Regenerative medicine: current research and perspective in pediatric surgery

The field of regenerative medicine, encompassing several disciplines including stem cell biology and tissue engineering, continues to advance with the accumulating research on cell manipulation technologies, gene therapy and new materials. Recent progress in preclinical and clinical studies may transcend the boundaries of regenerative medicine from laboratory research towards clinical reality. However, for the ultimate goal to construct bioengineered transplantable organs, a number of issues still need to be addressed. In particular, engineering of elaborate tissues and organs requires a fine combination of different relevant aspects; not only the repopulation of multiple cell phenotypes in an appropriate distribution but also the adjustment of the host environmental factors such as vascularisation, innervation and immunomodulation. The aim of this review article is to provide an overview of the recent discoveries and development in stem cells and tissue engineering, which are inseparably interconnected. The current status of research on tissue stem cells and bioengineering, and the possibilities for application in specific organs relevant to paediatric surgery have been specifically focused and outlined

Human gastro-intestinal organoid engineering: a state of the art

Gastrointestinal organ failure, from congenital or postnatally acquired pathologies, is a major cause of death across countries of all income levels. Organoids and engineered tissues have been widely investigated as tools to model organ functions and treat pathologies. In this review we aim to describe the progress in human organoid engineering applied to the gastrointestinal tract (namely esophagus, stomach, and intestine). Starting from the onset of the organoid culture technique, we illustrate genetic engineering, stem cell niche engineering, bioprinting, and microfluidics approaches used to integrate mechano-physiological parameters with human organoids. Thanks to these improvements, organoid technology allows disease modelling of patient-specific pathologies, and personalized treatment screening, also offering a cell source for autologous transplantation. We further present an overview of the advances of tissue engineering in animal systems, concerning novel materials and scaffolds to be combined with a variety of cell types to reconstitute a viable surrogate for implantation. The effort in this field sets organoids as an important tool in personalized and regenerative medicine. Their application combined with the advances in tissue engineering holds great potential for translational application

First steps to define murine amniotic fluid stem cell microenvironment

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells

Building gut from scratch — progress and update of intestinal tissue engineering

Short bowel syndrome (SBS), a condition defined by insufficient absorptive intestinal epithelium, is a rare disease, with an estimated prevalence up to 0.4 in 10,000 people. However, it has substantial morbidity and mortality for affected patients. The mainstay of treatment in SBS is supportive, in the form of intravenous parenteral nutrition, with the aim of achieving intestinal autonomy. The lack of a definitive curative therapy has led to attempts to harness innate developmental and regenerative mechanisms to engineer neo-intestine as an alternative approach to addressing this unmet clinical need. Exciting advances have been made in the field of intestinal tissue engineering (ITE) over the past decade, making a review in this field timely. In this Review, we discuss the latest advances in the components required to engineer intestinal grafts and summarize the progress of ITE. We also explore some key factors to consider and challenges to overcome when transitioning tissue-engineered intestine towards clinical translation, and provide the future outlook of ITE in therapeutic applications and beyond

Update on Foregut Molecular Embryology and Role of Regenerative Medicine Therapies

Esophageal atresia (OA) represents one of the commonest and most severe developmental disorders of the foregut, the most proximal segment of the gastrointestinal (GI) tract (esophagus and stomach) in embryological terms. Of intrigue is the common origin from this foregut of two very diverse functional entities, the digestive and respiratory systems. OA appears to result from incomplete separation of the ventral and dorsal parts of the foregut during development, resulting in disruption of esophageal anatomy and frequent association with tracheo-oesophageal fistula. Not surprisingly, and likely inherent to OA, are associated abnormalities in components of the enteric neuromusculature and ultimately loss of esophageal functional integrity. An appreciation of such developmental processes and associated defects has not only enhanced our understanding of the etiopathogenesis underlying such devastating defects but also highlighted the potential of novel corrective therapies. There has been considerable progress in the identification and propagation of neural crest stem cells from the GI tract itself or derived from pluripotent cells. Such cells have been successfully transplanted into models of enteric neuropathy confirming their ability to functionally integrate and replenish missing or defective enteric nerves. Combinatorial approaches in tissue engineering hold significant promise for the generation of organ-specific scaffolds such as the esophagus with current initiatives directed toward their cellularization to facilitate optimal function. This chapter outlines the most current understanding of the molecular embryology underlying foregut development and OA, and also explores the promise of regenerative medicine

Congenital diaphragmatic hernia subtypes: Comparing birth prevalence, occurrence by maternal age, and mortality in a national birth cohort

BACKGROUND: Population-based administrative data have rarely been used to compare the birth prevalence, risk factors for occurrence, and mortality of congenital diaphragmatic hernia (CDH) subtypes. OBJECTIVES: We used a national birth cohort to identify CDH subtypes and compared their birth prevalence, relationship with maternal age after accounting for sociodemographic factors, and 1-year mortality rates. METHODS: Linked hospital admission and death records were used to identify isolated and complex CDH cases (involving additional anomalies) among singleton livebirths in England between 2002 and 2018. The prevalence of each CDH subtype per 10,000 livebirths was estimated overall and by infant, birth and maternal characteristics. The relationship between maternal age and each subtype relative to no CDH was examined using multivariable log-binomial regression to estimate risk ratios (RRs). One-year mortality rates were examined using Kaplan-Meier curves and the hazard ratio (HR) of complex versus isolated CDH was calculated using Cox regression. RESULTS: Among 9.5 million livebirths, we identified 1285 with isolated CDH and 1150 with complex CDH. The overall prevalence of isolated and complex CDH was 1.4 (95% confidence interval [CI] 1.3, 1.4) and 1.2 (95% CI 1.1, 1.3) per 10,000 livebirths, respectively. Only complex CDH was associated with maternal age. Compared with maternal age 25-34 years, complex CDH risk was elevated for maternal age < 20 years (RR 1.31, 95% CI 1.00, 1.72). Risk was highest for maternal age ≥ 40 years (RR 1.61, 95% CI 1.21, 2.15) although accounting for chromosomal anomalies attenuated the risk (RR 1.39, 95% CI 1.00, 1.92). The 1-year mortality rate for complex CDH (33.1%, 95% CI 30.5, 35.9) was slightly higher than for isolated CDH (29.7%, 95% CI 27.3, 32.3) (HR 1.10, 95% CI 0.96, 1.27). CONCLUSIONS: Mechanisms of occurrence differed between and within CDH subtypes and 1-year mortality of complex CDH was slightly higher than for isolated CDH

Parents’ experiences of feeding children born with oesophageal atresia/tracheo-oesophageal fistula

Background: Feeding difficulties are widely acknowledged following oesophageal atresia/tracheo-oesophageal fistula repair. However, little is understood about the nature and severity of these difficulties. This study explored feeding in children with oesophageal atresia/tracheo-oesophageal fistula from the parent perspective. Methods: In collaboration with a patient support group, data were collected using a research-specific online discussion forum. Thematic analysis was used to code the data and identify themes. Results: One hundred and twenty-seven parents registered for the online forum, of whom 83 (65%) provided demographic data. Seventy-four (89%) of responders were mothers, 75 (90%) were of white ethnicity, 65 (78%) were from the UK. Six key themes were identified: feeding is a traumatic experience, feeding my child is scary, feeding is isolating and filled with uncertainty, feeding outside of the home is difficult, feeding associated emotions, developing coping strategies. Parents described features of medical, nutritional, feeding skill and psychosocial dysfunction across all stages of eating/drinking development. They described how their child's feeding difficulties had an impacted their own well-being. An interactional model of feeding difficulties in OA/TOF is proposed. Conclusions: Exploring parent experiences provides rich data from which to expand understanding of the complex nature of feeding difficulties in OA/TOF. Feeding should be viewed as a dyadic process, occurring within a family system. Intervention for feeding difficulties should be family-centred, addressing parental anxiety, trauma and uncertainty, as well as the child's underlying medical/surgical needs to optimise outcome. Further study of clinical correlates with parental experience is required

Thoracoscopic oesophageal atresia/tracheo-oesophageal fistula (OA/TOF) repair is associated with a higher stricture rate: a single institution’s experience

Purpose: Thoracoscopic OA/TOF repair was first described in 1999. Currently, less than 10% of surgeons routinely employ minimally access surgery. Our primary aim was to review our immediate-, early- and long-term outcomes with this technique compared with the open approach. Methods: A retrospective review of all patients undergoing primary OA/TOF (Type C) repair at our institution from 2009 was conducted. Outcome measures included length of surgery, conversion rate from thoracoscopy, early complications such as anastomotic leak and post-operative complications such as anastomotic strictures needing dilatations. Fisher’s exact and Kruskal–Wallis tests were used for statistical analysis. Results: 95 patients in total underwent OA/TOF repair during the study period of which 61 (64%) were completed via an open approach. 34 were attempted thoracoscopically of which 11 (33%) were converted. There was only one clinically significant anastomotic leak in our series that took place in the thoracoscopic group. We identified a significantly higher stricture rate in our thoracoscopic cohort (72%) versus open surgery (43%, P < 0.05). However, the median number of dilations (3) performed was not significantly different between the groups. There was one recurrent fistula in the thoracoscopic converted to open group. Our median follow-up was 60 months across the groups. Conclusion: In our experience, the clinically significant leak rate for both open and thoracoscopic repair as well as recurrent fistula is much lower than has been reported in the literature. We do not routinely perform contrast studies and are, thus, reporting clinically significant leaks only. The use of post-operative neck flexion, ventilation and paralysis is likely to be protective towards a leak. Thoracoscopic OA/TOF repair is associated with a higher stricture rate compared with open surgery; however, these strictures respond to a similar number of dilatations and are no more refractory. Larger, multicentre studies may be useful to investigate these finding further

Decellularized diaphragmatic muscle drives a constructive angiogenic response in vivo

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1\u3b1. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, \u3b1SMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration