Cross-linked Amniotic Membrane Research Articles

The Effect of Amniotic Tissue on Spinal Interventions: A Systematic Review.

Amniotic membrane tissue has been thought to potentiate healing in many soft tissue conditions. Specifically, recent studies have shown its therapeutic potential for treatment in the setting of spinal pathologies. The purpose of this study is to thoroughly review the existing scientific literature and evidence concerning the clinical use of amniotic membrane-derived biologic agents on postoperative outcomes following spinal surgery. A systematic review was conducted following preferred reporting items for systematic reviews and meta-analyses guidelines using PubMed, Embase, and Cochrane databases up to December 2020 to identify animal and clinical studies examining the therapeutic potential for amniotic membrane tissue in the setting of spinal pathologies (including disc herniation, prevention of epidural fibrosis, and spinal fusion). Studies were broken down into 2 categories: experimental model type and the type of amnion product being analyzed. A total of 12 studies (4 clinical studies and 8 studies utilizing animal models) met inclusion criteria. Additionally, the major types of amnion product were divided into cryopreserved/freeze-dried amniotic membrane, human amniotic fluid, human amniotic membrane, cross-linked amniotic membrane, and amnion-derived epithelial cells. While heterogeneity of study design precludes definitive specific results reporting, most studies showed positive benefits on healing/outcomes with amniotic augmentation. Specifically, amnion products have shown promising effects in reducing epidural adhesions and scar tissue after spine surgery, improving spinal fusion rate and postoperative pain scores, and promoting better functional outcomes after spine surgery. A review of the limited number of reported studies revealed a wide variety of amniotic membrane preparations, treatment regimens, and indications, which limit definitive conclusions. To date, while there is no definitive clinical proof that amniotic tissues enhance tissue repair or regeneration, the aggregate results demonstrate promising basic science and outcomes potential in spinal surgery. Further study is warranted to determine whether this application is appropriate in the clinical setting. This systematic review provides a summary of the existing literature regarding the use of amniotic membrane preparations, treatment regimens, and indications within spinal surgery. With the growing popularity and utilization of biologic agents such as amniotic membrane-derived products in orthopedic and neurologic surgery, this systematic review gives physicians a concise summary on the outcomes and indications associated with amniotic membrane products.

Evaluation and ultrastructural changes of amniotic membrane fragility after UVA/riboflavin cross-linking and its effects on biodegradation.

This study aims to evaluate the changes of fragility and ultrastructure of amniotic membrane after cross-linking by UVA/riboflavin.Forty-nine fresh amniotic membranes were randomly divided into 3 groups. Eighteen were in group A (CX group) and immersed in 0.1% riboflavin solution for 10 min for UVA/riboflavin cross-linking. Sixteen were in group B (B2 group), soaked for 10 min with 0.1% riboflavin. After soaking, membranes in group A and B were transferred into corneal preservation solution. Fifteen pieces were in group C, directly into corneal preservation solution. The biomechanical and ultrastructural changes of the amniotic tissue before and after cross-linking were examined (CX group = 13, B2 group = 11, C group = 15). The amniotic membrane tissue of group A (n = 5) and B (n = 5) was transplanted into 16 eyes of the rabbits, respectively, and the dissolution time of the amniotic membrane tissue was investigated.After cross-linking, compared with the control group, the elastic modulus of the low-stress area of the amniotic membrane (Elow) was higher, while the elastic modulus of the high-stress area of the amniotic membrane (Ehigh) was lower, with no significant difference in the tensile strength. Also, the collagen fibers showed coarse and bamboo-like changes. In group A, amniotic membranes began to dissolve 4 weeks after conjunctiva transplantation, and all amniotic membranes were dissolved and absorbed 6 weeks after conjunctiva transplantation. In group B, some amniotic membrane tissues were still visible 6 weeks after conjunctiva transplantation.This study suggested that after amniotic membrane cross-linking, the brittleness was increased, the hardness was enhanced, and the morphology of the collagen fiber was changed. The cross-linked amniotic membrane showed resistance to tissue dissolution.

Transparent, resilient human amniotic membrane laminates for corneal transplantation

This study evaluated a new technique to toughen and optically clarify human amniotic membrane (AM) tissue, which is naturally thin and clouded, and determined the suitability of the altered tissue for corneal transplantation. The technique created a tissue laminate by repeatedly depositing wet layers of AM and dehydrating them, followed by chemical cross-linking to tighten integration at the layer interfaces and within the layers, thereby improving the physical properties of the laminates by increasing light transmittance and mechanical strength. Interestingly, this improvement only occurred in laminates with at least 4 layers. Cross-linking also improved the resistance of the laminates to collagenase degradation, such as occurs in corneal melting. This study also confirmed that the AM tissue was biocompatible by inserting AM monolayers into the corneal stroma of rabbits, and by performing lamellar keratoplasty in rabbits with cross-linked AM laminates. The laminates were sufficiently thick and resilient to need only one set of sutures, whereas in previously described multi-layer AM transplantation technique, each layer required separate sutures. The current findings are a promising advance in the engineering of novel biomaterials and the alteration of existing tissues for medical use.

Optimization of dendrimer bridges in carbodiimide cross-linked amniotic membrane as a limbal epithelial stem cell niche

Optimization of dendrimer bridges in carbodiimide cross-linked amniotic membrane as a limbal epithelial stem cell niche

Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation

The Wnt pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects Wnt activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin β1, and integrin-linked kinase (ILK). Nuclear β-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear β-catenin, TCF-4 and deltaNp63α, whereas cytokeratin 12 expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin β1/ILK, which indirectly activated Wnt/β-catenin and subsequently deltaNp63α. Crosstalk between the integrin β1/ILK and Wnt/β-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM. Statement of significanceWe demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin β1 to the Wnt/β-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin β1, followed by activation of the ILK/Wnt/β-catenin/p63 cascade.

Carbodiimide cross-linking of amniotic membranes in the presence of amino acid bridges.

The purpose of this study was to investigate the carbodiimide cross-linking of amniotic membrane (AM) in the presence of amino acid bridges. The biological tissues were treated with glycine, lysine, or glutamic acid and chemically cross-linked to examine the role of amino acid types in collagenous biomaterial processing. Results of zeta potential measurements showed that the use of uncharged, positively and negatively charged amino acids dictates the charge state of membrane surface. Tensile strength and water content measurements demonstrated that the addition of lysine molecules to the cross-linking system can increase the cross-linking efficiency and dehydration degree while the introduction of glutamic acid in the AM samples decreases the number of cross-links per unit mass of chemically modified tissue collagen. The differences in the cross-linking density further determined the thermal and biological stability by differential scanning calorimetry and in vitro degradation tests. As demonstrated in matrix permeability studies, the improved formation of covalent cross-linkages imposed by lysine facilitated construction of stronger cross-linking structures. In contrast, the added glycine molecules were insufficient to enhance the resistances of the proteinaceous matrices to thermal denaturation and enzymatic degradation. The cytocompatibility of these biological tissue membranes was evaluated by using human corneal epithelial cell cultures. Results of cell viability, metabolic activity, and pro-inflammatory gene expression level showed that the AM materials cross-linked with carbodiimide in the presence of different types of amino acids are well tolerated without evidence of detrimental effect on cell growth. In addition, the amino acid treated and carbodiimide cross-linked AM implants had good biocompatibility in the anterior chamber of the rabbit eye model. Our findings suggest that amino acid type is a very important engineering parameter to mediate carbodiimide cross-linking of AM collagen.

Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering.

As a biological tissue material, amniotic membrane (AM) has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional (3D) spongy scaffold made of the extracellular matrix (ECM) of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering. In this experimental study, cellular components of human amniotic membrane (HAM) were removed with 0.03% (w/v) sodium dodecyl sulphate (SDS). Quantitative analysis was performed to determine levels of Glycosaminoglycans (GAGs), collagen, and deoxyribonucleic acid (DNA). To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid (HCl 0.1 M) with pepsin (1 mg/ml). In the present experiment 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin (H&E) staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scaffolds were cytocompatible. Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment (39 ± 4.06 μg/ml vs. 341 ± 29.60 μg/ml). Differences were observed between cellular and denude AM in matrix collagen (478 ± 18.06 μg/mg vs. 361 ± 27.47 μg/mg).With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the mechanical property, and resistance to collagenase digestion. The results of 2, 4, 6-Trinitrobenzenesulfonic acid (TNBS) test showed that cross-linking efficiency of AM derived ECM scaffolds was about 65% ± 10.53. Scaffolds treated with NHS/EDC cross-linker agent by 100 μg/ml collagenase, lost 75% of their dry weight after 14 days. The average pore size of 3D spongy scaffold was 160 µm measured from scanning electron microscope (SEM) images that it is suitable for cell penetration, nutrients and gas change. In addition, the NHS/ EDC cross-linked AM scaffolds were able to support human fetal fibroblast cell proliferation in vitro. Extracts and contact prepared from the 3D spongy scaffold of AM showed a significant increase in the attachment and proliferation of the human fetal fibroblasts cells. The extra-cellular matrix of denuded AM-based scaffold displays the main properties required for substitute skin including natural in vitro biodegradation, similar physical and mechanical characterization, nontoxic biomaterial and no toxic effect on cell attachment and cell proliferation.

Stabilization of collagen nanofibers with L-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells.

To overcome the drawbacks associated with limited cross-linking efficiency of carbodiimide modified amniotic membrane, this study investigated the use of l-lysine as an additional amino acid bridge to enhance the stability of a nanofibrous tissue matrix for a limbal epithelial cell culture platform. Results of ninhydrin assays and zeta potential measurements showed that the amount of positively charged amino acid residues incorporated into the tissue collagen chains is highly correlated with the l-lysine-pretreated concentration. The cross-linked structure and hydrophilicity of amniotic membrane scaffolding materials affected by the lysine molecular bridging effects were determined. With an increase in the l-lysine-pretreated concentration from 1 to 30 mM, the cross-linking density was significantly increased and water content was markedly decreased. The variations in resistance to thermal denaturation and enzymatic degradation were in accordance with the number of cross-links per unit mass of amniotic membrane, indicating l-lysine-modulated stabilization of collagen molecules. It was also noteworthy that the carbodiimide cross-linked tissue samples prepared using a relatively high l-lysine-pretreated concentration (ie, 30 mM) appeared to have decreased light transmittance and biocompatibility, probably due to the influence of a large nanofiber size and a high charge density. The rise in stemness gene and protein expression levels was dependent on improved cross-link formation, suggesting the crucial role of amino acid bridges in constructing suitable scaffolds to preserve limbal progenitor cells. It is concluded that mild to moderate pretreatment conditions (ie, 3–10 mM l-lysine) can provide a useful strategy to assist in the development of carbodiimide cross-linked amniotic membrane as a stable stem cell niche for corneal epithelial tissue engineering.

Effect of Matrix Nanostructure on the Functionality of Carbodiimide Cross-Linked Amniotic Membranes as Limbal Epithelial Cell Scaffolds

Carbodiimide cross-linked amniotic membrane (AM) can potentially serve as an artificial corneal epithelial stem cell niche in ocular surface wound healing. For the first time, this study was performed to investigate the relationship between nanostructure and functionality of carbodiimide cross-linked AM tissues as limbal epithelial cell (LEC) scaffold biomaterials. The triple-helical molecular conformation of AM collagen was checked after chemical treatment for varying cross-linking durations (1-4 h). Our data indicated that the unraveling of the helical structure into a more random globular state is accompanied by an increase in the cross-linking index of AM samples. The cross-linker-mediated alterations in tissue ultrastructure and substrate nanotopography of these proteinaceous matrices were confirmed by transmission electron and atomic force microscopy studies. With increasing treatment time, the chemically cross-linked AM possessed larger nanofiber diameter and exhibited rougher texture. Marked increases in the water content, light transmittance, and resistance to enzymatic degradation were found, probably due to collagen fibril aggregation in biological tissues. All the test AM materials were not toxic to the human corneal epithelial cell cultures and retained anti-inflammatory activity, indicating the tolerability and safety of carbodiimide (i.e., a zero-length cross-linker). In addition, the enhanced LEC growth and increased p63 and ABCG2 gene expressions were significantly noted on the AM samples with greater cross-linking degree. In summary, the findings reported in this paper suggest that a specific limbal epithelial stem cell-biomaterial interaction may occur in response to biophysical cue such as nanostructure of carbodiimide cross-linked AM matrix.

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

A prototype tissue engineered blood vessel using amniotic membrane as scaffold

In this study, we used amniotic membrane (AM), a natural extracellular matrix, as a scaffold for the fabrication of tissue engineered blood vessels (TEBVs). The inner surface of the denuded glutaraldehyde cross-linked AM tube was endothelialized with porcine vascular endothelial cells (ECs) and subjected to a physiological (12dynecm−2) shear stress (SS) for 2 and 4days. The results showed that after applying SS, an intact EC monolayer was maintained in the lumen surface of the TEBV. The ECs were aligned with their long axis parallel to the blood flow. The immunofluorescent microscopy showed that the intercellular junctional proteins, PECAM-1 and VE-cadherin, were surrounding the EC periphery and were better developed and more abundant in SS-treated TEBVs than the static controls. The Western blot indicated that the expressions of PECAM-1 and VE-cadherin were increased by 72±9% and 67±7%, respectively, after shear stress treatment. The distribution pattern of integrin β1 was mainly at the interface of ECs and AM in static TEBVs but it was extended to the cell–cell junctions after SS treatment. The SS promoted the expression of integrin αvβ3 without altering its distribution in TEBV. The results suggest that glutaraldehyde cross-linked AM tube can potentially be used as a scaffold biomaterial for TEBV fabrication. Most importantly, the use of an AM tube shortened the TEBV fabrication.

Use of a hyperdried cross-linked amniotic membrane as initial therapy for corneal perforations

To report the use of hyperdried cross-linked (HDCL) amniotic membrane (AM) patching with tissue adhesive as an initial therapy for corneal perforations. Cryopreserved AM was cross-linked with 0.1% glutaraldehyde and then dried using far-infrared rays and microwaves (hyperdry method). Three eyes of three patients with corneal perforations of up to 3 mm in diameter were included in this study. They were treated with a single-layer patch of HDCL-AM applied with a tissue adhesive (2-octyl-cyanoacrylate). We also evaluated the resistance of HDCL-AM to collagenases during in vitro digestion testing. In all three cases, the corneal perforations were repaired within 28 days (range, 17-28 days). No recurrence occurred during the follow-up period (3-6 months). In the collagenase digestion testing, the HDCL-AM did not dissolve until 48 h, whereas the cryopreserved AM completely dissolved within 60 min. Three cases of corneal perforations were successfully managed using HDCL-AM patching with tissue adhesive. The HDCL-AM was resistant to collagenases during in vitro digestion testing. The HDCL-AM was a useful substrate for corneal perforations. This simple surgical technique may be one of the initial therapeutic options for corneal perforations.

Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells

In ophthalmic tissue engineering, amniotic membrane (AM) is one of the most prevalent natural matrices used for limbal epithelial cell (LEC) cultivation and transplantation. However, the application of AM as a scaffold is limited by its low biomechanical strength and rapid biodegradation. The present study reports the development of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linked AM as an LEC carrier. The collagenous tissue materials were modified with varying cross-linker concentrations (0–0.25 mmol EDC/mg AM) and were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), ninhydrin assays, electron microscopy, light transmission measurements, mechanical and in vitro degradation tests, as well as diffusion permeability and cell culture studies. Our results showed that chemical cross-linking approaches saturation at concentrations of 0.05 mmol EDC/mg AM. The formation of cross-links (i.e., amide bonds) in the samples treated with 0.05 mmol EDC/mg AM may cause significant aggregation of tropocollagen molecules and collagen microfibrils without affecting cell morphology of biological tissues. With the optimum concentration of 0.05 mmol EDC/mg AM, chemical cross-linker could significantly enhance the mechanical and thermal stability, optical transparency, and resistance to collagenase digestion. Continuous permeation of albumin through the cross-linked AM would be helpful to cell growth over the matrix surface. In addition, the EDC cross-linked samples were able to support LEC proliferation and preserve epithelial progenitor cells in vitro and in vivo. It is concluded that the AM cross-linked with 0.05 mmol EDC/mg AM may be a potential biomaterial for regenerative medicine.

Implantation of amniotic membrane to reduce postlaminectomy epidural adhesions

Postlaminectomy epidural adhesion is implicated as a main cause of "failed back surgery syndrome" and associated with increased risk of complications during revision surgery. Various materials acting as mechanical barriers to reduce fibroblasts infiltration into epidural space have met with limited success. In present research, amniotic membrane (AM) was studied to investigate its effects on reducing epidural scar adhesion after laminectomy in a canine model. Laminectomy sites were created at L-1, L-3, L-5, and L-7 levels in 24 adult mongrel dogs. Freeze dried AM (FAM), cross-linked AM (CAM), and autologous free fat (AFF) were implanted, respectively, at a randomly assigned site in each dog with the remaining untreated site serving as internal control. The animals were sacrificed at 1, 6, and 12 weeks postoperatively. Then, gross pathologic observation including scar amount and adhesion tenacity, qualitative histology evaluation, and quantitative histology analysis were compared. Gross observation demonstrated that scar amount and adhesion tenacity of CAM group were significantly lower in comparison with those of FAM and non-treatment groups. A white, slightly vascularized CAM layer covered the dura mater without tenacious scar adhesion. The histology analysis also indicated reduced fibroblasts infiltration and consequent epidural fibrosis, which were similar to the results of AFF group. In conclusion, the CAM is effective in reducing epidural fibrosis and scar adhesion after laminectomy in canine model. It is a promising biomaterial for future clinical applications.