A tri-layer decellularized, dehydrated human amniotic membrane scaffold supports the cellular functions of human tenocytes in vitro.

Differences in scaffold design have the potential to influence cell-scaffold interactions. This study sought to determine whether a tri-layer design influences the cellular function of human tenocytes in vitro. The single-layer decellularized, dehydrated human amniotic membrane (DDHAM) and the tri-layer DDHAM (DDHAM-3L) similarly supported tenocyte function as evidenced by improved cell growth and migration, reduced dedifferentiation, and an attenuated inflammatory response. The tri-layer design provides a mechanically more robust scaffold without altering biological activity.

Placental-Derived Biomaterials and Their Application to Wound Healing: A Review.

Chronic wounds are associated with considerable patient morbidity and present a significant economic burden to the healthcare system. Often, chronic wounds are in a state of persistent inflammation and unable to progress to the next phase of wound healing. Placental-derived biomaterials are recognized for their biocompatibility, biodegradability, angiogenic, anti-inflammatory, antimicrobial, antifibrotic, immunomodulatory, and immune privileged properties. As such, placental-derived biomaterials have been used in wound management for more than a century. Placental-derived scaffolds are composed of extracellular matrix (ECM) that can mimic the native tissue, creating a reparative environment to promote ECM remodeling, cell migration, proliferation, and differentiation. Reliable evidence exists throughout the literature to support the safety and effectiveness of placental-derived biomaterials in wound healing. However, differences in source (i.e., anatomical regions of the placenta), preservation techniques, decellularization status, design, and clinical application have not been fully evaluated. This review provides an overview of wound healing and placental-derived biomaterials, summarizes the clinical results of placental-derived scaffolds in wound healing, and suggests directions for future work.

An in vitro comparison of human corneal epithelial cell activity and inflammatory response on differently designed ocular amniotic membranes and a clinical case study.

Amniotic membrane (AM) is a naturally derived biomaterial with biological and mechanical properties important to Ophthalmology. The epithelial side of the AM promotes epithelialization, while the stromal side regulates inflammation. However, not all AMs are equal. AMs undergo different processing with resultant changes in cellular content and structure. This study evaluates the effects of sidedness and processing on human corneal epithelial cell (HCEC) activity, the effect of processing on HCEC inflammatory response, and then a case study is presented. Three differently processed, commercially available ocular AMs were selected: (1) Biovance®3L Ocular, a decellularized, dehydrated human AM (DDHAM), (2) AMBIO2®, a dehydrated human AM (DHAM), and (3) AmnioGraft®, a cryopreserved human AM (CHAM). HCECs were seeded onto the AMs and incubated for 1, 4 and 7 days. Cell adhesion and viability were evaluated using alamarBlue assay. HCEC migration was evaluated using a scratch wound assay. An inflammatory response was induced by TNF-α treatment. The effect of AM on the expression of pro-inflammatory genes in HCECs was compared using quantitative polymerase chain reaction (qPCR). Staining confirmed complete decellularization and the absence of nuclei in DDHAM. HCEC activity was best supported on the stromal side of DDHAM. Under inflammatory stimulation, DDHAM promoted a higher initial inflammatory response with a declining trend across time. Clinically, DDHAM was used to successfully treat anterior basement membrane dystrophy. Compared with DHAM and CHAM, DDHAM had significant positive effects on the cellular activities of HCECs in vitro, which may suggest greater ocular cell compatibility in vivo.

A decellularized flowable placental connective tissue matrix supports cellular functions of human tenocytes in vitro.

PURPOSE: Injectable connective tissue matrices (CTMs) may promote tendon healing, given their minimally invasive properties, structural and biochemical extracellular matrix components, and capacity to fill irregular spaces. The purpose of this study is to evaluate the effects of placental CTMs on the cellular activities of human tenocytes. Decellularization, the removal of cells, cell fragments, and DNA from CTMs, has been shown to reduce the host's inflammatory response. Therefore, the authors hypothesize that a decellularized CTM will provide a more cell-friendly matrix to support tenocyte functions. METHODS: Three human placental CTMs were selected for comparison: AmnioFill® (A-CTM), a minimally manipulated, non-viable cellular particulate, BioRenew™ (B-CTM), a liquid matrix, and Interfyl® (I-CTM), a decellularized flowable particulate. Adhesion and proliferation were evaluated using cell viability assays and tenocyte migration using a transwell migration assay. Gene expression of tenocyte markers, cytokines, growth factors, and matrix metalloprotease (MMP) in tenocytes were assessed using quantitative polymerase chain reaction. RESULTS: Although A-CTM supported more tenocyte adhesion, I-CTM promoted significantly more tenocyte proliferation compared with A-CTM and B-CTM. Unlike A-CTM, tenocyte migration was higher in I-CTM than the control. The presence of I-CTM also prevented the loss of tenocyte phenotype, attenuated the expression of pro-inflammatory cytokines, growth factors, and MMP, and promoted the expression of antifibrotic growth factor, TGFß3. CONCLUSION: Compared with A-CTM and B-CTM, I-CTM interacted more favorably with human tenocytes in vitro. I-CTM supported tenocyte proliferation with reduced de-differentiation and attenuation of the inflammatory response, suggesting that I-CTM may support tendon healing and regeneration in vivo.

The impact of fertilization on the chicken egg yolk plasma and granule proteome 24 hours post-lay at room temperature: capitalizing on high-pH/low-pH reverse phase chromatography in conjunction with tandem mass tag (TMT) technology.

Chicken egg yolk is a rich source of nutrients providing high quality proteins, vitamins, minerals, carotenoids and antioxidants. Chicken egg yolk, recovered from whole egg within 24 hours post-lay has been utilized as a starting material in the preparation of a dietary supplement that has been demonstrated to lead to gains in muscle mass in a human clinical study. Further, an oil derived from chicken egg yolk has been utilized as a topical agent to treat third degree burn injury. The molecular changes that take place in fertilized, chicken egg yolk during the first 24 hours post-lay are not well understood. By studying how the protein composition of egg yolk varies with fertility status, one can utilize this knowledge to develop egg yolk-based products that have been optimized for specific applications. In this study, a direct quantitative comparison was made between the proteome of fertilized chicken egg yolk and the proteome of unfertilized chicken egg yolk, both maintained at 20 °C and analyzed within 24 hours post-lay. Egg yolk proteins from each fertility state were digested with trypsin, labeled with distinct chemical labels (tandem mass tag reagents) and then combined in a 1 : 1 ratio. A TMT-labeled tryptic digest derived from chicken egg yolk proteins (fertilized and unfertilized) was separated using high-pH/low-pH reverse-phase chromatography and analyzed using mass spectrometry. 225 protein identifications were made from this TMT-labeled tryptic digest based on a minimum of 2 unique peptides observed per protein. 9 proteins increased in abundance in fertilized egg yolk relative to unfertilized egg yolk and 9 proteins decreased in abundance in fertilized egg yolk relative to unfertilized egg yolk. Some proteins that increased in abundance in fertilized egg yolk play an important role in angiogenesis (pleiotrophin, histidine rich glycoprotein) and defense against pathogens (mannose-binding lectin, ß-defensin 11, serum amyloid P-component, ovostatin). Based on this study, fertilized chicken egg yolk may be more useful as a starting material relative to unfertilized chicken egg yolk for the purpose of enriching or isolating proteins with pro-angiogenic and anti-microbial properties.

Human placenta-derived cells (PDA-001) for the treatment of adults with multiple sclerosis: a randomized, placebo-controlled, multiple-dose study.

BACKGROUND: Infusion of PDA-001, a preparation of mesenchymal-like cells derived from full-term human placenta, is a new approach in the treatment of patients with multiple sclerosis. OBJECTIVE: This safety study aimed to rule out the possibility of paradoxical exacerbation of disease activity by PDA-001 in patients with multiple sclerosis. METHODS: This was a phase 1b, multicenter, randomized, double-blind, placebo-controlled, 2-dose ranging study including patients with relapsing-remitting multiple sclerosis or secondary progressive multiple sclerosis. The study was conducted at 6 sites in the United States and 2 sites in Canada. Patients were randomized 3:1 to receive 2 low-dose infusions of PDA-001 (150×10(6) cells) or placebo, given 1 week apart. After completing this cohort, subsequent patients received high-dose PDA-001 (600×10(6) cells) or placebo. Monthly brain magnetic resonance imaging scans were performed. The primary end point was ruling out the possibility of paradoxical worsening of MS disease activity. This was monitored using Cutter׳s rule (≥5 new gadolinium lesions on 2 consecutive scans) by brain magnetic resonance imaging on a monthly basis for six months and also the frequency of multiple sclerosis relapse. RESULTS: Ten patients with relapsing-remitting multiple sclerosis and 6 with secondary progressive multiple sclerosis were randomly assigned to treatment: 6 to low-dose PDA-001, 6 to high-dose PDA-001, and 4 to placebo. No patient met Cutter׳s rule. One patient receiving high-dose PDA-001 had an increase in T2 and gadolinium lesions and in Expanded Disability Status Scale score during a multiple sclerosis flare 5 months after receiving PDA-001. No other patient had an increase in Expanded Disability Status Scale score>0.5, and most had stable or decreasing Expanded Disability Status Scale scores. With high-dose PDA-001, 1 patient experienced a grade 1 anaphylactoid reaction and 1 had grade 2 superficial thrombophlebitis. Other adverse events were mild to moderate and included headache, fatigue, infusion site reactions, and urinary tract infection. CONCLUSION: PDA-001 infusions were safe and well tolerated in relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis patients. No paradoxical worsening of lesion counts was noted with either dose.

Safety and tolerability of human placenta-derived cells (PDA001) in treatment-resistant crohn's disease: a phase 1 study.

BACKGROUND: The clinical utility of cellular therapies is being investigated in a broad range of therapeutic areas. This phase 1 study represents the first exploration of PDA001, a preparation of cells cultured from human placental tissue, in subjects with Crohn's disease. METHODS: Twelve subjects with active, moderate-to-severe Crohn's disease unresponsive to previous therapy were given 2 intravenous infusions of PDA001 1 week apart, monitored weekly for 5 weeks, and assessed at 6 months, 1 year, and 2 years after infusion. Six subjects received 2 infusions of 2 × 10 cells (low dose), and 6 subjects received 2 infusions of 8 × 10 cells (high dose). RESULTS: Mean baseline Crohn's Disease Activity Index in the low-dose and high-dose groups was 305 and 364, respectively, and mean C-reactive protein was 8 mg/L and 49 mg/L, respectively. All subjects in the low-dose group achieved a clinical response (a Crohn's Disease Activity Index decrease of ≥70 points versus baseline), and 3 achieved remission (a Crohn's Disease Activity Index decrease of ≥100 to <150 points). Two subjects in the high-dose group achieved response, and none met remission criteria. Most adverse events were mild to moderate in severity and included headache, nausea, fever, and infusion site reactions. CONCLUSIONS: PDA001 infusions appear safe and well-tolerated in subjects with treatment-resistant Crohn's disease. A response was seen in all subjects in the low-dose group. The high-dose group, with a higher baseline disease activity, had only 2 responders, suggesting a more treatment-resistant population. A phase 2 study in this patient population is ongoing.

The anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions.

Lenalidomide (Revlimid) is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk Myelodysplastic Syndromes (MDS) associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy. Previous reports suggest that lenalidomide is anti-angiogenic and this property appears to be related to efficacy in patients with MDS. We have investigated the effect of lenalidomide on the formation of microvessels in a novel in vitro angiogenesis assay utilizing human umbilical arterial rings and in a capillary-like cord formation assay using cultured primary endothelial cells. We found that lenalidomide consistently inhibits both sprout formation by arterial rings and cord formation by endothelial cells in a dose-dependent manner. We also found an inhibitory effect of lenalidomide on the associations between cadherin 5, beta-catenin and CD31, adherens junction proteins whose interaction is critical for endothelial cell cord formation. Furthermore, lenalidomide inhibited VEGF-induced PI3K-Akt pathway signaling, which is known to regulate adherens junction formation. We also found a strong inhibitory effect of lenalidomide on hypoxia-induced endothelial cell formation of cords and HIF-1 alpha expression, the main mediator of hypoxia-mediated effects and a key driver of angiogenesis and metastasis. Anti-metastatic activity of lenalidomide in vivo was confirmed in the B16-F10 mouse melanoma model by a >40% reduction in melanoma lung colony counts versus untreated mice. Our results suggest that inhibitory effects on microvessel formation, in particular adherens junction formation and inhibition of hypoxia-induced processes support a potential anti-angiogenic and anti-metastatic mechanism for this clinically active drug.