Amniotic Membrane Tissue Research Articles

The Use of Cryo-Preserved Umbilical Cord Plus Amniotic Membrane Tissues in the Resection of Tarsal Coalition

Category: Hindfoot Introduction/Purpose: Tarsal coalition can be a common underlying cause of painful flatfoot deformity in adolescent and adult patient populations. When conservative options fail, surgical resection becomes the mainstay of treatment. Adequate resection leaves a void, often filled with bone wax, fat graft or adjacent muscle or tendon. Interposition grafting is designed to provide a physical barrier between the ends of the excised segment and deter recurrence or bony overgrowth. No definitive graft choice has been shown to be superior to another. The goal of this study was to evaluate the use commercially available umbilical cord plus amniotic membrane tissues as an interposition graft with functional and radiological outcomes at one year follow-up. Methods: Retrospective review was performed of patients who had resection of tarsal coalition at our institution. Patients who had interposition of umbilical cord plus amniotic membrane tissues (Amniox Medical, Atlanta GA) at the site of the resection were included in this review. Postoperative data collection included VAS scores, Foot Function index scores, and radiographic review. Radiographic review included postoperative CT scans in all patients. Assessment of hindfoot ROM was noted on physical examination at last follow up visit. Results: Fourteen patients (8 male and 6 female; median age 28.5 years at time of evaluation) were enrolled in the study. Of these patients, 7 were calcaneonavicular coalitions and 7 were talonavicular coalitions. Average follow up occurred at 18 months (range 9-32 months). Interposition of umbilical cord plus amniotic membrane tissues was performed at the site of the resection. Median improvement in pain visual analog score (VAS) was 89.75%, with no post-operative infection or wound complications. Post-operative CT scan review demonstrated evidence of osteoarthritis in two patients (one at talonavicular and one subtalar joint). Four of 14 patients (29%) were found to have recurrence at follow-up. The median post-operative Foot Function Index score was 46 (range 30-110) and 64% of patients had improvement in range of motion. Conclusion: Based on radiological and functional outcomes at an average 19 month follow up, the interposition of umbilical cord plus amniotic membrane tissues after tarsal coalition resection appears to be a viable option in treating these patients. No adverse reactions were found and recurrence rate was noted to be 29%.

Optimizing amniotic membrane tissue banking protocols for ophthalmic use.

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco's modified eagle's medium (DMEM) and glycerol at -80°C and at -196°C for a period of 6weeks and at 4°C in 70% alcohol for 6weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5×5cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at -196 and -80°C for 6 weeks. Similar sections were also stored at 4°C in 70% alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10% FBS in humidified atmosphere of 5% carbon dioxide at 37°C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8×10(6); 1.59, after 6weeks in DMSO:FBS at -196°C 3.0×10(6); 2.38 and at -80°C 2.1×10(6); 1.60, in DMEM:Glycerol at -196°C 3.6×10(6); 2.33 at -80°C 23×10(6); 1.66 and at 4°C 3.3×10(6); 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at -196°C showed morphology similar to fresh AM in both preservation media and AM stored at -80°C showed disruption of the stromal matrix. At 4°C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at -196°C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.

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.

Morselized Amniotic Membrane Tissue for Refractory Corneal Epithelial Defects in Cicatricial Ocular Surface Diseases.

PurposeTo evaluate the clinical efficacy of morselized amniotic membrane and umbilical cord tissue (MAU) in treating refractory corneal epithelial defect in ocular cicatricial diseases.MethodsRetrospective review of four patients with ocular cicatricial diseases treated with topical MAU for corneal epithelial defects refractory to conventional treatments including topical lubricants, autologous serum, bandage contact lens, and tarsorraphy. Their symptoms, corneal staining, conjunctival inflammation, and visual acuity were compared before and after treatment.ResultsAfter topical application of MAU twice daily, two patients demonstrated rapid corneal epithelialization with prompt visual acuity improvement at the first day. All patients showed corneal epithelialization in 7.3 ± 2.6 days accompanied by a significant relief of symptoms, reduction of ocular surface inflammation, and improvement of visual acuity.ConclusionThis pilot study suggests topical MAU can be developed into a novel treatment for treating refractory corneal epithelial defects.Translational RelevanceTopical MAU can be an effective novel treatment for refractory corneal epithelial defects.

Clinical comparison of coronally-advanced flap plus amniotic membrane or subepithelial connective tissue in the treatment of Miller's class I and II gingival recessions: A split-mouth study.

Background. The aim of the present study was to compare coronally advanced flap (CAF) plus amniotic membrane (AM) to CAF with connective tissue graft (CTG) in the treatment of Miller’s class I and II gingival recessions.Methods. Eleven healthy subjects with thirty Miller’s class І and ІІ gingival recessions ≥3 mm, were selevted for this research and randomly assigned to two groups in a split-mouth design. In the control group gingival recessions were treated with CAF and CTG; however, in the test group the lesions were treated with (AM) and CAF. The clinical parameters, including recession depth (RD), recession width (RW), keratinized tissue width (WKT), probing depth (PD) and clinical attachment level (CAL), were measured at baseline and 1, 3 and 6 months postoperatively. Statistical significance was set at P < 0.01.Results. Position changes of RD, RW, CAL, and MGJ were significant between baseline and one month after surgery (P < 0.01) in both the test and control groups and these values remained unchanged at 3- and 6-month follow-ups. There were no statistically significant differences in PD and WKT between baseline and 1-, 3- and 6-months intervals postoperatively. The mean root coverage values after 6 months were 75.5% and 63.1% for two groups, respectively. The mean recession depth reductions were 2.63±0.63 mm and 2±1.4 mm in the test and control groups, respectively. Conclusion. The results of this research showed that application of AM instead of connective tissue decreased surgical operation time and patient discomfort but the amount of root coverage was not significantly different between the two methods.

Radiation sterilization of tissue allografts: A review.

Tissue substitutes are required in a number of clinical conditions for treatment of injured and diseased tissues. Tissues like bone, skin, amniotic membrane and soft tissues obtained from human donor can be used for repair or reconstruction of the injured part of the body. Allograft tissues from human donor provide an excellent alternative to autografts. However, major concern with the use of allografts is the risk of infectious disease transmission. Therefore, tissue allografts should be sterilized to make them safe for clinical use. Gamma radiation has several advantages and is the most suitable method for sterilization of biological tissues. This review summarizes the use of gamma irradiation technology as an effective method for sterilization of biological tissues and ensuring safety of tissue allografts.

Pre-Differentiated Skin-Like Cells can Alleviate In Vitro Burn Injury

Considering importance of stem cell based therapies for wound healing, we have previously established protocols for successful differentiation of stem cells (isolated from placenta) into skin-like cells (keratinocytes and fibroblasts). In the current study we aim to evaluate the effect of pre-differentiated skin-like-cells on wound healing potential using an in vitro burn injury model. The amniotic epithelial cells (AECs) and umbilical cord mesenchymal stem cells (UCMSCs) obtained from amniotic membrane and umbilical cord tissue; respectively, were differentiated into skin-like cells (keratinocyte and fibroblasts respectively). In order to make an in vitro burn injury model, keratinocytes and fibroblasts were isolated from rats and insulted with high temperature (up to 50oC). The optimal temperature for burn injury models was determined using viability assay, cytotoxicity level, proliferation, and expression of pro-apoptotic. In order to assess effectiveness of pre-differentiated skin like cells, the keratinocyte- and fibroblasts-like cells were co-cultured with injured keratinocytes and injured fibroblasts, respectively. Results indicated that optimal temperature for the induction of heat injury for both keratinocytes and fibroblasts was 50oC. At this temperature both types of cells (keratinocytes and fibroblasts) showed modified morphology, drastic cellular injury, least viability, higher expression of pro-apoptotic markers and lower expression of proliferation genes. Co-culturing of pre-differentiated cells leads to an increase in viability and proliferation concurrent with decreased apoptosis. We have established successfully in vitro model of burn injury that might be helpful to evaluate the wound healing potential of cells. Further, predifferentiated skin-like cells are a potential source for the treatment of severe burn injuries.

Perforations cornéennes non traumatiques : modalités thérapeutiques

The treatment of non-traumatic perforations of the cornea is a real challenge for the choice of surgical technique as well as for management of the causal pathology. The goal of our study is to determine the anatomical and functional results of the management of non-traumatic perforation of the cornea in the absence of ready access to a corneal graft. This is a retrospective, non-comparative monocentric study of 23 consecutive cases diagnosed and treated between January 2011 and January 2013. We included 23 eyes with non-traumatic corneal perforation. Various surgical techniques were used depending on the size and location of the corneal perforation. The predominant etiology of the corneal perforations in our series was corneal abscess, found in 30.4% of cases (7 eyes). A conjunctival flap was performed in 43.5% of cases (10 eyes) followed by tarsorrhaphy in 21.7% of cases (5 eyes), autologous corneal patch in 17.4% of cases (4 eyes), amniotic membrane in 13% of cases (3 eyes) and finally cyanoacrylate glue in one case. Mean follow-up was 12.4 ± 2.1 months. Anatomical closure of the corneal perforation was achieved in 91.3% of cases (21 eyes), while the final visual acuity was not improved due to secondary opacities. The choice of surgical technique depends on the size of the corneal perforation, its location, its etiology and the resources available for emergencies. When amniotic membrane and corneal donor tissue are unavailable, conjunctival flap is an easy and effective technique, which is a good alternative to close corneal perforations less than 3mm. It improves ocular surface quality and prepares the eye for later penetrating keratoplasty. Conjunctival flap is a good technique that is still relevant today, especially in the absence of corneal donor tissue or amniotic membrane. The anatomical success rate is very satisfactory, but sometimes several interventions are needed to improve the visual prognosis.

Repair effect of human acellular dermal matrix on filtering blebs leakage in rabbit eye

Background Package and tissue patch implantation are common methods for repair of filtering bleb leaking after anti-glaucoma surgery.But the scarring or re-leakage of filtering bleb probably occur again. Objective This study was to investigate the repair effect of acellular dermal matrix (ADM) on filtering bleb leaking in rabbit model and compare the effectiveness among ADM, amniotic membrane and conjunctival overlap. Methods Trabeculectomy was performed on 48 eyes of 24 New Zealand rabbits, and models of filtering bleb leaking were established.The models were randomized into ADM group, amniotic membrane group and conjunctival covering group based on randomized number table.ADM patches with 4 mm×4 mm were implanted across lamellar cornea and sclera at a bridge in the ADM group, and the same size of amniotic membranes were used in the amniotic membrane group, and conjunctiva was sutured to limbus in the conjunctiva overlap group.The intraocular pressure (IOP) was measured before surgery and 1 day, 1 week, 1 month, 3 months and 6 months after surgery.The biocompatibility of materials above was assessed under the slit lamp microscope, and the status of filtering bleb was evaluated and compared with anterior segment optical coherent tomography (AS-OCT) 1 day, 1 week, 1 month, 3 months and 6 months after surgery. Results Before surgery and 1 day, 1 week, 1 month, 3 months and 6 months after surgery, the IOP was (26.9±4.3), (16.6±5.1), (22.1±6.2), (18.3±6.5), (22.7±2.5), (23.4±1.4) mmHg in the AMD group, (29.9±5.4), (14.9±6.4), (21.6±7.8), (26.3±4.1), (26.0±4.2) and (23.0±5.3) mmHg in the amniotic membrane group, and (28.7±4.3), (15.7±7.0), (22.0±6.3), (28.2±4.1), (24.7±4.1), (23.0±2.7) mmHg in the conjunctival overlap group, showing significant differences among different groups and various time points (Fgroup=8.419, P=0.011; Ftime=15.543, P=0.000). The IOPs were significantly lower from 1 day through 3 months after operation than those before operation in the AMD group (P=0.000, 0.000, 0.006, 0.045); while the IOPs were reduced only from 1 day through 1 week after operation in comparison with before operation in the amniotic membrane group and the conjunctival overlap group (P=0.000, 0.001). One month after surgery, the IOPs were significantly declined in the ADM group compared with the amniotic membrane group and the conjunctival overlap group (P=0.001, 0.000). The grafts were clear under the slit lamp microscope and exhibited the valid filtering bleb until 3 months after operation under the AS-OCT in the ADM group.However, the valid filtering bleb remained only 1 month after surgery in the amniotic membrane group and the conjunctival overlap group.Neovascularization on the filtering bleb was found 3 months in the AMD group but 1 month in the amniotic membrane group and the conjunctival overlap group. Conclusions Compared with amniotic membrane and conjunctival tissue, ADM patch for the repair of filtering bleb leakage can increase the survival duration of filtering bleb and remain lower IOP. Key words: Human acellular dermal matrix; Trabeculectomy/complication; Filtering blebs; Leakage; Repair; Amniotic membrane; Intraocular pressure; Rabbit

Development of a Sterile Amniotic Membrane Tissue Graft Using Supercritical Carbon Dioxide

Numerous techniques have been reported for preparing and sterilizing amniotic membrane (AM) for use in clinical applications. However, these preparations either do not produce completely sterile tissue or are detrimental to molecules unique to the tissue matrix, thus compromising beneficial wound-healing properties of the AM graft. The objective of this work was to produce a sterile human AM tissue graft using a novel preparation technique involving supercritical carbon dioxide (SCCO2). AM tissue was subjected to various sterilization treatment groups that optimized the duration of exposure to SCCO2 and the amount of peracetic acid (PAA) required to achieve a sterility assurance level of 10(-6) log reduction in bacterial load. Effects of sterilization treatment on the histological, biophysical, and biochemical properties of the sterile AM were evaluated and compared with those of native AM tissue. Exposure of the AM tissue to combined SCCO2 and PAA sterilization treatment did not significantly alter tissue architecture, the amounts of pertinent extracellular matrix proteins (type IV collagen, glycosaminoglycans, elastin) present in the tissue, or the biophysical properties of the tissue. AMs treated with SCCO2 were also found to be excellent substrates for adipose-derived stem cell (ASC) attachment and proliferation in vitro. Human ASCs, attached to all treatment groups after 24 h of culture and continued to proliferate over the next few days. The current study's results indicate that SCCO2 can be used to sterilize AM tissue grafts while simultaneously preserving their biological attributes. The preservation of these features make AM appealing for use in numerous clinical and tissue engineering applications.

In Vitro Differentiation Potential of Human Placenta Derived Cells into Skin Cells.

Skin autografting is the most viable and aesthetic technique for treatment of extensive burns; however, this practice has potential limitations. Harvesting cells from neonatal sources (such as placental tissue) is a simple, inexpensive, and noninvasive procedure. In the current study authors sought to evaluate in vitro potential of human placenta derived stem cells to develop into skin-like cells. After extensive washing, amniotic membrane and umbilical cord tissue were separated to harvest amniotic epithelial cells (AECs) and umbilical cord mesenchymal stem cells (UC-MSCs), respectively. Both types of cells were characterized for the expression of embryonic lineage markers and their growth characteristics were determined. AECs and UC-MSCs were induced to differentiate into keratinocytes-like and dermal fibroblasts-like cells, respectively. After induction, morphological changes were detected by microscopy. The differentiation potential was further assessed using immunostaining and RT-PCR analyses. AECs were positive for cytokeratins and E-Cadherin while UC-MSCs were positive for fibroblast specific makers. AECs differentiated into keratinocytes-like cells showed positive expression of keratinocyte specific cytokeratins, involucrin, and loricrin. UC-MSCs differentiated into dermal fibroblast-like cells indicated expression of collagen type 3, desmin, FGF-7, fibroblast activation protein alpha, procollagen-1, and vimentin. In conclusion, placenta is a potential source of cells to develop into skin-like cells.

A standardised ‘off the shelf’ substrate for enhanced tissue engineering

AbstractPurpose Amniotic membrane (AM) is a popular ophthalmic tissue engineering adjunct. We previously developed a highly effective thermolysin‐based denuding technique that preserves basement membrane integrity This technique has been validated clinically using fresh frozen AM (FAM). We now propose a standardised dry‐prepared ‘off the shelf’ AM tissue engineering substrate.Methods Dry preserved thermolysin denuded AM (DAM), and FAM denuded with ethylenediaminetetraacetic acid (EDTA), and dispase ‐based methodologies were prepared. Denuding efficiencies were compared using electron microscopy. The effect of denuding on AM molecular composition was investigated and characterised using proteomics. The propensity of DAM to support stem cells was explored using fluorescent immunohistochemistry for defined markers.Results Electron microscopy demonstrated thermolysin denuding efficiency was comparable in FAM and DAM. Proteomic analyses showed effective removal of epithelial cell proteins in DAM, but not EDTA–based denuding techniques. Whilst similar enzymatic activity to thermolysin, mechanical scraping reduces the efficacy of dispase denuding. Collagens IV, VI, periostin, βig‐h3 and VLA‐6 are targets of thermolysin activity. DAM maintains stem cell characteristics and is most effective in preventing differentiation.Conclusion Conventional EDTA and dispase procedures for preparing AM for tissue engineering are ineffective at removing cells whilst preserving the basement membrane. Combining our novel thermolysin denuding and dry preservation techniques improves the overall quality of AM for tissue‐engineered constructs. Therefore, we propose a fist of its kind, stable and dry‐stored ‘off the shelf’ construct for enhanced ocular surface tissue engineering.

Postoperative Changes in Amniotic Membrane as a Carrier for Allogeneic Cultured Limbal Epithelial Transplantation

To investigate the morphologic changes and outcomes of the amniotic membrane as a carrier for allogeneic cultivated limbal epithelial transplantation. Prospective, noncomparative, interventional study. A total of 16 eyes receiving allogeneic cultivated limbal epithelial transplantation with amniotic membrane as a carrier were enrolled. Morphologic changes in the amniotic membrane were observed by confocal microscopy and RTVue optical coherence tomography. The paired t test was employed to compare the mean best corrected visual acuity (BCVA) and corneal stromal thickness. Of the 16 eyes, 12 had stable ocular surfaces (group A), while the other 4 eyes had failed surgeries due to immune rejection (group B). Confocal microscopy showed residual amniotic membrane tissues in 8 eyes in group A at 1 year. However, the amniotic membrane was not detected in group B at 8-10 months. RTVue optical coherence tomography showed discontinuous amniotic membrane tissues in all eyes in group A at 1 year, while highly reflective opacity was seen in the corneal stroma in group B. There were no statistically significant differences in mean BCVA and corneal stromal thickness in group A at 1 month and 1 year after transplantation (P > 0.05), but the mean BCVA showed a statistically significant difference at 1 month and after the disappearance of the amniotic membrane in group B (P < 0.05). For eyes with stable ocular surfaces after cultivated limbal epithelial transplantation, the amniotic membrane can be present in the cornea for at least 1 year, with no impact on visual acuity or corneal stromal thickness. Chronic inflammation and neovascularization on the ocular surface may accelerate the disappearance of the amniotic membrane.

Structural and Biological Comparison of Cryopreserved and Fresh Amniotic Membrane Tissues

Structural and Biological Comparison of Cryopreserved and Fresh Amniotic Membrane Tissues

Comparative characteristics of amniotic membrane, endometrium and ovarian derived mesenchymal stem cells: A role for amniotic membrane in stem cell therapy

Abstract Objectives Isolation and comparison of cellular and molecular characteristics of MSCs derived from full term amniotic membrane (AM), endometrium and ovarian tissues. Investigation of a possible therapeutic potential for AM MSCs in the female reproductive tract. Study design & methods Human amniotic membranes (AMs) from term placentas ( N = 8), endometrial/stromal (by scrapping endometrium) and ovarian tissues from premenopausal hysterectomies ( N = 6) after mincing and enzymatic digestion were included in the study. Immunophenotyping by flow cytometry, Karyotyping, differentiation to mesoderm, RT-PCR analysis for transcription factors, WNT signaling pathways, pluripotency markers and HOXA genes were performed. Results Generated cells showed morphologically and phenotypically the characteristics of MSCs from other sources with fairly comparable results. The cumulative population doubling was significantly higher in endometrium MSCs 25 ± 5.2 compared to 17.2 ± 4.5 for AM MSCs and 10.7 ± 2.4 for ovarian MSCs ( P Intrauterine injection of GFP transfected AM MSCs into female Wistar rats ( N = 10), demonstrated the presence of AM cells with GFP expression after 14 days within several mice uterine and ovarian cryosections. AM MSCs highly expressed genes involved in patterning of the female reproductive tract. Conclusions AM, endometrium and ovarian derived MSCs share fairly similar morphological, cellular and molecular characteristics. They possess mesenchymal stemmness potentials and endometrial MSCs possess the highest expansion capacity. AM MSCs may have a potential for stem cell therapy in the female reproductive tract.

Molecular and chemical investigations and comparisons of biomaterials for ocular surface regeneration

This study investigated and compared the ultrastructural and chemical properties of representative biomaterials for ocular surface regeneration: a human amniotic membrane (AM) in a basal plate, a human AM in reflected chorion, a preserved AM, and a human corneo-scleral tissue. Assessments of the morphological differences in the extracellular matrices were evaluated by hematoxylin-eosin, Masson's trichrome (for total collagen), and picrosirius-red (for newly synthesized collagen) staining. Assessments of the changes in the molecular structures and chemical compositions of the biomaterials for ocular surface regeneration were evaluated by Raman spectroscopy. A placental AM (52 %) was a dense and thick collagenous structure compared to a reflected AM (23 %). The spectroscopy did not obtain any structural information for a preserved AM. The cornea group (100 %, control) and sclera group (104 %) showed the collagen lamellae and interfibrillar spacing, and a slight inflammatory reaction with more fibrous and granulomatous tissues. There was a formation of newly synthesized collagen in a placental AM, while there were few collagen components in a reflected AM. Human AM tissues showed consistent Raman spectra and the characteristic collagen bands, similar to the corneal and scleral tissues. Therefore, these findings suggest that human placental AM and reflected AM are structurally suitable for scleral and corneal surface regeneration, respectively, while human placental or preserved AM and reflected AM are molecularly and chemically suitable for corneal and scleral surface regeneration, respectively.

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.

Comparison of viscoelasticity between normal human sciatic nerve and amniotic membrane.

Sciatic nerve tissue was obtained from the gluteus maximus muscle segment of normal human cadavers and amniotic membrane tissue was obtained from healthy human puerperant placentas. Both tissues were analyzed for their stress relaxation and creep properties to determine suitability for transplantation applications. Human amniotic membrane and sciatic nerve tissues had similar tendencies for stress relaxation and creep properties. The stress value of the amniotic membrane stress relaxation group decreased to a greater extent compared with the sciatic nerve stress relaxation group. Similarly, the stress value of the amniotic membrane creep group increased to a greater extent compared with the sciatic nerve creep group. The stress relaxation curve for human amniotic membrane and sciatic nerve showed a logarithm correlation, while the creep curve showed an exponential correlation. These data indicate that amniotic membrane tissue has better stress relaxation and creep properties compared with sciatic nerve tissue.

“Triple-TEN” in the Treatment of Acute Ocular Complications From Toxic Epidermal Necrolysis

Toxic epidermal necrolysis (TEN) is a devastating form of Stevens-Johnson syndrome (SJS) with acute and chronic ocular complications. We present a novel aggressive combination strategy, termed "Triple-TEN", for the management of acute ocular TEN designed to minimize the risk of chronic, blinding sequelae. Two patients with life-threatening TEN accompanied by severe ocular surface defects and fulminant symblephara formation underwent "Triple-TEN" management of their acute ocular disease under aseptic techniques in the critical care setting, after failed treatment with intensive topical therapy and surgical division of symblephara. The Triple-TEN protocol comprises (1) subconjunctival triamcinolone (Kenalog 20 mg) administered into each of the fornices to curb the local inflammatory response without compromising systemic immunity, (2) placement of amniotic membrane tissue mounted on a polycarbonate skirt (ProKera) over the corneal and limbal regions to facilitate reepithelialization of the ocular surface, and (3) insertion of a steeply curved acrylic scleral shell spacer (Technovent, SC21) to vault the lids away from the globe providing a barrier to symblephara formation. In both cases, ocular surface inflammation resolved within 4 weeks with no progression of conjunctival cicatrization or evidence of limbal epithelial stem cell failure at 1 year follow-up. There were no long-term complications of the Triple-TEN regimen. Aggressive treatment with the Triple-TEN protocol for acute ocular TEN resistant to first-line therapy, may help prevent long-term blinding sequelae.

Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine.