College of Engineering

HistologicalAnalysisofNerveRegenerationinNerveConduitsEnhancedwithGrowthFactorsandAlignedFibers

Thank you to the College of Engineering for the Undergraduate Research Opportunity (UROP)

Grant.

Opportunity and SignificancePeripheral nerve injury can cause a complete loss offunctionality in the respective limb if the injured nerveimproperly regenerates. The current ‘gold standard’is an autograft but nerve growth conduits (NGC)have been developed to direct regeneration. In thisstudy, we fabricate a NGC with aligned fibers andgrowth-factor releasing microspheres. Histology isused to evaluate the extent of regeneration bymeasuring nerve fiber density and the myelination ofaxons in both exercise and without exercise groups.

Next Steps for Development and Test• We expect to see increased regeneration

histologically. Future steps consist of morestaining and analysis of longitudinally-cutnerves.

• Cross-sectioned paraformaldehyde-fixednerves have to be sliced and stained withtoluidine blue solution which dyes nucleicacids blue & polysaccharides purple.

• The gastrocnemius muscles will also behistologically analyzed for muscle fiberregeneration.

Technical ObjectivesDetermine if there is a synergistic effect on nervegrowth when physical therapy and extended growthfactor release NGC are combined throughhistological analysis.

Related Work [1]

References[1] Whitehead, T. J. & Sundararaghavan, Jove-J. Vis. Exp. e51517 (2014). [2] Xu, X. et al. Biomaterials 24, 2405–2412 (2003).

Monica Prasad Tonya Whitehead Harini Sundararaghavan, Ph.D Biomedical Engineering

Figure 2: NGC + GF

Figure 4: NGC + PT Group, with H&E

Figure 5: NGC without PT, Neurofilament stained with DAB

Figure 1: Aligned scaffolds fabricated w/ (right image) & w/out microspheres (left image) incorporated and cultured with DRGs

In Vitro• Cell culture studies about dorsal root ganglion (DRG)

with microspheres showed unidirectional neurite growth in aligned fibers (Figure 1)

• Growth is both directed and more robust with the GF microspheres & aligned fibers

Technical Approach, Accomplishments, and Results

• Conduits consisting of electrospun-fibers with growth-factor containing microspheres were inserted in Sciatic nerve injury model in Lewis Rats

In Vivo (3 month longevity)Five Surgical Treatments:• Group 1: Autograft • Group 2: NGC with growth factors and physical therapy

Group 3: NGC with growth factors but NO physical therapy (PT)

• Group 4: NGC without growth factors but WITH physical therapy

• Group 5: NGC without growth factors and without physical therapy

Post-Harvest• Paraformaldehyde-fixed nerves were paraffin embedded,

longitudinally sectioned, and stained w/ rabbit anti-neurofilament 200 antibody and hematoxylin-eosin (H&E)

• H&E stains nuclei and protein• Coloration with 3,3-diaminobenzidine (DAB) stains

neurofilament [2]

Figure 3: NGC + GF + PT