Cynthia R. Lee

Effects of immobilization and dynamic compression on intervertebral disc cell gene expression in vivo

Study Design. An in vivo analysis of the intervertebral disc’s cellular response to dynamic compression and immobilization was performed using a rat-tail model. Objective. To assess the effects of immobilization and short-term dynamic compression on intervertebral disc cell expression of anabolic and catabolic genes. Summary of Background Data. Static compressive loads applied in vivo alter the composition of the disc matrix and cell viability in a dose-dependent manner. The effects of in vivo dynamic compression, which is a more physiologic load, and reported risk factor for low back pain have not been investigated. Methods. An Ilizarov-type device was implanted on the rat tail and used to determine the effects from 72 hours of immobilization (n = 6), 2 hours of dynamic compression (1 MPa/0.2 Hz) (n = 8), and the coupled effect of immobilization followed by compression (n = 8). Real-time reverse transcription-polymerase chain reaction was used to measure changes in anabolic and catabolic gene levels relative to both internal control subjects and a sham-operated group (n = 7). Results. Immobilization and dynamic compression affect anabolic and catabolic genes, with an overall downregulation of types 1 and 2 collagen and upregulation of aggrecanase, collagenase, and stromelysin in the anulus. The effects of immobilization and compression appear to be additive for collagen types 1 and 2 in the anulus, but not in the nucleus, and not for catabolic genes. Conclusions. Short-duration dynamic compression and immobilization alter gene expression in the rat disc. In studying the response of the disc to loading, it is necessary to look at both anabolic and catabolic pathways, and to consider strain history.

Articular Cartilage Chondrocytes in Type I and Type II Collagen-GAG Matrices Exhibit Contractile Behavior in Vitro

Natural healing of articular cartilage defects generally does not occur, and untreated lesions may predispose the joint to osteoarthritis. To promote healing of cartilage defects, many researchers are turning toward a tissue engineering approach involving cultured cells and/or porous, resorbable matrices. This study investigated the contractile behavior of cultured canine chondrocytes seeded in a porous collagen-glycosaminoglycan (GAG) scaffold. Chondrocytes isolated from the knee joints of adult canines and expanded in monolayer culture were seeded into porous collagen-GAG scaffolds. Scaffolds were of two different compositions, with the predominant collagen being either type I or type II collagen, and of varying pore diameters. Over the 4-week culture period, the seeded cells contracted all of the type I and type II collagen-based matrices, despite a wide range of stiffness (145 +/- 23 Pa, for the type I scaffold, to 732 +/- 35 Pa, for the type II material). Pore diameter (25-85 microm, type I; and 53-257 microm, type II) did not affect cell-mediated contraction. Immunohistochemical staining revealed the presence of alpha-smooth muscle actin, an isoform responsible for contraction of smooth muscle cells and myofibroblasts, in the cytoplasm of the seeded cells and in chondrocytes in normal adult canine articular cartilage.

In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.

Study Design. Whole bovine coccygeal discs were cultured under static load, with or without vertebral endplates (VEPs), and assessed for cell viability, biochemical stability, biosynthetic activity, and biosynthetic responsiveness to changes in mechanical load. Objectives. To assess the effects of VEPs on biochemical and cellular stability of disc cells during in vitro culture of large disc explants. To determine whether cultured discs could respond to mechanical perturbation. Summary of Background Data. Previous methods for culturing the intervertebral disc have focused on rabbit and rat discs, but the small size of these discs limits the relevance of these culture systems to the human condition. Bovine coccygeal discs have similar dimensions to the human lumbar disc (i.e., similar size and nominal stresses), but long-term culture of these discs has not been reported. Methods. Bovine coccygeal discs were harvested with or without VEPs, cultured under static load (5 kg, ∼0.25 MPa, in situ swelling pressure) for up to 1 week, and evaluated for changes in hydration, glycosaminoglycan content, cell viability, and biosynthetic activity. Additionally, the biochemical and biosynthetic response of discs cultured without VEP to increasing the load to a 20-kg (∼1 MPa, the estimated stress in human lumbar disc during heavy lifting) static load for 6 hours was assessed. Results. During the first 24 hours, culturing discs with endplates was moderately better with regards to maintaining in situ anulus hydration and nucleus glycosaminoglycan levels. The endplates, however, obstructed media flow to the disc, resulting in a marked decrease in cell viability after 1 week of culture. Nucleus pulposus cell viability was maintained in discs cultured without endplates, but there was a significant drop in biosynthetic activity within 2 days of culture. Despite this drop, the disc cells in the discs without VEP remained biosynthetically responsive to changes in mechanical loading. Conclusions. It is possible to maintain cell viability and the biosynthetic responsiveness of large discs for up to 1 week in vitro when the discs are cultured under static load and without VEP.

Outgrowth of chondrocytes from human articular cartilage explants and expression of alpha-smooth muscle actin.

The objectives of this study were to investigate the effect of various enzymatic treatments on the outgrowth of chondrocytes from explants of adult human articular cartilage and the expression of a specific contractile protein isoform, α‐smooth muscle actin, known to facilitate wound closure in other connective tissues. Explants of articular cartilage were prepared from specimens obtained from patients undergoing total joint arthroplasty. The time to cell outgrowth in vitro was determined and the expression of α‐smooth muscle actin shown by immunohistochemistry. Treatment of the explants with collagenase for 15 minutes reduced the time to outgrowth from more than 30 days to 3 days. Hyaluronidase, chondroitinase ABC, and trypsin applied for the 15‐minute period had no effect on the time to cell outgrowth when compared with untreated controls. Pretreatment with hyaluronidase prior to collagenase reduced the time to outgrowth. A notable finding of this study was that the majority of chondrocytes in the adult human articular cartilage specimens and virtually all of the outgrowing cells contained α‐smooth muscle actin. We conclude that human articular chondrocytes have the capability to migrate through enzymatically degraded matrix and express a contractile actin isoform. Collagenase treatment reduces the time required for cell outgrowth.

Modulation of the Contractile and Biosynthetic Activity of Chondrocytes Seeded in Collagen–Glycosaminoglycan Matrices

Studies have demonstrated that articular chondrocytes can express the gene for alpha-smooth muscle actin (SMA) and can contract porous polymeric matrices employed for tissue engineering, thereby altering the pore structure and distorting the shape of the scaffold. The objectives of this study were to determine whether an agent known to disrupt microfilament organization in chondrocytes could reduce this contractility and to assess whether there was an association between the contractile behavior of chondrocytes and their biosynthetic activity. Staurosporine, an antibiotic known to inhibit protein kinase C and disrupt cytoskeletal structure, was used as the agent to modulate the chondrocytic phenotype and contractile and biosynthetic activity of serially passaged adult canine chondrocytes seeded in type 1 collagen-glycosaminoglycan scaffolds. Cells in monolayer culture treated with as little as 3 nM staurosporine for 4 days contained type II procollagen, whereas few cells in the untreated control cultures demonstrated type II procollagen synthesis. Treatment with staurosporine also led to a decrease in the amount of SMA synthesized by the cells. Consistent with this decreased expression of the contractile actin isoform, cells cultured in the collagen-glycosaminoglycan scaffolds and treated with 5 nM staurosporine contracted the scaffold significantly less than untreated cells (15% diameter contraction by treated cells, compared with more than 50% contraction by untreated cells). The staurosporine-treated cells were biosynthetically active, displaying higher rates of protein and glycosaminoglycan synthesis, as indicated by rates of incorporation of [(3)H]proline and [(35)S]sulfate, respectively, compared with untreated cells. The long-held notion that changes in cytoskeletal structure influence phenotypic characteristics of cultured chondrocytes may now be extended to relate expression of a specific muscle actin isoform to certain cell processes. Moreover, the finding that chondrocytes with a lower level of expression of SMA and reduced contractility display higher rates of biosynthesis warrants further study.

Organ Culture System for Mechanobiology Studies of the Intervertebral Disc

The development of in vitro models is critical for furthering understanding of the intervertebral disc and the development of disc regeneration/tissue engineering. An in vitro culture system targeted towards mechano-biology studies of the intervertebral disc (IVD) was built and validated using bovine coccygeal discs. Discs were maintained in culture for up to one week with and without vertebral endplates. Water content and glycosaminoglycan content were found to be stable and cells were metabolically active when cultured under a 5kg static load.Copyright