Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering

Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target; randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3m/s. Non-aligned scaffolds had an isotropic tensile modulus of 2.1+/-0.4MPa, compared to highly anisotropic scaffolds whose modulus was 11.6+/-3.1MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young's modulus in the fiber direction compared to perpendicular to the fiber direction when the rotation speed reached 8m/s. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.     

A structural model for the flexural mechanics of nonwoven tissue engineering scaffolds

The development of methods to predict the strength and stiffness of biomaterials used in tissue engineering is critical for load-bearing applications in which the essential functional requirements are primarily mechanical. We previously quantified changes in the effective stiffness (E) of needled nonwoven polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) scaffolds due to tissue formation and scaffold degradation under three-point bending. Toward predicting these changes, we present a structural model for E of a needled nonwoven scaffold in flexure. The model accounted for the number and orientation of fibers within a representative volume element of the scaffold demarcated by the needling process. The spring-like effective stiffness of the curved fibers was calculated using the sinusoidal fiber shapes. Structural and mechanical properties of PGA and PLLA fibers and PGA, PLLA, and 50:50 PGA/PLLA scaffolds were measured and compared with model predictions. To verify the general predictive capability, the predicted dependence of E on fiber diameter was compared with experimental measurements. Needled nonwoven scaffolds were found to exhibit distinct preferred (PD) and cross-preferred (XD) fiber directions, with an E ratio (PD/XD) of approximately 3:1. The good agreement between the predicted and experimental dependence of E on fiber diameter (R2 = 0.987) suggests that the structural model can be used to design scaffolds with E values more similar to native soft tissues. A comparison with previous results for cell-seeded scaffolds (Engelmayr, G. C., Jr., et al., 2005, Biomaterials, 26(2), pp. 175-187) suggests, for the first time, that the primary mechanical effect of collagen deposition is an increase in the number of fiber-fiber bond points yielding effectively stiffer scaffold fibers. This finding indicated that the effects of tissue deposition on needled nonwoven scaffold mechanics do not follow a rule-of-mixtures behavior. These important results underscore the need for structural approaches in modeling the effects of engineered tissue formation on nonwoven scaffolds, and their potential utility in scaffold design.     

Nanofibers and their applications in tissue engineering

Developing scaffolds that mimic the architecture of tissue at the nanoscale is one of the major challenges in the field of tissue engineering. The development of nanofibers has greatly enhanced the scope for fabricating scaffolds that can potentially meet this challenge. Currently, there are three techniques available for the synthesis of nanofibers: electrospinning, self-assembly, and phase separation. Of these techniques, electrospinning is the most widely studied technique and has also demonstrated the most promising results in terms of tissue engineering applications. The availability of a wide range of natural and synthetic biomaterials has broadened the scope for development of nanofibrous scaffolds, especially using the electrospinning technique. The three dimensional synthetic biodegradable scaffolds designed using nanofibers serve as an excellent framework for cell adhesion, proliferation, and differentiation. Therefore, nanofibers, irrespective of their method of synthesis, have been used as scaffolds for musculoskeletal tissue engineering (including bone, cartilage, ligament, and skeletal muscle), skin tissue engineering, vascular tissue engineering, neural tissue engineering, and as carriers for the controlled delivery of drugs, proteins, and DNA. This review summarizes the currently available techniques for nanofiber synthesis and discusses the use of nanofibers in tissue engineering and drug delivery applications.     

Functional 3‐D cardiac co‐culture model using bioactive chitosan nanofiber scaffolds

The in vitro generation of a three‐dimensional (3‐D) myocardial tissue‐like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long‐term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3‐D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long‐term cardiac function in the 3‐D co‐culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non‐toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono‐ and co‐cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha‐sarcomeric actin (SM‐actin) and gap junction protein, Connexin‐43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long‐term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono‐cultures resulted in loss of cardiomyocyte polarity and islands of non‐coherent contractions. However, the cardiomyocyte‐fibroblast co‐cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long‐term culture. The Cx43 expression in the fibroblast co‐culture was higher than the cardiomyocytes mono‐culture and endothelial cells co‐culture. In addition, fibroblast co‐cultures demonstrated synchronized contractions involving large tissue‐like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3‐D cardiac co‐culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3‐D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications. Biotechnol. Bioeng. 2013; 110: 637–647. © 2012 Wiley Periodicals, Inc.     

The influence of fibrous elastomer structure and porosity on matrix organization

Fibrous scaffolds are finding wide use in the field of tissue engineering, as they can be designed to mimic many native tissue properties and structures (e.g., cardiac tissue, meniscus). The influence of fiber alignment and scaffold architecture on cellular interactions and matrix organization was the focus of this study. Three scaffolds were fabricated from the photocrosslinkable elastomer poly(glycerol sebacate) (PGS), with changes in fiber alignment (non-aligned (NA) versus aligned (AL)) and the introduction of a PEO sacrificial polymer population to the AL scaffold (composite (CO)). PEO removal led to an increase in scaffold porosity and maintenance of scaffold anisotropy, as evident through visualization, mechanical testing, and mass loss studies. Hydrated scaffolds possessed moduli that ranged between ～3-240 kPa, failing within the range of properties (<300 kPa) appropriate for soft tissue engineering. CO scaffolds were completely degraded as early as 16 days, whereas NA and AL scaffolds had ～90% mass loss after 21 days when monitored in vitro. Neonatal cardiomyocytes, used as a representative cell type, that were seeded onto the scaffolds maintained their viability and aligned along the surface of the AL and CO fibers. When implanted subcutaneously in rats, a model that is commonly used to investigate in vivo tissue responses to biomaterials, CO scaffolds were completely integrated at 2 weeks, whereas ～13% and ～16% of the NA and AL scaffolds, respectively remained acellular. However, all scaffolds were completely populated with cells at 4 weeks post-implantation. Polarized light microscopy was used to evaluate the collagen elaboration and orientation within the scaffold. An increase in the amount of collagen was observed for CO scaffolds and enhanced alignment of the nascent collagen was observed for AL and CO scaffolds compared to NA scaffolds. Thus, these results indicate that the scaffold architecture and porosity are important considerations in controlling tissue formation.     

Formation of collagen-glycosaminoglycan blended nanofibrous scaffolds and their biological properties

The development of blended collagen and glycosaminoglycan (GAG) scaffolds can potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native extracellular matrix (ECM). In this study, we were able to obtain novel nanofibrous collagen-GAG scaffolds by electrospinning collagen blended with chondroitin sulfate (CS), a widely used GAG, in a mixed solvent of trifluoroethanol and water. The electrospun collagen-GAG scaffold with 4% CS (COLL-CS-04) exhibited a uniform fiber structure with nanoscale diameters. A second collagen-GAG scaffold with 10% CS consisted of smaller diameter fibers but exhibited a broader diameter distribution due to the different solution properties in comparison with COLL-CS-04. After cross-linking with glutaraldehyde vapor, the collagen-GAG scaffolds became more biostable and were resistant to collagenase degradation. This is evidently a more favorable environment allowing increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without cross-linking did not increase the biostability but still promoted cell growth. The potential of applying the nanoscale collagen-GAG scaffold in tissue engineering is significant since the nanodimension fibers made of natural ECM mimic closely the native ECM found in the human body. The high surface area characteristic of this scaffold may maximize cell-ECM interaction and promote tissue regeneration faster than other conventional scaffolds.     

Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration

The physical and spatial architectural geometries of electrospun scaffolds are important to their application in tissue engineering strategies. In this work, poly(epsilon-caprolactone) microfiber scaffolds with average fiber diameters ranging from 2 to 10 microm were individually electrospun to determine the parameters required for reproducibly fabricating scaffolds. As fiber diameter increased, the average pore size of the scaffolds, as measured by mercury porosimetry, increased (values ranging from 20 to 45 microm), while a constant porosity was observed. To capitalize on both the larger pore sizes of the microfiber layers and the nanoscale dimensions of the nanofiber layers, layered scaffolds were fabricated by sequential electrospinning. These scaffolds consisted of alternating layers of poly(epsilon-caprolactone) microfibers and poly(epsilon-caprolactone) nanofibers. By electrospinning the nanofiber layers for different lengths of time, the thickness of the nanofiber layers could be modulated. Bilayered constructs consisting of microfiber scaffolds with varying thicknesses of nanofibers on top were generated and evaluated for their potential to affect rat marrow stromal cell attachment, spreading, and infiltration. Cell attachment after 24 h did not increase with increasing number of nanofibers, but the presence of nanofibers enhanced cell spreading as evidenced by stronger F-actin staining. Additionally, increasing the thickness of the nanofiber layer reduced the infiltration of cells into the scaffolds under both static and flow perfusion culture for the specific conditions tested. The scaffold design presented in this study allows for cellular infiltration into the scaffolds while at the same time providing nanofibers as a physical mimicry of extracellular matrix.     

Fabrication,Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm⁻¹ can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.     

Bacterial cellulose-based materials and medical devices: current state and perspectives

Bacterial cellulose (BC) is a unique and promising material for use as implants and scaffolds in tissue engineering. It is composed of a pure cellulose nanofiber mesh spun by bacteria. It is remarkable for its strength and its ability to be engineered structurally and chemically at nano-, micro-, and macroscales. Its high water content and purity make the material biocompatible for multiple medical applications. Its biocompatibility, mechanical strength, chemical and morphologic controllability make it a natural choice for use in the body in biomedical devices with broader application than has yet been utilized. This paper reviews the current state of understanding of bacterial cellulose, known methods for controlling its physical and chemical structure (e.g., porosity, fiber alignment, etc.), biomedical applications for which it is currently being used, or investigated for use, challenges yet to be overcome, and future possibilities for BC.     

Release profiles of tricalcium phosphate nanoparticles from poly(L-lactic acid) electrospun scaffolds with single component, core-sheath, or porous fiber morphologies: effects on hASC viability and osteogenic differentiation

Functional PLA scaffolds are created with single component, core-sheath, or porous fiber morphology and doped with TCP nanoparticles to study the release profiles for use in bone tissue engineering applications. Pharmacokinetic analyses are performed for the three different nanofibrous structures after doping with TCP. Results indicate that single component and porous fiber scaffolds exhibit an initial-burst release profile whereas core-sheath fibers show a steady release. All scaffolds are then seeded with human adipose-derived stem cells (hASC), which remain viable and continue proliferation on all nanofibrous morphologies for up to 21 d. Osteogenic differentiation of hASC and cell-mediated calcium accretion are largest on porous fibers.     

A combination of bioactive and nonbioactive alkyl-peptides form a more stable nanofiber structure for differentiating neural stem cells: a molecular dynamics simulation survey

Self-assembling alkyl-peptides are important molecules due to their ability to construct nano-level structures such as nanofibers to be utilized as tissue engineering scaffolds. The bioactive epitope of FAQRVPP which acts as neural stem cells (NSCs) outgrowth inducing factor is used in nanofiber structures. Based on previous experimental studies the density and distribution pattern of the epitopes on the surface of the nanofibers plays an important role in the differentiation function efficiency. We decided to survey and compare the stability of two pre-constructed fiber structures in the forms of all-functionalized nanofiber (containing only bioactive alkyl-peptides) and distributed functionalized nanofiber (a combination of nonbioactive and bioactive alkyl-peptides with ratio 2:1). Our findings reveal that the all-functionalized fiber shows an unstable structure and is split into intermediate micelle-like structures to reduce compactness and steric hindrance of functional epitopes whereas the distributed functionalized fiber shows an integrated stable nanofiber with a more amount of beta sheets that are well-organized and oriented around the hydrophobic core. The hydrogen bonds and energy profiles of the structures indicate the role of hydrophobic interactions during the alkyl-chain core formation and the important role of electrostatic interactions and hydrogen bond network in the stability of the final structures. Finally, it seems that the possibility of the presence of intermediate structure is increased in the all-functionalized nanofiber environment, and it can reduce functional efficiency of the scaffolds. These findings can help to design more efficient nanofiber structures with different goals in scaffolds for tissue engineering. Abbreviations MD Molecular Dynamics

NSCs Neural Stem Cells

PME Particle mesh Ewald

RDF Radial Distribution Function

RG Radius of gyration

RASA Relative Accessible Surface Area

RMSD Root Mean Square Deviations

SASA Solvent Accessible Surface Area.

Communicated by Ramaswamy H. Sarma     

Electrospinning of collagen nanofibers

Electrospinning is a fabrication process that uses an electric field to control the deposition of polymer fibers onto a target substrate. This electrostatic processing strategy can be used to fabricate fibrous polymer mats composed of fiber diameters ranging from several microns down to 100 nm or less. In this study, we describe how electrospinning can be adapted to produce tissue-engineering scaffolds composed of collagen nanofibers. Optimizing conditions for calfskin type I collagen produced a matrix composed of 100 nm fibers that exhibited the 67 nm banding pattern that is characteristic of native collagen. The structural properties of electrospun collagen varied with the tissue of origin (type I from skin vs type I from placenta), the isotype (type I vs type III), and the concentration of the collagen solution used to spin the fibers. Electrospinning is a rapid and efficient process that can be used to selectively deposit polymers in a random fashion or along a predetermined and defined axis. Toward that end, our experiments demonstrate that it is possible to tailor subtle mechanical properties into a matrix by controlling fiber orientation. The inherent properties of the electrospinning process make it possible to fabricate complex, and seamless, three-dimensional shapes. Electrospun collagen promotes cell growth and the penetration of cells into the engineered matrix. The structural, material, and biological properties of electrospun collagen suggest that this material may represent a nearly ideal tissue engineering scaffold.     

Three-dimensional cancer cell culture in high-yield multiscale scaffolds by shear spinning

Polymeric scaffolds comprising two size scales of microfibers and submicron fibers can better support three-dimensional (3D) cell growth in tissue engineering, making them an important class of healthcare material. However, a major manufacturing barrier hampers their translation into wider practical use: scalability. Traditional production of two-scale scaffolds by electrospinning is slow and costly. For day-to-day cell cultures, the scaffolds need to be affordable, made in high yield to drive down cost. Combining expertise from academia and industry from the United Kingdom and United States, this study uses a new series of high-yield, low-cost scaffolds made by shear spinning for tissue engineering. The scaffolds comprise interwoven submicron fibers and microfibers throughout as observed under scanning electron microscopy and demonstrate good capability to support cell culturing for tumor modeling. Three model human cancer cell lines (HEK293, A549 and MCF-7) with stable expression of GFP were cultured in the scaffolds and found to exhibit efficient cell attachment and sustained 3D growth and proliferation for 30 days. Cryosection and multiphoton fluorescence microscopy confirmed the formation of compact 3D cell clusters throughout the scaffolds. In addition, comparative growth curves of 2D and 3D cultures show significant cell-type-dependent differences. This work applies high-yield shear-spun scaffolds in mammalian tissue engineering and brings practical, affordable applications of multiscale scaffolds closer to reality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2750, 2019.     

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.     

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.     

Patterning of polymer nanofiber meshes by electrospinning for biomedical applications

The end-product of the electrospinning process is typically a randomly aligned fiber mesh or membrane. This is a result of the electric field generated between the drop of polymer solution at the needle and the collector. The developed electric field causes the stretching of the fibers and their random deposition. By judicious selection of the collector architecture, it is thus possible to develop other morphologies on the nanofiber meshes. The aim of this work is to prepare fiber meshes using various patterned collectors with specific dimensions and designs and to evaluate how those patterns can affect the properties of the meshes relevant to biomedical applications. This study aims at verifying whether it is possible to control the architecture of the fiber meshes by tailoring the geometry of the collector. Three different metallic collector topographies are used to test this hypothesis. Electrospun nonwoven patterned meshes of polyethylene oxide (PEO) and poly( -caprolactone) (PCL) were successfully prepared. Those fiber meshes were analyzed by scanning electron microscopy (SEM). Both mechanical properties of the meshes and cell contacting experiments were performed to test the effect of the produced patterns over the properties of the meshes relevant for biomedical applications. The present study will evaluate cell adhesion sensitivity to the patterns generated and the effect of those patterns on the tensile properties of the fiber meshes.     

A novel electrospinning target to improve the yield of uniaxially aligned fibers

Electrospinning is a useful technique that can generate micro and nanometer‐sized fibers. Modification of the electrospinning parameters, such as deposition target geometry, can generate uniaxially aligned fibers for use in diverse applications ranging from tissue engineering to material fabrication. For example, meshes of fibers have been shown to mimic the extracellular matrix networks for use in smooth muscle cell proliferation. Further, aligned fibers can guide neurites to grow along the direction of the fibers. Here we present a novel electrospinning deposition target that combines the benefits of two previously reported electrodes: the standard parallel electrodes and the spinning wheel with a sharpened edge. This new target design significantly improves aligned fiber yield. Specifically, the target consists of two parallel aluminum plates with sharpened edges containing a bifurcating angle of 26°. Electric field computations show a larger probable area of aligned electric field vectors. This new deposition target allows fibers to deposit on a larger cross‐sectional area relative to the existing parallel electrode and at least doubles the yield of uniaxially aligned fibers. Further, fiber alignment and morphology are preserved after collection from the deposition target. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Scanning probe recognition microscopy investigation of tissue scaffold properties

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis.     

Controlled microchannelling in dense collagen scaffolds by soluble phosphate glass fibers

A problem with tissue engineering scaffolds is maintaining seeded cell viability and function due to limitations of oxygen and nutrient transfer. An approach to maintain suitable oxygen concentrations throughout the scaffold would be to controllably incorporate microchannelling within these scaffolds. This study investigated the incorporation of unidirectionally aligned soluble phosphate based glass fibers (PGF) into dense collagen scaffolds. PGF are degradable, and their degradation can be controlled through their chemistry and dimensions. Plastic compression was used to produce composite scaffolds at three different weight percentage while maintaining greater than 80% resident cell viability. PGF-collagen scaffold composition was quantified through thermogravimetric analysis as well as being morphologically and mechanically characterized. PGF degradation was measured through ion chromatography, and channel formation was verified with ultrasound imaging and SEM. The free movement of coated microbubble agents confirmed the channels to be continuous in nature and of 30-40 microm diameter. These microchannels in dense native collagen matrices could play an important role in hypoxia/perfusion limitations and also in the transportation of nutrients and potentially forming blood vessels through dense implants.     

Modeling interlamellar interactions in angle-ply biologic laminates for annulus fibrosus tissue engineering

Mechanical function of the annulus fibrosus of the intervertebral disc is dictated by the composition and microstructure of its highly ordered extracellular matrix. Recent work on engineered angle-ply laminates formed from mesenchymal stem cell (MSC)-seeded nanofibrous scaffolds indicates that the organization of collagen fibers into planes of alternating alignment may play an important role in annulus fibrosus tissue function. Specifically, these engineered tissues can resist tensile deformation through shearing of the interlamellar matrix as layers of collagen differentially reorient under load. In the present work, a hyperelastic constitutive model was developed to describe the role of interlamellar shearing in reinforcing the tensile response of biologic laminates, and was applied to experimental results from engineered annulus constructs formed from MSC-seeded nanofibrous scaffolds. By applying the constitutive model to uniaxial tensile stress–strain data for bilayers with three different fiber orientations, material parameters were generated that characterize the contributions of extrafibrillar matrix, fibers, and interlamellar shearing interactions. By 10 weeks of in vitro culture, interlamellar shearing accounted for nearly 50% of the total stress associated with uniaxial extension in the anatomic range of ply angle. The model successfully captured changes in function with extracellular matrix deposition through variations in the magnitude of model parameters with culture duration. This work illustrates the value of engineered tissues as tools to further our understanding of structure–function relations in native tissues and as a test-bed for the development of constitutive models to describe them.