**4. Aptamers to enhance scaffold biocompatibility**

As mentioned earlier in this chapter (see Section 2.1), one of the most investigated topics in TE is developing new methods to improve scaffold biocompatibility. To reach this goal, scaf‐ folds should be highly dynamic and possess surfaces capable to interact with cells, positive‐ ly modulating protein adsorption [36].

Several research groups have aimed to reach this goal by immobilizing the RGD peptide binding motif on scaffolds (see Section 2.1.2), by modifying chemically or physically scaffold surfaces (see Section 2.1.1) or by coating scaffolds with other highly biocompatible materials (see Section 2.1.3).

In this section we propose a new method to improve natural polymeric scaffold biocompati‐ bility, by using ssDNA aptamers screened for against human fibronectin as docking points, to ameliorate the adsorption of fibronectin, a naturally occurring molecule in damaged tissues, which is mainly involved in cell adhesion and in the regeneration process. The correct adsorption of fibronectin may lead to a faster colonization of the scaffold *in vitro* and to an acceleration of the regeneration process *in vivo*. **Figure 3** summarizes the rationale to use aptamers to enrich biomaterials with specific molecules.

**Figure 3.** Diagram representing the rationale of functionalizing substrates with aptamers to retain specific proteins. Un-functionalized scaffold adsorbs proteins from the environment based on their availability (A). Aptamer functional‐ ized scaffold specifically binds and retains target protein, by selectively enriching the adsorption for a specific protein (B).

Biomaterial functionalization with aptamers is not new in the literature. Wendel's group pioneered the field in 2007, by coating a vascular prosthesis with aptamers against circulat‐ ing endothelial progenitor cells (EPCs), to retain specific cells from the bloodstream and quickly create an autologous functional endothelium. Aptamers against EPCs were screened through the Cell-SELEX and covalently grafted on to polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE) substrates. Functionalized scaffolds were incubated with whole porcine blood, washed twice to remove non-specific debris, and stained for CD31 and CD144 by immunofluorescence to identify EPCs. EPCs were observed only on aptamer-grafted prosthesis, whereas no CD31 and CD144 positive cells were retained on control discs [144]. Five years later, Chen et al. designed an artificial ECM using aptamer-grafted polyethylene glycol (PEG) hydrogels: aptamers screened for against cell surface receptors were used as binding sites for cells and they were attached on to the gel through free radical polymeriza‐ tion. It was demonstrated that the amount of cells adhered to hydrogels was proportional to the amount of aptamer incorporated into the hydrogels [145].

Considering those results, we want to show the possibility of enriching natural synthetic scaffolds with aptamers against human Fibronectin to enhance cell adhesion and growth.

For this purpose, we used aptamer screened for against human fibronectin (Base Pair Biotechnologies, Pearland, TX) and modified at their 3′-end with a thiol group and at their 5′ end with biotin.

#### **4.1. Aptamers enhance cell adhesion and proliferation on polymeric scaffolds**

surfaces (see Section 2.1.1) or by coating scaffolds with other highly biocompatible materials

In this section we propose a new method to improve natural polymeric scaffold biocompati‐ bility, by using ssDNA aptamers screened for against human fibronectin as docking points, to ameliorate the adsorption of fibronectin, a naturally occurring molecule in damaged tissues, which is mainly involved in cell adhesion and in the regeneration process. The correct adsorption of fibronectin may lead to a faster colonization of the scaffold *in vitro* and to an acceleration of the regeneration process *in vivo*. **Figure 3** summarizes the rationale to use

**Figure 3.** Diagram representing the rationale of functionalizing substrates with aptamers to retain specific proteins. Un-functionalized scaffold adsorbs proteins from the environment based on their availability (A). Aptamer functional‐ ized scaffold specifically binds and retains target protein, by selectively enriching the adsorption for a specific protein

Biomaterial functionalization with aptamers is not new in the literature. Wendel's group pioneered the field in 2007, by coating a vascular prosthesis with aptamers against circulat‐ ing endothelial progenitor cells (EPCs), to retain specific cells from the bloodstream and quickly create an autologous functional endothelium. Aptamers against EPCs were screened through the Cell-SELEX and covalently grafted on to polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE) substrates. Functionalized scaffolds were incubated with whole porcine blood, washed twice to remove non-specific debris, and stained for CD31 and CD144 by immunofluorescence to identify EPCs. EPCs were observed only on aptamer-grafted prosthesis, whereas no CD31 and CD144 positive cells were retained on control discs [144]. Five years later, Chen et al. designed an artificial ECM using aptamer-grafted polyethylene glycol (PEG) hydrogels: aptamers screened for against cell surface receptors were used as binding sites for cells and they were attached on to the gel through free radical polymeriza‐ tion. It was demonstrated that the amount of cells adhered to hydrogels was proportional to

the amount of aptamer incorporated into the hydrogels [145].

aptamers to enrich biomaterials with specific molecules.

(see Section 2.1.3).

342 Advanced Techniques in Bone Regeneration

(B).

Two natural polymer scaffolds were used as substrates: a thiolate hyaluronic acid/polyethy‐ lene glycol hydrogel (tHA/PEGDA) and a chitosan modified with D-(+)-raffinose film. tHA/ PEGDAgels are 3D matrices normally usedfor stem cell culture and which offer scant adhesion to cells. For this reason, they are often enriched with adhesion molecules, such as RGD peptides, when firmer adhesion is required. Aptamers were bound to these hydrogels by exploiting the acrylate functional groups of PEGDA, which can easily bind thiol groups on aptamers. Five microliters of aptamer at increasing concentration were mixed to each 50 μl gel.

Chitosan is one of the most investigated natural polymers for TE applications, because it is highly biocompatible [146]. However, some cell types grow slowly on chitosan films, and consequently they were chosen as substrates to be enriched with aptamers. Aptamers were immobilized on 2% chitosan films (*r* = 3.0 mm; *h* = 0.25 mm) at increasing concentration by exploiting the spontaneous ability of chitosan to bind sulfur-containing substances [147].

**Figure 4.** Study of aptamer ability to enhance the proliferation of human osteoblasts (hOB) on tHA/PEGDA hydrogel. Microphotographs, taken with an inverted microscope, showing hOB cells on tHA/PEGDA after 48 h of culture (A–D). The rate of cell growth is proportional to the quantity of aptamer used for the functionalization (E).

Five thousand hOB cells (human osteoblasts) on tHA/PEGDA gels and 5000 MC3T3-E1 cells (murine preosteoblasts from bone/calvaria) on 2% chitosan films were cultured for 7 days. Cells were monitored day by day with an inverted microscope.

Cell proliferation on tHA/PEGDA and chitosan substrates is shown in **Figures 4** and **5**.

**Figure 5.** Study of aptamer ability to enhance the proliferation of murine osteoblastic cells (MC3T3-E1) on 2% chitosan films. Microphotographs, taken with an inverted microscope, showing MC3T3-E1 cells on 2% chitosan films after 48 h of culture and stained with the Trypan Blue to discriminate viable and dead cells. The rate of cell growth is proportion‐ al to the quantity of aptamer used for the functionalization (E).

In both the cases, aptamers increase the number of adhering cells and the rate of cell growth is proportional to the amount of aptamer used. Cell morphology appear round both in control groups and in aptamer-rich samples, unlike the flattened spindle shape morphology that is normally observed on tissue culture plastic substrates, and is routinely associated to firm cell adhesion. Although cell adhesion does appear improved in the presence of aptamers, as indicated by a significantly higher number of cells, the culture substrate is mechanically elastic and the normal morphological features of a good adhesion cannot be achieved.

**Figure 6.** Histograms representing the amount of protein adsorbed on polymeric scaffolds with or without aptamers. Scaffolds were incubated for 2 h with 30 μg of proteins. The amount of protein bind by the scaffold was quantitated through the Bradford.

Although aptamers act on both substrates presumably in comparable ways, by binding fibronectin, the rationale for their use is possibly different, as suggested by results of protein adsorption assays reported in **Figure 6**, where 30 μg of serum protein were incubated for 2 h on different scaffolds with or without aptamers and quantitated with Bradford.

The presence of aptamers on tHA/PEGDA quantitatively increases the amount of fibronec‐ tin on the gel and this may explain the improved cell adhesion and proliferation. Cells on control gels do not get in contact with adsorbed proteins and lack good attachment points for their integrins. Availability of a higher amount of adhesive protein then results in more firmly adhering cells. On the other hand, chitosan is known to bind massive amounts of protein from the supernatant and aptamers do not affect the quantity of adsorbed proteins. A viable hypothesis for the effects of aptamers on chitosan is therefore that aptamers may affect the quality of adsorbed protein. Aptamers may preserve the natural conformation of fibronectin on films, without unfolding it and maintaining a favorable exposure of adhesion sequences for cells.

Evidences reported show that aptamers are a viable approach to improve the biocompatibili‐ ty of scaffolds, ameliorating the process of adhesive protein adsorption on surfaces both quantitatively and qualitatively, and should further investigated to create tissue-specific scaffold for tissue engineering.
