**4. Biomaterial and cellular system association – discussion and final remarks**

Using the rat model, we recently tested *in vivo* the efficacy of biomaterials and cellular sys‐ tem association in treatment of sciatic nerve axonotmesis and neurotmesis injury. Following transection, axons show staggered regeneration and may take substantial time to cross the injured site and enter the distal nerve stump [119]. However delayed axonal elongation might be caused by growth inhibition originated from the distal nerve itself, growth-stimu‐ lating influences may overcome axons stagger. As a potential source of growth promoting signals, MSCs transplantation is expected to give a positive outcome. Our results showed that the use of either undifferentiated or differentiated MSCs in axonotmesis lesion boosted the recovery of sensory and motor function. In both cell-enriched experimental groups we observed that the myelin sheath was thicker, this suggests that MSCs might apply their posi‐ tive effects on SCs, the key element in Wallerian degeneration and the following axonal re‐ generation [120]. Also results from *in vivo* testing previously performed by our research group showed that infiltration of MSCs from the Wharton's jelly, or the combination of chi‐ tosan type III membrane enwrapment and MSCs enrichment after nerve crush injury pro‐ vide an advantage to post-traumatic nerve regeneration [56, 57]. Chitosan type III was developed as a hybrid of chitosan by adding GPTMS. A synergistic effect of an extra perme‐ ability and physicochemical properties of chitosan type III and the presence of silica ions Warburg effect and glycolysis stimulation. MSCs do not require oxidative phosphorylation to survive as alternative, hypoxia extends the lifespan, increases their proliferative ability and reduces differentiation [118]. The morphologic and biochemical characteristics of neu‐ ral-like cells are already described but the mechanism by which stem cells differentiate into neural-like cells is still unknown. In our research work, MSCs that undergone differentiation into neural-like cells, consumed significantly less glucose and produced significantly less lactate than MSCs that undergone only expansion. These major differences allow us to con‐ clude that during MSCs differentiation in neural-like cells the glycolytic process, which proved to be the crucial metabolic mechanism during MSCs expansion, is switched to oxida‐

Our results show clear evidences that MSCs expansion is dependent of glycolysis while their differentiation in neural-like cells requires the switch of the metabolic profile to oxida‐ tive metabolism. Also important may be the role of oxidative stress during this process. This work is a first step to identify key metabolic-related mechanisms responsible for human

The lack of standardization of MSCs isolated from the Wharton's jelly culture conditions has limited some progress in scientific and clinical research. Understanding these MSCs metabo‐ lism during expansion, as well as determining molecular and biochemical mechanisms for differentiation is of great significance to develop new effective stem cell-based therapies.

**4. Biomaterial and cellular system association – discussion and final**

Using the rat model, we recently tested *in vivo* the efficacy of biomaterials and cellular sys‐ tem association in treatment of sciatic nerve axonotmesis and neurotmesis injury. Following transection, axons show staggered regeneration and may take substantial time to cross the injured site and enter the distal nerve stump [119]. However delayed axonal elongation might be caused by growth inhibition originated from the distal nerve itself, growth-stimu‐ lating influences may overcome axons stagger. As a potential source of growth promoting signals, MSCs transplantation is expected to give a positive outcome. Our results showed that the use of either undifferentiated or differentiated MSCs in axonotmesis lesion boosted the recovery of sensory and motor function. In both cell-enriched experimental groups we observed that the myelin sheath was thicker, this suggests that MSCs might apply their posi‐ tive effects on SCs, the key element in Wallerian degeneration and the following axonal re‐ generation [120]. Also results from *in vivo* testing previously performed by our research group showed that infiltration of MSCs from the Wharton's jelly, or the combination of chi‐ tosan type III membrane enwrapment and MSCs enrichment after nerve crush injury pro‐ vide an advantage to post-traumatic nerve regeneration [56, 57]. Chitosan type III was developed as a hybrid of chitosan by adding GPTMS. A synergistic effect of an extra perme‐ ability and physicochemical properties of chitosan type III and the presence of silica ions

MSCs from the Wharton's jelly expansion and differentiation [55].

tive metabolism [55].

486 Advances in Biomaterials Science and Biomedical Applications

**remarks**

may be responsible for the good results in post-traumatic nerve regeneration promotion ob‐ served in the sciatic nerve after axonotmesis and neurotmesis [57, 91]. The substantial im‐ provement of axonal regeneration found in sciatic nerve crush enwrapped by chitosan type III membranes and for bridging nerve gaps after neurotmesis [57, 91], suggests that this bio‐ material may not just work as a simple mechanical device but instead may induce nerve re‐ generation. The neuroregenerative properties of chitosan type III may be explained by the effect on SCs proliferation, axon elongation and myelinization [55, 91]. Our data also showed that PLC does not deleteriously interfere with the nerve regeneration process, as a matter of fact, the information on the effectiveness of PLC membranes and tube-guides for allowing nerve regeneration was already provided experimentally and with patients [82]. The MSCs from the Wharton's jelly may be a valuable source in the repair of the peripheral nervous system with capacity to differentiate into neuroglial-like cells. The transplanted MSCs are also able to promote local blood vessel formation and release the neurotrophic fac‐ tors brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) [55]. Previous results obtained by our research group using N1E-115 cells *in vitro* differentiated into neuroglial-like cells to promote regeneration of axonotmesis and neuro‐ tmesis lesions in the rat model showed that there was no significant effect in promoting ax‐ on regeneration and, when N1E-115 cells were cultured inside a PLGA scaffold used to bridge a nerve defect, they can even exert negative effects on nerve fiber regeneration. The presence of transplanted N1E-115 cells in nerve scaffolds competing for the local blood sup‐ ply of nutrients and oxygen and by space-occupying effect could have hindered the positive effect of local neurotrophic factor release leading a negative outcome on nerve regeneration. Thus, N1E-115 cells did not prove to be a suitable candidate cellular system for treatment of nerve injury after axonotmesis and neurotmesis and their application is limited only to re‐ search purposes as a basic scientific step for the development of other cell delivery systems, due to its neoplastic origin [57-59, 91, 93]. The MSCs isolated from the Wharton´s jelly through PLC and chitosan type III membranes might be a potentially valuable tool to im‐ prove clinical outcome especially after trauma to sensory nerves, such as digital nerves. The results from our experimental work [55, 56] showed that the use of either undifferentiated or neuroglial-like differentiated MSCs enhanced the recovery of sensory and motor function of the rat sciatic nerve. The observation that in both cell-enriched experimental groups myelin sheath was thicker, suggest that MSCs might exert their positive effects on SCs, the key ele‐ ment in Wallerian degeneration and the following axonal regeneration [120]. In addition, these cells represent a non-controversial source of primitive mesenchymal progenitor cells that can be harvested after birth, cryogenically stored, thawed, and expanded for therapeu‐ tic uses, including nerve injuries like axonotmesis and neurotmesis. The time and tempera‐ ture of the transport (and the saline solution used) of the UC units from the hospital / clinic to the laboratory is crucial for a successful outcome considering MSCs isolation and prolifer‐ ation from fresh and cryopreserved UCT. It is highly recommend that the transport from the clinic or hospital to the laboratory should be refrigerated, and the UC units should be imme‐ diately immersed in a sterile saline solution like HBSS or DPBS.
