A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid.

BACKGROUND/PURPOSE: Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases. METHODS: Exosomes were isolated by reagent-enhanced centrifugation from cell culture media of banked human bone marrow (bm) and amniotic fluid (af) MSCs after serum starvation. Characterization was by flow exometry for tetraspanin markers CD9, CD63, and CD81, transmission electron microscopy for size and morphology, and tunable resistive pulse sensing for size distribution and concentration. Statistical comparisons of count data were made by Poisson regression modeling and Student's T-test. RESULTS: Exosomes of appropriate size and morphology were isolated with comparable expressions of CD9 (96% vs. 94%), CD63 (88% vs. 66%), and CD81 (71% vs. 63%) for bmMSC and afMSC, respectively. Total exosome yield (particles/mL) adjusted for number of cells was higher from afMSCs than bmMSCs by an estimated 25% (P < 0.001). CONCLUSIONS: While bone marrow and amniotic fluid mesenchymal stem cells are comparable sources of exosomes in size distribution, morphology, and expression of typical surface markers, yield may be higher from amniotic fluid cells. The amniotic fluid appears to be a preferable source of exosomes for clinical applications. LEVEL OF EVIDENCE: N/A (bench laboratory study).

Transamniotic stem cell therapy (TRASCET) in a rabbit model of spina bifida.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with select mesenchymal stem cells (MSCs) has been shown to induce partial or complete skin coverage of spina bifida in rodents. Clinical translation of this emerging therapy hinges on its efficacy in larger animal models. We sought to study TRASCET in a model requiring intra-amniotic injections 60 times larger than those performed in the rat. METHODS: Rabbit fetuses (n = 65) with surgically created spina bifida were divided into three groups. One group (untreated) had no further manipulations. Two groups received volume-matched intra-amniotic injections of either saline or a concentrated suspension of amniotic fluid MSCs (afMSCs) at the time of operation. Infused afMSCs consisted of banked heterologous rabbit afMSCs with mesenchymal identity confirmed by flow cytometry, labeled with green fluorescent protein. Defect coverage at term was blindly categorized only if the presence of a distinctive neoskin was confirmed histologically. Statistical comparisons were by logistic regression and the likelihood ratio test. RESULTS: Among survivors with spina bifida (n = 19), there were statistically significant higher rates of defect coverage (all partial) in the afMSC group when compared with the saline and untreated groups (0-50%; p = 0.022-0.036), with no difference between the saline and untreated groups (p = 1.00). Donor afMSCs were identified locally, though sparsely and not in the neoskin. CONCLUSIONS: Concentrated intra-amniotic injection of amniotic mesenchymal stem cells can induce partial coverage of experimental spina bifida in a leporine model. Transamniotic stem cell therapy may become a feasible strategy in the prenatal management of spina bifida. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Donor mesenchymal stem cell linetics after transamniotic stem cell therapy (TRASCET) for experimental spina bifida.

PURPOSE: We sought to examine donor mesenchymal stem cell (MSC) kinetics after transamniotic stem cell therapy (TRASCET) in experimental spina bifida. METHODS: Pregnant Sprague-Dawley dams exposed to retinoic acid for the induction of fetal neural tube defects received volume-matched intra-amniotic injections on gestational day 17 (E17; term=E22): either amniotic fluid MSCs (afMSCs) labeled with a luciferase reporter gene (n=78), or luciferase protein alone (n=66). Samples from twelve organ systems from each surviving fetus with spina bifida (total n=60) were screened via microplate luminometry at term. RESULTS: Donor afMSCs were identified exclusively in the placenta, umbilical cord, spleen, bone marrow, hip bones, defect, and brain. Luminometry was negative in control fetuses receiving luciferase alone (p<0.001). Signal intensity in relative light units (RLUs) was moderately correlated between the defect and the hip bones (rho=0.38, p=0.048), and between the placenta and the brain (rho=0.40, p=0.038). CONCLUSIONS: Amniotic mesenchymal stem cells engraft to specific sites after concentrated intra-amniotic injection in the setting of spina bifida. A hematogenous route encompassing the bone marrow as well as distant central nervous system homing are fundamental constituents of cell trafficking. These findings must be considered during eventual patient selection for transamniotic stem cell therapy in the prenatal management of spina bifida.

Fetal bone marrow homing of donor mesenchymal stem cells after transamniotic stem cell therapy (TRASCET).

PURPOSE: Donor cell engraftment patterns following transamniotic stem cell therapy (TRASCET) with amniotic fluid mesenchymal stem cells (afMSCs) are incompatible with solely direct amniotic seeding. We sought to determine whether fetal bone marrow is a component of such engraftment and to examine the chronology of afMSC placental trafficking. METHODS: Two groups of Sprague-Dawley rat fetuses received volume-matched intraamniotic injections on gestational day 17 (E17; term E22): either afMSCs labeled with a luciferase reporter gene or luciferase protein alone. Placental samples were procured at daily time points thereafter until term. Fetal bone marrow was obtained at term only owing to size constraints. Specimens were screened for luminescence via microplate luminometry. RESULTS: Donor afMSCs were identified in the bone marrow and placenta of fetuses receiving labeled afMSCs, but not in those receiving luciferase alone (P<0.001). Luminescence was significantly higher in placentas at E18 compared to E19 (P<0.001), E20 (P=0.007), and E21 (P=0.004), with no difference with E22/term (P=0.97). CONCLUSIONS: Donor mesenchymal stem cells home to the fetal bone marrow after intraamniotic injection. The chronology of placental trafficking is suggestive of controlled cell routing rather than plain cell clearance. Fetal bone marrow engraftment of donor cells significantly expands potential applications of transamniotic stem cell therapy.

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.

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 3-8 weeks post-implantation. Statistical analysis was by the Student t-test and Fisher's exact test (P < 0.05). 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.

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.

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. © 2014 S. Karger AG, Basel.

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.

A comparative analysis of human mesenchymal stem cell response to hypoxia in vitro: Implications to translational strategies.

PURPOSE: Mesenchymal stem cells (MSCs) are particularly valuable for structural tissue replacement. We compared the response to hypoxia among human MSCs derived from four different clinically relevant sources as an adjunct to translational developments. METHODS: Immunophenotypically indistinguishable human MSC lineages derived from bone marrow (bmMSCs), adipose tissue (adMSCs), amniotic fluid (afMSCs), and umbilical cord blood (cbMSCs) were submitted to either room air or 1% O2, under otherwise standard culture conditions. Cell expansion and quantitative RT-PCR data were obtained at different time points. Statistical analysis was by two-way mixed model and the F-test (P<0.05). RESULTS: The effect of hypoxia on expansion kinetics was dependent on cell source. Only prenatal sources of MSCs - afMSCs (P=0.002) and cbMSCs (P<0.001) - proliferated significantly faster under hypoxia than normoxia. Increased HIF1-alpha expression correlated consistently with increased cell expansion only among afMSCs. There were no significant variabilities in Survivin, Oct-4, and VEGF expressions. CONCLUSIONS: Mesenchymal stem cell tolerance to hypoxia in vitro varies with cell source. Prenatal cells, particularly those derived from amniotic fluid, are more robust than their postnatal counterparts. HIF1-alpha may play a role in the amniotic fluid-derived cells' enhanced response. These findings should inform the choice of mesenchymal stem cells for prospective regenerative strategies.

Targeted quantitative amniotic cell profiling: a potential diagnostic tool in the prenatal management of neural tube defects.

PURPOSE: We sought to determine whether amniotic cell profiles correlate quantitatively with neural tube defect (NTD) type and/or size. METHODS: Sprague-Dawley fetuses exposed to retinoic acid (n=61) underwent amniotic fluid sample procurement before term. 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 included ANOVA and post-hoc Bonferroni adjusted comparisons (P<0.05). RESULTS: There was a statistically significant increase in the proportion of aNSCs in fetuses with spina bifida (6.78%± 1.87%) when compared to those with exencephaly (0.64%± 0.23%) or with both spina bifida and exencephaly (0.22%± 0.09%). Conversely, there was a statistically significant decrease in the proportion of aMSCs in fetuses with exencephaly, either isolated (1.09%± 0.42%) or in combination defects (2.37%± 0.63%) when compared with normal fetuses (8.83%± 1.38%). In fetuses with isolated exencephaly, there was a statistically significant inverse correlation between the proportion of aNSCs and defect size. CONCLUSIONS: The proportions of neural and mesenchymal stem cells in the amniotic fluid correlate with the type and size of experimental NTDs. Targeted quantitative amniotic cell profiling may become a useful diagnostic tool in the prenatal evaluation of these anomalies.

Intra-amniotic delivery of amniotic-derived neural stem cells in a syngeneic model of spina bifida.

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.

The amniotic fluid as a source of neural stem cells in the setting of experimental neural tube defects.

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, 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, for example, NSCs in the setting of experimental NTDs. The amniotic fluid may be a practical source of autologous NSCs applicable to novel forms of therapies for spina bifida.

Development of students' critical thinking: the educators' ability to use questioning skills in the baccalaureate programmes in nursing in Pakistan.

OBJECTIVE: To enhance the Critical Thinking skills of educators associated with the nursing baccalaureate programmes in Pakistan. By focusing on the type and level of questions asked by the educators. METHODS: Ninety-one faculty members from 14 out of 17 schools participated in the study. Data on the faculty's questioning skills was obtained through classroom observations and field notes. The duration of the observations was 45-60 minutes. Using Bloom's Taxonomy for cognitive thinking, questions were categorised into high and low categories. RESULTS: Most of the questions (68.9 %) asked by the participants were of lower levels, while some (5.37 %) were ambiguous. In many instances, the participants did not allow a sufficient wait-time for students to think and respond. CONCLUSION: The findings suggest that educators must learn to use the questioning strategy effectively. They should ask higher level questions if they wish to inculcate Critical Thinking in students.

Prenatal tracheal reconstruction with a hybrid amniotic mesenchymal stem cells-engineered construct derived from decellularized airway.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Craniofacial repair with fetal bone grafts engineered from amniotic mesenchymal stem cells.

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.

A large animal model of the fetal tracheal stenosis/atresia spectrum.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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.

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.