The influence of chemical treatment and suture on the elastic behavior of calf pericardium utilized in the construction of cardiac bioprostheses

Poor mechanical properties of biological tissue are known to cause wear, leading to the failure of cardiac bioprostheses made of calf pericardium. Different chemical agents such as sodium dodecyl sulfate (SDS) are presently being tested as possible inhibitors of the calcification process. The objective of this report was to determine the mechanical behavior of calf pericardium treated with SDS for 24 h and the influence of the suture on the mechanical properties of the tissue. Forty-eight samples were tested: 24 subjected to a standard treatment with glutaraldehyde (12 sewn with 4/0 silk suture thread) and 24 incubated with SDS for 24 h (12 sewn with the same suture thread). Each sutured and nonsutured sample was cut into two strips to yield paired samples. All were subjected to tensile stress to breaking point. The mean stress at breaking point in the nonsutured series treated with glutaraldehyde alone was 16.42 and 13.85 MPa, depending on the region of the pericardium, while in the sutured samples subjected to glutaraldehyde the mean stress was 7.50 and 7.63 MPa, respectively, differences which were statistically significant (p=0.03 and p=0.003, respectively) when the means for nonsutured samples from equivalent regions treated with glutaraldehyde were compared. The stress at breaking point was lower in the SDS-treated series, ranging between 2.60 and 3.56 MPa. The mathematical functions that govern the stress/strain or deformation were obtained. In the series of pericardium treated with SDS, deformations of 10% were produced with stresses of under 0.4 MPa, an outcome that is intolerable from the constructive point of view. We established a regression model that enabled us to determine the mechanical behavior of a sutured sample by testing a contiguous piece of tissue, with a high correlation coefficient (r > 0.99). We consider this finding to be of interest in the selection of pericardium for use in the construction of leaflets for cardiac bioprostheses. ©2000 Kluwer Academic Publishers     

Analysis of shearing stress in the limited durability of bovine pericardium used as a biomaterial

The objective of the study was to determine the shearing stress exerted by the suture thread under conditions of normal working stress. Thirty-six samples of calf pericardium, similar to that employed in the manufacture of bioprosthetic cardiac valve leaflets, were subjected to tensile testing. Prior to the trial, a continuous suture was sewn in the central zone of each sample, at a 45° angle to the longest axis of the sample, using commercially-available threads (silk, Gore-Tex, Surgilene and nylon). Application of the Mohr circle for combined wear revealed that the shearing stress ranged between 2.68-fold greater (for samples sewn with silk) and 5.48-fold greater (for samples sewn with nylon) than the working tensile stress in the region of the suture. It is concluded that the shearing stress is responsible for the limited durability of sutured samples of calf pericardium prepared to simulate bioprosthetic cardiac valve leaflets. © 1998 Chapman & Hall     

Sutured calf pericardium: influence of the type and angle of the suture on mechanical behavior

Careful selection of the biological tissue to be used in cardiac bioprostheses and a thorough knowledge of its mechanical behavior, facilitating both the prediction of this behavior and the interactions between the tissue and the other materials employed, is the best approach to designing a durable implant. For this purpose, a study involving uniaxial tensile testing of calf pericardium was carried out. Two sets of three contiguous strips of tissue were obtained from each pericardial membrane, to perform a total of 144 trials. Two samples were sewn with one of four commercially available suture materials: Gore-Tex, nylon, Prolene and silk. In each set of three samples, the center strip remained intact and unsutured to serve as a control, while the left-hand strip was sutured at a 45° angle with respect to the longitudinal axis and the right-hand strip was sewn at a 90° angle. All the samples were tested until rupture. The results demonstrated a significant loss of mean load (p<0.01) in the sutured samples at rupture. The angle of the suture had no influence on these results, although the stress/strain curves showed that, as the tensile stress increased, the mechanical behaviors were less uniform. The rupture of the collagen fibers could explain this phenomenon. The pericardium sutured with Gore-Tex presented a greater strain, or deformation (elongation), at lower levels of stress, regardless of the angle of the suture. The tissue selection criteria, based on the use of paired samples, enabled a correct prediction of the mechanical behavior of the tissue, with excellent correlation coefficients (>0.98) and a high degree of homogeneity in the results.     

Hysteresis of a biomaterial: Influence of sutures and biological adhesives

We studied the changes in energy consumption of samples of calf pericardium, when joined or not joined by sutures and adhesives, by means of hysteretic cycles. Sixty-four samples were subsequently subjected to tensile stress until rupture. An overlapping suture sewn in the form of a rectangle presented an acceptable mean resistance to rupture of over 10 MPa, although lower than the mean values in an unsutured control series where the mean resistance surpassed 15 MPa. The contribution of an acrylic adhesive to the resistance to rupture was negligible. The sutured samples that were reinforced with adhesives and had not been subjected to hysteretic cycles prior to rupture showed an anisotropic behavior. This behavior appeared to be lost in all the samples that underwent hysteretic cycles. We found an inflection point in the stress/strain curve following the stepwise increase in the load, with a value greater than and proximate to the final load applied. This inflection should be analyzed by means of microscopy. Finally, the mathematical relationship between the energy consumed and the stress applied, the strain or deformation produced and the number of cycles of hysteresis to which the samples were subjected was established as the ultimate objective of this study. The bonding systems provoked a greater consumption of energy, with the greatest consumption corresponding to the first cycle in all the series assayed. An equation relating the energy consumption in a sample to the number of hysteretic cycles to which it was subjected was obtained. Its asymptote on the x-axis indicates the energy consumption for a theoretical number of cycles, making it possible to estimate the durability of the sample.     

Porcine pericardial membrane subjected to tensile testing: preliminary study of the process of selecting tissue for use in the construction of cardiac bioprostheses

The durability of cardiac bioprostheses is limited fundamentally by structural failure due to mechanical fatigue and calcification. In the present report, we analyze, using an in vitro hydraulic simulator to test tensile strength, the mechanical behavior of porcine pericardium for the purpose of establishing the criteria for selecting the biomaterial, taking into account both morphological criteria (thickness and homogeneity of the specimens) and mechanical criteria (stress at breaking point), using the epidemiological model of paired samples. The stress at breakage was found to range widely from 24.07 MPa to 100.29 MPa, although we observed no statistically significant differences when comparing the mean results in the different regions and zones of the pericardium being studies. The application of the selection criteria in the present series resulted in an excellent mathematical fit in terms of the stress/elongation (R ² > 0.95), making it possible to establish, by means of linear regression, the prediction of the tensile strength in one zone on the basis of the values observed in its paired specimen. © 2001 Kluwer Academic Publishers     

Comparative study of the mechanical behaviour of a cyanoacrylate and a bioadhesive

We compared the mechanical resistance of 18 samples of calf pericardium bonded with a 100 mm2 overlap, by two types of glues: a cyanoacrylate (Loctite 4011) and a bioadhesive (BioGlue). Comparative tensile testing was also carried out in 40 paired samples, 20 bonded with the cyanoacrylate and 20 unbonded controls. The findings at rupture showed a greater resistance of the calf pericardium glued with cyanoacrylate, with a mean tensile strength of 0.15 MPa vs. 0.04 MPa for the biological glue (p= 0.000). They also demonstrated a loss of resistance of the samples bonded with cyanoacrylate when compared with that of the unbonded other halves of the pairs: 0.20 MPa and 0.27 MPa vs. 19.47 MPa and 24.44 MPa (p < 0.001). The method of selection by means of paired samples made it possible to establish the equations that relate the stress and strain, or deformation, with excellent coefficients of determination (R2). These equations demonstrate the marked elastic behaviour of the bonded samples. Moreover, these findings show the cyanoacrylate to be superior to the biological glue, leading to the examination of the compatibility, inalterability over time and mechanical behaviour of the cyanoacrylate in sutured samples, as well as the study of the anisotropy of the biomaterial when bonded with a bioadhesive.     

A new method for selecting calf pericardium for use in cardiac bioprostheses on the basis of morphological and mechanical criteria

The durability of existing calf pericardium bioprostheses is limited by phenomena such as mechanical stress and calcification, the factors most frequently implicated in valve failure. Varying the preferred direction of the collagen fibers influences the mechanical behavior of the pericardial membrane. Given this possible variation, a strict control of the selection of the biomaterial employed in the construction of valve leaflets is essential, but a reliable method of selection has yet to be established.This study describes the development of a new system of in vitro selection involving a hydraulic simulator that reproduces the mechanical behavior of pericardial membranes subjected to the stress of continuous flow.By combining morphological criteria such as thickness and homogeneity with those of mechanical behavior, and by selecting paired samples from different parts of the pericardium, we obtained excellent mathematical fits. Linear regression analysis provided the mode of predicting the tensile strength in a given sample when this value had been determined in its twin. The upper zones of calf pericardium, corresponding to either right or left ventricle but at a distance from ligamentous structures, showed the best mean results at rupture (60 MPa) and permitted the most reliable prediction. The expected stress for an elongation of 30% was 1.12 MPa, as was previously observed, with a 95% confidence interval of between 1.11 and 1.14 MPa.These trials, together with the careful selection of the pairs, should help to establish definitive selection criteria. © 2001 Kluwer Academic Publishers     

Mechanical effects of increases in the load applied in uniaxial and biaxial tensile testing. Part II. Porcine pericardium

The mechanical behavior of porcine pericardium was analyzed to compare it with that of calf pericardium employed in valve leaflets for cardiac bioprostheses. Forty samples of pericardium were subjected to uniaxial tensile testing, 20 as controls and 20 exposed to loads increasing stepwise from 0.5 to 1.5 kg and to 3 kg, and thereafter to rupture, with a return to zero load between each new increment. Another 20 samples were used in biaxial tensile tests involving the application of loads increasing stepwise (to 0.5, 1.5, 3 and 5 kg) until rupture with a zero-load interval before each increment. The ultimate stresses were very similar, showing no statistically significant differences when compared in terms of type of assay, controls and study samples or region of pericardial tissue being tested. In the stepwise biaxial assays, the mean stresses at rupture were also very homogeneous. Using morphological and mechanical criteria for sample selection, it was possible to obtain mathematical fits for the stress/strain relationship, with excellent coefficients of determination. The relationship between the area under the stress/strain curve and the load applied or the strain observed was also studied in the biaxial assay as an equivalent to the cycles of hysteresis produced in the test. The increment in the area under the curve (the energy consumed) may be a good parameter for assessing the changes in the collagen fiber architecture of the pericardial tissue, changes that may help to detect early failure.     

Mechanical effects of increases in the load applied in uniaxial and biaxial tensile testing: Part I. Calf pericardium

The authors analyzed the mechanical behavior of the calf pericardium employed in the construction of valve leaflets for cardiac bioprostheses. Forty samples of pericardium were subjected to uniaxial tensile testing, 20 as controls and 20 exposed to loads increasing stepwise until rupture, with a return to zero load between each new increment. Another 20 samples were used similarly in biaxial tensile tests involving loads increasing stepwise until rupture, again returning to zero load between steps. The ultimate stresses in the uniaxial study were very similar and were not influenced by the region of pericardial tissue being tested or the increments in load to which the tissue was exposed. The mean stresses at rupture in the stepwise biaxial assays were significantly greater (p<0.01). Using morphological and mechanical criteria for sample selection, it was possible to obtain mathematical fits for the stress/strain relationship in both types of assays, with excellent coefficients of determination (R ²>0.90). In uniaxial tests in which the selection criteria were not applied, the correlation improved as the load increased, a phenomenon that did not occur in the biaxial studies. The values varied throughout the different cycles, adopting exponential forms when the strain was greatest. These variations, which demonstrate that the increase in the energy consumed is a function of the stress applied and of the strain produced, should be good parameters for assessing the changes in the collagen fiber architecture of pericardial tissue subjected to cyclic stress, and may help to detect early failure.     

Bovine pericardium for heart valve bioprostheses:in vitro andin vivo characterization of new chemical treatments

This study is aimed at investigating the bovine pericardium treated with different chemical procedures applied to prevent dystrophic calcification; decellularization of the fresh pericardium (samples B and C); fixation of the pericardium with glutaraldehyde (samples A, B, C, D and E); detoxification with aminoacids (samples A and B) and storage in a solution of benzoic acid esters. Pericardial sacs were harvested and delivered to the laboratories to be submitted to the chemical treatments. The samples E have been treated as the samples D but before the implantation they were exposed to the surgical lamp in order to promote some drying. The samples were tested for their mechanical properties and shrinkage temperature (at 1 week and after 36 months). Thein vivo tests were performed by means of implantation in a paravertebral subcutaneous position in rats. At the explant (2, 4 and 8 weeks) the samples were submitted to histological assay and the calcium content determined by spectroscopic atomic absorption. All the samples showed loss of tensile strength and elongation at 36 months (except for the sample A), as well as a moderate diminution of the shrinkage temperature. The histology showed that the decellularized samples (B, C) were thicker than the others and the collagen fibres were extensively homogenated. The cell colonization was macrophagic for the samples A and D while it was also composed of giant cells in the samples B, C and E at 8 weeks. The von Kossa's staining was positive only for the samples D and E after 4 weeks of implantation. The calcium content of the samples D was 285.3 mg/g at 8 weeks while in E it was 44.4 mg/g dry tissue at 4 weeks; for the remaining samples the calcium content did not increase with the time (2.1–2.3 mg/g at 8 weeks). In conclusion, the pericardium decellularization and detoxification associated with its storage in a glutaraldehyde-free solution is a promising method in order to realize more durable pericardial bioprostheses. The investigated tissue treatments applied to the bovine pericardium permit removal of the calcium nucleation sites, and hence the avoidance of the severe drawback of the aldehyde crosslink, but at the same time maintain the necessary and well known tissue stability.     

Inhibition of bovine pericardium calcification: A comparative study of Al3+ and lipid removing treatments

The biological heart prostheses present midterm and long term problems owing to the progressive deterioration and calcification of the tissue. In the attempt to study the latter problem, we have compared the effectiveness of anticalcification treatments in calf pericardium samples implanted into female Wistar rats after undergoing the following procedures: Group I, control, treatment with glutaraldehyde; group II, treated with 0.1m Al³⁺ for 24 h; and group III, subjected to lipid removal by chemical treatment with chloroform/methanol for 1 h. Positive results were obtained with both treatments, but the results after 60 days of implantation were more favourable with lipid removal than with Al³⁺ treatment.     

Polyethylene glycol-grafted bovine pericardium: a novel hybrid tissue resistant to calcification

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium (GATBP). An investigation was made of the grafting of different molecular weight polyethylene glycol (PEG 600, 1500, 4000 and 6000) via glutaraldehyde (GA) linkages to bovine pericardium (BP) and of their stability and calcification. The process of the calcification profile was studied by in vitro experiments via incubating pericardial samples in a metastable solution of calcium phosphate. Calcification of bovine pericardium grafted with PEG 6000 was significantly decreased compared to low molecular weight PEG grafts or Sodium dodecyl sulphate- (SDS) and GA-treated tissues. The mechanical properties of these modified tissues after enzyme (Trypsin) digestion and calcification were investigated. The biocompatibility aspects of grafted tissues were also established by monitoring the platelet adhesion, octane contact angle and water of hydration. PEG 6000-grafted tissues retained the maximum strength in trypsin buffer and calcium phosphate solutions. Scanning electron micrographs revealed that the PEG-grafted bovine pericardium had substantially inhibited the platelet–surface attachment and their spreading. It is conceivable that high molecular weight polyethylene glycol-grafted pericardium (a hybrid tissue) may be a suitable calcium-resistant material for developing prosthetic valves due to their stability and biocompatibility. © 1999 Kluwer Academic Publishers     

Calcification and identification of metalloproteinases in bovine pericardium after subcutaneous implantation in rats

The purpose of this study was to evaluate the influence of two anticalcification pre-treatments (chloroform/methanol and ethanol) and serum conditioning of glutaraldehyde-crosslinked bovine pericardium on the calcification degree and the presence of gelatinase activities in a subcutaneous implantation model in rats. Regarding calcification of the implants, glutaraldehyde control treatments showed a significatively higher calcification degree than pericardium treated with anticalcification reagents. Serum conditioning of glutaraldehyde treated tissues did not influence the calcification degree; moreover, no differences were found in these samples with the time of implantation (30 and 90 days). On the other hand, anticalcification treatments resulted in a very significant decrease in the calcium content in the implanted membranes. Gelatinase activities were detected by gelatin zymography in almost all the implanted samples. However, control tissues with and without serum conditioning showed less gelatinase activities than those samples pre-treated with anticalcification treatments. Metalloproteinase (MMP-2) activity was detected in all the samples analyzed but a higher expression of MMP-9 was detected in those implants treated with chloroform/methanol and ethanol. Additional gelatinase activities showing lower molecular weight than MMP-2 were also detected in both anticalcification treated samples. The presence of these gelatinase activities is probably due to host cellular infiltrates and could contribute to the biomaterial degradation.     

The mechanical response of glutaraldehyde-fixed bovine pericardium to uniaxial load

Bovine pericardium, treated with glutaraldehyde, is used in the construction of heart valve substitutes. This study examines the mechanical properties of this tissue by using a continuum physics approximation of the material. A consideration of the relative magnitudes of the characteristic deformation time of a heart valve leaflet and the measured relaxation time of the tissue suggests that it can be effectively represented by a non-linear elastic solid. A compressible isotropic strain energy function is used to characterize the homogeneous deformation of the tissue when it is subjected to uniaxial load. The initial elastic material which is characterized by only two elastic constants, undergoes a transition to a second elastic material which is governed by a strain energy function of different magnitude by the same functional form as that associated with the initial elastic solid. This model is used to investigate the pericardial sac-to-sac and within-sac directional variation of the response to load in the unstrained state. Analysis of variance shows that glutaraldehyde treated pericardium possesses no preferred directional strength properties in the unstrained state. Any observed differences in the mechanical properties of different test specimens can be attributed to random biological variation alone.     

高强度聚苯胺-聚丙烯酸/聚丙烯酰胺导电水凝胶的制备与性能

将导电聚合物引入到水凝胶网络中的导电高分子基导电水凝胶,因结合了水凝胶的三维网络结构、良好的生物相容性、优异的力学性能等和导电高分子良好电学性能等优点而被广泛研究,特别是以聚苯胺(PANI)为导电高分子的导电水凝胶。但PANI不溶于水,因此很难制备PANI基导电水凝胶。本文以制备高强度PANI基导电水凝胶为目的,尝试将PANI接枝在亲水性聚合物聚丙烯酸(PAA)上,获得能在水中均匀分散的PANI-PAA导电复合物,再使其与丙烯酰胺(AM)聚合得到高强度的PANI-PAA/PAM导电水凝胶。通过力学性能及电化学性能测试,发现该导电水凝胶具有良好的力学性能和电化学性能。当以十二烷基硫酸钠(SDS)为分散剂时,其电导率可达4.63 S·m?1,可承受压缩应力1.33 MPa (压缩耗散能为85.50 kJ·m?3),拉伸断裂伸长率达964%,相应的断裂强度为0.25 MPa;而以NaOH为分散剂时,凝胶的电导率可达4.19 S·m?1,可承受压缩应力1.13 MPa (压缩耗散能为73.45 kJ·m?3),拉伸断裂伸长率达896%;相应的断裂强度为 0.14 MPa。该研究为高强度聚苯胺基导电水凝胶的制备提供了思路。      

Evaluation of alternative glutaraldehyde stabilization strategies for collagenous biomaterials

A range of alternative crosslinking conditions based on glutaraldehyde were examined for their effectiveness for stabilizing collagen-based materials using test samples of a collagen-polymer composite tube. Stabilization of collagen was performed with various concentrations of glutaraldehyde at acid pH, in the absence or presence of 0.7 m NaCl to control collagen swelling. For each condition, some samples were further treated at neutral pH. These test samples were compared with samples treated with glutaraldehyde at neutral pH and with samples of Omniflow vascular prosthesis. The effectiveness of the stabilization was examined by amino acid analysis, to assess the extent of modification, isometric tension analysis, to evaluate the extent of crosslinking, compliance and accelerated fatigue testing, to evaluate mechanical properties, and by a rat subcutaneous model to evaluate tissue response and propensity to calcification. The data indicated that effective crosslinking could be achieved at low pH and that this can be increased slightly by the presence of NaCl. At low pH, the extent of calcification was low compared to samples treated at pH 7. Subsequent treatment at pH 7 of samples given an initial low pH glutaraldehyde (GA) treatment generally did not alter shrinkage temperatures although the extent of lysine modification and calcification did increase. In general, a more inflammatory response was observed in samples tanned at low pH, although this was not as severe as responses to untreated tissue implants. The Omniflow vascular prosthesis showed excellent chemical and mechanical properties and did not show any calcification.     

A uniaxially reinforced zircon-silicon carbide composite

Zircon matrix composites uniaxially reinforced with as-supplied and BN-coated silicon carbide filaments were fabricated, and their mechanical properties were measured in flexure mode. A toughened-composite behaviour was displayed by the reinforced samples with strengths between 681 and 700 MPa and toughness between 24 and 41 kJ m^–2. In comparison, the monolithic zircon failed in a brittle manner, had an average strength of 280 MPa and toughness of 0.95 kJ m^–2. Influence of fibre-matrix interfacial shear stress on the first matrix cracking stress, ultimate failure strength and strain, toughness, and mode of failure were studied. The composite toughness was found to be dependent on the interfacial shear stress whereas the first matrix cracking stress was independent of the interfacial shear stress. These results on mechanical properties are compared with predictions from composite models.     

Crosslinking of tissue-derived biomaterials in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)

In contrast to bifunctional reagents such as glutaraldehyde or polyfunctional reagents such as polyepoxides, carbodiimides belong to the class of zero-length crosslinkers which modify amino acid side-groups to permit crosslink formation, but do not remain as part of that linkage. The authors have compared the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and glutaraldehyde (the de facto industrial standard crosslinker) on the hydrothermal, biochemical, and uniaxial mechanical properties of bovine pericardium. EDC crosslinking was optimized for maximum increase in collagen denaturation temperature using variables of pH, concentration, and ratio of EDC to N-hydroxysuccinimide (NHS): a reagent for formation of activated esters. EDC and glutaraldehyde crosslinked materials were subjected to hydrothermal denaturation tests, biochemical degradation by enzymes (collagenase, trypsin) and CNBr, amino acid analysis for unreacted lysine, and to high strain rate mechanical tests including: large deformation stress-strain studies (0.1 to 10 Hz), stress relaxation experiments (loading time 0.1 s) and small deformation forced vibration (1 and 10 Hz). A protocol for EDC crosslinking was developed which used 1.15% EDC (2:1 EDC:NHS) at pH 5.5 for 24 h. The increase in denaturation temperature for EDC (from 69.7±1.2°C to 86.0±0.3°C) was equivalent to that produced by glutaraldehyde (85.3±0.4°C). Both treatments equivalently increased resistance to collagenase and CNBr degradation; however, after denaturation, the EDC-treated tissue was slightly more resistant to collagenase, and markedly more resistant to trypsin. EDC-treated materials were more extensible and more elastic than glutaraldehyde-treated materials. Despite the differences in crosslinking mechanism, EDC and glutaraldehyde-treated materials are very similar. Subtle but intriguing differences in biochemical structure remain to be investigated.     

Artificial midline-fascia of the human abdominal wall for testing suture strength

To reduce testing of human abdominal wall closure-modalities in test animals, a fibre reinforced rubber with identical mechanical properties compared to the human midline fascia (linea alba: LA) was developed. The microscopic structure of the human LA, stress-strain behaviour, maximum tensile force and macroscopic failure mechanism in tensile tests with human LA were defined as indicators for the required properties of the fibre reinforced rubber. A composite consisting of latex rubber and cotton fibres was developed that shows mechanical properties comparable to the human abdominal wall. The results of the tensile tests on sutured artificial LA were highly similar to those performed on sutured human LA. The material presented in this study is proposed as a substitute for human and animal tissues presently used to test suture techniques. A protocol for an approach to develop artificial fibrous soft tissue like fascie and tendon was drawn up.     

Evaluation of cross-linked gelatin membranes as delivery carriers for retinal sheets

The delivery of intact sheet transplants to the subretinal space can prevent cell loss that is generally associated with the injection of cell suspensions or cell aggregates. The aim of this study was to develop chemically modified gelatin matrices that enhance the delivery efficiency and analyze whether the gelatin membranes cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) can be considered as potential carriers for retinal sheets. The characteristics of EDC cross-linked gelatin membranes were determined by mechanical and in vitro degradation tests, melting point measurements, cell proliferation assays, cytokine expression analyses, and tissue delivery studies. Gelatin membranes without cross-linking and glutaraldehyde cross-linked gelatin samples were used for comparison. Results of this study indicated that introduction of cross-links is capable of rendering the gelatin network more stable against mechanical stresses and deformations as well as rapid hydrolysis during intraocular delivery of delicate tissue sheets. In comparison with the glutaraldehyde treated samples, the EDC cross-linked gelatin membranes showed a better degradation profile and a relatively higher cytocompatibility. In addition, after EDC cross-linking, the gelatin matrices having an acceptable melting point could be used for the fabrication of a sandwich-like carrier with a high transfer and encapsulation efficiency. These findings suggest that the cross-linking agent type gives an influence on delivery functionality of gelatin membranes. In summary, the EDC cross-linked gelatin is an ideal candidate for use as a carrier material in retinal sheet delivery applications.