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author:("Kim, inwood")
1.  Brief mechanical ventilation impacts airway cartilage properties in neonatal lambs 
Pediatric Pulmonology  2011;47(8):763-770.
Ultrasound imaging allows in vivo assessment of tracheal kinetics and cartilage structure. To date, the impact of mechanical ventilation (MV) on extracellular matrix (ECM) in airway cartilage is unclear, but an indication of its functional and structural change may support the development of protective therapies. The objective of this study was to characterize changes in mechanical properties of the neonatal airway during MV with alterations in cartilage ECM. Trachea segments were isolated in a neonatal lamb model; ultrasound dimensions and pressure-volume relationships were measured on sham (no MV; n = 6) and MV (n = 7) airways for 4 h. Tracheal cross-sections were harvested at 4 h, tissues were fixed and stained, and Fourier transform infrared imaging spectroscopy (FT-IRIS) was performed. Over 4 h of MV, bulk modulus (28%) and elastic modulus (282%) increased. The MV tracheae showed higher collagen, proteoglycan content, and collagen integrity (new tissue formation); whereas no changes were seen in the controls. These data are clinically relevant in that airway properties can be correlated with MV and changes in cartilage extracellular matrix. Mechanical ventilation increases the in vivo dimensions of the trachea, and is associated with evidence of airway tissue remodeling. Injury to the neonatal airway from MV may have relevance for the development of tracheomalacia. We demonstrated active airway tissue remodeling during MV using a FT-IRIS technique which identifies changes in ECM.
PMCID: PMC3362668  PMID: 22170596
neonatal airway; elastic modulus; ultrasound; Fourier transform infrared imaging spectroscopy (FT-IRIS)
2.  High Mesenchymal Stem Cell Seeding Densities in Hyaluronic Acid Hydrogels Produce Engineered Cartilage with Native Tissue Properties 
Acta Biomaterialia  2012;8(8):3027-3034.
Engineered cartilage based on adult mesenchymal stem cells (MSCs) is an alluring goal for the repair of articular defects. However, efforts to date have failed to generate constructs with sufficient mechanical properties to function in the demanding environment of the joint. Our findings with a novel photocrosslinked hyaluronic acid (HA) hydrogel suggest that stiff gels (high HA concentration, 5% w/vol) foster chondrogenic differentiation and matrix production, but limit overall functional maturation due to the inability of formed matrix to diffuse away from the point of production and form a contiguous network. In the current study, we hypothesized that increasing the MSC seeding density would decrease the required diffusional distance, and so expedite the development of functional properties. To test this hypothesis, bovine MSCs were encapsulated at seeding densities of either 20 or 60 million cells per mL in 1%, 3%, and 5% (w/vol) hyaluronic acid (HA) hydrogels. Counter our hypothesis, higher concentration HA gels (3% and 5%) did not develop more rapidly with increased MSC seeding density. However, the biomechanical properties of low concentration (1%) HA constructs increased markedly (nearly 3-fold with a 3-fold increase in seeding density). To ensure that optimal nutrient access was delivered, we next cultured these constructs under dynamic culture conditions (orbital shaking) for 9 weeks. Under these conditions, 1% HA seeded at 60 million MSCs per mL reached a compressive modulus in excess of 1 MPa (compared to 0.3-0.4MPa for free swelling constructs). This is the highest level we have reported to date in this HA hydrogel system, and represents a significant advance towards functional stem cell-based tissue engineered cartilage.
PMCID: PMC3389207  PMID: 22546516
cartilage; hydrogel; tissue engineering; hyaluronic acid / hyaluronan; mesenchymal stem cell
3.  Transient exposure to TGF-β3 improves the functional chondrogenesis of MSC-laden hyaluronic acid hydrogels 
Tissue engineering with adult stem cells is a promising approach for the restoration of focal defects in articular cartilage. For this, progenitor cells would ideally be delivered to (and maintained within) the defect site via a biocompatible material and in combination with soluble factors to promote initial cell differentiation and subsequent tissue maturation in vivo. While growth factor delivery methods are continually being optimized, most offer only a short (days to weeks) delivery profile at high doses. To address this issue, we investigated mesenchymal stem cell (MSC) differentiation and maturation in photocrosslinkable hyaluronic acid (HA) hydrogels with transient exposure to the pro-chondrogenic molecule transforming growth factor-beta3 (TGF-β3), at varying doses (10, 50 and 100 ng/mL) and durations (3, 7, 21 and 63 days). Mechanical, biochemical, and histological outcomes were evaluated through 9 weeks of culture. Results showed that a brief exposure (7 days) to a very high level (100 ng/mL) of TGF-β 3 was sufficient to both induce and maintain cartilage formation in these 3D constructs. Indeed, this short delivery resulted in constructs with mechanical and biochemical properties that exceeded that of continuous exposure to a lower level (10 ng/mL) of TGF-β 3 over the entire 9-week time course. Of important note, the total TGF delivery in these two scenarios was roughly equivalent (200 vs. 180 ng), but the timing of delivery differed markedly. These data support the idea that acute exposure to a high dose of TGF will induce functional and long-term differentiation of stem cell populations, and furthers our efforts to improve cartilage repair in vivo.
PMCID: PMC3367258  PMID: 22658158
Hyaluronic Acid; Mesenchymal Stem Cells; Cartilage Tissue Engineering
European cells & materials  2010;19:72-85.
Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering given their ability to undergo chondrogenesis in 3D culture systems. Mechanical forces play an important role in regulating both cartilage development and MSC chondrogenic gene expression, however, mechanical stimulation has yet to enhance the mechanical properties of engineered constructs. In this study, we applied long-term dynamic compression to MSC-seeded constructs and assessed whether varying pre-culture duration, loading regimens and inclusion of TGF-β3 during loading would influence functional outcomes and these phenotypic transitions. Loading initiated before chondrogenesis decreased functional maturation, although chondrogenic gene expression increased. In contrast, loading initiated after chondrogenesis and matrix elaboration further improved the mechanical properties of MSC-based constructs, but only when TGF-β3 levels were maintained and under specific loading parameters. Although matrix quantity was not affected by dynamic compression, matrix distribution, assessed histologically and by FT-IRIS analysis, was significantly improved on the micro- (pericellular) and macro- (construct expanse) scales. Further, whole genome expression profiling revealed marked shifts in the molecular topography with dynamic loading. These results demonstrate, for the first time, that dynamic compressive loading initiated after a sufficient period of chondro-induction and with sustained TGF-β exposure enhances matrix distribution and the mechanical properties of MSC-seeded constructs.
PMCID: PMC3486923  PMID: 20186667
Cartilage; tissue engineering; mesenchymal stem cells; chondrogenesis; mechanical stimulation
5.  The Validation of Ultrasound-Guided Lumbar Facet Nerve Blocks as Confirmed by Fluoroscopy 
Asian Spine Journal  2012;6(3):163-167.
Study Design
This is a prospective study.
To develop a methodological approach for conducting ultrasound-guided lumbar facet nerve block by defining essential ultrasound-guided landmarks in order to assess the feasibility of this method.
Overview of Literature
The current role of ultrasound guidance for musculoskeletal intervention treatments has been reported upon in previous literature.
Ultrasound-guided facet nerve block was done in 95 segments for 50 patients with chronic back pain by facet arthropathy. After the surface landmarks of the spinous process and iliac crest line were confirmed, longitudinal facet views were obtained by a curved array transducer to identify the different spinal segments. The spinous process and facet joint with transverse process were delineated by transverse sonograms at each level and the target point for the block was defined as lying on the upper edge of the transverse process. The needle was inserted toward the target point. After a contrast injection, the placement of the needle and contrast was checked by fluoroscopy.
Eighty-seven segments (91.6%) could be guided successfully to the right facet nerve block by using ultrasound. After fluoroscopic control, 8 needles had to be corrected because of problems with other segments (3 cases) and lamina placements (5 cases). For the 42 patients who underwent successful block by ultrasound, however, the mean visual analogue score for back pain was improved from 6.2 ± 0.9 before the block to 4.0 ± 1.0 after the block (p = 0.001).
Ultrasound-guided longitudinal facet view and the surface landmarks of the spinous process and iliac crest line seems to be a promising guidance technique for the lumbar facet nerve block technique.
PMCID: PMC3429606  PMID: 22977695
Lumbosacral region; Nerve block; Ultrasonography
6.  Material and mechanical properties of bones deficient for fibrillin-1 or fibrillin-2 microfibrils 
The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of fibrillin-1 or fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in fibrillin-1 or fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that fibrillin-1 plays a predominantly greater role than fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by fibrillin proteins. Our study therefore advance our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in fibrillin-1 or -2.
PMCID: PMC3097426  PMID: 21440062
bone material and mechanical properties; congenital contractural arachnodactyly; fibrillin; Marfan syndrome; TGFβ
7.  Evaluation of Early Osteochondral Defect Repair in a Rabbit Model Utilizing Fourier Transform–Infrared Imaging Spectroscopy, Magnetic Resonance Imaging, and Quantitative T2 Mapping 
Evaluation of the morphology and matrix composition of repair cartilage is a critical step toward understanding the natural history of cartilage repair and efficacy of potential therapeutics. In the current study, short-term articular cartilage repair (3 and 6 weeks) was evaluated in a rabbit osteochondral defect model treated with thrombin peptide (TP-508) using magnetic resonance imaging (MRI), quantitative T2 mapping, and Fourier transform–infrared imaging spectroscopy (FT-IRIS).
Three-mm-diameter osteochondral defects were made in the rabbit trochlear groove and filled with either TP-508 plus poly-lactoglycolidic acid microspheres or poly-lactoglycolidic acid microspheres alone (placebo). Repair tissue and adjacent normal cartilage were evaluated at 3 and 6 weeks postdefect creation. Intact knees were evaluated by magnetic resonance imaging for repair morphology, and with quantitative T2 mapping to assess collagen orientation. Histological sections were evaluated by FT-IRIS for parameters that reflect collagen quantity and quality, as well as proteoglycan (PG) content.
Results and Conclusion
There was no significant difference in volume of repair tissue at either time point. At 6 weeks, placebo repair tissue demonstrated longer T2 values (p < 0.01) than TP-508 did. Although both placebo and TP-508 repair tissue demonstrated longer T2 values than adjacent normal cartilage did, the 6-week T2 values of the TP-508 specimens were closer to those of the adjacent normal cartilage than were the placebo values. FT-IRIS analysis demonstrated a significant increase in collagen content, integrity, and PG content of the TP-508 repair tissue from 3 to 6 weeks (p ≤ 0.05). In addition, the collagen and PG content of the TP-508 samples were closer to normal cartilage at 3 weeks than were the placebo samples. Further, there was a significant inverse correlation between the T2 relaxation values and collagen orientation in the normal cartilage. However, there were no significant correlations between T2 relaxation values and any FT-IRIS parameter in the repair tissue. Together, the data demonstrate that MRI and FT-IRIS assessment of cartilage repair tissue provide molecular information that furthers understanding of the cartilage repair process.
PMCID: PMC2945312  PMID: 19586313

Results 1-7 (7)