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 GFPpositive 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. PaPer

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.

The ethically justifiable indications of fetal tissue engineering as a strategy for 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. Amniotic mesenchymal stem cell (aMSC)-engineered bone has been shown experimentally to be a viable alternative for craniofacial repair. In this study, we sought to compare aMSC-derived bone grafts engineered from two fundamentally distinct biodegradable matrices in a leporine model of craniofacial reconstruction, as a necessary step towards preclinical optimization of this therapeutic concept.

PURPOSE:The primary purpose of this study was to investigate the relationship between Pediatric Ulcerative Colitis Activity Index (PUCAI) and operative management. We also specifically evaluated those patients receiving tacrolimus for their disease.METHODS:A retrospective review (1/06-1/11) identified ulcerative colitis patients (≤21 years old) undergoing restorative proctocolectomy with rectal mucosectomy and ileal pouch-anal anastomosis. Main outcomes included pre-operative PUCAI, combined versus staged procedure, and postoperative complications. Patients receiving tacrolimus within 3 months of surgical intervention were identified. PUCAI at tacrolimus induction and medication side effects were also noted.RESULTS:Sixty patients were identified. Forty-two (70%) underwent combined and 18 (30%) had staged procedures. Pre-operative PUCAI was lower for combined versus staged patients (p = < 0.001). Furthermore, a higher pre-operative PUCAI strongly correlated with the likelihood of undergoing a staged procedure (p < 0.001). Forty-four patients (73%) received tacrolimus. Significant improvement in their PUCAI was noted from induction to pre-operative evaluation (p < 0.001). Minor and reversible side effects occurred in 46% of patients receiving tacrolimus, but complication rates were not significantly different.CONCLUSIONS:There is a very strong correlation between the PUCAI and the likelihood of undergoing a staged procedure. A significant improvement in PUCAI occurs following preoperative tacrolimus therapy.

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.

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. (C) 2013 Elsevier Inc. All rights reserved.

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. (C) 2012 Elsevier Inc. All rights reserved.

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. (C) 2012 Elsevier Inc. All rights reserved.

Purpose: The purpose of this study was to evaluate recurrence and survival outcomes in pediatric adrenal cortical neoplasms.Methods: A 90-year retrospective review of children with adrenal cortical neoplasms was performed using multivariate Cox regression analysis to identify factors associated with recurrence and tumor-related mortality.Results: The evaluable cohort included 29 patients. Twenty-seven underwent resection. Twenty-two (81%) had localized disease, and 5 (19%) had locally advanced disease (all received chemotherapy and 2 of 5 were cured). Two patients presenting with metastatic disease died despite treatment. There were 4 recurrences; all patients died. Tumor-related mortality was 24% (7/29). Kaplan-Meier freedom from recurrence was 85% at 1 year (95% confidence interval, 75%-95%). Multivariate Cox regression revealed that older age (P = .01), higher mitotic rate (P = .005), and necrosis (P < .001) were independent predictors of tumor-related death. Higher mitotic rate (P = .007) and larger tumor size (P = .03) were significant predictors of tumor recurrence.Conclusion: Risk factors for poor outcomes in patients with adrenocortical tumors include older age, higher mitotic rate, higher percent necrosis, and larger tumor size. Therefore, the presence of these factors may warrant consideration of adjuvant chemotherapy, even in the absence of advanced disease. (C) 2011 Elsevier Inc. All rights reserved.

Tracheomalacia (TM) refers to a softening of the cartilaginous tracheal rings. The weakened cartilage allows a narrowing in the anteroposterior tracheal diameter. Its incidence is estimated to be 1 in 1445 infants. Both congenital and acquired forms have been identified. Congenital TM is most commonly associated with tracheoesophageal fistula/esophageal atresia in children.1Blair G.K. Cohen R. Filler R.M. Treatment of tracheomalacia: eight years' experience.J Pediatr Surg. 1986; 21: 781-785Abstract Full Text PDF PubMed Scopus (102) Google Scholar The effected tracheal rings have been found to contain a shortened segment of cartilage with a compensatory lengthening of the membranous portion.2Wailoo M.P. Emery J.L. The trachea in children with tracheo-oesophageal fistula.Histopathology. 1979; 3: 329-338Crossref PubMed Scopus (136) Google Scholar This is thought to be caused by abnormal embryonic separation of the trachea from the esophagus, leaving the trachea with too much tissue.1Blair G.K. Cohen R. Filler R.M. Treatment of tracheomalacia: eight years' experience.J Pediatr Surg. 1986; 21: 781-785Abstract Full Text PDF PubMed Scopus (102) Google Scholar Primary TM has also been associated with immature cartilage in premature infants, primary cartilage disorders such as polychondritis and chondromalacia, and several syndromes including the mucopolysaccharidoses and Down syndrome. The acquired or secondary forms of TM are more common than the primary forms and result from prolonged endotracheal intubation or tracheostomy. Secondary TM can also be the result of external compression from surrounding cardiovascular anomalies, skeletal disorders such as scoliosis, and space-occupying tumors or cysts.3Carden K.A. Boiselle P.M. Waltz D.A. et al.Tracheomalacia and tracheobronchomalacia in children and adults: an in-depth review.Chest. 2005; 127: 984-1005Crossref PubMed Scopus (500) Google Scholar The weakened trachea is subsequently susceptible to collapse with increased intrathoracic pressure, as in expiration, coughing, and crying. It is also vulnerable to external pressures created by food boluses, dilations proximal to esophageal strictures following tracheoesophageal fistula/esophageal atresia repair, and regurgitation due to gastroesophageal reflux disease. Patients present with noisy breathing, barking or “TOF” (tracheo-oesphageal fistula) cough, recurrent respiratory infections, feeding difficulties, and apneic spells.1Blair G.K. Cohen R. Filler R.M. Treatment of tracheomalacia: eight years' experience.J Pediatr Surg. 1986; 21: 781-785Abstract Full Text PDF PubMed Scopus (102) Google Scholar Mild to moderate cases generally resolve as the child grows and the cartilage strengthens. Severe cases, however, can have a mortality rate as high as 80%.3Carden K.A. Boiselle P.M. Waltz D.A. et al.Tracheomalacia and tracheobronchomalacia in children and adults: an in-depth review.Chest. 2005; 127: 984-1005Crossref PubMed Scopus (500) Google Scholar Treatment of TM can involve symptomatic medical management, tracheal stents, which have little reported success,4Valerie E.P. Durrant A.C. Forte V. et al.A decade of using intraluminal tracheal/bronchial stents in the management of tracheomalacia and/or bronchomalacia: is it better than aortopexy?.J Pediatr Surg. 2005; 40 ([discussion 907]): 904-907Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar and aortopexy. Aortopexy is currently considered the best option for the most severe cases and can be performed from a right, left, or anterior approach, using an open or thoracoscopic technique. Several criteria must be met before consideration for surgical repair. Once tracheomalacia is suspected as the cause of a child's respiratory distress, an awake direct laryngobronchoscopy (DLB) should be performed for definitive diagnosis. If TM is identified and it is intrathoracic, a computed tomographic angiogram can evaluate the relationship of the vasculature to the affected area. A dynamic computed tomography can help further isolate the specific region of the trachea that collapses as well as any surrounding structures that may aid in surgical planning.5Lee E.Y. Tracy D.A. Bastos M. et al.Expiratory volumetric MDCT evaluation of air trapping in pediatric patients with and without tracheomalacia.AJR Am J Roentgenol. 2010; 194: 1210-1215Crossref PubMed Scopus (22) Google Scholar Pre- and postoperative echocardiography evaluates the relationships and vascular integrity of the aorta and pulmonary arteries. If the workup reveals the aorta lying over the affected trachea, surgical intervention is warranted. Decisions regarding an open versus thoracoscopic approach, as well as where to enter the chest, will depend on the child's previous surgical history and imaging. Although we initially performed the thoracoscopic procedures via a left-sided approach,6Perger L. Kim H.B. Jaksic T. et al.Thoracoscopic aortopexy for treatment of tracheomalacia in infants and children.J Laparoendosc Adv Surg Tech A. 2009; 19: S249-S254Crossref PubMed Scopus (29) Google Scholar we now prefer a right-sided approach to reduce possible complications from working near the pulmonary artery. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12, Figure 13Figure 2Tracheomalacia with (inset) tracheal collapse seen on bronchoscopy. Here, with TM, the weakened cartilaginous rings are collapsing under the great vessels. On bronchoscopy, the tracheal lumen is narrowed with expiration, revealing the obstruction underlying the symptomatology.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Patient positioning. The patient is positioned supine with the right side elevated to 20°-30°, to adequately expose the right anterolateral chest. The right arm is secured away from the operative field.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Bronchial blocker. Using a bronchial blocker inflated inside the right main stem bronchus, the right lung is deflated to create more exposure and working space within the right thoracic cavity. ET = endotracheal; RMSB = right main stem bronchus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Trocar placement. Three- and 4-mm trocars are placed in the fourth and fifth mid-axillary and the sixth anterior axillary intercostal spaces. A 4-mm trocar is placed in the fifth intercostal space for the camera and 3-mm trocars are placed in the fourth and sixth intercostals spaces for instrumentation. Placement of these trocars should be such that they are aiming toward the manubrium. Adjustments to trocar location should be made as needed depending on the patient's anatomy. Insufflation pressures are set between 0 and 7 mm Hg.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Initial view. On entering the right chest, the view consists mainly of the thymus, the heart, and the great vessels. The sternum can be seen partially obstructed by the large thymus. The phrenic nerve runs posteriorly along the pericardium; it should be identified and carefully preserved throughout the case. n. = nerve.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Thymus. The thymus is dissected free from the sternum and the pericardium and divided down the midline. The right lobe of the thymus is then swept superiorly into the apex of the right chest. The thymus can also be resected if adequate exposure is unattainable. This gives a more direct view of the aorta lying underneath the pericardium.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Pericardiopexy. In some cases, a pericardiopexy may adequately treat the TM and should always be attempted. The pericardium is lifted using 2 graspers and pulled toward the sternum. Simultaneously (inset), the bronchoscope is used to evaluate the degree of improvement in TM. This will be a more viable option in patients with less severe TM or TM that involves the distal trachea. If this allows acceptable tracheal opening, sutures are placed from the pericardium to the periostium of the sternum. The first suture is placed at the most superior aspect of the pericardium and work continues inferiorly. Another bronchoscopy is performed holding the sutures taut to confirm improved tracheal narrowing before securing the knots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Opening the pericardium. If pericardiopexy does not provide adequate elevation or the affected trachea is too proximal, the pericardium needs to be opened to expose the underlying aorta. This can be done using the laparoscopic scissors, starting at the aortic root and extending as far as is necessary to attain adequate exposure.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Aortopexy suture placement. Sutures are placed through the tunica media of the aorta and the sternal periostium. We use anywhere from 3 to 8 braided polyester sutures starting superiorly along the most anterior aspect of the aorta. It is important to place sutures precisely along the anterior aorta so that, once tied down, there is no aortic twisting. Sutures can be placed using a forehand or backhand technique depending on the surgeon's preference. If necessary, sutures may extend onto the innominate artery. It should also be noted that stitches placed too proximal (near the heart) may damage the aortic valve or involve the coronary arteries.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 11Bronchoscopic confirmation. Holding the sutures taut, bronchoscopy (inset) again confirms adequate tracheal opening before tying down the sutures. Once all sutures are secured, another bronchoscopy is performed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 12Knots and closure. Knots are secured down using a knot pusher. The inset shows the finished aortopexy with the aorta snugly against the sternum. A final DLB is performed with spontaneous breathing to confirm improved TM. If it was not removed, the thymus is replaced in a more natural position from the apex of the chest. A small chest tube can be placed through 1 of the port sites. The bronchial blocker is removed and the right lung is allowed to re-expand before closure. The remaining port sites are closed and sterile dressings are placed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 13Things to avoid. (A) If there is too much anterior pull on the trachea, it can start to kink. On bronchoscopy, this will cause a narrowing in the anterior-posterior direction similar to that seen with TM. If this occurs, sutures will need to be removed and replaced with less anterior pull. (B) Sutures should be placed through the tunica media of the aorta. They should not enter the lumen as this causes profuse bleeding. (C) If sutures are not placed along the most anterior aspect of the aorta, there can be significant aortic twisting. Sutures are most likely to be placed too far along the right lateral aspect of the aorta, rotating it toward the patient's left. (D) Here is another view of the torqued aorta. The proximity of the right pulmonary artery is also visible here. It is of note that, although the pulmonary artery is less visible during a right-sided approach, it is still imperative to maintain awareness of its location and to avoid injuring it. PT = pulmonary trunk; RPA = right pulmonary artery.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Symptoms should be expected to improve quickly on recovery from anesthesia. Improvement after open aortopexy has been reported to be anywhere from immediately postprocedure up to 6 months postoperatively.7Vazquez-Jimenez J.F. Sachweh J.S. Liakopoulos O.J. et al.Aortopexy in severe tracheal instability: short-term and long-term outcome in 29 infants and children.Ann Thorac Surg. 2001; 72: 1898-1901Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Dave S. Currie B.G. The role of aortopexy in severe tracheomalacia.J Pediatr Surg. 2006; 41: 533-537Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar It should be noted that frequently some patients maintain some residual TOF cough and are persistently susceptible to upper respiratory infections. Generally, all patients need intensive care monitoring immediately following surgery, but once extubated should be able to transfer to the ward shortly thereafter. The chest tube can be placed to water seal once the patient has recovered from anesthesia and can usually be removed on postoperative day 1 if there are no complications. We routinely perform a postoperative echocardiogram as previously mentioned. Discharge and follow-up are generally determined by the severity of any residual symptoms.