Biodegradable biocomposite non-woven matrices based on PDLLA- and elastin-solubilized proteins/elastin

Poly(D,L-lactide) (PDLLA) was synthesized by ring-opening polymerization of D,L-lactide. Non-woven PDLLA matrices were prepared by an extrusion/winding process. The process conditions were optimized and the surfaces of these matrices were modified by glow-discharge treatment and/or glutaraldehyde incorporation for immobilization of elastin-derived proteins (ESP) to the matrix to increase the biocompatibility and also to improve the bioactivity of the matrix. Glow-discharge conditions were optimized. Ethylene diamine (EDA) and Ar were used as the active monomers in the plasma phase. When EDA was used, the glow-discharge treated PDLLA matrices were first allowed to be reacted with glutaraldehyde, although, when Ar used, the treated matrices were used directly for ESP immobilization. The higher degree of immobilization was obtained for EDA and glutaraldehyde. The ESP-incorporated PDLLA matrices were further treated with elastin by cross-reaction of the ESP molecules on the matrix surfaces with elastin. Scanning electron microscopy (SEM) studies showed that ESP were homogeneously deposited the surface of the matrix.     

Copper nanoparticle cues for biomimetic cellular assembly of crosslinked elastin fibers

Elastin, a structural protein distributed in the extracellular matrix of vascular tissues, is critical to maintaining the elastic stability and mechanical properties of blood vessels, as well as regulating cell-signaling pathways involved in vascular injury response and morphogenesis. Pathological degradation of vascular elastin or its malformation within native vessels and the poor ability to tissue-engineer elastin-rich vascular replacements due to innately poor elastin synthesis by adult vascular cells can compromise vascular homeostasis, and must thus be addressed. Our recent studies attest to the utility of hyaluronan (HA) oligomers for elastin synthesis and organization by adult vascular smooth muscle cells (SMCs), though the elastin matrix yields in these cases were quite low relative to total elastin produced. Thus, in this study, we investigated the utility of copper (Cu(2+)) ions to enhance cellular elastin deposition, crosslinking and maturation into structural fibers. Copper nanoparticles (CuNPs; 80-100 nm) in the dose range of 1-100 ng ml(-1) were tested for Cu(2+) ion release, and based on mathematical modeling of their release profiles, CuNPs (1, 10, and 400 ng ml(-1)) were chosen for supplementation to adult SMC cultures. The 400 ng ml(-1) dose of CuNPs cumulatively delivered Cu(2+) doses in the range of 0.1 M, over the 21 day culture period. It was observed that while exogenous CuNP supplements do not up-regulate tropoelastin production by vascular SMCs, they promoted formation of crosslinked elastin matrices. The deposition of crosslinked matrix elastin was further improved by the additional presence of HA oligomers in these cultures. Immunofluorescence imaging and structural analysis of the isolated elastin matrices indicate that amorphous elastin clumps were formed within non-additive control cultures, while aggregating elastin fibrils were observed within SMC cultures treated with CuNPs (1-10 ng ml(-1)) alone or together with HA oligomers. The presence of 400 ng ml(-1) of CuNPs concurrent with HA oligomers furthered aggregation of these elastin fibrils into mature fibers with diameters ranging from 200 to 500 nm. Ultrastructural analysis of elastin matrix within cultures treated with HA oligomers and 400 ng ml(-1) of CuNPs suggest that elastin matrix deposition as stimulated by Cu(2+) ions proceeds via a fibrillin-mediated assembly process, with enhanced crosslinking occurring via stimulation of lysyl oxidase. Overall, the data suggest that CuNPs and HA oligomers are highly useful for regenerating crosslinked, fibrillar elastin matrices by adult vascular SMCs. These results have immense utility in tissue-engineering vascular replacements.     

A strategy to establish a gene-activated matrix on titanium using gene vectors protected in a polylactide coating

Bioactive implants are promising tools in regenerative medicine. Here we describe a versatile procedure for preparing a gene-activated matrix on titanium. Lyophilized copolymer-protected gene vectors (COPROGs) suspended in poly(d,l-lactide) (PDLLA) solutions in ethyl acetate were used to varnish solid surfaces. The gene-activated PDLLA surfaces were first established on polypropylene 96-well plates. Vector release from these surfaces in aqueous buffer, cell viability and gene transfer efficiency to NIH 3T3 fibroblasts was strongly dependent on the vector dose and its ratio to PDLLA film thickness. A detailed analysis of these relationships allowed establishing correlations which can be used to calculate suitable combinations of COPROGs and PDLLA yielding optimal gene transfer efficiency. This was verified with COPROG-activated PDLLA coatings on titanium foils. HEK 293 and mesenchymal stem cells expressed the BMP-2 gene comprised in the gene-activated surface in a manner that was consistent with the predicted dose-response and toxicity profiles found in NIH 3T3 cells. The systematic procedure presented here for identifying optimal coating compositions can be applied to any combination of vector type and coating material.     

Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types

We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface.     

模压挤出条件对聚(D,L-乳酸)及其复合材料力学性能的影响

在辛酸亚锡的催化下由丙交酯开环聚合制备了聚（D，L）-乳酸（PDLLA），并用二苯基甲烷二异氰酸酯（MDI）作为扩链剂分别合成了MDI扩链聚（D，L）-乳酸（PDLLA/MDI）和MDI扩链聚（D，L）-乳酸/羟基磷灰石（PDLLA/HA/MDI）复合材料，采用自行设计的模压挤出设备着重研究了成型加工条件对这两类可生物降解材料力学性能的影响，实验结果表明，在最佳条件下PDLLA和PDLLA/MDI的弯曲强度分别为35.1MPa和51.3MPa，弯曲模量分别为2413.6MPa和1830.9MPa,PDLLA/HA和PDLLA/HA/MDI复合材料的弯曲强度分别为31.2MPa和55.4MPa。弯曲模量分别为1735.0MPa和2068.5MPa，可见，MDI扩链可显著提高PDLLA和PDLLA/HA复合材料的力学性能。     

Novel porous aortic elastin and collagen scaffolds for tissue engineering

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.     

Modulation of osteoblast function using poly(D,L-lactic acid) surfaces modified with alkylation derivative of chitosan

Poly(D,L-lactic acid) (PDLLA) was modified with alkylated chitosan (N-butyl chitosan and N-cetyl chitosan), and the effects of modified films on the functions of rat osteoblasts were investigated. The characteristics of surfaces(both modified and control) were examined by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). Cell morphologies on these surfaces were taken using scanning electron microscopy (SEM). Cell attachment and proliferation were used to assess cell behavior on modified surface and control. MTT assay was used to determined cell viability, and alkaline phosphatase (ALP) activity was taken to evaluate differentiated cell function. Compared with the untreated films, no significant difference in cell attachment of osteoblasts was found on the modified films at a period of 8 h (p > 0.05). However, cell proliferation of N-butyl chitosan rather than N-cetyl chitosan modified PDLLA films was significantly higher than that found on control one (p < 0.05) at the end of the 4th and 7th days. The cell viability of osteoblasts on N-butyl chitosan modified PDLLA films were found higher than that on control (p < 0.05). These results suggested that N-butyl chitosan contributed greater than N-cetyl chitosan when used to modify PDLLA films for improving its biocompatibility.     

Modulation of osteoblast function using poly(D,L-lactic acid) surfaces modified with alkylation derivative of chitosan

Poly(D,L-lactic acid) (PDLLA) was modified with alkylated chitosan (N-butyl chitosan and N-cetyl chitosan), and the effects of modified films on the functions of rat osteoblasts were investigated. The characteristics of surfaces (both modified and control) were examined by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). Cell morphologies on these surfaces were taken using scanning electron microscopy (SEM). Cell attachment and proliferation were used to assess cell behavior on modified surface and control. MTT assay was used to determined cell viability, and alkaline phosphatase (ALP) activity was taken to evaluate differentiated cell function. Compared with the untreated films, no significant difference in cell attachment of osteoblasts was found on the modified films at a period of 8 h (p > 0.05). However, cell proliferation of N-butyl chitosan rather than N-cetyl chitosan modified PDLLA films was significantly higher than that found on control one (p < 0.05) at the end of the 4th and 7th days. The cell viability of osteoblasts on N-butyl chitosan modified PDLLA films were found higher than that on control (p < 0.05). These results suggested that N-butyl chitosan contributed greater than N-cetyl chitosan when used to modify PDLLA films for improving its biocompatibility.     

Poly(D,L-lactic acid) surfaces modified by silk fibroin: effects on the culture of osteoblast in vitro.

The objective of this study was to modify the surface of poly(D,L-lactic acid) (PDLLA) with different molecular weight of silk fibroins, and assess the effects of the modified surfaces on the functions of rat osteoblasts cultured in vitro. The properties of the modified PDLLA surface and the control one were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA). The former indicated the variation of hydrophilicity and the latter suggested that the modified PDLLA film using silk fibroin is enriched with nitrogen atoms. The biocompatibility of the PDLLA film may be altered and in turn affects the seeded cell functions. Therefore, attachment and proliferation of osteoblasts seeded on the modified PDLLA films and the control one were examined. Cell morphologies on these films were studied by scanning electron microscopy (SEM) and cell viability was evaluated by MTT assay. In addition, differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. These results suggest that the silk fibroin-modified PDLLA surface can improve the interaction between osteoblasts and the PDLLA films.     

Three-dimensional topography of synthetic scaffolds induces elastin synthesis by human coronary artery smooth muscle cells

Due to the important structural and signaling roles of elastin in vascular stability, engineered human vascular tissues must incorporate elastin. However, despite considerable progress toward engineering of elastin-containing vascular tissues from animal cells, currently engineered vascular tissues using human cells largely lack elastin. In this study, we evaluated the effect of scaffold topography (two dimensional [2D] vs. three dimensional [3D]) on elastogenesis in adult human coronary artery smooth muscle cells (HCASMCs). We report that elastin gene expression by HCASMCs was increased by twofold after 4 days of culture in porous 3D polyurethane scaffolds. Transforming growth factor β1 (TGF-β1) further increased elastin gene expression in 3D cultures but not in 2D cultures. To evaluate if gene expression is translated into elastin synthesis, both 2D and 3D cultures were analyzed using Western blots. We show that only HCASMCs in 3D scaffolds produced elastin, suggesting that scaffold geometry itself is an important cue for elastogenesis. Moreover, TGF-β1 enhanced elastin synthesis in 3D, but had no effect on cells grown on 2D surfaces. TGF-β1, known to induce vascular smooth muscle cells (VSMC) differentiation, upregulated contractile VSMC marker proteins smooth muscle-α-actin and calponin in cells on 2D surfaces. Interestingly, in 3D scaffolds, TGF-β1 failed to upregulate these differentiation marker proteins for at least 7 days, but did so in cells cultured for 14 days, whereas elastin synthesis was not affected. To our knowledge this study is the first to successfully demonstrate that adult human VSMC can produce elastin when seeded on 3D scaffolds and to directly compare the effect of scaffold topography on elastin synthesis. Knowledge about the conditions required to regulate the phenotype of human VSMCs is paramount to engineer elastin-containing autologous human vascular substitutes.     

Elastin Calcification and its Prevention with Aluminum Chloride Pretreatment

Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3).     

Characterization of collagen gel solutions and collagen matrices for cell culture

The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%.     

Role of elastin in pathologic calcification of xenograft heart valves

Bioprosthetic heart valves fabricated from glutaraldehyde crosslinked porcine aortic valves often fail because of calcific degeneration. Calcification occurs in both cusp and aortic wall portions of bioprosthetic heart valves. The purpose of this study was to discern the role of different aortic wall components in the calcification process. Thus, we selectively extracted cells and other extracellular matrix proteins from porcine aorta using trypsin/DNase/RNase, cyanogen bromide (CNBr), and sodium hydroxide (NaOH) treatments and subdermally implanted these pretreated aortas in young rats. Total DNA and phospholipid data showed complete removal of cells by CNBr and NaOH treatments, whereas trypsin/DNase/RNase treatment was effective in removing DNA but not phospholipids. As shown by amino acid data and Masson's trichrome staining, collagen was removed in CNBr and NaOH treatments. Control fresh porcine aorta calcified significantly after 21 days of implantation (Ca 26.4 +/- 2.4 microg/mg). Removal of cells and collagen from the aorta by CNBr treatment did not lead to a statistically significant reduction in aortic calcification (Ca 20.8 +/- 3.0 microg/mg). Moreover, partial degradation of elastin fibers caused by NaOH (during extraction) and trypsin treatment (after implantation) of the aorta significantly increased elastin-oriented calcification (Ca 94.4 +/- 9.3 and 58.4 +/- 4.6 microg/mg, respectively). Our results indicate that the elastin component of the aorta may undergo independent calcification irrespective of devitalized cell-mediated calcification observed in glutaraldehyde crosslinked aortas. Our results also demonstrate the importance of studying elastin-oriented calcification in decellularized elastin-rich aortic matrices currently used in tissue-engineering applications.     

Matrix-assisted pulsed laser evaporation of poly(D,L-lactide) for biomedical applications: effect of near infrared radiation

The deposition of thin films of poly(D,L-lactide) (PDLLA) by using the matrix-assisted pulsed laser evaporation (MAPLE) technique is investigated. PDLLA is a highly biocompatible and biodegradable polymer, with wide applicability in the biomedical field. The laser wavelength used in the MAPLE process is optimized to obtain a good-quality deposition. The structure of the polymer film is analyzed by Fourier transform infrared spectroscopy (FTIR). It is found that the chemical structure of PDLLA undergoes little or no damage during deposition with near-infrared laser radiation (1064 nm). It is thus confirmed that at this wavelength, the MAPLE technique can be applied for fragile biopolymer molecules, which are easily damaged by other laser radiations (UV radiation). This method allows future development of tailored polymer coatings for biomedical applications.     

ESCA investigation of low-temperature ammonia plasma-treated polyethylene substrate for immobilization of protein.

A low-temperature radiofrequency plasma excited in anhydrous ammonia was used to modify polyethylene substrate surfaces for covalent immobilization of proteins. Electron spectroscopy for chemical application (ESCA) was used for surface characterization of polyethylene to a depth scale of 7 nm. The data revealed that surface modification is extensive and occurs in seconds at low discharge power. Primary amino functionalities were detected on the polyethylene surface and the level is dependent on plasma parameters. 125I-labelled antibodies covalently attached to amino groups via glutaraldehyde allowed the conditions for optimum level of primary amine to be established. Both ESCA data and protein loadings are in excellent agreement.     

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.     

Diamine-plasma treated and Cu(II)-incorporated poly(hydroxyethylmethacrylate) microbeads for albumin adsorption

Poly(2-hydroxyethylmethacrylate) (PHEMA) microbeads prepared by suspension polymerization were treated with diamine-plasmas (i.e. ethylene-diamine (EDA) and hexamethylene diamine (HMDA)) in a glow-discharge reactor in which the exposure time and glow-discharge power were changed between 5 and 30 min and 5 and 20 W, respectively. The amount of nitrogen deposition increased both with increase in exposure time and glow-discharge power. The maximum amounts of nitrogen deposition on the microbeads were 22.3 and 23.4 micromol g(-1) with the EDA- and HMDA-plasmas. Then, Cu(II) ions were incorporated onto the PHEMA microbeads by chelating with the nitrogen-carrying functional groups. Different amounts of Cu(II) ions (2.4-6.8 mg g(-1)) were incorporated on the PHEMA microbeads by changing the initial concentration of Cu(II) ions. Bovine serum albumin (BSA) adsorption onto the unmodified PHEMA, diamine-plasma treated PHEMA, and diamine-plasma treated Cu(II)-incorporated PHEMA microbeads was investigated. The non-specific adsorption of BSA on the unmodified microbeads was very low (0.22 mg BSA g(-1)). Deposition of nitrogen increased the BSA adsorption (9.3 mg g(-1) for EDA-plasma and 12.7 mg g(-1) for HMDA-plasma). Cu(II)-incorporation significantly increased the BSA adsorption (154 mg g(-1) for EDA-plasma and 178 mg g(-1) for HMDA-plasma). Further increases in the albumin adsorption capacities of the polymer microbeads (185 mg g(-1) for EDA-plasma and 208 mg g(-1) for HMDA-plasma) were observed when human plasma was used. More than 92% of the adsorbed albumin molecules was desorbed in 1 h in the desorption medium containing 0.5 M NaSCN at pH 8.0. Repeated adsorption-desorption cycles showed the feasibility of these plasma-modified polymer microbeads.     

Impact of delivery mode of hyaluronan oligomers on elastogenic responses of adult vascular smooth muscle cells

Our prior studies demonstrated that exogenous supplements of pure hyaluronan (HA) tetramers (HA4) dramatically upregulate elastin matrix synthesis by adult vascular smooth muscle cells (SMCs). Some studies suggest that exogenous HA likely only transiently contacts and signals cells, and may elicit different cell responses when presented on a substrate (e.g., scaffold surface). To clarify such differences, we used a carbodiimide-based chemistry to tether HA4 onto glass, and compared elastin matrix synthesis by SMCs cultured on these substrates, with those cultured with equivalent amounts of exogenous HA4. Tethered HA4-layers were first characterized for homogeneity, topography, and hydrolytic stability using SEM, XPS, AFM, and FACE. In general, mode of HA4 presentation did not influence its impact on SMC proliferation, or cell synthesis of tropoelastin and matrix elastin, relative to non-HA controls; however, surface-tethered HA4 stimulated SMCs to generate significantly greater amounts of elastin-stabilizing desmosine crosslinks, which partially accounts for the greater resistance to enzymatic breakdown of elastin derived from these cultures. Elastin derived from both sets of cultures contained peptide masses that correspond to the predominant peptides present in rat aortic elastin. SEM and TEM showed that HA4-stimulated fibrillin-mediated elastin matrix deposition, and organization into fibrils. Surface-immobilized HA4 was particularly conducive to organization of elastin into aggregating fibrils, and their networking to form closely woven sheets of elastin fibers, as seen in cardiovascular tissues. The results suggest that incorporation of elastogenic HA4 mers onto cell culture substrates or scaffolds is a better approach than exogenous supplementation for in vitro or in vivo regeneration of architecturally and compositionally faithful-, and more stable mimics of native vascular elastin matrices.