Combining Chondrocytes and Smooth Muscle Cells to Engineer Hybrid Soft Tissue Constructs

Successful approaches to tissue engineering smooth muscle tissues utilize biodegradable scaffolds seeded with autologous cells. One common problem in using biological scaffolds specifically is the difficulty of inducing cellular penetration and controlling de novo extracellular matrix deposition=remodeling in vitro. Our hypothesis was that small intestinal submucosa (SIS) exposed to specific mechanical stimulation regimes would modulate the synthesis of de novo collagen and elastin by bladder smooth muscle cells (BSMC) within the SIS matrix. We further hypothesized that the cytokines vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), two key growth factors involved in epithelial mesenchymal signaling, will promote the cellular penetration into SIS necessary for mechanical stimulation. BSMC were seeded at 0.5Â10 6 cells=cm 2 onto the luminal side of SIS specimens. VEGF (10 ng=mL) and FGF-2 (5 ng=mL) were added to each insert in the media every other day for up to 7 days in static culture. Following static culture, specimens were stretched stripbiaxially under 15% peak strain at either 0.5 or 0.1 Hz for an additional 7 days. Following the culture period, specimens were assayed histologically and biochemically for cellular penetration, proliferation, elastin, collagen, and protease activity. Histological analyses demonstrated that in standard culture media, BSMC remained on the surface of the SIS while both FGF-2 and VEGF profoundly promoted ingrowth of the BSMC into the SIS. The penetration of the cells in response to these cytokines was confirmed using a Transwell assay. Following cellular penetration, BSMC produced significant amounts of elastic fibers under cyclic mechanical stretching at 0.1 Hz under 15% stretch, as evidenced by colorimetric assay and histology using a Verhoeff-Van Gieson stain. Protease activity was assessed in the media and found to be statistically increased in static culture following FGF-2 treatment. These findings demonstrate, for the first time, the capability of BSMC to produce histologically apparent elastin fibers in vitro. Moreover, our results suggest that a strategy involving growth factors and controlled mechanical stimulation may be used to engineer functional, elastin-rich tissue replacements using decellularized biologically derived scaffolds.

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“…Collagen and elastin concentrations were determined based on the techniques adapted from Brown et al, 24 which 3952 LONG HEISE ET AL.…”

Section: Collagen and Elastin Assessmentmentioning

confidence: 99%

Generating Elastin-Rich Small Intestinal Submucosa–Based Smooth Muscle Constructs Utilizing Exogenous Growth Factors and Cyclic Mechanical Stimulation

Heise

Ivanova

Parekh

et al. 2009

Tissue Engineering Part A

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“…Water in engineered and native arteries was calculated as (4) Volume fractions of polymer (area of PGA as a fraction of vessel area) were measured from histological images of engineered vessels at suture line and non-suture line regions using NIH Image. See Appendix for stress concentration calculations.…”

Section: Tissue Compositionmentioning

confidence: 99%

Mechanical Properties and Compositions of Tissue Engineered and Native Arteries

et al. 2007

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With the goal of mimicking the mechanical properties of a given native tissue, tissue engineers seek to culture replacement tissues with compositions similar to those of native tissues. In this report, differences between the mechanical properties of engineered arteries and native arteries were correlated with differences in tissue composition. Engineered arteries failed to match the strengths or compliances of native tissues. Lower strengths of engineered arteries resulted partially from inferior organization of collagen, but not from differences in collagen density. Furthermore, ultimate strengths of engineered vessels were significantly reduced by the presence of residual polyglycolic acid polymer fragments, which caused stress concentrations in the vessel wall. Lower compliances of engineered vessels resulted from minimal smooth muscle cell contractility and a lack of organized extracellular elastin. Organization of elastin and collagen in engineered arteries may have been partially hindered by high concentrations of sulfated glycosaminoglycans. Tissue engineers should continue to regulate cell phenotype and promote synthesis of proteins that are known to dominate the mechanical properties of the associated native tissue. However, we should also be aware of the potential negative impacts of polymer fragments and glycosaminoglycans on the mechanical properties of engineered tissues.

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“…Total sulfated GAG was then quantified using the Blyscan Glycosaminoglycan Assay kit (Biocolor), based on 1,9-dimethylmethylene blue binding. 24,25 After being hydrolyzed by 2 N NaOH for 20 min at 1108C, samples were assayed for total collagen content by a chloramine-T hydroxyproline assay. 26 …”

Section: Quantitative Biochemistrymentioning

confidence: 99%

Effects of confinement on the mechanical properties of self‐assembled articular cartilage constructs in the direction orthogonal to the confinement surface

Elder

Athanasiou

2007

Journal Orthopaedic Research

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This study examined the effects of radial confinement and passive axial compressioninduced vertical confinement, on the biomechanical, biochemical, and histological properties of selfassembled chondrocyte constructs. The self-assembled constructs, engineered without the use of an exogenous scaffold, exhibited significant increases in stiffness in the direction orthogonal to that of the confinement surface. With radial confinement, the significantly increased aggregate modulus was accompanied by increased collagen organization in the direction perpendicular to the articular surface, with no change in collagen or glycosaminoglycan (GAG) content. Additionally, radial confinement was most beneficial when applied before 2 weeks. With passive axial compression, the significantly increased Young's modulus and ultimate tensile strength were accompanied by a significant increase in collagen production. This study is the first to demonstrate the beneficial effects of confinement on tissue engineered constructs in the direction orthogonal to that of the confinement surface. ß

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Combining Chondrocytes and Smooth Muscle Cells to Engineer Hybrid Soft Tissue Constructs

Cited by 76 publications

References 24 publications

Generating Elastin-Rich Small Intestinal Submucosa–Based Smooth Muscle Constructs Utilizing Exogenous Growth Factors and Cyclic Mechanical Stimulation

Generating Elastin-Rich Small Intestinal Submucosa–Based Smooth Muscle Constructs Utilizing Exogenous Growth Factors and Cyclic Mechanical Stimulation

Mechanical Properties and Compositions of Tissue Engineered and Native Arteries

Effects of confinement on the mechanical properties of self‐assembled articular cartilage constructs in the direction orthogonal to the confinement surface

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