14% and 46, 20, and 33μm2, respectively, relative to the analogous PEO microfiber scaffold. The tensile properties of the more porous PLGA scaffolds after PEO microparticle removal, remained stable for all extrusion rates of PEO tested, relative to the PLGA scaffolds after PEO microfiber removal. Histological analysis revealed that removal of PEO microparticles significantly increased the depth of cellular migration through the PLGA scaffolds, relative to PEO microfiber scaffolds, with maximum migratory depths of 1120μm versus 150μm over 28 days, respectively. Additionally, depth of cellular infiltration responded dose-dependently in the PEO microparticle scaffolds, whereas in the PEO microfiber scaffolds there was no correlation. Further analysis with Masson's Trichrome staining and electron microscopy revealed that collagen density and depth of deposition substantially increased in PLGA scaffolds after removal of PEO microparticles relative to PEO microfibers. Thus, this study demonstrates an effective strategy to control the porous fraction of electrospun scaffolds via the incorporation of sacrificial PEO microparticles, without significant decreases in mechanical properties, thereby enhancing cellular infiltration and subsequent extracellular matrix deposition.

Immobilization of nanocarriers within a porous chitosan scaffold for the sustained delivery of growth factors in bone tissue engineering applications

To guide the natural bone regeneration process, bone tissue engineering strategies rely on the development of a scaffold architecture that mimics the extracellular matrix and incorporates important extracellular signaling molecules, which promote fracture healing and bone formation pathways. Incorporation of growth factors into particles embedded within the scaffold can offer both protection of protein bioactivity and a sustained release profile. In this work, a novel method to immobilize carrier nanoparticles within scaffold pores is proposed. A biodegradable, osteoconductive, porous chitosan scaffold was fabricated via the "freeze-drying method," leading to scaffolds with a storage modulus of 8.5 kPa and 300 μm pores, in line with existing bone scaffold properties. Next, poly(methyl methacrylate-co-methacrylic acid) nanoparticles were synthesized and immobilized to the scaffold via carbodiimide-crosslinker chemistry. A fluorescent imaging study confirmed that the conventional methods of protein and nanocarrier incorporation into scaffolds can lead to over 60% diffusion out of the scaffold within the first 5 min of implantation, and total disappearance within 4 weeks. The novel method of nanocarrier immobilization to the scaffold backbone via carbodiimide-crosslinker chemistry allows full retention of particles for up to 4 weeks within the scaffold bulk, with no negative effects on the viability and proliferation of human umbilical vein endothelial cells.

Etching anisotropic surface topography onto fibrin microthread scaffolds for guiding myoblast alignment.

To regenerate functional muscle tissue, engineered scaffolds should impart topographical features to induce myoblast alignment by a phenomenon known as contact guidance. Myoblast alignment is an essential step towards myotube formation, which is guided in vivo by extracellular matrix structure and micron-scale grooves between adjacent muscle fibers. Fibrin microthread scaffolds mimic the morphological architecture of native muscle tissue and have demonstrated promise as an implantable scaffold for treating skeletal muscle injuries. While these scaffolds promote modest myoblast alignment, it is not sufficient to generate highly functional muscle tissue. The goal of this study is to develop and characterize a new method of etching the surface of fibrin microthreads to incorporate aligned, sub-micron grooves that promote myoblast alignment. To generate these topographic features, we placed fibrin microthreads into 2-(N-morpholino)ethane-sulfonic acid (MES) acidic buffer and evaluated the effect of buffer pH on the generation of these features. Surface characterization with atomic force microscopy and scanning electron microscopy indicated the generation of aligned, sub-micron sized grooves on microthreads in MES buffer with pH 5.0. Microthreads etched with surface features had tensile mechanical properties comparable to controls, indicating that the surface treatment does not inhibit scaffold bulk properties. Our data demonstrate that etching threads in MES buffer with pH 5.0 enhanced alignment and filamentous actin stress fiber organization of myoblasts on the surface of scaffolds. The ability to tune topographic features on the surfaces of scaffolds independent of mechanical properties provides a valuable tool for designing microthread-based scaffolds to enhance regeneration of functional muscle tissue.

Collagen and bone morphogenetic protein-2 functionalized hydroxyapatite scaffolds induce osteogenic differentiation in human adipose-derived stem cells.

Surface modification is one important way to fabricate successful biocompatible materials in bone tissue engineering. Hydroxyapatite (HAp) materials have received considerable attention as suitable bioceramics for manufacturing osseous implants because of their similarity to bone mineral in terms of chemical composition. In this study, the surface of porous HAp scaffold was modified by collagen treatment and bone morphogenetic protein-2 (BMP-2) conjugation. The surface modification did not affect the HAp scaffold's bulk properties. No significant difference in compressive strength was found among different scaffolds, with HAp, collagen modified HAp, and collagen-BMP-2-functionalized HAp having compressive strengths of 45.8 ± 3.12, 51.2 ± 4.09, and 50.7 ± 3.98 MPa, respectively. In vitro studies were performed to compare adhesion and osteogenic differentiation between human adipose-derived stem cells (hADSCs) with modified surfaces and those unmodified HAp surfaces. Collagen or BMP-2 alone was insufficient and that both collagen and BMP-2 are necessary to get the desired results. The findings suggest the possibility of using three-dimensional HAp scaffold treated with gold-standard collagen coating and highly researched BMP-2 growth factor as a platform to deliver hADSCs. Results of this study could be used to develop treatment strategy for regenerating completely transected models using more synergistic approaches.

Argon plasma improves the tissue integration and angiogenesis of subcutaneous implants by modifying surface chemistry and topography.

BackgroundTissue integration and vessel formation are important criteria for the successful implantation of synthetic biomaterials for subcutaneous implantation.ObjectiveWe report the optimization of plasma surface modification (PSM) using argon (Ar), oxygen (O2) and nitrogen (N2) gases of a polyurethane polymer to enhance tissue integration and angiogenesis.MethodsThe scaffold’s bulk and surface characteristics were compared before and after PSM with either Ar, O2 and N2. The viability and adhesion of human dermal fibroblasts (HDFs) on the modified scaffolds were compared. The formation of extracellular matrix by the HDFs on the modified scaffolds was evaluated. Scaffolds were subcutaneously implanted in a mouse model for 3 months to analyze tissue integration, angiogenesis and capsule formation.ResultsSurface analysis demonstrated that interfacial modification (chemistry, topography and wettability) achieved by PSM is unique and varies according to the gas used. O2 plasma led to extensive changes in interfacial properties, whereas Ar treatment caused moderate changes. N2 plasma caused the least effect on surface chemistry of the polymer. PSM-treated scaffolds significantly (P<0.05) enhanced HDF activity and growth over 21 days. Among all three gases, Ar modification showed the highest protein adsorption. Ar-modified scaffolds also showed a significant upregulation of adhesion-related proteins (vinculin, focal adhesion kinase, talin and paxillin; P<0.05) and extracellular matrix marker genes (collagen type I, fibronectin, laminin and elastin) and deposition of associated proteins by the HDFs. Subcutaneous implantation after 3 months demonstrated the highest tissue integration and angiogenesis and the lowest capsule formation on Ar-modified scaffolds compared with O2- and N2-modified scaffolds.ConclusionPSM using Ar is a cost-effective and efficient method to improve the tissue integration and angiogenesis of subcutaneous implants.

Electrospun vein grafts with high cell infiltration for vascular tissue engineering

Demand is increasing for functional small-diameter vascular grafts (diameter<6mm) for clinical arterial replacement. In the present study, we develop a bilayer poly(ε-caprolactone, PCL) fibrous vascular graft consisting of a thin internal layer made of longitudinally aligned fibers and a relatively thick highly porous external layer. The internal layer provides a scaffold with the necessary mechanical strength and enhances the growth of endothelial cells, whereas the external layer enhances cell motility through the scaffold bulk. The biocompatibility and biological performance of bilayer fibrous scaffolds are evaluated by in vivo experiments, molecular biology, and histology studies. Our bilayer scaffolds demonstrate much better fiber alignment and higher porosity than do normal electrospun vascular grafts with randomly distributed fibers. The results suggest that the proposed grafts can overcome limitations owing to the inadequate porosity, small pores, and poor cell infiltration of scaffolds fabricated by conventional electrospinning. The unique structure of bilayer scaffolds is satisfactory and promotes cell proliferation, collagen–fiber deposition, and ingrowth of smooth muscle cells and endothelial cells in vivo. The results of this study illustrate the strong potential of such bilayer fibrous scaffolds for vascular tissue engineering and regeneration.

Poly-l-lactic acid: Pellets to fiber to fused filament fabricated scaffolds, and scaffold weight loss study

Poly-l-lactic acid (PLLA) is a bioresorbable polymer used in a variety of biomedical applications. Many 3D printers employ the fused filament fabrication (FFF) approach with the ubiquitous low-cost poly-lactic acid (PLA) fiber. However, use of the FFF approach to fabricate scaffolds with medical grade PLLA polymer remains largely unexplored. In this study, high molecular weight PL-32 pellets were extruded into ∼1.7mm diameter PLLA fiber. Melt rheometric data of the PLLA polymer was analyzed and demonstrated pseudo-plastic behavior with a flow index of n=0.465 (<1). Differential scanning calorimetry (DSC) was conducted using samples from the extruded fiber to obtain thermal properties. DSC of the 3D printed struts was also analyzed to assess changes in thermal properties due to FFF. The DSC and rheometric analysis results were subsequently used to define appropriate FFF process parameters. Constant porosity scaffolds were FFF 3D printed with 4 distinct laydown patterns; 0/90° rectilinear (control), 45/135° rectilinear, Archimedean chords, and honeycomb using the in-house developed custom multi-modality 3D bioprinter (CMMB). The effect of laydown pattern on scaffold bulk erosion (weight loss) was studied by immersion in phosphate-buffered saline (PBS) over a 6-month period and measured monthly. A repeated measures analysis of variance (ANOVA) was performed to identify statistically significant differences between mean percent weight loss of the four laydown patterns at each time point (1–6 months). The resulting data follows distinct temporal trends, but no statistically significant differences between means at individual time points were found. Cross-sectional scanning electron microscope (SEM) images of the 6-month degraded scaffolds showed noticeable structural deterioration. The study demonstrates successful processing of PLLA fiber from PL-32 pellets and FFF-based 3D printing of bioresorbable scaffolds with pre-defined laydown patterns using medical grade PLLA polymer which could prove beneficial in biomedical applications.

Arrayed Hollow Channels in Silk-based Scaffolds Provide Functional Outcomes for Engineering Critically-sized Tissue Constructs.

In the field of regenerative medicine there is a need for scaffolds that support large, critically-sized tissue formation. Major limitations in reaching this goal are the delivery of oxygen and nutrients throughout the bulk of the engineered tissue as well as host tissue integration and vascularization upon implantation. To address these limitations we previously reported the development of a porous scaffold platform made from biodegradable silk protein that contains an array of vascular-like structures that extend through the bulk of the scaffold. Here we report that the hollow channels play a pivotal role in enhancing cell infiltration, delivering oxygen and nutrients to the scaffold bulk, and promoting in vivo host tissue integration and vascularization. The unique features of this protein biomaterial system, including the vascular structures and tunable material properties, render this scaffold a robust and versatile tool for implementation in a variety of tissue engineering, regenerative medicine and disease modeling applications.

Engineered Hypopharynx from Coculture of Epithelial Cells and Fibroblasts Using Poly(ester urethane) as Substratum

Porous polymeric scaffolds have been much investigated and applied in the field of tissue engineering research. Poly(ester urethane) (PEU) scaffolds, comprising pores of 1–20 μm in diameter on one surface and ≥200 μm on the opposite surface and in bulk, were fabricated using phase separation method for hypopharyngeal tissue engineering. The scaffolds were grafted with silk fibroin (SF) generated from natural silkworm cocoon to enhance the scaffold's hydrophilicity and further improve cytocompatibility to both primary epithelial cells (ECs) and fibroblasts of human hypopharynx tissue. Coculture of ECs and fibroblasts was conducted on the SF-grafted PEU scaffold (PEU-SF) to evaluate its in vitro cytocompatibility. After co-culture for 14 days, ECs were lined on the scaffold surface while fibroblasts were distributed in scaffold bulk. The results of in vivo investigation showed that PEU porous scaffold possessed good biocompatibility after it was grafted by silk fibroin. SF grafting improved the cell/tissue infiltration into scaffold bulk. Thus, PEU-SF porous scaffold is expected to be a good candidate to support the hypopharynx regeneration.

A silk-based scaffold platform with tunable architecture for engineering critically-sized tissue constructs

In the field of tissue engineering and regenerative medicine there is significant unmet need for critically-sized, fully degradable biomaterial scaffold systems with tunable properties for optimizing tissue formation in vitro and tissue regeneration in vivo. To address this need, we have developed a silk-based scaffold platform that has tunable material properties, including localized and bioactive functionalization, degradation rate, and mechanical properties and that provides arrays of linear hollow channels for delivery of oxygen and nutrients throughout the scaffold bulk. The scaffolds can be assembled with dimensions that range from millimeters to centimeters, addressing the need for a critically-sized platform for tissue formation. We demonstrate that the hollow channel arrays support localized and confluent endothelialization. This new platform offers a unique and versatile tool for engineering ‘tailored’ scaffolds for a range of tissue engineering and regenerative medicine needs.

Modulation of gene expression using electrospun scaffolds with templated architecture

The fabrication of biomimetic scaffolds is a critical component to fulfill the promise of functional tissue-engineered materials. We describe herein a simple technique, based on printed circuit board manufacturing, to produce novel templates for electrospinning scaffolds for tissue-engineering applications. This technique facilitates fabrication of electrospun scaffolds with templated architecture, which we defined as a scaffold's bulk mechanical properties being driven by its fiber architecture. Electrospun scaffolds with templated architectures were characterized with regard to fiber alignment and mechanical properties. Fast Fourier transform analysis revealed a high degree of fiber alignment along the conducting traces of the templates. Mechanical testing showed that scaffolds demonstrated tunable mechanical properties as a function of templated architecture. Fibroblast-seeded scaffolds were subjected to a peak strain of 3 or 10% at 0.5 Hz for 1 h. Exposing seeded scaffolds to the low strain magnitude (3%) significantly increased collagen I gene expression compared to the high strain magnitude (10%) in a scaffold architecture-dependent manner. These experiments indicate that scaffolds with templated architectures can be produced, and modulation of gene expression is possible with templated architectures. This technology holds promise for the long-term goal of creating tissue-engineered replacements with the biomechanical and biochemical make-up of native tissues.

Numerical Fluid-Dynamic Optimization of Microchannel-Provided Porous Scaffolds for the Co-Culture of Adherent and Non-Adherent Cells

Computational fluid dynamic (CFD) techniques were used to optimize the microenvironment inside scaffolds for hematopoietic stem cell (HSC) culture in a perfusion bioreactor. These matrices are meant to be seeded with adherent bone marrow stromal cells and then co-cultivated with HSCs; the scaffold micro-architecture and the fluid-dynamic conditions have to be optimized to avoid non-adherent stem cells being dragged away while ensuring adequate nutrient supply. The insertion of longitudinal microchannels was tested as a tool to improve perfusion in a homogeneous porous scaffold. Models of microchannel-provided scaffolds, characterized by different values of geometric parameters concerning pores and channels, were built, and numerical fluid-dynamic and oxygen-transfer analyses were carried out. The results of the computations indicated that the microchannels created preferential paths for culture medium flow, causing low shear stresses and drag forces within the pores; meanwhile, they improved oxygen delivery by forcing its penetration into the scaffold bulk. In particular, an 85% porous, 3-mm-thick scaffold with 175-microm-diameter pores was considered; at a constant average drag force guaranteeing stem cell suspension inside this porous bulk, the addition of approximately 260-microm-diameter, 700-microm-spaced channels resulted in 34% higher oxygen partial pressure at the exit (approximately 135 vs 101 mmHg), maintaining a wall shear stress median value of approximately 0.14 mPa. The present work demonstrates the capacity of microchannel-provided scaffolds to ensure suitable conditions for HSC culture and shows that CFD methods are a valuable tool to retrieve significant clues for the design of the culture environment.