טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBerkovitch Yulia
SubjectPrecisely Controlled Platform for the In Vitro and In Vivo
Regeneration of Peripheral Nerves
DepartmentDepartment of Biotechnology
Supervisor Professor Dror Seliktar
Full Thesis textFull thesis text - English Version


Abstract

Treatment of peripheral nerve injuries has evolved over the past several decades to include the use of sophisticated new materials endowed with trophic and topographical cues that are essential for in vivo nerve fiber regeneration. Control over cell behavior is achieved by mimicking the same signals that direct nerve development during embryogenesis, or nerve fiber regeneration during natural tissue repair. These interdependent signals include spatiotemporal delivery of neurotropic factors, extracellular matrix interactions, and cell-cell communications. In this research, we explored the use of an advanced engineering design strategy for peripheral nerve repair, using semi-synthetic biomaterials that enable precisely controlled environmental stimuli to regenerate neurons and glial cells after peripheral nerve injury. We created a provisional nerve scaffold comprised of natural fibrinogen, gelatin, or albumin and synthetic polyethylene glycol (PEG) constituents.  This combination allowed us to control the degradation rate, protein composition, optical properties, and structural features of the matrix and thus to establish a more precise platform for the in vitro and in vivo regeneration of peripheral nerves.  We demonstrated the capability of the PEG-Fibrinogen, PEG-Gelatin, and PEG-Albumin hydrogels to support three-dimensional (3D) neurite and Schwann cell invasion using a neonatal chick dorsal root ganglia (DRG) assay. We also used a photo-patterning technique that is applied to these semitransparent 3D hydrogel biomaterials in order to create microscopic guidance patterns in the form of microchannels. The DRG assay was then used to investigate how physical and biochemical attributes of the microchannels govern the directional outgrowth of neurites and glial cells into the 3D patterned biomaterials. We provided a full characterization of the effects of microchannel diameter and protein constituents on the neurite extension and glial cell migration. We found that the laser energy of the ablation can be used in order to control the dimensions of the channels and the subsequent outgrowth of neuronal cells into them. However, the biomolecular signaling from the fibrinogen backbone of the hydrogel had an equally pronounced effect on neuronal outgrowth into microchannels. These results indicated that trophic and topographical cues are indeed essential aspects in the design of neural engineering matrices that can be used for in vivo axonal regeneration or for basic neuronal morphogenesis research. Finally, we showed that PEG-fibrinogen and PEG-gelatin hydrogels could just as well support nerve regeneration in rat sciatic nerve resection model.