Robert E. Akins

Production of heparin-containing hydrogels for modulating cell responses

Successful tissue regeneration requires that biomaterials have optimal bioactivity and mechanical properties. Heparin-containing hydrogels that can be crosslinked in situ were designed to contain tunable amounts of biological components (e.g. heparin, arginine-glycine-aspartate (RGD)) as well as to exhibit controlled mechanical properties (e.g. shear modulus). These gel parameters can also be tuned to provide controlled delivery of proteins, such as growth factors, for regulating cellular behavior. Maleimide-functionalized low-molecular-weight heparin (LWMH) was conjugated to a poly(ethylene glycol) (PEG) hydrogel. The elastic shear modulus, as assessed via oscillatory rheology experiments, could be tuned by the concentration of polymer in the hydrogel, and by the end group functionality of PEG. Hydrogels of two different moduli (2.8 and 0.4kPa) were used to study differences in the response of human aortic adventitial fibroblasts (AoAF) in two-dimensional cell culture experiments. These experiments indicated that the AoAFs show improved adhesion to materials with the higher modulus. Evaluation of cell responses to hydrogels with RGD linked to the hydrogels via conjugation to PEG or to LMWH indicated improved cellular responses to these materials when the bioactive ligands were chemically attached through linkage to the PEG rather than to the LMWH. These results highlight important design considerations in the tailoring of these materials for cardiovascular tissue engineering applications.

Three-Dimensional Culture Alters Primary Cardiac Cell Phenotype

The directed formation of complex three-dimensional (3D) tissue architecture is a fundamental goal in tissue engineering and regenerative medicine. The growth of cells in 3D structures is expected to influence cellular phenotype and function, especially relative cell distribution, expression profiles, and responsiveness to exogenous signals; however, relatively few studies have been carried out to examine the effects of 3D reaggregation on cells from critical target organs, like the heart. Accordingly, we cultured primary cardiac ventricular cells in a 3D model system using a serum-free medium to test the hypothesis that expression profiles, multicellular organizational pathways, tissue maturation markers, and responsiveness to hormone stimulation were significantly altered in stable cell populations grown in 3D versus 2D culture. We found that distinct multi-cellular structures formed in 3D in conjunction with changes in mRNA expression profile, up-regulation of endothelial cell migratory pathways, decreases in the expression of fetal genes (Nppa and Ankrd1), and increased sensitivity to tri-iodothyronine stimulation when compared to parallel 2D cultures comprising the same cell populations. These results indicate that the culture of primary cardiac cells in 3D aggregates leads to physiologically relevant alterations in component cell phenotype consistent with cardiac ventricular tissue formation and maturation.

Neuromuscular junctions in Cerebral palsy: Presence of extrajunctional acetylcholine receptors

Background Cerebral palsy (CP) is the most prevalent neurologic disease in children. A primary deficit in CP is neuromuscular dysfunction; however, neuromuscular junctions in children with CP have not been studied. Evidence exists that up-regulation of acetylcholine receptors (AChRs) may be present in children with CP, and the current study was undertaken to examine this possibility. Methods Thirty-nine children with spastic CP and 25 neurologically normal children were enrolled in the study. Paraspinal muscles underwent biopsy during scheduled spinal fusion surgery. Two sets of assessments were performed on the biopsy specimens: (1) reverse-transcription polymerase chain reaction and Western blotting to evaluate the expression of the &ggr; subunit of the AChR; and (2) histologic evaluation using a double-stain technique for AChR and acetylcholinesterase, wherein acetylcholinesterase staining defined the limits of the neuromuscular junction, and AChR staining that appeared outside of these limits indicated an abnormal distribution of AChRs. Results Reverse-transcription polymerase chain reaction and Western blot analyses showed that neither the CP nor non-CP samples had detectable &ggr;-AChR subunit. Histologic analysis indicated that 11 of 39 children with CP and none of 20 children with idiopathic scoliosis scored positive for the presence of AChR outside of the neuromuscular junction (P = 0.0085). Conclusion A subset of children with CP have an abnormal distribution of AChR relative to the acetylcholinesterase found at the neuromuscular junction. The altered distribution of AChR in CP was not associated with a detectable presence of the &ggr;-AChR subunit, suggesting that the nonjunctional AChRs in CP does not contain the &ggr; subunit.

Differential effects of substrate modulus on human vascular endothelial, smooth muscle, and fibroblastic cells †

Regenerative medicine approaches offer attractive alternatives to standard vascular reconstruction; however, the biomaterials to be used must have optimal biochemical and mechanical properties. To evaluate the effects of biomaterial properties on vascular cells, heparinized poly(ethylene glycol) (PEG)-based hydrogels of three different moduli, 13.7, 5.2, and 0.3 kPa, containing fibronectin and growth factor were utilized to support the growth of three human vascular cell types. The cell types exhibited differences in attachment, proliferation, and gene expression profiles associated with the hydrogel modulus. Human vascular smooth muscle cells demonstrated preferential attachment on the highest-modulus hydrogel, adventitial fibroblasts demonstrated preferential growth on the highest-modulus hydrogel, and human umbilical vein endothelial cells demonstrated preferential growth on the lowest-modulus hydrogel investigated. Our studies suggest that the growth of multiple vascular cell types can be supported by PEG hydrogels and that different populations can be controlled by altering the mechanical properties of biomaterials.

Altered Expression of Muscle- and Cytoskeleton-Related Genes in a Rat Strain With Inherited Cryptorchidism

Development of the fetal gubernaculum is a prerequisite for testicular descent and dependent on insulin-like 3 and androgen, but knowledge of downstream effectors is limited. We analyzed transcript profiles in gubernaculum and testis to address changes occurring during normal and abnormal testicular descent in Long Evans wild-type (wt) and cryptorchid (orl) fetuses. Total RNA from male wt and orl gubernacula (gestational days [GD]18-20), wt female gubernacula (GD18), and testis (GD17 and 19) was hybridized to Affymetrix GeneChips. Statistical analysis of temporal, gender, and strain-specific differences in gene expression was performed with the use of linear models analysis with empirical Bayes statistics and analysis of variance (gubernaculum) and linear analysis (testis). Overrepresented common gene ontology functional categories and pathways were identified in groups of differentially expressed genes with the Database for Annotation, Visualization, and Integrated Discovery. Transcript profiles were dynamic in wt males between GD18-19 and GD20, comparatively static in orl GD18-20 gubernaculum, and similar in wt and orl testis. Functional analysis of differentially expressed genes in wt and orl gubernaculum identified categories related to metabolism, cellular biogenesis, small GTPase-mediated signal transduction, cytoskeleton, muscle development, and insulin signaling. Genes involved in androgen receptor signaling, regulated by androgens, or both were overrepresented in differentially expressed gubernaculum and testis gene groups. Quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed differential expression of genes related to muscle development, including Myog, Tnnt2, Fst, Igf1, Igfbp5, Id2, and Msx1. These data suggest that the orl mutation results in a primary gubernacular defect that affects muscle development and cytoskeletal function and might alter androgen-regulated pathways.

Dysmorphic neuromuscular junctions associated with motor ability in cerebral palsy.

Cerebral palsy (CP) is the most prevalent neurologic disease in children and a leading cause of severe physical disability. Research and clinical experience indicate that children with CP have abnormal neuromuscular junctions (NMJs), and we present evidence that nonapposition of neuromuscular junction components is associated with the severity of motor system deficit in CP. Leg muscle biopsies collected from ambulatory (n = 21) or nonambulatory (n = 38) CP patients were stained in order to detect acetylcholine receptor (AChR) and acetylcholine esterase (AChE). Image analysis was used to calculate the extra‐AChE spread (EAS) of AChR staining to estimate the amount of AChR occurring outside the functional, AChE‐delimited NMJ. Nonambulatory children exhibited higher average EAS (P = 0.025) and had a greater proportion of their NMJs with significantly elevated EAS (P = 0.023) than ambulatory children. These results indicate that physical disability in children with CP is associated with structurally dysmorphic NMJs, which has important implications for the management of CP patients, especially during surgery and anesthesia. Muscle Nerve, 2005

Cryptorchidism in the Orl Rat Is Associated with Muscle Patterning Defects in the Fetal Gubernaculum and Altered Hormonal Signaling

ABSTRACT Cryptorchidism, or undescended testis, is a common male genital anomaly of unclear etiology. Hormonal stimulation of the developing fetal gubernaculum by testicular androgens and insulin-like 3 (INSL3) is required for testicular descent. In studies of the orl fetal rat, one of several reported strains with inherited cryptorchidism, we studied hormone levels, gene expression in intact and hormone-stimulated gubernaculum, and imaging of the developing cremaster muscle facilitated by a tissue clearing protocol to further characterize development of the orl gubernaculum. Abnormal localization of the inverted gubernaculum was visible soon after birth. In the orl fetus, testicular testosterone, gubernacular androgen-responsive transcript levels, and muscle-specific gene expression were reduced. However, the in vitro transcriptional response of the orl gubernaculum to androgen was largely comparable to wild type (wt). In contrast, increases in serum INSL3, gubernacular INSL3-responsive transcript levels, expression of the INSL3 receptor, Rxfp2, and the response of the orl gubernaculum to INSL3 in vitro all suggest enhanced activation of INSL3/RXFP2 signaling in the orl rat. However, DNA sequence analysis did not identify functional variants in orl Insl3. Finally, combined analysis of the present and previous studies of the orl transcriptome confirmed altered expression of muscle and cellular motility genes, and whole mount imaging revealed aberrant muscle pattern formation in the orl fetal gubernaculum. The nature and prevalence of developmental muscle defects in the orl gubernaculum are consistent with the cryptorchid phenotype in this strain. These data suggest impaired androgen and enhanced INSL3 signaling in the orl fetus accompanied by defective cremaster muscle development.

Ultrafast protein determinations using microwave enhancement

We present here microwave-based modifications of standard protein assays that dramatically reduce the time required to determine protein concentrations. Typical protein determinations involve incubation times ranging from 15-60 min. Microwave irradiation of specimens reduces this time requirement to 10-20 s without compromising accuracy or reliability. The remarkable speed with which protein determinations may be carried out using microwave enhancement greatly simplifies general laboratory procedures that depend on the estimation of protein concentrations.

Phenotype Specific Association of the TGFBR3 Locus with Nonsyndromic Cryptorchidism

PURPOSE Based on a genome-wide association study of testicular dysgenesis syndrome showing a possible association with TGFBR3, we analyzed data from a larger, phenotypically restricted cryptorchidism population for potential replication of this signal. MATERIALS AND METHODS We excluded samples based on strict quality control criteria, leaving 844 cases and 2,718 controls of European ancestry that were analyzed in 2 separate groups based on genotyping platform (ie Illumina® HumanHap550, version 1 or 3, or Human610-Quad, version 1 BeadChip in group 1 and Human OmniExpress 12, version 1 BeadChip platform in group 2). Analyses included genotype imputation at the TGFBR3 locus, association analysis of imputed data with correction for population substructure, subsequent meta-analysis of data for groups 1 and 2, and selective genotyping of independent cases (330) and controls (324) for replication. We also measured Tgfbr3 mRNA levels and performed TGFBR3/betaglycan immunostaining in rat fetal gubernaculum. RESULTS We identified suggestive (p ≤ 1× 10(-4)) association of markers in/near TGFBR3, including rs9661103 (OR 1.40; 95% CI 1.20, 1.64; p = 2.71 × 10(-5)) and rs10782968 (OR 1.58; 95% CI 1.26, 1.98; p = 9.36 × 10(-5)) in groups 1 and 2, respectively. In subgroup analyses we observed strongest association of rs17576372 (OR 1.42; 95% CI 1.24, 1.60; p = 1.67 × 10(-4)) with proximal and rs11165059 (OR 1.32; 95% CI 1.15, 1.38; p = 9.42 × 10(-4)) with distal testis position, signals in strong linkage disequilibrium with rs9661103 and rs10782968, respectively. Association of the prior genome-wide association study signal (rs12082710) was marginal (OR 1.13; 95% CI 0.99, 1.28; p = 0.09 for group 1), and we were unable to replicate signals in our independent cohort. Tgfbr3/betaglycan was differentially expressed in wild-type and cryptorchid rat fetal gubernaculum. CONCLUSIONS These data suggest complex or phenotype specific association of cryptorchidism with TGFBR3 and the gubernaculum as a potential target of TGFβ signaling.

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure.

Hydrogels derived from resilin-like polypeptides (RLPs) have shown outstanding mechanical resilience and cytocompatibility; expanding the versatility of RLP-based materials via conjugation with other polypeptides and polymers would offer great promise in the design of a range of materials. Here, we present an investigation of the biochemical and mechanical properties of hybrid hydrogels composed of a recombinant RLP and a multiarm PEG macromer. These hybrid hydrogels can be rapidly cross-linked through a Michael-type addition reaction between the thiols of cysteine residues on the RLP and vinyl sulfone groups on the multiarm PEG. Oscillatory rheology and tensile testing confirmed the formation of elastomeric hydrogels with mechanical resilience comparable to aortic elastin; hydrogel stiffness was easily modulated through the cross-linking ratio. Macromolecular phase separation of the RLP-PEG hydrogels offers the unique advantage of imparting a heterogeneous microstructure, which can be used to localize cells, through simple mixing and cross-linking. Assessment of degradation of the RLP by matrix metalloproteinases (MMPs) illustrated the specific proteolysis of the polypeptide in both its soluble form and when cross-linked into hydrogels. Finally, the successful encapsulation and viable three-dimensional culture of human mesenchymal stem cells (hMSCs) demonstrated the cytocompatibility of the RLP-PEG gels. Overall, the cytocompatibility, elastomeric mechanical properties, microheterogeneity, and degradability of the RLP-PEG hybrid hydrogels offer a suite of promising properties for the development of cell-instructive, structured tissue engineering scaffolds.