Poly (D,L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord.
Freeze-dried poly(D,L-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/10(6) cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 h, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at 1 week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved.
Hurtado
Andres
A
The Miami Project to Cure Paralysis, University of Miami School of Medicine, P.O. Box 016960, R-48, Miami, FL 33136, USA.
Moon
Lawrence D F
LD
Maquet
Véronique
V
Blits
Bas
B
Jérôme
Robert
R
Oudega
Martin
M
D000818:Animals; D001369:Axons / Q000737:chemistry / Q000502:physiology; D001808:Blood Vessels / Q000502:physiology; D049108:Cell Enlargement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002809:Chondroitin Sulfates / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D048091:Guided Tissue Regeneration / Q000379:methods*; D006614:Hindlimb / Q000187:drug effects / Q000503:physiopathology; D020007:Implants, Experimental; D019344:Lactic Acid / Q000737:chemistry / Q000627:therapeutic use*; D018919:Neovascularization, Physiologic; D009413:Nerve Fibers, Myelinated / Q000502:physiology; D009414:Nerve Growth Factors / Q000235:genetics* / Q000378:metabolism; D009416:Nerve Regeneration; D020933:Neurotrophin 3 / Q000235:genetics / Q000378:metabolism; D011091:Polyesters; D011108:Polymers / Q000737:chemistry / Q000627:therapeutic use*; D051381:Rats; D011916:Rats, Inbred F344; D012583:Schwann Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D013116:Spinal Cord / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D013119:Spinal Cord Injuries / Q000473:pathology / Q000628:therapy*; D013904:Thoracic Vertebrae; D014162:Transfection
D017077:Culture Media, Conditioned; D005904:Glial Fibrillary Acidic Protein; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D011091:Polyesters; D011108:Polymers; C038142:chondroitin sulfate glycosaminoglycan; D049452:Green Fluorescent Proteins; D019344:Lactic Acid; C033616:poly(lactide); D002809:Chondroitin Sulfates