Steven B. Nicoll

Synergistic action of growth factors and dynamic loading for articular cartilage tissue engineering.

It has previously been demonstrated that dynamic deformational loading of chondrocyte-seeded agarose hydrogels over the course of 1 month can increase construct mechanical and biochemical properties relative to free-swelling controls. The present study examines the manner in which two mediators of matrix biosynthesis, the growth factors TGF-beta1 and IGF-I, interact with applied dynamic deformational loading. Under free-swelling conditions in control medium (C), the [proteoglycan content][collagen content][equilibrium aggregate modulus] of cell-laden (10 x 10(6) cells/mL) 2% agarose constructs reached a peak of [0.54% wet weight (ww)][0.16% ww][13.4 kPa]c, whereas the addition of TGF-beta1 or IGF-I to the control medium led to significantly higher peaks of [1.18% ww][0.97% ww][23.6 kPa](C-TGF) and [1.00% ww][0.63% ww][19.3 kPa](C-IGF), respectively, by day 28 or 35 (p<0.01). Under dynamic loading in control medium (L), the measured parameters were [1.10% ww][0.52% ww][24.5 kPa]L, and with the addition of TGF-beta1 or IGF-I to the control medium these further increased to [1.49% ww][1.07% ww][50.5 kPa](L-TGF) and [1.48% ww][0.81% ww][46.2 kPa](L-IGF), respectively (p<0.05). Immunohistochemical staining revealed that type II collagen accumulated primarily in the pericellular area under free-swelling conditions, but spanned the entire tissue in dynamically loaded constructs. Applied in concert, dynamic deformational loading and TGF-beta1 or IGF-I increased the aggregate modulus of engineered constructs by 277 or 245%, respectively, an increase greater than the sum of either stimulus applied alone. These results support the hypothesis that the combination of chemical and mechanical promoters of matrix biosynthesis can optimize the growth of tissue-engineered cartilage constructs.

In vitro release kinetics of biologically active transforming growth factor-β1 from a novel porous glass carrier

Sol-gel silica-based porous glass (xerogel) was used as a novel carrier material for recombinant human transforming growth factor-beta 1 (TGF-beta 1). Room temperature synthesis procedures included sol preparation, the addition of TGF-beta 1 solution to the sol, subsequent gelation and drying. After determination of optimal synthesis parameters, the material was assayed in vitro for its ability to release biologically active TGF-beta 1 in a controlled manner. Sustained release of TGF-beta 1 over a 7-day period was demonstrated. On the basis of published TGF-beta 1 potency, the amount released is capable of eliciting bone tissue reactivity. These findings suggest that this novel glass-growth factor composite may serve as an effective bone graft material for the repair of osseous defects.

Quantification of cartilage biomechanical and biochemical properties via T1ρ magnetic resonance imaging

The aim of this study is to develop T1ρ as an MR marker of the compositional and functional condition of cartilage. Specifically, we investigate the correlation of changes in cartilage biomechanical and biochemical properties with T1ρ relaxation rate in a cytokine‐induced model of degeneration. Bovine cartilage explants were cultured with 30 ng/mL of interleukin‐1β to mimic the cartilage degradation of early osteoarthritis. The average rate of T1ρ relaxation was calculated from T1ρ maps acquired on a 4.7 T research scanner. Stress‐relaxation biomechanical tests were conducted with a confined compression apparatus to measure uniaxial aggregate modulus (HA) and hydraulic permeability (k0) using linear biphasic theory. Proteoglycan, collagen, and water content were measured via biochemical assays. Average T1ρ relaxation rate was strongly correlated with proteoglycan content (R2 = 0.926), HA (R2 = 0.828), and log10 k0 (R2 = 0.862). Results of this study demonstrate that T1ρ MRI can detect changes in proteoglycan content and biomechanical properties of cartilage in a physiologically relevant model of cartilage degeneration. The T1ρ technique can potentially be used to noninvasively and quantitatively assess the biochemical and biomechanical characteristics of articular cartilage in humans during the progression of osteoarthritis. Magn Reson Med, 2005.

Characterization of novel photocrosslinked carboxymethylcellulose hydrogels for encapsulation of nucleus pulposus cells.

Back pain is a significant clinical concern often associated with degeneration of the intervertebral disc (IVD). Tissue engineering strategies may provide a viable IVD replacement therapy; however, an ideal biomaterial scaffold has yet to be identified. One candidate material is carboxymethylcellulose (CMC), a water-soluble derivative of cellulose. In this study, 90 and 250 kDa CMC polymers were modified with functional methacrylate groups and photocrosslinked to produce hydrogels at different macromer concentrations. At 7 days, bovine nucleus pulposus (NP) cells encapsulated in these hydrogels were viable, with values for the elastic modulus ranging from 1.07 + or - 0.06 to 4.29 + or - 1.25 kPa. Three specific formulations were chosen for further study based on cell viability and mechanical integrity assessments: 4% 90 kDa, 2% 250 kDa and 3% 250 kDa CMC. The equilibrium weight swelling ratio of these formulations remained steady throughout the 2 week study (46.45 + or - 3.14, 48.55 + or - 2.91 and 42.41 + or - 3.06, respectively). The equilibrium Youngs modulus of all cell-laden and cell-free control samples decreased over time, with the exception of cell-laden 3% 250 kDa CMC constructs, indicating an interplay between limited hydrolysis of interchain crosslinks and the elaboration of a functional matrix. Histological analyses of 3% 250 kDa CMC hydrogels confirmed the presence of rounded cells in lacunae and the pericellular deposition of chondroitin sulfate proteoglycan, a phenotypic NP marker. Taken together, these studies support the use of photocrosslinked CMC hydrogels as tunable biomaterials for NP cell encapsulation.

Photo-crosslinked alginate hydrogels support enhanced matrix accumulation by nucleus pulposus cells in vivo

OBJECTIVE Intervertebral disc (IVD) degeneration is a major health concern in the United States. Replacement of the nucleus pulposus (NP) with injectable biomaterials represents a potential treatment strategy for IVD degeneration. The objective of this study was to characterize the extracellular matrix (ECM) assembly and functional properties of NP cell-encapsulated, photo-crosslinked alginate hydrogels in comparison to ionically crosslinked alginate constructs. METHODS Methacrylated alginate was synthesized by esterification of hydroxyl groups with methacrylic anhydride. Bovine NP cells were encapsulated in alginate hydrogels by ionic crosslinking using CaCl(2) or through photo-crosslinking upon exposure to long-wave UV light in the presence of a photoinitiator. The hydrogels were evaluated in vitro by gross and histological analysis and in vivo using a murine subcutaneous pouch model. In vivo samples were analyzed for gene expression, ECM localization and accumulation, and equilibrium mechanical properties. RESULTS Ionically crosslinked hydrogels exhibited inferior proteoglycan accumulation in vitro and were unable to maintain structural integrity in vivo. In further studies, photo-crosslinked alginate hydrogels were implanted for up to 8 weeks to examine NP tissue formation. Photo-crosslinked hydrogels displayed temporal increases in gene expression and assembly of type II collagen and proteoglycans. Additionally, hydrogels remained intact over the duration of the study and the equilibrium Youngs modulus increased from 1.24+/-0.09 kPa to 4.31+/-1.39 kPa, indicating the formation of functional matrix with properties comparable to those of the native NP. CONCLUSIONS These findings support the use of photo-crosslinked alginate hydrogels as biomaterial scaffolds for NP replacement.

Characterization of photocrosslinked alginate hydrogels for nucleus pulposus cell encapsulation.

Intervertebral disc (IVD) degeneration is a significant health concern in the USA. Tissue engineering strategies have the potential to provide a viable alternative to current treatments. Nevertheless, such approaches require a suitable biomaterial scaffold for IVD tissue regeneration. Calcium crosslinked alginate has traditionally been used for in vitro culture of nucleus pulposus (NP) cells of the IVD. However, such ionically crosslinked hydrogels lose structural integrity over time. Recently, various polymers have been modified with photopolymerizable functional groups to create covalently crosslinked hydrogels. This technology may be employed to maintain the structural and mechanical integrity of three-dimensional alginate hydrogels. In this study, photocrosslinkable alginate was synthesized and evaluated for material properties and the ability to maintain the viability of encapsulated NP cells. Photocrosslinked alginate at varying percent modifications and weight/volume percentages displayed equilibrium swelling ratios and Youngs moduli of 30.52 +/- 1.782 to 43.50 +/- 1.345 and 0.5850 +/- 0.1701 to 8.824 +/- 0.6014 kPa, respectively. The viability of encapsulated NP cells was highest in hydrogels at lower percent modifications, and decreased with time in culture. Taken together, this study is the first to demonstrate that photocrosslinked alginate can be used for cellular encapsulation and synthesized with tunable material properties that may be tailored for specific applications.

Effects of Applied DC Electric Field on Ligament Fibroblast Migration and Wound Healing

Applied electric fields (static and pulsing) are widely used in orthopedic practices to treat nonunions and spine fusions and have been shown to improve ligament healing in vivo. Few studies, however, have addressed the effect of electric fields (EFs) on ligament fibroblast migration and biosynthesis. In the current study, we applied static and pulsing direct current (DC) EFs to calf anterior cruciate ligament (ACL) fibroblasts. ACL fibroblasts demonstrated enhanced migration speed and perpendicular alignment to the applied EFs. The motility of ligament fibroblasts was further modulated on type I collagen. In addition, type I collagen expression increased in ACL fibroblasts after exposure to pulsing EFs. In vitro wound-healing studies showed inhibitory effects of static EFs, which were alleviated with a pulsing EF. Our results demonstrate that applied EFs augment ACL fibroblast migration and biosynthesis and provide potential mechanisms by which EFs may be used for enhancing ligament healing and repair.

Cartilage Interstitial Fluid Load Support in Unconfined Compression Following Enzymatic Digestion

Interstitial fluid pressurization plays an important role in cartilage biomechanics and is believed to be a primary mechanism of load support in synovial joints. The objective of this study was to investigate the effects of enzymatic degradation on the interstitial fluid load support mechanism of articular cartilage in unconfined compression. Thirty-seven immature bovine cartilage plugs were tested in unconfined compression before and after enzymatic digestion. The peak fluid load support decreased significantly (p < 0.0001) from 84 +/- 10% to 53 +/- 19% and from 80 +/- 10% to 46 +/- 21% after 18-hours digestion with 1.0 u/mg-wet-weight and 0.7 u/mg-wet-weight of collagenase, respectively. Treatment with 0.1 u/ml of chondroitinase ABC for 24 hours also significantly reduced the peak fluid load support from 83 +/- 12% to 48 +/- 16% (p < 0.0001). The drop in interstitial fluid load support following enzymatic treatment is believed to result from a decrease in the ratio of tensile to compressive moduli of the solid matrix.

The effect of serial monolayer passaging on the collagen expression profile of outer and inner anulus fibrosus cells

Study Design. Sheep outer and inner anulus fibrosus cells were isolated and analyzed to determine the effect of serial monolayer passaging on their phenotype. Objectives. To characterize the effect of sequential serial passage on outer and inner anulus cells to determine at which point passaged cells are significantly different from freshly isolated cells. Summary of Background Data. Previous studies show that chondrocytic cells lose their differentiated phenotype with sequential monolayer passage. Although intervertebral disc cells are similar, to our knowledge, a complete characterization of passage effects has not been performed. Methods. Sheep outer and inner anulus cells were isolated, serially passaged, and evaluated for changes in cellular morphology, collagen I and II gene expression and protein elaboration, and total protein and deoxyribonucleic acid content. Results. Outer anulus cells displayed an elongated morphology, while inner anulus cells were initially polygonal and became more fibroblast-like with passage. At low passage, outer anulus cells showed higher collagen I expression, while inner anulus cells indicated higher collagen II expression. At high passage, collagen I expression increased for inner anulus cells and decreased for outer anulus cells, whereas collagen II expression decreased for both cell types. Immunohistochemical staining confirmed gene expression results. Conclusions. The differences in expression profiles of outer and inner anulus cells support previous findings that zonal differences exist between the cell types. Up to passage 2, both cell types were not significantly different from freshly isolated cells and maintained distinct phenotypic characteristics. However, after 6 sequential passages, outer and inner anulus cells became morphologically indistinguishable, and displayed no significant differences in collagen I gene and protein expression, thus becoming a more homogeneous population. As such, serial monolayer passaging has a marked effect on disc cell behavior, and is an important factor to consider when designing and evaluating in vitro studies and for potential cell-based therapies for disc repair.

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.