Evaluation of Mesenchymal Stem Cells from Multiple Sources for Optimal Osteoinduction on Nanofiber Scaffolds
John A. van Aalst, MD, MA, FACS1, Courtney R. Reed, BS1, Monteserrat Caballero, PhD1, Anthony Andrady, PhD2, Li Han, PhD2, Elizabeth G. Loboa, PhD3, Salim C. Saba, MD1.
1University of North Carolina, Chapel Hill, Chapel Hill, NC, USA, 2RTI, International, Durham, NC, USA, 3North Carolina State University, Raleigh, NC, USA.
PURPOSE: Tissue engineering requires a reliable source of pleuripotential cells and a predictable scaffold. Adult abdominoplasty fat is an increasingly utilized source of mesenchymal stem cells; an emerging, yet under-utilized, source is the umbilical cord. Both have the advantage of being an unlimited, and generally discarded, supply of stem cells. Palate periosteum is another, untapped, source of mesenchymal stem cells. Nanofibers are ideal scaffolds because the submicron fibers have an exponentially large surface-to-volume ratio that improves cell-scaffold interaction. Nanofibers can be created from multiple polymers, including polycaprolactone (PCL; monocryl suture), and poly (lactic-co-glycolytic) acid (PLGA; absorbable plates and screws). The most common, predictable technique for creating nanofibrils is electrospinning, whereby a viscous polymer is injected across a voltage potential toward a collecting plate, creating the nanofiber.
The objective of this work is to compare osteoinduction of mesenchymal stem cells derived from human adipose-derived adult mesenchymal stem cells (hADAS), umbilical cord-derived stem cells (UCSCs), and palate periosteum (PPSCs). The combination of nanofiber scaffolds with mesenchymal stem cell osteoinduction provides unique solutions for craniofacial bone defects.
METHODS: IRB approval was obtained for all studies. Nanofibers were electrospun with PCL and PLGA in methylene chloride (fiber diameter, 200 to 400 nm). hADAS harvested from abdominoplasty specimens were grown to passage 3. UCSCs were harvested by explant technique, and PPSCs were harvested by 4 mm2 punch biopsy and grown to passage 2. Surface markers were determined; cells were seeded onto PCL and PLGA nanofibers (density of 5.3 x 104 cells/cm2) and grown to confluence in appropriate media; cells were osteoinduced in media containing dexamethasone, ascorbic acid, and b-glycerophosphate. At 3 weeks, Alizarin red staining for calcium formation and real time polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP-2) was performed.
RESULTS: hADASs were CD73+, CD105+, CD166+, CD34-, and CD45-; hUCSCs and PPSCs were CD73+, CD105+, CD34-, CD90+, and SSEA-4+. However, there were differences in percent positivity when comparing hUCSCs and PPSCs: hUCSCs were 97% CD 90+ and 85% SSEA-4+, while PPSCs were 60% and 20% +, respectively. hADASs showed higher proliferation on PLGA while hUCSCs showed similar growth and viability on both PLGA and PCL scaffolds. All three cell types showed significant calcium deposition following osteoinduction. However, BMP-2 expression in hADASs was significantly greater than control on PCL (443%) as compared to PLGA (39%) nanoscaffolds. BMP-2 expression in hUCSCs was greater than control on PCL (70%) and on PLGA (56%) nanoscaffolds. ALP expression in hADASs was significantly greater than control on PCL (592%) than on PLGA (72%) nanoscaffolds.
CONCLUSION: These studies suggest variations in percent positivity for mesenchymal stem cell surface markers on hADASs, hUCSCs and PPSCs. Though proliferation appeared to be equivalent on both polymer scaffolds, hADASs and hUCSCs showed greater osteoinduction on PCL nanoscaffolds. This finding suggests that the scaffold polymer itself may be an active participant in osteoinduction. Optimizing the source of mesenchymal stem cells for reliable, robust osteogenesis, on nanoscaffolds that increase osteogensis will lead to improved solutions for craniofacial bone defects.