Multilayer cell-seeded polymer nanofiber constructs for soft-tissue reconstruction.
OBJECTIVE
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.


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


METHODS
Academic research laboratory.


METHODS
Thirty-six Sprague-Dawley rats.


METHODS
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).


METHODS
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
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.
Department of Otolaryngology-Head & Neck Surgery, University of Virginia, Charlottesville.
Daniel A
DA
Barker
Daniel T
DT
Bowers
Brian
B
Hughley
Elizabeth W
EW
Chance
Kevin J
KJ
Klembczyk
Kenneth L
KL
Brayman
Stephen S
SS
Park
Edward A
EA
Botchwey
D062088:Acellular Dermis; D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D006085:Graft Survival; D006801:Humans; D057139:Nanofibers; D011108:Polymers / Q000737:chemistry / Q000494:pharmacology*; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D019651:Plastic Surgery Procedures / Q000379:methods; D012038:Regeneration; D012680:Sensitivity and Specificity; D017695:Soft Tissue Injuries / Q000601:surgery*; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
D001672:Biocompatible Materials; D011108:Polymers