Interobserver variability in physician-modified endograft planning by comparison with a three-dimensional printed aortic model.

BACKGROUND: With the increasing application of fenestrated and physician-modified endografting for aneurysm repair, there is increasing concern about the accuracy of vessel position measurements based on computed tomography scans. Inaccuracies in measurements may result in a "window-shutter" or "eclipsing" phenomenon whereby the fenestration may not overlie the vessel ostium completely. We hypothesized that vessel position measurements from reconstructed imaging do not represent the true vessel position as obtained from a three-dimensional (3D) printed physical model of the visceral aortic segment. METHODS: Medical 3D modeling software was used to develop the 3D reconstructions, which were then exported to the 3D printing software. This allowed 3D models to be physically generated. The distances to the top and bottom and the angle of each of the celiac, superior mesenteric, right renal, and left renal arteries were recorded. These same measurements were obtained by each of the blinded reviewers in addition to the aortic diameter at the midpoint of each of these vessels. Measurements were compared with intraclass correlation coefficient, nonparametric Spearman rank correlation test, and one-sample t-test to assess accuracy and precision. Statistical significance was set at P < .05 for all tests. RESULTS: Both the individual measurements and the average of the measurements were statistically accurate (significant) for the bottom of the superior mesenteric artery and the top and bottom of both the right and left renal arteries. There was variability and inaccuracy in all visceral vessel angles and in the bottom of the celiac artery (the top and the angle of the celiac artery were the arbitrary referents). CONCLUSIONS: Whereas the visceral vessel orifices are largely accurately assessed and measured, the vessel angles are not. This may lead to an eclipsing phenomenon, which may contribute to branch or fenestrated vessel failure and therefore reintervention. Further efforts should assess the clinical significance of the eclipsing phenomenon and should target accurate and appropriate fenestration construction to prevent long-term morbidity.

Structural and biomechanical characterizations of porcine myocardial extracellular matrix.

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson's trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat's pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering.

Structural and biomechanical characterizations of acellular porcine mitral valve scaffolds: anterior leaflets, posterior leaflets, and chordae tendineae.

Mitral valve (MV) tissue engineering is still in its early stage, and one major challenge in MV tissue engineering is to identify appropriate scaffold materials. With the potential of acellular MV scaffolds being demonstrated recently, it is important to have a full understanding of the biomechanics of the native MV components and their acellular scaffolds. In this study, we have successfully characterized the structural and mechanical properties of porcine MV components, including anterior leaflet (AL), posterior leaflet (PL), strut chordae, and basal chordae, before and after decellularization. Quantitative DNA assay showed more than 90% reduction in DNA content, and Griffonia simplicifolia (GS) lectin immunohistochemistry confirmed the complete lack of porcine α-Gal antigen in the acellular MV components. In the acellular AL and PL, the atrialis, spongiosa, and fibrosa trilayered structure, along with its ECM constitutes, i.e., collagen fibers, elastin fibers, and portion of GAGs, were preserved. Nevertheless, the ECM of both AL and PL experienced a certain degree of disruption, exhibiting a less dense, porous ECM morphology. The overall anatomical morphology of the strut and basal chordae were also maintained after decellularization, with longitudinal morphology experiencing minimum disruption, but the cross-sectional morphology exhibiting evenly-distributed porous structure. In the acellular AL and PL, the nonlinear anisotropic biaxial mechanical behavior was overall preserved; however, uniaxial tensile tests showed that the removal of cellular content and the disruption of structural ECM did result in small decreases in maximum tensile modulus, tissue extensibility, failure stress, and failure strain for both MV leaflets and chordae.

Lectin and antibody-based histochemical techniques for cardiovascular tissue engineering.

Tissue engineering holds immense potential for treatment of cardiovascular diseases by creating living structures to replace diseased blood vessels, heart valves, and cardiac muscle. In a traditional approach, scaffolds are seeded with stem cells and subjected to stimuli in bioreactors that mimic physiologic conditions or are directly implanted into target sites in animal models. The expected results are significant cell changes, extensive remodeling of the scaffolds and creation of surrogate structures that would be deemed acceptable for tissue regeneration. Histochemical techniques are increasingly becoming essential tools in tissue engineering research. In our studies, we used lectin and antibody-based techniques to characterize novel collagen and elastin scaffolds and to ensure efficient removal of xenoantigens. Scaffolds were implanted in animals and infiltrated host cells were identified using antibodies to activated fibroblasts, macrophages, and lymphocytes. Stem cell-seeded scaffolds were subjected to mechanical strains and tested for differentiation into cardiovascular cells using antibody-based double immunofluorescence methods. Finally, living heart valves were constructed from scaffolds and stem cells, subjected to conditioning in a bioreactor and stem cell differentiation evaluated by immunofluorescence. Overall, these techniques have proven to be outstanding companions to biochemical, molecular biology and cell analysis methods used in tissue engineering research and development.

Small noncytotoxic carbon nano-onions: first covalent functionalization with biomolecules.

Small carbon nano-onions (CNOs, 6-8 shells) were prepared in high yield and functionalized with carboxylic groups by chemical oxidation. After functionalization these nanostructures were soluble in aqueous solutions. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 tetrazolium (MTS) tests showed excellent cytocompatibility of all CNOs analyzed at 30 and 300 microg mL(-1), so these carbon nanostructures can be safely used for biological applications. The first covalent functionalization of oxidized CNOs (ox-CNOs) with biomolecules, by using biotin-avidin interactions is reported here. Multilayers were prepared on a gold surface by layer-by-layer assembly and the process was monitored by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Covalent binding of molecules to the short amine-terminated organosulfur monolayers was assessed by Fourier transform infrared spectroscopy using total attenuated reflactance mode (FT-IR/HATR).

Challenges in Perioperative Animal Care for Orthotopic Implantation of Tissue-Engineered Pulmonary Valves in the Ovine Model.

BACKGROUND: Development of valvular substitutes meeting the performance criteria for surgical correction of congenital heart malformations is a major research challenge. The sheep is probably the most widely used animal model in heart valves regenerative medicine. Although the standard cardiopulmonary bypass (CPB) technique and various anesthetic and surgical protocols are reported to be feasible and safe, they are associated with significant morbidity and mortality rates. The premise of this paper is that the surgical technique itself, especially the perioperative animal care and management protocol, is essential for successful outcomes and survival. METHODS: Ten juvenile and adult female sheep aged 7.8-37.5 months and weighing 32.0-58.0 kg underwent orthotopic implantation of tissue-engineered pulmonary valve conduits on beating heart under normothermic CPB. The animals were followed-up for 6 months before scheduled euthanasia. RESULTS: Based on our observations, we established a guide for perioperative care, follow-up, and treatment containing information regarding the appropriate clinical, biological, and ultrasound examinations and recommendations for feasible and safe anesthetic, surgical, and euthanasia protocols. Specific recommendations were also included for perioperative care of juvenile versus adult sheep. CONCLUSION: The described surgical technique was feasible, with a low mortality rate and minimal surgical complications. The proposed anesthetic protocol was safe and effective, ensuring both adequate sedation and analgesia as well as rapid recovery from anesthesia without significant complications. The established guide for postoperative care, follow-up and treatment in sheep after open-heart surgery may help other research teams working in the field of heart valves tissue regeneration.

Pentagalloyl Glucose and Its Functional Role in Vascular Health: Biomechanics and Drug-Delivery Characteristics.

Pentagalloyl glucose (PGG) is an elastin-stabilizing polyphenolic compound that has significant biomedical benefits, such as being a free radical sink, an anti-inflammatory agent, anti-diabetic agent, enzymatic resistant properties, etc. This review article focuses on the important benefits of PGG on vascular health, including its role in tissue mechanics, the different modes of pharmacological administration (e.g., oral, intravenous and endovascular route, intraperitoneal route, subcutaneous route, and nanoparticle based delivery and microbubble-based delivery), and its potential therapeutic role in vascular diseases such as abdominal aortic aneurysms (AAA). In particular, the use of PGG for AAA suppression and prevention has been demonstrated to be effective only in the calcium chloride rat AAA model. Therefore, in this critical review we address the challenges that lie ahead for the clinical translation of PGG as an AAA growth suppressor.

Elastin stabilization for treatment of abdominal aortic aneurysms.

BACKGROUND: Maintaining the integrity of arterial elastin is vital for the prevention of abdominal aortic aneurysm (AAA) development. We hypothesized that in vivo stabilization of aortic elastin with pentagalloyl glucose (PGG), an elastin-binding polyphenol, would interfere with AAA development. METHODS AND RESULTS: Safety and efficacy of PGG treatment were first tested in vitro using cytotoxicity, elastin stability, and PGG-elastin interaction assays. For in vivo studies, the efficacy of PGG was evaluated within a well-established AAA model in rats on the basis of CaCl2-mediated aortic injury. With this model, PGG was delivered periadventitially at 2 separate time points during the course of AAA development; aortic diameter, elastin integrity, and other pathological aspects were monitored and evaluated in PGG-treated aortas compared with saline-treated control aortas. Our results show that a one-time periadventitial delivery of noncytotoxic levels of PGG inhibits elastin degeneration, attenuates aneurysmal expansion, and hinders AAA development in rats without interfering with the pathogenic mechanisms typical of this model, namely inflammation, calcification, and high metalloproteinase activities. PGG binds specifically to arterial elastin and, in doing so, preserves the integrity of elastic lamellae despite the presence of high levels of proteinases derived from inflammatory cells. CONCLUSIONS: Periadventitial administration of PGG hinders the development of AAA in a clinically relevant animal model. Stabilization of aortic elastin in aneurysm-prone arterial segments offers great potential toward the development of safe and effective therapies for AAAs.

Detergent-based decellularization strategy preserves macro- and microstructure of heart valves.

OBJECTIVES: Biological tissue has great potential to function as bioprostheses in patients for heart valve replacement. As these matrices are mainly xenogenic, the immunogenicity needs to be reduced by decellularization steps. Reseeding of bioscaffolds has tremendous potential to prevent calcification upon implantation, so intact microstructure of the material is mandatory. An optimal decellularization protocol of heart valves resulting in adequate preservation of the extracellular architecture has still not been developed. Biological scaffolds must be decellularized to remove the antigenic potential while preserving the complex mixture of structural and functional proteins that constitute the extracellular matrix. METHODS: Here, we compared 3 different decellularization strategies for their efficiency to remove cells completely while preserving the porcine heart valve ultrastructure. Porcine pulmonary heart valves were treated either with trypsin-ethylenediaminetetraacetic acid (TRP), a protocol using detergents in combination with nucleases (DET + ENZ), or with Accutase® solution followed by nuclease treatment (ACC + ENZ). The treated heart valves then were subjected to histological, DNA and scanning electron microscopic analyses. RESULTS: All DNA fragments were removed after ACC + ENZ treatment, whereas cellular removal was incomplete in the TRP group. TRP and ACC + ENZ-treated valves were enlarged and showed a disrupted architecture and degraded ultrastructure. In contrast, fully acellular heart valves with intact architecture, layer composition and surface topography were achieved with DET + ENZ treatment. DET + ENZ treatment yielded excellent results in terms of preservation of material architecture and removal of DNA content. CONCLUSIONS: Compared to TRP and ACC + ENZ procedures, DET + ENZ-treated porcine pulmonary heart valves demonstrated well-preserved macroscopic structures and microscopic matrix components and represent an excellent scaffold for further application in tissue engineering.

Neomycin prevents enzyme-mediated glycosaminoglycan degradation in bioprosthetic heart valves.

Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, BHVs have limited durability and fail either due to degeneration or calcification. Glycosaminoglycans (GAGs), one of the integral components of heart valve cuspal tissue, are not stabilized by conventional glutaraldehyde crosslinking. Previously we have shown that valvular GAGs could be chemically fixed with GAG-targeted chemistry. However, chemically stabilized GAGs were only partially stable to enzymatic degradation. In the present study an enzyme inhibitor was incorporated in the cusps to effectively prevent enzymatic degradation. Thus, neomycin trisulfate, a known hyaluronidase inhibitor, was incorporated in cusps via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry followed by glutaraldehyde crosslinking (NEG). Controls included cusps crosslinked with either EDC/NHS followed by glutaraldehyde (ENG) or only with glutaraldehyde (GLUT). NEG group showed improved resistance to in vitro enzymatic degradation as compared to GLUT and ENG groups. All groups showed similar collagen stability, measured as a thermal denaturation temperature by differential scanning calorimetry (DSC). The cusps were implanted subdermally in rats to study in vivo degradation of GAGs. NEG group preserved significantly more GAGs than ENG and GLUT. NEG and ENG groups showed reduced calcification than GLUT.

In vivo cellular repopulation of tubular elastin scaffolds mediated by basic fibroblast growth factor.

In vivo tissue engineering has been explored as a method to repopulate scaffolds with autologous cells to create a functional, living, and non-immunogenic tissue substitute. In this study, we describe an approach to in vivo cellular repopulation of a tissue-derived tubular elastin scaffold. Pure elastin scaffolds were prepared from porcine carotid arteries (elastin tubes). Elastin tubes were filled with agarose gel containing basic fibroblast growth factor (bFGF) to allow sustained release of growth factor. These tubes were implanted in subdermal pouches in adult rats. The elastin tubes with growth factor had significantly more cell infiltration at 28 days than those without growth factor. Immunohistochemical staining indicated that most of these cells were fibroblasts, of which a few were activated fibroblasts (myofibroblasts). Microvasculature was also observed within the scaffolds. Macrophage infiltration was seen at 7 days, which diminished by 28 days of implantation. None of the elastin tubes with bFGF calcified. These results demonstrated that the sustained release of bFGF brings about repopulation of elastin scaffolds in vivo while inhibiting calcification. Results showing myofibroblast infiltration and vascularization are encouraging since such an in vivo implantation technique could be used for autologous cell repopulation of elastin scaffolds for vascular graft applications.

Glycosaminoglycan-targeted fixation for improved bioprosthetic heart valve stabilization.

Numerous crosslinking chemistries and methodologies have been investigated as alternative fixatives to glutaraldehyde (GLUT) for the stabilization of bioprosthetic heart valves (BHVs). Particular attention has been paid to valve leaflet collagen and elastin stability following fixation. However, the stability of glycosaminoglycans (GAGs), the primary component of the spongiosa layer of the BHV, has been largely overlooked despite recent evidence provided by our group illustrating their structural and functional importance. In the present study we investigate the ability of two different crosslinking chemistries: sodium metaperiodate (NaIO(4)) followed by GLUT (PG) and 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) followed by GLUT (ENG) to stabilize GAGs within BHV leaflets and compare resulting leaflet characteristics with that of GLUT-treated tissue. Incubation of fixed leaflets in GAG-degrading enzymes illustrated in vitro resistance of GAGs towards degradation in PG and ENG treated tissue while GLUT fixation alone was not effective in preventing GAG loss from BHV leaflets. Following subdermal implantation, significant amounts of GAGs were retained in leaflets in the ENG group in comparison to GLUT-treated tissue, although GAG loss was evident in all groups. Utilizing GAG-targeted fixation did not alter calcification potential of the leaflets while collagen stability was maintained at levels similar to that observed in conventional GLUT-treated tissue.

Toward cell therapy for vascular calcification: osteoclast-mediated demineralization of calcified elastin.

BACKGROUND: Elastin-oriented vascular calcification is a clinically significant feature, which involves formation of ectopic bone-like structures. Taking advantage of the similarities between arterial calcification and bone regulation, our hypothesis was that therapeutic approaches for limitation of vascular calcification could be developed using site-specific delivery of autologous osteoclasts. In the present paper, we tested the hypothesis that bone-marrow-derived osteoclasts have the ability to demineralize calcified elastin, without significant alterations in elastin integrity. METHODS: Active, multinucleated osteoclasts were obtained by in vitro maturation of rat bone-marrow-derived progenitor cells in the presence of vitamin D(3) and retinoic acid. Cell phenotype was validated by staining for tartrate-resistant acid phosphatase, formation of resorption pits on hydroxyapatite-coated disks, and RT-PCR for identification of cathepsin K gene expression. Calcified aortic elastin was seeded with osteoclasts and calcium, and phosphorous levels were monitored in gels and culture media to detect demineralization of elastin. Soluble elastin peptides were also monitored in culture media for elastin degradation. For in vivo experiments, pure aortic elastin was coimplanted with allogenic osteoclasts subdermally into rats, and the degree of elastin calcification and degradation was evaluated using mineral analysis and desmosine quantitation. RESULTS: Bone-marrow-derived osteoclasts reduced mineral content of calcified elastin in vitro by 80%. Moreover, in vivo implantation of allogenic osteoclasts in the vicinity of calcifying elastin limited elastin mineralization by almost 50%, in the absence of detectable elastin degradation. CONCLUSIONS: Osteoclasts have the ability to demineralize calcified elastin, without significant alterations in elastin integrity.

Structural requirements for stabilization of vascular elastin by polyphenolic tannins.

Elastin-associated degeneration and calcification are potential causes of long-term failure of glutaraldehyde (Glut) fixed tissue bioprostheses used in cardiovascular surgery. This vulnerability may be attributed to the inability of Glut to cross-link and adequately protect vascular elastin from enzymatic attack. Tannic acid (TA), a poly galloyl glucose (Glc), is compatible with Glut fixation, binds to vascular elastin, improves resistance to degradation and reduces in vivo calcification. While these results provided evidence of a beneficial interaction between elastin and TA, the nature and mechanisms of these interactions are unclear; moreover, TA-elastin binding exhibits a partial instability after long-term interaction with vascular elastin which could contribute to issues of implant toxicity. In present studies, we used resistance to elastase, mechanical properties, and cell viability assays to evaluate the elastin-stabilizing potential and cytotoxicity of TA derivatives and individual TA components such as acetylated TA (AcTA), pentagalloylglucose (PGG), free gallic acid (Gall) and Glc. Our comparative study demonstrates that polyphenolic hydroxyl groups are the main structural groups essential to the interaction between TA and elastin. Furthermore, we show that PGG, the core structure of TA, possesses the same unique elastin-stabilizing qualities of TA, yet it is much less cytotoxic than TA and thus could be potentially useful as an elastin-stabilizing agent for cardiovascular bioprostheses.

Stability and function of glycosaminoglycans in porcine bioprosthetic heart valves.

Glycosaminoglycans (GAGs) are important structural and functional components in native aortic heart valves and in glutaraldehyde (Glut)-fixed bioprosthetic heart valves (BHVs). However, very little is known about the fate of GAGs within the extracellular matrix of BHVs and their contribution to BHV longevity. BHVs used in heart valve replacement surgery have limited durability due to mechanical failure and pathologic calcification. In the present study we bring evidence for the dramatic loss of GAGs from within the BHV cusp structure during storage in saline and both short- and long-term Glut fixation. In order to gain insight into role of GAGs, we compared properties of fresh and Glut-fixed porcine heart valve cusps before and after complete GAG removal. GAG removal resulted in significant morphological and functional tissue alterations, including decreases in cuspal thickness, reduction of water content and diminution of rehydration capacity. By virtue of this diminished hydration, loss of GAGs also greatly increased the "with-curvature" flexural rigidity of cuspal tissue. However, removal of GAGs did not alter calcification potential of BHV cups when implanted in the rat subdermal model. Controlling the extent of pre-implantation GAG degradation in BHVs and development of improved GAG crosslinking techniques are expected to improve the mechanical durability of future cardiovascular bioprostheses.

Biocompatibility and remodeling potential of pure arterial elastin and collagen scaffolds.

Surgical therapy of cardiovascular disorders frequently requires replacement of diseased tissues with prosthetic devices or grafts. In typical tissue engineering approaches, scaffolds are utilized to serve as templates to support cell growth and remodeling. Decellularized vascular matrices have been previously investigated as scaffolds for tissue engineering. However, cell migration into these scaffolds was inadequate due to the very tight matrix organization specific to the aortic structure. To address this problem, we prepared two types of decellularized scaffolds from porcine vascular tissues. Pure elastin scaffolds and pure collagen scaffolds were prepared by selectively removing the collagen component or elastin, respectively. In the current study, we use a subdermal implantation model to demonstrate that arterial elastin and collagen scaffolds exhibit enhanced potential for repopulation by host cells in vivo. Notably, numerous new collagen fibers and bundles were found within the remodeled elastin scaffolds and new elastin fibers within collagen scaffolds, respectively, clearly indicating their ability to support de novo extracellular matrix synthesis. We also show that biological cues such as growth factors are required for efficient repopulation of elastin and collagen scaffolds. Finally, we bring evidence that these scaffolds can be endothelialized in vitro for thrombosis resistance and thus can serve as promising candidates for cardiovascular tissue engineering.

Immunomodulatory effects of adipose tissue-derived stem cells on elastin scaffold remodeling in diabetes.

Diabetes is a major risk factor for the progression of vascular disease, contributing to elevated levels of glycoxidation, chronic inflammation and calcification. Tissue engineering emerges as a potential solution for the treatment of vascular diseases however there is a considerable gap in the understanding of how scaffolds and stem cells will perform in patients with diabetes. We hypothesized that adipose tissue-derived stem cells (ASCs) by virtue of their immunosuppressive potential would moderate the diabetes-intensified inflammatory reactions and induce positive construct remodeling. To test this hypothesis, we prepared arterial elastin scaffolds seeded with autologous ASCs and implanted them subdermally in diabetic rats and compared inflammatory markers, macrophage polarization, matrix remodeling, calcification and bone protein expression to control scaffolds implanted with and without cells in nondiabetic rats. ASC-seeded scaffolds exhibited lower levels of CD8+ T-cells and CD68+ pan-macrophages and higher numbers of M2 macrophages, smooth muscle cell-like and fibroblast-like cells. Calcification and osteogenic markers were reduced in ASCseeded scaffolds implanted in non-diabetic rats but remained unchanged in diabetes, unless the scaffolds were first pre-treated with penta-galloyl glucose (PGG), a known anti-oxidative elastin-binding polyphenol. In conclusion, autologous ASC seeding in elastin scaffolds is effective in combating diabetes-related complications. To prevent calcification, the oxidative milieu needs to be reduced by elastin-binding antioxidants such as PGG.

Longitudinal Stretching for Maturation of Vascular Tissues Using Magnetic Forces.

Cellular spheroids were studied to determine their use as "bioinks" in the biofabrication of tissue engineered constructs. Specifically, magnetic forces were used to mediate the cyclic longitudinal stretching of tissues composed of Janus magnetic cellular spheroids (JMCSs), as part of a post-processing method for enhancing the deposition and mechanical properties of an extracellular matrix (ECM). The purpose was to accelerate the conventional tissue maturation process via novel post-processing techniques that accelerate the functional, structural, and mechanical mimicking of native tissues. The results of a forty-day study of JMCSs indicated an expression of collagen I, collagen IV, elastin, and fibronectin, which are important vascular ECM proteins. Most notably, the subsequent exposure of fused tissue sheets composed of JMCSs to magnetic forces did not hinder the production of these key proteins. Quantitative results demonstrate that cyclic longitudinal stretching of the tissue sheets mediated by these magnetic forces increased the Young's modulus and induced collagen fiber alignment over a seven day period, when compared to statically conditioned controls. Specifically, the elastin and collagen content of these dynamically-conditioned sheets were 35- and three-fold greater, respectively, at seven days compared to the statically-conditioned controls at three days. These findings indicate the potential of using magnetic forces in tissue maturation, specifically through the cyclic longitudinal stretching of tissues.