Myosin II activity regulates neurite outgrowth and guidance in response to chondroitin sulfate proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in the CNS that inhibit axonal regeneration after CNS injury. Signaling pathways in neurons triggered by CSPGs are still largely unknown. In this study, using well-characterized in vitro assays for neurite outgrowth and neurite guidance, we demonstrate a major role for myosin II in the response of neurons to CSPGs. We found that the phosphorylation of myosin II regulatory light chains is increased by CSPGs. Specific inhibition of myosin II activity with blebbistatin allows growing neurites to cross onto CSPG-rich areas and increases the length of neurites of neurons growing on CSPGs. Using specific gene knockdown, we demonstrate selective roles for myosin IIA and IIB in these processes. Time lapse microscopy and immunocytochemistry demonstrated that CSPGs also inhibit cell adhesion and cell spreading. Inhibition of myosin II selectively accelerated neurite initiation without altering cell adhesion and spreading on CSPGs.

Delivery of adipose-derived precursor cells for peripheral nerve repair.

To test the hypothesis that the transplantation of adipose precursor cells (APCs) improves nerve regeneration and functional recovery, human APCs were transplanted into the lumen of a nerve guide in a 6-mm unilateral sciatic nerve defect in athymic rats. The three control groups for the study were biodegradable, polycaprolactone-based nerve conduit without APCs, autograft, and empty defect. Behavioral tests were performed every 3 weeks, and the sciatic functional index (SFI) was calculated based on measurements from the hindlimb prints. After 12 weeks, the nerve as well as right and left gastrocnemius muscles were removed and preserved for histological evaluation. Full regeneration of the sciatic nerve occurred on the rats that received the autograft, the guide, and the guide with APCs; no regeneration was observed on any of the rats in which the defect was left untreated (empty defect). APCs survived transplantation for up to 12 weeks in the injured peripheral nerve. No significant colocalization was observed between the immunostaining for glial fibrillary protein and anti-human lamin A/C, implying that the APCs did not differentiate into Schwann cells at the site of injury. In comparison with the rats with untreated defects, a decrease in muscle atrophy was observed on those rats that received the autograft and the guide with cells as indicated by the gastrocnemius muscle weight ratio and the muscle fiber ratio. Significant differences in SFI were observed 3 weeks postinjury between the rats in which the guide was left empty and those that received the guide with APCs; however, these differences were not observed at 12 weeks. The transplantation of APCs promoted the formation of a more robust nerve as evidenced by the results from the cross-sectional area of regenerated nerve, and the transplantation of APCs produced a decrease in muscle atrophy.

Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications.

Human adipose tissue has been recognized as a source of adult stem cells for tissue engineering applications such as bone, cartilage, and soft tissue repair. For the success of these tissue-engineering approaches, a cell delivery vehicle such as a hydrogel or scaffold is required to position the stem cells at the site of need. Surface modification techniques have been instrumental in the development of scaffolds that promote cell-surface interactions. In this study, poly(caprolactone) (PCL), surfaces were modified in order to promote the attachment and proliferation of adipose-derived stem cells (ASCs). RGD, YIGSR, and IKVAV peptide sequences derived from the extracellular matrix protein laminin were each covalently attached to an aminated polymer surface using carbodiimide chemistry. The surface was characterized using scanning electron microscopy (SEM), goniometry and X-ray photoelectron spectroscopy (XPS). The attachment and proliferation of ASCs was assessed on the different peptide-treated surfaces. XPS analysis confirmed the presence of the peptide sequences on the surface of the polymer as indicated by the increase in the nitrogen/carbon ratio on the surface of the polymer. Among all peptide sequences tested, IKVAV-treated surfaces had a significantly greater number of ASCs bound 2 and 3 days after cell seeding. SEM confirmed differences in the morphology of the cells attached to the three peptide-treated surfaces. These results indicate that IKVAV is a suitable peptide sequence for use in surface modification techniques aimed at improving the attachment of ASCs to a tissue-engineered scaffold.

Comparison of biodegradable conduits within aged rat sciatic nerve defects.

BACKGROUND: Considering that little is known about the peripheral nerve regenerative capacity of elderly patients, the authors studied nerve regenerative capacity in aged rats and compared the effect of three synthetic nerve guides with different material characteristics and porosity. The authors hypothesized that the use of a biodegradable composite nerve guide (CultiGuides) would promote nerve regeneration and functional recovery in a manner similar to treatment with autografts or U.S. Food and Drug Administration-approved polyglycolic acid Neurotubes in an aged rat sciatic nerve defect model. METHODS: Aged Sprague-Dawley rats (11 months old) underwent a 1-cm sciatic nerve resection in the right leg [group 1, control (contralateral leg samples), n = 10; group 2, negative (nerve gap defect), n = 6; group 3, autograft, n = 10; group 4, polycaprolactone, n = 10; group 5, CultiGuides, n = 10; and group 6, Neurotube, n = 10]. RESULTS: After 12 weeks, the negative group did not demonstrate any nerve regeneration. In the regenerated and distal nerve, all treated groups had increased myelinated areas compared with the negative control. In the regenerated nerve, there was a significant increase in myelination in the Neurotube group compared with the polycaprolactone group (p < 0.001). However, in the distal nerve, there were no differences among the treatment groups. Walking track analyses and gastrocnemius muscle weight ratios were not different among treatment groups 3 through 6. CONCLUSIONS: The results showed differences in myelination; Neurotubes promoted the highest degree of myelination (p < 0.001) as compared with all groups. The authors found no improvement in function of the repaired nerve as measured by percentage of autotomy, the sciatic function index, and gastrocnemius muscle weight. No group was able to recover function in this aged model.

Development of an assay for ozone-specific antioxidant capacity.

A method of determining the ozone-specific antioxidant capacity (OZAC) of lavage samples from the respiratory system was developed: Gaseous ozone (O(3)) was produced in cuvettes by irradiation with an ultraviolet lamp; aliquots of sample or of a saline control were then added and sufficient time was allowed for ozonation to reach completion; and an aliquot of indigo trisulfonate (ITS) was added to react with excess O(3). Because each molecule of O(3) rapidly bleaches one molecule of the deeply colored ITS, an OZAC value in concentration units was computed from the difference in light absorbance between the sample and the saline control multiplied by the extinction coefficient of ITS. Experiments in 0-40 micro M antioxidant solutions indicated that the OZAC values of uric acid and ascorbic acid were close to their actual concentrations and were independent of O(3) concentration. On the other hand, the OZAC of reduced glutathione and possibly human nasal lavage were nonlinearly related to antioxidant concentration and were directly related to O(3) concentration.