Christopher L. Gilchrist

Expression of Laminin Isoforms, Receptors and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin gamma1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin alpha6 subunit, as compared to adjacent anulus fibrosus region. This result suggests that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors, and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e., integrins), and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc. Our goal was to reveal features of cellular environment and cell-matrix interactions in the immature NP. Results from both immunohistochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin alpha5 chain, laminin receptors (integrin alpha3, alpha6, beta4 subunit, and CD239), and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors, and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.

Extracellular Matrix Ligand and Stiffness Modulate Immature Nucleus Pulposus Cell-Cell Interactions

The nucleus pulposus (NP) of the intervertebral disc functions to provide compressive load support in the spine, and contains cells that play a critical role in the generation and maintenance of this tissue. The NP cell population undergoes significant morphological and phenotypic changes during maturation and aging, transitioning from large, vacuolated immature cells arranged in cell clusters to a sparse population of smaller, isolated chondrocyte-like cells. These morphological and organizational changes appear to correlate with the first signs of degenerative changes within the intervertebral disc. The extracellular matrix of the immature NP is a soft, gelatinous material containing multiple laminin isoforms, features that are unique to the NP relative to other regions of the disc and that change with aging and degeneration. Based on this knowledge, we hypothesized that a soft, laminin-rich extracellular matrix environment would promote NP cell-cell interactions and phenotypes similar to those found in immature NP tissues. NP cells were isolated from porcine intervertebral discs and cultured in matrix environments of varying mechanical stiffness that were functionalized with various matrix ligands; cellular responses to periods of culture were assessed using quantitative measures of cell organization and phenotype. Results show that soft (<720 Pa), laminin-containing extracellular matrix substrates promote NP cell morphologies, cell-cell interactions, and proteoglycan production in vitro, and that this behavior is dependent upon both extracellular matrix ligand and substrate mechanical properties. These findings indicate that NP cell organization and phenotype may be highly sensitive to their surrounding extracellular matrix environment.

Transfer of Macroscale Tissue Strain to Microscale Cell Regions in the Deformed Meniscus

Cells within fibrocartilaginous tissues, including chondrocytes and fibroblasts of the meniscus, ligament, and tendon, regulate cell biosynthesis in response to local mechanical stimuli. The processes by which an applied mechanical load is transferred through the extracellular matrix to the environment of a cell are not fully understood. To better understand the role of mechanics in controlling cell phenotype and biosynthetic activity, this study was conducted to measure strain at different length scales in tissue of the fibrocartilaginous meniscus of the knee joint, and to define a quantitative parameter that describes the strain transferred from the far-field tissue to a microenvironment surrounding a cell. Experiments were performed to apply a controlled uniaxial tensile deformation to explants of porcine meniscus containing live cells. Using texture correlation analyses of confocal microscopy images, two-dimensional Lagrangian and principal strains were measured at length scales representative of the tissue (macroscale) and microenvironment in the region of a cell (microscale) to yield a strain transfer ratio as a measure of median microscale to macroscale strain. The data demonstrate that principal strains at the microscale are coupled to and amplified from macroscale principal strains for a majority of cell microenvironments located across diverse microstructural regions, with average strain transfer ratios of 1.6 and 2.9 for the maximum and minimum principal strains, respectively. Lagrangian strain components calculated along the experimental axes of applied deformations exhibited considerable spatial heterogeneity and intersample variability, and suggest the existence of both strain amplification and attenuation. This feature is consistent with an in-plane rotation of the principal strain axes relative to the experimental axes at the microscale that may result from fiber sliding, fiber twisting, and fiber-matrix interactions that are believed to be important for regulating deformation in other fibrocartilaginous tissues. The findings for consistent amplification of macroscale to microscale principal strains suggest a coordinated pattern of strain transfer from applied deformation to the microscale environment of a cell that is largely independent of these microstructural features in the fibrocartilaginous meniscus.

Aligned multilayered electrospun scaffolds for rotator cuff tendon tissue engineering.

UNLABELLED The rotator cuff consists of several tendons and muscles that provide stability and force transmission in the shoulder joint. Whereas most rotator cuff tears are amenable to suture repair, the overall success rate of repair is low, and massive tears are prone to re-tear. Extracellular matrix (ECM) patches are used to augment suture repair, but they have limitations. Tissue-engineered approaches provide a promising solution for massive rotator cuff tears. Previous studies have shown that, compared to nonaligned scaffolds, aligned electrospun polymer scaffolds exhibit greater anisotropy and exert a greater tenogenic effect. Nevertheless, achieving rapid cell infiltration through the full thickness of the scaffold is challenging, and scaling to a translationally relevant size may be difficult. Our goal was to evaluate whether a novel method of alignment, combining a multilayered electrospinning technique with a hybrid of several electrospinning alignment techniques, would permit cell infiltration and collagen deposition through the thickness of poly(ε-caprolactone) scaffolds following seeding with human adipose-derived stem cells. Furthermore, we evaluated whether multilayered aligned scaffolds enhanced collagen alignment, tendon-related gene expression, and mechanical properties compared to multilayered nonaligned scaffolds. Both aligned and nonaligned multilayered scaffolds demonstrated cell infiltration and ECM deposition through the full thickness of the scaffold after only 28days of culture. Aligned scaffolds displayed significantly increased expression of tenomodulin compared to nonaligned scaffolds and exhibited aligned collagen fibrils throughout the full thickness, the presence of which may account for the increased yield stress and Youngs modulus of cell-seeded aligned scaffolds along the axis of fiber alignment. STATEMENT OF SIGNIFICANCE Rotator cuff tears are an important clinical problem in the shoulder, with over 300,000 surgical repairs performed annually. Re-tear rates may be high, and current methods used to augment surgical repair have limited evidence to support their clinical use due to inadequate initial mechanical properties and slow cellular infiltration. Tissue engineering approaches such as electrospinning have shown similar challenges in previous studies. In this study, a novel technique to align electrospun fibers while using a multilayered approach demonstrated increased mechanical properties and development of aligned collagen through the full thickness of the scaffolds compared to nonaligned multilayered scaffolds, and both types of scaffolds demonstrated rapid cell infiltration through the full thickness of the scaffold.

Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin‐mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM‐111 and LM‐511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM‐binding integrins was quantified for human NP cells using flow cytometry. The cell‐ECM attachment mechanism was determined by quantifying cell attachment to LM‐111, LM‐511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM‐111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell‐based therapeutics.

Universally Conserved Relationships between Nuclear Shape and Cytoplasmic Mechanical Properties in Human Stem Cells

The ability of cells to proliferate, differentiate, transduce extracellular signals and assemble tissues involves structural connections between nucleus and cytoskeleton. Yet, how the mechanics of these connections vary inside stem cells is not fully understood. To address those questions, we combined two-dimensional particle-tracking microrheology and morphological measures using variable reduction techniques to measure whether cytoplasmic mechanics allow for discrimination between different human adherent stem cell types and across different culture conditions. Here we show that nuclear shape is a quantifiable discriminant of mechanical properties in the perinuclear cytoskeleton (pnCSK) of various stem cell types. Also, we find the pnCSK is a region with different mechanical properties than elsewhere in the cytoskeleton, with heterogeneously distributed locations exhibiting subdiffusive features, and which obeys physical relations conserved among various stem cell types. Finally, we offer a prospective basis to discriminate between stem cell types by coupling perinuclear mechanical properties to nuclear shape.

Nano-Scale and Micro-Scale Substrate Architectures Direct Collagen Alignment in Tendon Neo-Tissue Formation

Biomaterial scaffolds that present defined microenvironmental cues (e.g., nano-topography) to cells have shown promise for a variety of tissue engineering applications. However, the specific cues best suited for promoting the formation of aligned, fibrous tissues such as tendon are not fully understood. In this study, we utilize a micro-photopatterning (μPP) model system to precisely arrange scaffold-mimicking microenvironmental cues and investigate their role in the formation of tendon-like neo-tissues. Our data show that scaffold architectural features at both nano- and micro-length scales may be important parameters for directing tendon-like cell organization and the formation of aligned, fibrillar collagen.Copyright

Cell Morphology and Migration of Nucleus Pulposus Cells Depends on Substrate Stiffness and Ligand

Changes in nucleus pulposus (NP) cell phenotype and morphology are implicated in the progression of intervertebral disc (IVD) disorders. Understanding how changes in the NP cell microenvironment influence cell behavior and function is important for revealing how pathology-related changes in IVD extracellular matrix may affect NP cell biology. In this study, live-cell imaging techniques were utilized to study changes in cell migration and morphology when cultured upon substrates of different matrix proteins and stiffnesses. Results indicate that soft substrates containing matrix proteins promote cell clustering and cell-cell interactions which mimic in vivo conditions of healthy NP cells.Copyright