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

Disruption of Basal Lamina Components in Neuromotor Synapses of Children with Spastic Quadriplegic Cerebral Palsy

<div><p>Cerebral palsy (CP) is a static encephalopathy occurring when a lesion to the developing brain results in disordered movement and posture. Patients present with sometimes overlapping spastic, athetoid/dyskinetic, and ataxic symptoms. Spastic CP, which is characterized by stiff muscles, weakness, and poor motor control, accounts for ∼80% of cases. The detailed mechanisms leading to disordered movement in spastic CP are not completely understood, but clinical experience and recent studies suggest involvement of peripheral motor synapses. For example, it is recognized that CP patients have altered sensitivities to drugs that target neuromuscular junctions (NMJs), and protein localization studies suggest that NMJ microanatomy is disrupted in CP. Since CP originates during maturation, we hypothesized that NMJ disruption in spastic CP is associated with retention of an immature neuromotor phenotype later in life. Scoliosis patients with spastic CP or idiopathic disease were enrolled in a prospective, partially-blinded study to evaluate NMJ organization and neuromotor maturation. The localization of synaptic acetylcholine esterase (AChE) relative to postsynaptic acetylcholine receptor (AChR), synaptic laminin β2, and presynaptic vesicle protein 2 (SV2) appeared mismatched in the CP samples; whereas, no significant disruption was found between AChR and SV2. These data suggest that pre- and postsynaptic NMJ components in CP children were appropriately distributed even though AChE and laminin β2 within the synaptic basal lamina appeared disrupted. Follow up electron microscopy indicated that NMJs from CP patients appeared generally mature and similar to controls with some differences present, including deeper postsynaptic folds and reduced presynaptic mitochondria. Analysis of maturational markers, including myosin, syntrophin, myogenin, and AChR subunit expression, and telomere lengths, all indicated similar levels of motor maturation in the two groups. Thus, NMJ disruption in CP was found to principally involve components of the synaptic basal lamina and subtle ultra-structural modifications but appeared unrelated to neuromotor maturational status.</p></div

NMJs from CP Patients by Electron Microscopy.

<p>A) Top panel shows an electron micrograph of a CP NMJ. A zoomed out image is shown in the upper right corner with a red line marking a Schwann cell nucleus, cyan marking the Schwann cell membrane, magenta marking the nerve terminal, yellow marking the postsynaptic folds, and green marking the postsynaptic nucleus. Scale bars = 1 µm. B) A zoomed image of primary folds in the CP NMJ is also shown. The blue lines indicate the general depth of postsynaptic fold penetrance into the muscle. For data analysis, each fold was measured separately using ImageJ software to draw trace lines for individual folds following the methods outlined by Banks et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070288#pone.0070288-Banks1" target="_blank">[27]</a>. C) A CP NMJ with a single mitochondrion in the nerve terminal (arrow). The total area occupied by mitochondria was calculated with ImageJ software by tracing each mitochondrion segment and measuring the summed mitochondrial area relative to the total area of the terminal.</p

CP NMJs have a reduced mitochondrial load.

<p>A) Confocal images showing double staining for acetylcholine receptor by α-bungarotoxin (red) and mitochondria (green) using a mouse monoclonal antibody for complex VI subunit I of cytochrome c oxidase (Mitosciences, Oregon). Images represent the mean intensities of fluorescence signal across 10 z-stacked images by confocal microscopy. CP NMJs show a decreased intensity for synaptic mitochondria. B) Average mitochondrial fluorescence intensities of NMJs of CP (93.25±8.017 SEM, n = 25 NMJs) and control (156.70±12.035 SEM, n = 25 NMJs) samples. Bars represent the mean ± SEM of immunofluorescence intensity in arbitrary fluorescence units based on the signal from the mitochondrial stain at the NMJs of CP and control samples.</p

Determination of appositional score.

<p>Sample images are shown for a single NMJ from a <i>spinalis</i> biopsy of a child with CP. The distribution of AChR (Panel A), AChE (B), and the composite image of both AChR and AChE (C) are shown. Each image was thresholded using the standard algorithm provided in the Image Pro Software, which objectively identifies foreground versus background pixels based on the distribution of intensities in the image, resulting in images D, E, and F. Distinct boundaries were reproducibly identifiable relative to background using this approach. A line drawn through the NMJ (G) provided the corresponding histogram (H) which displayed the intensities of AChR and AChE staining across the NMJ. The pixels above the preset threshold levels were categorized and counted to determine appositional scores.</p

Comparison of neuromotor maturation in idiopathic scoliosis (IS) and CP patients.

<p>A) <i>Spinalis</i> sample from a control patient showing distinctly stained type I (dark) and type II (light) fibers using an ATPase assay at low pH (4.65). B) <i>Spinalis</i> sample from a patient with CP showing type I and type II fibers with an overall mature appearance similar to that seen in panel A, except for an increased predominance of type I fibers. C) Analysis of fiber typing: at least 100 fibers were enumerated as either type I or type II fibers for each patient sample (n = 10 per group) and compared for fiber type predominance. The average frequency of each type is shown. Patients with CP had a significantly higher frequency of type I fibers than patients with IS (p = 0.013 by Mann-Whitney) and a significantly lower frequency of type II fibers (p = 0.044 by Mann-Whitney). D) CP (lanes 6–8) and IS (lanes 1–4) muscles were analyzed for the presence of fast, slow, and embryonic MYHs by Western blot. Fetal muscle (lane 5) was used as a positive control for immaturity and the presence of embryonic MYH. Antibodies used are as follows: A4.74 (fast) for MYH1, MYH2, and MYH4; F1.652 (embryonic) for MYH3; A4.951 (slow type I) for MYH7. Neither CP nor control muscle contained the embryonic MYH isoform. E) RNA was extracted from <i>spinalis</i> muscle and subjected to real time RT-PCR. Data are presented as mean (± SD) fold differences compared to fetal tissue (n = 12 CP, 12 IS). AChRα1 (CHRNA1), AChRδ (CHRND), AChRε (CHRNE), AChRγ (CHRNG), AChRα7 (CHRNA7), myogenin (MYOG), AChE (ACHE), and β2 syntrophin (SNTB2), did not show significant differences between the CP and control groups. β1 syntrophin (SNTB1) expression was significantly higher in CP samples than control samples (p<0.001 by Mann-Whitney).</p

Median values and ranges for NMJ component comparisons; patients pooled by diagnosis.

<p>R = AChR; O = outside of; E = AChE; L = laminin β2; V = SV2.</p>*<p>represents p values<0.05 by Mann-Whitney.</p

Telomere lengths expressed as T/S ratio relative to normal leg muscle in patients with CP, idiopathic scoliosis, or DMD.

<p>T/S Ratio Relative to Normal.</p

Median values and ranges for NMJ non-colocalization scores; each patients received a score equivalent to the median values within their NMJs.

<p>R = AChR; O = outside of; E = AChE; L = laminin β2; V = SV2.</p>*<p>represents p values<0.05 by Mann-Whitney.</p

Comparison of individual CP patients with the idiopathic scoliosis group.

<p>Pair-wise Mann-Whitney tests were run to determine how individual CP patients compared to the idiopathic scoliosis groups. R = AChR; O = outside of; E = AChE; L = laminin β2; V = SV2. +++ indicates that the child had significantly higher values than the control group, while −−− indicates that the child had significantly lower values than the control group (p≤0.002; note that this level of significance was selected because there were 25 CP children compared to the control/IS group).</p