Beatrice Dionigi

Partial or complete coverage of experimental spina bifida by simple intra-amniotic injection of concentrated amniotic mesenchymal stem cells

PURPOSE We sought to determine whether simple intra-amniotic delivery of concentrated amniotic mesenchymal stem cells (afMSCs) may elicit prenatal coverage of experimental spina bifida. METHODS Time-dated pregnant Sprague-Dawley dams (n=24) exposed to retinoic acid for the induction of fetal neural tube defects were divided in three groups. Group I had no further manipulations. Groups II and III received volume-matched intra-amniotic injections of either saline (Group II) or a suspension of syngeneic afMSCs labeled with green fluorescent protein (Group III) in all fetuses (n=202) on gestational day 17 (term=21-22 days). Animals were killed before term. Statistical comparisons were by ANOVA (P<0.05). RESULTS Of 165 fetuses viable at euthanasia, a spina bifida was present in 58% (96/165), with no significant differences in defect dimension across the groups (P=0.19). However, variable degrees of coverage of the defect by a rudimentary skin confirmed histologically were only present in Group III (P<0.001), in which donor afMSCs were documented, with no differences between Groups I and II (P=0.98). CONCLUSIONS Amniotic mesenchymal stem cells can induce partial or complete coverage of experimental spina bifida after concentrated intra-amniotic injection. Trans-amniotic stem cell therapy (TRASCET) may become a practical option in the prenatal management of spina bifida.

Trans-amniotic stem cell therapy (TRASCET) minimizes Chiari-II malformation in experimental spina bifida

PURPOSE We sought to study the impact of trans-amniotic stem cell therapy (TRASCET) in the Chiari-II malformation in experimental spina bifida. METHODS Sprague-Dawley fetuses (n=62) exposed to retinoic acid were divided into three groups at term (21-22 days gestation): untreated isolated spina bifida (n=21), isolated spina bifida treated with intra-amniotic injection of concentrated, syngeneic, labeled amniotic fluid mesenchymal stem cells (afMSCs) on gestational day 17 (n=28), and normal controls (n=13). Analyses included measurements of brainstem and cerebellar placement on high resolution MRI and histology. Statistical comparisons included ANOVA. RESULTS In parallel to the expected induced coverage of the spina bifida in the afMSC-treated group (P<0.001), there were statistically significant differences in brainstem displacement across the groups (P<0.001), with the highest caudal displacement in the untreated group. Significant differences in cerebellar displacement were also noted, albeit less pronounced. Pairwise comparisons were statistically significant, with P=0.014 between treated and normal controls in caudal brainstem displacement and P<0.001 for all other comparisons. Labeled afMSCs were identified in 71% of treated fetuses. CONCLUSIONS Induced coverage of spina bifida by TRASCET minimizes the Chiari-II malformation in the retinoic acid rodent model, further suggesting it as a practical alternative for the prenatal management of spina bifida.

Extraluminal distraction enterogenesis using shape-memory polymer

PURPOSE Although a few techniques for lengthening intestine by mechanical stretch have been described, they are relatively complex, and the majority involve placement of an intraluminal device. Ideally, techniques applicable to humans would be easy to perform and extraluminal to avoid the potential for mucosal injury. This study of distraction enterogenesis used an extraluminal, radially self-expanding shape-memory polymer cylinder and a simple operative approach to both elongate intestine and grow new tissue. METHODS Young Sprague Dawley rats (250-350 g) underwent Roux-en-Y isolation of a small intestinal limb and were divided in three groups: no further manipulation (Control 1, C1); placement of a nonexpanding device (Control 2, C2); or placement of a radially expanding device by the limb (Experimental, Exp). For C2 and Exp animals, the blind end of the limb was wrapped around the radially expanding cylindrical device with the limb-end sutured back to the limb-side. Bowel length was measured at operation and at necropsy (14 days) both in-situ and ex-vivo under standard tension (6g weight). Change in length is shown as mean ± standard deviation. A blinded gastrointestinal pathologist reviewed histology and recorded multiple measures of intestinal adaptation. The DNA to protein ratio was quantified as a surrogate for cellular proliferation. Changes in length, histologic measures, and DNA:protein were compared using analysis of variance, with significance set at P<0.05. RESULTS The length of the Roux limb in situ increased significantly in Exp animals (n=8, 29.0 ± 5.8mm) compared with C1 animals (n=5, -11.2 ± 9.0mm, P<0.01). The length of the Roux limb ex vivo under standard tension increased in the Exp group (25.8 ± 4.2mm) compared with the C2 group (n=6, -4.3 ± 6.0, P<0.01). There were no differences in histologic measures of bowel adaptation between the groups, namely villous height and width, crypt depth, crypt density, and crypt fission rate (all P ≥ 0.08). Muscularis mucosal thickness was also not different (P=0.25). There was no difference in DNA:protein between groups (P=0.47). CONCLUSION An extraluminally placed, radially expanding shape-memory polymer cylinder successfully lengthened intestine, without damaging mucosa. Lack of difference in muscularis thickness and a constant DNA:protein ratio suggests that this process may be related to actual growth rather than mere stretch. This study demonstrated a simple approach that warrants further study aiming at potential clinical applicability.

Contrast enhanced ultrasound for the evaluation of blunt pediatric abdominal trauma

INTRODUCTION Blunt abdominal trauma is a common problem in children. Computed tomography (CT) is the gold standard for imaging in pediatric blunt abdominal trauma, however up to 50% of CTs are normal and CT carries a risk of radiation-induced cancer. Contrast enhanced ultrasound (CEUS) may allow accurate detection of abdominal organ injuries while eliminating exposure to ionizing radiation. METHODS Children aged 7-18years with a CT-diagnosed abdominal solid organ injury underwent grayscale/power Doppler ultrasound (conventional US) and CEUS within 48h of injury. Two blinded radiologists underwent a brief training in CEUS and then interpreted the CEUS images without patient interaction. Conventional US and CEUS images were compared to CT for the presence of injury and, if present, the injury grade. Patients were monitored for contrast-related adverse reactions. RESULTS Twenty one injured organs were identified by CT in eighteen children. Conventional US identified the injuries with a sensitivity of 45.2%, which increased to 85.7% using CEUS. The specificity of conventional US was 96.4% and increased to 98.6% using CEUS. The positive predictive value increased from 79.2% to 94.7% and the negative predictive value from 85.3% to 95.8%. Two patients had injuries that were missed by both radiologists on CEUS. In a 100kg, 17year old female, a grade III liver injury was not seen by either radiologist on CEUS. Her accompanying grade I kidney injury was not seen by one of the radiologist on CEUS. The second patient, a 16year old female, had a grade III splenic injury that was missed by both radiologists on CEUS. She also had an adjacent grade II kidney injury that was seen by both. Injuries, when noted, were graded within 1 grade of CT 33/35 times with CEUS. There were no adverse reactions to the contrast. CONCLUSION CEUS is a promising imaging modality that can detect most abdominal solid organ injuries in children while eliminating exposure to ionizing radiation. A multicenter trial is warranted before widespread use can be recommended. LEVEL OF EVIDENCE Level II; Diagnostic Prospective Study.

The Impact of Gestational Age on Targeted Amniotic Cell Profiling in Experimental Neural Tube Defects

Purpose: The proportions of select stem cells in term amniotic fluid have been shown to correlate with the type and size of experimental neural tube defects (NTDs). We sought to determine the impact of gestational age upon this form of targeted amniotic cell profiling. Methods: Sprague-Dawley fetuses with retinoic acid-induced NTDs (n = 110) underwent amniotic fluid procurement at four time points in gestation. Samples were analyzed by flow cytometry for the presence of cells concomitantly expressing Nestin and Sox-2 (neural stem cells, aNSCs) and cells concomitantly expressing CD29 and CD44 (mesenchymal stem cells, aMSCs). Statistical analysis was by nonparametric Kruskal-Wallis ANOVA (p < 0.05). Results: There was a statistically significant impact of gestational age on the proportions of both aMSCs (p = 0.01) and aNSCs (p < 0.01) in fetuses with isolated spina bifida. No such impact was noted in normal fetuses (p > 0.10 for both cells), in isolated exencephaly (p > 0.10 for both cells), or in combination defects (p > 0.10 for both cells). Gestational age had no effect on aNSC/aMSC ratios. Conclusions: Targeted quantitative amniotic cell profiling varies with gestational age in experimental isolated spina bifida. This finding should be considered prior to the eventual translation of this diagnostic adjunct into the prenatal evaluation of these anomalies.

Restoring esophageal continuity following a failed colonic interposition for long-gap esophageal atresia.

The Foker process is a method of esophageal lengthening through axial tension-induced growth, allowing for subsequent primary reconstruction of the esophagus in esophageal atresia (EA). In this unique case, the Foker process was used to grow the remaining esophageal segment long enough to attain esophageal continuity following failed colonic interpositions for long-gap esophageal atresia (LGEA). Initially developed for the treatment of LGEA in neonates, this case demonstrates that (i) an active esophageal lengthening response may still be present beyond the neonate time-period; and, (ii) the Foker process can be used to restore esophageal continuity following a failed colonic interposition if the lower esophageal segment is still present.

Extraluminal helicoidal stretch (Helixtretch): A novel method of intestinal lengthening

PURPOSE We sought to test a novel, extraluminal method of intestinal lengthening that precludes violation of the intestinal wall. METHODS Sprague-Dawley rats (n=45) with size-matched bowel segments isolated by Roux-en-Y reconstruction were divided into three groups. Group 1 (n=14) had no further manipulations. In Groups 2 (n=12) and 3 (n=19), the isolated segment was wrapped around a length-matched device in a helicoidal fashion. In Group 2, the device consisted of plain polyurethane tubing. In Group 3, it consisted of a gradually expanding hygroscopic hydrogel (12.5mm final diameter). Euthanasia was performed at 8-21 days. Statistical analysis was by two-way ANOVA (P<0.05). RESULTS Overall survival was 87% (39/45). There was a statistically significant increase in bowel length in Group 3 compared to the other two groups (P<0.001). This increase correlated with the number of helicoidal coils (P=0.018), but not with post-operative time (P>0.50). There were no significant differences in total DNA/protein ratio across the groups (P=0.65). Histologically, there was an apparent increase in the goblet cell density in Group 3. CONCLUSIONS Measured extraluminal helicoidal stretch (Helixtretch) is tolerated by the intestine. Helixtretch induces bowel lengthening in a rodent model. Further analysis of this novel, minimally invasive alternative for intestinal augmentation is warranted.

Transamniotic Stem Cell Therapy (TRASCET)

Trans-Amniotic Stem Cell Therapy (TRASCET) is a novel therapeutic paradigm for the treatment of congenital anomalies. It is based on the principle of harnessing and enhancing the biological role of select population of stem cells that either naturally occur in the amniotic fluid or are present therein in the setting of disease for therapeutic benefit. It has been recently shown that amniotic fluid-derived mesenchymal stem cells (afMSCs) play a central role in the widely known enhanced ability of the fetus to repair tissue damage. This germane finding was not only the first demonstration of a biological role for any amniotic cell, it has also provided validation for the use of afMSCs in regenerative strategies, in that these cells already play a regenerative role in nature. More recently, it has also first been shown experimentally that the intra-amniotic delivery of afMSCs in large numbers can either elicit the repair, or significantly mitigate the effects associated with major congenital anomalies by boosting the activity that these cells normally have. For example, concentrated amounts of these cells injected into the amniotic cavity can induce partial or complete coverage of experimental spina bifida, the most prevalent neural tube defect compatible with life, by promoting the local formation of host-derived skin, thus protecting the spinal cord from damage. In another example, intra-amniotic administration of afMSCs in large numbers can also significantly alleviate the bowel damage associated with gastroschisis, one of the most common major abdominal wall defects. Other applications involving different congenital anomalies and/or other stem cells present in the amniotic fluid in diseased pregnancies are currently under investigation in this still very young facet of fetal stem cell therapy. Combined, these discoveries point to simple, easily accessible intra-amniotic administration of afMSCs and/or other cells as practical, minimally invasive alternatives for the management of different congenital anomalies, with significant therapeutic gain at minimal to no risk to the mother and fetus.