Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration

Abstract

Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.

Fig. 1

PCL/col/HA scaffolds (“TRI”), or scaffolds composed of 100% PCL, were implanted into a cortical defect in a rat tibia for seven days. A) Low magnification images of transverse sections stained with Goldner’s Trichrome show new bone stained light blue-green, soft tissue stained red, and cell nuclei stained black. TRI scaffolds supported robust new bone formation throughout the defect (A), especially in direct contact with the scaffold surface (B). However, endogenous cells can be seen lining the edge of scaffolds (B inset). The small pore sizes of electrospun TRI scaffolds hinder cell infiltration and tissue-ingrowth. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

SEM images of selective cleavage of collagen I present in TRI scaffolds. A) Treatment with collagenase solution in vitro (b, e) or exposure to endogenous collagenases in a rat subcutaneous skin pouch (c, f) are able to cleave the collagen within fibers of TRI scaffolds creating larger pores (red circles), but have no effect on PCL scaffolds. B) Weighing the scaffolds before and after soaking in collagenase solution verified cleavage of collagen fibers in TRI scaffolds. An ** denotes p < .001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Pre-treating scaffolds with collagenase does not facilitate cell infiltration of MSCs in vitro. GFP-expressing MSCs were seeded onto TRI scaffolds previously treated with collagenase to create larger pores, or untreated scaffolds as a control. After two weeks, samples were fixed and sectioned to evaluate MSC infiltration into the scaffolds (a and b). Collagenase treatment did not facilitate significant cellular infiltration. In attempt to stimulate cellular infiltration, PDGF-BB, a known MSC chemoattractant, was added to the underside of scaffolds after collagenase treatment and prior to cell seeding. This was done to create a chemoattractive gradient through the scaffold. MSC infiltration remained minimal (c).

Fig. 4

Decreasing the packing density of electrospun fibers. A unique collecting plate was used in order to decrease the packing density of electrospun fibers and therefore create larger pores (A). Although 100% PCL scaffolds formed in loosely packed layers (B) and possessed a favorable 3-dimensional architecture with deep channels (C), these results were not observed when TRI scaffolds were electrospun using the same collecting plate.

Fig. 5

Removal of sacrificial electrospun fibers. A) As an alternative method to increase pore sizes, water-soluble fibers of PEO were incorporated into TRI scaffolds. Fluorescent dyes confirmed that separate fibers of PEO (Red) and TRI (Green) were intermixed in the scaffold (a). After washing scaffolds in water, PEO fibers were removed (b). B) Weighing the scaffolds before and after washing indicated removal of PEO fibers by mass loss. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Mean pore size analysis of electrospun TRI and TRI/PEO scaffolds. A) Fluorescently stained TRI/PEO scaffolds (washed to remove PEO) or TRI scaffolds electrospun without PEO fibers were visualized using a Zeiss confocal microscope. The area of 25 pores manually selected at random were measured using the auto area detect feature of NIS-Elements software. B) The mean pore size of TRI/PEO scaffolds was significantly greater than TRI scaffolds. An ** denotes p < .0001.

Fig. 7

Electrospun scaffolds created with PEO fibers support cell infiltration of MSCs in vitro. After removal of PEO fibers from TRI/PEO scaffolds by washing, MSCs were seeded for one week. A) Scaffolds were sectioned and stained with DAPI to show cellular nuclei location. MSCs were able to infiltrate into the TRI/PEO scaffolds, but not TRI scaffolds, as seen by presence of nuclei within TRI/PEO scaffolds. B) Cell infiltration was quantified using a custom MatLab script. On average, MSCs seeded on TRI/PEO scaffolds migrated 45.59 μm into the scaffold, significantly greater than infiltration on TRI scaffolds (6.13 μm). An ** denotes p < .0001.

Fig. 8

Electrospun scaffolds created with PEO fibers support infiltration of endogenous cells from calvarial organ cultures. After removal of PEO fibers from TRI/PEO scaffolds by washing, scaffolds were placed directly on top of excised calvaria from neonatal mice. After 8 days in culture, the scaffold/calvaria constructs were fixed and vertical sections were stained with DAPI to show cellular nuclei location (it should be noted that the apparent gap between the scaffold and the calvaria is an artifact introducing during processing and sectioning of the samples). A) Endogenous cells were observed within the TRI/PEO scaffolds, while remaining largely on the surface of TRI scaffolds. B) Cell infiltration was quantified using a custom MatLab script. On average, endogenous bone cells migrated 63.15 μm into TRI/PEO scaffolds, which was significantly greater than infiltration levels observed on TRI scaffolds (20.06 μm). An ** denotes p < .0001