Amniotic Tissue Research Articles

MiR-16a-5p antagonizes FGF-2 in ligamentogenic differentiation of MSC: a new therapeutic perspective for tendon regeneration

With the increasing demand for exercise, the population of patients with ankle sprain to anterior talofibular ligament injury has the characteristics of a large base and high requirements for returning to sports, and how to promote the repair of damaged ligaments from a microscopic perspective is an urgent problem to be solved. In many studies, human amniotic mesenchymal stem cells have strong differentiation ability, and can be induced to continuously differentiate into ligament cells to achieve the purpose of repairing damaged ligaments. Human amniotic stem cells were extracted and cultured from human amniotic tissues, evaluated by cell identification and other techniques, and evaluated into ligament differentiation by toluidine blue, alizarin red, oil red O staining and detection of ligament cell differentiation, protein detection by Western blot, mRNA level by qPCR, and finally, the targeted binding relationship between miR-16a-5p and mRNA FGF2 was verified by double luciferase reporter assay. The expression of collagen type 1 (COL 1), collagen type 3 (COL3), SCX and MKX was increased by overexpression of mRNA FGF2, respectively, and miR-16a-5p had a targeted effect on FGF2 and regulated the ligamentous differentiation of human amniotic mesenchymal stem cells. We found that the regulatory effect of overexpressed mRNA FGF2 on mesenchymal stem cells could be inhibited by up-regulation of miR-16a-5p, while the knockdown of FGF2 could reverse the regulatory effect of miR-16a-5p inhibition on ligament-forming differentiation of human amniotic mesenchymal stem cells. In this study, we discovered the existence of the miR-16a-5p–FGF2 axis in human amniotic mesenchymal stem cells, and the differentiation of human amniotic mesenchymal stem cells into ligamentous cells can be regulated by regulating various links in this axis.

Umbilical Amniotic Tissue Graft as an Alternative Approach for Eyelid Reconstruction After Necrotizing Fasciitis Debridement

A 54-year-old female with myelodysplastic syndrome on chemotherapy presented with 10 days of periocular erythema and edema worsening on oral antibiotics. Computed Tomography scan showed periorbital soft tissue swelling without postseptal extension or abscess. Intravenous broad-spectrum antibiotics were administered. However, she developed necrosis of the upper eyelid requiring aggressive debridement. Nine days after debridement, the defect measuring 5.5 × 3 cm was covered using an umbilical amniotic tissue graft (AmnioGuard, BioTissue, Miami, FL). At postoperative week 4, 5-fluorouracil was injected to prevent the shortening of anterior lamella. At postoperative week 20, the graft had dissolved and been replaced by regenerated skin. Final eyelid exam demonstrated normal eyelid elevation and minimal lagophthalmos. Herein, we present a case of umbilical amniotic membrane as a substrate graft to support the healing of the eyelid defect by secondary intention.

Revision Meniscal Repair With Amniotic Membrane Augmentation

Meniscal injury is common, and despite modern techniques, the failure rate following repair remains high. While there are recent treatment advances in the form of biologics, there is limited evidence and agreement on these emerging therapies and their role in meniscal healing. Amniotic tissue (umbilical cord allograft) is a biologic augmentation therapy that has been utilized in other musculoskeletal applications but has not been reported for use in meniscal repair. We describe a technique to deliver an allograft amniotic membrane into a meniscus tear repair site, potentially optimizing healing.

Decellularized amniotic membrane hydrogel promotes mesenchymal stem cell differentiation into smooth muscle cells.

Previous studies showed that the bladder extracellular matrix (B-ECM) could increase the differentiation efficiency of mesenchymal cells into smooth muscle cells (SMC). This study investigates the potential of human amniotic membrane-derived hydrogel (HAM-hydrogel) as an alternative to xenogeneic B-ECM for the myogenic differentiation of the rabbit adipose tissue-derived MSC (AD-MSC). Decellularized human amniotic membrane (HAM) and sheep urinary bladder (SUB) were utilized to create pre-gel solutions for hydrogel formation. Rabbit AD-MSCs were cultured on SUB-hydrogel or HAM-hydrogel-coated plates supplemented with differentiation media containing myogenic growth factors (PDGF-BB and TGF-β1). An uncoated plate served as the control. After 2 weeks, real-time qPCR, immunocytochemistry, flow cytometry, and western blot were employed to assess the expression of SMC-specific markers (MHC and α-SMA) at both protein and mRNA levels. Our decellularization protocol efficiently removed cell nuclei from the bladder and amniotic tissues, preserving key ECM components (collagen, mucopolysaccharides, and elastin) within the hydrogels. Compared to the control, the hydrogel-coated groups exhibited significantly upregulated expression of SMC markers (p ≤ .05). These findings suggest HAM-hydrogel as a promising xenogeneic-free alternative for bladder tissue engineering, potentially overcoming limitations associated with ethical concerns and contamination risks of xenogeneic materials.

Application of a practical amniotic membrane ring made on-site for restoration of ocular surface health in dry eye disease

Purpose: Dry eye disease (DED) is one of the most common ocular surface disorders worldwide. Despite different underlying pathogenic processes, DED is characterized by ocular surface inflammation, which in turn induces further damage to the corneal epithelium and its underlying structures. Amniotic membrane transplants are known to have potent anti-inflammatory effects and also have the ability to enhance epithelial healing. In this study, we aimed to evaluate the clinical efficacy of amniotic membrane ring (AMR) application in treating refractory dry eye disease.Methods: A retrospective analysis of 22 patients treated with contact lens-like amniotic membrane rings was performed. This amniotic membrane ring was formed using an aspiration catheter covered by a large piece of amniotic membrane graft. The amniotic membrane was sutured to the catheter using eight sutures. In this way, a customized amniotic membrane ring was prepared for each patient. Patients’ demographics, symptoms, use of medications, conjunctival inflammation, corneal staining, and visual acuity were compared before and after treatment. In addition, the amniotic membrane retention duration, the amniotic membrane's effect on ocular surface healing, follow-up time, and complications were evaluated.Results: Twenty-eight eyes of 22 patients (18 females and 4 males) aged 53.32 ± 13.36 (6-73) years were included. The AMR retention duration ranged from 5 to 16 days, with a mean of 11 ± 3.09 days, at which time the amniotic membrane had dissolved or been removed inadvertently by the patient. Discomfort with the ring was seen in 1 of 28 eyes (3.6 %). The patients reported symptomatic relief for a period of 3.64 ± 1.25 months. Symptomatic relief was accompanied by a reduction of OSDI scores (from 63.39 ± 17.24 to 33.19 ± 12.45) (p<0.001), use of topical medications (from 4.21 ± 1.03 to 2.42 ± 0.50) (p<0.001), conjunctival hyperemia (from 1.57 ± 1.19 to 0.35 ± 0.48) (p<0.001), corneal staining (from 2.89 ± 1.16 to 0.57 ± 0.74) (p<0.001), and improvement in visual acuity (from 0.23 ± 0.16 to 0.16 ± 0.25 logMAR) (p=0.001).Conclusions: Amniotic membrane ring treatment might be used to treat refractory dry eye disease. This technique has an economic advantage over other commercially available amniotic bandage tissues and can be easily removed and replaced during a follow-up examination.

The improvement of inflammatory infiltration and pregnancy outcome in mice with recurrent spontaneous abortion by human amniotic mesenchymal stem cells.

Recurrent spontaneous abortion is thought to be mostly triggered by immune-related causes. Mesenchymal stem cells, which exhibit the traits of multi-directional differentiation capacity and low immunogenicity, have recently been recommended as a viable treatment for spontaneous abortion-prone mice to increase the success of pregnancy. Amniotic membrane tissue is a byproduct of pregnancy and delivery that has a wide range of potential uses due to its easy access to raw materials and little ethical constraints. To construct an abortion-prone mouse model for this investigation, CBA/J female mice were coupled with male DBA/2 mice, while CBA/J female mice were paired with male BALB/c mice as a control. The identical volume of human amniotic mesenchymal stem cells or phosphate buffer was injected intraperitoneally on the 4.5th day of pregnancy. CBA/J female mice were sacrificed by cervical dislocation on the 13.5th day of pregnancy, the embryo absorption rate was calculated, and the uterus, decidua tissues and placenta were gathered for examination. Through detection, it was discovered that human amniotic mesenchymal stem cells significantly increased the expression of interleukin 10 and transforming growth factor beta, while they significantly decreased the expression of interleukin 1 beta and interleukin 6, improved vascular formation and angiogenesis, and minimized the embryo absorption rate and inflammatory cell infiltration in the recurrent spontaneous abortion + human amniotic mesenchymal stem cells group. In any case, human amniotic mesenchymal stem cells regulate inflammatory factors and cell balance at the maternal-fetal interface, which result in a reduction in the rate of embryo absorption and inflammatory infiltration and provide an innovative perspective to the clinical therapy of recurrent spontaneous abortion.

A Novel Approach to The Use of Flowable Human Amniotic Fluid for Diabetic Foot Ulcers in High-Risk Veterans Affairs Patients

As of 2017, there are 425 million people worldwide who suffer and are affected by Diabetes Mellitus. Among those 425 million people, approximately 34% of people go on to develop diabetic foot ulcers with more than 50% becoming infected. There are many causes of ulcers, including metabolic, biomechanical, traumatic, or post-operative complications. The use of human amniotic fluid has the potential to be an additional treatment for pedal wounds in patients with diabetes mellitus. Indications may include wounds which have prolonged or delayed healing as a result of biomechanical complications, co-morbidities, or failed conservative treatment. This case series explores the effectiveness at topical application of flowable human amniotic fluid in 3 different high risk diabetic patients who all suffered from a pedal ulcer. Each patient had a different location and circumstance for their wound allowing for some variety in surgical planning and wound care decision making. Two of the three patients were able to heal completely within 3 months of initial consult with the remaining patient healing within 4 months of initial consult. Per chart reviewing, all three patients have remained healed at the 1 year follow-up with no evidence of reoccurrence. This series hopes to open a discussion regarding the novel use of topical application of human amniotic tissue as an advanced wound care option for those who may have failed conservative therapy or may be looking for alternative methods to help heal pedal ulcers.

Comparative Analysis of Fluorescent Labeling Techniques for Tracking Canine Amniotic Stem Cells.

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.

Comparative, Controlled, Retrospective Study of Patient-Reported Outcomes After Meniscectomy With Adjunctive Use of Platelet-Rich Plasma or Amniotic Umbilical Cord Tissue.

Background: Meniscal tears are one of the most frequent injuries to the knee, with an estimated incidence of 222 per 100,000 individuals aged 18 to 55 years based on magnetic resonance imaging. Poor outcomes following meniscal surgical interventions are common and have led many surgeons to use biologic augmentation strategies to enhance the healing. Methods: We conducted a single-center, retrospective, observational study of patients who underwent arthroscopic meniscectomy with and without adjunctive platelet-rich plasma (PRP) or the particulate form of amniotic umbilical cord (AMUC) tissue. We evaluated patient-reported outcomes on the visual analog scale for pain, International Knee Documentation Committee (IKDC) Subjective Knee Evaluation Form, Lysholm Knee Scoring Scale, 12-Item Short Form Survey, and Knee Injury and Osteoarthritis Outcome Score (KOOS) during a 1-year postoperative period. Complications and follow-up procedures were also evaluated. Results: We evaluated 113 patients who underwent meniscectomies from November 2010 to March 2017. Pain severity was significantly decreased only in the AMUC group at 6 months (P=0.0143). Patients in the AMUC group demonstrated significant improvement in functional recovery based on the IKDC and the KOOS subscales of pain, symptoms, activities of daily living, and sport and recreation function at 6 months. Patients in the PRP group had a significant benefit in the KOOS subscales of pain, symptoms, sport and recreation function, and knee-related quality of life at 3 months. Improvement in the control group was less substantial. Patients in the PRP group had more complications and follow-up procedures (30.0%) than patients in the AMUC group (8.3%). Conclusion: In our study population, arthroscopic meniscectomy with adjunctive use of AMUC tissue improved patient-reported outcomes and reduced the reoperation rate compared to conventional technique or adjunctive use of PRP.

Functional Recovery after the Application of Amniotic Tissues and Methylene Blue during Radical Prostatectomy-A Pilot Study.

Amniotic tissues and methylene blue (MB) provide the ability for neuroregeneration, and MB enables intraoperative neurostaining. We first combined the techniques to explore a neuroprotective effect on early functional outcomes in a retrospective proof-of-concept trial of 14 patients undergoing radical prostatectomy (RP). The patients were followed up at a median of 13 months, and the continence and potency rates were reported. Early recovery of continence was found after three months. No effect on potency was detected. The findings indicate the feasibility of this tissue-engineering strategy, and justify prospective comparative studies.

Micronuclei and nuclear buds in amniotic tissue of rats treated with cyclophosphamide.

Fetal development can be altered by DNA damage caused by maternal exposure to chemical, physical, or biological agents during gestation. One method of assessing genotoxicity is to detect micronuclei (MNs) and/or nuclear abnormalities. This can be performed in vivo and requires only frequently dividing tissues, such as amniotic tissue (AT), which is in contact with the fetal environment and is composed of very thin layers of cells. This study evaluated the presence of MNs, nucleoplasmic bridges, and nuclear buds (NBs) in the fetal AT following maternal exposure to cyclophosphamide (CP) during pregnancy. Pregnant Wistar rats were divided into a negative control group and an experimental group that was orally administered CP (10mg/kg). Daily blood smears were obtained from pregnant rats on days 14-19 of gestation. The rats were dissected, and fetal ATs were obtained on the 19th day of gestation. The MN and NB frequencies in AT cells were analyzed using a fluorescence microscope (100×). Micronucleated erythrocytes in the peripheral blood of the control rats were also assessed. Micronucleated polychromatic erythrocyte frequencies were significantly higher than those in the controls. Polychromatic erythrocyte frequencies were lower in CP-treated rats than in controls at 48-120h. Fetuses in the CP-treated group also showed a significant increase in MNs and NBs in AT cells. In conclusion, AT could be used for analyzing MNs and NBs in rats following maternal exposure to a genotoxic agent and as a viable alternative for analyzing the integrity of fetal DNA during gestation.

High Apoptotic Index in Amniotic Membrane of Pregnant Women is A Risk Factor for Preterm Labor

This study aims to prove a high apoptotic index in the amniotic membrane as a risk factor for preterm Labor. Case-control study, comparing preterm and term groups (n=54) who underwent labor in Obstetrics Emergency Room, Sanglah Central General Hospital Denpasar, Bali. Apoptotic index is a method of identifying and assessing the quantity of tissue undergoing apoptosis, which in this study was the amniotic membrane tissue, calculated and stained using the TUNEL method. Data collected was subjected to further statistical tests. Normality test with Shapiro Wilk test, followed by parametric independent T-test and Mann Whitney test was performed. Cut off apoptotic index was determined using the Receiver Operating Characteristic (ROC) curve. Bivariate analysis with Chi-square test and calculation of odds ratio was performed to assess the high apoptotic index to the risk of preterm Labor. Statistical significance was obtained with p value &lt;0.05. Based on the cut-off value of ROC, the apoptotic index was classified into high (≥37.5%) and low (&lt;37.5%) apoptotic index. A high apoptotic index is a risk factor for preterm Labor and increases the risk 6 times greater than the group with a low apoptotic index (p=0.003; OR 5.714; 95% CI 1.764–18.507). High apoptotic index in the amniotic membranes of pregnant women is a risk factor for preterm Labor.

Poster 378: The Availability and Price of Orthobiologic Injections in the Chicagoland Area: A Market Assessment Study

Objectives: Orthobiologic therapies have recently gained popularity in orthopaedic surgery as potential treatment options that can be used to manage the symptoms of musculoskeletal conditions. However, the market for these products remains unregulated by the Food and Drug Administration (FDA), giving rise to a wide range of available yet untested products. There are growing concerns related to patient safety and product efficacy, but also in regard to the amount of money being charged by providers for these treatments Thus, the purpose of this study was to investigate the current market for orthobiologic injections for knee osteoarthritis in a large, metropolitan city and determine 1) the willingness of providers to disclose the cost of an injection prior to an appointment; 2) the cost of injections if disclosed; 3) the cellular source of injections being advertised as “stem cell” products; and 4) whether there are differences in the market for orthobiologics within and outside of orthopaedic sports medicine practices. Methods: Orthopaedic sports medicine providers in the Chicagoland area were identified through the American Orthopaedic Society for Sports Medicine (AOSSM) and Arthroscopy Association of North America (AANA) registries of sports medicine physicians seeing patients in at least one location within the Chicagoland area. Non orthopaedic clinics (including chiropractors, pain management physicians, and self-advertised “regenerative medicine” clinics) offering orthobiologic injections were compiled using Yelp! and Google search for listings and reviews containing the following key words: “PRP” or “platelet-rich plasma” or “platelet rich plasma,” “SCT” or “stem cells” or “amniotic tissue injections” or “adipose stem cell injections,” and “BMAC” or “bone marrow aspirate concentrate.” A single author [BLINDED] contacted every clinic via a common telephone script, which was modified from an existing study. Data was collected for each site on the types of injections offered, whether price was disclosed over the phone, the price of injections offered, and the source of alternative “stem cell” injections if they were provided. Prices were explicitly specified as being for one injection of one joint. Results: A total of 25 orthopaedic sports medicine practices (comprising 80 AOSSM/AANA surgeons) and 40 non orthopaedic clinics offered at least one of either PRP, “stem cell,” and/or BMAC injections over the phone (Table 1). There was no difference in the likelihood of providing PRP injections (p = 0.698) or BMAC injections (p = 0.340). However, non orthopaedic clinics were significantly more likely to offer “stem cell” injections (p &lt; 0.001). There was no difference in the willingness to disclose the price of PRP without an appointment (p = 0.269), willingness to disclose the price of “stem cell” injections (p = 0.302), willingness to disclose price of BMAC (p = 0.528), or willingness to disclose the source of what each clinic advertised as “stem cell” injections (p = 0.302). While the average price of a BMAC injection was not different between provider types (p = 0.238), the average price of a single PRP injection was significantly higher (p = 0.011) and significantly more variable (F statistic = 0.139; p &lt; 0.001) at non orthopaedic clinics (Figure 1). Similarly, the average price of a single “stem cell” injection was significantly higher (p &lt; 0.001) and significantly more variable (F statistic = 0.080; p = 0.025) at non orthopaedic clinics. Most commonly, alternative “stem cell” products included amniotic-based injections, which are currently discouraged for distribution or use based upon the most recent FDA guidance document. Conclusions: When compared to orthopaedic sports medicine providers, non orthopaedic clinics providing orthobiologic treatments in the Chicagoland area are more likely to offer alternative “stem cell” injections and charge a significantly greater and more variable amount per PRP and “stem cell” injection. Unfortunately, there was liberal use of the terms “regenerative medicine” and “stem cell” therapies in the face of increased scrutiny related to the FDA’s current position on cell-based therapies. Future studies should examine these trends on a national scale to assess compliance with FDA regulations on orthobiologics, as well as establish standards of price for these treatments. [Table: see text]

519-P: Randomized Clinical Trial to Compare Cryopreserved and Lyopreserved Placental Cord Tissue to Treat Complex Diabetic Foot Surgical Wounds—A Proof of Concept Study

Objective: To compare the incidence of infection, healing and time to heal in patients treated with cryopreserved (CPAT) and lyopreserved amniotic tissue (LPAT) in complex diabetic surgical wound (CDFW). Methods: This single-blinded 12-week randomized clinical trial compared cryopreserved and lyopreserved amniotic cord tissue to treat complex diabetic foot wounds (CDFW). LPAT or CRAT was applied at baseline and again after four weeks. We enrolled subjects with UT2A-D and 3A-D wounds (depth to tendon, muscle, or bone with infection and/or PAD) and excluded subjects with ABI&amp;lt;0.5 or TBI&amp;lt;0.3, untreated osteomyelitis, and autoimmune diseases. We used a 3-D camera to evaluate wound area and volume. The Chi-squared test was used to compare clinical outcomes and the Wilcoxon Ranked Sum Test to compare wound area and volume. Results: The mean baseline wound areas were 12.9cm2 ±10.7 for CPAT and 11.7cm2 ±7.0 for LPAT. The mean baseline wound volume were 7.5cm3 ±8.1 for CPAT and 9.2cm3±10.2 for LPAT. There was no difference between CPAT and LPAT in healing (36.8% vs. 19.0%, p=0.21) and infection (10.5% vs. 9.5%, p=0.72). There was no difference in mean wound area reduction between CPAT and LPAT (75.9± 32.3% vs. 65.5±38.4%, p=0.41), nor in mean volume reduction (85.0±30.8% vs. 79.9±31.9%, p=0.61). When we stratified wound dimensions, there was no difference between CPAT and LPAT in subjects that had 90-99% wound volume reduction (36.8% vs. 47.6%), 70-89% wound volume reduction (15.8% vs 19.0%) and 0-69% wound volume reduction (10.5% vs. 19.0%). There was no difference between CPAT and LPAT in subjects that had 90-99% wound area reduction 90-99% (10.5% vs. 23.8%), 70-89% (26.3% vs. 19.0%) and 0-69% (26.3% vs. 38.1%). Conclusion: Cryopreserved and Lyopreserved amniotic tissue provided similar results in patients with complex diabetic foot wounds. Disclosure L.A.Lavery: Research Support; AstraZeneca, Tissue tech, American Diabetes Association, PolarityTE, Inc., Microbion, Jarvis, Integra Lifesciences Holdings Corp, Smith+Nephew. A.L.Killeen: Research Support; 3M/KCI, AstraZeneca, BioTissue surgical, Hologenix, LLC, Integra LifeSciences, Kent Imaging Inc., Microbion, PolarityTE, Inc., Smith+Nephew, Stark Enterprises, LLC, Xyken, VasoActiv, ViOptix. M.A.Suludere: None. P.Crisologo: None. Funding Smith &amp; Nephew

Investigating the regenerative effects of folic acid on human amniotic epithelial stem cells and amniotic pore culture technique (APCT) model in vitro using an integrated pharmacological-bioinformatic approach

IntroductionDisruption of fetal membranes before the onset of labor is referred to as premature rupture of membranes (PROM). Lack of maternal folic acid (FA) supplementation reportedly leads to PROM. However, there is a lack of information on the location of FA receptors in the amniotic tissue. Additionally, the regulatory role and potential molecular targets of FA in PROM in vitro have rarely been investigated. MethodsThe three FA receptors (folate receptor α isoform [FRα], transporter of reduced folate [RFC], and proton-coupled folate transporter [PCFT]) in human amniotic epithelial stem cells (hAESCs) and amniotic tissue were localized using immunohistochemistry and immunocytochemistry staining. Effect and mechanism analyses of FA were performed in hAESCs and amniotic pore culture technique (APCT) models. An integrated pharmacological-bioinformatics approach was utilized to explore the potential targets of FA for the treatment of PROM. ResultsThe three FA receptors were widely expressed in human amniotic tissue, especially in the hAESC cytoplasm. FA stimulated the amnion regeneration in the in vitro APCT model. This mimics the PROM status, in which cystathionine-β-synthase, an FA metabolite enzyme, may play an important role. The top ten hub targets (STAT1, mTOR, PIK3R1, PTPN11, PDGFRB, ABL1, CXCR4, NFKB1, HDAC1, and HDAC2) of FA for preventing PROM were identified using an integrated pharmacological–bioinformatic approach. DiscussionFRα, RFC, and PCFT are widely expressed in human amniotic tissue and hAESCs. FA aids the healing of ruptured membrane.

Adipose and amnion-derived mesenchymal stem cells: Extracellular vesicles characterization and implication for reproductive biotechnology

The stem cell-based research for reproductive biotechnology has been widely studied and shows promise for repairing defective tissue or degenerated cells to treat different diseases. The adipose tissue and amniotic membrane have awakened great interest in regenerative medicine and arises as a promising source of mesenchymal stem cells. Both types, adipose and amniotic derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages.. We aimed to evaluate the effect of basal supplementation of exosomes in cell cultures with canine amniotic mesenchymal stem cells (MSCs). Mesenchymal stem cells derived from canine amniotic and adipose tissue were isolated and cultured performing cell passages until 80–90% confluence was reached. The growth curve was determined and peak cell growth was observed in the second passage. The cells were then characterized and differentiated into adipogenic, chondrogenic and osteogenic lineages. Extracellular vesicles from amnion were isolated using an ultracentrifugation protocol and characterized by nanosight analysis. To evaluate their ability to improve cellular viability in naturally inefficient passages, exosomes were co-cultures to the MSC cells. The results showed a 15–20% increase in the expansion rate of cultures supplemented with vesicles extracted in the first and second passages when compared to the control group. Statistical analysis using the Dunnett test (p ≤ 0.05) corroborated this result, showing a positive correlation between supplementation and expansion rate. These results indicate not only the importance of exosomes in the cell communication process but also the feasibility of the culture supplementation protocol for therapeutic purposes. The potential of the AMSCs for reproductive biotechnology is undoubted, however, their application to repair reproductive disorders and the involved mechanisms remain elusive. The strategies to enable the Adipose Stem Cells and AMSCs application in reproductive biotechnology and optimize their use for tissue regeneration open new venues using exosomes interactions.

Outcome following local injection of a liquid amnion allograft for treatment of equine tendonitis or desmitis – 100 cases

BackgroundTendon and ligament injuries are significant causes of loss of use and early retirement in performance horses. Amniotic fluid and tissue are excellent sources of growth factors and cytokines important in tendon and ligament healing. Thus, an equine-origin liquid amnion allograft (ELAA) may be beneficial in the treatment of equine tendonitis and desmitis. Objectives of this study were to report the outcome achieved (i.e. ability to return to work) for horses diagnosed with tendonitis or desmitis lesions treated with local injection of ELAA and to compare these outcomes to those reported for other regenerative medicine modalities.MethodsA prospective, multi-center, non-blinded clinical trial was conducted. Equine veterinarians at 14 sites were selected to participate in the data collection for the trial. Criterion for inclusion was a horse presenting with lameness which was attributed to tendonitis or desmitis by diagnostic anesthesia and/or imaging. These horses were subsequently treated by local injection of the lesion with ELAA by the attending veterinarian. Standardized questionnaires describing each horse’s signalment, discipline, ability to return to work, and any adverse events were completed and submitted by the attending veterinarian following a minimum of six months of follow-up. The current literature was reviewed to identify clinical studies reporting outcomes of equine tendonitis/desmitis lesions treated with other regenerative therapies. Contingency table analyses were performed comparing outcomes.ResultsQuestionnaires for 100 horses with 128 tendonitis and desmitis lesions met the inclusion criteria. Of these, 72 horses with 94 lesions returned to or exceeded their original level of work, 10 horses with 13 lesions returned to work but could not perform to previous standards, and 18 horses with 20 lesions did not return to work as a result of the injury. No differences were observed when outcome of horses treated with ELAA were compared to those of similar studies using other regenerative therapies.ConclusionsTreatment of tendonitis and desmitis lesions by local injection of ELAA resulted in similar outcomes for horses returning to previous level of performance as other regenerative modalities such as mesenchymal stem cells, platelet-rich plasma, and autologous conditioned serum; however, blinded placebo-controlled studies are indicated.

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro

Objectives: The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro. Design: Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes. Materials: Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study. Settings: To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control. Methods: Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05. Results: As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 μm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 μm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8). Limitations: In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested. Conclusions: We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

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.

Biological Evaluation of a Native Amniotic Membrane Allograft

Category: Basic Sciences/Biologics; Other Introduction/Purpose: Placental tissue allografts have long been used as effective wound dressings for hard to heal wounds and are demonstrating increasing utility in the field of foot and ankle surgeries. Previous observations of the PURION-processed dehydrated amnion/chorion membrane (dHACM) demonstrated retention of regulatory proteins inherent to amniotic tissues and preservation of the bioactivity to stimulate cellular activities. Novel and patent-pending processing techniques have been developed to introduce a thicker and more robust allograft, as compared to dHACM. Lyophilized human amnion chorion membrane (LHACM) will confer versatility to clinicians with improved handling while maintaining the natural tissue composition and intrinsic biological activities. This study characterizes the biological properties of LHACM which may support the healing cascade. Methods: LHACM was processed using a modified PURION process which incorporates an optimized cleansing process, enabling retention of the intermediate layer, and lyophilization. Tissue architecture was visualized with hematoxylin and eosin staining. The hygroscopic contribution of the intermediate layer was appraised with absorbency measurements. Cleansing efficiency was determined through quantification of residual hemin remaining in the tissue. Regulatory proteins were quantified using a multiplex ELISA. The cellular response to LHACM treatment was assessed using multiple in vitro assays targeting cellular proliferation and analysis of signaling pathways. Adult dermal fibroblasts (HDFs) cultured over a 3-day period in LHACM eluates were assessed for cellular proliferation. In vitro models were developed to assess the regulation of relevant signaling pathways, including TGF-β1 and Wnt/β-catenin for fibrotic and proliferative processes, respectively. Cellular responses were evaluated through quantitative PCR and cell reporter lines. Results: Histological evaluation showed that processing maintains the tissue structure. Absorption testing demonstrated LHACM is more hygroscopic than dHACM. Hemin analysis demonstrated low levels of residual hemin which were comparable for LHACM and dHACM allografts. Screening for regulatory proteins indicate that LHACM contains a diverse array of regulatory proteins, inherent to amniotic membranes. In vitro treatment of primary cells with LHACM eluates promoted dermal fibroblast proliferation as well as regulation of elevated TGFβ- and Wnt/β-catenin signaling. Conclusion: The processing method of LHACM maintains the intrinsic properties of amniotic membranes with similar levels of biological activity as compared to dHACM, while providing an option for a thicker allograft with alternative handling characteristics. LHACM represents a novel product concept which increases versatility of amniotic membrane application in varied outpatient and surgical uses.