Jin Nam

Compressive forces induce osteogenic gene expression in calvarial osteoblasts

Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Youngs modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.

The Role of Changes in Extracellular Matrix of Cartilage in the Presence of Inflammation on the Pathology of Osteoarthritis

Osteoarthritis (OA) is a degenerative disease that affects various tissues surrounding joints such as articular cartilage, subchondral bone, synovial membrane, and ligaments. No therapy is currently available to completely prevent the initiation or progression of the disease partly due to poor understanding of the mechanisms of the disease pathology. Cartilage is the main tissue afflicted by OA, and chondrocytes, the sole cellular component in the tissue, actively participate in the degeneration process. Multiple factors affect the development and progression of OA including inflammation that is sustained during the progression of the disease and alteration in biomechanical conditions due to wear and tear or trauma in cartilage. During the progression of OA, extracellular matrix (ECM) of cartilage is actively remodeled by chondrocytes under inflammatory conditions. This alteration of ECM, in turn, changes the biomechanical environment of chondrocytes, which further drives the progression of the disease in the presence of inflammation. The changes in ECM composition and structure also prevent participation of mesenchymal stem cells in the repair process by inhibiting their chondrogenic differentiation. This review focuses on how inflammation-induced ECM remodeling disturbs cellular activities to prevent self-regeneration of cartilage in the pathology of OA.

Fabrication of Skeletal Muscle Constructs by Topographic Activation of Cell Alignment

Skeletal muscle fiber construction for tissue‐engineered grafts requires assembly of unidirectionally aligned juxtaposed myotubes. To construct such a tissue, a polymer microchip with linearly aligned microgrooves was fabricated that could direct myoblast adaptation under stringent conditions. The closely spaced microgrooves fabricated by a modified replica molding process guided linear cellular alignment. Examination of the myoblasts by immunofluorescence microscopy demonstrated that the microgrooves with subcellular widths and appropriate height‐to‐width ratios were required for practically complete linear alignment of myoblasts. The topology‐dependent cell alignment encouraged differentiation of myoblasts into multinucleate, myosin heavy chain positive myotubes. The monolayer of myotubes formed on the microstructured chips allowed attachment, growth and differentiation of subsequent layers of linearly arranged myoblasts, parallel to the primary monolayer of myotubes. The consequent deposition of additional myoblasts on the previous layer of myotubes resulted in three‐dimensional multi‐layered structures of myotubes, typical of differentiated skeletal muscle tissue. The findings demonstrate that the on‐chip device holds promise for providing an efficient means for guided muscle tissue construction. Biotechnol. Bioeng. 2009;102: 624–631.

Structuring electrospun polycaprolactone nanofiber tissue scaffolds by femtosecond laser ablation

Meshes of electrospun (ES) polycaprolactone (PCL) and polyethylene terephthalate nanofiber meshes were structured by ablation of linear grooves with a scanned femtosecond laser. Focus spot size, pulse energy, and scanning speed were varied to determine their affects on groove size and the characteristics of the electrospun fiber at the edges of these grooves. The femtosecond laser was seen to be an effective means for flexibly structuring the surface of ES PCL scaffolds. Femtosecond ablation resulted in much more uniformly ablated patterns compared to Q-switched nanosecond pulse laser ablation. Also, the width of the ablated grooves was well controlled by laser energy and focus spot size, although the grooves were significantly larger than the spot size. Also, some melting of fibers was observed at the edges of grooves. These affects were attributed to optical radiation from laser-induced plasma at higher pulse energies and melting of fibers at laser fluences lower than the ablation threshold. The ablatio...

Biomechanical Thresholds Regulate Inflammation through the NF-κB Pathway: Experiments and Modeling

Background During normal physical activities cartilage experiences dynamic compressive forces that are essential to maintain cartilage integrity. However, at non-physiologic levels these signals can induce inflammation and initiate cartilage destruction. Here, by examining the pro-inflammatory signaling networks, we developed a mathematical model to show the magnitude-dependent regulation of chondrocytic responses by compressive forces. Methodology/Principal Findings Chondrocytic cells grown in 3-D scaffolds were subjected to various magnitudes of dynamic compressive strain (DCS), and the regulation of pro-inflammatory gene expression via activation of nuclear factor-kappa B (NF-κB) signaling cascade examined. Experimental evidences provide the existence of a threshold in the magnitude of DCS that regulates the mRNA expression of nitric oxide synthase (NOS2), an inducible pro-inflammatory enzyme. Interestingly, below this threshold, DCS inhibits the interleukin-1β (IL-1β)-induced pro-inflammatory gene expression, with the degree of suppression depending on the magnitude of DCS. This suppression of NOS2 by DCS correlates with the attenuation of the NF-κB signaling pathway as measured by IL-1β-induced phosphorylation of the inhibitor of kappa B (IκB)-α, degradation of IκB-α and IκB-β, and subsequent nuclear translocation of NF-κB p65. A mathematical model developed to understand the complex dynamics of the system predicts two thresholds in the magnitudes of DCS, one for the inhibition of IL-1β-induced expression of NOS2 by DCS at low magnitudes, and second for the DCS-induced expression of NOS2 at higher magnitudes. Conclusions/Significance Experimental and computational results indicate that biomechanical signals suppress and induce inflammation at critical thresholds through activation/suppression of the NF-κB signaling pathway. These thresholds arise due to the bistable behavior of the networks originating from the positive feedback loop between NF-κB and its target genes. These findings lay initial groundwork for the identification of the thresholds in physical activities that can differentiate its favorable actions from its unfavorable consequences on joints.

Transcriptome-wide gene regulation by gentle treadmill walking during the progression of monoiodoacetate-induced arthritis.

OBJECTIVE Physiotherapies are the most widely recommended conservative treatment for arthritic diseases. The present study was undertaken to examine the molecular mechanisms underlying the effects of gentle treadmill walking (GTW) on various stages of monoiodoacetate-induced arthritis (MIA) to elucidate the basis for the success or failure of such therapies in joint damage. METHODS Knees were obtained from untreated control rats, rats with MIA that did not undergo GTW, rats with MIA in which GTW regimens were started 1 day post-MIA induction, and rats with MIA in which GTW regimens were started after cartilage damage had progressed to grade 1 or grade 2. The cartilage was examined macroscopically, microscopically, and by microfocal computed tomography imaging. Transcriptome-wide gene expression analysis was performed, and microarray data were assessed by Ingenuity Pathways Analysis to identify molecular functional networks regulated by GTW. RESULTS GTW intervention started on day 1 post-MIA induction significantly prevented the progression of MIA, but its efficacy was reduced when implemented on knees exhibiting close to grade 1 cartilage damage. GTW accelerated cartilage damage in knees with close to grade 2 damage. Transcriptome-wide gene expression analysis revealed that GTW intervention started 1 day post-MIA inception significantly suppressed inflammation-associated genes and up-regulated matrix-associated gene networks. However, delayed GTW intervention after grade 1 damage had occurred was less effective in suppressing proinflammatory genes or up-regulating matrix synthesis. CONCLUSION The present findings suggest that GTW suppresses proinflammatory gene networks and up-regulates matrix synthesis to prevent progression of cartilage damage in MIA-affected knees. However, the extent of cartilage damage at the initiation of GTW may be an important determinant of the success or failure of such therapies.

Novel Electrospun Scaffolds for the Molecular Analysis of Chondrocytes Under Dynamic Compression

Mechanical training of engineered tissue constructs is believed necessary to improve regeneration of cartilaginous grafts. Nevertheless, molecular mechanisms underlying mechanical activation are not clear. This is partly due to unavailability of appropriate scaffolds allowing exposure of cells to dynamic compressive strains (DCS) in vitro while permitting subsequent molecular analyses. We demonstrate that three-dimensional macroporous electrospun poly(epsilon-caprolactone) scaffolds can be fabricated that are suitable for the functional and molecular analysis of dynamically loaded chondrocytes. These scaffolds encourage chondrocytic proliferation promoting expression of collagen type II, aggrecan, and Sox9 while retaining mechanical strength after prolonged dynamic compression. Further, they exhibit superior infiltration of exogenous agents into the cells and permit easy retrieval of cellular components postcompression to allow exploration of molecular mechanisms of DCS. Using these scaffolds, we observed that chondrocytes responded to DCS in a magnitude-dependent manner exhibiting antiinflammatory and proanabolic responses at low physiological magnitudes. Proinflammatory responses and decreased cellular viability were observed at hyperphysiological magnitudes. These scaffolds provide a means of unraveling the mechanotransduction-induced transcriptional and posttranslational activities involved in cartilage regeneration and repair.

Interactions between endothelial cells and electrospun methacrylic terpolymer fibers for engineered vascular replacements

A compliant terpolymer made of hexylmethacrylate (HMA), methylmethacrylate (MMA), and methacrylic acid (MAA) intended for use in small diameter vascular graft applications has been developed. The mechanical properties and in vitro biostability of this terpolymer have been previously characterized. The goal of this investigation was to examine the interactions between endothelial cells and the new terpolymer and to evaluate endothelial cell function. Electrospinning was used to produce both oriented and random terpolymer fiber scaffolds. Smooth solution cast films and tissue culture polystyrene were used as negative and positive controls, respectively. Human blood outgrowth endothelial cells and human umbilical vein endothelial cells were incubated with the test and control samples and characterized with respect to initial cell attachment, proliferation, viability, and maintenance of the endothelial cell phenotype. It was found that the terpolymer is cytocompatible allowing endothelial cell growth, with random fibers being more effective in promoting enhanced cellular activities than oriented fibers. In addition, endothelial cells cultured on these substrates appeared to maintain their phenotype. The results from this study demonstrate that electrospun HMA:MMA:MAA terpolymer has the potential to be used successfully in fabricating small diameter blood vessel replacements.

Polyaniline/poly(ε-caprolactone) composite electrospun nanofiber-based gas sensors: optimization of sensing properties by dopants and doping concentration

Electrospinning was utilized to synthesize a polyaniline (PANI)/poly(ε-caprolactone) (PCL) composite in the form of nanofibers to examine its gas sensing performance. Electrical conductivity of the composite nanofibers was tailored by secondary doping with protonic acids including hydrochloride (HCl) or camphorsulfonic acid (HCSA). FT-IR and diffuse reflectance UV-vis spectroscopy were utilized to examine doping-dependent changes in the chemical structure and the protonation state of the nanofibers, respectively. The oxidation and protonation state of the composite nanofibers were shown to strongly depend on the doping agent and duration, demonstrating a simple way of controlling the electrical conductivity of the composite. PANI/PCL electrospun nanofibers having various electrical conductivities via varying dopants and doping concentrations, were configured to chemiresistors for sensing various analytes, including water vapor, NH3, and NO2. Secondary doping with Cl(-) and CSA differentially affected sensing behaviors by having distinctive optimal sensitivities. Biphasic sensitivity with respect to electrical conductivity was observed, demonstrating a facile method to enhance gas sensitivity by optimizing secondary doping. A balance between Debye length of the nanofibers and overall charge conduction may play an important role for modulating such an optimal sensitivity.

Size-dependent piezoelectric and mechanical properties of electrospun P(VDF-TrFE) nanofibers for enhanced energy harvesting

Piezoelectricity-based energy harvesting from wasted mechanical energies has garnered an increasing attention as a clean energy source. Especially, flexible organic piezoelectric materials provide an opportunity to exploit their uses in mechanically challenging areas where brittle inorganic counterparts have mechanical limitations. In this regard, electrospinning has shown its advantages of producing poly(vinylidene fluoride) (PVDF)-based nanofibrous structures without the necessity of a secondary processing to induce/increase piezoelectric properties. However, the effects of electrospun fiber dimension, one of the main morphological parameters in electrospun fibers, on piezoelectricity have not been fully understood. In this study, two dependent design of experiments (DOEs) were utilized to systematically control the dimensions of electrospun poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) to produce nanofibers having their diameter ranging from 1000 to sub-100 nm. Such a dimensional reduction resulted in the increase of piezoelectric responsible electroactive phase content and the degree of crystallinity. These changes in crystal structure led to approximately 2-fold increase in piezoelectric constant as compared to typical P(VDF-TrFE) thin films. More substantially, the dimensional reduction also increased the Youngs modulus of the nanofibers up to approximately 80-fold. The increases in piezoelectric constant and Youngs modulus collectively enhanced piezoelectric performance, resulting in the exponential increase in electric output of nanofiber mats when the fiber diameters were reduced from 860 nm down to 90 nm. Taken together, the results suggest a new strategy to improve the piezoelectric performance of electrospun P(VDF-TrFE) via optimization of their electromechanical and mechanical properties.