Increasing the Pore Sizes
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Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised
of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration
As the average age of population increases, the need for bone grafts to repair skeletal defects will rise. Massive bone defects are a great challenge to reconstructive surgery. The preferred treatment is a bone autografting. But the supply of suitable bone is limited and its collection is painful. There are numerous drawbacks with this procedure, including increased surgery time, donor site pain, and limited quantity of bone needed for this procedure. The use of biomaterials an alternative is currently being researched to tackle this issue. Some of the vital features of biomaterials thought to be crucial for effective bone regeneration include: (i) a biochemical composition and structure that supports osteogenic cell responses, (ii) appropriate kinetics of biodegradability, without any release of toxic byproducts, and (iii) a highly interconnected porous network that allows for proper tissue ingrowth and vascularization of the biomaterial. Many researchers have turned toward the process of electrospinning.
Electrospinning is a method which uses an electrical charge to draw very fine fibers, usually on the micro or nano scale, from a liquid or a solution. Electrospun scaffolds have a nano-firbous structure with interconnecting pores and a large surface to volume ratio, resembling natural extracellular matrix (ECM). Previous researchers have developed electrospun scaffolds that combine degradable polymers such as polycaprolactone (PCL) with native bone matrix molecules including collagen I and hydroxyapatite (HA). They have reported that scaffolds composed of blended PCL/collagen I nanofibers, with nanoparticles of HA distributed throughout the thickness of the matrix, promoted greater mesenchymal stem cell (MSC) adhesion, cell spreading, and cell proliferation, as compared with scaffolds composed of PCL alone. However, this method had one known limitation and that is the pore sizes within the matrices are too small to allow efficient cell infiltration. Migration of cells into the scaffold is an essential step in overall healing of the bone defect. Several methods have been proposed to increase the pore sizes of electrospun scaffolds. One such method was to alternate layers of microfibers with nanofibers, however cell infiltration under static culture conditions was minimal. Another methods used were salt leaching or cryogenic electrospinning. However, these methods achieved moderate success, and these techniques required advanced electrospinning setups. Previous researches have focused on scaffolds composed solely of synthetic polymers, which offer minimal biological cues for cells. The goal of this study was to compare various methods for increasing the pore size of bone-mimetic, PCL/col/HA (“TRI”) electrospun scaffolds in order to facilitate the infiltration of osteogenic cells. In this article, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col/HA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water soluble polymer PEO.
Previous studies have shown that TRI electrospun scaffolds support MSC responses in vitro that are promising for new bone formation. However, the small pore sizes within these scaffolds restrict cellular infiltration. To solve the problem of small pore sizes in TRI electrospun scaffolds, researchers in this article have performed three separate techniques that tackle this issue. They have tried to increase the pore sizes by controlling the degradation of collagen fibers, reducing the packing density of electrospun fibers, and inclusion of sacrificial PEO fibers in scaffolds.
To address the issue of pore sizes, the authors first tested whether the collagen present in TRI scaffolds could be used as a target for controlled degradation, thereby creating larger pores in order to increase cell infiltration. The authors hypothesized that a limited treatment with collagenase solution could be employed to introduce selective fiber breaks. TRI scaffolds, and PCL scaffolds as a control, were treated with collagenase, and substrates where then analyzed by SEM to display the presence of fiber breakages. The second method used by the authors to increase the pore sizes was to reduce the packing density of electrospun fibers. In this method, a plastic petri dish was used to cover the normal aluminum collecting plate, and twenty evenly spaced holes were created in the petri dish, followed by the placement of 19 needles though the holes touching the grounded collecting plate as seen in figure 1A. The limited amount of grounded points for fiber attachment reduced the packing density of the electrospun fibers, creating a more three-dimensional scaffold with larger pore sizes. The last method used to increase the pore sizes of TRI scaffolds, the inclusion of sacrificial fibers of the water-soluble polymer poly ethylene oxide (PEO) was
Figure 1
used. By electrospinning separate solutions of PEO and TRI, a mixed scaffold was created. After the scaffold was created, it was soaked in water and that washed away the sacrificial fibers, leaving just the TRI scaffolds with gaps where the PEO fibers had been. By adding fluorescent dyes to the electrospinning solutions