A cost-efficient, compact tool for two-photon cross-linking advances research into the generation of biomimetic scaffolds and artificial organs.
The use of artificial soft tissues in medical applications has been a subject of intense investigation for many years. There is a growing need in pharmaceutical, cosmetic and chemical research for in vitro experimental platforms that mimic natural tissues for the replacement, reduction and refinement of animal experiments (the three R principle). In the European Union, the Cosmetics Directive and REACH regulation have increased demand for substance testing while simultaneously limiting the feasibility of animal testing. Thus, the development of appropriate in vitro assays to answer complex questions—such as the take-up rate of active agents through the skin into the blood system—are of great interest. Additionally artificial tissue replacements hold the key to many innovations in the health sector; the use of artificial skin as a dressing for burn and chronic wounds is but one example. Furthermore, whole organ replacement can be enabled by the generation of tissue-engineered body parts such as heart valves or connective tissue.
All of those applications require artificial tissue and tissue grafts that closely mimic the structure of natural soft tissue. This biomimetic approach favours the development of reliable and accurate in vitro test systems as well as fully functional and long-term implantable therapeutic soft-tissue replacements. Soft tissue, however, is a complex three-dimensional system of different cell types, proteins and biologically active compounds. To efficiently mimic the natural environment of cells, three main aspects have to be considered.
Three facets of biomimetics
First, the mechanical properties of the tissue surrounding the cells have a huge impact on their function and behaviour. The differentiating behaviour of progenitor cells is strongly influenced by the hardness and elasticity of the surroundings. Hence, mesenchymal stem cells may differentiate into bone, cartilage or fatty tissue cells, depending on the hardness of the surfaces with which they are in contact. A highly controlled in vitro test system must adapt to each relevant cell type; most cell experiments, however, are performed in standard polystyrene Petri dishes with a nonelastic surface.
Second, an important factor in mimicking nature is the three-dimensional structure mostly governed by extracellular matrix (ECM) proteins such as collagen and elastin. Additionally, different cell types form certain patterns for a functional 3-D tissue.
For example, skin consists of the upper epidermis, the dermis and the hypodermis. Each layer has its typical function. The epidermis has a dense construction composed of different cells that provide protection from the environment: keratinocytes safeguard from mechanical stress, melanocytes shield against UV radiation and Langerhans cells fight germs. In the second layer, elasticity is imparted by a fibrous ECM that forms a dense connective tissue. The following subcutis layer is a loose connective tissue containing large blood vessels, which support the capillary system, and pressure-sensitive cells. Sweat glands, hair follicles and nerves exist in the dermis and hypodermis along with a dense network of blood vessels, regulating nutritional supply and disposal of metabolites. These different parts of the tissue are built by varying cell types and surrounding extracellular matrix proteins.
Third, in addition to the proper mechanical and structural surroundings, cells require biochemical information. Their fate can be influenced by neighbouring cells (integrins), cells in close vicinity (cytokines) and even cells from long distances (hormones). Also the previously mentioned ECM sends a biochemical signal to the cells, which are connected via integrins. In summary, the path towards an engineered soft tissue requires a scaffold with suitable mechanical, structural and biochemical properties.
An understanding of the complexity of this three-dimensional natural environment of living tissue forms the basis for modelling an artificial system. Most research on cell adhesion and cell growth is still performed in a two-dimensional, nonelastic environment. Basic research concerning adhesion has yielded much insight into the complexity of this topic and revealed the need for three-dimensional biomimetic cell-culture methods. Several techniques have proven their worth in the development of 3-D cell cultures: these include the casting of protein hydrogels, the creation of sponge-like structures by cryogenic processes and the production of nanostructured nonwovens by means of electrospinning. These methods usually ensure that biochemical requirements are met. Even the mechanical and raw structural properties can be mimicked, like a fibrous ECM system. But these methods do not allow the freeform construction of a defined and reproducible 3-D scaffold. One way to overcome this drawback is the use of two-photon induced cross-linking (TPC).
|Figure 1: In the two-photon cross-linking process, a tightly focused laser beam cross-links a small volume of material inside a liquid resin. The resulting voxel measures approximately 1 µm.|
In TPC, a laser beam initiates a photochemical reaction inside a liquid resin (Figure 1). The resin consists of photosensitive polymers, which undergo laser-induced cross-linking. This method is unique in its inherent three-dimensional character and its ability to achieve high resolutions. Microstructures down to the submicrometre range are feasible, which makes TPC a valuable tool for 3-D cell cultures.
The physical basis of TPC is a nonlinear optical effect induced by high-intensity laser irradiation. Because of the intensity, the density of photons is sufficiently high that two low-energy photons, instead of one high-energy photon, can be absorbed simultaneously by the photosensitive molecule. By replacing one high-energy photon with two low-energy photons, laser systems emitting in the visible or near-infrared band can be used to initiate photochemical processes in the UV spectrum. Many photosensitive materials are transparent in these wavelengths, thus allowing the laser beam to target not just the surface but the volume of the liquid resin. The high intensities, necessary for initiating TPC, are only present in ultrashort laser pulses tightly focused by high numerical aperture objectives. Thus, only a small volume, called a volume pixel (voxel), inside the liquid is cross-linked. In all three spatial directions, the achievable resolutions can be better than the diffraction limit of the focusing objective, which is usually 2 µm or smaller.
|Figure 2: A model of elastic capillaries is fabricated with a tube that has an inner diameter of 20 µm.|
In our approach we used a synthetic, biocompatible, elastic polymer developed by the Fraunhofer Institute for Applied Polymer Research (IAP) to generate capillaries with mechanical properties that resemble natural blood vessels (Figure 2). The artificial capillaries have an inner diameter measuring approximately 20 µm. Pores measuring a few micrometres are feasible, with the aim of enabling the nutritional supply of surrounding tissue. The 3-D scaffolds can be fabricated from various materials, ranging from elastic to nonelastic polymers and even biopolymers such as proteins from the extracellular matrix. Microstructured protein scaffolds may even represent a way to copy the natural properties of soft tissue, thus allowing the generation of biomimetic in vitro test systems.
Many proteins can be cross-linked by means of TPC, including collagen, fibronectin, concanavalin A, avidin and albumin. Although TPC-cross-linked proteins do not entirely preserve their native state, protein microstructures demonstrate a distinctive cell guiding capability, showing that important biological functions are conserved after cross-linking. The main challenge of protein microstructures resides in their mechanical stability. Because of their hydrogel-like character, the mostly elastic microstructures tend to deform in solution and shrink after drying. This mechanical weakness makes it almost impossible to produce complex three-dimensional scaffolds.
Figure 3: A cell cage is made from two modified protein materials. By adding a protein membrane to the top of the structure, the cage can be sealed.
One possible way to overcome this is by using subsequent wet chemical fixation techniques established for cell biology. However, these techniques use chemical cross-linking agents, such as formaldehyde, which have an extreme effect on protein functionality. For this reason, the Fraunhofer Institute for Laser Technology (ILT) in Aachen, Germany, has established a process that combines the mechanical properties of synthetic polymers with the biochemical properties of proteins to form polymer-protein hybrid structures. Combining different material classes in a single scaffold significantly increases its functional diversity. By using this novel technique, scaffolds that combine mechanical, structural and biochemical characteristics resembling natural tissue can be produced. A first hybrid scaffold test structure for in vitro assays was successfully generated.
A cell cage was constructed for studying cell interactions with vertical protein membranes (Figure 3). The polymer posts along the edges of the test structure support 20-µm long protein membranes, which show almost no deformation or shrinkage even after excessive drying. A similar structure was generated for horizontal protein membranes (Figure 4) composed of a specially designed material by Fraunhofer-IGB. Here, the polymer forms a hollow barrel, supporting the approximately 1-µm-thick protein membrane.
Figure 4: A hollow polymer barrel supports a free-hanging protein membrane. An opening in the front of the barrel allows rinsing of the non-cross-linked protein residue.
Compact, affordable two-photon cross-linking system
Equipment capable of performing TPC is often large and expensive, which hinders widespread use of the technique. The machines also typically are not compatible with sterile processing requirements. To overcome these shortcomings, Fraunhofer ILT has developed a cost-efficient and compact TPC module (Figure 5).
TPC technology typically employs laser sources emitting pulses that are several hundred femtoseconds long in near-infrared wavelengths. Experiments at ILT showed that frequency-doubled microchip-laser sources are a viable alternative for many materials. These laser sources emit pulses that are several hundred picoseconds long in the visible range. Experiments showed that these laser sources engender more efficient cross-linking than femtosecond-laser sources. Furthermore, they are less expensive and more compact.
At Fraunhofer ILT, a TPC prototype has been developed using a µ-chip laser source. The high accuracy of TPC processing can be obtained within a relatively large working field measuring 25 x 25 x 0.5 mm3.
As a stand-alone tabletop machine, the prototype facilitates full beam manipulation and optical inspection via real time monitoring. Additionally, this module can be integrated into larger processing tools since beam steering is incorporated and, thus, is independent from sample handling. It is even small enough to fit within a standard cleanroom bench.
|Figure 5: A prototype tool for two-photon cross-linking developed by Fraunhofer ILT features a small footprint and affordability.|
By contrast with other TPC setups, the focused laser beam can move freely in all three spatial directions throughout the working field. Since the sample and built structure are at rest and, therefore, not subject to acceleration, this prototype is well suited for liquid resins or delicate materials. Furthermore, the translational decoupling allows for an easy combination of pre- and postfabrication steps in a closed processing unit.
In the future, this technology will be used to generate more complex biomimetic scaffolds. As a first application, a TPC module will be integrated into a small-scale in vitro test system to study the human skin’s vascularisation process.