Michel Spina

Thermal analysis characterization of aortic tissues for cardiac valve bioprostheses

Two multistep extractions were achieved on porcine aortic tissues to obtain acellular matrices used for cardiac bioprostheses. The evaluation of structural modifications and the possible damage of extracellular matrix fibrous proteins were investigated by means of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Protein-water interactions and degradation temperatures were determined by TGA. DSC was used to characterize protein thermal transitions (glass transition and denaturation), which provided information on the dynamic structure of the aortic tissue components. Sodium dodecyl sulfate (SDS) extraction had a destructuring effect, while Triton and cholate treatments did not affect the structural integrity of either elastin and collagen. A DSC comparison showed that SDS destabilizes the collagen triple helical domain and swells the elastin network.

Decellularized Allogeneic Heart Valves Demonstrate Self-Regeneration Potential after a Long-Term Preclinical Evaluation

Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.

The use of thermal techniques for the characterization and selection of natural biomaterials.

In this paper we explore the ability of thermal analysis to check elastin and collagen integrity in different biomaterial applications. Differential Scanning Calorimetry (DSC) has been used to analyze the first and second order transitions of the biological macromolecules in the hydrated and dehydrated state. First, we report the characterization of control cardiovascular tissues such as pericardium, aortic wall and valvular leaflet. Their thermal properties are compared to pure elastin and pure collagen. Second, we present results obtained on two collagen rich tissues: pericardia with different chemical treatments and collagen with physical treatments. Finally, more complex cardiovascular tissues composed of elastin and collagen are analyzed and the effect of detergent treatment on the physical structure of collagen and elastin is brought to the fore.

Characterisation of elastin and collagen in aortic bioprostheses

Porcine aortic valves used as cardiac valve bioprostheses are well adapted to physiological functions in the short term, but they lack long-term durability. Several multi-step extractions have been performed to obtain a perfectly acellular matrix. A new physical methodology is proposed to evaluate the resulting fibrous protein damage after biochemical extraction (TRI-COL and SDS). Thermal analysis techniques are adapted to collagen and elastin characterisation in the solid state. The aortic tissue thermal transitions are determined by differential scanning calorimetry (DSC): elastin glass transition is observed around 200°C, and collagen denaturation is observed around 230°C. These parameters are characteristic of the elastin network arrangement and of collagen triple-helix stability. The technique of thermostimulated currents (TSC) is well suited to specify the chain dynamics of proteins. The lowtemperature relaxations observed in both collagen and elastin are associated with localised motions, whereas the high-temperature modes are attributed to more delocalised motions of the chains. Therefore TSC and DSC spectrometries allow physical parameters specific to collagen and elastin to be obtained and their interaction in aortic tissues to be determined. According to the significant evolution of these parameters on SDS samples, the destabilising effect of this detergent is highlighted.

Differential distribution of structural components and hydration in aortic and pulmonary heart valve conduits: impact of detergent-based cell removal

Evaluation of the physiological performance of biological scaffolds for tissue engineering applications has been mostly based on biophysical and morphological methods, with limited attention paid to the quantitative contribution of the main structural components to native and/or treated valve assemblies. In the present study quantitation addressed the porcine leaflet, sinus and adjacent wall of aortic and pulmonary valved conduits before and after detergent-based cell removal. Collagen, elastin, glycosaminoglycan, lipid and water contents were expressed in terms of relative concentration and volume fraction in order to assess their effective contribution to the native tissue and to changes following decellularization procedures. The main findings were recognition of unexpectedly large water and underestimated collagen contents, differential distribution of elastin between the sectors and of glycosaminoglycan along the conduits and pulmonary scaffold destabilization upon cell removal, not found in the aortic case. Simultaneous investigations allowed consistent comparisons between native and decellularized tissues and added analytical knowledge crucial for designing realistic constitutive models. We have provided a quantitative structural foundation for earlier biomechanical findings in pulmonary leaflets and the basis for validation of theoretical assumptions still lacking the support of experimental evidence in both conduits. Future insights into the distribution of load-bearing components in human conduits are likely to provide indications important to optimize the surgical positioning of valvular grafts.

Dielectric characterization of collagen, elastin, and aortic valves in the low temperature range.

The low temperature dielectric relaxation of porcine aortic valves and its main macromolecular proteins, i.e. elastin and collagen, have been investigated in the dry state and at low levels of hydration by thermally stimulated currents spectrometry, with an equivalent frequency of 10-3 Hz. Two secondary relaxation modes, labeled γ and β with increasing temperature, are found for the three materials. Since the γ-mode is independent upon hydration while the β-mode is strongly plasticized by water, these relaxation modes have been attributed to localized motions of the polypeptidic chains containing apolar and polar residues, respectively. The deconvolution of the β-mode by fractional polarization gives the experimental distribution of the dielectric relaxation times of the three materials, and allows us to deduce the activation parameters of each elementary process. These analyses shows the existence of compensation phenomena between the activation parameters, implying cooperative mechanisms. The occurrence of these phenomena with their characteristic parameters are used to specify the origin of the localized relaxation modes in collagen and elastin, and to assign the specific role of each protein in the aortic valves.

Characterization of aneurysmal aortas by biochemical, thermal, and dielectric techniques

Abdominal aortic aneurysms (AAA) are characterized by structural alterations of the aortic wall resulting from the degradation of elastic fibres and an increase of collagen/elastin ratio. In this study we investigated the chain dynamics of AAA tissues by two techniques generally used for the characterization of polymers, Differential scanning calorimetry (DSC) and thermally stimulated currents (TSC), and we correlated the obtained data with biochemical analyses. The thermal denaturation of collagen observed by DSC allowed us to evaluate the thermal stability of the triple helix domain: notable modifications were evidenced between collagen from control tissue and collagen from AAA, particularly concerning the thermal denaturation. The dielectric analysis of pathologic aortic walls by TSC revealed a relevant change of collagen mobility in AAA, with the occurrence of a specific mode of relaxation between -60 and -40°C. Biochemical, thermal, and dielectric results are compatible with increase of new collagen deposition and/or impairment of the collagen phase stability in the extracellular matrix of AAAs.

Analysis of the molecular mobility of collagen and elastin in safe, atheromatous and aneurysmal aortas.

AIM OF THE STUDY In this study, we propose to use a thermal technique, Differential Scanning Calorimetry (DSC) to follow the evolution of elastin and collagen in safe and pathological cardiovascular tissues. PATIENTS AND METHODS The first part of this study deals with the analysis of the elastin network and associated proteins during ageing (from children to old persons) in aortic walls. The second part is devoted to the characterization of the collagenic phase in aneurysms. In both cases, physical data are correlated with biochemical analyses. RESULTS AND CONCLUSION For old persons aortas with atheromatous stades, elastin and associated proteins are found to interpenetrate to form a homogenous phase. Abdominal aortic aneurysms (AAA) are characterized by structural alterations of the aortic wall resulting from the degradation of elastic fibers and an increase of collagen/elastin ratio. Notable modifications are evidenced between collagen from control tissue and collagen from AAA, particularly concerning the thermal denaturation. Biochemical and thermal results are compatible with the increase of new collagen deposition and/or impairment of the collagen phase stability in the extracellular matrix of AAAs.