3D-printed nerve guidance conduits multi-functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats

Abstract

Background Nerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration. Methods 3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve. Results The inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair. Conclusions 3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

A biocomplex to repair experimental critical size defects associated with photobiomodulation therapy

“…The surgical cavities in which the fibrin biopolymer (XS and XS-PBM groups) were used showed intense angiogenesis as early as 14 days, as well as the occasional presence of granulation tissue and the permanence of vascular spaces at 42 days [ 75 ]. This biological response demonstrates that the biopolymer is biocompatible, in agreement with previous studies [ 31 , 76 , 77 ], because in addition to not having a foreign body reaction, it favored the insertion in the surgical bed, the deposition and proliferation of osteoblastic cells, functioning as a scaffold for bone regeneration [ 78 - 82 ].…”

“…It can also be highlighted the binding function of the fibrin biopolymer, since in its use with the Bio-Oss TM particles, it allowed the permanence in the implantation site forming multiple layers. As the biomaterials particulate in granules can be easily displaced from the surgical bed, even by the action of the local bleeding itself, it allows the use of these agents to be inducted with the purpose of forming a firm and mechanically stable network, with good adhesive properties [ 22 , 26 , 82 ]. The clinical applicability and benefits of commercially available fibrin sealants, derived from human blood, for soft tissues are well documented, but their contribution to bone surgery and oral and periodontal surgery remains controversial [ 83 ].…”

“…Finally, snake venom purified fibrin biopolymer is the only totally heterologous sealant in the world, with several beneficial properties in multiple areas of regenerative medicine. It has a low production cost, has proven to be clinically safe and effective in the treatment of chronic venous ulcers, and, through pre-clinical research carried out, a great potential for clinical applications such as scaffolding and drug delivery systems is foreseen [ 27 , 34 , 38 , 82 , 84 - 89 ]. The idea of using a biocomplex associated with PBM therapy by our research group opens a new opportunity to develop an innovative therapy to treat critical-size defects.…”

Carbon-nanotube yarns induce axonal regeneration in peripheral nerve defect

“…In the cross-sections of the 16-week evaluation, the numbers of neurofilament, the numbers of S100 positive cells and the IR of S100 was measured at the proximal and distal nerve end. The immunoreactivity was calculated using the ratio of the immunoreactivity area at the P5, D5, and D nerve sites to the P site, referring to the previously described methods 30 , 31 . For cell count, the numbers of S100 stainable structures with DAPI stained nuclei were counted respectively by manual cell counting.…”

In vitro study of decellularized rat tissues for nerve regeneration

“…This is always characteristic of low antigenicity, supports vascularization, is porous for oxygen diffusion, and avoids long-term compression and tissue adhesion. Nerve-guiding conduits made of various materials have been previously investigated, and their ability to facilitate peripheral nerve regeneration has been demonstrated in animals ( 9 , 10 ). Not only natural biomaterials, such as collagen ( 11 ), silk ( 12 ), and gelatin ( 13 ), but also synthetic neural conduits such as polylactic acid and poly (lactic-co-glycolic acid) ( 14 , 15 ), play an important role in nerve regeneration with additional cells or growth factors.…”