Fabrication of Large Size Ex Vivo-Produced Oral Mucosal Equivalents for Clinical Application

The soft tissue reconstruction of significant avulsed and/or surgically created tissue defects requires the ability to manufacture substantial soft tissue constructs for repair of the resulting wounds. In this study, we detail the issues that need to be addressed in upsizing the manufacture of larger tissue-engineered devices (ex vivo-produced oral mucosa equivalent [EVPOME]) in vitro from a methodology previously used for smaller constructs. The larger-sized EVPOME, consisting of autologous human oral keratinocytes and a dermal substitute, AlloDerm?, was fabricated for the purpose of reconstructing large clinical defects. Regulated as an autologous somatic cell therapy product, the fabrication process abided by current Good Manufacturing Practices and current Good Tissue Practices as required by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration (FDA). Successful fabrication of large EVPOMEs utilized a higher cell seeding density (5.3?105 cells/cm2) with a relatively thinner AlloDerm, ranging from 356.6 to 508.0??m in thickness. During the air?liquid interface culture, the thickness of the scaffold affected the medium diffusion rate, which, in turn, resulted in changes of epithelial stratification. Histologically, keratinocyte progenitor (p63), proliferation (Ki-67), and late differentiation marker (filaggrin) expression showed differences correlating with the expression of glucose transporter-1 (GLUT1) in the EVPOMEs from the thickest (550?1020??m) to the thinnest (228.6?330.2??m) AlloDerm scaffold. Glucose consumption and 2-deoxyglucose (2DG) uptake showed direct correlation with scaffold thickness. The scaffold size and thickness have an impact on the cellular phenotype and epithelial maturation in the manufacturing process of the EVPOME due to the glucose accessibility influenced by the diffusion rate. These outcomes provide basic strategies to manufacture a large-sized, healthy EVPOME graft for reconstructing large mucosa defects.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140251/1/ten.tec.2014.0600.pd

Differentiation of Human Embryonic Stem Cells to a Parathyroid-Like Phenotype

Iatrogenic hypoparathyroidism is the most common complication of cervical endocrine surgery. Current management is limited and palliative. As the molecular steps in parathyroid development have been defined, they may be replicable in vitro, with a goal of cellular replacement therapy. Human embryonic stem cell (hESC) lines were investigated as a model for parathyroid regeneration in vitro. BG01 was selected as a model based on expression of genes of interest in embryoid bodies (EBs). Established strategies for mouse embryonic stem cell differentiation into definitive endoderm were modified and extended to maximize the expression of definitive markers of parathyroid development. The optimal approach included the use of Activin A at 100 ng/mL with BG01 cells grown on murine embryonic fibroblasts for 5 days under conditions of increasing serum concentration. After 5 days, the cells were allowed to mature further in tissue culture without murine fibroblasts but with continuous Activin A. Our strategy produced differentiated cell cultures that expressed intermediate markers of endoderm and parathyroid development (CXCR4, EYA1, Six1, and Pax1), as well as markers of committed parathyroid precursors or developed parathyroid glands (glial cell missing-2 [Gcm2], CCL21, calcium sensing receptor [CaSR], and parathyroid hormone [PTH]). We further characterized the cells by testing conditioned medium from various time points in our differentiation scheme for the presence of PTH. We found that by keeping the cells in culture 2 weeks after the withdrawal of Activin A, the cells were able to produce PTH. Further in vivo work will be needed to demonstrate proper functionality of the cells developed in this way.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78132/1/scd.2008.0337.pd

Comparison of two decellularized dermal equivalents

Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature‐derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p = .01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co‐culture, separated by a physical barrier to create a cell‐free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143690/1/term2530.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143690/2/term2530_am.pd

Spectrum of color gene deletions and phenotype in patients with blue cone monochromacy

Blue cone monochromacy (BCM) is an X-linked ocular disease characterized by poor visual acuity, nystagmus, and photodysphoria in males with severely reduced color discrimination. Deletions, rearrangements and point mutations in the red and green pigment genes have been implicated in causing BCM. We assessed the spectrum of genetic alterations in ten families with BCM by Southern blot, polymerase chain reaction, and sequencing analysis, and the phenotype was characterized by ophthalmoscopy, fluorescein angiography, and a battery of tests to assess color vision in addition to routine ophthalmological examination. All families showed clinical features associated with BCM. Acuities were reduced in all affected males, and photopic b-wave was reduced by more than 90% in seven families. In three families, however, the photopic b-wave response showed uncharacteristic relative preservation of 30–80% (of the clinical low-normal value). The color vision was unusually preserved in two affected males, but this was not correlated with photopic electroretinography retention. Progressive macular atrophy was observed in affected members of two BCM families while the rest of the families presented with normal fundus. In nine families deletions were identified in the gene encoding the red-sensitive photopigment and/or in the region up to 17.8 kb upstream of the red gene which contains the locus control region and other regulatory sequences. In the same nine families the red pigment gene showed a range of deletions from the loss of a single exon to loss of the complete red gene. In one family no mutation was found in the exons of the red gene or the locus control region but showed loss of the complete green gene. No association was observed between the phenotypes and genotypes in these families.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42266/1/439-107-1-75_s004390000338.pd

Novel mutations in XLRS1 causing retinoschisis, including first evidence of putative leader sequence change

Juvenile retinoschisis is an X-linked recessive disease caused by mutations in the XLRS1 gene. We screened 31 new unrelated patients and families for XLRS1 mutations in addition to previously reported mutations for 60 of our families (Retinoschisis Consortium, Hum Mol Genet 1998;7:1185–1192). Twenty-three different mutations including 12 novel ones were identified in 28 patients. Mutations identified in this study include 19 missense mutations, two nonsense mutations, one intragenic deletion, four microdeletions, one insertion, and one intronic sequence substitution that is likely to result in a splice site defect. Two novel mutations, c.38T→C (L13P) and c.667T→C (C223R), respectively, present the first genetic evidence for the functional significance of the putative leader peptide sequence and for the functional significance at the carboxyl terminal of the XLRS1 protein beyond the discoidin domain. Mutations in 25 of the families were localized to exons 4–6, emphasizing the critical functional significance of the discoidin domain of the XLRS1 protein. Hum Mutat 14:423–427, 1999. © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35178/1/8_ftp.pd

A randomised trial of observational learning from 2D and 3D models in robotically assisted surgery

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: Advances in 3D technology mean that both robotic surgical devices and surgical simulators can now incorporate stereoscopic viewing capabilities. While depth information may benefit robotic surgical performance, it is unclear whether 3D viewing also aids skill acquisition when learning from observing others. As observational learning plays a major role in surgical skills training, this study aimed to evaluate whether 3D viewing provides learning benefits in a robotically assisted surgical task. METHODS: 90 medical students were assigned to either (1) 2D or (2) 3D observation of a consultant surgeon performing a training task on the daVinci S robotic system, or (3) a no observation control, in a randomised parallel design. Subsequent performance and instrument movement metrics were assessed immediately following observation and at one-week retention. RESULTS: Both 2D and 3D groups outperformed no observation controls following the observation intervention (ps < 0.05), but there was no difference between 2D and 3D groups at any of the timepoints. There was also no difference in movement parameters between groups. CONCLUSIONS: While 3D viewing systems may have beneficial effects for surgical performance, these results suggest that depth information has limited utility during observational learning of surgical skills in novices. The task constraints and end goals may provide more important information for learning than the relative motion of surgical instruments in 3D space.This research was supported by an Intuitive Surgical grant awarded to Dr G Buckingha

Functional implications of the spectrum of mutations found in 234 cases with X-linked juvenile retinoschisis (XLRS)

X-linked retinoschisis (XLRS) is the most common cause of juvenile macular degeneration in males, resulting in vision loss early in life. The gene involved in XLRS was identified recently. It encodes a protein with a disoidin domain, suggested to be involved in cell-cell interactions. We have screened the gene for mutations in 234 familial and sporadic retinoschisis cases and identified 82 different mutations in 214 (91%). Thirty one mutations were found more than once, i.e. 2-10 times, with the exception of the 214G→A mutation which was found in 34 apparently unrelated cases. The origin of the patients, the linkage data and the site of the mutations (mainly CG dinucleotides) indicate that most recurrent mutations had independent origins and thus suggest the existence of a significant new mutation rate in XLRS1. The mutations identified cover the entire spectrum, from small intra-genic deletions (7%), to nonsense (6%), missense (75%), small frameshifting insertions/deletions (6%) and splice site mutations (6%). Since, regardless of the mutation type, no females with a typical RS phenotype were identified, RS seems to be caused by loss-of-function mutations only. Mutations occurred non-randomly, with hotspots at several CG dinudeotides and a C6 stretch. Exons 1-3 contained few, mainly translation-truncating mutations, arguing against an important functional role for this segment of the protein. Exons 4-6, encoding the discoidin domain, contained most, mainly missense mutations. An alignment of 32 discoidin domain proteins was constructed to reveal the consensus sequence and to deduce the functional importance of the missense mutations identified. The mutation analysis revealed a high preponderance of mutations involving or creating cysteine residues, pointing to sites important for the tertiary folding and/or protein function, and highlights several amino acids which may be involved in XLRS1-specific protein-protein interactions. Despite the enormous mutation heterogeneity, patients have relatively uniform clinical manifestations although with great intra-familial variation in age at onset and progression