Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus

The annulus fibrosus (AF) of the intervertebral disc is a multi-lamellar fibrocartilage that, together with the nucleus pulposus, confers mechanical support and flexibility to the spine. Function of the AF is predicated on a high degree of structural organization over multiple length scales: aligned collagen fibers reside within each lamella, and the direction of alignment alternates between adjacent lamellae from +30° to −30° with respect to the transverse axis of the spine. Electrospinning permits fabrication of scaffolds consisting of aligned arrays of nanofibers, and has proven effective for directing the alignment of both cells and extracellular matrix (ECM) deposition [1–3]. We recently employed electrospinning to engineer the primary functional unit of the AF, a single lamella [4]. However, it remains a challenge to engineer a multi-lamellar tissue that replicates the cross-ply fiber architecture of the native AF. Moreover, relatively few studies have considered functional properties of engineered AF, and, when measured, tensile properties of these constructs have been inferior to native AF [4]. In this study, mesenchymal stem cells (MSCs) were seeded onto aligned nanofibrous scaffolds organized into bi-lamellar constructs with opposing or parallel fiber orientations, and their functional maturation was evaluated with time. Additionally, we determined a novel role for inter-lamellar ECM in reinforcing the tensile response of bilayers, and confirmed this mechanism by testing acellular bilayers with controllable interface properties.

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Towards intervertebral disc engineering: Bio-mimetics of form and function of the annulus fibrosus lamellae

Journal of the Mechanical Behavior of Biomedical Materials ◽

10.1016/j.jmbbm.2019.03.023 ◽

2019 ◽

Vol 94 ◽

pp. 298-307 ◽

Cited By ~ 3

Author(s):

Mirit Sharabi ◽

Shir Wertheimer ◽

Kelly R. Wade ◽

Fabio Galbusera ◽

Dafna Benayahu ◽

...

Keyword(s):

Intervertebral Disc ◽

Annulus Fibrosus ◽

Form And Function ◽

And Function

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Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715912115 ◽

2017 ◽

Vol 115 (3) ◽

pp. 477-482 ◽

Cited By ~ 28

Author(s):

Bibhas K. Bhunia ◽

David L. Kaplan ◽

Biman B. Mandal

Keyword(s):

Intervertebral Disc ◽

Annulus Fibrosus ◽

Human Mesenchymal Stem Cells ◽

Collagen Type I ◽

Hierarchical Architecture ◽

Elastic Fibers ◽

Type I ◽

Form And Function ◽

Tissue Organization ◽

And Function

Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col Iα 1, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

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Modeling Inter-Lamellar Interactions in Angle-Ply Nanofibrous Biologic Laminates for Annulus Fibrosus Tissue Engineering

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19479 ◽

2010 ◽

Cited By ~ 1

Author(s):

Nandan L. Nerurkar ◽

Robert L. Mauck ◽

Dawn M. Elliott

Keyword(s):

Tissue Engineering ◽

Annulus Fibrosus ◽

Uniaxial Extension ◽

Direct Consequence ◽

Continuum Models ◽

Form And Function ◽

Multiple Length Scales ◽

And Function ◽

High Degree

Function of the annulus fibrosus (AF) of the intervertebral disc is predicated on a high degree of structural organization over multiple length scales. Recently, we have employed aligned electrospun scaffolds to engineer nanofibrous biologic laminates that replicate the form and function of the AF [1]. Further, we determined that interlamellar shearing — a direct consequence of the +/−30° angle-ply architecture — plays an important role in reinforcing the tensile response of these materials (Fig. 1). Although we have utilized fiber-reinforced continuum models to characterize the evolving mechanics of single-lamellar AF constructs with in vitro culture [2, 3], these models are not capable of capturing the interlamellar interactions observed in bi-lamellar constructs. Indeed, continuum models of the native AF typically do not account for the organization of fiber populations into discrete, alternating planes of alignment, and so these models, too, do not account for inter-lamellar shearing interactions [4–6]. Therefore, in the present work we propose a novel constitutive model for the reinforcing role of interlamellar shearing during uniaxial extension of angle-ply biologic laminates and employ this model to evaluate the functional evolution of bilayers for AF tissue engineering.

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Scanning tunneling microscopy (STM) of DNA and RNA

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100152148 ◽

1989 ◽

Vol 47 ◽

pp. 34-35

Author(s):

Patricia G. Arscott ◽

Gil Lee ◽

Victor A. Bloomfield ◽

D. Fennell Evans

Keyword(s):

Molecular Structures ◽

Inorganic Materials ◽

Resolving Power ◽

Scanning Tunneling ◽

Tunneling Microscopy ◽

Form And Function ◽

Dna And Rna ◽

Calf Thymus ◽

And Function ◽

The Relationship

STM is one of the most promising techniques available for visualizing the fine details of biomolecular structure. It has been used to map the surface topography of inorganic materials in atomic dimensions, and thus has the resolving power not only to determine the conformation of small molecules but to distinguish site-specific features within a molecule. That level of detail is of critical importance in understanding the relationship between form and function in biological systems. The size, shape, and accessibility of molecular structures can be determined much more accurately by STM than by electron microscopy since no staining, shadowing or labeling with heavy metals is required, and there is no exposure to damaging radiation by electrons. Crystallography and most other physical techniques do not give information about individual molecules.We have obtained striking images of DNA and RNA, using calf thymus DNA and two synthetic polynucleotides, poly(dG-me5dC)·poly(dG-me5dC) and poly(rA)·poly(rU).

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