Ingo Heschel

Human preadipocytes seeded on freeze-dried collagen scaffolds investigated in vitro and in vivo

Currently, there is no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, after tumor resection and in hereditary and congenital defects (e.g. Rombergs disease, Poland syndrome). The autologous transplantation of mature adipose tissue has poor results. In this study human preadipocytes of young adults were isolated and cultured. 10(6) preadipocytes were seeded onto collagen sponges with uniform 40 microm pore size and regular lamellar structure and implanted into immunodeficient mice. Collagen sponges without preadipocytes were used in the controls. Macroscopical impression, weight, thickness, histology, immunohistochemistry (scaffold structure, cellularity, penetration depth of the seeded cells) and ultrastructure were assessed after 24 h in vitro and after explantation at 3 and 8 weeks. Preadipocytes penetrated the scaffolds 24 h after seeding at a depth of 299+/-55 microm before implantation. Macroscopically after 3 and 8 weeks in vivo layers of adipose tissue accompanied by new vessels were found on all preadipocyte/collagen grafts. The control grafts appeared unchanged without vessel ingrowth. There was a significant weight loss of all grafts between 24 h in vitro and 3 weeks in vivo (p < 0.05), whereas there was only a slight weight reduction from week 3 to 8. The thickness decreased in the first 3 weeks (p < 0.05) in all grafts. The preadipocyte/collagen grafts were thinner but had a higher weight than the controls at this point in time. The histology showed adipose tissue and a rich vascularisation adherent to the scaffolds under a capsule. The control sponges contained only few cells and a capsule but no adipose tissue. Human-vimentin positive cells were found in all preadipocyte/collagen grafts but not in the controls, penetrating 1188+/-498 microm (3 weeks) and 1433+/-685 microm (8 weeks). Ultrastructural analysis showed complete in vivo differentiation of viable adipocytes in the sponge seeded with preadipocytes. Formation of extracellular matrix was more pronounced in the preadipocyte/collagen grafts. The transplantation of isolated and cultured preadipocytes within a standardised collagen matrix resulted in well-vascularised adipose-like tissue. It is assumed that a pore size greater than 40 microm is required, as preadipocytes enlarge during differentiation due to incorporation of lipids.

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.

Dendritic ice morphology in unidirectionally solidified collagen suspensions

Collagen is a fibrillar protein which is commonly used as a biodegradable biomaterial. A typical application of such a biomaterial is a freeze-dried collagen sponge which is primarily used as a permanent skin replacement for the treatment of deep dermal burns. Especially to diminish scar formation caused by a severe loss of skin, it is necessary to induce a cell migration into the sponges and the regeneration of endogenous tissue within the sponges. Thereby, the three-dimensional structure and the pore sizes of the collagen sponges strongly influence the wound healing. In order to study this influence, the development of a process to manufacture collagen sponges with an adjustable and homogeneous three-dimensional structure is necessary. The porous structure of freeze-dried sponges corresponds to the ice crystal morphology after freezing. In order to form an adjustable and homogeneous ice morphology, the unidirectional solidification of collagen suspensions was investigated. All experiments were performed in a cryomicroscope according to the Bridgman technique. To induce a constitutional supercooling leading to the breakdown of the planar ice front and the formation of a stable dendritic ice crystal morphology (without side branches), the addition of solutes to the basic collagen suspension is required. We used acetic acid and ethanol as soluble additives, because they are suitable for medical application. The effects of the temperature gradient, the ice front velocity, and the solute concentration on the primary spacing and the dendrite sizes were examined. In order to find a simplified experimental system, acetic acid solutions without collagen were solidified under the same freezing conditions. Although the primary spacings and the dendrite sizes almost varied in the same range for solutions and suspensions, they were influenced in a different manner by the freezing parameters and the solute concentrations. Varying the aforementioned parameters the primary spacing could be adjusted in the range from 40 to 60 μm. Furthermore, the ice crystal sizes, which correspond to the pore size after freeze-drying, could be varied between 30 and 50 μm.

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.

Motor outcome and allodynia are largely unaffected by novel olfactory ensheathing cell grafts to repair low-thoracic lesion gaps in the adult rat spinal cord.

Olfactory ensheathing cells (OEC) are a promising graftable cell population for improving functional outcomes after experimental spinal cord injury. However only few studies have focused on experimental models with large cavitations, which require bridging substrates to transfer and maintain the donor cells within the lesion site. Here, a state-of-the-art collagen-based multi-channeled three dimensional scaffold was used to deliver olfactory ensheathing cells to 2 mm long unilateral low-thoracic hemisection cavities. For a period of 10 weeks, allodynia of the hindpaws was monitored using the von Frey hair filament test, while an extensive analysis of motor ability was performed with use of the CatWalk gait analysis system and the BBB locomotor scale. No substantial improvement or deterioration of motor functions was induced and there was no effect on lesion-induced allodynia. On the basis of these data, we conclude that relatively large spinal cord lesions with cavitation may present additional hurdles to the therapeutic effect of OEC. Future studies are needed to address the nature that such lesion cavities place on cell grafts.

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.

Analysis of desorption and diffusion during secondary drying in vacuum freeze-drying of hydroxyethyl starch

Abstract Freeze-drying is a common process for the long-term preservation of biochemicals derived from aqueous solutions. One of the important factors involved in a successful freeze-drying procedure is the secondary drying step. The effect of temperature (254–293 K) and pressure (5–44 Pa) on water mass transport during the secondary drying stage has been examined for concentrated, aqueous solutions of hydroxyethyl starch, a stabilizing agent common in the freeze-drying of biological material. Desorption equilibria were reached after evacuation for 5–8 d. The equilibrium water content W showed a significant variation with water activity aw. Based on a Freundlich Sorption model, the equilibrium water content may be described by the equation W = 0.578 · aw0.515. Water diffusion coefficients were derived from the equilibration behaviour according to Ficks diffusion law and were found to vary over the range 1.0–2.8 × 10−11m2s−1. The mass transport properties and their dependence on temperature, pressure and composition are discussed in detail. The importance of these results for a comprehensive understanding of the freeze-drying process, efficient process design and final product quality are emphasized.

Technical description of a new low-temperature freeze-drying device

Abstract A new freeze-drying device was built to allow lyophilisation at very low temperatures. This paper gives a short description of both the construction and performance data. The device is based on a simple single-chamber principle. The condenser, as well as the sample tray, consist of copper pipes cooled respectively by liquid nitrogen and the external ethanol flow of a cryostat. Thus, the minimal temperature of the condenser is below −190°C and the sample tray reaches about −80°C, both measured by Pt100 resistance temperature detectors. For higher drying temperatures, the device can optionally operate in an alternative mode without liquid nitrogen. The data acquisition and the dynamic control of the freeze-drying device is computer-assisted with specially designed software modules based on LabVIEW® (National Instruments).

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 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.