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ABSTRACT: Objective: Neural stem cells (NSCs) may promote spinal cord repair in fetuses with experimental spina bifida. We sought to determine the fate of amniotic-derived NSCs (aNSCs) after simple intra-amniotic injection in a syngeneic model of spina bifida. Methods: Fetal neural tube defects were induced on 20 pregnant Lewis dams by prenatal administration of retinoic acid. Ten dams served as amniotic fluid donors for epigenetic isolation of aNSCs, which were expanded and labeled with 5-bromo-2'-deoxyuridine. The remaining 10 dams received intra-amniotic injections of the processed aNSCs, blindly in all their fetuses (n = 37) on gestational day 17 (term = E21-22). Fetuses with spina bifida underwent screening for the presence of donor aNSCs in the spinal cord at term. Results: Donor cells were identified in 93.3% of the animals with spina bifida, selectively populating the neural placode, typically in clusters, retaining an undifferentiated morphology, and predominantly on exposed neural surfaces, though some were detected deeper in neighboring neural tissue. Conclusions: The amniotic cavity can serve as a route of administration of NSCs in experimental spina bifida. Simple intra-amniotic delivery of NSCs may be a practical adjuvant to regenerative strategies for the treatment of spina bifida.
Fetal Diagnosis and Therapy 04/2013; · 1.05 Impact Factor
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ABSTRACT: BACKGROUND: Postoperative intussusception (POI) is a sporadic complication whose mechanisms and risk factors remain poorly understood. Its epidemiology in the minimally invasive surgery era has yet to be well described, particularly in children. We sought to examine risk factors, demographics, and anatomic patterns of pediatric POI in recent years. STUDY DESIGN: This was a 13-year retrospective review from a single tertiary pediatric center. Variables analyzed included patient demographics, time of occurrence, type of intussusception, type of anesthesia, and triggering surgical procedure. The latter variable was divided into 2 groups: abdominal and nonabdominal interventions. Statistical analysis was by 2-tailed Fisher's exact test with significance set at p < 0.05. RESULTS: Among 822 cases of intussusception in 718 patients, 22 documented cases of POI were identified. Twelve of them occurred after abdominal procedures; there was a statistically significant difference in the incidence of POI after open surgery (0.091%; 11 of 12,126) when compared with minimally invasive interventions (0.013%; 1 of 7,610; p = 0.036). As expected, ileoileal and jejunojejunal intussusceptions were the most common forms of POI after abdominal operations (12 of 12; 100%); however, ileocolic intussusceptions were common forms of POI after nonabdominal cases (5 of 10; 50%; p = 0.01). Epidural anesthesia did not appear to be a risk factor for POI. CONCLUSIONS: Although rare, postoperative intussusception can occur after a multitude of interventions, including those performed at a distance from the abdomen. Although small bowel intussusception is the predominant variant of this complication after abdominal procedures, ileocolic intussusception is prevalent after other interventions. Minimally invasive abdominal access may protect against postoperative intussusception in children.
Journal of the American College of Surgeons 04/2013; · 4.55 Impact Factor
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ABSTRACT: We sought to determine whether neural stem cells (NSCs) can be isolated from the amniotic fluid in the setting of neural tube defects (NTDs), as a prerequisite for eventual autologous perinatal therapies. Pregnant Sprague-Dawley dams (n=62) were divided into experimental (n=42) and control (n=20) groups, depending on prenatal exposure to retinoic acid for the induction of fetal NTDs. Animals were killed before term for analysis (n=685 fetuses). Amniotic fluid samples from both groups underwent epigenetic selection for NSCs, followed by exposure to neural differentiation media. Representative cell samples underwent multiple morphological and phenotypical analyses at different time points. No control fetus (n=267) had any structural abnormality, whereas at least one type of NTD developed in 52% (217/418) of the experimental fetuses (namely isolated spina bifida, n=144; isolated exencephaly, n=24; or a combination of the two, n=49). Only amniotic samples from fetuses with a NTD yielded cells with typical neural progenitor morphology and robust expression of both Nestin and Sox-2, primary markers of NSCs. These cells responded to differentiation media by displaying typical morphological changes, along with expression of Beta-Tubulin III, Glial Fibrillary Acidic Protein (GFAP), and/or O4, markers for immature neurons, astrocytes, and oligodendrocytes, respectively. This was concurrent with downregulation of Nestin and Sox-2. We conclude that the amniotic fluid can harbor disease-specific stem cells, e.g. neural stem cells in the setting of experimental neural tube defects. The amniotic fluid may be a practical source of autologous neural stem cells applicable to novel forms of therapies for spina bifida.
Stem cells and development 09/2012; · 4.15 Impact Factor
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ABSTRACT: This study was aimed at examining an airway construct engineered from autologous amniotic mesenchymal stem cells (aMSCs) and a xenologous decellularized airway scaffold as a means for tracheal repair.
Fetal lambs (N = 13) with a tracheal defect were divided into 2 groups. One group (acellular, n = 6) was repaired with a decellularized leporine tracheal segment. The other group (engineered, n = 7) received an identical graft seeded with expanded/labeled autologous aMSCs. Newborns were euthanized for multiple analyses.
Eleven lambs survived to term, 10 of which could breathe at birth. Engineered grafts showed a significant increase in diameter in vivo (P = .04) unlike acellular grafts (P = .62), although variable stenosis was present in all implants. Engineered constructs exhibited full epithelialization, compared with none of the acellular grafts (P = .002). Engineered grafts had a significantly greater degree of increase in elastin levels after implantation than acellular implants (P = .04). No such differences were noted in collagen and glycosaminoglycan contents. Donor cells were detected in engineered grafts, which displayed a pseudostratified columnar epithelium.
Constructs engineered from aMSCs and decellularized airway undergo enhanced remodeling and epithelialization in vivo when compared with equivalent acellular implants. Amniotic mesenchymal stem cell-engineered airways may become an alternative for perinatal airway repair.
Journal of Pediatric Surgery 06/2012; 47(6):1072-9. · 1.45 Impact Factor
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ABSTRACT: BACKGROUND: Ethically acceptable applications of fetal tissue engineering as a perinatal therapy can be expanded beyond life-threatening anomalies by amniotic fluid cell-based methods, in which cell procurement poses no additional risk to the mother. We sought to start to determine whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be an adjunct to craniofacial repair. METHODS: New Zealand rabbits (n = 12) underwent creation of a full-thickness diploic nasal bone defect. We then equally divided animals into two groups based on how the defect was repaired: namely, size-matched implants of electrospun biodegradable nanofibers with or without nuclear labeled, allogeneic aMSCs maintained in osteogenic medium. We killed animals 8 wk post-implantation for multiple analyses. Statistical analysis included analysis of variance, post-hoc Bonferroni adjusted comparisons, and Levene's F-test, as appropriate (P < 0.05), with significance set at P < 0.05. RESULTS: Micro-computed tomography scanning (two- and three-dimensional) showed no significant differences in defect radiodensity between groups. However, extracellular calcium levels were significantly higher in engineered grafts than in acellular implants (P = 0.003). There was significantly greater variability in mineralization in acellular implants than in engineered grafts by both direct calcium (P = 0.008) and micro-computed tomography measurements (P = 0.032). There were no significant differences in alkaline phosphatase activity or variance between groups. We documented labeled cells in the engineered grafts. CONCLUSIONS: Craniofacial repair with osseous grafts engineered from aMSCs lead to enhanced and more consistent mineralization compared with an equivalent acellular prosthetic repair. Amniotic fluid-derived engineered bone may become a practical adjunct to perinatal craniofacial reconstruction.
Journal of Surgical Research 05/2012; · 2.25 Impact Factor
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ABSTRACT: Under a Food and Drug Administration directive, we examined definite long-term safety and efficacy aspects of an engineered diaphragmatic tendon graft as a regulatory prerequisite for clinical trials.
Newborn lambs (N = 27) underwent partial diaphragmatic replacement with a Teflon patch, a composite acellular bioprosthesis, or the same bioprosthesis seeded with autologous amniotic mesenchymal stem cells processed under Good Manufacturing Practice guidelines. Multiple safety and efficacy analyses were performed at different time points up to 14 months of age (ovine adulthood).
There was no mortality. None of the blood tests or full body autopsy specimens showed any abnormality. There was a significantly higher failure rate in animals that received an acellular bioprosthetic graft vs an engineered graft, with no significant differences between Teflon and acellular bioprosthetic implants. Tensile strength and total collagen levels were significantly higher in engineered grafts than in acellular bioprosthetic grafts. On histology, lysozyme and myeloperoxidase stainings were unremarkable in all grafts.
Diaphragmatic repair with a clinically viable autologous tendon engineered with amniotic mesenchymal stem cells leads to improved outcomes when compared with an equivalent acellular bioprosthesis, with no local or systemic adverse effects. Clinical trials of engineered diaphragmatic repair appear practicable within regulatory guidelines.
Journal of Pediatric Surgery 01/2011; 46(1):57-61. · 1.45 Impact Factor
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ABSTRACT: Severe neonatal pulmonary hypoplasia incurs mortality rates approaching 71% to 95%. We sought to determine the utility of serial amnioinfusions through a subcutaneously implanted intraamniotic catheter to prevent pulmonary hypoplasia in fetal obstructive uropathy.
Fetal lambs (n = 32) were divided into 3 groups. Group I (n = 12) underwent a sham operation, group II (n = 15) underwent a complete urinary tract obstruction via ligation of the urachus and urethra with a subcutaneous tunneled intraamniotic port-a-cath without amnioinfusions, and group III (n = 5) underwent a creation of a complete urinary tract obstruction with a port-a-cath as described in group II with serial amnioinfusions. Lung tissue was analyzed by lung volume to body weight ratios and stereology. Statistical analysis was performed by analysis of variance and Bonferroni comparisons (P < .05).
Obstructed fetuses grossly had smaller lungs than treated and control animals. Lung volume to body weight ratios were statistically significant between groups. Airspace fractions were comparable between groups I and III (average = 0.53 and 0.55, respectively), although both were significantly greater than group II (average = 0.48) (P = .049).
Serial amnioinfusions through an intraamniotic port-a-cath prevented pulmonary hypoplasia in an ovine model of complete obstructive uropathy. The use of an easily accessible device for amnioinfusions may be a viable option to treat oligohydramnios.
Journal of Pediatric Surgery 01/2011; 46(1):67-71. · 1.45 Impact Factor
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ABSTRACT: The amniotic fluid and placenta are sources of diverse progenitor cell populations, including -mesenchymal, hematopoietic, trophoblastic, and possibly more primitive stem cells. Given that much of the amniotic cavity and placenta share a common origin, namely the inner cell mass of the morula, perhaps it is not surprising that most types of progenitor cells that can be isolated from these two sources also share many characteristics. This chapter focuses solely on the most abundant and easy to isolate progenitor cell population found therein, the mesenchymal stem cells (MSCs). Unlike some of the other stem cell types, MSCs are present throughout gestation. Methods of isolation, expansion, freezing, and thawing of these cells will be presented with preference given to the simplest methods available for any given procedure.
Methods in molecular biology (Clifton, N.J.) 01/2011; 698:75-88.
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ABSTRACT: Fetal wound healing involves minimal inflammation and limited scarring. Its mechanisms, which remain to be fully elucidated, hold valuable clues for wound healing modulation and the development of regenerative strategies. We sought to determine whether fetal wound healing includes a hitherto unrecognized cellular component. Two sets of fetal lambs underwent consecutive experiments at midgestation. First, fetuses received an intra-amniotic infusion of labeled autologous amniotic mesenchymal stem cells (aMSCs), in parallel to different surgical manipulations. Subsequently, fetuses underwent creation of 2 symmetrical, size-matched skin wounds, both encased by a titanium chamber. One of the chambers was left open and the other covered with a semipermeable membrane that allowed for passage of water and all molecules, but not any cells. Survivors from both experiments had their wounds analyzed at different time points before term. Labeled aMSCs were documented in all concurrent surgical wounds. Covered wounds showed a significantly slower healing rate than open wounds. Paired comparisons indicated significantly lower elastin levels in covered wounds at the mid time points, with no significant differences in collagen levels. No significant changes in hyaluronic acid levels were detected between the wound types. Immunohistochemistry for substance P was positive in both open and covered wounds. We conclude that fetal wound healing encompasses an autologous yet exogenous cellular component in naturally occurring aMSCs. Although seemingly not absolutely essential to the healing process, amniotic cells expedite wound closure and enhance its extracellular matrix profile. Further scrutiny into translational implications of this finding is warranted.
Stem cells and development 10/2010; 20(6):969-76. · 4.15 Impact Factor
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Grace A Nicksa,
Edward O'Neil,
David C Yu,
Adam S Curatolo,
Brendan L McNeish,
Carol E Barnewolt,
David Zurakowski,
Terry L Buchmiller,
Marsha A Moses,
Seymour Rosen, Dario O Fauza
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ABSTRACT: We aimed to determine whether the profile of matrix metalloproteinase (MMP) activity in fetal urine correlates with the degree of kidney damage in the setting of congenital obstructive uropathy.
Fetal lambs underwent either a sham operation or creation of a complete urinary tract obstruction. Necropsies were performed before term, when urinary MMP profiling was performed by zymography; and kidney damage was assessed histologically by multiple semiquantitative analyses and histomorphometric measurements.
There was a significant correlation between inner medullary thickness and MMP-9 (P = .005) and 63-kd MMP-2 (P = .019) activities. In like manner, the only MMPs associated with kidney fibrosis were MMP-9 and 63-kd MMP-2. Matrix metalloproteinase-9 activity was a highly significant independent predictor of the total combined kidney fibrosis score (P < .001) as well as of higher fibrosis grades in each of 6 kidney areas analyzed (all with P < .01). The activity of 63-kd MMP-2 correlated significantly with higher fibrosis in select areas.
In a fetal ovine model, urinary MMP activity correlates with the degree of kidney damage. The presence of MMP-9 (in particular) and that of 63-kd MMP-2 are independent predictors of severity. Prenatal urinary MMP profiling may enhance patient stratification and counseling in the setting of congenital obstructive uropathy.
Journal of Pediatric Surgery 06/2010; 45(6):1120-5. · 1.45 Impact Factor
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ABSTRACT: We sought to compare the efficacy of engineered fetal bone grafts with acellular constructs in an autologous model of chest wall repair.
Rabbits (n = 10) with a full-thickness sternal defect were equally divided in 2 groups based on how the defect was repaired, namely, either with an autologous bone construct engineered with amniotic mesenchymal stem cells on a nanofibrous scaffold or a size-matched identical scaffold with no cells. Animals were killed at comparable time-points 18 to 20 weeks postimplantation for multiple analyses.
Gross evidence of nonunion confirmed by micro-computed tomography scanning was present in 3 (60%) of 5 of the acellular implants but in no engineered grafts. Histology confirmed the presence of bone in both types of repair, albeit seemingly less robust in the acellular grafts. Mineral density in vivo was significantly higher in engineered grafts than in acellular ones, with more variability among the latter. There was no difference in alkaline phosphatase activity between the groups.
Chest wall repair with an autologous osseous graft engineered with amniotic mesenchymal stem cells leads to improved and more consistent outcomes in the midterm when compared with an equivalent acellular prosthetic repair in a leporine model. Amniotic fluid-derived engineered bone may become a practical alternative for perinatal chest wall reconstruction.
Journal of Pediatric Surgery 06/2010; 45(6):1354-60. · 1.45 Impact Factor
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ABSTRACT: Treatment of congenital tracheal stenosis/atresia remains essentially unresolved. Previous models of this disease entity have been restricted to rodents and the chick. We sought to establish the principles of a large, surgical animal model of this spectrum of fetal anomalies.
Fetal lambs (n = 8) underwent open surgery at 90-112 days gestation. Their cervical tracheas were encircled by a biocompatible polytetrafluoroethylene wrap, so as to extrinsically restrict their external diameter by 25%. Survivors (n = 7) were killed at different time points post-operatively before term. The manipulated tracheal segments were compared with their respective proximal portions (controls). Analyses included morphometry, histology and quantitative extracellular matrix measurements.
At necropsy, the typical gross appearance of tracheal stenosis/atresia was present in all manipulated tracheal segments. Histological findings included the virtual disappearance of the membranous portion of the trachea, along with infolding, fragmentation, and/or posterior fusion of cartilaginous rings, often with disappearance of the airway mucosa. There were significant decreases in diameter (P < 0.001) and total collagen levels (P = 0.005) on the manipulated trachea compared with the control portions. No significant differences were observed in overall elastin or glycosaminoglycan contents. A significant time-dependent increase in elastin was noted on the control, but not the experimental side.
In a surgical ovine model, controlled extrinsic compression of the fetal trachea leads to morphological and biochemical findings compatible with the congenital tracheal stenosis/atresia spectrum. This simple and easily reproducible prenatal model can be instrumental in the development of emerging therapies for these congenital anomalies.
Journal of Surgical Research 03/2010; 171(1):164-9. · 2.25 Impact Factor
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ABSTRACT: The diagnostic evaluation, patient stratification, and prenatal counseling for congenital obstructive uropathy remain sub-optimal. Matrix metalloproteinase (MMP) expression profiles are emerging as a valuable diagnostic tool in assorted disease processes. We sought to determine whether congenital obstructive uropathy impacts MMP expression in fetal urine.
Fetal lambs (n = 25) were divided in two groups: group I (n = 12) underwent a sham operation and group II (n = 13) underwent creation of a complete urinary tract obstruction. Gelatin zymography panels for 4 MMP species were performed on fetal urine in both groups at comparable times post-operatively. Statistical analysis was by the Fisher's exact test (P < .05).
Overall fetal survival was 80% (20/25). A variety of significant differences in MMP expression between the two groups were identified. The following profiles were present only in obstructed animals: any MMP other than MMP-2 (P = .029), including any MMP other than 63 kDa and 65 kDa (P = .009); 2 or more MMPs excluding MMP-2s (0.029); and 3 or more MMPs (P = .029).
Limited matrix metalloproteinase expression is present in the urine of normal ovine fetuses. Fetal obstructive uropathy impacts urinary MMP expression in various distinguishable patterns. Prenatal urinary MMP profiling may become a practical and valuable diagnostic tool in the evaluation of congenital obstructive uropathy.
Journal of Pediatric Surgery 01/2010; 45(1):70-3. · 1.45 Impact Factor
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ABSTRACT: Videofetoscopy typically demands the substitution of oft-turbid amniotic fluid with clear crystalloid. This maneuver can be cumbersome and may lead to complications. We sought to determine the optical properties of the amniotic fluid, as a pre-requisite for optimizing video image processing during videofetoscopy and eventually avoid amniotic fluid replacement.
Human amniotic fluid samples (n = 21) were procured at 19-36 weeks of gestation. Optical refraction and reflection indices were recorded as percentages of light transmission through the fluid using an integrated spectrometer covering wavelengths of 400-950 nm, with 1.0 nm resolution. Statistical analysis was by one-way ANOVA (p < 0.05).
Peak optical refraction fell within a relatively limited window of the near-infrared spectrum, at 848.1 +/- 52.3 nm, regardless of gestational age or overall light absorbance. Within the visible spectrum, transmission was highest at the highest wavelengths. A statistically significant inverse relationship existed between gestational age and overall light transmission. Light reflection was negligible in all samples.
Light transmission through amniotic fluid is optimal in the near-infrared spectrum and at the highest visible wavelengths, regardless of gestational age. Overall light transmission through amniotic fluid decreases throughout gestation. The light source and camera of videofetoscopy systems should be designed accordingly, possibly obviating the need for routine intraoperative amniotic fluid exchange.
Fetal Diagnosis and Therapy 11/2009; 27(2):87-90. · 1.05 Impact Factor
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ABSTRACT: We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be used in postnatal sternal repair.
Leporine aMSCs were isolated, identified, transfected with green fluorescent protein (GFP), expanded, and seeded onto biodegradable electrospun nanofibrous scaffolds (n = 6). Constructs were dynamically maintained in an osteogenic medium and equally divided into 2 groups with respect to time in vitro as follows: 14.6 or 33.9 weeks. They were then used to repair full-thickness sternal defects spanning 2 to 3 intercostal spaces in allogeneic kits (n = 6). Grafts were submitted to multiple analyses 2 months thereafter.
Chest roentgenograms showed defect closure in all animals, confirmed at necropsy. Graft density as assessed by microcomputed tomographic scans increased significantly in vivo, yet there were no differences in mineralization by extracellular calcium measurements preimplantation and postimplantation. There was a borderline increase in alkaline phosphatase activity in vivo, suggesting ongoing graft remodeling. Histologically, implants contained GFP-positive cells and few mononuclear infiltrates. There were no differences between the 2 construct groups in any comparison.
Engineered osseous grafts derived from amniotic mesenchymal stem cells may become a viable alternative for sternal repair. The amniotic fluid can be a practical cell source for engineered chest wall reconstruction.
Journal of Pediatric Surgery 07/2009; 44(6):1120-6; discussion 1126. · 1.45 Impact Factor
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ABSTRACT: Attempts at harnessing the prospective benefits of the therapeutic use of fetal cells or tissues date many decades before the modern era of transplantation. The first reported transplantation of human fetal tissue took place in 1922. Fetal cells or tissues also have been used as helpful investigational tools since the 1930s. Still, it was only in the last three decades that fetal tissue transplantation in people has started to lead to favorable outcomes, yet by and large anecdotally. This article offers an outlook on a relatively new dimension in fetal cell-based therapies, namely the engineering of tissues in the laboratory, along with its prospective applications.
Clinics in perinatology 07/2009; 36(2):473-88, xii. · 1.54 Impact Factor
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ABSTRACT: We aimed to identify risk factors for neonatal surgical airway intervention among fetuses with prenatally diagnosed cervical masses.
An 8-year retrospective review identified 23 consecutive patients with a prenatal diagnosis of a neck mass, managed at a single tertiary center. Variables analyzed included anticipated diagnosis, extent of the mass, need for any surgical airway intervention in the neonatal period, final histopathology data, and survival. Statistical analysis was based on the Fisher and Fisher-Freeman-Halton exact tests (significance set at P < or = .05) and exact 95% confidence intervals for risk differences.
Eight patients underwent termination of pregnancy or were lost to follow-up. The imaging-based prenatal diagnosis was confirmed postnatally in 93% (14/15) of the remaining patients. Final diagnoses included lymphatic malformation (8), teratoma (6), and esophageal duplication (1). Teratomas were associated with a significantly higher risk for neonatal airway intervention than lymphatic malformations (67% vs 11%, P = .02). The majority of such procedures were performed under ex utero intrapartum treatment. Survival was 93% (14/15).
Cervical teratomas are significantly more likely to demand surgical airway intervention in the neonate, typically under ex utero intrapartum treatment, than cervical lymphatic malformations. These findings should be considered in the prenatal counseling for fetal cervical masses.
Journal of Pediatric Surgery 01/2009; 44(1):76-9. · 1.45 Impact Factor
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ABSTRACT: Serial transverse enteroplasty (STEP) has been shown to improve bowel function in short bowel syndrome. The effect of the STEP procedure on intestinal motility is not known, but some have hypothesized that it could disrupt bowel innervation and thus impair intestinal motility.
Growing Yorkshire pigs (n = 7) underwent 3 operations at 6-week intervals: (1) reversal of 50 cm of jejunum, (2) 90% bowel resection +/- STEP to the proximal dilated bowel (4 STEP, 3 control), and (3) implantation of serosal strain gauges. At each operation, baseline and post-octreotide small intestinal motility was studied with continuously perfused manometry catheters using non-anticholinergic anesthesia. In addition, awake monitoring was performed using strain gauge analysis 1 week after the third operation. Characteristics of phase III of the migrating motor complex (MMC) were compared between and within groups using t test, chi(2), and analysis of variance, with significance set at P < .05.
Manometry data from the third surgery revealed no differences between groups or compared with baseline within groups for the presence and characteristics of phase III of the MMC. Specifically, the mean amplitude and frequency of phase III after octreotide, and both the mean baseline and mean octreotide-stimulated motility indices were equivalent. The duration of phase III after octreotide stimulation was significantly increased in the STEP animals, suggesting a potential benefit of the STEP procedure. Strain gauge analysis, performed in awake animals, confirmed no differences between the groups for basal and octreotide-stimulated characteristics of phase III of the MMC.
These preliminary data suggest that the STEP procedure in a porcine model of short bowel syndrome does not interfere with baseline or hormonally stimulated motility within the small bowel. These findings further support the STEP procedure as a safe option for the surgical management of short bowel syndrome.
Journal of Pediatric Surgery 01/2009; 44(1):229-35; discussion 235. · 1.45 Impact Factor
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ABSTRACT: We introduce the notion of prenatal neural stem cell (NSC) delivery to the spinal cord as an adjuvant to fetal repair of spina bifida.
Fetal lambs with experimental myelomeningocele (MMC; n = 25) were divided in 3 groups: group I, no repair; group II, standard surgical MMC coverage; and group III, MMC coverage plus delivery of a murine NSCs clone into the spinal cord defect. Donor cells constitutively expressed lacZ encoding for Escherichia coli beta-galactosidase, yet they were further labeled by exposure to either BrdU and/or to the fluorescent membrane dye PKH-26. Blinded initial clinical evaluations and multiple spinal cord analyses were undertaken soon after birth.
Both survival and the incidence of major paraparesis were significantly worse in group I compared with groups II and III. In group III, NSC density was highest within the most damaged areas of the spinal cord, with selective engraftment within those regions. Donor NSCs retained an undifferentiated state in vivo, producing neurotrophic factors within the defect. No animals in group III had a worsened condition following this intervention.
Neural stem cells retain an undifferentiated state and produce neurotrophic factors in the short term after delivery to the fetal spinal cord, in the setting of experimental MMC. Further scrutiny of NSC delivery to the spinal cord as a therapeutic strategy against spina bifida is warranted.
Surgery 10/2008; 144(3):367-73. · 3.10 Impact Factor
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ABSTRACT: Because of the 4 to 6-month interval between a diagnostic amniocentesis and birth, clinical application of amniotic mesenchymal stem cell (AMSC)-based therapies demands a 3-stage cell manufacturing process, including isolation/primary expansion, cryopreservation, and thawing/secondary expansion. We sought to determine the feasibility and cell yield of such a staged cell manufacturing process, within regulatory guidelines.
Human AMSCs isolated from diagnostic amniocentesis samples (n = 11) were processed under Food and Drug Administration-accredited good manufacturing practice. Expanded cells were characterized by flow cytometry and cryopreserved for 3 to 5 months. Cell release criteria included more than 90% CD29+, CD73+, and CD44+; less than 5% CD34+ and CD45+; negative mycoplasma quantitative polymerase chain reaction (QPCR) and endotoxin assay; and at least 70% viability.
Isolation and ample expansion of AMSCs was achieved in 54.5% (6/11) of the samples. Early processing and at least a 2-mL sample were necessary for reliable cell manufacturing. Cell yield before cryopreservation was 223.2 +/- 65.4 x 10(6) cells (44.6-fold expansion), plus a 14.7 x 10(6)-cell backup, after 36.3 +/- 7.8 days. Cell viability postthaw was 88%. Expanded cells maintained a multipotent mesenchymal progenitor profile.
Human amniotic mesenchymal stem cells can be manufactured in large numbers from diagnostic amniocentesis, by an accredited staged processing, under definite procurement guidelines. These data further support the viability of clinical trials of amniotic mesenchymal stem cell-based therapies.
Journal of Pediatric Surgery 07/2008; 43(6):1164-9. · 1.45 Impact Factor
