Research and Studies

EFFICACY

A prospective, randomized comparative study of weekly versus biweekly application of dehydrated human amnion/chorion membrane allograft in the management of diabetic foot ulcers

The aim of this study is to determine if weekly application of dehydrated human amnion/chorion membrane allograft reduce time to heal more effectively than biweekly application for treatment of diabetic foot ulcers.
This was an institutional review board-approved, registered, prospective, randomized, comparative, non-blinded, single-centre clinical trial. Patients with non-infected ulcers of ≥ 4 weeks duration were included for the study. They were randomized to receive weekly or biweekly application of allograft in addition to a non-adherent, moist dressing with compressive wrapping. All wounds were offloaded. The primary study outcome was mean time to healing.
Overall, during the 12-week study period, 92·5% (37/40) ulcers completely healed. Mean time to complete healing was 4·1 ± 2·9 versus 2·4 ± 1·8 weeks (P = 0·039) in the biweekly versus weekly groups, respectively. Complete healing occurred in 50% versus 90% by 4 weeks in the biweekly and weekly groups, respectively (P = 0·014). Number of grafts applied to healed wounds was similar at 2·4 ± 1·5 and 2·3 ± 1·8 for biweekly versus weekly groups, respectively (P = 0·841).
These results validate previous studies showing that the allograft is an effective treatment for diabetic ulcers and show that wounds treated with weekly application heal more rapidly than with biweekly application. More rapid healing may decrease clinical operational costs and prevent long-term medical complications.
Keywords: Amniotic membrane allograft, Diabetic ulcer, Dehydrated amnion/chorion

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An evaluation of dehydrated human amniotic membrane allografts in patients with DFUs.

OBJECTIVE: To evaluate healing of chronic diabetic foot ulcers (DFUs) with use of dehydrated human amniotic membrane (dHAM) in patients failing under standard of care treatment.

METHOD: The study population was identified from patients with chronic DFUs enrolled in a randomised trial comparing standard care with standard care with the addition of dHAM. The present study included patients that failed to heal (defined as < 50% decrease in wound size after 6 weeks, or not completely healed by 12 weeks of treatment) with standard care. One week after withdrawal from the randomised trial, these patients were offered treatment with standard care and bi-weekly application of dHAM. Subsequent evaluation of clinical records was made with IRB approval and patient consent. Each patient was used as their own control to compare wound size reduction between treatment periods, and healing rates within the 12-week dHAM treatment period.

RESULTS: Eleven patients were included in the study. Mean wound chronicity was 21.1 +/- 12.4 weeks (range 11-54 weeks) and mean wound size was 4.7 +/- 5.0cm2, at baseline. Complete healing was achieved in 55% by 4 weeks, 64% by 6 weeks and 91% by 12 weeks with bi-weekly dHAM application. Mean weeks to complete healing was 4.2 +/- 3.1 weeks for the 10 patients healed. After 4 weeks of standard care, wounds had decreased in size an average of 26.8 +/- 45.3% versus 87.6 +/- 16.0% after 4 weeks of dHAM treatment (two applications; p < 0.001).

CONCLUSION: This study provides evidence that the incorporation of dHAM into standard of care for patients with chronic DFUs can be beneficial. The results suggest that dHAM is a viable option for the treatment of recalcitrant DFUs.

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Chart Review Study of Subjects Administered Amnionic Membrane for Treatment of Joint Pain

Context: Treatment of joint pain with injection of amnionic membrane has not been adequately studied.

Objectives: Determine if patients who received cryopreserved particulate amnionic membrane (CPAM) injected into painful back and knee joints experience less back or knee pain, improve physical ability, and use less opiates and NSAIDs over a 12 week time period.

Methods: Charts were reviewed for 20 consenting patients receiving CPAM, clinically available from tissue banks, injected into joints to relieve pain consistent with the clinical practice at a single center. Ten subjects had back pain, and 10 subjects had knee pain.

Results: VAS pain scores improved from 7.5 to 1.1 over 12 weeks (p<0.001). WOMAC daily activity function score improved from 46 to 11over 12 weeks (p<0.001). Opiate usage decreased from 55% to 15% over 12 weeks (p<0.001). NSAID usage decreased from 80% to 10% over 12 weeks (p<0.001). Location of injection was not a significant covariate factor for any outcome.

Conclusion: Thus, amnionic membrane injection into painful back and knee joints improves pain and physical function, and decreases opiate and NSAID usage for at least 12 weeks.

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A prospective randomized comparative parallel study of amniotic membrane wound graft in the management of diabetic foot ulcers.

Our purpose was to compare healing characteristics of diabetic foot ulcers treated with dehydrated human amniotic membrane allografts (EpiFix®, MiMedx, Kennesaw, GA) versus standard of care.

An IRB-approved, prospective, randomized, single-centre clinical trial was performed. Included were patients with a diabetic foot ulcer of at least 4-week duration without infection having adequate arterial perfusion. Patients were randomized to receive standard care alone or standard care with the addition of EpiFix. Wound size reduction and rates of complete healing after 4 and 6 weeks were evaluated. In the standard care group (n = 12) and the EpiFix group (n = 13) wounds reduced in size by a mean of 32.0% ± 47.3% versus 97.1% ± 7.0% (P < 0.001) after 4 weeks, whereas at 6 weeks wounds were reduced by -1.8% ± 70.3% versus 98.4% ± 5.8% (P < 0.001), standard care versus EpiFix, respectively. After 4 and 6 weeks of treatment the overall healing rate with application of EpiFix was shown to be 77% and 92%, respectively, whereas standard care healed 0% and 8% of the wounds (P < 0.001), respectively. Patients treated with EpiFix achieved superior healing rates over standard treatment alone.

These results show that using EpiFix in addition to standard care is efficacious for wound healing.

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Initial Clinical Experience with the Use of Human Amniotic Membrane Tissue During Repair of Posterior Tibial and Achilles Tendons

Connective tissues that become damaged or diseased can result in reduced mobility and contribute to the development of chronic pain. While conservative treatment approaches are often helpful, many patients become debilitated and require surgical intervention. As a result of the widespread prevalence of these conditions, procedures to repair and reconstruct damaged connective tissue are commonplace. While these procedures are usually successful, the healing of damaged tendons and ligaments following surgery is particularly difficult often resulting in the failure of the tendon or ligament to regain its original strength.

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Cryopreserved human amniotic membrane injection for plantar fasciitis: a randomized, controlled, double-blind pilot study.

BACKGROUND: Treatment options for plantar fasciitis have resulted in varied patient outcomes. The aim of this study was to compare a novel treatment, cryopreserved human amniotic membrane (c-hAM), to a traditional treatment, corticosteroid. Our hypothesis was that c-hAM would be safe and comparable to corticosteroids for plantar fasciitis in regard to patient outcomes.

METHODS: A randomized, controlled, double-blind, single-center pilot study was completed. Patients were randomized into one of 2 treatment groups: c-hAM or corticosteroid. Patients received an injection at their initial baseline visit with an option for a second injection at their first 6-week follow-up. Total follow-up was obtained for 12 weeks after the most recent injection. The primary outcome measurement was the Foot Health Status Questionnaire (FHSQ). The secondary outcome measurements were the Visual Analog Scale (VAS) and verbally reported percentage improvement. Data were analyzed between groups for the 2 different cohorts (1 injection versus 2 injections). Twenty-three patients had complete follow-up. Fourteen were randomized to receive corticosteroid and 9 were randomized to receive c-hAM.

RESULTS: Three patients in each group received second injections. With the numbers available, the majority of outcome measurements showed no statistical difference between groups. The corticosteroid did, however, have greater FHSQ shoe fit improvement (P = .0244) at 6 weeks, FHSQ general health improvement (P = .0132) at 6 weeks, and verbally reported improvement (P = .041) at 12 weeks in the one-injection cohort. Cryopreserved hAM had greater FHSQ foot pain improvement (P = .0113) at 18 weeks in the 2-injection cohort.

CONCLUSION: Cryopreserved hAM injection may be safe and comparable to corticosteroid injection for treatment of plantar fasciitis. This is a pilot study and requires further investigation.

LEVEL OF EVIDENCE: Level I, prospective randomized trial.

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Cryopreserved Amniotic Suspension for the Treatment of Knee Osteoarthritis.

There are few treatment options for symptomatic knee osteoarthritis (OA). Human amniotic suspension allografts (ASA) have anti-inflammatory and chondroregenerative potential and thus represent a promising treatment strategy. In anticipation of a large, placebo-controlled trial of intra-articular ASA for symptomatic knee OA, an open-label prospective feasibility study was performed. Six patients with Kellgren-Lawrence grades 3 and 4 tibiofemoral knee OA were administered a single intra-articular ASA injection containing cryopreserved particulated human amnion and amniotic fluid cells. Patients were followed for 12 months after treatment. No significant injection reactions were noted. Compared with baseline there were (1) no significant effect of the ASA injection on blood cell counts, lymphocyte subsets, or inflammatory markers and (2) a small, but statistically significant increase in serum IgG and IgE levels. Patient-reported outcomes including International Knee Documentation Committee, Knee Injury and Osteoarthritis Outcome, and Single Assessment Numeric Evaluation scores were collected throughout the study and evaluated for up to 12 months. Overall, this study demonstrates the feasibility of a single intra-articular injection of ASA for the treatment of knee OA and provides the foundation for a large placebo-controlled trial of intra-articular ASA for symptomatic knee OA.

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A Randomized Controlled Single-Blind Study Demonstrating Superiority of Amniotic Suspension Allograft Injection Over Hyaluronic Acid and Saline Control for Modification of Knee Osteoarthritis Symptoms.

Placental-derived tissues are a known source of anti-inflammatory and immune modulating factors. Published pilot data on amniotic suspension allograft (ASA) for the treatment of osteoarthritis (OA) demonstrated safety and trends for improved pain and function. A multicenter randomized controlled trial was designed to evaluate the efficacy of symptom modulation with ASA compared with saline and hyaluronic acid (HA) in subjects with knee OA. A total of 200 subjects were randomized 1:1:1 to ASA, HA, or saline, with subjects blinded to their allocation. Changes from baseline of patient-reported outcomes (PROs)-EQ-5D-5L, Knee Osteoarthritis Outcome Score (KOOS), visual analog scale (VAS), Tegner, and Single Assessment Numerical Evaluation (SANE)-were compared between groups. Patients reporting unacceptable pain at 3 months were considered treatment failures and withdrawn from the study. Statistical analysis was completed by comparing changes in PROs from baseline to 3 and 6 months for all groups. Comparison of demographics between treatment groups showed no significant differences between groups. Patients reporting unacceptable pain at 3 months in each group were ASA (13.2%), HA (68.8%), and saline (75%). Patients receiving ASA demonstrated significantly greater improvements from baseline for overall pain (VAS), KOOS pain, and KOOS-activities of daily living scores compared with those in the HA group (3 months) and both groups (6 months). ASA patients had significantly greater improvements in KOOS symptom scores compared with HA and saline at 3 and 6 months, respectively. OMERACT-OARSI responder rates for ASA, HA, and saline groups were 69.1, 39.1, and 42.6%, respectively (p = 0.0007). Subjects receiving ASA treatment showed greater improvements in PROs and fewer patients reported unacceptable pain compared with HA and saline. The evidence presented in this Level I Randomized Controlled Trial suggests that ASA injection is an effective treatment for the nonoperative management of symptomatic knee OA.

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SAFETY

Conditioned medium from horse amniotic membrane-derived multipotent progenitor cells: immunomodulatory activity in vitro and first clinical application in tendon and ligament injuries in vivo.

We have recently demonstrated that heterologous transplantation of horse amniotic membrane-derived mesenchymal cells (AMCs) can be useful for cell therapy applications in tendon diseases, and hypothesized that these cells may promote tendon repair via paracrine-acting molecules targeting inflammatory processes. To test this hypothesis, here we examined the immunomodulatory characteristics of AMCs and of their conditioned medium (AMC-CM) in vitro, and studied the potential therapeutic effect of AMC-CM in thirteen different spontaneous horse tendon and ligament injuries in vivo. Our results demonstrate that AMCs are capable of inhibiting peripheral blood mononuclear cell (PBMC) proliferation after allogenic stimulation either when cocultured in cell-to-cell contact, or when the two cell types are physically separated by a transwell membrane, suggesting that soluble factors are implicated in this phenomenon. Our hypothesis is further supported by the demonstration that PBMC proliferation is inhibited by AMC-CM. In our in vivo studies, no significant adverse effects were observed in treated tendons, and clinical and ultrasonographical evaluation did not reveal evidence of inappropriate tissue or tumor formation. Clinical outcomes were favorable and the significantly lower rate (15.38%) of reinjuries observed compared to untreated animals, suggests that treatment with AMC-CM is very efficacious. In conclusion, this study identifies AMC-CM as a novel therapeutic biological cell-free product for treating horse tendon and ligament diseases.

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The tumorigenicity of human embryonic and induced pluripotent stem cells.

The unique abilities of human pluripotent stem cells to self-renew and to differentiate into cells of the three germ layers make them an invaluable tool for the future of regenerative medicine. However, the same properties also make them tumorigenic, and therefore hinder their clinical application. Hence, the tumorigenicity of human embryonic stem cells (HESCs) has been extensively studied. Until recently, it was assumed that human induced pluripotent stem cells (HiPSCs) would behave like their embryonic counterparts in respect to their tumorigenicity. However, a rapidly accumulating body of evidence suggests that there are important genetic and epigenetic differences between these two cell types, which seem to influence their tumorigenicity.

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Application of Amniotic Tissue in Orthopedic Surgery

The use of amniotic tissue in orthopedic surgery has increased in recent years. While more studies are needed to fully understand the therapeutic potential and define the appropriate applications of these tissues, basic science and clinical studies are available that indicate promising results. This review will highlight these studies as they relate to the musculoskeletal system.

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Immunogenicity of human amniotic epithelial cells after transplantation into volunteers.

Human amniotic epithelial cells do not express on their surfaces HLA-A, B, C, and DR antigens, or beta 2-microglobulin. In vitro these cells synthesise the enzymes lacking in patients with selected enzymatic deficiencies: the survival of a transplanted monolayer of human amniotic epithelial cells was therefore investigated in seven volunteers. None of the volunteers showed clinical signs of acute rejection, and amniotic epithelial cells were demonstrated by biopsy up to 7 weeks after implantation. HLA antibodies were not detected in samples of serum from four volunteers thoroughly investigated, and there was no in-vitro lymphocyte reaction to the amniotic cells in two of them. The results suggest that acute immune rejection does not occur after the transplantation of human amniotic epithelial cells.

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Amnion-derived stem cells: in quest of clinical applications.

In the promising field of regenerative medicine, human perinatal stem cells are of great interest as potential stem cells with clinical applications. Perinatal stem cells could be isolated from normally discarded human placentae, which are an ideal cell source in terms of availability, the fewer number of ethical concerns, less DNA damage, and so on. Numerous studies have demonstrated that some of the placenta-derived cells possess stem cell characteristics like pluripotent differentiation ability, particularly in amniotic epithelial (AE) cells. Term human amniotic epithelium contains a relatively large number of stem cell marker-positive cells as an adult stem cell source. In this review, we introduce a model theory of why so many AE cells possess stem cell characteristics. We also describe previous work concerning the therapeutic applications and discuss the pluripotency of the AE cells and potential pitfalls for amnion-derived stem cell research.

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Amnion-derived pluripotent/multipotent stem cells

Amniotic epithelium is derived from the epiblast by approx 8 d after fertilization. Other parts of the placenta are derived from extraembryonic tissue. In addition to this developmental difference, amniotic epithelial (AE) cells are known to have unique characteristics, such as low level expression of major histocompatibility complex antigens, and a less restricted differentiation potential. The differentiation of the AE cells to the neural lineage is well documented. Recently, we reported that AE cells from term placenta express several stem cell surface markers that are commonly found on pluripotent stem cells such as embryonic stem cells, and that in culture, AE cells differentiate into cell types from all three germ layers. In this review, we describe the unique characteristics of the AE stem cells and summarize previous work concerning the stem cell nature of cells from amnion.

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RELIABILITY

Quantitative analysis of composite umbilical cord tissue health using a standardized explant approach and an assay of metabolic activity.

BACKGROUND: Umbilical cord (UC) tissue can be collected in a noninvasive procedure and is enriched in progenitor cells with potential therapeutic value. Mesenchymal stromal cells (MSCs) can be reliably harvested from fresh or cryopreserved UC tissue by explant outgrowth with no apparent impact on functionality. A number of stem cell banks offer cryopreservation of UC tissue, alongside cord blood, for future cell-based applications. In this setting, measuring and monitoring UC quality is critical.

MATERIALS AND METHODS: UC explants were evaluated using a plating and scoring system accounting for cell attachment and proliferation. Explant scores for fresh and cryopreserved-then-thawed tissue from the same UC were compared. Metabolic activity of composite UC tissue was also assayed after exposure of the tissue to conditions anticipated to affect UC quality and compared with explant scores within the same UC.

RESULTS: All fresh and cryopreserved tissues yielded MSC-like cells, and cryopreservation of the tissue did not prevent the ability to isolate MSCs by the explant method. Thawed UC tissue scores were 91% (±0.6%; P = 0.0009) that of the fresh, biologically identical tissue. Within the same UC, explant scores correlated well to both cell yield (R2 = 0.85) and tissue metabolic activity (R2 = 0.69).

DISCUSSION: A uniform explant scoring assay can provide information about the quality of composite UC tissue. Such quantitative measurement is useful for analysis of tissue variability and process monitoring. Additionally, a metabolic assay of UC tissue health provides results that correlate well to explant scoring results.

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Characterization and Comparison of Cryopreserved Umbilical Cord Products

Umbilical cord (UC) is a placental organ that provides nutrients from mother to fetus. UC is structurally soft and spongy, allowing it to mechanically buffer physical impacts to the fetus [1, 2]. The UC contains Wharton's jelly (WJ), which is rich in collagen and hyaluronic acid. The UC has unique immune invasive properties which enables the tissue to evade host rejection and makes it a valuable tool for tissue therapy [3]. Moreover, UC tissue is well known to have anti-inflammatory properties and have been shown to promote healing in various orthopedic indications [4]. This study characterizes and defines the viability of the cellular and molecular components of cryopreserved Signature Cord and an external UC product.

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Minimally Manipulated Amniotic Membrane Highly Retains Cellular Structure and Molecular Components as in Native Tissue

Amniotic membrane (AM) is a placental organ that protects the fetus and keeps it isolated from the organs of the mother. Rich in collagen and hyaluronic acid, the AM is structurally elastic, soft and spongy, allowing it to mechanically buffer physical impacts to the fetus [1-3]. Another relevant property of AM is the unique immunosuppressive and angiogenic properties of enabling this tissue to evade host rejection and induce vessel regeneration, respectively, making it a valuable tool for tissue therapy [4-6]. Moreover, it is well known to have anti-inflammatory properties and has been shown to promote healing in various orthopedic indications [7]. This study characterizes and defines the viability of Amniotic Membrane cellular and molecular components observed in cryopreserved Signature Matrix.

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Comparative analysis of human Wharton's jelly mesenchymal stem cells derived from different parts of the same umbilical cord.

Easy isolation, lack of ethical issues, high proliferation, multi-lineage differentiation potential and immunomodulatory properties of umbilical cord (UC)-derived mesenchymal stem cells (MSCs) make them a valuable tool in stem cell research. Recently, Wharton's jelly (WJ) was proven as the best MSC source among various compartments of UC. However, it is still unclear whether or not Wharton's jelly-derived MSCs (WJMSCs) from different parts of the whole cord exhibit the same characteristics. There may be varied MSCs present in different parts of WJ throughout the length of the UC. For this purpose, using an explant attachment method, WJMSCs were isolated from three different parts of the UC, mainly present towards the placenta (mother part), the center of the whole cord (central part) and the part attached to the fetus (baby part). WJMSCs from all three parts were maintained in normal growth conditions (10% ADMEM) and analyzed for mesenchymal markers, pluripotent genes, proliferation rate and tri-lineage differentiation potential. All WJMSCs were highly proliferative, positively expressed CD90, CD105, CD73 and vimentin, while not expressing CD34, CD45, CD14, CD19 or HLA-DR, differentiated into adipocytes, osteocytes and chondrocytes and expressed pluripotency markers OCT-4, SOX-2 and NANOG at gene and protein levels. Furthermore, MSCs derived from all the parts were shown to have potency towards hepatocyte-like cell differentiation. Human bone marrow-derived MSCs were used as a positive control. Finally, we conclude that WJMSCs derived from all the parts are valuable sources and can be efficiently used in various fields of regenerative medicine.

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