Effects of Modified Instructions on Peabody Developmental Motor Scales, Second Edition, Gross Motor Scores in Children with Typical Development.

Aims: The current study assessed whether modifying instructions on the Peabody Developmental Motor Scales, Second Edition (PDMS-2) affected scores in children with typical development. Methods: The gross motor portion of the PDMS-2 was administered twice, 2-10 days apart, to 38 children. Age- and gender-matched groups received instructions in both standard and modified formats, with order depending on group assignment. Results: Gross Motor Quotient results showed an effect for instruction type (p = .03) and an interaction between instruction type and order (p = .02). Improved scores for those given modified instructions during the second session indicated the interaction favored modifications. Stationary scores showed an effect for instruction type (p = .01) and an interaction between instruction type and age (p = .02). Object Manipulation scores showed an interaction between instruction type and order only (p =.002); Locomotion scores showed no significant changes (p = .25). Percentile rank changes ranged from 9% to 22% across subtests. Conclusions: Findings suggested instruction modifications may change PDMS-2 gross motor scores, even in children with typical development. Findings also suggested normative scores should not be reported if modifications were used during testing. Research is needed to determine optimal cues for the best representation of true motor ability during standardized assessment.

Effect of Urea and Thiourea on Generation of Xenogeneic Extracellular Matrix Scaffolds for Tissue Engineering.

Effective solubilization of proteins by chaotropes in proteomic applications motivates their use in solubilization-based antigen removal/decellularization strategies. A high urea concentration has previously been reported to significantly reduce lipophilic antigen content of bovine pericardium (BP); however, structure and function of the resultant extracellular matrix (ECM) scaffold were compromised. It has been recently demonstrated that in vivo ECM scaffold fate is determined by two primary outcome measures as follows: (1) sufficient reduction in antigen content to avoid graft-specific adaptive immune responses and (2) maintenance of native ECM structural proteins to avoid graft-specific innate responses. In this work, we assessed residual antigenicity, ECM architecture, ECM content, thermal stability, and tensile properties of BP subjected to a gradient of urea concentrations to determine whether an intermediate concentration exists at which both antigenicity and structure-function primary outcome measures for successful in vivo scaffold outcome can simultaneously be achieved. Alteration in tissue structure-function properties at various urea concentrations with decreased effectiveness for antigen removal makes use of urea-mediated antigen removal unlikely to be suitable for functional scaffold generation.

In vivo xenogeneic scaffold fate is determined by residual antigenicity and extracellular matrix preservation.

The immunological potential of animal-derived tissues and organs is the critical hurdle to increasing their clinical implementation. Glutaraldehyde-fixation cross-links proteins in xenogeneic tissues (e.g., bovine pericardium) to delay immune rejection, but also compromises the regenerative potential of the resultant biomaterial. Unfixed xenogeneic biomaterials in which xenoantigenicity has been ameliorated and native extracellular matrix (ECM) architecture has been maintained have the potential to overcome limitations of current clinically utilized glutaraldehyde-fixed biomaterials. The objective of this work was to determine how residual antigenicity and ECM architecture preservation modulate recipient immune and regenerative responses towards unfixed bovine pericardium (BP) ECM scaffolds. Disruption of ECM architecture during scaffold generation, with either SDS-decellularization or glutaraldehyde-fixation, stimulated recipient foreign body response and resultant fibrotic encapsulation following leporine subpannicular implantation. Conversely, BP scaffolds subjected to stepwise removal of hydrophilic and lipophilic antigens using amidosulfobetaine-14 (ASB-14) maintained native ECM architecture and thereby avoided fibrotic encapsulation. Removal of hydrophilic and lipophilic antigens significantly decreased local and systemic graft-specific, adaptive immune responses and subsequent calcification of BP scaffolds compared to scaffolds undergoing hydrophile removal only. Critically, removal of antigenic components and preservation of ECM architecture with ASB-14 promoted full-thickness recipient non-immune cellular repopulation of the BP scaffold. Further, unlike clinically utilized fixed BP, ASB-14-treated scaffolds fostered rapid intimal and medial vessel wall regeneration in a porcine carotid patch angioplasty model. This work highlights the importance of residual antigenicity and ECM architecture preservation in modulating recipient immune and regenerative responses towards xenogeneic biomaterial generation.

Stepwise solubilization-based antigen removal for xenogeneic scaffold generation in tissue engineering.

The ability of residual antigens on decellularized tissue to elicit the immune response upon implantation motivates development of a more rigorous antigen removal (AR) process for xenogeneic scaffold generation. Antigen removal strategies promoting solubilization of hydrophilic proteins (predominantly cytoplasmic) enhance the reduction of hydrophilic antigenicity in bovine pericardium (BP); however, the diversity of protein antigens within a tissue necessitates development of AR strategies capable of addressing a spectrum of protein antigen solubilities. In the present study, methods for promoting solubilization of lipophilic proteins (predominantly membrane) were investigated for their ability to reduce lipophilic antigenicity of BP when applied as a second AR step following our previously described hydrophilic AR method. Bovine pericardium following AR (BP-AR) was assessed for residual hydrophilic and lipophilic antigenicity, removal of known lipophilic xenoantigens, tensile properties, and extracellular matrix structure and composition. Facilitating hydrophile solubilization (using dithiothreitol and potassium chloride) followed by lipophile solubilization (using amidosulfobetaine-14 (ASB-14)), in a two-step sequential, differential AR strategy, significantly reduces residual hydrophilic and lipophilic antigenicity of BP-AR beyond that achieved with either one-step hydrophilic AR or decellularization using 1% (w/v) sodium dodecyl sulfate. Moreover, use of 1% (w/v) ASB-14 for lipophilic AR eliminates the two most critical known barriers to xenotransplantation (galactose-α(1,3)-galactose and major histocompatibility complex I)) from BP-AR without compromising the structure-function properties of the biomaterial. This study demonstrates the importance of a sequential, differential protein solubilization approach to reduce biomaterial antigenicity in the production of a xenogeneic scaffold for heart valve tissue engineering.

Localized decrease of beta-catenin contributes to the differentiation of human embryonic stem cells.

Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, beta-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of beta-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down beta-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of beta-catenin contributes to the spatial pattern of differentiation in hESC colonies.