Andrew K. Pappa

Analysis of Human Auricular Cartilage to Guide Tissue-Engineered Nanofiber-Based Chondrogenesis: Implications for Microtia Reconstruction

Objective. Nanofiber-supported, in vitro–generated cartilage may represent an optimal starting material for the development of a cartilage implant for use in microtia reconstruction. To do so, the authors aim to first characterize the molecular composition of endogenous auricular cartilage and determine if human umbilical cord mesenchymal stem cells (hUCMSCs) can be differentiated into cartilage in vitro. Study Design. Prospective, controlled. Setting. Academic research laboratory. Subjects and Methods. Human ear cartilage from normal adults, pediatric patients with microtia, and pediatric patients with preauricular appendages (n = 2) was analyzed for collagens I, II, and X and elastin expression. In parallel, hUCMSCs were cultured on either polycaprolactone (PCL) or D, L-lactide-co-glycolic acid (PLGA) nanofiber scaffolds for 21 days under chondrogenic conditions. Cells were harvested for histologic, biochemical, and quantitative polymerase chain reaction analysis. Control cells were grown under both chondrogenic and nonchondrogenic conditions in the absence of nanofiber scaffolds. Results. Histological analysis of human ear cartilage revealed similar levels and distribution of collagens I and X and elastin. Collagen II was not highly expressed in the microtia samples. hUCMSC cultures stained positively for glycosaminosglycans (GAG) and sulfated proteoglycans. Compared to control cells, hUCMSCs grown on PLGA nanofiber scaffolds had a higher differentiation index (P ≤ .012) and higher levels of collagen X mRNA expression (P ≤ .006). Conclusion. These data provide information regarding the composition of endogenous ear cartilage and suggest that hUCMSCs grown on PLGA nanofiber scaffolds may represent an optimal starting material for the development of a cartilage implant for use in microtia reconstruction.

The Compound Action Potential in Subjects Receiving a Cochlear Implant.

Hypothesis: The compound action potential (CAP) is a purely neural component of the cochleas response to sound, and may provide information regarding the existing neural substrate in cochlear implant (CI) subjects that can help account for variance in speech perception outcomes. Background: Measurement of the “total response” (TR), or sum of the magnitudes of spectral components in the ongoing responses to tone bursts across frequencies, has been shown to account for 40 to 50% of variance in speech perception outcomes. The ongoing response is composed of both hair cell and neural components. This correlation may be improved with the addition of the CAP. Methods: Intraoperative round window electrocochleography (ECochG) was performed in adult and pediatric CI subjects (n = 238). Stimuli were tones of different frequencies (250 Hz–4 kHz) at 90 dB nHL. The CAP was assessed in two ways, as an amplitude and with a scaling factor derived from a function fitted to the response. The results were correlated with consonant-nucleus-consonant (CNC) word scores at 6 months post-implantation (n = 51). Results: Only about half of the subjects had a measurable CAP at any frequency. The CNC word scores correlated weakly with both amplitude (r2 = 0.20, p < 0.001) and scaling factor (r2 = 0.25, p < 0.01). In contrast, the TR alone accounted for 43% of the variance, and addition of either CAP measurement in multiple regression did not account for additional variance. Conclusions: The underlying pathology in CI patients causes the CAP to be often absent and highly variable when present. The TR is a better predictor of speech perception outcomes than the CAP.

Biochemical Properties of Tissue-Engineered Cartilage

ObjectiveMicrotia is treated with rib cartilage sculpting and staged procedures; though aesthetically pleasing, these constructs lack native ear flexibility. Tissue-engineered (TE) elastic cartilage may bridge this gap; however, TE cartilage implants lead to hypertrophic changes with calcification and loss of flexibility. Retaining flexibility in TE cartilage must focus on increased elastin, maintained collagen II, decreased collagen X, with prevention of calcification. This study compares biochemical properties of human cartilage to TE cartilage from umbilical cord mesenchymal stem cells (UCMSCs). Our goal is to establish a baseline for clinically useful TE cartilage. MethodsDiscarded cartilage from conchal bowl, microtic ears, preauricular tags, rib, and TE cartilage were evaluated for collagen I, II, X, calcium, glycosaminoglycans, elastin, and fibrillin I and III. Human UCMSCs were chondroinduced on 2D surfaces and 3D D,L-lactide-co-glycolic acid (PLGA) fibers. ResultsCartilage samples demonstrated similar staining for collagens I, II, and X, elastin, and fibrillin I and III, but differed from rib. TE pellets and PLGA-supported cartilage were similar to auricular samples in elastin and fibrillin I staining. TE samples were exclusively stained for fibrillin III. Only microtic samples demonstrated calcium staining. ConclusionsTE cartilage expressed similar levels of elastin, fibrillin I, and collagens I and X when compared to native cartilage. Microtic cartilage demonstrated elevated calcium, suggesting this abnormal tissue may not be a viable cell source for TE cartilage. TE cartilage appears to recapitulate the embryonic development of fibrillin III, which is not expressed in adult tissue, possibly providing a strategy to control TE elastic cartilage phenotype.

Osteoinduction of umbilical cord and palate periosteum-derived mesenchymal stem cells on poly(Lactic-Co-Glycolic) acid Nanomicrofibers

AbstractThe need for tissue-engineered bone to treat complex craniofacial bone defects secondary to congenital anomalies, trauma, and cancer extirpation is sizeable. Traditional strategies for treatment have focused on autologous bone in younger patients and bone substitutes in older patients. However, the capacity for merging new technologies, including the creation of nanofiber and microfiber scaffolds with advances in natal sources of stem cells, is crucial to improving our treatment options. The advantages of using smaller diameter fibers for scaffolding are 2-fold: the similar fiber diameters mimic the in vivo extracellular matrix construct and smaller fibers also provide a dramatically increased surface area for cell-scaffold interactions. In this study, we compare the capacity for a polymer with Federal Drug Administration approval for use in humans, poly(lactic-co-glycolic) acid (PLGA) from Delta polymer, to support osteoinduction of mesenchymal stem cells (MSCs) harvested from the umbilical cord (UC) and palate periosteum (PP). Proliferation of both UC- and PP-derived MSCs was improved on PLGA scaffolds. The PLGA scaffolds promoted UC MSC differentiation (indicated by earlier gene expression and higher calcium deposition), but not in PP-derived MSCs. Umbilical cord–derived MSCs on the PLGA nanomicrofiber scaffolds have potential clinical utility in providing solutions for craniofacial bone defects, with the added benefit of earlier availability.

Effects of connective tissue growth factor on the regulation of elastogenesis in human umbilical cord-derived mesenchymal stem cells

BackgroundA key to clinical microtia reconstruction is construct flexibility. The most significant current limitation to engineered elastic cartilage is maintaining an elastic phenotype, which is principally dependent on elastin production (although other parameters, including maintenance of a ratio above 1 for collagens II to I, minimizing collagen X content, and presence of adequate matrix fibrillin for elastin binding, all play supporting roles). Connective tissue growth factor (CTGF), a compound secreted by chondrocytes, has been shown to promote an elastic phenotype in mature rabbit chondrocytes; however, CTGF effect on undifferentiated mesenchymal stem cells (MSCs) has not been characterized. The principal aim of this study is to analyze CTGF effect on elastin production in umbilical cord (UC)-derived MSCs and to determine optimal timing of treatment to maximize elastin production. MethodsHuman UCMSCs (hUCMSCs) were isolated from Wharton jelly using an explant technique, grown to passage 3, seeded onto nanofiber scaffolds, and chondroinduced for 21 days. Nanofiber scaffolds were electrospun using solubilized poly L-lactide/D-lactide/glycolide (PLGA). Chondrogenic media was supplemented with 25 &mgr;g/mL CTGF starting at day 0 or 7. Messenger RNA (mRNA) for Collagen I, II, X, fibrillin, and elastin was quantified by RT-PCR; glycosaminoglycan (GAG) matrix deposition was assessed and normalized by cellular DNA content. Elastin protein was assessed by Western blot analysis. All experiments were performed in triplicate with MSCs from 4 distinct cords. Multiway analysis of variance with Newman-Keuls post test was used to determine statistical significance. ResultsConnective tissue growth factor treatment results in increased GAG/DNA ratio; the differentiation index was maintained above 1 in all conditions, with increased collage II noted at days 7 and 14 in CTGF conditions; no difference in collagen X or fibrillin mRNA was noted. Increased elastin mRNA and protein were noted at day 14 in conditions treated with CTGF at day 7 after differentiation. ConclusionsConnective tissue growth factor leads to maximal elastin increase in UCMSCs after 7 days of chondroinduction and not in undifferentiated MSCs. With appropriately timed treatment, CTGF may be a useful adjunct in maintaining an elastic cartilage phenotype in engineered cartilage from human UCMSCs.

The organization of frequency and binaural cues in the gerbil inferior colliculus

The inferior colliculus (IC) is the common target of separate pathways that transmit different types of auditory information. Beyond tonotopy, little is known about the organization of response properties within the 3‐dimensional layout of the auditory midbrain in most species. Through study of interaural time difference (ITD) processing, the functional properties of neurons can be readily characterized and related to specific pathways. To characterize the representation of ITDs relative to the frequency and hodological organization of the IC, the properties of neurons were recorded and the sites recovered histologically. Subdivisions of the IC were identified based on cytochrome oxidase (CO) histochemistry. The results were plotted within a framework formed by an MRI atlas of the gerbil brain. The central nucleus was composed of two parts, and lateral and dorsal cortical areas were identified. The lateral part of the central nucleus had the highest CO activity in the IC and a high proportion of neurons sensitive to ITDs. The medial portion had lower CO activity and fewer ITD‐sensitive neurons. A common tonotopy with a dorsolateral to ventromedial gradient of low to high frequencies spanned the two regions. The distribution of physiological responses was in close agreement with known patterns of ascending inputs. An understanding of the 3‐dimensional organization of the IC is needed to specify how the single tonotopic representation in the IC central nucleus leads to the multiple tonotopic representations in core areas of the auditory cortex.

Predictors of Adverse Outcomes in Free Flap Reconstruction: A Single-Institution Experience

Objective Understanding the independent predictors of poor outcomes in free flap surgery is essential for patient selection. We aim to determine the independent predictors of major complications, flap survival, and extended hospital stays. Study Design Retrospective cohort study. Setting Tertiary medical center. Subjects and Methods We reviewed medical records from all vascularized tissue transfers in the head and neck between 2007 and 2014 at our institution. We recorded demographics, medical comorbidities, disease characteristics, flap characteristics, and intraoperative events. We defined outcomes as major complications in the 30-day postoperative period, flap death or partial flap survival, and a length of stay ≥14 days. We used bivariate and multivariate methods to test for associations. Results Of 170 free flap operations, 44% had major complications; 11% fully or partially failed; and 27% required an extended hospital stay. Independent predictors of major complications were age ≥60 years (odds ratio [OR], 3.7; P = .001), revision surgery (OR, 3.5; P = .004), and a prior neck dissection (OR, 3.5; P = .004). Independent predictors of flap failure were revision surgery (OR, 4.1, P = .01) and the use of a plate (OR, 3.7; P = .03). Revision surgery was independently associated with a longer stay (OR, 3.0; P = .01), and the use of a radial forearm flap was associated with a shorter stay (OR, 0.3, P = .047). Conclusion These results underscore that caution is warranted in revision flap surgery, patients with prior neck operations, and patients aged ≥60 years.

Simultaneous Incidental Parathyroid Carcinoma and Intrathyroid Parathyroid Gland in Suspected Renal Failure Induced Hyperparathyroidism

Hyperparathyroidism is a common disorder affecting more than hundreds of thousands of people annually. While most commonly secondary to an adenoma, it may also arise from four-gland hyperplasia or malignancy. In the case of primary hyperparathyroidism, the number of glands involved may be unknown prior to surgery. In contrast, the metabolic disorder associated with renal failure induced hyperparathyroidism ensures a hyperplasia picture. Despite the uniform hyperplasia seen in tertiary disease and the preoperative expectation for four-gland exploration, our case demonstrates the continued need for a surgeons vigilance during dissection to identify all glands and appropriately use intraoperative parathyroid hormone (PTH) testing. In addition, while intraoperative PTH assessment is an effective method for confirming adequacy of treatment for hyperparathyroidism, only surgical pathology can confirm malignancy, which should be considered with PTH levels > 1,000. The case also underscores the importance of comprehensive surgery management and mindful interpretation of intraoperative PTH levels in the management of hyperparathyroidism. Standard surgical technique includes complete exploration of the central compartment, and thyroid lobectomy when the aforementioned exploration fails to reveal the necessary parathyroid tissue, especially with a persistently elevated PTH. Without a standardized progressive compartment exploration and judicious use of intraoperative hormone testing, intrathyroidal parathyroid glands can be missed.

A pilot study comparing mechanical properties of tissue-engineered cartilages and various endogenous cartilages

Background: Mechanical properties of tissue‐engineered cartilage and a variety of endogenous cartilage were measured. The main goal was to evaluate if the tissue‐engineered cartilage have similar mechanical characteristics to be replaced with rib cartilage in microtia reconstruction. Such study lays the foundation for future human clinical trials for microtia reconstruction. Method: Atomic force microscopy and compression testing were used to measure the viscoelasticity of tissue‐engineered cartilage (stem cell seeded on Poly lactic co‐glycolytic acid nanofibers and Pellet) and endogenous cartilage: conchal bowl, microtic ears, preauricular remnants, and rib. Atomic force microscopy, calculates biomaterial elasticity through force‐deformation measurement and Hertz model. Compression testing determines the stress relaxation by measuring slope of stress reduction at 10% strain. Finding: Tissue‐engineered cartilage demonstrated elasticity (4.6 kPa for pellet and 6.6 kPa for PLGA) and stress relaxation properties (7.6 (SD 1.1) kPa/s for pellet) most similar to those of native conchal bowl cartilage (31.8 (SD 18) kPa for the elasticity and 15.1 (SD 2.1) kPa/s for stress relaxation factor). Rib cartilage was most dissimilar from the mechanical characteristics of conchal cartilage and demonstrated the highest elastic modulus (361 (SD 372) kPa). Moreover, except preauricular cartilage samples, the level of elastic modulus increased with age. Interpretation: The use of tissue‐engineered cartilage developed via PLGA and Pellet methods, may be an appropriate substitute for rib cartilage in the reconstruction of microtic ears, however their mechanical characteristics still need to be improved and require further validation in animal studies. HighlightsSurgical reconstruction for microtia involves usage of costal and rib cartilage.Mechanical properties of Tissue Engineered and human cartilages were evaluated.Rib cartilage has most dissimilar mechanical characteristics to conchal cartilage.Tissue Engineered cartilage may be used in the reconstruction of microtic ears.