Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

Abstract

Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorbs membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96 h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorbs membrane, the [Ca 2þ ]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorbs membrane proved to be adequate and used as scaffold associated with undiffer-entiated HMSCs or neuroglial-differentiated HMSCs. In vivo testing was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorbs membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL).Stereological analysis was carried out on regenerated nerve fibers. In vitro investigation showed the formation of typical neuroglial cells after differentiation, which were positively stained for the typical specific neuroglial markers such as the GFAP, the GAP-43 and NeuN.NMR showed clear evidence that HMSCs expansion is glycolysis-dependent but their differentiation requires the switch of the metabolic profile to oxidative metabolism. In vivo studies showed enhanced recovery of motor and sensory function in animals treated with transplanted undifferentiated and differentiated HMSCs that was accompanied by an increase in myelin sheath. Taken together, HMSC from the umbilical cord Wharton jelly might be useful for improving the clinical outcome after peripheral nerve lesion.

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“…These genes were not expressed, or expressed at very low levels, in the undifferentiated MSCs transcriptome. Overall, these results support the fact that the MSCs used in vivo were not differentiated but presented a neuro-glial potential for differentiation confirming previous results where the same MSCs undifferentiated and in vitro differentiated into neuro-glial cells were tested in axonotmesis and neurotmesis injuries associated to other biomaterials namely poly(DL-lactide-ε-caprolactone), hybrid chitosan and collagen [3,25,31] .…”

“…The histological nerve preparation followed the previously reported protocol [3,9,17,25,31,33,35] and a systematic random sampling and D-disector were adopted [41][42][43][44] . At the end of the healing period tested (week 20), tibialis anterior (TA) muscles of all experimental (PVA, PVA-CNTs, PVA-PPy, Graft, End-to-End, and PVA-CNTsMSCs) and Control (no lesion) groups were collected, and the tissue samples were fixed in 10% buffered formalin, routinely processed, dehydrated and embedded in paraffin wax.…”

“…Also, it is considered by the scientific bibliography that rats without sciatic nerve injury withdraw their paws from the hotplate within 4.3 s or less [9,17,31,33] . If there was no paw withdrawal after 12 s, the animal was assigned the maximal WRL of 12 s and the heat stimulus was removed to prevent tissue damage [34][35][36] .…”

“…The force in grams (g) applied is recorded individually and the results for both hind-limbs (affected and un-affected hind-limb) are taken for equation 1 calculation. The normal (unaffected limb) EPT (NEPT) and experimental EPT (EEPT) values considered for equation (1) and the percentage of functional deficit is calculated [9,[28][29][30][31] .…”

“…To assess the nociceptive WRL, the hotplate test described by Masters et al [32] was used with some modifications and previously described by the authors [3,9,17,25,31,33] . Also, it is considered by the scientific bibliography that rats without sciatic nerve injury withdraw their paws from the hotplate within 4.3 s or less [9,17,31,33] .…”