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Original Investigation |

Multilayer Cell-Seeded Polymer Nanofiber Constructs for Soft-Tissue Reconstruction

Daniel A. Barker, MD1; Daniel T. Bowers, BS2; Brian Hughley, MD1; Elizabeth W. Chance, MD3; Kevin J. Klembczyk, BS2; Kenneth L. Brayman, MD, PhD4; Stephen S. Park, MD1; Edward A. Botchwey, PhD2,5
[+] Author Affiliations
1Department of Otolaryngology–Head & Neck Surgery, University of Virginia, Charlottesville
2Biomedical Engineering, University of Virginia, Charlottesville
3Department of Otolaryngology, Martha Jefferson Hospital, Charlottesville, Virginia
4Department of Surgery, University of Virginia, Charlottesville
5Departments of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta
JAMA Otolaryngol Head Neck Surg. 2013;139(9):914-922. doi:10.1001/jamaoto.2013.4119.
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Importance  Cell seeding throughout the thickness of a nanofiber construct allows for patient-specific implant alternatives with long-lasting effects, earlier integration, and reduced inflammation when compared with traditional implants. Cell seeding may improve implant integration with host tissue; however, the effect of cell seeding on thick nanofiber constructs has not been studied.

Objective  To use a novel cell-preseeded nanofiber tissue engineering technique to create a 3-dimensional biocompatible implant alternative to decellularized extracellular matrix.

Design  Animal study with mammalian cell culture to study tissue engineered scaffolds.

Setting  Academic research laboratory.

Participants  Thirty-six Sprague-Dawley rats.

Interventions  The rats each received 4 implant types. The grafts included rat primary (enhanced green fluorescent protein–positive [eGFP+]) fibroblast-seeded polycaprolactone (PCL)/collagen nanofiber scaffold, PCL/collagen cell–free nanofiber scaffold, acellular human cadaveric dermis (AlloDerm), and acellular porcine dermis (ENDURAGen). Rats were monitored postoperatively and received enrofloxacin in the drinking water for 4 days prophylactically and buprenorphine (0.2-0.5 mg/kg administered subcutaneously twice a day postoperatively for pain for 48 hours).

Main Outcomes and Measures  The viability of NIH/3T3 fibroblasts cultured on PCL electrospun nanofibers was evaluated using fluorescence microscopy. Soft-tissue remodeling was examined histologically and with novel ex vivo volume determinations of implants using micro–computed tomography of cell-seeded implants relative to nanofibers without cells and commonly used dermal grafts of porcine and human origin (ENDURAGen and AlloDerm, respectively). The fate and distribution of eGFP+ seeded donor fibroblasts were assessed using immunohistochemistry.

Results  Fibroblasts migrated across nanofiber layers within 12 hours and remained viable on a single layer for up to 14 days. Scanning electron microscopy confirmed a nanoscale structure with a mean (SD) diameter of 158 (72) nm. Low extrusion rates demonstrated the excellent biocompatibility in vivo. Histological examination of the scaffolds demonstrated minimal inflammation. Cell seeding encouraged rapid vascularization of the nanofiber implants. Cells of donor origin (eGFP+) declined with the duration of implantation. Implant volume was not significantly affected for up to 8 weeks by the preseeding of cells (P > .05).

Conclusions and Relevance  Polymer nanofiber–based scaffolds mimic natural extracellular matrix. Preseeding the nanofiber construct with cells improved vascularization without notable effects on volume. An effect of cell preseeding on scaffold vascularization was evident beyond the presence of preseeded cells. This 3-dimensional, multilayer method of cell seeding throughout a 1-mm-thick construct is simple and feasible for clinical application. Further development of this technique may affect the clinical practice of facial plastic and reconstructive surgeons.

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Figure 1.
Experimental Methods

Electrospun nanofibers made of polycaprolactone (PCL)/collagen (A) were imaged under a scanning electron microscope. The constructs were lifted from the collector and sterilized. An example 10 × 10-cm prepared construct is pictured next to a ruler measuring about 16 cm long (B). Primary fibroblasts were harvested (C) and seeded on the scaffold in a monolayer. After cells were allowed to attach and culture on the fibers, the scaffold was folded and cut (D). A cross section of a scaffold shown below. The cell-preseeded scaffolds were compared with unseeded scaffolds and decellularized dermis as controls in a single rat (E). Rats had implants for 4, 8, or 12 weeks. Each animal received 1 of each of the 4 implant types in distinct subcutaneous pockets on the dorsum. Implants were scanned (1), implanted (2), explanted, scanned a final time (3), and processed for histologic analysis (4). Postexplant scanning (3) was completed after fixation in paraformaldehyde; therefore, the samples were soaked in 70% ethanol at the second scanning, while implants for the first scan were soaked in aqueous media. Fibroblast antibody staining (F and G). Fibroblasts were identified using P4HB antibody. Histologic specimens are shown with antibody (F) and without antibody (G). Scale bar indicates 200 μm. C indicates cells (in part A, C indicates concentration), d, distance between needle and collector; ECM, extracellular matrix; eGFP, enhanced green fluorescent protein; f, flow rate; H, human; N (in part E) or NF, nanofiber; P, porcine; V+, positive applied voltage; and WD, working distance.

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Figure 2.
Viability of NIH/3T3 Fibroblasts on a Single Layer of Nanofibers and Volume Changes

A, Quantification of cell (C) viability on nanofibers (NFs) vs tissue culture polystyrene (TCPS). Data represent the mean of at least 3 samples in each group at each time point. B, Representative images of cell viability. Calcein AM is converted to the fluorescent calcein in the cytoplasm of viable cells, while propidium iodide (PI) stains the nuclear material in cells that do not have a patent cell membrane. Notice in the overlay that calcein-stained cells were not stained by PI, showing the accuracy of the stains. Calcein also reveals a spindlelike cell footprint on the nanofiber substrate compared with the TCPS. C, Percent change in volume at 4, 8, and 12 weeks of implantation based on micro–computed tomographic scan numerical evaluation. Each implant was referenced to that individual implant prior to surgery. The porcine extracellular matrix (ECM) was significantly more stable. The seeding of cells did not have a significant effect until 12 weeks after implantation. At 4 weeks, 10 samples were available for all implants; at 8 weeks, 8, 9, 9, and 8 samples were available for NFs, NFs with cells, human ECM, and porcine ECM, respectively; and at 12 weeks, 10 samples were available for all implants. Limit lines indicate standard error. D, Renderings of thresholded reconstruction of implants from micro–computed tomographic scans before (left) and after (right) weeks in a subcutaneous pocket. Radiographic density became more punctate following implant.aP < .05 vs porcine ECM.bP < .05 with a connecting line shows the indicated comparison.

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Figure 3.
Histological Analysis of Tissue Layers and Blood Vessel Investment

A, Thickness of host tissue (arrows) was measured on cross sections of the implants (original magnification ×40). Limit lines indicate the standard deviation of the means of all the layers in 6 implants. Host-tissue deposition between the nanofiber (NF) layers was variable but not statistically significant (P > .10 for all comparisons of NFs vs NFs + cells [NF + C]). B, Area (shown as a percentage) of blood vessels outside the implant border (top graph). Cell seeding increased blood vessels early, but NFs without cells sustained blood vessels longer. Limit lines indicate standard error (n = 30). Histological score of blood vessel penetration (bottom graph) (0 indicates sparse; 1, on the periphery; 2, scattered; 3, well distributed; and 4, very dense). The bar indicates the median value, with individual data points shown by open circles, which are all integer numbers. A slight offset was used to allow individual data points to be resolved. H indicates human; P, porcine. Hematoxylin-eosin–stained slides show little inflammation and cellular residence as well as host extracellular matrix (ECM). C, D, and E, NFs at 4, 8, and 12 weeks, respectively. F, G, and H, NF + C at 4, 8, and 12 weeks, respectively. (original magnification ×40).aP < .05 vs NF.bP < .05 vs NF + C by Mann-Whitney test (n = 9).cP < .05 vs P by Mann-Whitney test (n = 9).

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Figure 4.
Fate of Seeded Enhanced Green Fluorescent Protein (eGFP)–Positive (eGFP+) Donor Cells

A, Images of sections showing eGFP+ cells (original magnification ×40). Scale bar indicates 100 μm. The inset shows an electronic enlargement of the area indicated (original magnification ×130). The black arrow indicates a cell with a fibroblastic appearance; the white arrow, a cell with a macrophage appearance. B, A large area of an implant shows the regions referred to as inside, interface, and outside. The eGFP+ cells in histologic sections are highlighted by immunohistochemistry. The eGFP+ cells were absent within the implants. C, Fibroblasts were apparent at the interface of the implants they were originally seeded on and, to some extent, at other implants. D, Macrophages that took up eGFP+ donor cells migrated to other implants but did not tend to infiltrate the implant, remaining outside or at the interface instead. Limit lines indicate the standard error of 20 high-power fields. NF indicates nanofiber; NF + C, NFs + cells.aP < .01 vs NF or as the connecting line indicates.

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Figure 5.
Immune Reaction to Nanofiber (NF)–Based Implants

The thickness of the capsule was visualized using Masson trichrome–stained sections on nanofibers (NFs) (A) and NFs with cells (B) (original magnification ×40). The capsule thickness was measured (C). Immunostaining for CD8 was performed (D). Scale bar indicates 100 μm. The CD8+ cells were quantified (E). Significant reductions in the NFs with cells group was apparent at 12 weeks. Limit lines indicate standard error (n = 20) for each bar. The number of foreign body giant cells (FBGCs) per slide area was greatest at the NF implants with few exceptions (F). At 12 weeks, there were fewer FBGCs on the NFs than there were on the NFs with cells. The area of the individual FBGCs followed almost the same trend, with the NF groups having larger FBGCs than the decellularized extracellular matrix groups at 4 and 8 weeks (G). By 12 weeks, however, the cell-seeded NF implants were surrounded by larger FBGCs than were the NF-only implants. Limit lines indicate standard error (n = 30) for each bar.aP < .05 (≥25 measurements were made for each group).bP < .02 vs NF.cP < .05.dP < .05, NF or NF + C vs human or porcine.

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