Patient factors associated with novel EAR-Q appearance, psychosocial, and social scales: A cross-sectional study and regression analysis.

INTRODUCTION: The EAR-Q is a rigorously validated patient-reported outcome measure, which evaluates ear appearance and health-related quality of life (HRQL) in patients with congenital or acquired ear conditions. The aim of this study was to conduct an exploratory analysis to examine the factors associated with EAR-Q appearance and HRQL scale scores. METHODS: In this study, 862 participants, aged 8-29 years, with congenital or acquired ear conditions, completed the EAR-Q as part of an international field-test study. Patients responded to demographic and clinical questions as well as the EAR-Q. Univariable and multivariable linear regression analyses were used to determine factors that were significant predictors for the scores on the EAR-Q Appearance, Psychological, and Social scales. RESULTS: Most participants were men (57.4%), awaiting treatment (55.0%), and had a microtia diagnosis (70.4%), with a mean age of 13 (±4) years. Worse ear appearance scores (p < 0.02) were associated with male gender, microtia, no history of treatment, ear surgery within 6 months, unilateral involvement, and greater self-reported ear asymmetry. Decreased psychological scores (p < 0.01) were associated with increasing participant age, no treatment history, recent ear surgery, and dissatisfaction with ears matching or overall dissatisfaction. Lower social scores (p ≤ 0.04) were associated with no treatment history, those awaiting surgery, ear surgery within the last 6 months, bilateral involvement, and self-reported ears matching or overall appearance. CONCLUSION: This analysis identified patient factors that may influence ear appearance and HRQL scale scores. These findings provide evidence of patient factors that should be adjusted for when undertaking future observational research designs using the EAR-Q in this patient population.

Mosaic BRAF Fusions Are a Recurrent Cause of Congenital Melanocytic Nevi Targetable by MAPK Pathway Inhibition.

Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 19. Recurrently, fusions involved the loss of the 5´ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro, and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.

Management of the Congenital Cleft Earlobe with a Conchal Cartilage Graft.

Congenital earlobe clefts are the most common lower auricular malformations. They represent a unique reconstructive challenge. The goal of surgery includes restoration of a natural lobular contour and volume as well as a fine surgical scar. Several surgical techniques have been described, most of which only address the cleft deformity but not the soft tissue deficiency. We hereby describe a technique which addresses both the cleft and improves soft tissue deficiency through a conchal cartilage graft.

Management of helical adhesion malformation.

Congenital auricular anomalies are common, however very few articles in the English literature address helical adhesion malformation, whereby the helix is adhered to the scapha. We describe a surgical approach to correct helical adhesion malformation which, unlike previously described methods, is simpler and results in an inconspicuous scar on the back of the ear. We illustrate our technique with an 11-year-old girl with bilateral helical adhesion malformations. Satisfactory ear shape was achieved bilaterally without complication. Meticulous dissection over the helix and exposure beyond the adhesion is of paramount importance in successfully correcting this malformation. Through a posterior auricular incision, this wide exposure and visualisation of the helix can be attained, with the added benefit of a scar hidden on the back of the ear.

Management of the infected preauricular sinus.

INTRODUCTION: The preauricular sinus is a common finding in children and may often be complicated by infection. Complete excision of the sinus is the only definitive cure. Failure to recognise the presence of a sinus, particularly when infection erupts away from the sinus, may lead to inadequate management and unnecessary surgery. AIM: We report our experience in managing infected preauricular sinuses and highlight important points in our surgical technique. METHODS: A retrospective review was performed of our electronic patient database for all paediatric patients who underwent surgical excision of preauricular sinuses by the senior author at Great Ormond Street Hospital for Children between January 2013 and October 2022. RESULTS: A total of 10 patients underwent surgical management of 11 preauricular sinuses with a median follow-up time of 40 months (range 1-136). Eight patients underwent excision of the preauricular sinus due to infection. All infected cases affected the preauricular cheek skin and had previously undergone at least one unsuccessful surgical drainage prior to a referral to our unit. All cases were successfully operated on at our unit with no reported complications or recurrence. CONCLUSION: A lack of awareness of the presence of a sinus and identification of a preauricular pit by the inexperienced clinician will result in inadequate treatment of this condition and potentially unnecessary surgical procedures. Our paper highlights the importance of correctly identifying the extent of the sinus and describes a safe and reliable technique to ensure complete removal of the preauricular sinus with satisfactorily low recurrence rates.

Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro.

Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.

Human platelet lysate enhances proliferation but not chondrogenic differentiation of pediatric mesenchymal progenitors.

BACKGROUND AIMS: Cell therapies have the potential to improve reconstructive procedures for congenital craniofacial cartilage anomalies such as microtia. Adipose-derived stem cells (ADSCs) and auricular cartilage stem/progenitor cells (CSPCs) are promising candidates for cartilage reconstruction, but their successful use in the clinic will require the development of xeno-free expansion and differentiation protocols that can maximize their capacity for chondrogenesis. METHODS: We assessed the behavior of human ADSCs and CSPCs grown either in qualified fetal bovine serum (FBS) or human platelet lysate (hPL), a xeno-free alternative, in conventional monolayer and 3-dimensional spheroid cultures. RESULTS: We show that CSPCs and ADSCs display greater proliferation rate in hPL than FBS and express typical mesenchymal stromal cell surface antigens in both media. When expanded in hPL, both cell types, particularly CSPCs, maintain a spindle-like morphology and lower surface area over more passages than in FBS. Both media supplements support chondrogenic differentiation of CSPCs and ADSCs grown either as monolayers or spheroids. However, chondrogenesis appears less ordered in hPL than FBS, with reduced co-localization of aggrecan and collagen type II in spheroids. CONCLUSIONS: hPL may be beneficial for the expansion of cells with chondrogenic potential and maintaining stemness, but not for their chondrogenic differentiation for tissue engineering or disease modeling.

Lop Ear to Conchal Microtia: An Algorithmic Surgical Approach.

BACKGROUND: The lop ear deformity is defined by a deficient helix and scapha, underdeveloped anthelix, and downfolding of the helix. The terminology used is still confusing, and the treatment is not entirely structured. The aim of this study was to provide a new systematic surgical approach of this deformity based on our center's experience. MATERIALS AND METHODS: All patients undergoing surgical correction of lop ears between 2007 and 2019 at Great Ormond Street Hospital were included. Patients' data, surgical techniques, and postoperative complications were recorded. RESULTS: Based on our records, we identified 3 surgical techniques for the correction of lop ears, based on the degree of deformity encountered. In a mild lop ear, correction was achieved with a modified otoplasty technique by improving the definition of the antihelix and superior crus. In a moderate deformity, additional remodeling of the lidded helix was performed (extended otoplasty), whereas for the severe lop ear, the amount of cupping and the deficient cartilage required formal reconstruction using a carved rib cartilage framework. There were a total of 109 patients and 146 lop ears: 58 mild, 27 moderate, and 61 severe lop ears. CONCLUSION: We feel that there is a point in the spectrum of congenital ear deformity when a severe lop ear becomes a conchal microtia and recommend this approach to simplify the management of these cases. This is intended to bring greater clarity to how to deal with lop ears, based on the severity of the deformity and the surgical techniques used.

Intrathoracic prosthesis in children in preventing post pneumonectomy syndrome: Its role in congenital single lung and post pneumonectomy situations.

BACKGROUND: Postpneumopnectomy syndrome (PPS) is an extreme rotation and malposition of mediastinum causing dynamic and symptomatic central airway compression, arisingafter pneumonectomy or more uncommonly, in congenital single lung physiology. Affected patients present with severe respiratory compromise. Intrathoracic prosthesis placement is an evolving technique in children that mitigate the effects of thoracic dead space. RESEARCH QUESTION: Assessment of clinical recovery and functional benefit in children undergoing placement of intrathoracic prosthesis following pneumonectomy or in congenital single lung situations. STUDY DESIGN AND METHODS: Retrospective chart review of patients at Great Ormond Street Hospital from 2010 to 2020 was performed of all patients who underwent intrathoracic tissue expander placement. We summarize the outcomes of twenty four children, including those with both congenital and postpneumonectomy PPS etiology. RESULTS: 24 Children who underwent placement of intrathoracic prosthesis for PPS in the study period with median age of 3.5 months and weight of 5 kg. Single lung etiology was congenital in 15 children (6 agenesis, 9 hypoplasia), and postpneumonectomy in 9 children. In seven patients, there was associated long segment tracheal stenosis. Pre operative ECMO was required in 2 patients, and pre operative ventilation was required in 12 patients all of whom had congenital single lung. Intrathoracic prosthesis placement was concurrent with intracardiac repair in 5 patients. There were no operative deaths, but one early postoperative death related to septicaemia. Median follow up was 75 months with 10 patients on continued respiratory support and 3 on nocturnal support with good quality of life. Two children needed reoperations for replacement of prosthesis. CONCLUSION: The use of tissue expanders is within the armamentarium of most plastic surgeons' practice. We also therefore advocate for a collaborative team approach involving Plastic and Cardiothoracic Surgery for surgical treatment of these patients. This multidisciplinary strategy has improved management of this rare and debilitating condition of PPS, thereby offering significant improvements in general progress of these sick children having single lung physiology. Evidence is still lacking on functional outcomes in these children and further work is necessary to prove that this is indeed achievable.

The Impact of COVID-19 on Plastic Surgery Training in the United Kingdom, Canada and Australia-A Cross-Sectional Study.

Background Surgical trainees worldwide have been thrust into a period of uncertainty, with respect to the implications COVID-19 pandemic will have on their roles, training, and future career prospects. It is currently unclear how plastic surgery trainees are being affected by COVID-19. This study examined the experience of plastic surgery trainees in Canada, the UK, and Australia to determine trainee roles during the early COVID-19 emergency response and how training changed during this time. Methods A cross-sectional survey-based study was designed for plastic surgery trainees in the UK, Canada and Australia. In total, 110 trainees responded to the survey. Statistical tests were conducted to determine differences in responses, based on year of training and country of residence. Results In total, 9.7% (10/103) of respondents reported being deployed to cover another service. There was a significant difference between redeployment based on country ( p = 0.001). Within the UK group, 28.9% of respondents were redeployed. For trainees not deployed, 95.5% (85/89) reported that there has been a reduction in operative volume. Ninety-seven (94.1%) respondents reported that there were ongoing teaching activities offered by their program. The majority of trainees (66.4%) were concerned about their training. There was a significant difference between overall concern and country ( p < 0.05). Conclusion In these unprecedented times, training programs in plastic surgery should be aware of the major impact that COVID-19 has had on trainees and will have on their training. The majority of plastic surgery trainees have experienced a reduction in surgical exposure but have maintained some form of regular teaching.

FACE-Q Craniofacial Module: Part 1 validation of CLEFT-Q scales for use in children and young adults with facial conditions.

BACKGROUND: The CLEFT-Q includes 12 independently functioning scales that measure appearance (face, nose, nostrils, teeth, lips, jaws), health-related quality of life (psychological, social, school, speech distress), and speech function, and an eating/drinking checklist. Previous qualitative research revealed that the CLEFT-Q has content validity in noncleft craniofacial conditions. This study aimed to examine the psychometric performance of the CLEFT-Q in an international sample of patients with a broad range of facial conditions. METHODS: Data were collected between October 2016 and December 2019 from 2132 patients aged 8 to 29 years with noncleft facial conditions. Rasch measurement theory (RMT) analysis was used to examine Differential Item Function (DIF) by comparing the original CLEFT-Q sample and the new FACE-Q craniofacial sample. Reliability and validity of the scales in a combined cleft and craniofacial sample (n=4743) were examined. RESULTS: DIF was found for 23 CLEFT-Q items when the datasets for the two samples were compared. When items with DIF were split by sample, correlations between the original and split person locations showed that DIF had negligible impact on scale scoring (correlations ≥0.995). In the combined sample, RMT analysis led to the retention of original content for ten CLEFT-Q scales, modification of the Teeth scale, and the addition of an Eating/Drinking scale. Data obtained fit with the Rasch model for 11 scales (exception School, p=0.04). Person Separation Index and Cronbach alpha values met the criteria. CONCLUSION: The scales described in this study can be used to measure outcomes in children and young adults with cleft and noncleft craniofacial conditions.

FACE-Q craniofacial module: Part 2 Psychometric properties of newly developed scales for children and young adults with facial conditions.

BACKGROUND: The FACE-Q Craniofacial Module is a patient-reported outcome measure designed for patients aged 8 to 29 years with conditions associated with a facial difference. In part 1, we describe the psychometric findings for the original CLEFT-Q scales tested in patients with cleft and noncleft facial conditions. The aim of this study was to examine psychometric performance of new FACE-Q Craniofacial Module scales. METHODS: Data were collected between December 2016 and December 2019 from patients aged 8 to 29 years with conditions associated with a visible or functional facial difference. Rasch measurement theory (RMT) analysis was used to examine psychometric properties of each scale. Scores were transformed from 0 (worst) to 100 (best) for tests of construct validity. RESULTS: 1495 participants were recruited with a broad range of conditions (e.g., birthmarks, facial paralysis, craniosynostosis, craniofacial microsomia, etc.) RMT analysis resulted in the refinement of 7 appearance scales (Birthmark, Cheeks, Chin, Eyes, Forehead, Head Shape, Smile), two function scales (Breathing, Facial), and an Appearance Distress scale. Person separation index and Cronbach alpha values met criteria. Three checklists were also formed (Eye Function, and Eye and Face Adverse Effects). Significantly lower scores on eight of nine scales were reported by participants whose appearance or functional difference was rated as a major rather than minor or no difference. Higher appearance distress correlated with lower appearance scale scores. CONCLUSION: The FACE-Q Craniofacial Module scales can be used to collect and compare patient reported outcomes data in children and young adults with a facial condition.

An international study to develop the EAR-Q patient-reported outcome measure for children and young adults with ear conditions.

BACKGROUND: There is currently a lack of patient-reported outcome measures for ear reconstruction. We developed the EAR-Q to measure ear appearance and post-operative adverse effects from the patient perspective. METHODS: Field-test data were collected from children and young adults in eight countries between 13 May 2016 and 12 December 2019. Rasch measurement theory (RMT) analysis was used to refine the scales and to examine their psychometric properties. RESULTS: Participants had microtia (n = 607), prominent ears (n = 145) or another ear condition (n = 111), and provided 960 assessments for the Appearance scale (e.g., size, shape, photos), and 137 assessments for the Adverse Effects scale (e.g., itchy, painful, numb). RMT analysis led to the reduction of each scale to 10-items. Data fit the Rasch model for the Appearance (X2(80) = 90.9, p = 0.19) and Adverse Effects (X2(20) = 24.5, p = 0.22) scales. All items in each scale had ordered thresholds and good item fit. There was no evidence of differential item function for the Appearance scale by age, gender, language, or type of ear condition. Reliability was high for the Appearance scale, with person separation index (PSI) and Cronbach alpha values with and without extremes ≥0.92. Reliability for the Adverse Effects scale was adequate (i.e., PSI and Cronbach alpha values ≥0.71). Higher scores (liked appearance more) correlated with higher scores (better) on Psychological, Social and School scales. INTERPRETATION: The EAR-Q can be used in those 8-29 years of age to understand the patient perspective in clinical practice and research, and in addition, can be used to benchmark outcomes for ear reconstruction internationally.

Modification of the cartilaginous framework for autologous ear reconstruction: Construction of a stable complete ring framework with grander highs and lows.

BACKGROUND: Successful microtia reconstruction involves fabrication of a framework with well-defined ear subunits. Tragal definition and deep conchal bowl are key elements to produce a natural well-defined and contoured ear. We describe a modification in the cartilage framework with the purpose of increasing framework stability, tragal definition and conchal bowl depth. METHODS: The tragus is placed on two cartilaginous bars (L-shaped), which are fixed to the framework base block creating a complete ring. These increase tragal projection and conchal depth. The tragus is carved angled posteriorly to have a shadowing effect on the absent auditory canal. Aesthetic outcome was assessed, at least 6 months after the second stage, on a 5-point ordinal scale (1-5) using the scoring system published in the UK microtia care standards and agreed on by the International Society for Auricular Reconstruction (ISAR). RESULTS: A total of 406 auricular reconstructions were performed in 363 patients (206 right, 114 left, 43 bilateral). After excluding cases who did not have second stage and those without complete photographs, 290 cases were assessed. The described modification in the framework carving was performed in 255 ears out of these 290 ears. The mean aesthetic score before and after the modification was 2.4 and 3.09 respectively for the tragus (p < 0.001), 2.2 and 2.95 for the intertragal notch (p < 0.001) and 2.77 and 3.49 for the concha (p < 0.001). CONCLUSIONS: This technical refinement has resulted in increased permanence and definition of the tragus and deeper and more aesthetic concha.

Concepts Important to Patients With Facial Differences: A Qualitative Study Informing a New Module of the FACE-Q for Children and Young Adults.

OBJECTIVE: The concepts important to children and young adults who undergo treatments for facial differences are not well-defined. Measurement of treatment outcomes from the patient's perspective is necessary to ensure goals of treatment are met. We aimed to identify concepts important to children and young adults with facial differences through a qualitative study. DESIGN: An interpretive description qualitative approach was followed. Semistructured interviews were conducted, transcribed verbatim, and coded using a line-by-line approach. Qualitative analysis led to the development of a conceptual framework of outcomes important to patients. SETTING: Interviews were conducted in Canada and the United Kingdom at home, by telephone, or in the hospital. PARTICIPANTS: Participants (N = 72) were recruited between May and June 2014 from craniofacial clinics at the Hospital for Sick Children (Toronto) and Great Ormond Street Hospital (London). Participants included anyone with a visible and/or functional facial difference aged 8 to 29 years and fluent in English, excluding patients with a cleft. The sample included 38 females and 34 males, with a mean age of 13.9 years, and included 28 facial conditions (11 facial paralysis, 18 ear anomalies, 26 skeletal conditions, and 17 soft tissue conditions). RESULTS: Analysis led to identification of important concepts within 4 overarching domains: facial appearance, facial function, adverse effects of treatment, and health-related quality of life (psychological, social, and school function). CONCLUSIONS: Our study provides an understanding of concepts important to children and young adults with facial differences.

Arterio-venous malformations of the ear: Description of distinct anatomical presentation and multidisciplinary management approach.

BACKGROUND: Arterio-venous malformations (AVMs) of the ear are a rare entity and their management should be decided in a dedicated multidisciplinary team (MDT) setting. The aim of this study is to describe the distinct anatomical patterns of the auricular AVMs in our unit and propose a combined interventional radiological and surgical approach. MATERIALS AND METHODS: All consecutive patients presenting with AVMs of the ear and reviewed by the Vascular Anomalies MDT between 2014 and 2019 were included in this study. Signs, symptoms, diagnostic investigations and operative findings were collected prospectively. RESULTS: After reviewing our nine patients, we identified four anatomical patterns of auricular AVMs: I: involves just a component of the ear and should undergo embolization followed by excision and reconstruction without significant loss of form; II: affects the superior two-thirds of the ear, sparing the lobule and part of the conchal bowl; these patients should undergo embolization, excision and monitoring before formal reconstruction of the ear is offered; III: involves the entire ear and should undergo embolization and pinnectomy; if there is no recurrence, the patients can be offered either a carved-rib cartilage reconstruction or a prosthesis, depending on the quality of the surrounding soft-tissues; IV: involves the ear and surrounding tissue, making surgical management and subsequent reconstruction extensive. CONCLUSION: The management of auricular AVMs is based on the extent of the ear involved. We feel that our combined interventional radiological and surgical approach will aid the management of these complex patients.

Modeling Normal and Pathological Ear Cartilage in vitro Using Somatic Stem Cells in Three-Dimensional Culture.

Microtia (underdeveloped ear) is a rare congenital dysmorphology affecting the development of the outer ear. Although human microtic cartilage has not been fully characterized, chondrogenic cells derived from this tissue have been proposed as a suitable source for autologous auricular reconstruction. The aim of this study was to further characterize native microtic cartilage and investigate the properties of cartilage stem/progenitor cells (CSPCs) derived from it. Two-dimensional (2D) systems are most commonly used to assess the chondrogenic potential of somatic stem cells in vitro, but limit cell interactions and differentiation. Hence here we investigated the behavior of microtic CSPCs in three-dimensional spheroid cultures. Remarkable similarities between human microtic cartilages from five patients, as compared to normal cartilage, were observed notwithstanding possibly different etiologies of the disease. Native microtic cartilage displayed poorly defined perichondrium and hyper-cellularity, an immature phenotype that resembled that of the normal developing human auricular cartilage we studied in parallel. Crucially, our analysis of microtic ears revealed for the first time that, unlike normal cartilage, microtic cartilages are vascularized. Importantly, CSPCs isolated from human microtic and normal ear cartilages were found to recapitulate many characteristics of pathological and healthy tissues, respectively, when allowed to differentiate as spheroids, but not in monolayer cultures. Noteworthily, starting from initially homogeneous cell pellets, CSPC spheroids spontaneously underwent a maturation process in culture, and formed two regions (inner and outer region) separated by a boundary, with distinct cell types that differed in chondrogenic commitment as indicated by expression of chondrogenic markers. Compared to normal ear-derived spheroids, microtic spheroids were asymmetric, hyper-cellularized and the inner and outer regions did not develop properly. Hence, their organization resembled that of native microtic cartilage. Together, our results identify novel features of microtic ears and highlight the importance of 3D self-organizing in vitro systems for better understanding somatic stem cell behavior and disease modeling. Our observations of ear-derived chondrogenic stem cell behavior have implications for choice of cells for tissue engineered reconstructive purposes and for modeling the etiopathogenesis of microtia.

Three-dimensional environment and vascularization induce osteogenic maturation of human adipose-derived stem cells comparable to that of bone-derived progenitors.

While human adipose-derived stem cells (hADSCs) are known to possess osteogenic differentiation potential, the bone tissues formed are generally considered rudimentary and immature compared with those made by bone-derived precursor cells such as human bone marrow-derived mesenchymal stem cells (hBMSCs) and less commonly studied human calvarium osteoprogenitor cells (hOPs). Traditional differentiation protocols have tended to focus on osteoinduction of hADSCs through the addition of osteogenic differentiation media or use of stimulatory bioactive scaffolds which have not resulted in mature bone formation. Here, we tested the hypothesis that by reproducing the physical as well as biochemical bone microenvironment through the use of three-dimensional (3D) culture and vascularization we could enhance osteogenic maturation in hADSCs. In addition to biomolecular characterization, we performed structural analysis through extracellular collagen alignment and mineral density in our bone tissue engineered samples to evaluate osteogenic maturation. We further compared bone formed by hADSCs, hBMSCs, and hOPs against mature human pediatric calvarial bone, yet not extensively investigated. Although bone generated by all three cell types was still less mature than native pediatric bone, a fibrin-based 3D microenvironment together with vascularization boosted osteogenic maturation of hADSC making it similar to that of bone-derived osteoprogenitors. This demonstrates the important role of vascularization and 3D culture in driving osteogenic maturation of cells easily available but constitutively less committed to this lineage and suggests a crucial avenue for recreating the bone microenvironment for tissue engineering of mature craniofacial bone tissues from pediatric hADSCs, as well as hBMSCs and hOPs.