Dario O. Fauza

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

PURPOSEnWe sought to determine whether simple intra-amniotic delivery of concentrated amniotic mesenchymal stem cells (afMSCs) may elicit prenatal coverage of experimental spina bifida.nnnMETHODSnTime-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).nnnRESULTSnOf 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).nnnCONCLUSIONSnAmniotic 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

PURPOSEnWe sought to study the impact of trans-amniotic stem cell therapy (TRASCET) in the Chiari-II malformation in experimental spina bifida.nnnMETHODSnSprague-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.nnnRESULTSnIn 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.nnnCONCLUSIONSnInduced 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.

A comparison between placental and amniotic mesenchymal stem cells for transamniotic stem cell therapy (TRASCET) in experimental spina bifida.

PURPOSEnWe compared placental-derived and amniotic fluid-derived mesenchymal stem cells (pMSCs and afMSCs, respectively) in transamniotic stem cell therapy (TRASCET) for experimental spina bifida.nnnMETHODSnPregnant dams (n=29) exposed to retinoic acid for the induction of fetal spina bifida were divided into four groups. Three groups received volume-matched intraamniotic injections of either saline (n=38 fetuses) or a suspension of 2×10(6) cells/mL of syngeneic, labeled afMSCs (n=73) or pMSCs (n=115) on gestational day 17 (term=21-22days). Untreated fetuses served as controls. Animals were killed before term. Statistical comparisons were by Fishers exact test (p<0.05).nnnRESULTSnSurvival was similar across treatment groups (p=0.08). In fetuses with isolated spina bifida (n=100), there were higher percentages of defect coverage (either partial or complete) in both afMSC and pMSC groups compared with saline and untreated groups (p<0.001-0.03 in pairwise comparisons). There were no differences in coverage rates between afMSC and pMSC groups (p=0.94) or between saline and untreated groups (p=0.98).nnnCONCLUSIONSnBoth pMSC and afMSC can induce comparable rates of coverage of experimental spina bifida after concentrated intraamniotic injection in the rodent model. This broadens the options for timing and cell source for TRASCET as a potential alternative in the prenatal management of spina bifida.

Extraluminal distraction enterogenesis using shape-memory polymer

PURPOSEnAlthough 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.nnnMETHODSnYoung 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.nnnRESULTSnThe 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).nnnCONCLUSIONnAn 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.

Transamniotic stem cell therapy (TRASCET) mitigates bowel damage in a model of gastroschisis

PURPOSEnWe sought to determine whether intraamniotic delivery of concentrated amniotic-derived mesenchymal stem cells (afMSCs) could reduce damage to exposed bowel in experimental gastroschisis.nnnMETHODSnRat fetuses (n=117) with surgically created gastroschisis were divided into three groups: untreated animals (n=62) and two groups receiving volume-matched intraamniotic injections of either saline (n=25) or 2 × 10(6) cells/mL of syngeneic, labeled afMSCs (n=30). Animals were killed before term, along with normal controls (NL). Blinded observers performed computerized measurements of total and segmental (serosa, muscularis, and mucosa) intestinal wall thicknesses. Statistical comparisons were by ANOVA (P<0.05).nnnRESULTSnAmong survivors with gastroschisis, there were statistically significant decreases in total bowel wall, serosal, muscular, and mucosal thicknesses in the afMSC group vs. the untreated group (P=0.001/0.035/0.001/0.005, respectively) and vs. the saline group (P=0.003/0.05/<0.001/0.026, respectively). There were no such significant differences between the untreated and saline groups. There were no differences between the afMSC group and NL, except for a significantly thicker muscular layer in the afMSC group (P=0.014). Labeled afMSCs were scarcely identified, suggesting a paracrine effect.nnnCONCLUSIONSnAmniotic mesenchymal stem cells mitigate bowel damage in experimental gastroschisis after concentrated intraamniotic injection. Transamniotic stem cell therapy (TRASCET) may become a practical component of the treatment of gastroschisis.

Transamniotic stem cell therapy (TRASCET) in a leporine model of gastroschisis.

BACKGROUND/PURPOSEnTransamniotic stem cell therapy (TRASCET) with amniotic fluid mesenchymal stem cells (afMSCs) has been shown to mitigate bowel damage in a rodent model of gastroschisis. As a prerequisite to clinical translation, we sought to study TRASCET in a larger animal model.nnnMETHODSnNew Zealand rabbit fetuses (n=64) with surgically created gastroschisis were divided into three groups. One group (untreated) had no further manipulations. Two groups received volume-matched intraamniotic injections of either saline or a suspension of afMSCs. Nonmanipulated fetuses served as controls. Histomorphologic measurements of intestinal damage, along with biochemical profiling of inflammation markers, were performed at term. Statistical comparisons were by Fishers exact test, ANOVA and the Wald test (P<0.05).nnnRESULTSnOverall survival was 62.5%. Segmental and total intestinal wall thicknesses were significantly decreased in the afMSC group compared with the untreated and saline groups (all P<0.001), with no significant differences between untreated and saline groups (P=0.24 to 1.00, depending on layer). Muscularis and serosal layers were significantly thicker in the afMSC group than in normal controls (P=0.045 and P<0.001, respectively).nnnCONCLUSIONSnConcentrated intraamniotic injection of afMSC lessens, yet does not prevent, intestinal damage in a leporine model of gastroschisis. TRASCET may become a valuable strategy in the management of gastroschisis.nnnLEVEL OF EVIDENCEnN/A - animal/experimental studies.

Donor mesenchymal stem cells home to maternal wounds after transamniotic stem cell therapy (TRASCET) in a rodent model.

PURPOSEnTransamniotic stem cell therapy (TRASCET) with amniotic fluid-derived MSCs (afMSCs) has emerged experimentally as a practical treatment strategy for congenital anomalies. In this study, we sought to determine whether afMSCs migrate to the mother following TRASCET.nnnMETHODSnPregnant rat dams were divided into three groups. Two groups received volume-matched injections into all amniotic cavities of either a suspension of afMSCs labeled with a luciferase reporter gene or the luciferase protein alone. In a third group, a suspension of labeled cells was aliquoted onto the serosal surface of the uterus. Maternal samples from the laparotomy scar (fascia and skin separately), bone marrow, and peripheral blood were procured, along with placenta and umbilical cord. Specimens were screened for luminescence via microplate luminometry.nnnRESULTSnLuminescence was detected in 60% (9/15) of the fascial scars from the group receiving intraamniotic injection of afMSCs, but in none of the other groups (P<0.001). There was a direct correlation between the presence of donor cells in the placenta and their presence in maternal fascia (Wald test=10.2; P=0.001).nnnCONCLUSIONSnAmniotic mesenchymal stem cells migrate to maternal sites of injury after intraamniotic injection. Maternal homing of donor cells must be considered in the setting of transamniotic stem cell therapy.nnnLEVEL OF EVIDENCEnN/A (animal and laboratory study).

Limb reconstruction with decellularized, non-demineralized bone in a young leporine model.

Limb salvage from a variety of pathological processes in children is often limited by the unavailability of optimal allograft bone, or an appropriate structural bone substitute. In this study, we sought to examine a practical alternative for pediatric limb repair, based on decellularized, non-demineralized bone grafts, and to determine whether controlled recellularization prior to implantation has any impact on outcome. Growing New Zealand rabbits (n = 12) with a complete, critical-size defect on the left tibiofibula were equally divided into two groups. One group received a decellularized, non-demineralized leporine tibiofibula graft. The other group received an equivalent graft seeded with mesenchymal stem cells labeled with green fluorescent protein (GFP), at a fixed density. Animals were euthanized at comparable time points 3-8u2009weeks post-implantation. Statistical analysis was by the Student t-test and Fishers exact test (Pxa0<xa00.05). There was no significant difference in the rate of non-union between the two groups, including on 3D micro-CT. Incorporated grafts achieved adequate axial bending rigidity, torsional rigidity, union yield and flexural strength, with no significant differences or unequal variances between the groups. Correspondingly, there were no significant differences in extracellular calcium levels, or alkaline phosphatase activity. Histology confirmed the presence of neobone in both groups, with GFP-positive cells in the recellularized grafts. It was shown that osseous grafts derived from decellularized, non-demineralized bone undergo adequate remodeling in vivo after the repair of critical-size limb defects in a growing leporine model, irrespective of subsequent recellularization. This methodology may become a practical alternative for pediatric limb reconstruction.

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

PURPOSEnWe sought to test a novel, extraluminal method of intestinal lengthening that precludes violation of the intestinal wall.nnnMETHODSnSprague-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).nnnRESULTSnOverall 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.nnnCONCLUSIONSnMeasured 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.

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement

BACKGROUND/PURPOSEnWe sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair.nnnMETHODSnHuman afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05).nnnRESULTSnThere was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96).nnnCONCLUSIONSnAmong clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair.nnnLEVEL OF EVIDENCEnN/A (animal and laboratory study).