David J. Schreyer

Differential hypertrophy and atrophy among all types of cutaneous innervation in the glabrous skin of the monkey hand during aging and naturally occurring type 2 diabetes

Diabetic neuropathy (DN) is a common severe complication of type 2 diabetes. The symptoms of chronic pain, tingling, and numbness are generally attributed to small fiber dysfunction. However, little is known about the pathology among innervation to distal extremities, where symptoms start earliest and are most severe, and where the innervation density is the highest and includes a wide variety of large fiber sensory endings. Our study assessed the immunochemistry, morphology, and density of the nonvascular innervation in glabrous skin from the hands of aged nondiabetic rhesus monkeys and from age‐matched monkeys that had different durations of spontaneously occurring type 2 diabetes. Age‐related reductions occurred among all types of innervation, with epidermal C‐fiber endings preferentially diminishing earlier than presumptive Aδ‐fiber endings. In diabetic monkeys epidermal innervation density diminished faster, became more unevenly distributed, and lost immunodetectable expression of calcitonin gene‐related peptide and capsaicin receptors, TrpV1. Pacinian corpuscles also deteriorated. However, during the first few years of hyperglycemia, a surprising hypertrophy occurred among terminal arbors of remaining epidermal endings. Hypertrophy also occurred among Meissner corpuscles and Merkel endings supplied by Aβ fibers. After longer‐term hyperglycemia, Meissner corpuscle hypertrophy declined but the number of corpuscles remained higher than in age‐matched nondiabetics. However, the diabetic Meissner corpuscles had an abnormal structure and immunochemistry. In contrast, the expanded Merkel innervation was reduced to age‐matched nondiabetic levels. These results indicate that transient phases of substantial innervation remodeling occur during the progression of diabetes, with differential increases and decreases occurring among the varieties of innervation. J. Comp. Neurol. 501:543–567, 2007.

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and Talpha1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced.

Constitutive expression of GAP-43 correlates with rapid, but not slow regrowth of injured dorsal root axons in the adult rat.

It has been postulated that the neuronal growth-associated protein GAP-43 plays an essential role in axon elongation. Although termination of developmental axon growth is generally accompanied by a decline in expression of GAP-43, a subpopulation of dorsal root ganglion (DRG) neurons retains constitutive expression of GAP-43 throughout adulthood. Peripheral nerve regeneration occurring subsequent to injury of the peripheral axon branches of adult DRG neurons is accompanied by renewed elevation of GAP-43 expression. Lesions of DRG central axon branches in the dorsal roots are also followed by some regenerative growth, but little or no increase in GAP-43 expression above the constitutive level is observed. To determine whether dorsal root axon regeneration occurs only from neurons which constitutively express GAP-43, we have used retrograde fluorescent labeling to identify those DRG neurons which extend axons beyond a crush lesion of the dorsal root. Only GAP-43 immunoreactive neurons supported axon regrowth of 7 mm or greater within the first week. At later times, axon regrowth is seen to occur from neurons both with and without GAP-43 immunoreactivity. We conclude that regeneration of injured axons within the dorsal root is not absolutely dependent on the presence of GAP-43, but that expression of GAP-43 is correlated with a capacity for rapid growth.

Influence of Calcium Ions on Cell Survival and Proliferation in the Context of an Alginate Hydrogel

One goal of biofabrication is to incorporate living cells into artificial scaffolds in order to repair damaged tissues or organs. Although there are many studies on various biofabrication techniques, the maintenance of cell viability during the biofabrication process and cell proliferation after the process is still a challenging issue. Construction of scaffolds using hydrogels composed of natural materials can avoid exposure of cells to harsh chemicals or temperature extremes but can still entail exposure to non-physiological conditions, causing cell damage or even death. This paper presents an experimental investigation into the influence on Schwann cell survival and proliferation of calcium used for ionic crosslinking of alginate hydrogel during the biofabrication process. The experimental results obtained show the viability and proliferation capacity of cells, either suspended in cell culture medium or encapsulated in hydrogel, and vary with the calcium concentration and the time period of cells exposed to the calcium environment. The experimental results also show the alginate concentration and cell density, that have profound influence on cell survival and proliferation, and solution viscosity as well. This study suggests the incorporation of living cells in calcium-crosslinked hydrogel in the biofabrication process can be regulated for controlled cell survival and proliferation.

Cyclic AMP Prevents an Increase in GAP-43 but Promotes Neurite Growth in Cultured Adult Rat Dorsal Root Ganglion Neurons

High expression of the growth-associated protein GAP-43 in neurons is correlated with developmental and regenerative axon growth. It has been postulated that during development and after injury, GAP-43 expression is elevated due to the unavailability of a target-derived repressive signal, but that GAP-43 expression then declines upon target contact. Here we examine the cyclic AMP second messenger signaling pathway to determine if it might mediate retrograde transmission of a signal which represses GAP-43 expression and inhibits growth. Cultures of adult rat dorsal root ganglia were chronically exposed to membrane-permeable analogs of cyclic AMP and activators of adenyl cyclase. These treatments caused GAP-43 protein levels to decrease in a dose-dependent manner, although neuronal survival was not affected. GAP-43 mRNA was also decreases by cyclic AMP. GAP-43 protein levels were not repressed by neurotrophins, cytokines, or other agents. Surprisingly, cyclic AMP caused an increase in the rate of neurite outgrowth, even though the neurons were partially depleted of GAP-43. Growth stimulation was quickly inducible and reversible, could occur in the presence of transcription inhibitors, and did not entail alterations in branching pattern. These findings suggest that axon growth involving high levels of GAP-43 is distinct from the growth stimulation which is rapidly induced by cyclic AMP.

PLGA/alginate composite microspheres for hydrophilic protein delivery.

Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate.

X-ray diffraction enhanced imaging as a novel method to visualize low-density scaffolds in soft tissue engineering.

Scaffold visualization is challenging yet essential to the success of various tissue engineering applications. The aim of this study was to explore the potential of X-ray diffraction enhanced imaging (DEI) as a novel method for the visualization of low density engineered scaffolds in soft tissue. Imaging of the scaffolds made from poly(L-lactide) (PLLA) and chitosan was conducted using synchrotron radiation-based radiography, in-line phase-contrast imaging (in-line PCI), and DEI techniques as well as laboratory-based radiography. Scaffolds were visualized in air, water, and rat muscle tissue. Compared with the images from X-ray radiography and in-line PCI techniques, DEI images more clearly show the structure of the low density scaffold in air and have enhanced image contrast. DEI was the only technique able to visualize scaffolds embedded in unstained muscle tissue; this method could also define the microstructure of muscle tissue in the boundary areas. At a photon energy of 20 KeV, DEI had the capacity to image PLLA/chitosan scaffolds in soft tissue with a sample thickness of up to 4 cm. The DEI technique can be applied at high X-ray energies, thus facilitating lower in vivo radiation doses to tissues during imaging as compared to conventional radiography.

Effects of laminin blended with chitosan on axon guidance on patterned substrates

Axon guidance is a crucial consideration in the design of tissue scaffolds used to promote nerve regeneration. Here we investigate the combined use of laminin (a putative axon adhesion and guidance molecule) and chitosan (a leading candidate base material for the construction of scaffolds) for promoting axon guidance in cultured adult dorsal root ganglion (DRG) neurons. Using a dispensing-based rapid prototyping (DBRP) technique, two-dimensional grid patterns were created by dispensing chitosan or laminin-blended chitosan substrate strands oriented in orthogonal directions. In vitro experiments illustrated DRG neurites on these patterns preferentially grew upon and followed the laminin-blended chitosan pathways. These results suggest that an orientation of neurite growth can be achieved in an artificially patterned substrate by creating selectively biofunctional pathways. The DBRP technique may provide improved strategies for the use of biofunctional pathways in the design of three-dimensional scaffolds for guidance of nerve repair.

Experimental approaches to vascularisation within tissue engineering constructs

Tissue engineering opens up a new area to restore the function of damaged tissue or replace a defective organ. Common strategies in tissue engineering to repair and form new tissue containing a functional vascular network include the use of cells, growth factors, extracellular matrix proteins, and biophysical stimuli. Yet, formation of well-distributed, interconnected, and stable vascular network still remains challenging. In addition, anastomoses with host vasculature upon implantation and long-time survival of the new blood vessel in vivo are other critical issues to be addressed. This paper presents a brief review of recent advances in vascularization in vitro as well as in vivo for tissue engineering, along with suggestions for future research.

Preparation and characterization of alginate microspheres for sustained protein delivery within tissue scaffolds

Tissue engineering scaffolds are designed not only to provide structural support for the repair of damaged tissue, but can also serve the function of bioactive protein delivery. Here we present a study on the preparation and characterization of protein-loaded microspheres, either alone or incorporated into mock tissue scaffolds, for sustained protein delivery. Alginate microspheres were prepared by a novel, small-scale water-in-oil emulsion technique and loaded with fluorescently labeled immunoglobulin G (IgG). Microsphere size appears to be influenced by the magnitude and distribution of force generated by mechanical stirring during emulsion. Protein release studies show that sustained IgG release from microspheres could be achieved and that application of a secondary coating of chitosan could further slow the rate of protein release. Preservation of bioactivity of released IgG protein was confirmed using an immunohistochemical assay. When IgG-loaded microspheres were incorporated into mock scaffolds, initial protein release was diminished and the overall time course of release was extended. The present study demonstrates that protein-loaded microspheres can be prepared with a controlled release profile and preserved biological activity, and can be incorporated into scaffolds to achieve sustained and prolonged protein delivery in a tissue engineering application.