Functional recovery not correlated with axon regeneration through olfactory ensheathing cell-seeded scaffolds in a model of acute spinal cord injury.

The implantation of bioengineered scaffolds into lesion-induced gaps of the spinal cord is a promising strategy for promoting functional tissue repair because it can be combined with other intervention strategies. Our previous investigations showed that functional improvement following the implantation of a longitudinally microstructured collagen scaffold into unilateral mid-cervical spinal cord resection injuries of adult Lewis rats was associated with only poor axon regeneration within the scaffold. In an attempt to improve graft-host integration as well as functional recovery, scaffolds were seeded with highly enriched populations of syngeneic, olfactory bulb-derived ensheathing cells (OECs) prior to implantation into the same lesion model. Regenerating neurofilament-positive axons closely followed the trajectory of the donor OECs, as well as that of the migrating host cells within the scaffold. However, there was only a trend for increased numbers of regenerating axons above that supported by non-seeded scaffolds or in the untreated lesions. Nonetheless, significant functional recovery in skilled forelimb motor function was observed following the implantation of both seeded and non-seeded scaffolds which could not be correlated to the extent of axon regeneration within the scaffold. Mechanisms other than simple bridging of axon regeneration across the lesion must be responsible for the improved motor function.

Development of a Bioreactor to Culture Tissue Engineered Ureters Based on the Application of Tubular OPTIMAIX 3D Scaffolds.

Regenerative medicine, tissue engineering and biomedical research give hope to many patients who need bio-implants. Tissue engineering applications have already been developed based on bioreactors. Physiological ureter implants, however, do not still function sufficiently, as they represent tubular hollow structures with very specific cellular structures and alignments consisting of several cell types. The aim of this study was to a develop a new bioreactor system based on seamless, collagenous, tubular OPTIMAIX 3D prototype sponge as scaffold material for ex-vivo culturing of a tissue engineered ureter replacement for future urological applications. Particular emphasis was given to a great extent to mimic the physiological environment similar to the in vivo situation of a ureter. NIH-3T3 fibroblasts, C2C12, Urotsa and primary genitourinary tract cells were applied as co-cultures on the scaffold and the penetration of cells into the collagenous material was followed. By the end of this study, the bioreactor was functioning, physiological parameter as temperature and pH and the newly developed BIOREACTOR system is applicable to tubular scaffold materials with different lengths and diameters. The automatized incubation system worked reliably. The tubular OPTIMAIX 3D sponge was a suitable scaffold material for tissue engineering purposes and co-cultivation procedures.

Functional improvement following implantation of a microstructured, type-I collagen scaffold into experimental injuries of the adult rat spinal cord.

The formation of cystic cavitation following severe spinal cord injury (SCI) constitutes one of the major barriers to successful axonal regeneration and tissue repair. The development of bioengineered scaffolds that assist in the bridging of such lesion-induced gaps may contribute to the formulation of combination strategies aimed at promoting functional tissue repair. Our previous in vitro investigations have demonstrated the directed axon regeneration and glial migration supporting properties of microstructured collagen scaffold that had been engineered to possess mechanical properties similar to those of spinal cord tissues. Here, the effect of implanting the longitudinally orientated scaffold into unilateral resection injuries (2mm long) of the mid-cervical lateral funiculus of adult rats has been investigated using behavioural and correlative morphological techniques. The resection injuries caused an immediate and long lasting (up to 12 weeks post injury) deficit of food pellet retrieval by the ipsilateral forepaw. Implantation of the orientated collagen scaffold promoted a significant improvement in pellet retrieval by the ipsilateral forepaw at 6 weeks which continued to improve up to 12 weeks post injury. In contrast, implantation of a non-orientated gelatine scaffold did not result in significant functional improvement. Surprisingly, the improved motor performance was not correlated with the regeneration of lesioned axons through the implanted scaffold. This observation supports the notion that biomaterials may support functional recovery by mechanisms other than simple bridging of the lesion site, such as the local sprouting of injured, or even non-injured fibres.

Vascular potency of Sus scrofa bone marrow-derived mesenchymal stem cells: a progenitor source of medial but not endothelial cells.

Short-term thrombotic occlusion and compliance mismatch hamper clinical use of synthetic small-diameter tissue engineered vascular grafts. It is felt that preconditioning of the graft with intimal (endothelial) and medial (vascular smooth muscle) cells contributes to patency of the graft. Autologous, non-vessel-derived cells are preferred because of systemic vascular pathology and immunologic concerns. We tested in a porcine model whether cultured bone marrow-derived mononuclear cells, also referred to as mesenchymal stem cells (MSC), are a potential source of intimal or medial cells in vascular tissue engineering. We show that MSC cultured in endothelial medium do not gain an endothelial phenotype or functional characteristics, even after enrichment for CD31, culturing under flow, treatment with additional growth factors (vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2), or co-culture with microvascular endothelial cells (EC). On the other hand, we show that MSC cultured in MSC medium, but not in smooth muscle cell medium, show phenotypical and functional characteristics of vascular smooth muscle cells. We conclude that bone marrow-derived MSCs can be used as a bona fide source of medial, but not EC in small-diameter vascular tissue engineering.

Cytocompatibility of a novel, longitudinally microstructured collagen scaffold intended for nerve tissue repair.

Traumatic injury to the nervous system induces functional deficits as a result of axonal destruction and the formation of scar tissue, cystic cavitation, and physical gaps. Bioengineering bridging materials should ideally act as cell carriers for the implantation of axon growth-promoting glia, as well as supporting integration with host cell types. Here, we describe the cytocompatibility of a novel, micro-structured porcine collagen scaffold containing densely packed and highly orientated channels that, in three-dimensional (3D) tissue culture, supports attachment, proliferation, aligned process extension, and directed migration by populations of glial cells (olfactory nerve ensheathing cells and astrocytes) and orientated axonal growth by neurons (differentiated human SH-SY5Y neuroblastoma cell line). The seeded glia required several weeks to penetrate deeply into the highly porous scaffold, where they adopted an orientated morphology similar to that displayed in simple 2D cultures. The direct interaction between SH-SY5Y-derived nerve fibers and the collagen scaffold also resulted in highly orientated axonal growth. It is likely that biocompatible scaffolds that are capable of promoting glial cell attachment, migration, and highly orientated process outgrowth will be important for future repair strategies for traumatically injured nervous tissues.

In vitro cell alignment obtained with a Schwann cell enriched microstructured nerve guide with longitudinal guidance channels.

Therapeutic benefits of autologous nerve grafting in repair of peripheral nerve lesions have not been reached using any alternative nerve guide. Nevertheless, issues of co-morbidity and limited availability of donor nerves urgently ask for a need of bioartificial nerve guides which could either replace or complement autologous nerve grafts. It is increasingly appreciated that optimal nerve guides comprise both physical and molecular cues in support of peripheral axon regeneration. Now, we present a collagen-based microstructured 3D nerve guide containing numerous longitudinal guidance channels with dimensions resembling natural endoneurial tubes. Moreover, these nerve guides could be functionalized by Schwann cell (SC) seeding. Viable SCs did not only adhere to the nerve guide, but also migrated throughout the guidance channels. Of particular importance was the observation that SCs within the guidance channels formed cellular columns reminiscent of "Bands of Büngner", which are crucial structures in the natural process of peripheral nerve regeneration during the Wallerian degeneration. We, therefore, conclude that our orientated 3D nerve guides (decorated with SCs) with their physical and molecular properties may hold great promise in the repair of peripheral nerve lesion and serve as a basis for future experimental regeneration studies.

In vitro assessment of axonal growth using dorsal root ganglia explants in a novel three-dimensional collagen matrix.

The goal of this study was the development of a bioartificial nerve guide to induce axonal regeneration in the peripheral nervous system (PNS). In this in vitro study, the ability of a novel, 3-dimensional (3D), highly oriented, cross-linked porcine collagen scaffold to promote directed axonal growth has been studied. Collagen nerve guides with longitudinal guidance channels were manufactured using a series of chemical and mechanical treatments with a patented unidirectional freezing process, followed by freeze-drying (pore sizes 20-50 microm). Hemisected rat dorsal root ganglia (DRG) were positioned such that neural and non-neural elements could migrate into the collagen scaffold. After 21 days, S100-positive Schwann cells (SCs) migrated into the scaffold and aligned within the guidance channels in a columnar fashion, resembling "Bands of Büngner." Neurofilament-positive axons (mean length +/- SD 756 microm +/- 318 microm, maximum 1496 microm) from DRG neurons entered the scaffold where the growth within the guidance channels was closely associated with the oriented SCs. This study confirmed the importance of SCs in the regeneration process (neurotrophic theory). The alignment of SCs within the guidance channels supported directional axonal growth (contact guidance theory). The microstructural properties of the scaffold (open, porous, longitudinal pore channels) and the in vitro data after DRG loading (axonal regeneration along migrated and columnar-aligned SCs resembling "Band of Büngner") suggest that this novel oriented 3D collagen scaffold serves as a basis for future experimental regeneration studies in the PNS.

Impact of sterilization on the porous design and cell behavior in collagen sponges prepared for tissue engineering.

This study investigates the impact of different sterilization processes on structural integrity and stability of collagen sponges designed for tissue engineering. Collagen sponges with uniform pore size (20 microm) were sterilized either with ethylene oxide (EO) or gamma irradiation (2.5 Mrad). Gamma-sterilized sponges showed a dramatic decrease of resistance against enzyme degradation and severe shrinkage after cell seeding. Collapsed porosity inhibited fibroblasts and barred completely the human umbilical vein endothelial cell ingrowth into the sponges. On the contrary, the porous structure and stability of EO-sterilized sponges remained almost unaltered. Fibroblasts and endothelial cells exhibited favorable proliferation and migration within sponges with normal morphology. Tubular formation by seeded endothelial cells occurred early in the first week. Therefore, we emphasize that the impact of sterilization of biomaterials is substantial and any new procedure has to be evaluated by correlating the impact of the procedure on the porous structure with cell proliferation behavior.