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.

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.

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.

Divergent regulation of GAP-43 expression and CNS neurite outgrowth by cyclic AMP.

Robust process outgrowth and high expression of the growth-associated protein GAP-43 seem to be intrinsic features of neurons, but both are down-regulated after axonal contact of target cells. We report that chronic exposure of the serotonergic CNS cell line RN46A to cyclic AMP analogs, forskolin, or cholera toxin represses GAP-43 expression in a dose dependent manner. Thus, cAMP could mediate a GAP-43 repressive signal that is initiated extracellularly. Activation of the cyclic AMP pathway by these same reagents, however, enhances the rate that RN46A cells extend neurites. This stimulation of neurite growth can occur during inhibition of new transcription, and in the absence of high levels of GAP-43. These findings demonstrate that a GAP-43-repressing intracellular signaling pathway exists, that repression of GAP-43 expression by cAMP is not directly coupled to inhibition of neurite growth, and that acceleration of growth cone advancement by cAMP is not dependent on the presence of GAP-43.

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.

Quantitative analysis of GAP-43 expression by neurons in microcultures using cell-ELISA.

A cell-ELISA technique is described which allows the quantification of GAP-43 protein in a large number of microcultures of adult dorsal root ganglion neurons. GAP-43 is measured in the 1-10 ng range, corresponding to the amount of GAP-43 present in fewer than 500 DRG neurons. Specificity of the assay is confirmed using Western blotting and immunocytochemistry. The GAP-43 content of adult DRG microcultures rises during 2 weeks in culture, although the number of surviving neurons decreases. The GAP-43 content of cultured adult DRG neurons is not increased by chronic exposure to added nerve growth factor after 7 days in vitro. However, GAP-43 is increased in DRG taken from animals with prior peripheral nerve injury, and is decreased by chronic exposure to dibutyryl cyclic AMP after 7 days in vitro. The method affords the sensitivity and statistical power to document modest changes in GAP-43 protein abundance in complex cultures.

Magnitude of GAP-43 induction following peripheral axotomy of adult rat dorsal root ganglion neurons is independent of lesion distance.

Regenerative axon growth in peripheral neurons is accompanied by increased expression of the growth-associated protein GAP-43. We examined the increase of GAP-43 immunoreactivity in DRG neurons following lesions at different distances along the sciatic nerve, using immunocytochemistry. To control for the variable involvement of DRG axons following injury at different sites, injured neurons were identified by retrograde labeling with Fluoro-Gold. GAP-43 labeling was similar for proximal, distal, and far-distal injuries when only injured neurons are considered. Our results stand in contrast to studies which show that GAP-43 upregulation in neurons of the central nervous system occurs only when lesions are made close to the cell body. This suggests that the mechanisms which control GAP-43 expression following injury differ between central and peripheral neurons.

The effects of a lesion or a peripheral nerve graft on GAP-43 upregulation in the adult rat brain: an in situ hybridization and immunocytochemical study.

We have sought to determine (1) if thalamic neurons upregulate the growth associated protein GAP-43 as a response to injury, or if a peripheral nerve graft is required to induce, enhance or sustain such a response, and (2) if thalamic neurons with different regenerative potentials also display different GAP-43 responses. Levels of GAP-43 protein (detected by LM and EM immunohistochemistry) and of GAP-43 mRNA (detected by in situ hybridization) were compared in the thalamus of adult rats between 1 d and 180 d after making a stab lesion or after implanting a peripheral nerve autograft. Stab injury is a sufficient stimulus to cause a transient upregulation in GAP-43 expression by neurons in the thalamus (both around the graft tip and in particular in the thalamic reticular nucleus) in the first week after injury but this response is both prolonged, and enhanced in the presence of a peripheral nerve graft. In addition, we demonstrate directly, by double labelling, that neurons of the thalamic reticular nucleus displaying high levels of the mRNA for GAP-43, have axons regenerating in the distal portion of the graft. These findings lend direct support to the hypothesis that upregulation of the GAP-43 gene is essential for prolonged regenerative axonal growth. We also demonstrate GAP-43 protein in graft Schwann cells and in brain astrocytes close to the site of graft implantation.

The downregulation of GAP-43 is not responsible for the failure of regeneration in freeze-killed nerve grafts in the rat.

Freeze-killed nerve grafts in rats are able to support limited axonal regeneration from severed peripheral nerves, but by 6 weeks postoperation, axonal elongation through the grafts ceases. To find out whether this limited regeneration may be related to GAP-43 expression, 4-cm freeze-killed nerve grafts were attached to the proximal stumps of severed tibial nerves in adult inbred Fischer rats. For comparison, tibial nerve crush, to allow functional regeneration, or section and ligation, which allows only abortive axonal sprouting, were also performed. After survival for 3 or 6 weeks, the lumbar spinal cord and L4 dorsal root ganglia were stained for GAP-43 mRNA. Freeze-killed grafts of 3-8 weeks duration were processed for GAP-43 immunocytochemistry. Three weeks after all three operations, comparable numbers of axotomized spinal motorneurons and primary sensory DRG neurons reexpressed high levels of GAP-43 mRNA. Six weeks after tibial nerve crush, the number of tibial motorneurons and DRG cells expressing GAP-43 mRNA returned to control levels but after section and ligation or freeze-killed nerve grafting many positively stained cells were still visible. GAP-43 immunoreactivity was detectable using immunocytochemistry in many unmyelinated axons which had regenerated into the freeze-killed grafts at all times. Both axonal profiles in contact with Schwann cells and those which lacked such contact were GAP-43 positive. These results suggest that the cessation of axonal regeneration into freeze-killed tibial nerve grafts is not the result of a down-regulation of GAP-43. Furthermore, the presence of high levels of GAP-43 alone is not sufficient to ensure prolonged axonal regeneration.

Astrocytes play a major role in the control of neuronal proliferation in vitro.

The elements that control neuronal proliferation are largely unknown. Proliferating neurons in cultures of goldfish brain were studied in an attempt to identify the cell types involved. Neuronal proliferation was found to occur only when the neuronal stem cells were in direct contact with astrocytes, and never directly on the substrate. The regulation of neuronal proliferation thus appears to be mediated, at least in part, by contact with astrocytes. In addition, neurite extension was inhibited by medium conditioned by fish astrocytes. Since neurite extension and neuronal proliferation are mutually exclusive processes, inhibition of neurite extension by soluble substances derived from the astrocytes is probably one of the mechanisms controlling neuronal proliferation. The complex reciprocal relationship between neurons and astrocytes is also demonstrated by an observed inhibition of astrocytic proliferation by medium conditioned by differentiating fish neurons. This inhibition of astrocytic proliferation might be part of a mechanism through which interference with neuronal differentiation by astrocytes is avoided. The results of this study thus suggest that astrocytes, in addition to their known roles in controlling neuronal migration, neuronal differentiation and neurite elongation, may also play a role in the control of neuronal proliferation.

Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons.

Peripheral nerve injury results in the increased synthesis and axonal transport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine whether the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se.

Axonal regeneration is associated with glial migration: comparison between the injured optic nerves of fish and rats.

The central nervous systems of mammals and fish differ significantly in their ability to regenerate. Central nervous system axons in the fish readily regenerate after injury, while in mammals they begin to elongate but their growth is aborted at the site of injury, an area previously shown to contain no glial cells. In the present study we compared the ability of glial cells to migrate and thus to repopulate the injured area in fish and rats, and used light and electron microscopy in an attempt to correlate such migration with the ability of axons to traverse this area. One week after the optic nerve was crushed, both axonal and glial responses to injury were similar in fish and rat. In both species glial cells were absent in the injured area (indicated by the disappearance of glial fibrillary acidic protein and vimentin immunoreactive cells from the site of injury in rat and fish, respectively), while at the same time axonal growth, indicated by expression of the growth-associated protein GAP-43, was restricted to the proximal part of the nerve. In fish, 2 weeks after the crush, GAP-43 staining (i.e., growing axons) was seen at the site of injury, in association with migrating vimentin-positive glial cells. One week later the site of injury in the fish optic nerve was repopulated by vimentin-positive glial cells, and GAP-43-positive axons had already traversed the site of injury and reached the distal part of the nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Fate of GAP-43 in ascending spinal axons of DRG neurons after peripheral nerve injury: delayed accumulation and correlation with regenerative potential.

Proteins characteristic of growing axons often fail to be induced or transported along axons that have been interrupted far from their cell bodies in the adult mammalian CNS. Here, we inquire whether long axons in the mammalian CNS can support efficient axonal transport and deposition of one such protein, GAP-43, when the protein is induced in neuron cell bodies. We have used immunocytochemistry to follow the fate of GAP-43 in dorsal column axons ascending the rat spinal cord from dorsal column axons ascending the rat spinal cord from dorsal root ganglion (DRG) neurons, after synthesis of the protein is induced in these cells by peripheral nerve injury. Sciatic nerve lesions do lead to an accumulation of GAP-43 in dorsal column axons derived from the lumbar DRG. However, in distal segments of these CNS axons, accumulation of GAP-43 is apparent only after a delay of 1-2 weeks, in contrast to its rapid accumulation in axon segments within the PNS environment, suggesting that deposition and stabilization of GAP-43 can be limited by local, posttranslational regulation. GAP-43 immunoreactivity subsides to control levels within 8 weeks after crush lesions that permit peripheral axon regeneration, but remains robust 8 weeks after resection lesions that prevent peripheral regeneration. Accumulation of GAP-43 in cervical dorsal column axons after peripheral nerve injury is closely correlated with the ability of these axons to respond to local cues capable of eliciting axon growth (Richardson and Verge, 1986).

Changes in the distribution of GAP-43 during the development of neuronal polarity.

GAP-43, a neuron specific growth-associated protein, is selectively distributed to the axonal domain in developing neurons; it is absent from dendrites and their growth cones. Using immunofluorescence microscopy, we have further examined the distribution of GAP-43 during the development of hippocampal neurons in culture, in order to determine when this polarized distribution arises. Cultured hippocampal neurons initially extend several short processes which have the potential to become either axons or dendrites. At this stage, before the morphological expression of polarity, GAP-43 is concentrated in the growth cones of these processes but is distributed more or less equally among them. Polarity becomes established when one of these processes elongates to become the axon. At the earliest stage when the emerging axon can be identified, GAP-43 is preferentially concentrated in its growth cone. During the next few days, as the remaining processes take on dendritic properties, they lose their residual GAP-43 immunoreactivity. Throughout development, GAP-43 remains highly concentrated in the axonal growth cone, but the concentration of GAP-43 in the axon shaft increases, beginning near the growth cone and progressing proximally until GAP-43 is uniformly distributed along the entire axon. At all stages of development, GAP-43 is also concentrated in the region of the Golgi apparatus. These results suggest that the selective sorting of at least one membrane protein into the axon coincides with the morphological expression of polarity. These results also raise the possibility that GAP-43 may play an important role in the early phases of axonal outgrowth, by which the functional polarity of neurons is established.

Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones.

Outgrowth of distinct axonal and dendritic processes is essential for the development of the functional polarity of nerve cells. In cultures of neurons from the hippocampus, where the differential outgrowth of axons and dendrites is readily discernible, we have sought molecules that might underlie the distinct modes of elongation of these two types of processes. One particularly interesting protein is GAP-43 (also termed B-50, F1 or P-57), a neuron-specific, membrane-associated phosphoprotein whose expression is dramatically elevated during neuronal development and regeneration. GAP-43 is among the most abundant proteins in neuronal growth cones, the motile structures that form the tips of advancing neurites, but its function in neuronal growth remains unknown. Using immunofluorescence staining, we show that GAP-43 is present in axons and concentrated in axonal growth cones of hippocampal neurons in culture. Surprisingly, we could not detect GAP-43 in growing dendrites and dendritic growth cones. These results show that GAP-43 is compartmentalized in developing nerve cells and provide the first direct evidence of important molecular differences between axonal and dendritic growth cones. The sorting and selective transport of GAP-43 may give axons and axonal growth cones certain of their distinctive properties, such as the ability to grow rapidly over long distances or the manner in which they recognize and respond to cues in their environment.

Axon elimination in the developing corticospinal tract of the rat.

Quantitative ultrastructural analysis of the corticospinal tract (CST) at the mid-thoracic spinal level in a series of early postnatal and young adult rats reveals that the tract is initially composed primarily of morphologically immature axon shafts, growth cones, and pale neuroglial processes. The total number of axons in the tract rises quickly to a peak level up to 90% greater than that present in the adult tract; it then declines, contemporaneously with the restriction of the areal extent of the set of spinally projecting cells in the cerebral cortex. During the time of axon elimination, axons remain small and morphologically immature, and small numbers of growth cones persist. Glial cells take on more mature forms within the tract several days before axon outgrowth ceases and myelination begins at the end of the second postnatal week. The fully mature CST retains a large complement of small, unmyelinated axons.

Topographic sequence of outgrowth of corticospinal axons in the rat: a study using retrograde axonal labeling with Fast blue.

The retrogradely transported dye, Fast blue, was injected into cervical or lumbar segments of the spinal cord of rats during the first days of life in order to label the cell bodies of origin of the corticospinal tract which is growing down the cord during that period. The first corticospinal axons arrive at cervical levels immediately after birth and all arise from a circumscribed group of layer V pyramidal cells in a small region of dorsal parietal cortex. This same cell group provides the corticospinal projection to lumbar segments of the spinal cord, the axons reaching those segments at the end of the first postnatal week. The area of lumbar projecting cells undergoes relatively little expansion and no diminution during subsequent weeks and into adulthood. The area occupied by cortical cells projecting to the spinal cord expands during the first postnatal week, but the axons of all these additional cells do not appear to invade the lower sequents of the spinal cord. By the end of the first week, corticospinal cells can be labeled in a continuous sheet throughout most of the extent of the frontal, parietal and cingulate cortex. During the second and third postnatal weeks, the area sending axons to the upper levels of the spinal cord diminishes and large areas bereft of retrogradely labeled corticospinal cells appear: laterally, in lateral frontal and lateral parietal cortex; dorsally, at the border of frontal and parietal cortex; medially, in medial frontal and cingulate cortex. The more restricted adult pattern is established at the end of the third week. Hence, the first cortical axons to advance down the spinal cord are those that will innervate the lumbar segments in the adult. Later addition of corticospinal axons involves only those projecting to upper cord segments. Within this group there are those which will establish persistent connections from appropriate cortical areas and others that will shortly be eliminated from inappropriate areas.

Growth of corticospinal axons on prosthetic substrates introduced into the spinal cord of neonatal rats.

Nitrocellulose implants treated with biological materials known to support neurite growth in vitro were introduced at thoracolumbar levels of the neonatal rat spinal cord before the arrival of growing corticospinal tract (CST) axons. Implant placement was designed to interrupt the normal CST growth path and provide a potential, alternative growth path. Subsequent growth of CST axons within the spinal cord in the vicinity of the implants was evaluated by labeling the axons with the anterograde transport of horseradish peroxidase. Untreated implants either blocked further CST axon growth or deflected CST axons to abnormal positions. Implants bearing living cells from spinal cord primary cultures were able to support the adhesion and growth of CST axons. Similarly, acellular implants coated with laminin, but not with poly-L-lysine supported the adhesion and growth of CST axons, suggesting that laminin or some other adhesive factor produced by immature neuroglial cells may be normally involved in CST axon growth and guidance.

Growing corticospinal axons by-pass lesions of neonatal rat spinal cord.

The anterograde transport of horseradish peroxidase was used to label newly growing corticospinal axons after they had entered lesioned regions of the neonatal rat spinal cord. Two types of lesions were made at thoracic and lumbar levels before the arrival of the first corticospinal axons. (1) Thermal lesions were produced by the brief application of a heated rod to the vertebral column and could destroy the normal growth path over several spinal segments. Corticospinal axons, when successful in growing distal to thermal lesions, did so at the same rate as in normal controls and retaining their normal relative positions and morphology, especially fasciculation. (2) Surgical lesions were produced by cutting the spinal cord and were limited to one segment but could result in a barrier in the normal growth path composed of a cyst or glial scar. Corticospinal axons that succeeded in growing distal to a surgical lesion did so by being deflected to unusual positions, became defasciculated, and sometimes their normal growth rate was slowed. That corticospinal axons could in many instances grow past the two types lf lesion suggests that a morphologically stereotyped glial scaffolding is not necessary for axon growth. The role of fasciculation in normal axon growth is highlighted by the disparate effects of the two types of lesion.