From macroscopic mechanics to cell-effective stiffness within highly aligned macroporous collagen scaffolds.

In the design of macroporous biomaterial scaffolds, attention is payed predominantly to the readily accessible macroscopic mechanical properties rather than to the mechanical properties experienced by the cells adhering to the material. However, the direct cell mechanical environment has been shown to be of special relevance for biological processes such as proliferation, differentiation and extracellular matrix formation both in vitro and in vivo. In this study we investigated how individual architectural features of highly aligned macroporous collagen scaffolds contribute to its mechanical properties on the macroscopic vs. the microscopic scale. Scaffolds were produced by controlled freezing and freeze-drying, a method frequently used for manufacturing of macroporous biomaterials. The individual architectural features of the biomaterial were carefully characterized to develop a finite element model (FE-model) that finally provided insights in the relation between the biomaterial's mechanical properties on the macro-scale and the properties on the micro-scale, as experienced by adhering cells. FE-models were validated by experimental characterization of the scaffolds, both on the macroscopic and the microscopic level, using mechanical compression testing and atomic force microscopy. As a result, a so-called cell-effective stiffness of these non-trivial scaffold architectures could be predicted for the first time. A linear dependency between the macroscopic scaffold stiffness and the cell-effective stiffness was found, with the latter being consistently higher by a factor of 6.4 ±â€¯0.6. The relevance of the cell-effective stiffness in controlling progenitor cell differentiation was confirmed in vitro. The obtained information about the cell-effective stiffness is of particular relevance for the early stages of tissue regeneration, when the cells first populate and interact with the biomaterial. Beyond the specific biomaterial investigated here, the introduced method is transferable to other complex biomaterial architectures. Design-optimization in 3D macroporous scaffolds that are based on a deeper understanding of the mechanical environment provided to the cells will help to enhance biomaterial-based tissue regeneration approaches.

A biomaterial with a channel-like pore architecture induces endochondral healing of bone defects.

Biomaterials developed to treat bone defects have classically focused on bone healing via direct, intramembranous ossification. In contrast, most bones in our body develop from a cartilage template via a second pathway called endochondral ossification. The unsolved clinical challenge to regenerate large bone defects has brought endochondral ossification into discussion as an alternative approach for bone healing. However, a biomaterial strategy for the regeneration of large bone defects via endochondral ossification is missing. Here we report on a biomaterial with a channel-like pore architecture to control cell recruitment and tissue patterning in the early phase of healing. In consequence of extracellular matrix alignment, CD146+ progenitor cell accumulation and restrained vascularization, a highly organized endochondral ossification process is induced in rats. Our findings demonstrate that a pure biomaterial approach has the potential to recapitulate a developmental bone growth process for bone healing. This might motivate future strategies for biomaterial-based tissue regeneration.

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.

Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts.

Tissue engineering of skeletal muscle from cultured cells has been attempted using a variety of synthetic and natural macromolecular scaffolds. Our study describes the application of artificial scaffolds (collagen sponges, CS) consisting of collagen-I with parallel pores (width 20-50 microm) using the permanent myogenic cell line C(2)C(12). CS were infiltrated with a high-density cell suspension, incubated in medium for proliferation of myoblasts prior to further culture in fusion medium to induce differentiation and formation of multinucleated myotubes. This resulted in a parallel arrangement of myotubes within the pore structures. CS with either proliferating cells or with myotubes were grafted into the beds of excised anterior tibial muscles of immunodeficient host mice. The recipient mice were transgenic for enhanced green fluorescent protein (eGFP) to determine a host contribution to the regenerated muscle tissue. Histological analysis 14-50 days after surgery showed that donor muscle fibres had formed in situ with host contributions in the outer portions of the regenerates. The function of the regenerates was assessed by direct electrical stimulation which resulted in the generation of mechanical force. Our study demonstrated that biodegradable CS with parallel pores support the formation of oriented muscle fibres and are compatible with force generation in regenerated muscle.

'UroMaix' scaffolds: novel collagen matrices for application in tissue engineering of the urinary tract.

Reconstruction of bladder and ureter tissue is indicated in cases of injury, stenosis, infection or tumor. Substitution by ileum, colon or pure synthetic polymers generates a variety of complications. Biohybrid tissue mimicking structural and functional attributes of the multilayered wall architecture of the urinary conduit may be the solution to current problems. This study reports on porcine urinary tract cells isolated and placed on UroMaix matrices with different degrees of cross-linking produced from highly purified type I collagen from medically approved porcine tissue. A patented procedure revealed membrane structures composed of a dense fibrous side and an open fibrous side. These scaffolds with the porcine urinary tract cells were incubated in a batch culture system for up to 14 days. Cell growth and topographical orientation were examined. Urothelial cells showed maximum attachment and a significant increase of living cells on the dense fiber layer of UroMaix-1. No attachment of urothelial cells occurred on the other prototypes. Smooth muscle cells showed similar behavior within the open fiber layer of all UroMaix matrices. Both urothelial and smooth muscle cells retained their phenotypes as demonstrated by the immunostaining of epithelial cytokeratin 18 and the smooth muscle myosin heavy chain respectively. Thus we could show that UroMaix scaffolds support the attachment and proliferation of urinary tract cells. The elastomeric properties of the collagenous matrices promise attractive applications in the tissue engineering of the urinary tract with its high mechanical demands.

Preparation of collagen scaffolds and their applications in tissue engineering.

Freeze-dried collagen scaffolds can be used for a variety of medical tissue engineering applications. The pore structure of the scaffolds might play a decisive role for the inoculation, growth and differentiation of the cells. For a controlled 3D-cell growth the pore structure needs to be homogeneous and the pore size individually adjustable. For lyophilised scaffolds, the pore structure is determined by the ice crystal morphology during freezing under steady conditions. Scaffolds with a homogeneous pore structure and a range of pore size between 25 and 100 microns were reached. Cells such as preadipocytes, keratinocytes, and fibroblasts showed to adhere well to the collagen matrix.

Magnification of the pore size in biodegradable collagen sponges.

In tissue engineering cells are often combined with a carrying structure with collagen being a suitable material to form a 3D-scaffold. A process to manufacture collagen sponges with an adjustable and homogeneous structure has been developed at the Helmholtz-Institute. Using this process, collagen suspensions are frozen directionally and subsequently vacuum-dried. One clinical application in which these scaffolds can be used is soft tissue reconstruction. Various soft tissue defects require an adequate replacement, e.g. in the case of severe burn wounds, or after tumour resections. Collagen (type I) sponges, which are cultured with preadipocytes, may be used to regenerate such defects. In this case, pore sizes of approximately 100 microm are desired to allow a complete differentiation of preadipocytes into adipocytes. Based on known technology to manufacture collagen sponges with an adjustable and homogeneous pore structure, research on the increase of pore size beyond the previous limit of 40 microm was necessary in order to enable soft tissue replacement. A scaffold with an average pore size of 100 microm was obtained.

Control of pore structure and size in freeze-dried collagen sponges.

Because of many suitable properties, collagen sponges are used as an acellular implant or a biomaterial in the field of tissue engineering. Generally, the inner three-dimensional structure of the sponges influences the behavior of cells. To investigate this influence, it is necessary to develop a process to produce sponges with a defined, adjustable, and homogeneous pore structure. Collagen sponges can be produced by freeze-drying of collagen suspensions. The pore structure of the freeze-dried sponges mirrors the ice-crystal morphology after freezing. In industrial production, the collagen suspensions are solidified under time- and space-dependent freezing conditions, resulting in an inhomogeneous pore structure. In this investigation, unidirectional solidification was applied during the freezing process to produce collagen sponges with a homogeneous pore structure. Using this technique the entire sample can be solidified under thermally constant freezing conditions. The ice-crystal morphology and size can be adjusted by varying the solute concentration in the collagen suspension. Collagen sponges with a very uniform and defined pore structure can be produced. Furthermore, the pore size can be adjusted between 20-40 microm. The thickness of the sponges prepared during this research was 10 mm.

Experimental MR imaging-guided interstitial cryotherapy of the brain.

BACKGROUND AND PURPOSE: Hyperthermal ablation techniques such as laser or RF ablation require dedicated heat-sensitive MR imaging sequences for monitoring MR imaging--guided interventions. Because cryotherapy does not have these limitations, the purpose of this study was to evaluate the feasibility of MR imaging--guided percutaneous cryotherapy of the brain. METHODS: An experimental cryoprobe with an outer diameter of 2.7 mm was inserted into the right frontal lobe of 11 healthy pigs under MR imaging control. Freezing procedures were monitored by using an interventional 1.5-T magnet and a gradient-echo sequence with radial k-space trajectories, a fast T2-weighted single-shot spin-echo sequence, and a T1-weighted single-shot gradient-echo sequence. In three animals, the procedure was also monitored by using dynamic CT. A freeze-thaw cycle with a duration of 3 minutes was repeated three times per animal. Follow-up MR images were obtained 3, 7, and 14 days after cryotherapy by using conventional MR sequences. Six animals were killed 7 days after intervention, and five animals were killed 14 days after intervention. The brains were sectioned, and the histologic findings of the lesions were compared with the MR imaging appearance. RESULTS: No artifacts due to the probe were observed on the MR images or CT scans. The ice formation (mean diameter, 12.5 mm) was very well delineated as a signal-free sphere. MR monitoring of the freezing procedure yielded a significantly higher ice:tissue contrast than did CT. The size of the ice ball as imaged by MR imaging and CT during the intervention correlated well with the MR imaging appearance of the lesions at the 14-day follow-up examination and with the histologic findings. Histologically, coagulation necrosis and gliosis were found, surrounded by a transition zone of edema and a disrupted blood-brain barrier, corresponding to a contrast-enhancing rim around the lesions on follow-up MR images. CONCLUSION: MR imaging-guided cryotherapy of the brain is possible and allows a precise prediction of the resulting necrosis. MR imaging of the freezing process does not require heat-sensitive sequences and is superior to CT for monitoring of cryoablation.

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. Romberg's 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.

Variation of the HES concentration for the cryopreservation of keratinocytes in suspensions and in monolayers.

It has recently been shown that keratinocytes, both in suspension and in monolayers, can be successfully cryopreserved with hydroxyethyl starch (HES) (6, 9). HES is a nontoxic biodegradable macromolecule which is clinically approved as a plasma expander and which has already been used for the cryopreservation of red blood cells (10, 11). In this study we varied the HES concentration between 0 and 10 wt% in 2% steps for suspended cells and between 0, 4, 6, 8, and 10 wt% for monolayer cells in order to determine the effect on the survival rate and metabolic activity after cryopreservation. The experiments with the suspended cells were performed both with and without NCS. Cryopreserved keratinocytes can be transplanted onto patients for the treatment of deep dermal burns and leg ulcers. In this study, we achieved a survival rate of 80% for the suspended cells (10 wt% HES, 3 degrees C/min) and a survival rate of even 88% when the cells were cryopreserved as a monolayer using the same parameters. The addition of NCS did not improve the results for the suspended cells significantly.

Freeze-drying of red blood cells: how useful are freeze/thaw experiments for optimization of the cooling rate?

A red blood cell suspension, prepared according to a high-yield HES cryopreservation protocol, was frozen at selected cooling rates of 50, 220, 1250, 4200, and 13,500 K/min. After either thawing or vacuum-drying, the cell recovery was determined using a modified saline stability test. As expected, the recovery of thawed samples followed the theory of Mazur's two-factor hypothesis. The best result was found at a cooling rate of 220 K/min. In contrast, the recovery of freeze-dried and rehydrated samples was very poor at that rate, but maximal at 4200 K/min where thawing caused almost complete hemolysis. This discrepancy is attributed to different damaging mechanisms involved with the respective sample processing subsequent to freezing. While thawing leads to increased devitrification and recrystallization at supraoptimal cooling rates for cryopreservation, the resultant almost vitreous sample structure seems to be advantageous for vacuum-drying. It can be concluded that freeze/thaw experiments are not sufficient for optimization of the cooling rate for freeze-drying.

Cryopreservation of keratinocytes in a monolayer.

The cryopreservation of cells in tissues is one of the major challenges in current cryobiology, especially with regard to the progressively increasing field of tissue engineering. It is very questionable whether protocols which were developed for the cryopreservation of isolated cells are also applicable for cells in more complex structures, such as tissues. As a starting point toward cryopreservation of these three-dimensional structures, the aim of this study was to find an optimum cryopreservation protocol for keratinocytes in a monolayer (two-dimensional structure). These epidermal cells can be transplanted as a monolayer grown on an appropriate matrix for the treatment of deep-dermal burns and leg ulcers. The successful cryopreservation of such transplants would offer the advantage of long-term storage and immediate availability of the transplant. In our study, the variables investigated were the cryoprotective solution and the cooling rate. In order to find a nontoxic cryoprotective agent (CPA) which could be transplanted without an additional washing step, we included hydroxyethyl starch (HES) as a possible CPA in our experimental protocol with the commonly used CPAs Me(2)SO, glycerol, and ethylene glycol. For the evaluation, the cell survival rate was determined by dye exclusion (trypan blue) and the cell metabolism was investigated by cell activity assay (alamarBlue). In conclusion, the cryopreservation protocol with 10 wt.-% HES resulted not only in the highest survival rate (72%) but also in the highest metabolic activity of the cells after thawing; comparable values for the other CPAs were: Me(2)SO, 48%; glycerol, 8%; and ethylene glycol, 10%.

Imaging of interstitial cryotherapy--an in vitro comparison of ultrasound, computed tomography, and magnetic resonance imaging.

RATIONALE AND OBJECTIVES: To evaluate the imaging capabilities of ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI) in monitoring interstitial cryotherapy and to compare them with visual control. METHODS: An experimental MR-compatible, vacuum-insulated and liquid nitrogen-cooled cryoprobe was inserted under in vitro conditions into a porcine liver, which was kept at a temperature of 37 +/- 1 degrees C, in a water bath with continuous stirring. The freezing procedure was controlled macroscopically, by US (Toshiba Sonolayer, 7.5-MHz linear array transducer), by CT (Siemens Somatom Plus, slice thickness 2-8 mm, 165-210 mA at 120 kV), and by MRI (Philips Gyroscan ACS-NT, FFE TR/TE/FA = 15/5.4/25 degrees, T1-SE 550/20, T2-TSE 1800/100) after the iceball reached its maximum size. RESULTS: The maximum iceball diameter around the probe tip was 12.0 mm by visual control, 12.4 mm by US, 12.7 mm by CT, and within 12.8 mm by spin echo sequences and 11 mm by gradient echo sequence. Due to the nearly signal-free appearance of the frozen tissue on MR images, the ice/tissue contrast on T1-weighted and gradient echo images was superior to T2-weighted images and CT images. Sonographically, the ice formation appeared as a hyperechoic sickle with nearly complete acoustic shadowing. CONCLUSION: Due to the better ice/tissue contrast, T1-weighted or gradient echo MR images were superior to CT and US in monitoring interstitial cryotherapy. Gradient echo sequences generally underestimated the ice diameter by 15%.

Freeze-drying of red blood cells at ultra-Low temperatures.

The hemolysis of human red blood cells (RBCs) after freeze-drying and resuspension depends on the vacuum-drying temperature. In an experimental study, RBCs were first solidified based on a modified high-yield cryopreservation protocol in the presence of hydroxyethyl starch and maltose. Afterward, they were vacuum-dried in a special low-temperature freeze-drying device at selected shelf temperatures between -5 and -65 degrees C. Subsequently, the dried samples were resuspended in an isotonic, phosphate-buffered saline solution. The hemolysis was determined according to a modified saline stability test. It decreases with a decreasing shelf temperature until a minimum is reached at -35 degrees C. A further decrease of the shelf temperature has no beneficial effect; the hemolysis even increases. To interpret these results, we assume that the hemolysis depends on two contrary damaging effects: (1) the higher the shelf temperature, the higher the probability of structural damages occurring during drying; (2) the lower the shelf temperature, the lower the driving force for water transport; this may lead to an incomplete intracellular dehydration which means that the cells are not in a glassy state at ambient temperature.

The effect of storage temperature on the stability of frozen erythrocytes.

A systematic study on the stability of frozen erythrocytes was performed. Washed and concentrated erythrocytes were mixed with an equal volume of cryoprotective solution containing 24% (w/w) hydroxyethyl starch (HES) and 60 mmol/liter NaCl according to an established protocol. Volumes of 250 microliters of this mixture were filled into polypropylene tubes and cooled to -196 degrees C with a rate of 293 degrees C/min by immersion in liquid nitrogen. The storage temperature was then varied from -10 to -75 degrees C and could be identified as the predominant factor influencing hemolysis kinetics. The effect of storage temperature on the frozen erythrocytes after thawing was evaluated by measuring the hemolysis in a dilute, isotonic NaCl solution (saline stability). A strong time dependence was found within the temperature range studied and could be described by an exponential kinetic law. A stability prediction was made for storage temperatures lower than those examined. Temperature ranges of qualitatively different hemolysis kinetics were identified and compared to devitrification behavior of intra-and extracellular solutions. The intracellular solution was simulated by a concentrated mixture of dried erythrocytes and water. The devitrification behavior was studied using DSC techniques. A rapidly frozen mixture was annealed at selected temperatures which fall into the range of storage temperatures for frozen erythrocytes. This paper tentatively interprets the devitrification data with respect to the means for cell damage during storage. The results are reviewed with respect to the design of a safe storage procedure.