Segmental mandibular reconstruction poses a considerable challenge in head and neck surgery. When segmental defects are critical, and thus unable to heal on their own, the use of fibular free tissue transfer has become the gold standard. However, this procedure has multiple drawbacks including donor site morbidity, increased recovery time, and training in microvascular techniques.
Tissue engineering may provide a realistic alternative to free tissue transfer for the replacement of bone following segemental mandibulectomy. Periosteum has previously been shown to heal large, even critical defects, in long and flat bones alike. Within the head and neck region, the pericranium represents a large sheet of well vascularized periosteum that is easy to harvest with low morbidity. Given these characteristics, we set out to grow these cells in 3D culture as a step toward regeneration of critical bony defects.
Pericranial tissue was obtained from neurosurgical patients requiring open craniotomy. Mesenchymal stem cells were then prepared using standard collagenase digestion and plastic adherence methods. The presence of stem cells with three-way differentiation potential (adipogenic, chondrogenic, and osteogenic) was confirmed, respectively, by Oil Red-O, Alcian Blue, and Alizarin Red staining.
To investigate osteogenesis and vasculogenesis in 3D culture media, 1x106 pericranial cells were grown in Matrigel media for 21 days. FGF, IL-1, IL-6, PDGF, TNF-α, VEGF, a mix of all these cytokines or none of the cytokines were added to individual samples. Furthermore, to establish optimal growth conditions, we compared two regimens: a) osteogenic media supplemented with the above cytokines, and b) a mixed chondrogenic/osteogenic media, also supplemented with cytokines, as follows: days 1-7, chondrogenic media only; days 8-10, 50:50 chondrogenic + osteogenic media; and days 11-21, osteogenic media only.
Immunohistochemistry visualization of osteo/chondrogenesis was performed with differentiation-specific dyes as above. Vasculogenesis was visualized with a fluorescent antibody to Von Willenbrand factor (Abcam-8822). Cell nuclei were stained with DAPI. Slides were quantitated by intensity staining with Image J software. vWF/DAPI ratios were normalized to zero mean and unit variance to allow group comparisons.
To further investigate the osteogenic and vasculogenic potential of pericranium derived cells, polypropylene-fumarate (PPF) scaffolds were cross-linked with 0.03 mg/mL jelly-fish collagen by overnight incubation under a UV source, followed by incubation with pericranial cells, cytokines and mixed chondrogenic/osteogenic media as above.
Each sample demonstrated osteogenic and vasculogenic differentiation in 3D culture media, and high power field cell counts were performed. Overall, there was a trend for greater cell proliferation in the samples grown in chondrogenic media first, and transitioned into osteogenic media compared with samples grown entirely in osteogenic media.
Preliminary scanning electron microscopy of cells grown within the PPF scaffolds revealed ~90% occlusion of scaffold pores with osteocytes and extensive vWF staining.
In summary, we show that periosteum derived cells can be induced to differentiate into osteocytes and vascular cells when grown in 3D culture media under various cytokine milieus. Furthermore, we anticipate these cells may be used to create new bone along a PPF scaffold, pre-coated with collagen.