Photocrosslinkable human amniotic membrane hydrogel for recovery from spinal cord injury

The recovery and reconstruction of central nervous system function after spinal cord injury(SCI)is a worldwide problem.The difficulty lies in the feasibility issue of new axons passing through the injured area and the negative effect of scarring after injury.As a biological material,the human amniotic membrane(HAM)has the advantages of protecting nerve growth,inhibiting scar formation,and promoting neovascularization,but its weak physical properties are difficult to apply in treating SCI.In this study,HAMs were first decellularized and then chemically grafted with methacrylic anhydride.Next,the composite was photocrosslinked with gelatin methacrylate to prepare a cross-network biological complex.The final complexes prepared by appeal were used for in vitro and in vivo studies of SCI in rats,separately.In the in vitro experiment,the composite scaffold inherited abundant biological factors from the amniotic membrane and had the physical properties of a hydrogel,thus providing a favorable environment for the growth and development of neurons and blood vessels.In the in vivo experiment,the composite reduced scarring and promoted the growth of new nerves.Overall,the composite scaffolds can stably simulate the extracellular microenvironment in SCI defects,regulate pathological changes,and promote the generation of new neurons.Therefore,decellularized HAM hydrogels are promising biocomposite materials for central nerve repair after SCI.

Effect of autologous serum after amniotic membrane transplantation for persistent corneal ulcers

AIM:To investigate the effect of adding autologous serum eye drops to the postoperative regime after amniotic membrane transplantation for severe persistent corneal ulcers.METHODS:Forty eyes of 40 patients with persistent corneal ulcers were randomly assigned to artificial tears(sodium hyaluronate 0.2%,ATs group,n=20)or autologous serum eye drops(ASEDs,n=20)following treatment with amniotic membrane transplantation.Digital slit lamp images were acquired from all patients before and 30d post treatment.The area with fibrovascular tissue was calculated using Image J.Central corneal sensitivity was assessed by Cochet-Bonnet aesthesiometry before and one month after treatment.Scar tissue transparency was assessed with a novel optical densitometry.RESULTS:Mean age of patients was 61.65±16.47y and 57.3±19.11y in the ATs group and ASEDs group,respectively.Twenty-two male and 18 female patients were included in the study.The improvement in visual acuity was significantly greater in the ASEDs group(0.14±0.04)than the ATs(0.08±0.04;P=0.00046).Cochet-Bonnet aesthesiometry improved significantly after treatment with a similar rate between groups.There were no statistically significant differences in the area of postoperative fibrovascular tissue between the two groups(P=0.082).The success rate in the two groups was similar.The difference in densitometry between the ATs and ASEDs group was statistically significant(P=0.042)with greater reduction from baseline in the ASEDS group.CONCLUSION:Autologous serum eye drops can lead to better visual acuity,more stable results and improved densitometry and should be considered in the postoperative care following amniotic membrane transplantation.

Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane

AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM). METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors, yielding an HCEP cell line which was its growth performance, chromosome morphology, tumorigenicity and expression of marker proteins analyzed. In addition, the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light, electron and slit-lamp microscopies. RESULTS: HCEP cells proliferated to confluence in 3 weeks, which have been subcultured to passage 160. A continuous untransfected HCEP cell line, designated as utHCEPC01, was established with a population doubling time of 45.42 hours as was determined at passage 100. The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3. They, with no tumorigenicity, formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture, maintained expression of marker proteins including keratin 3 and integrin p 1 and attached tightly to dAMs. The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original. CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study. The cells maintained expression of marker proteins. The cell line was biocompatible with dAM. It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP, promising for the treatment of diseases caused by corneal epithelial disorders.

Autologous nerve anastomosis versus human amniotic membrane anastomosisA rheological comparison following simulated sciatic nerve injury

The sciatic nerve is biological viscoelastic solid,with stress relaxation and creep characteristics.In this study,a comparative analysis of the stress relaxation and creep characteristics of the sciatic nerve was conducted after simulating sciatic nerve injury and anastomosing with autologous nerve or human amniotic membrane.The results demonstrate that,at the 7 200-second time point,both stress reduction and strain increase in the human amniotic membrane anastomosis group were significantly greater than in the autologous nerve anastomosis group.Our findings indicate that human amniotic membrane anastomosis for sciatic nerve injury has excellent rheological characteristics and is conducive to regeneration of the injured nerve.

Clinical and impression cytology findings of amniotic membrane and oral mucosal membrane transplantation for the management of socket contracture

AIM:To investigate and compare the cytopathological and clinical effects of amniotic membrane transplantation(AMT)and oral mucosal membrane transplantation(OMMT)in socket contraction.METHODS:Twelve patients who could not be fitted with ocular prosthesis due to socket contracture were included in this study.Seven patients underwent AMT and 5 patients underwent OMMT.Thirteen patients who had healthy sockets were included as control group.Depth of inferior fornix,degree of inflammation,extent of the socket contracture and socket volume were measured in the preoperative period and at sixth and twelfth weeks postoperatively.Impression cytology of conjunctival fornices and tear transforming growth factor beta-1(TGFβ1)levels were determined.RESULTS:In the AMT group,socket volume and lower fornix depth values were significantly higher(P=0.030and P=0.004 respectively)and inflammation levels and impression cytology stages(P=0.037 and P=0.022respectively)were significantly lower in postoperative period compared to preoperative period.In the OMMT group,no statistical differences were found in terms of clinical parameters,inflammation levels and impression cytology stages of preoperative versus postoperative values.Preoperative tear TGFβ1 levels were higher in AMT and OMMT groups compared to the control group(25.5 ng/mL,26.3 ng/mL and 21.7 ng/mL respectively).Decreased tear TGFβ1 levels were observed in both the AMT and OMMT groups postoperatively(median decrease value=2.1 ng/mL and 2.7 ng/mL respectively).CONCLUSION:AMT is associated with postoperative improvement in inferior fornix depth,socket volume,inflammation and impression cytology levels and may bea more proper alternative method than OMMT in the management of socket contracture.

Homemade lyophilized cross linking amniotic sustained-release drug membrane with anti-scarring role after filtering surgery in rabbit eyes

AIM: To investigate the antifibrotic effect of the freeze-dried bilayered fibrin-binding amniotic membrane as a drug delivery system on glaucoma surgery in rabbit model. The aim of this study was to prepare a novel local delivery system for the sustained and controllable release of 5-Fu. METHODS: Twenty-four Japanese white rabbits were randomized into three groups: the experimental group (ocular trabeculectomy in combination with 5-Fu loaded freeze-dried bilayered fibrin-binding amniotic membrane transplantation), the control group (ocular trabeculectomy in combination with 5-Fu) and the blank group (single trabeculectomy). HE staining, massion staining and immunohistochemistry for alpha -SMA were performed on days 7, 14, 21 and 30 following surgery. The concentration of 5-Fu in rabbit aqueous humor was examined by high performance liquid chromatography (HPLC) 3 days after the surgery. RESULTS: Statistical differences were noted in intraocular pressure among groups on day 7, 14, 21 and 30 following surgery. Histology further demonstrated that trabeculectomy in combination with freeze-dried bilayered fibrin-binding amniotic membrane yielded well wound healing and no scar formation and was beneficial for long term effect. CONCLUSION: HPLC showed a good slow-release effect with freeze-dried bilayered fibrin-binding amniotic membrane.

United membrane biological reactor in the treatment of wastewater

The united membrane biological reactor(UMBR) was studied for the treatment of some simulate and municipal wastewater. The removal efficiency for COD and turbidity are greater than 80% and 99% respectively. Effluent COD is less than 100 mg/L while turbidity less than 5 The removal of LAS in bath wastewater is greater than 70%. In treatment of dinning hall wastewater, removal of fatty oil is greater than 90%, and its concentration in effluent is less than 5 mg/L. The match of biological reactor and the membrane separation component were calculated. The stable performance of wastewater treatment can be maintained by the optimization of operation conditions and the cleanout of membranes.

Sutureless amniotic membrane transplantation following excision of ocular surface neoplasia

【正】Dear Sir,S uccess of surgical management of conjunctival neoplasias primarily depends on the complete excision of the mass and eradication of tumor cells from the ocular surface[1-3].Especially in cases that extensive resections are required,primary closure may not be feasible;so ocular surface reconstruction can be a challenging problem for the surgeons.For conjunctival tissue defects different surgical

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Many researchers have employed the cryopreserved amniotic membrane（CAM） and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane （AM）. The lyophilized amniotic membrane（LAM） has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings（ Ahn＇s supporter） were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, foUowed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

Human amniotic membrane and nerve tissue engineering

Totally three articles focusing on ＂the effects of microenvironment for human amniotic epithelial cell transplantation on cell survival and differentiation, the migration of human amniotic epithelial cells after in vitro transplantation, and the rheological characteristics of anastomosing injured sciatic nerve with human amniotic membrane without amniotic epithelial cells＂ are published in three issues. We hope that our readers find these papers useful to their research.

Group B Streptococcal Chorioamnionitis with Intact Amniotic Membranes. A Rare?

Group B streptococcus infection (GBS) is an important cause of perinatal infection. Maternal colonization by GBS can cause amniotic infection and thus fetal infection, sometimes causing fetal death. This infection generally occurs in association with premature rupture of membranes. We present here an infrequent case of GBS chorioamnionitis in full-term gestation with intact amniotic membranes. The curiosity of the case lies in the integrity of the amniotic membranes, the asymptomatic clinical presentation, and the adverse result (term fetal death).

Significant impact of cryopreserved amniotic membrane on acute ocular Stevens-Johnson syndrome and toxic epidermal necrolysis

The ocular surface is covered by an epithelium encompassing an area including the cornea,the limbus and the conjunctiva bordered by the upper and lower lids.The healthy state of the ocular surface epithelium depends on a stable and protective preocular tear film when the eye is open.A stable preocular tear film is governed by sound ocular surface defense that involves effective

Comment on amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis

【正】Dear Editor,We congratulate Zeng et al[1]for their study entitled"Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis".The authors endeavored to present an alternative method for ophthalmologists in the treatment of a challenging case.We would like to express our reservations and ask for the attitudes of the authors about

Clinical outcome of combined conjunctival autograft transplantation and amniotic membrane transplantation in pterygium surgery

AIM： To compare long-term outcome of primary and recurrent pterygium surgery with three different techniques： combined conjunctival autograft and overlay amniotic membrane transplantation （CAT with AMT）, conjunctival autograft transplantation （CAT） alone and amniotic membrane transplantation （AMT） alone. METHODS： In this retrospective study, 142 eyes of 142 pterygium patients （104 primary, 38 recurrent）who underwent CAT （group A）, AMT （group B） or CAT with AMT （group C） respectively following surgical excision were reviewed and compared based on the recurrences and post-operative complications. RESULTS： The number of recurrence post-surgery were 17 （9 from primary, 8 from recurrent; the same description below）, 18 （10, 8） and 2 （1, 1） in groups A, B, and C respectively; dry eyes were 22 （16, 6）, 27 （18, 9） and 7 （3, 4）; conjunctival inflammations were 30 （17, 13）, 27 （16, 11） and 11 （6, 5）. Patients in group C （either pdmary or recurrent or both） mainly showed significantly better results than those in group A or B （P〈0.05） regarding above-mentioned clinical effects. CONCLUSION： Combined CAT and overly AMT have significantly lower rates of recurrence and postoperative complications for primary and recurrent pterygium surgery than CAT or AMT alone.

Strain and stress variations in the human amniotic membrane and fresh corpse autologous sciatic nerve anastomosis in a model of sciatic nerve injury

A 10-mm long sciatic nerve injury model was established in fresh normal Chinese patient cadavers. Amniotic membrane was harvested from healthy maternal placentas and was prepared into multilayered,coiled,tubular specimens.Sciatic nerve injury models were respectively anastomosed using the autologous cadaveric sciatic nerve and human amniotic membrane.Tensile test results showed that maximal loading,maximal displacement,maximal stress,and maximal strain of sciatic nerve injury models anastomosed with human amniotic membrane were greater than those in the autologous nerve anastomosis group.The strain-stress curves of the human amniotic membrane and sciatic nerves indicated exponential change at the first phase,which became elastic deformation curves at the second and third phases,and displayed plastic deformation curves at the fourth phase,at which point the specimens lost their bearing capacity.Experimental findings suggested that human amniotic membranes and autologous sciatic nerves exhibit similar stress-strain curves, good elastic properties,and certain strain and stress capabilities in anastomosis of the injured sciatic nerve.

Human umbilical cord mesenchymal stem cell-loaded amniotic membrane for the repair of radial nerve injury

In this study, we loaded human umbilical cord mesenchymal stem cells onto human amniotic membrane with epithelial cells to prepare nerve conduits, i.e., a relatively closed nerve regeneration chamber. After neurolysis, the injured radial nerve was enwrapped with the prepared nerve conduit, which was fixed to the epineurium by sutures, with the cell on the inner surface of the conduit. Simultaneously, a 1.0 mL aliquot of human umbilical cord mesenchymal stem cell suspension was injected into the distal and proximal ends of the injured radial nerve with 1.0 cm intervals. A total of 1.75 x 107 cells were seeded on the amniotic membrane. In the control group, patients received only neurolysis. At 12 weeks after cell transplantation, more than 80% of patients exhibited obvious improvements in muscular strength, and touch and pain sensations. In contrast, these improvements were observed only in 55-65% of control patients. At 8 and 12 weeks, muscular electrophysiological function in the region dominated by the injured radial nerve was significantly better in the transplantation group than the control group. After cell transplantation, no immunological rejections were observed. These findings suggest that human umbilical cord mesenchymal stem cell-loaded amniotic membrane can be used for the repair of radial nerve injury.

Human amniotic membrane transplantation: Different modalities of its use in ophthalmology

The amniotic membrane(AM) is the inner layer of the fetal membranes and consist of 3 different layers: the epithelium, basement membrane and stroma which further consists of three contiguous but distinct layers: the inner compact layer, middle fibroblast layer and the outermost spongy layer. The AM has been shown to have anti-inflammatory, anti-fibrotic, anti-angiogenic as well as anti-microbial properties. Also because of its transparent structure, lack of immunogenicity and the ability to provide an excellent substrate for growth, migration and adhesion of epithelial corneal and conjunctival cells, it is being used increasingly for ocular surface reconstruction in a variety of ocular pathologies including corneal disorders associated with limbal stem cell deficiency, surgeries for conjunctival reconstruction, as a carrier for ex vivo expansion of limbal epithelial cells, glaucoma surgeries and sceral melts and perforations. However indiscriminate use of human AM needs to be discouraged as complications though infrequent can occur. These include risk of transmission of bacterial, viral or fungal infections to the recipient if the donors are not adequately screened for communicable diseases, if the membrane is not processed under sterile condi-tions or if storage is improper. Optimal outcomes can be achieved only with meticulous case selection. This review explores the ever expanding ophthalmological indications for the use of human AM.

Comparison of trabeculectomy and trabeculectomy with amniotic membrane transplantation in the same patient with bilateral glaucoma

AIM:To observe effects of trabeculectomy with amniotic membrane transplantation(AMT) in controlling postoperative intraocular pressure(IOP) in patients with medically uncontrolled glaucoma.·METHODS:This study included adult patients with requiring bilateral glaucoma surgery.Each patient underwent trabeculectomy(Non-AMT group) in one eye and with AMT(AMT group) in the other eye according to randomized principle.Success was defined as intraocular pressure(IOP) 【21mmHg without any anti-glaucoma medications at 24 months follow-up.The two groups were compared in terms of IOP,complications and success rate.·RESULTS:Thirty-four eyes of 17 patients were investigated in this study.There was no statistically signifcant difference in pre-operative IOP between the two groups.The mean IOP was lower in AMT group compared with Non-AMT group on follow up months 12,18,and 24.Postoperative complications were more frequent in Non-AMT group(35.3%,6/17) compared with AMT group(5.9%,1/17).The success rate of surgery was88.2%(15/17) in Non-AMT group and 100%(17/17) in AMT group.·CONCLUSION:Trabeculectomy with AMT is an effective procedure to reduce IOP and complications,thereby improving surgical success rates.

Effects of Honghua preserved amniotic membrane on scar healing in experimental glaucoma surgery

AIM:To investigate the efficacy and safety of Honghua preserved amniotic membrane(AM)for preventing scar formation of the filtering bleb in a rabbit model of glaucoma trabeculectomy surgery.METHODS:Totally 36 rabbits(36 eyes)were randomly divided into 3 groups:the experimental group(ocular trabeculectomy in combination with Honghua preserved AM transplantation),the control group(ocular trabeculectomy surgery in combination with AM implantation),and the blank group(single trabeculectomy).Clinical observations[including intraocular pressure(IOP),filtering blebs and complications],MassonTrichrome staining,real-time quantitative reverse transcription-polymerase chain reaction(real-time PCR),Western blot were performed on different time points(D1,D7,D14,D21 and D56)after the surgery.RESULTS:After operated for 14d,there were statistically significant differences in the filtering blebs compared to the situation before operation(P【0.05),whereas no statistically difference on that among three groups(P】0.05).After 21d,the IOP of experimental group was lowest(P【0.05).There was significant difference between control group and blank group(P【0.05).On postoperative D14,the mean number of fibroblasts in the experimental group was significantlylower(40.6±10.2)compared to those in the control group(54.4±10.8)and blank group(68.2±11.6)(P【0.05,respectively).The mean numbers of the macrophage in the experimental and control groups were respcitively significantly lower versus the blank group(P【0.05,P【0.05,respectively).Compared to that in blank group,the level of transforming growth factor-β(TGF-β1)expression in sclera and conjunctival areas was reduced in the experimental and control groups on protein and mRNA level(P【0.05),but not significant difference between these two groups(P】0.05).CONCLUSION:The trabeculectory surgery with Honghua preserved AM can control IOP,sustain the functional filtration bleb,inhibit the proliferation of fibroblasts and open the filtrating pathway on the rabbit glaucoma models.

Fresh human amniotic membrane effectively promotes the repair of injured common peroneal nerve

Suture and autologous nerve transplantation are the primary therapeutic measures for completely severed nerves. However, imbalances in the microenvironment and adhesion of surrounding tissues can affect the quality of nerve regeneration and repair. Previous studies have shown that human amniotic membrane can promote the healing of a variety of tissues. In this study, the right common peroneal nerve underwent a 5-mm transection in rats. Epineural nerve repair was performed using 10/0 non-absorbable surgical suture. The repair site was wrapped with a two-layer amniotic membrane with α-cyanoacrylate rapid medical adhesive after suture. Hindlimb motor function was assessed using footprint analysis. Conduction velocity of the common peroneal nerve was calculated by neural electrical stimulation. The retrograde axoplasmic transport of the common peroneal nerve was observed using fast blue BB salt retrograde fluorescent staining. Hematoxylin- eosin staining was used to detect the pathological changes of the common peroneal nerve sputum. The mRNA expression of axon regeneration-related neurotrophic factors and inhibitors was measured using real-time polymerase chain reaction. The results showed that the amniotic membrane significantly improved the function of the injured nerve;the toe spread function rapidly recovered, the nerve conduction velocity was restored, and the number of fast blue BB salt particles were increased in the spinal cord. The amniotic membrane also increased the recovery rate of the tibialis anterior muscle and improved the tissue structure of the muscle. Meanwhile, mRNA expression of nerve growth factor, growth associated protein-43, collapsin response mediator protein-2, and brain-derived neurotrophic factor recovered to near-normal levels, while Lingo-1 mRNA expression decreased significantly in spinal cord tissues. mRNA expression of glial-derived neurotrophic factor did not change significantly. Changes in mRNA levels were more significant in amniotic-membrane-wrapping-treated rats compared with model and nerve sutured rats. These results demonstrate that fresh amniotic membrane wrapping can promote the functional recovery of sutured common peroneal nerve via regulation of expression levels of neurotrophic factors and inhibitors associated with axonal regeneration. The study was approved by the Committee on Animal Research and Ethics at the Affiliate Hospital of Zunyi Medical University, China (approval No. 112) on December 1, 2017.