Ugrás a tartalomhoz

Three dimensional tissue cultures and tissue engineering

Dr. Domokos Bartis, Dr. Judit Pongrácz (2011)

University of Pécs



The requirements for biomaterials used in tissue engineering are quite strictly defined. Biocompatibility for example is high on the agenda, as scaffold and bioreactor materials have to be tissue friendly and not eliciting immunoresponse. Moreover, at best, the biomaterial should support cellular and tissue functions like adhesion, differentiation and proliferation via its special surface chemistry. Porosity is an important requirement concerning scaffolds. Generally the porosity should reach and even exceed 90% to allow even seeding of cells and to support vascular ingrowth after implantation. Controlled biodegradation is also an important issue in some cases when the healthy tissue replaces the implanted biomaterial and the biomaterial gradually degrades in the body of the host. Biomaterials can be divided into natural and synthetic biomaterials.

Natural biomaterials

The advantages of natural biomaterials (Figure IV-1) are that they mostly come from an in vivo source therefore large quantities are constantly available at a reasonable price.

Figure IV-1: Types of natural biomaterials

Further advantages of natural biomaterials are that they already have binding sites for cells and adhesion molecules so the biocompatibility is not a major issue. However, there are also some disadvantages. Due to natural variability in the in vivo source, the lot-to-lot variability is always a concern. Additionally, potential impurities may also result in unwanted immune reactions, while their mechanical properties are also limited.

Collagen is the most frequently used and therefore the most studied biomaterial. There are rich in vivo sources, as all connective tissues of animals are rich in this ubiquitous protein. Collagen has a fibrous structure, and its amino acid composition is unique. It provides binding sites for integrins called RGD sites (from the amino acid sequence arginine-glycine-aspartic acid). Collagen has a superior biocompatibility being a conserved protein. Additional benefit, that the immune system well tolerates collagen. It is capable of supporting a large spectrum cellular differentiation types, therefore collagen is well preferred as scaffold.

Another, easily accessible type of scaffold is fibrinogen. Fibrinogen is obtained from (human) plasma. Although in its uncleaved form is a soluble protein, upon cleavage with thrombin fibrinogen sets as a gel and forms a 3D meshwork which is 100% biocompatible and physiological in wound healing. It is often used as a biological glue when cells to be seeded onto scaffolds (e.g. non-woven mesh or fleece or other porous materials) are suspended first in a fibrinogen-containing solution. Then the solution is applied onto the scaffold and upon the addition of thrombin, a hydrogel is formed which enhances the cells’ ability to attach to the 3D scaffold. Fibrin is also suitable for supporting ES cell differentiation as well as keeping differentiated cells in culture. Recent applications of fibrin include cardiovascular, cartilage, bone and neuronal tissue engineering.

Silk that is also used as scaffold material, is a protein produced within specialized glands of some arthropods. It has a special tertiary structure consisting of repeating amino acid motifs forming an overlapping beta-sheet structure giving the unique sturdiness for this protein. There have been many industrial attempts to mimic the features of silk. As a result the availability of recombinant analogues is increasing.

The silkworm’s (Bombix mori) silk consists of two different protein components, namely Fibroin and Sericin. Sericin forms the outer layer on the fibroin core making it slippery and elastic. Fibroin that is biocompatible and possesses excellent mechanical properties is also used as tissue engineering scaffold. Its use is widespread in bone, cartilage and ligament engineering. Additionally, silk fibroin can be modified chemically, e.g. the attachment of RGD groups provides binding sites for osteoblasts and enhances Ca++ deposition and bone cell differentiation. Moreover, silk promotes more intensive chondrogenesis than collagen used as a scaffold material for cartilage engineering. The degradation rate of silk is very slow but finally bone tissue gradually replaces the silk scaffold.

Polysaccharide-based biomaterials are polymers consisting of sugar monomers. Those used for tissue engineering purposes are of plant (seaweed) or animal origin. Some polysaccharides may trigger unwanted immune reactions so a careful selection is advised considering polysaccharide scaffold materials. Polysaccharides are most frequently used as hydrogels, which per se form a 3D meshwork so they can provide a scaffold for seeded cells. These hydrogels are frequently used as injectons: they can be dispensed directly to the site of injury so it supports wound healing and also cell growth and differentiation.

Main source of agarose, another scaffold material for tissue engineering, are red algae and seaweed. Agarose is the most frequently used polysaccharide scaffold consisting of a galactose-based backbone. It is immunologically inert, so no immune response is triggered. One of its great advantages lies in its versatility: the stiffness and mechanical parameters can be easily manipulated of agarose gels. It has been used for scaffolding cartilage, heart, nerve tissues and it also supports stem cell differentiation.

Alginate is the polysaccharide component of the cell walls of brown algae. It is an acidic compound, so in tissue engineering various cationic alginate salts are used. Sodium-alginate is a frequently used food additive (E-401) and its use is also widespread in gastronomy. Besides of gastronomic applications, sodium alginate is used in industry as a heavy metal-binding or fat-binding agent. The potassium salt is also used in the food industry as an emulsifier and stabilizer. In tissue engineering its calcium salt or Calcium-alginate has gained widespread application. Calcium alginate is a water-insoluble gel-like material, and it is generally used in the industry or laboratory for enzyme immobilization or encapsulation. In tissue engineering calcium-alginate proved to be useful for encapsulation of whole living cells, thus isolating them from the immune system preventing rejection after transplantation. In a clinical trial, calcium-alginate was used for the encapsulation of pancreatic islet cells which preserved their ability to produce insulin according to the needs of the host as the encapsulation prevented the immune reaction against the grafted cells.

Hyaluronan which is also termed as Hyaluronic acid is an animal-derived polysaccharide which is extensively used as a scaffold material in tissue engineering. It is a non-sulfatated Glucose-Amino-Glycan (GAG) molecule and exists as a major component of the ECM in hyalinic cartilage and skin but it is present in other organs as well. As a natural ECM component, multiple cell surface receptor binding and cell adhesion sites available on the macromolecular complex. Hyaluronan has an important role in wound healing and tissue repair. Moreover, it supports embryonic stem cell differentiation, survival and proliferation. Like other polysaccharides, hyaluronan is used as a gel in nerve, cartilage and skin tissue engineering.

Chitosan is derived from the deacetylation of chitin which is a strongly cationic polysaccharide which is the main component of the arthropod exoskeleton. Chitosan is commercially derived from sea-dwelling crustaceans and it is widely used for bandages and wound dressing, utilizing its ability to enhance blood clotting. Chitosan scaffolds are mainly used in bone tissue engineering, as chitosan supports osteoblast differentiation. Moreover, chitosan-calcium-phosphate composite scaffolds form a pliable, injectable hydrogel at slightly acidic pH. Upon transition to physiological pH, it gels thus anchoring osteocytes to the scaffold. Native and collagen-modified chitosan can be used for tissue engineering, as both forms support progenitor differentiation into osteoblasts.

Synthetic biomaterials

In tissue engineering a large scale of synthetic biomaterials (Figure IV-2) are used besides that of natural origin.

Figure IV-2: Types of synthetic biomaterials

Their main advantages over natural biomaterials are the high reproducibility, availability on demand and constant quality supporting industrial-scale production. Moreover, by application of slight changes of production, an easy control of mechanical properties, degradation rate, shape, composition, etc. can be adjusted to current needs. However, synthetic materials often lack sites for cell adhesion and the biocompatibility is frequently questionable. Biocompatibility and support of stem cell differentiation is not clear either, while immune reactions are also possible.

Poly (lactic-co-glycolic acid) PLGA is an FDA approved scaffold material. PLGA is a mixed polymer consisting of lactic and glycolic acids in various ratios. PLGA is a biodegradable material and its degradation rate can be modulated. It is one of the most frequently used biomaterials applied in neural tissue, bone and cartilage tissue engineering. PLGA is biocompatible, triggers no immune reaction and supports embryonic stem cell differentiation, proliferation and survival. However one has to consider that the degradation products are acidic, therefore may alter cell metabolism.

Poly (ethylene glycol) PEG is a commonly used biocompatible polymer. This molecule has amphophilic properties having a hydrophilic head and a variably long hydrophobic tail. The PEGylation of proteins is a commonly used pharmaceutical technology to modulate the degradation and absorbtion of bioactive proteins like interferons. The chemical modification of PEG is also available e.g. heparin, peptides, or RGD motifs. PEG is also a frequently used scaffold material in bone, cartilage, nerve, liver and vascular tissue engineering. Application of PEG-formed hydrogels is very versatile. The chemical nature, the extent of crosslinking, and the potential application of modifications, like RGD or other amino-acid groups, makes PEG widely used. PEG-based gels can be applied not only to entrap or anchor cells but also in storing and delivering bioactive molecules like BMP or TGFβ.

Peptide-based biomaterials consist of short amino acid sequences which usually are ampholitic thus making peptides capable of self-assembly. The application of these peptides allows the combination of the advantages of synthetic materials with that of natural scaffolds. Synthetic nature of the peptides eliminates batch-to-batch variability and consistent purity and quality can be achieved. Moreover, known binding sites for cell-surface adhesion molecules can be included in the sequence. For example, IKVAV amino acid sequence is from laminin and it facilitates neurite outgrowth. RGD sequence is a binding site for integins which promotes cellular adherence and migration.

Ceramic-based biomaterials are used in bone tissue engineering only. These scaffolds are totally or partly inorganic. Mostly they are shaped with heat forming porous, brittle materials. For example, bioactive glass is slowly biodegradable. With an ion-exchange mechanism it slowly turns to natural hydroxyapathite via surface degradation. So bioglass is biocompatible and used as an implant material. Hydroxyapatite is the inorganic compound of bone. Sometimes it is used as a completely inorganic, porous scaffold, more often it is combined with other biocompatible, organic polymers, like PLGA, collagen, or chitosan. This combination also enhances the drug delivery capacity of these scaffolds to enhance bone formation.

Metals are used as implant materials. Mostly alumina and titanium alloys are used because of their biocompatibility. The high durability of metals is needed where implants are subjected to extreme mechanical load, like articular prostheses, dental implants or heart valves. These metals are bioinert materials, however, sometimes metallic implants may cause immunological reactions like metal allergy in sensitive individuals. Also, as they are not biodegradable it is questionable whether metals can be listed amongst tissue enginnering scaffolds.