Circumferential Alignment Research Articles

An X-Ray Scattering Study into the Structural Basis of Corneal Refractive Function in an Avian Model

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3–4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.

Digital micromirror device projection printing system for meniscus tissue engineering

Meniscus degeneration due to age or injury can lead to osteoarthritis. Although promising, current cell-based approaches show limited success. Here we present three-dimensional methacrylated gelatin (GelMA) scaffolds patterned via projection stereolithography to emulate the circumferential alignment of cells in native meniscus tissue. Cultured human avascular zone meniscus cells from normal meniscus were seeded on the scaffolds. Cell viability was monitored, and new tissue formation was assessed by gene expression analysis and histology after 2weeks in serum-free culture with transforming growth factor β1 (10ngml−1). Light, confocal and scanning electron microscopy were used to observe cell–GelMA interactions. Tensile mechanical testing was performed on unseeded, fresh scaffolds and 2-week-old cell-seeded and unseeded scaffolds. 2-week-old cell–GelMA constructs were implanted into surgically created meniscus defects in an explant organ culture model. No cytotoxic effects were observed 3weeks after implantation, and cells grew and aligned to the patterned GelMA strands. Gene expression profiles and histology indicated promotion of a fibrocartilage-like meniscus phenotype, and scaffold integration with repair tissue was observed in the explant model. We show that micropatterned GelMA scaffolds are non-toxic, produce organized cellular alignment, and promote meniscus-like tissue formation. Prefabrication of GelMA scaffolds with architectures mimicking the meniscus collagen bundle organization shows promise for meniscal repair. Furthermore, the technique presented may be scaled up to repair larger defects.

Microstructural characterization of CPPD and hydroxyapatite crystal depositions on human menisci

AbstractMeniscus is a fibrocartilaginous tissue composed mainly of water and a dense elaborate collagen network with a predominantly circumferential alignment. Crystal formation and accumulation on meniscal tissue is frequently observed especially in elderly. In this study, we used X‐ray diffraction (XRD), FTIR and FT‐Raman for the structural identification of the depositions and Optical microscopy, Scanning Electron microscopy (SEM/EDX) and Atomic Force microscopy (AFM), in order to investigate the structural relationship between the crystal deposits and the collagen fibers of human meniscal tissues. We are reporting on the formation of intercalary “colonies” of Calcium Pyrophosphate Dihydrate (CPPD) crystals with two distinct morphologies corresponding to the monoclinic and the triclinic phase, as well as the formation of micro‐aggregations composed of nano‐crystalline HAP aggregations which are developed along the longitudinal axis of collagen fibers without extensively disturbing the collagens arrangement.

Tubular hydrogels of circumferentially aligned nanofibers to encapsulate and orient vascular cells

There is a great clinical need for tissue engineered blood vessels that could be used to replace or bypass damaged arteries. The success of such grafts will depend strongly on their ability to mimic the cellular and matrix organization found in native arteries, but currently available cell scaffolds such as electrospun fibers or hydrogels lack the ability to simultaneously encapsulate and align cells. Our laboratory has recently developed liquid crystalline solutions of peptide amphiphile nanofibers that form aligned domains at exceedingly low concentrations (<1wt%), and can be trapped as gels with macroscopic alignment using low shear rates and ionic crosslinking. We describe here the use of these systems to fabricate tubes with macroscopic circumferential alignment and demonstrate their potential as arterial cell scaffolds. The nanofibers in these tubes were circumferentially aligned by applying small amounts of shear in a custom built flow chamber prior to gelation. Small angle X-ray scattering confirmed that the direction of nanofiber alignment was the same as the direction of shear flow. We also show the encapsulation of smooth muscle cells during the fabrication process without compromising cell viability. After two days in culture the encapsulated cells oriented their long axis in the direction of nanofiber alignment thus mimicking the circumferential alignment seen in native arteries. Cell density roughly doubled after 12 days demonstrating the scaffold's ability to facilitate necessary graft maturation. Since these nanofiber gels are composed of >99% water by weight, the cells have abundant room for proliferation and remodeling. In contrast to previously reported arterial cell scaffolds, this new material can encapsulate cells and direct cellular organization without the requirement of external stimuli or gel compaction.

Abstract 351: Collagen Alignment Correlates with Differential Biaxial Stiffness in Nonhuman Primate Carotid and Femoral Arteries

Introduction: There are known differences in arterial compliance in different regions of the arterial tree. These differences are thought to be determined by pressure and flow demands of their capacitance artery and their respective tissue bed perfusion requirements, and they may play a role in certain arteries having a greater or lesser prevalence of disease as well as favorable response to intervention. Both the femoral and carotid arteries are susceptible to peripheral arterial disease. Here we define the differences in compliance and analyze the microstructural collagen alignment between these arteries to assess the hypothesis that arterial stiffness, defined as the inverse of compliance, will correlate with collagen alignment. Methods: In-vitro biaxial mechanical tests using a custom system were performed on carotid and femoral arteries excised from non-human primates at necroscopy. Pressure-diameter and force-axial stretch responses were measured, and the arterial compliance ( C , units of %/mmHg x 10 -2 ) at the physiological pressure range to calculate the axial stretch. To characterize the microstructure, planar sections of the arteries were imaged under two-photon confocal microscopy. Three-dimensional, z-stack images were taken at two different locations across the medial thickness for each artery. The images were post-processed using a fast Fourier transform analysis to the quantify fiber angle distributions. Circular descriptive statistics was then used to calculate a fiber dispersion index ( FDI ), where 0≤ FDI ≤1. FDI =0 signifies random fiber dispersion and FDI =1 signifies complete circumferential fiber alignment. T-test statistical analysis was used to compare groups. P&lt;.05 was considered significant. Results: Biaxial testing identified femoral arteries as being more than twice as stiff as the carotid artery (P=.01; Figure). Microstructural fiber analysis demonstrated significantly increased collagen alignment in the femoral compared to carotid artery ( FDI =0.31, n =4 for femoral versus FDI =0.26, n=4 for carotid, p =0.03).Conclusion: Arterial stiffness in the femoral and carotid artery of nonhuman primates correlates with collagen alignment in the vessel media. The absence of a cellular requirement in the observed mechanical behavior of these arteries suggests that not only matrix composition but also alignment contribute to arterial stiffness. Differential arterial responses to procedures, particularly those endovascular in nature, may be expected by these results.

FtsZ rings and helices: physical mechanisms for the dynamic alignment of biopolymers in rod-shaped bacteria

In many bacterial species, the protein FtsZ forms a cytoskeletal ring that marks the future division site and scaffolds the division machinery. In rod-shaped bacteria, most frequently membrane-attached FtsZ rings or ring fragments are reported and occasionally helices. By contrast, axial FtsZ clusters have never been reported. In this paper, we investigate theoretically how dynamic FtsZ aggregates align in rod-shaped bacteria. We study systematically different physical mechanisms that affect the alignment of FtsZ polymers using a computational model that relies on autocatalytic aggregation of FtsZ filaments at the membrane. Our study identifies a general tool kit of physical and geometrical mechanisms by which rod-shaped cells align biopolymer aggregates. Our analysis compares the relative impact of each mechanism on the circumferential alignment of FtsZ as observed in rod-shaped bacteria. We determine spontaneous curvature of FtsZ polymers and axial confinement of FtsZ on the membrane as the strongest factors. Including Min oscillations in our model, we find that these stabilize axial and helical clusters on short time scales, but promote the formation of an FtsZ ring at the cell middle at longer times. This effect could provide an explanation to the long standing puzzle of transiently observed oscillating FtsZ helices in Escherichia coli cells prior to cell division.

Magnetic resonance in tissue engineering

Magnetic resonance in tissue engineering

Anaplastic astrocytoma with angiocentric ependymal differentiation

Angiocentric glioma (AG) is an epileptogenic benign cerebral tumor primarily affecting children and young adults, and characterized histopathologically by an angiocentric pattern of growth of monomorphous bipolar cells with features of ependymal differentiation (WHO grade I). We report an unusual cerebral glial tumor in a 66-year-old woman with generalized tonic-clonic seizure; the patient also had a 6-year history of headache. On MRI, the tumor appeared as a large T2-hyperintense lesion involving the right insular gyri-anterior temporal lobe, with post-contrast enhancement in the insula region. Histopathologically, the tumor involving the insular cortex-subcortical white matter was composed of GFAP-positive glial cells showing two different morphologies: one type had monomorphous bipolar cytoplasm and was angiocentric with circumferential alignment to the blood vessels, with dot-like structures positive for epithelial membrane antigen and a Ki-67 labeling index of <1%, and the other was apparently astrocytic, being diffusely and more widely distributed in the parenchyma, showing mitoses and a Ki-67 labeling index of >5%. In the anterior temporal lobe, a diffuse increase in the number of astrocytic cells was evident in part of the cortex and subcortical white matter. On the basis of these findings, we considered whether the present tumor may represent an unusual example of AG with infiltrating astrocytic cells showing primary anaplastic features (AG with anaplastic features), or anaplastic astrocytoma showing primary vascular-associated ependymal differentiation (anaplastic astrocytoma with angiocentric ependymal differentiation). At present, the latter appears to be the more appropriate interpretation.

Implantable arterial grafts from human fibroblasts and fibrin using a multi-graft pulsed flow-stretch bioreactor with noninvasive strength monitoring

Tissue-engineered arteries based on entrapment of human dermal fibroblasts in fibrin gel yield completely biological vascular grafts that possess circumferential alignment characteristic of native arteries and essential to their mechanical properties. A bioreactor was developed to condition six grafts in the same culture medium while being subjected to similar cyclic distension and transmural flow resulting from pulsed flow distributed among the graft lumens via a manifold. The lumenal pressure and circumferential stretch were noninvasively monitored and used to calculate stiffness in the range of 80–120 mmHg and then to successfully predict graft burst strength. The length of the graft was incrementally shortened during bioreactor culture to maintain circumferential alignment and achieve mechanical anisotropy comparable to native arteries. After 7–9 weeks of bioreactor culture, the fibrin-based grafts were extensively remodeled by the fibroblasts into circumferentially-aligned tubes of collagen and other extracellular matrix with burst pressures in the range of 1400–1600 mmHg and compliance comparable to native arteries. The tissue suture retention force was also suitable for implantation in the rat model and, with poly(lactic acid) sewing rings entrapped at both ends of the graft, also in the ovine model. The strength achieved with a biological scaffold in such a short duration is unprecedented for an engineered artery.

Electrospinning of small diameter 3-D nanofibrous tubular scaffolds with controllable nanofiber orientations for vascular grafts

The control of nanofiber orientation in nanofibrous tubular scaffolds can benefit the cell responses along specific directions. For small diameter tubular scaffolds, however, it becomes difficult to engineer nanofiber orientation. This paper reports a novel electrospinning technique for the fabrication of 3-D nanofibrous tubular scaffolds with controllable nanofiber orientations. Synthetic absorbable poly-ε-caprolactone (PCL) was used as the model biomaterial to demonstrate this new electrospinning technique. Electrospun 3-D PCL nanofibrous tubular scaffolds of 4.5mm in diameter with different nanofiber orientations (viz. circumferential, axial, and combinations of circumferential and axial directions) were successfully fabricated. The degree of nanofiber alignment in the electrospun 3-D tubular scaffolds was quantified by using the fast Fourier transform (FFT) analysis. The results indicated that excellent circumferential nanofiber alignment could be achieved in the 3-D nanofibrous PCL tubular scaffolds. The nanofibrous tubular scaffolds with oriented nanofibers had not only directional mechanical property but also could facilitate the orientation of the endothelial cell attachment on the fibers. Multiple layers of aligned nanofibers in different orientations can produce 3-D nanofibrous tubular scaffolds of different macroscopic properties.

Self-Assembly of Aligned Tissue-Engineered Annulus Fibrosus and Intervertebral Disc Composite Via Collagen Gel Contraction

Many cartilaginous tissues such as intervertebral disc (IVD) display a heterogeneous collagen microstructure that results in mechanical anisotropy. These structures are responsible for mechanical function of the tissue and regulate cellular interactions and metabolic responses of cells embedded within these tissues. Using collagen gels seeded with ovine annulus fibrosus cells, constructs of varying structure and heterogeneity were created to mimic the circumferential alignment of the IVD. Alignment was induced within gels by contracting annular gels around an inner boundary using both a polyethylene center and alginate center to create a composite engineered IVD. Collagen alignment and heterogeneity were measured using second harmonic generation microscopy. Decreasing initial collagen density from 2.5 mg/mL to 1 mg/mL produced greater contraction of constructs, resulting in gels that were 55% and 6.2% of the original area after culture, respectively. As a result, more alignment occurred in annular-shaped 1 mg/mL gels compared with 2.5 mg/mL gels (p < 0.05). This alignment was also produced in a composite-engineered IVD with alginate nucleus pulposus. The resulting collagen alignment could promote further aligned collagen development necessary for the creation of a mechanically functional tissue-engineered IVD.

Structural inhomogeneity and fiber orientation in the inner arterial media

The microstructural orientation of vascular wall constituents is of interest to scientists and clinicians because alterations in their native states are associated with various cardiovascular diseases. In the arterial media, the orientation of these constituents is often described as circumferential. However, it has been noted that, just below the endothelial surface, the vascular wall constituents are oriented axially. To further study this reported change in orientation, and to resolve previous observations (which were made under conditions of no load), we used nonlinear optical microscopy to examine the orientation of collagen and elastin fibers in the inner medial region of bovine common carotid arteries. Images were obtained from this part of the arterial wall under varying degrees of mechanical strain: 0%, 10% axial, 10% circumferential, and 10% biaxial. We observed that close to the endothelium these components are aligned in the axial direction but abruptly change to a circumferential alignment at a depth of approximately 20 mum from the endothelial surface. The application of mechanical strain resulted in a significantly greater degree of fiber alignment, both collagen and elastin, in the strain direction, regardless of their initial unloaded orientation. Furthermore, variations in strain conditions resulted in an increase or a decrease in the overall degree of fiber alignment in the subendothelial layer depending on the direction of the applied strain. This high-resolution investigation adds more detail to existing descriptions of complex structure-function relationships in vascular tissue, which is essential for a better understanding of the pathophysiological processes resulting from injury, disease progression, and interventional therapies.

Ultrastructural changes in the retinopathy, globe enlarged (rge) chick cornea

In the cornea, the precise organisation of fibrillar collagen and associated proteoglycans comprising the stromal extracellular matrix plays a major role in governing tissue form and function. Recently, abnormal collagen alignment was noted in the misshapen corneas of mature chickens affected by the retinopathy, globe enlarged (rge) mutation. Here we further characterize corneal ultrastructural changes as the rge eye develops post-hatch. Wide-angle X-ray scattering disclosed alteration to dominant collagen lamellae directions in the rge chick cornea, compared to age-matched controls. These changes accompanied eye globe enlargement and corneal flattening in affected birds, manifesting as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Collagen intermolecular separation was unchanged in rge. However, small-angle X-ray scattering results suggest collagen fibril separation and diameter increase more rapidly towards the corneal periphery in rge at 3 months post-hatch compared to controls, although central collagen fibril diameter was unchanged. By transmission electron microscopy utilising cuprolinic blue stain, the morphology and distribution of stromal proteoglycans were unaltered in rge corneas otherwise demonstrating abnormal collagen fibril organisation. From a numerical simulation of tissue mechanics, progressive remodelling of stromal collagen in rge during globe enlargement post-hatch appears to be related to the corneal morphometric changes presented by the disease.

An in vitro assay of collagen fiber alignment by acupuncture needle rotation

BackgroundDuring traditional acupuncture therapy, soft tissues attach to and wind around the acupuncture needle. To study this phenomenon in a controlled and quantitative setting, we performed acupuncture needling in vitro.MethodsAcupuncture was simulated in vitro in three-dimensional, type I collagen gels prepared at 1.5 mg/ml, 2.0 mg/ml, and 2.5 mg/ml collagen, and either crosslinked with formalin or left untreated. Acupuncture needles were inserted into the gels and rotated via a computer-controlled motor at 0.3 rev/sec for up to 10 revolutions while capturing the evolution of birefringence under cross-polarization.ResultsSimulated acupuncture produced circumferential alignment of collagen fibers close to the needle that evolved into radial alignment as the distance from the needle increased, which generally matched observations from published tissue explant studies. All gels failed prior to 10 revolutions, and the location of failure was near the transition between circumferential and radial alignment. Crosslinked collagen failed at a significantly lower number of revolutions than untreated collagen, whereas collagen concentration had no effect on gel failure. The strength of the alignment field increased with increasing collagen concentration and decreased with crosslinking. Separate studies were performed in which the gel thickness and depth of needle insertion were varied. As gel thickness increased, gels failed at fewer needle revolutions. For the same depth of insertion, alignment was greater in thinner gels. Alignment increased as the depth of insertion increased.ConclusionThese results indicate that the mechanostructural properties of soft connective tissues may affect their response to acupuncture therapy. The in vitro model provides a platform to study mechanotransduction during acupuncture in a highly controlled and quantitative setting.

Full-field deformation of bovine cornea under constrained inflation conditions

The viscoelastic response of bovine corneas was characterized using in vitro inflation (bulge) experiments combined with spatially-resolved deformation mapping via digital image correlation. A complex fixture conforming to the limbal annulus was developed to hold the attached sclera rigid while allowing deformation only in the cornea. A statistical set of experiments was performed for a pressure range of 3.6–8 kPa (27–60 mmHg), representing nominal bovine intraocular pressure (IOP) to acute glaucoma conditions. A broader pressure range of 0–32 kPa (0–240 mmHg) was also examined to characterize the nonlinear finite deformation behavior of the tissue. Results showed that for pressures near and above IOP, the majority of the deformation was localized in the limbus and peripheral regions, which left the central cornea largely undeformed. This observation was consistent with the known preferred circumferential alignment of collagen fibrils outside of the central cornea. In general, the inflation experiments observed viscoelastic behavior in the form of rate-dependent hysteresis in the pressure–deformation response of the apex of the cornea, creep in the apex deformation at a constant inflation pressure, and relaxation in the pressure response at a constant inflation volume. The 3.6–8 kPa (27–60 mmHg) pressure range produced small viscoelastic deformations and a nearly linear pressure–deformation response, which suggests that for physiological pressure ranges, the cornea can be approximated as a linear viscoelastic or linear pseudo-elastic material.

Functional Tissue-Engineered Valves from Cell-Remodeled Fibrin with Commissural Alignment of Cell-Produced Collagen

Heart valve replacements composed of living tissue that can adapt, repair, and grow with a patient would provide a more clinically beneficial option than current inert replacements. Bioartificial valves were produced by entrapping human dermal fibroblasts within a fibrin gel. Using a mold design that presents appropriate mechanical constraints to the cell-induced fibrin gel compaction, gross fiber alignment (commissure-to-commissure alignment in the leaflets and circumferential alignment in the root) and the basic geometry of a native aortic valve were obtained. After static incubation on the mold in complete medium supplemented with transforming growth factor beta 1, insulin, and ascorbate, collagen fibers produced by the entrapped cells were found to coalign with the fibrin based on histological analyses. The resultant tensile mechanical properties were anisotropic. Ultimate tensile strength and tensile modulus of the leaflets in the commissural direction were 0.53 and 2.34 MPa, respectively. The constructs were capable of withstanding backpressure commensurate with porcine aortic valves in regurgitation tests (330 mmHg) and opened and closed under physiological pressure swings of 10 and 20 mmHg, respectively. These data support proof of principle of using cell-remodeled fibrin gel to produce tissue-engineered valve replacements.

Tissue-Engineered Valves with Commissural Alignment

We present a novel approach to producing bioartificial valves using the tissue-equivalent method of entrapping cells within a biopolymer gel and using a mold design that presents appropriate mechanical constraints to the cell-induced gel compaction to yield both the fibril alignment and the geometry of a native valve. Bileaflet valves were fabricated from bovine collagen and neonatal human dermal fibroblasts as proof of principle. The resultant valves possessed both commissure-tocommissure alignment of collagen fibers in the leaflets and circumferential alignment in the root. While this alignment was manifested in planar biaxial tensile mechanical properties, histology of the leaflets revealed an aligned collagen matrix but lacking other extracellular matrix (ECM) components present in the native valve. The apparent lack of ECM production by the fibroblasts after contracting and aligning the collagen fibrils is consistent with peak loads during biaxial testing being only approximately 10% of native leaflet values and a 0:1 coupling index that was only approximately 50% of native leaflet values despite exhibiting comparable values for the anisotropy index.

Separate pathways of RNA recruitment lead to the compartmentalization of the zebrafish germ plasm

The maternal RNAs vasa, dead end, nanos1, and daz-like all become localized to the peripheral ends of the first and second cleavage furrows, where they form part of the zebrafish germ plasm. We show that aggregates of a first class of germ plasm components, which include dead end, nanos1, and vasa RNAs, are initially present in a wide cortical band at the animal pole. Aggregates containing these three RNAs appear to be associated with f-actin, which during the first cell cycle undergoes a microtubule-dependent movement towards the periphery as well as circumferential alignment. These cytoskeletal rearrangements lead to the further aggregation of particles containing these RNAs and their concomitant recruitment to the forming furrow. Aggregates containing a second class of germ plasm RNA components, which include the transcript for daz-like, translocate along the plane of the cortex towards the animal pole, where they are recruited to the germ plasm. After recruitment to the furrow, these two classes of RNAs occupy overlapping yet distinct regions of the germ plasm, and this arrangement is maintained during the early cleavage stages. Our observations suggest that separate pathways of RNA recruitment facilitate the compartmentalization of the zebrafish germ plasm.

Robust Design of a Natural Gas Transmission Pipeline Inspection Tool using the Taguchi Method

ABSTRACT The capability of a natural gas transmission pipeline inspection tool to detect cracks and corrosion is affected by a number of factors, namely, magnetization level, sensor spacing, radial liftoff, and circumferential alignment of the sensors relative to a defect. The first two factors are design parameters and are controllable factors. The last two factors are hard to control on site and are treated as noise factors. The Taguchi method is utilized to obtain an efficient set of design parameters that are robust with respect to noise and other variations in test parameters. This article first introduces the basic idea underlying the Taguchi method. Procedures for designing and simulation of experiments are given along with simulation results, which are analyzed using the main effects and analysis of variance techniques. Conclusive remarks based on the analysis are finally summarized.

Development of a three-dimensional physiological model of the internal anal sphincter bioengineered in vitro from isolated smooth muscle cells

Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is largely attributed to decreased mechanical efficiency of the internal anal sphincter (IAS), yet little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. The object of this study was to develop a three-dimensional (3-D) physiological model of the IAS bioengineered in vitro from isolated smooth muscle cells. Smooth muscle cells isolated from the IAS of rabbits were seeded in culture on top of a loose fibrin gel, where they migrated and self-assembled in circumferential alignment. As the cells proliferated, the fibrin gel contracted around a 5-mm-diameter SYLGARD mold, resulting in a 3-D cylindrical ring of sphincteric tissue. We found that 1) the bioengineered IAS rings generated a spontaneous basal tone, 2) stimulation with 8-bromo-cAMP (8-Br-cAMP) caused a sustained decrease in the basal tone (relaxation) that was calcium-independent, 3) upon stimulation with ACh, bioengineered IAS rings showed a calcium- and concentration-dependent peak contraction at 30 s that was sustained for 4 min, 4) addition of 8-Br-cAMP induced rapid relaxation of ACh-induced contraction and force generation of IAS rings, and 5) bioengineered sphincter rings show striking functional differences when compared with bioengineered rings made from isolated colonic smooth muscle cells. This is the first report of a 3-D in vitro model of a gastrointestinal smooth muscle IAS. Bioengineered IAS rings demonstrate physiological functionality and may be used in the elucidation of the mechanisms causing sphincter malfunction.