Evaluation of the Use of Human Umbilical Cord for In-Utero Spina Bifida Repair

LAS VEGAS (Jan. 23, 2017)—In two studies to be presented Saturday, Jan. 28, in the oral concurrent session at 8:45 a.m. PST, at the Society for Maternal-Fetal Medicine’s annual meeting, The Pregnancy Meeting™, researchers evaluated a possible regenerative patch by using human umbilical cord in two studies titled Cryopreserved Human Umbilical Cord (HUC) vs Acellular Dermal Matrix (ADM) for In-Utero Spina Bifida Repair and the study Conventional vs cryopreserved human umbilical cord (HUC) patch based on repair for in-utero spina bifida in a sheep model.

Spina Bifida is a birth defect where there is an incomplete closure of the backbone and the coverings around the spinal cord. It affects more than 4,000 children born each year in the United States and is associated with hydrocephalus (fluid in the brain), developmental delay, lifelong disability and death.

Spina Bifida is also associated with a need for shunt placement. In-utero surgery has been shown to reduce the need for shunting of hydrocephalus and to improve a child’s ability to walk. However, more than half of the children who undergo such surgery do not benefit from the in-utero repair, which failure has been attributed to suboptimal repair leading to persistent leakage of cerebrospinal fluid and spinal cord scar formation at the repair site.

Researchers are trying to identify a regenerative patch material for repair that would further reduce morbidity after repair through decreased spinal cord damage from reduced inflammation and scar formation.

In the first study, conducted in a pregnant rat model, the researchers sought to compare two types of patches: a cryopreserved human umbilical cord patch and an acellular dermal matrix, a currently used graft material in clinical practice. The scientists aimed to explore the cellular response in both inflammatory and regenerative properties after in-utero repair in a fetus.  The cryopreserved human umbilical cord patch was determined to promote organized cellular migration of epidermal and meningeal cells and decrease acute inflammatory response and cell death compared to the acellular dermal matrix.

In the second study, conducted in a pregnant sheep model, the researchers compared the cryopreserved human umbilical cord patch to the current method of closure using sutures on the defect to test the functional and structural preservation of spinal cord at the repair site. The lambs repaired using cryopreserved human umbilical cord showed improved spinal cord function with decreased meningeal scar formation and better preservation of the spinal cord tracts.

Although these studies were preclinical, the human umbilical cord patch shows promising results to improve outcomes in spina bifida birth defects. Researchers worked with experienced veterinary staff who observed the animals daily for any complications. These animal studies were reviewed and approved by The University of Texas Health Science Center at Houston (UTHealth) Animal Welfare Committee as scientifically justified and appropriate.

“The unmet clinical need in in-utero spina bifida repair is the optimal closure method to reduce the ongoing damage to the spinal cord from inflammation and scar formation and to promote continued fetal spinal cord development. Based on the safety and efficacy of the cryopreserved human umbilical cord patch for in-utero spina bifida in pregnant rats and sheep, we have performed four cases of in-utero human repair under FDA approval. The early results have been promising, and the studies are ongoing to evaluate the long-term benefits in these patients. We believe that this is an initial step toward a safe and minimally invasive in-utero spina bifida repair,” said Ramesha Papanna, M.D., M.P.H., the principal investigator of the projects at The Fetal Center at Children’s Memorial Hermann Hospital and McGovern Medical School at UTHealth, who is also the presenter of one of the studies at the SMFM annual conference.

Lovepreet Mann, M.B.B.S., is a research instructor in the department of obstetrics and gynecology at McGovern Medical School and the co-investigator and the primary author who conducted experiments in the pregnant rat model in collaboration with Papanna.  Mann will present one of the studies at the SMFM annual meeting.

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A copy of the abstract is available at http://www.smfmnewsroom.org and below.  For interviews please contact Vicki Bendure at Vicki@bendurepr.com 202-374-9259 (cell).

About the Society for Maternal-Fetal Medicine

The Society for Maternal-Fetal Medicine (est. 1977) is the premiere membership organization for obstetricians/gynecologists who have additional formal education and training in maternal-fetal medicine.  The society is devoted to reducing high-risk pregnancy complications by sharing expertise through continuing education to its 2,000 members on the latest pregnancy assessment and treatment methods. It also serves as an advocate for improving public policy, and expanding research funding and opportunities for maternal-fetal medicine. The group hosts an annual meeting in which groundbreaking new ideas and research in the area of maternal-fetal medicine are shared and discussed.  For more information visit www.smfm.org.

Abstract 84  Cryopreserved human umbilical cord (HUC) vs acellular dermal matrix (ADM) for in-utero spina bifida repair

Lovepreet K. Mann¹ , Jong H. Won¹ , Saul Snowise² , Marissa Onanian³ , Stephen Fletcher³ , Scheffer CG. Tseng⁴ , Raymond J. Grill⁵ , Ramesha Papanna²   ¹Dept. OB/GYN, McGovern Medical School at UTHealth, Houston, TX,  ²Dept. OB/GYN, The Fetal Center at Children’s Memorial Hermann Hospital, McGovern Medical School at UTHealth, Houston, TX,  ³Dept. Pediatric Neurosurgery, McGovern Medical School at UTHealth, Houston, TX,  ⁴Ocular Surface Center, Miami, FL,  ⁵Department of Neurobiology and Anatomical Sciences, University of Mississippi Medical Center, Jackson, MS

OBJECTIVE: Optimal regenerative patch for in-utero spina bifida repair is an unmet need to further improve neurological outcomes by decreasing scar formation. To investigate the acute inflammatory response and regenerative ingrowth of native cells at repair site after HUC vs. ADM sutured over spina bifida lesions (SB) in a retinoic acid (RA) rat model.

STUDY DESIGN: Pregnant time-dated Sprague-Dawley rats were gavaged with RA (40 mg/kg) on gestational day (GD) 10 to induce SB in fetuses. In-utero repairs were performed on GD20 and HUC or ADM were sutured over the SB. After 48-52 hours, fetuses were harvested and fixed in 4% paraformaldehyde. Trichrome staining was used to assess cellular migration within the patches. Immunofluorescence was then performed to assess inflammatory response using myeloperoxidase (MPO:neutrophils), and F4/80 (macrophages). To evaluate regenerative ingrowth of cells, native cell markers including cytokeratin (Cyt 5/6: epidermal), glial fibrillary acidic protein (GFAP: astrocytes) and chondroitin sulfate-56 (CS-56: meningeal layer) were used. TUNEL (Apoptosis) staining were also performed. Total cells in the patch including outer surface and inner surface (towards the placode) and in the exudate were counted using Z-Pro software.

RESULTS: Fetal survival with intact patches was equal: HUC 8/12 vs ADM 7/12, (p=0.5). The cellular migration was not significantly different between the two groups (p=0.4). Neutrophils were lower in the HUC compared to ADM (Figure 1). Macrophages cells were not significantly different in both groups. Epidermal cells were present on the outer surface in both groups, however, the cells were positive on the inner surface of the patch in 71% ADM compared to 0% in HUC (p=0.03). Meningeal cells were positive in the inner surface of both patches, whereas, these cells were present on the outer surfaces of 71%) ADM compared to none in HUC (p¼0.03). Astrocyte cells were attached to the inner surface of 86% ADM compared to only 25% HUC; p=0.01 (Figure 2). The patterns suggests disorganized regeneration in ADM. TUNEL positive cells were lower in the HUC compared to ADM (p=0.03).

CONCLUSION: Cryopreserved human umbilical cord promotes lower acute inflammatory response, better organized cellular migration and lower cell death as compared to acellular dermal matrix at the in-utero spina bifida repair site.

Abstract 85   Conventional vs cryopreserved human umbilical cord (HUC) patch based repair for in-utero spina bifida in a sheep model

Ramesha Papanna¹ , Lovepreet K. Mann¹ , Jong H. Won² , Yisel Morales¹ , Stephen Fletcher³ , Ranu R. Jain⁴ , Suzanne M. Lopez⁵ , Kenneth J. Moise Jr.¹ , Raymond J. Grill⁶   ¹Dept. OB/GYN, The Fetal Center at Children’s Memorial Hermann Hospital, McGovern Medical School at UTHealth, Houston, TX,  ²University of Texas McGovern Medical School, Houston, TX,  ³Dept. Pediatric Neurosurgery, McGovern Medical School at UTHealth, Houston, TX,  ⁴Dept. Anesthesia, McGovern Medical School at UTHealth, Houston, TX,  ⁵Neonatology, Dept. Pediatrics, McGovern Medical School at UTHealth, Houston, TX,  ⁶Department of Neurobiology and Anatomical Sciences, University of Mississippi Medical Center, Jackson, MS

OBJECTIVE: We have previously demonstrated that in-utero spina bifida repair with a HUC patch can reverse hindbrain herniation. The objective of this study was to evaluate the efficacy of in-utero repair of spina bifida using single layer HUC patch compared to conventional repair (CR) in order to decrease scar formation and improve offspring spinal cord function in a sheep model.

STUDY DESIGN: On gestational day (GD) 75, spina bifida without myelotomy (remove dura matter only) was created in the 7 timed pregnant ewe (11 fetuses). Repeat hysterotomy was performed on GD 95 and the surviving fetuses were randomly assigned to CR or HUC. Three lambs without spina bifida served as controls. The ewes were delivered at GD 140 by C-section and neurological assessments of the newborns were performed on day 2 of life using Texas Spinal Cord Injury Scale (TSCIS) to assess the gait, proprioception and nociception. Bladder control was assessed using ultrasound. MRI with digital tensor imaging (DTI) of the spinal cord was performed to quantify the disruption of spinal tracts. Masson’s Trichrome was performed to assess presence of scar tissue at the repair site followed by immunofluorescence for myofibroblast activation using ɑ-smooth muscle actin (ɑSMA) and for meningeal cells (chondroitin sulfate-56) and astrocytes (glial fibrillar associated protein).

RESULTS: There were 7/11 (64%) fetuses with spina bifida that survived to the repair stage; of which 3 underwent CR and 4 were repaired with HUC; all seven survived to delivery. The combined hind limbs TSCIS scores for the control lambs were 200, HUC were 16 7 and CR were 12.7 4 (Control vs. CR p=0.03; CR vs. HUC p=0.5 and Control vs. HUC p=0.4). CR had predominant loss of proprioception and mild to moderate ataxia. HUC repaired lambs had normal proprioception and mild ataxia in the hind limbs. There was no difference in bladder control between three groups. DTI showed disruption of dorsal column in the spinal cord in 3/3 CR compared to 1/4 HUC (Figure 1; p=0.1). Histology showed thickened arachnoid layer with activation of ɑSMA in CR 3/3 compared to 0/4 in HUC (Figure 2: p=0.03) suggesting scar formation in meningeal layers.

CONCLUSION: In our spina bifida sheep model, HUC preserved the integrity of meningeal layers after in-utero repair as compared to CR repair, with improvements in the spinal cord function.

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