Shaun A. Steigman

Sternal repair with bone grafts engineered from amniotic mesenchymal stem cells

PURPOSE We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be used in postnatal sternal repair. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Preclinical regulatory validation of a 3-stage amniotic mesenchymal stem cell manufacturing protocol

PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Preclinical regulatory validation of an engineered diaphragmatic tendon made with amniotic mesenchymal stem cells

PURPOSE 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. METHODS 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). RESULTS 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. CONCLUSIONS 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.

Amniotic Mesenchymal Stem Cells Enhance Normal Fetal Wound Healing

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.

Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta

Diverse progenitor cell populations, including mesenchymal, hematopoietic, trophoblastic, and possibly more primitive stem cells can be isolated from the amniotic fluid and the placenta. At least some of the amniotic and placental cells share a common origin, namely the inner cell mass of the morula. Indeed, most types of progenitor cells that can be isolated from these two sources share many characteristics. This unit will focus solely on the mesenchymal stem cells, the most abundant progenitor cell population found therein and, unlike some of the other stem cell types, present all through gestation. Protocols for isolation, expansion, freezing, and thawing of these cells are presented. Preference is given to the simplest methods available for any given procedure.

Use of White Blood Cell Count and Negative Appendectomy Rate

BACKGROUND: Despite increased utilization of laboratory, radiologic imaging, and scoring systems, negative appendectomy (NA) rates in children remain above 3% nationwide. We reviewed the clinical data of patients undergoing appendectomy to further reduce our NA rate. METHODS: A retrospective review was conducted of all appendectomies performed for suspected appendicitis at a tertiary children’s hospital during a 42-month period. Preoperative clinical, laboratory, and radiographic data were collected. Variables absent or normal in more than half of NAs were further analyzed. Receiver operating characteristic curves were constructed for continuous variables by using appropriate cutoff points to determine sensitivity and false-positive rates. The results were validated by analyzing the 12 months immediately after the establishment of these rules. RESULTS: Of 847 appendectomies performed, 22 (2.6%) had a pathologically normal appendix. The only variables found to be normal in more than half of NAs were white blood cell (WBC) count (89%) and neutrophil count (79%). A receiver operating characteristic curve indicates that using WBC cutoffs of 9000 and 8000 per µL yielded sensitivities of 92% and 95%, respectively, and reduction in NA rates by 77% and 36%, respectively. Results observed in the subsequent 12 months confirmed these expected sensitivities and specificities. CONCLUSIONS: Absence of an elevated WBC count is a risk factor for NA. Withholding appendectomy for WBC counts <9000 and 8000 per µL reduces the NA rate to 0.6% and 1.2%, respectively. Missed true appendicitis in patients with normal WBC counts can be mitigated by a trial of observation in those presenting with early symptom onset.

Chest wall repair with engineered fetal bone grafts: an efficacy analysis in an autologous leporine model.

PURPOSE We sought to compare the efficacy of engineered fetal bone grafts with acellular constructs in an autologous model of chest wall repair. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Differential risk for neonatal surgical airway intervention in prenatally diagnosed neck masses

PURPOSE We aimed to identify risk factors for neonatal surgical airway intervention among fetuses with prenatally diagnosed cervical masses. METHODS 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. RESULTS 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). CONCLUSIONS 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.

Structural and biomechanical characteristics of the diaphragmatic tendon in infancy and childhood: an initial analysis

BACKGROUND Engineered tendon grafts have been shown, experimentally, to be promising alternatives for partial diaphragmatic replacement. This study was aimed at determining the cellularity, extracellular matrix composition, and biomechanical characteristics of the diaphragmatic tendon in infants and children to be used as a reference for proper diaphragmatic graft engineering. METHODS The left diaphragmatic tendon was procured at autopsy from 13 patients divided into 2 groups. Group I (n = 9) consisted of newborns and infants. Group II (n = 4) consisted of children and adolescents. Samples underwent quantitative assays for total DNA, glycosaminoglycans, collagen, and elastin contents. Biomechanical measurements included modular and ultimate tensile strength analyses. Statistical comparisons were by the 2-sample Students t test. RESULTS Group I showed significantly higher levels of total DNA, glycosaminoglycans, collagen, and elastin than group II. Conversely, group II tended to have higher modular and ultimate tensile strengths. CONCLUSIONS In neonates and infants, the diaphragmatic tendon has increased cell density and higher levels of major extracellular matrix components than in older children, in whom the diaphragmatic tendon tends to have higher tensile strength. Engineered diaphragmatic constructs should be tailored to the distinct anatomical, functional, and biomechanical characteristics of the diaphragmatic tendon at different age groups.

Optical Properties of Human Amniotic Fluid: Implications for Videofetoscopic Surgery

Objective: 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. Methods: 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). Results: 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. Conclusions: 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.