Age-dependent changes of stress and strain in the human heart valve and their relation with collagen remodeling.

In order to create tissue-engineered heart valves with long-term functionality, it is essential to fully understand collagen remodeling during neo-tissue formation. Collagen remodeling is thought to maintain mechanical tissue homeostasis. Yet, the driving factor of collagen remodeling remains unidentified. In this study, we determined the collagen architecture and the geometric and mechanical properties of human native semilunar heart valves of fetal to adult age using confocal microscopy, micro-indentation and inverse finite element analysis. The outcomes were used to predict age-dependent changes in stress and stretch in the heart valves via finite element modeling. The results indicated that the circumferential stresses are different between the aortic and pulmonary valve, and, moreover, that the stress increases considerably over time in the aortic valve. Strikingly, relatively small differences were found in stretch with time and between the aortic and pulmonary valve, particularly in the circumferential direction, which is the main determinant of the collagen fiber stretch. Therefore, we suggest that collagen remodeling in the human heart valve maintains a stretch-driven homeostasis. Next to these novel insights, the unique human data set created in this study provides valuable input for the development of numerical models of collagen remodeling and optimization of tissue engineering. STATEMENT OF SIGNIFICANCE: Annually, over 280,000 heart valve replacements are performed worldwide. Tissue engineering has the potential to provide valvular disease patients with living valve substitutes that can last a lifetime. Valve functionality is mainly determined by the collagen architecture. Hence, understanding collagen remodeling is crucial for creating tissue-engineered valves with long-term functionality. In this study, we determined the structural and material properties of human native heart valves of fetal to adult age to gain insight into the mechanical stimuli responsible for collagen remodeling. The age-dependent evolutionary changes in mechanical state of the native valve suggest that collagen remodeling in heart valves is a stretch-driven process.

Mechanics of the pulmonary valve in the aortic position.

Mathematical models can provide valuable information to assess and evaluate the mechanical behavior and remodeling of native tissue. A relevant example when studying collagen remodeling is the Ross procedure because it involves placing the pulmonary autograft in the more demanding aortic valve mechanical environment. The objective of this study was therefore to assess and evaluate the mechanical differences between the aortic valve and pulmonary valve and the remodeling that may occur in the pulmonary valve when placed in the aortic position. The results from biaxial tensile tests of pairs of human aortic and pulmonary valves were compared and used to determine the parameters of a structurally based constitutive model. Finite element analyzes were then performed to simulate the mechanical response of both valves to the aortic diastolic load. Additionally, remodeling laws were applied to assess the remodeling of the pulmonary valve leaflet to the new environment. The pulmonary valve showed to be more extensible and less anisotropic than the aortic valve. When exposed to aortic pressure, the pulmonary leaflet appeared to remodel by increasing its thickness and reorganizing its collagen fibers, rotating them toward the circumferential direction.

Characterization of the calcitonin gene-related peptide receptor antagonist telcagepant (MK-0974) in human isolated coronary arteries.

The sensory neuropeptide calcitonin gene-related peptide (CGRP) plays a role in primary headaches, and CGRP receptor antagonists are effective in migraine treatment. CGRP is a potent vasodilator, raising the possibility that antagonism of its receptor could have cardiovascular effects. We therefore investigated the effects of the antimigraine CGRP receptor antagonist telcagepant (MK-0974) [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridine-1-yl)piperidine-1-carboxamide] on human isolated coronary arteries. Arteries with different internal diameters were studied to assess the potential for differential effects across the coronary vascular bed. The concentration-dependent relaxation responses to human alphaCGRP were greater in distal coronary arteries (i.d. 600-1000 microm; E(max) = 83 +/- 7%) than proximal coronary arteries (i.d. 2-3 mm; E(max) = 23 +/- 9%), coronary arteries from explanted hearts (i.d. 3-5 mm; E(max) = 11 +/- 3%), and coronary arterioles (i.d. 200-300 microm; E(max) = 15 +/- 7%). Telcagepant alone did not induce contraction or relaxation of these coronary blood vessels. Pretreatment with telcagepant (10 nM to 1 microM) antagonized alphaCGRP-induced relaxation competitively in distal coronary arteries (pA(2) = 8.43 +/- 0.24) and proximal coronary arteries and coronary arterioles (1 microM telcagepant, giving pK(B) = 7.89 +/- 0.13 and 7.78 +/- 0.16, respectively). alphaCGRP significantly increased cAMP levels in distal, but not proximal, coronary arteries, and this was abolished by pretreatment with telcagepant. Immunohistochemistry revealed the expression and colocalization of the CGRP receptor elements calcitonin-like receptor and receptor activity-modifying protein 1 in the smooth muscle cells in the media layer of human coronary arteries. These findings in vitro support the cardiovascular safety of CGRP receptor antagonists and suggest that telcagepant is unlikely to induce coronary side effects under normal cardiovascular conditions.

Human adipose tissue-derived cells delay re-epithelialization in comparison with skin fibroblasts in organotypic skin culture.

BACKGROUND: Wound healing of deep and extensive burns can induce hypertrophic scar formation. During the early steps of wound healing fibroblasts migrate into the wounded area. Fibroblastic cells present in tissues other than dermis may also migrate into the wounded area and participate in the wound healing process. OBJECTIVES: To examine the influence of human fibroblastic cells derived from subcutaneous fat or dermis on epidermal morphogenesis in vitro. METHODS: We prepared human skin equivalents (HSEs) made of a collagen type I matrix populated either with dermal fibroblasts or adipose tissue-derived cells (ADCs), on top of which keratinocytes were seeded and subsequently grown at the air-liquid interface. RESULTS: A fully differentiated epidermis was formed on matrices populated with ADCs. However, the HSE formed differed in a number of features from HSE generated with dermal fibroblasts. The major differences included: marked contraction of the dermal matrix, low lateral migration of keratinocytes, high keratin 17 expression indicating increased keratinocyte activation, delayed deposition of collagen IV at the epidermal/matrix junction, accumulation of alpha-smooth muscle actin-positive cells only underneath the epidermal compartment and positioning of these cells in a direction parallel to the epidermal compartment. The latter two phenomena have also been found in scar tissue. CONCLUSIONS: The possibility of generating HSEs with different cell types represents an attractive approach for in vitro studies focusing on the mechanism of wound healing.

Porcine wound models for skin substitution and burn treatment.

Skin regeneration is an important field of tissue engineering. Especially in larger burns and chronic wounds, present treatments are insufficient in preventing scar formation and promoting healing. Initial screening of potentially interesting products for skin substitution is usually done by in vitro tests. Before entering the clinic, however, in vivo studies in immunocompetent animals are necessary to prove efficacy and provide information on safety aspects. We have obtained extensive experience using the domestic pig as test animal for studies on skin replacement materials, including tissue engineered skin substitutes, and burn wound treatment. Two models are described: an excisional wound model for testing of dermal and epidermal substitutes and a burn wound model for contact and scald burns, which allows testing of modern wound dressings in comparison to the present gold standards in burn treatment. The results of these experiments show that in vivo testing was able to reveal (dis)advantages of the treatments which were not detected during in vitro studies.

The suitability of cells from different tissues for use in tissue-engineered skin substitutes.

Tissue-engineered skin substitutes may be a future remedy for burn wounds and chronic wounds, as wound contraction and scar formation cannot be prevented with the current standard treatment. The aim of this study therefore was to identify readily available sources of fibroblasts suitable for dermal substitution. Three different tissues were studied: dermal tissue from split-skin graft, subcutaneous fat tissue and eschar tissue obtained through debridement of burn wounds. We determined the cellular profile and the cell numbers immediately after isolation and after 2 and 14 days of fibroblast culture using flow cytometry and cell counting with a cytometer. In addition, parts of the isolated cell suspensions were seeded directly into a porous collagen dermal substitute to investigate contraction over time. Various cell types were isolated from the three different tissues, but after 14 days of culturing predominantly fibroblasts (>90%) were detected. Keratinocytes, granulocytes and macrophages, if present, disappeared within 14 days. In the cell populations derived from dermal tissue, the percentage of myofibroblasts had decreased significantly by day 14 (from 8% to 3%, P=0.028). In contrast, this percentage had increased in the cell populations derived from fat and eschar (from 23% to 40% and from 20% to 38%, respectively). The fibroblast yield from dermal tissue after 2 weeks of culturing (50 x 10(6) cells/g of tissue) was significantly higher than the yield from fat and eschar tissue (2 x 10(6) cells/g of each tissue, P=0.029). Immunohistochemistry of collagen matrices seeded and cultured with fat- and eschar-derived cells revealed a high prevalence of myofibroblasts, whereas hardly any myofibroblasts were detected in the matrices seeded with dermal cells. The contraction of the eschar matrices was highest (74+/-6% remaining area), whereas dermal matrices contracted significantly less (92+/-7% remaining area, P=0.029) with intermediate contraction for fat matrices. We conclude that fibroblast cultures can be established from dermal tissue, fat tissue and eschar tissue. Dermis is the best fibroblast source for use in skin substitutes as it yields the highest numbers of fibroblasts with minimal numbers of myofibroblasts.