Sounok Sen

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

Successful engineering of load-bearing tissues requires recapitulation of their complex mechanical functions. Given the intimate relationship between function and form, biomimetic materials that replicate anatomic form are of great interest for tissue engineering applications. However, for complex tissues such as the annulus fibrosus, scaffolds have failed to capture their multi-scale structural hierarchy. Consequently, engineered tissues have yet to reach functional equivalence with their native counterparts. Here we present a novel strategy for annulus fibrosus tissue engineering that replicates this hierarchy with anisotropic nanofibrous laminates seeded with mesenchymal stem cells. These scaffolds directed the deposition of organized, collagen-rich extracellular matrix that mimicked the angle-ply, multi-lamellar architecture and achieved mechanical parity with native tissue after 10 weeks of in vitro culture. Further, we identified a novel role for inter-lamellar shearing in reinforcing the tensile response of biologic laminates, a mechanism that has not previously been considered for these tissues.

Comparison of animal discs used in disc research to human lumbar disc: axial compression mechanics and glycosaminoglycan content.

Study Design. Experimental measurement and normalization of in vitro disc axial compression mechanics and glycosaminoglycan and water content for several animal species used in intervertebral disc research. Objective. To compare normalized axial mechanical properties and glycosaminoglycan and water content from other species to those of the human disc to aid in selection and interpretation of results in animal disc studies. Summary of Background Data. There is a lack of mechanical and biochemical comparative data from animal intervertebral discs with respect to the human disc. Methods. Intervertebral disc axial mechanical properties, glycosaminoglycan, and water content were evaluated for 9 disc types in 7 mammalian species: the calf, pig, baboon, sheep, rabbit, rat and mouse lumbar, and the cow and rat tail. Disc area and height were used for calculation of the normalized mechanical parameters. Glycosaminoglycan content was normalized by dry weight. Results. Many directly measured mechanical parameters varied by orders of magnitude. However, these parameters became comparable and often did not show significant differences after geometric normalization. Both glycosaminoglycan and water content revealed similarity across species. Conclusion. Disc axial mechanics are very similar across animal species when normalizing by the geometric parameters of disc height and area. This suggests that the disc tissue material properties are largely conserved across animal species. These results provide a reference to compare disc axial mechanics and glycosaminoglycan and water composition of experimental animal models to the human lumbar disc, to aid in both selection and interpretation of experimental disc research.

Engineered Disc-Like Angle-Ply Structures for Intervertebral Disc Replacement

Study Design. To develop a construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement. Objective. To engineer a disc-like angle-ply structure (DAPS) that replicates the multiscale architecture of the intervertebral disc. Summary of Background Data. Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multiscale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues. Methods. A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus (NP) and oriented electrospun nanofibrous scaffolds to form the anulus fibrosus region (AF). Bovine MSCs were seeded into both regions and biochemical, histologic, and mechanical maturation were evaluated with in vitro culture. Results. We show that mechanical testing in compression and torsion, loading methods commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into both AF and NP regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposit matrix that is organized in an angle-ply configuration. Finally, constructs demonstrate functional development with long-term in vitro culture. Conclusion. These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically relevant composite disc analogue for eventual disc replacement.

Dynamic culture enhances stem cell infiltration and modulates extracellular matrix production on aligned electrospun nanofibrous scaffolds.

Electrospun nanofibrous scaffolds have become widely investigated for tissue engineering applications, owing to their ability to replicate the scale and organization of many fiber-reinforced soft tissues such as the knee meniscus, the annulus fibrosus of the intervertebral disc, tendon, and cartilage. However, due to their small pore size and dense packing of fibers, cellular ingress into electrospun scaffolds is limited. Progress in the application of electrospun scaffolds has therefore been hampered, as limited cell infiltration results in heterogeneous deposition of extracellular matrix and mechanical properties that remain below native benchmarks. In the present study, dynamic culture conditions dramatically improved the infiltration of mesenchymal stem cells into aligned nanofibrous scaffolds. While dynamic culture resulted in a reduction of glycosaminoglycan content, removal from dynamic culture to free-swelling conditions after 6 weeks resulted recovery of glycosaminoglycan content. Dynamic culture significantly increased collagen content, and collagen was more uniformly distributed throughout the scaffold thickness. While mechanical function was assessed and tensile modulus increased with culture duration, dynamic culture did not result in any additional improvement beyond free-swelling culture. Transient dynamic (6 weeks dynamic followed by 6 weeks free-swelling) culture significantly enhanced cell infiltration while permitting GAG accumulation. In this study, we demonstrated that a simple modification to standard in vitro culture conditions effectively improves cellular ingress into electrospun scaffolds, resolving a challenge which has until now limited the utility of these materials for various tissue engineering applications.

An in vivo model of reduced nucleus pulposus glycosaminoglycan content in the rat lumbar intervertebral disc.

Study Design. An in vivo model resembling early stage disc degeneration in the rat lumbar spine. Objective. Simulate the reduced glycosaminoglycan content and altered mechanics observed in intervertebral disc degeneration using a controlled injection of chondroitinase ABC (ChABC). Summary of Background Data. Nucleus glycosaminoglycan reduction occurs early during disc degeneration; however, mechanisms through which degeneration progresses from this state are unknown. Animal models simulating this condition are essential for understanding disease progression and for development of therapies aimed at early intervention. Methods. ChABC was injected into the nucleus pulposus, and discs were evaluated via micro-CT, mechanical testing, biochemical assays, and histology 4 and 12 weeks after injection. Results. At 4 weeks, reductions in nucleus glycosaminoglycan level by 43%, average height by 12%, neutral zone modulus by 40%, and increases in range of motion by 40%, and creep strain by 25% were found. Neutral zone modulus and range of motion were correlated with nucleus glycosaminoglycan. At 12 weeks, recovery of some mechanical function was detected as range of motion and creep returned to control levels; however, this was not attributed to glycosaminoglycan restoration, because mechanics were no longer correlated with glycosaminoglycan. Conclusion. An in vivo model simulating physiologic levels of glycosaminoglycan loss was created to aid in understanding the relationships between altered biochemistry, altered mechanics, and altered cellular function in degeneration.

Reduced nucleus pulposus glycosaminoglycan content alters intervertebral disc dynamic viscoelastic mechanics.

The intervertebral disc functions over a range of dynamic loading regimes including axial loads applied across a spectrum of frequencies at varying compressive loads. Biochemical changes occurring in early degeneration, including reduced nucleus pulposus glycosaminoglycan content, may alter disc mechanical behavior and thus may contribute to the progression of degeneration. The objective of this study was to determine disc dynamic viscoelastic properties under several equilibrium loads and loading frequencies, and further, to determine how reduced nucleus glycosaminoglycan content alters dynamic mechanics. We hypothesized that (1) dynamic stiffness would be elevated with increasing equilibrium load and increasing frequency, (2) the disc would behave more elastically at higher frequencies, and finally, (3) dynamic stiffness would be reduced at low equilibrium loads under all frequencies due to nucleus glycosaminoglycan loss. We mechanically tested control and chondroitinase ABC injected rat lumbar motion segments at several equilibrium loads using oscillatory loading at frequencies ranging from 0.05 to 5Hz. The rat lumbar disc behaved non-linearly with higher dynamic stiffness at elevated compressive loads irrespective of frequency. Phase angle was not affected by equilibrium load, although it decreased as frequency was increased. Reduced glycosaminoglycan decreased dynamic stiffness at low loads but not at high equilibrium loads and led to increased phase angle at all loads and frequencies. The findings of this study demonstrate the effect of equilibrium load and loading frequencies on dynamic disc mechanics and indicate possible mechanical mechanisms through which disc degeneration can progress.

MULTI-LAMELLAR AND MULTI-AXIAL MATURATION OF CELL-SEEDED FIBER- REINFORCED TISSUE ENGINEERED CONSTRUCTS

The architecture of load-bearing fibrous tissues is optimized to enable a specific set of mechanical functions. This organization arises from a complex process of cell patterning, matrix deposition, and functional maturation [1]. In their mature state, these tissues span multiple length scales, encompassing nanoscale interactions of cells with extracellular matrix to the centimeter length scales of the anatomic tissue volume and shape. Two structures that typify dense fibrous tissues are the meniscus of the knee and the annulus fibrosus (AF) of the intervertebral disc (IVD). The mechanical function of the wedge-shaped knee meniscus is based on its stiff prevailing circumferential collagen architecture that resists tensile deformation [2,3]. Adding to its complexity, radial tie fibers and sheets are interwoven amongst these fibers, increasing stiffness in the transverse direction and binding the tissue together [4]. In the annulus fibrosus, multiple anisotropic lamellae are stacked in concentric rings with their prevailing fiber directions alternating above and below the horizontal axis in adjacent layers [5]. The high circumferential tensile properties of this laminate structure allow it to resist bulging of the nucleus pulposus with compressive loading of the spine. Given their structural properties, unique form, and demanding mechanical environments, the knee meniscus and the AF region of the IVD represent two of the most challenging tissues to consider for functional tissue engineering.Copyright

Effect of Degeneration on the Dynamic Viscoelastic Properties of Human Annulus Fibrosus in Tension

Physiological cyclic loading of the intervertebral disc generally occurs between 1 and 5 Hz [1]. However, most mechanical assessments of disc AF tissue have relied on uniaxial tensile tests under quasi-static conditions [2]. Furthermore, the few studies that have addressed AF viscoelasticity have reported only static viscoelastic properties such as creep or stress-relaxation [3]. As such, there exists almost no data characterizing the dynamic viscoelastic properties of AF tissue in tension. Such data would be critical for several applications: to elucidate the mechanical progression of intervertebral disc degeneration, to develop and validate structural finite element models, and to provide native tissue benchmarks for regenerative approaches that aim to restore mechanical function to diseased or degenerate tissue. Therefore, the objectives of this study are to: (1) quantify human AF tissue frequency-dependent and strain-dependent viscoelastic properties of human AF tissue in circumferential tension, and (2) determine the effects of disc degeneration on these properties.Copyright

Mesenchymal Stem Cell Seeded Nanofibrous Laminates Mimic the Multi-Scale 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.Copyright

Intervertebral Disc Function Is Restored in an In Vivo Model of Chronic Nucleus Pulposus Glycosaminoglycan Reduction

Reduced nucleus pulposus glycosaminoglycan (GAG) content is one of the earliest clinically detectable changes during the course of intervertebral disc degeneration [1,2]. Depletion of nucleus GAG by small percentages consistent with this early loss has been experimentally linked to altered motion segment mechanical function, and thus, potentially increases the risk of damage accumulation directly due to elevated stresses and strains and through altered cellular function [3]. Recently, our laboratory has established an in vivo model in a rat lumbar disc which moderately decreases nucleus GAG to levels observed in early human degeneration. In this model, GAG loss is accompanied by a state of hypermobility at both 4 and 12 weeks post treatment [4], potentially making the disc susceptible to mechanical failure. The objective of this study was to determine the long term effects of nucleus GAG depletion and to determine if altered discs demonstrate hallmark features of disc degeneration. We hypothesized that GAG will remain depleted 24 weeks post treatment, potentially decreasing to lower levels, and further that geometrical and mechanical changes consistent with degeneration will be observed.Copyright