**3. Regeneration and in vivo testing**

thus supporting the suitability of our cell culture and differentiation procedures. This con‐ cern also resulted from our previous experience with N1E-115 neoplastic cell line and the negative results we obtained in the treatment of axonotmesis and neurotmesis injuries [57-59]. Nevertheless, undifferentiated MSCs from the Wharton's jelly culture (obtained from either protocol or from the Promocell cell line) showed normal morphology when in‐

The differentiation was tested based on the expression of typical neuronal markers such as GFAP, GAP-43 and NeuN by neural-like cells attained from MSCs. Undifferentiated MSCs were negatively labeled to GFAP, GAP-43 and NeuN. After 96 hours of differentiation the attained cells were positively stained for glial protein GFAP and for the growth-associated protein GAP-43. All nucleus of neural-like cells were also labeled with the neuron specific nuclear protein called NeuN showing that differentiation of MSCs in neural-like cells was successfully achieved for MSCs obtained from UCT (fresh and cryopreserved) and for the

MSCs express nestin, a maker for neural and other stem cells [60, 61] and can be differentiat‐ ed in adipose tissue, bone, cartilage, skeletal muscle cells, cardiomyocyte-like cells, and neu‐ roglial-like cells [54, 55, 60, 62], presenting great potential to biomedical engineering applications. These cells fit into the category of primitive stromal cells and because they are abundant and inexpensive, they might be very useful for regenerative medicine and biotech‐

By employing neuron-conditioned media, sonic hedgehog and fibroblast growth factor 8, MSCs isolated from the Wharton's jelly can be induced toward dopaminergic neurons. These cells have been transplanted into hemiparkinsonian rats where they prevented the progressive degeneration/behavioral deterioration seen in these rats [63]. Rat MSCs isolated from the Wharton's jelly when transplanted into brains of rats with global cerebral ischemia significantly reduced neuronal loss, apparently due to a rescue phenomenon [64]. Neuronal differentiation of human MSCs could also provide cells to replace neurons lost due to neuro‐ degenerative diseases. Recent studies showed that transplanted MSCs-derived neurons be‐ come electrophysiologically integrated within the host neural tissue [65]. However, all these

A consequence of cell metabolism during *in vitro* expansion is that culture conditions are constantly changing. The comprehension and optimization of the expansion and differentia‐ tion process will contribute to maximization of cell yield, reduced need of cell culture, and a decrease in total processing costs [66, 67]. Elucidation of regulatory mechanisms of MSCs differentiation will allow optimization of *in vitro* culture and their clinical use in the treat‐ ment of neural-related diseases. Research is being performed to optimize expansion process parameters in order to grow MSCs in a controlled, reproducible, and cost-effective way [68].

therapeutic applications need uniform and reproducible regulation.

Metabolism is certainly one of these parameters.

spected with an inverted microscope (Figure 7).

476 Advances in Biomaterials Science and Biomedical Applications

Promocell MSC cell line (Figure 8) [55].

nology applications.

*2.1.2. Differentiation into neuroglial-like cells*

With the world wide global increase in life expectancy, a variety of disabling diseases with large impact on human population are arising. This includes cardiovascular, neurological, musculoskeletal, and malignancies. Therefore, it is imperative that new and more effective treatment methods are developed to correct for these changes. Further research with experi‐ mental animal systems is required to translate to in vivo cell-based therapy that has been extensively investigated in vitro [1]. Stem cell biology is probably the golden key for cell therapies and regenerative medicine. Regeneration is the physical process where remaining tissues organize themselves to replace missing or injured tissues *in vivo* [39].

It has been speculated that once MSCs have the potential to differentiate into several tissues, they might be responsible for turnover and maintenance of adult tissues, just like hemato‐ poietic stem cells have this role in blood cells [69]. First, it was believed that after injection of MSCs, these were able to migrate to the damaged site and to differentiate into ones with the appropriate function for repairing, so MSCs could mediate tissue repair through there mul‐ tilineage capacity replacing damaged cells. Subsequent studies have suggested that the mechanism used by MSCs for tissue repairing is not really this way. This new idea was rein‐ forced by the confirmation that this cells homed to damaged site, particularly to spots of hy‐ poxia, inflammation and apoptosis [70, 71].

Recent studies demonstrated that transplanted MSCs modified the surrounding tissue mi‐ croenvironment, promoting repair with functional improvement by secretion factors (known as paracrine effect), stimulation of preexisting stem cells in the original tissue and decreasing of inflammation and immune response [72]. Other studies have demonstrated that MSC-conditioned media by itself could have therapeutic effects. All this data suggest that MSC apply a reparative effect on injured side through its paracrine effects [73].

It is necessary to overcome some barriers before a cell-based therapy becomes routine in clinics, including the cell number and the administration way of treatment. MSCs are diffi‐ cult to be maintained stable in culture for long time, but due to their short doubling time, if at the outset many cells are harvested they may be properly scaled up in primary culture, never forgetting the ideal seeding number [39].

MSCs are an attractive candidate for cell-based regenerative therapy; the evidence is that currently there are 139 trial registries for MSC therapy 27 of which are based on umbilical cord MSCs [74].

#### **3.1. Nerve regeneration**

After Central Nervous System (CNS) lesions, Peripheral Nervous System (PNS) injuries are the ones with minor successes in terms of functional recovery. These kinds of injuries are frequent in clinical practice. About two centuries ago it was assumed that these nerves would never regenerate. Indeed, scientific and clinical knowledge greatly increased in this area. Nevertheless, a full understanding of axonal recovery and treatment of nerve defects, especially complete functional achievement and organ reinnervation after nerve injury, still remains the principle challenge of regenerative biology and medicine [75, 76].

#### *3.1.1. Nerve repair*

Many peripheral nerve injuries can only be dealt through reconstructive surgical proce‐ dures. Despite continuous refinement of microsurgery techniques, peripheral nerve repair still stands as one of the most challenging tasks in neurosurgery, as functional recovery is rarely satisfactory in these patients [76]. Direct repair should be the procedure of choice whenever tension-free suturing is possible; however, patients with loss of nerve tissue, re‐ sulting in a nerve gap, are considered for a nerve graft procedure. In these cases, the donor nerves used for grafting are commonly expendable sensory nerves. This technique, howev‐ er, has some disadvantages, with the most prominent being donor site morbidity, that may lead to a secondary sensory deficit and occasionally neuroma and pain. In addition, no do‐ nor and recipient nerve diameters often occurs which might be the basis for poor functional recovery. Alternatives to peripheral nerve grafts include cadaver nerve segments allografts, end-to-side neurorrhaphy, and entubulation by means of autologous non-nervous tissues, such as vein and muscles [76]. One advantage of these allografts compared with the auto‐ grafts is the absence of donor site morbidity and theoretically the unlimited length of tissue available [77]. Experimental work from a number of laboratories has emphasized the impor‐ tance of entubulation for peripheral nerve repair to manage nerve defects that cannot be bridged without tension (neurotmesis with loss of nerve tissue). Nerves will regenerate from the proximal nerve stump towards the distal one, whereas neuroma formation and in‐ growth of fibrous tissue into the nerve gap are prevented [78]. The reliability of animal mod‐ els is crucial for PN research, including therapeutic strategies using biomaterials and cellular systems. As a matter of fact, rodents, particularly the rat and the mouse, have become the most frequently used animal models for the study of peripheral nerve regeneration because of the widespread availability of these animals as well as the distribution of their nerve trunks which is similar to humans [79]. Because of its PN size, the rat sciatic nerve has been the most commonly experimental model used in studies concerning the PN regeneration and possible therapeutic approaches [80]. Functional recovery after PN injury is frequently incomplete, even with adequate microsurgery, so, many research and clinical studies have been performed including biomaterials for tube-guides. Since the 80's, Food and Drug Ad‐ ministration (FDA) has approved a variety of these biomaterials both natural and synthetic. The ideal biomaterial nerve graft should increase number, length and speed of axon regen‐ eration [77]. It should be:


#### **v.** technically reproducible, transparent, easy to manipulate, and sterilize [81].

Currently 3 types of materials are available for nerve reconstruction: non-resorbable, natural resorbable and synthetic resorbable. Polyvinil alcohol hydrogel (PVA) is an example of a non-absorbable biomaterial. It combines water in similar proportions to human tissue, with PVA providing a stable structure easy to sterilize, which is a main advantage of this materi‐ als, but has some limitations such as: nerve compression and suture tension after regenera‐ tion due to its non-resorbable nature [77]. Collagen type I from humans or animals, is an example of a natural resorbable device, which has some advantages such as:

**i.** easy to isolate and purify,

especially complete functional achievement and organ reinnervation after nerve injury, still

Many peripheral nerve injuries can only be dealt through reconstructive surgical proce‐ dures. Despite continuous refinement of microsurgery techniques, peripheral nerve repair still stands as one of the most challenging tasks in neurosurgery, as functional recovery is rarely satisfactory in these patients [76]. Direct repair should be the procedure of choice whenever tension-free suturing is possible; however, patients with loss of nerve tissue, re‐ sulting in a nerve gap, are considered for a nerve graft procedure. In these cases, the donor nerves used for grafting are commonly expendable sensory nerves. This technique, howev‐ er, has some disadvantages, with the most prominent being donor site morbidity, that may lead to a secondary sensory deficit and occasionally neuroma and pain. In addition, no do‐ nor and recipient nerve diameters often occurs which might be the basis for poor functional recovery. Alternatives to peripheral nerve grafts include cadaver nerve segments allografts, end-to-side neurorrhaphy, and entubulation by means of autologous non-nervous tissues, such as vein and muscles [76]. One advantage of these allografts compared with the auto‐ grafts is the absence of donor site morbidity and theoretically the unlimited length of tissue available [77]. Experimental work from a number of laboratories has emphasized the impor‐ tance of entubulation for peripheral nerve repair to manage nerve defects that cannot be bridged without tension (neurotmesis with loss of nerve tissue). Nerves will regenerate from the proximal nerve stump towards the distal one, whereas neuroma formation and in‐ growth of fibrous tissue into the nerve gap are prevented [78]. The reliability of animal mod‐ els is crucial for PN research, including therapeutic strategies using biomaterials and cellular systems. As a matter of fact, rodents, particularly the rat and the mouse, have become the most frequently used animal models for the study of peripheral nerve regeneration because of the widespread availability of these animals as well as the distribution of their nerve trunks which is similar to humans [79]. Because of its PN size, the rat sciatic nerve has been the most commonly experimental model used in studies concerning the PN regeneration and possible therapeutic approaches [80]. Functional recovery after PN injury is frequently incomplete, even with adequate microsurgery, so, many research and clinical studies have been performed including biomaterials for tube-guides. Since the 80's, Food and Drug Ad‐ ministration (FDA) has approved a variety of these biomaterials both natural and synthetic. The ideal biomaterial nerve graft should increase number, length and speed of axon regen‐

**i.** biocompatible, not toxic neither present undesired immunologic response;

**ii.** permeable enough to permit nutrient and oxygen diffusion and allows cell support

**iv.** biodegradable, the ideal rate is to remain intact during axon regeneration across

remains the principle challenge of regenerative biology and medicine [75, 76].

*3.1.1. Nerve repair*

478 Advances in Biomaterials Science and Biomedical Applications

eration [77]. It should be:

systems;

**iii.** flexible and soft to avoid compression;

nerve gap and after degrade softly and


On the other hand, offers some immune response requiring the use of immunosuppres‐ sive drugs or pre-treatment of the material before clinical use [77]. Poly (DL-lactide-ε-capro‐ lactone) (PLC) a synthetic resorbable material is the only transparent device approved by FDA, important characteristic for the surgeon that facilitates the insertion of the nerve stumps across the nerve gap, but, on the other hand it is not flexible [77]. Chitosan, PLC, colla‐ gen, poly(L-lactide) and poly(glycolide) copolymers (PLGA) and others, some of them, pre‐ viously studied by our group [57, 58, 82] were associated to cellular systems, which are able to differentiate into neuroglial-like cells or capable of modulating the inflammatory proc‐ ess, improved nerve regeneration, in terms of motor and sensory recovery, and also shorten‐ ing the healing period after axonotmesis and neurotmesis, avoiding regional muscular atrophy [57, 58, 82].

Researches with acellular nerve allografts, as alternative for repairing peripheral nerve de‐ fects have been reported. These nerve allografts remove the immunoreactive SCs and mye‐ lin however preserve the internal structure of original nerve, containing vital components such as collagen I, laminin and growth factors essential for repairmen of the lesions [83]. Acellu‐ lar grafts remain insufficient, due to the increasing extent of nerve damages. Also, viable cells are necessary for debris removal and environmental regeneration reestablishment [83].

Cell transplantation, such as Schwann cells (SCs) transplantation has been proposed as a method of improving peripheral nerve regeneration [84]. SCs are peripheral glial cells that enwrap axons to form myelin with a central role in neuronal function. When there is dam‐ age in PNS, SCs are induced to mislay myelin, proliferate and segregate numerous factors, including cytokines responsible for reproducing a microenvironment suitable for support‐ ing axon regeneration [85, 86]. They also have a vital participation in endogenous repair, re‐ constructing myelin, which are essential for functional recovery [85, 86]. SCs, MSCs, ESCs, marrow stromal cells are the most studied support cells candidates. SCs transplantation en‐ hance axon outgrowth both *in vitro* [87] and *in vivo* [88]. Although to achieve an adequate amount of autologous SC, a donor nerve is necessary and a minimum of 4-8 weeks for *in vitro* expansion. Umbilical cord MSCs may be the perfect cell model as supplement for nerve grafts, once they are easily obtained, with no ethical controversy and can differentiate into neuglial-like cells [83]. Matuse and collaborators induced MSCs from the umbilical cord into SCs capable of supporting peripheral nerve regeneration and myelin reconstruction *in vivo*. They transplanted these SCs into injured sciatic nerve, and proved that these cells main‐ tained their differentiated phenotype *in vivo*, and contributed for axonal regeneration and functional recovery [89].

In our studies we aimed to explore the therapeutic value of human umbilical cord matrix (Wharton's jelly) derived MSCs, undifferentiated and differentiated in neuroglial-like cells, both *in vitro* and *in vivo*, associated to a variety of biomaterials such as, Poly (DL-lactide-εcaprolactone) PLC (Vivosorb®) membrane, and Chitosan type III on rat sciatic nerve axo‐ notmesis and neurotmesis experimental model. For cell transplantation into injured nerves (with axonotmesis and neurotmesis injuries), there are two main techniques. The cellular system may be directly inoculated into the neural scaffold which has been interposed be‐ tween the proximal and distal nerve stumps or around the crush injury (in neurotmesis and axonotmesis injuries, respectively); or the cells can be pre-added to the neural scaffold via inoculation or co-culture (in most of the cellular systems, it is allowed to form a monolayer) and then the biomaterial with the cellular system is implanted in the injured nerve [82].

Our PLC studies [55] demonstrated that this biomaterial does not interfere negatively with the nerve regeneration process, in fact, the information on the effectiveness of PLC mem‐ branes and tube-guides for allowing nerve regeneration was already provided experimen‐ tally and with patients [82]. PLC becomes hydrophilic by water uptake, which increases the permeability of the polymer. This is essential for the control of nutrient and other metabolite transportation to the surrounding healing tissue. A few weeks after implantation, the me‐ chanical power gradually decreases and there is a loss of molecular weight as a result of the hydrolysis process. Nearly in 24 months, PLC degrades into lactic acid and hydroxycaproic acid which are both safely metabolized into water and carbon dioxide and/or excreted through the urinary tract. In contrast to other biodegradable polymers, PCL has the advant‐ age of not creating an acidic and potentially disturbing micro-environment, which is favora‐ ble to the surrounding tissue [90]. Chitosan has attracted particular attention in medical areas due to its biocompatibility, biodegradability, and low toxicity, low cost, improvement of wound-healing and antibacterial properties. Moreover, the potential use of chitosan in nerve regeneration has been demonstrated both *in vitro* and *in vivo* [57, 91]. Chitosan is a partially deacetylated polymer of acetyl glucosamine obtained after the alkaline deacetyla‐ tion of chitin [57, 82]. While chitosan matrices have low mechanical strength under physio‐ logical conditions and are unable to maintain a predefined shape after transplantation, their mechanical properties can be improved by modification with a silane agent, namely γ-glyci‐ doxypropyltrimethoxysilane (GPTMS), one of the silane-coupling agents which has epoxy and methoxysilane groups. The epoxy group reacts with the amino groups of chitosan mole‐ cules, while the methoxysilane groups are hydrolyzed and form silanol groups. Finally, the silanol groups are subjected to the construction of a siloxane network due to the condensa‐ tion. Thus, the mechanical strength of chitosan can be improved by the cross-linking be‐ tween chitosan, GPTMS and siloxane network. By adding GPTMS and employing a freezedrying technique, we have previously obtained chitosan type III membranes (hybrid chitosan membranes) with pores of about 110 μm diameter and about 90% of porosity, and which were successful in improving sciatic nerve regeneration after axonotmesis and neuro‐ tmesis [56, 57, 82].

neuglial-like cells [83]. Matuse and collaborators induced MSCs from the umbilical cord into SCs capable of supporting peripheral nerve regeneration and myelin reconstruction *in vivo*. They transplanted these SCs into injured sciatic nerve, and proved that these cells main‐ tained their differentiated phenotype *in vivo*, and contributed for axonal regeneration and

In our studies we aimed to explore the therapeutic value of human umbilical cord matrix (Wharton's jelly) derived MSCs, undifferentiated and differentiated in neuroglial-like cells, both *in vitro* and *in vivo*, associated to a variety of biomaterials such as, Poly (DL-lactide-εcaprolactone) PLC (Vivosorb®) membrane, and Chitosan type III on rat sciatic nerve axo‐ notmesis and neurotmesis experimental model. For cell transplantation into injured nerves (with axonotmesis and neurotmesis injuries), there are two main techniques. The cellular system may be directly inoculated into the neural scaffold which has been interposed be‐ tween the proximal and distal nerve stumps or around the crush injury (in neurotmesis and axonotmesis injuries, respectively); or the cells can be pre-added to the neural scaffold via inoculation or co-culture (in most of the cellular systems, it is allowed to form a monolayer) and then the biomaterial with the cellular system is implanted in the injured nerve [82].

Our PLC studies [55] demonstrated that this biomaterial does not interfere negatively with the nerve regeneration process, in fact, the information on the effectiveness of PLC mem‐ branes and tube-guides for allowing nerve regeneration was already provided experimen‐ tally and with patients [82]. PLC becomes hydrophilic by water uptake, which increases the permeability of the polymer. This is essential for the control of nutrient and other metabolite transportation to the surrounding healing tissue. A few weeks after implantation, the me‐ chanical power gradually decreases and there is a loss of molecular weight as a result of the hydrolysis process. Nearly in 24 months, PLC degrades into lactic acid and hydroxycaproic acid which are both safely metabolized into water and carbon dioxide and/or excreted through the urinary tract. In contrast to other biodegradable polymers, PCL has the advant‐ age of not creating an acidic and potentially disturbing micro-environment, which is favora‐ ble to the surrounding tissue [90]. Chitosan has attracted particular attention in medical areas due to its biocompatibility, biodegradability, and low toxicity, low cost, improvement of wound-healing and antibacterial properties. Moreover, the potential use of chitosan in nerve regeneration has been demonstrated both *in vitro* and *in vivo* [57, 91]. Chitosan is a partially deacetylated polymer of acetyl glucosamine obtained after the alkaline deacetyla‐ tion of chitin [57, 82]. While chitosan matrices have low mechanical strength under physio‐ logical conditions and are unable to maintain a predefined shape after transplantation, their mechanical properties can be improved by modification with a silane agent, namely γ-glyci‐ doxypropyltrimethoxysilane (GPTMS), one of the silane-coupling agents which has epoxy and methoxysilane groups. The epoxy group reacts with the amino groups of chitosan mole‐ cules, while the methoxysilane groups are hydrolyzed and form silanol groups. Finally, the silanol groups are subjected to the construction of a siloxane network due to the condensa‐ tion. Thus, the mechanical strength of chitosan can be improved by the cross-linking be‐ tween chitosan, GPTMS and siloxane network. By adding GPTMS and employing a freezedrying technique, we have previously obtained chitosan type III membranes (hybrid

functional recovery [89].

480 Advances in Biomaterials Science and Biomedical Applications

The induction of a crush injury in rat sciatic nerve provides a very realistic and useful model of damage for the study of the role of numerous factors in regenerative processes [57]. Focal crush causes axonal interruption but preserves the connective sheaths (axonotmesis). After axonotmesis injury regeneration is usually successful, after a short (1-2 day) latency, axons regenerate at a steady rate towards the distal nerve stump, supported by the reactive SCs and the preserved endoneural tubules enhance axonal elongation and facilitate adequate re‐ innervation [92]. Our research group has been testing the efficacy of combining biomaterials and cellular systems in the treatment of sciatic nerve crush injury [57-59, 82, 90, 91, 93-95]. Following transection, axons show staggered regeneration and may take substantial time to actually cross the injury site and enter the distal nerve stump [60]. Although delayed axonal elongation might be caused by growth inhibition originating from the distal nerve itself, growth-stimulating influences may overcome axons stagger. More robust and fast nerve re‐ generation is expected to result in better reinnervation and functional recovery. As a poten‐ tial source of growth promoting signals, MSCs transplantation is expected to have a positive outcome. Our results showed that the use of either undifferentiated or differentiated HMSCs enhanced the recovery of sensory and motor function in axonotmesis lesion of the rat sciatic nerve [56]. Neurotmesis must be surgically treated by direct end-to-end suture of the two nerve stumps or by a nerve graft harvested from elsewhere in the body in case of tissue loss. To avoid secondary damage due to harvesting of the nerve graft, a tube-guide can be used to bridge the nerve gap. Acutely after sciatic nerve transection there is a com‐ plete loss of both motor and thermal sensory function. Sensory and motor deficit then pro‐ gressively decrease along the post-operative. From a morphological point of view, nerve regeneration occurs if Wallerian degeneration is efficient and is substituted by re-growing axons and the accompanying viable SCs [96, 97]. The axon regeneration pattern is improved by using appropriate biomaterials for the tube-guide design, like chitosan type III and PLC and cellular systems like MSCs from the Wharton jelly [57, 90, 91, 95]. The surgical techni‐ que and the time for the reconstructive surgery is also crucial for the nerve regeneration af‐ ter neurotmesis [57, 90, 91, 95].

#### **3.2. Assessment of nerve regeneration in the sciatic nerve rat model**

Although both morphological and functional data have been used to assess neural regenera‐ tion after induced crush injuries, the correlation between these two types of assessment is usually poor [94, 98-100]. Classical and newly developed methods of assessing nerve recov‐ ery, including histomorphometry, retrograde transport of horseradish peroxidase and retro‐ grade fluorescent labeling [79] do not necessarily predict the reestablishment of motor and sensory functions [100-103]. Although such techniques are useful in studying the nerve re‐ generation process, they generally fail in assessing functional recovery [100]. In this sense, research on peripheral nerve injury needs to combine both functional and morphological as‐ sessment. The use of biomechanical techniques and rat's gait kinematic evaluation is a prog‐ ress in documenting functional recovery [104]. Indeed, the use of biomechanical parameters has given valuable insight into the effects of the sciatic denervation/reinnervation, and thus represents an integration of the neural control acting on the ankle and foot muscles, which is very useful and accurate to evaluate different therapeutic approaches [103-105].

#### *3.2.1. Functional Assessment*

After injury and treatment of animals, follow-up results are very important for analysis of functional recovery. Animals are tested preoperatively (week 0), and every week during 12 and 20 weeks, for axonotmesis and neurotmesis of the rat sciatic nerve, respectively. Motor performance and nociceptive function are evaluated by measuring extensor postural thrust (EPT) and withdrawal reflex latency (WRL), respectively [55, 58, 94]. For EPT test, the affect‐ ed and normal limbs are tested 3 times, with an interval of 2 minutes between consecutive tests, and the 3 values are averaged to obtain a final result. The normal (unaffected limb) EPT (NEPT) and experimental EPT (EEPT) values are incorporated into an equation (Equa‐ tion (1)) to derive the percentage of functional deficit, as described in the literature [106]:

$$\% \text{ Motor deficit} = \left[ \left( \text{NEPT} - \text{EEPT} \right) \text{ / NEPT} \right] \times 100 \tag{1}$$

The nociceptive withdrawal reflex (WRL) was adapted from the hotplate test developed by Masters et al. [107]. Normal rats withdraw their paws from the hotplate within 4s or less. The cutoff time for heat stimulation is set at 12 seconds to avoid skin damage to the foot.

For Sciatic Functional Index (SFI), animals are tested in a confined walkway that they cross, measuring 42 cm long and 8.2 cm wide, with a dark shelter at the end. Several measure‐ ments are taken from the footprints:


In the static evaluation (SSI) only the parameters TS and ITS, are measured. For SFI and SSI, all measurements are taken from the experimental (E) and normal (N) sides. Prints for meas‐ urements are chosen at the time of walking based on precise, clear and completeness of foot‐ prints. The mean distances of three measurements are used to calculate the following factors (dynamic and static):

$$\text{Tose spread factor (TSF)} = \text{(ETS -- NTS) } / \text{ NTS} \tag{2}$$

$$\text{Intermediate toe spread factor (ITSF)} \ = \begin{pmatrix} \text{EITS} \ - \text{NTS} \end{pmatrix} \ / \text{ NTS} \tag{3}$$

Mesenchymal Stem Cells from Extra-Embryonic Tissues for Tissue Engineering – Regeneration of the Peripheral Nerve http://dx.doi.org/10.5772/53336 483

$$\text{Pprint length factor (PLF)} \;= \; \text{(EPL} - \text{NPL)} \; / \; \text{NPL} \tag{4}$$

SFI is calculated as described by Bain et al. [108] according to the following equation:

$$\begin{aligned} \text{SFI} &= -38.3 \text{(EPL - NPL) } / \text{NPL + 109.5} \text{(ETS - NTS) } / \text{NTS + 13.3} \text{(EIT - NIT) } / \text{NTI - 8.8} \\ &= \left( -38.3 \times \text{PLF} \right) + \left( 109.5 \times \text{TSF} \right) + \left( 13.3 \times \text{ITSF} \right) - 8.8 \end{aligned} \tag{5}$$

For SFI and SSI, an index score of 0 is considered normal and an index of -100 indicates total impairment. When no footprints are measurable, the index score of -100 is given [109]. In each walking track 3 footprints are analyzed by a single observer, and the average of the measurements is used in SFI calculations.

Ankle kinematics analysis is carried out prior nerve injury, at week-2 and every 4 weeks during the 12 or the 20-week follow-up time, for axonotmesis and neurotmesis lesions, re‐ spectively. The motion capture is performed with 2 digital high speed cameras (Oqus, Qual‐ ysis®) at a rate of 200 images per second, and Qualisys Track Manager software (QTM, Qualysis®). The cameras operate on a infra-red light frequency ensuring a high level of ac‐ curacy on the determination of reflective marker position and a position residual of less than 2.7 mm was obtained. Cameras are usually positioned to not recorder significant signal de‐ flection during the test and four reflective markers were placed at the skin of the rat right hindlimb at the proximal edge of the tibia, the lateral malleolus and the fifth metatarsal head. Advanced analysis of the 2-D movement (sagittal plan) data is performed with Visu‐ al3D software (C-Motion®, Inc). The rats' ankle angle is determined using the scalar product between a vector representing the foot and a vector representing the lower leg. With this model, positive and negative values of position of the ankle joint (θ°) indicate dorsiflexion and plantarflexion, respectively. For each step cycle the following time points are identified: midswing, midstance, initial contact (IC) and toe-off (TO) [104, 109-113] and are time nor‐ malized for 100% of step cycle. The normalized temporal parameters are averaged over all recorded trials. Angular velocity of the ankle joint (Ω °/s) is also determined where negative values correspond to dorsiflexion. A total of 6 walking trials for each animal with stance phases lasting between 150 and 400 ms are considered for analysis, since this corresponds to the normal walking velocity of the rat (20–60 cm/s) [104]. Animals walk on a Perspex track with length, width and height of respectively 120, 12, and 15 cm. In order to ensure locomo‐ tion in a straight direction, the width of the apparatus is adjusted to the size of the rats dur‐ ing the experiments.

#### *3.2.2. Morphologic Assessment*

ress in documenting functional recovery [104]. Indeed, the use of biomechanical parameters has given valuable insight into the effects of the sciatic denervation/reinnervation, and thus represents an integration of the neural control acting on the ankle and foot muscles, which is

After injury and treatment of animals, follow-up results are very important for analysis of functional recovery. Animals are tested preoperatively (week 0), and every week during 12 and 20 weeks, for axonotmesis and neurotmesis of the rat sciatic nerve, respectively. Motor performance and nociceptive function are evaluated by measuring extensor postural thrust (EPT) and withdrawal reflex latency (WRL), respectively [55, 58, 94]. For EPT test, the affect‐ ed and normal limbs are tested 3 times, with an interval of 2 minutes between consecutive tests, and the 3 values are averaged to obtain a final result. The normal (unaffected limb) EPT (NEPT) and experimental EPT (EEPT) values are incorporated into an equation (Equa‐ tion (1)) to derive the percentage of functional deficit, as described in the literature [106]:

The nociceptive withdrawal reflex (WRL) was adapted from the hotplate test developed by Masters et al. [107]. Normal rats withdraw their paws from the hotplate within 4s or less. The cutoff time for heat stimulation is set at 12 seconds to avoid skin damage to the foot.

For Sciatic Functional Index (SFI), animals are tested in a confined walkway that they cross, measuring 42 cm long and 8.2 cm wide, with a dark shelter at the end. Several measure‐

**iii.** distance from the second to the fourth toe, the intermediary toe spread (ITS).

In the static evaluation (SSI) only the parameters TS and ITS, are measured. For SFI and SSI, all measurements are taken from the experimental (E) and normal (N) sides. Prints for meas‐ urements are chosen at the time of walking based on precise, clear and completeness of foot‐ prints. The mean distances of three measurements are used to calculate the following factors

Toe spread factor TSF ETS – NTS / NTS ( ) = ( ) (2)

Intermediate toe spread factor ITSF EITS – NITS / NITS ( ) = ( ) (3)

**i.** distance from the heel to the third toe, the print length (PL);

**ii.** distance from the first to the fifth toe, the toe spread (TS); and

% Motor deficit NEPT – EEPT / NEPT 100 = ´ é ù ( ) ë û (1)

very useful and accurate to evaluate different therapeutic approaches [103-105].

*3.2.1. Functional Assessment*

482 Advances in Biomaterials Science and Biomedical Applications

ments are taken from the footprints:

(dynamic and static):

Nerve samples are processed for quantitative morphometry of myelinated nerve fibers [114]. Fixation is usually carried out using 2.5% purified glutaraldehyde and 0.5% saccarose in 0.1M Sorensen phosphate buffer for 6-8 hours and resin embedding is obtained following Glauerts' procedure (Scipio et al., 2008). Series of 2-μm thick semi-thin transverse sections are cut using a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained by Toluidine blue. Stereology is carried out on a DM4000B microscope equip‐ ped with a DFC320 digital camera and an IM50 image manager system (Leica Microsystems, Wetzlar, Germany). Systematic random sampling and D-disector is always adopted using a protocol previously described [115, 116]. Fiber density and total number of myelinated fi‐ bers is estimated together with fiber and axon diameter and myelin thickness.

#### **3.3. Results**

#### *3.3.1. Differentiation and metabolism of MSCs from Wharton's jelly*

In our experimental studies we expanded undifferentiated MSCs from human umbilical cord Wharton's jelly that exhibited a normal star-like shape with a flat morphology in cul‐ ture (Figures 4 and 5). To prevent the possibility of eventual mutations due to expansion ar‐ tifacts, a total of 20 Giemsa-stained metaphases of these cells, were analyzed for numerical aberrations. Sporadic, non-clonal aneuploidy was found in 3 cells (41-45 chromosomes). The other 17 metaphases had 46 chromosomes (Figure 7). The karyotype was determined in a completely analyzed G-banding metaphase. No structural alterations were found. The kar‐ yotype analysis to the MSCs cell line derived from Human Wharton jelly demonstrated that this cell line has not neoplastic characteristics and is stable during the cell culture proce‐ dures in terms of number and structure of the somatic and sexual chromosomes [55].

**Figure 7.** Selected metaphases from undifferentiated MSC cells isolated from Wharton's jelly, showing the normal number of chromosomes (46, XY). Magnification: 1000X.

We differentiated MSC from Wharton's Jelly into neuroglial-like cells. After 96 hours of in‐ cubation in neurogenic medium, we observed a morphological change. The cells became ex‐ ceedingly long and there was a formation of typical neural-like cells with multi-branches and secondary branches (Figure 6). The differentiation was tested based on the expression of typical neuronal markers such as GFAP, GAP-43 and NeuN by neural-like cells attained from HwMSCs. Undifferentiated MSCs were negatively labeled to GFAP, GAP-43 and NeuN (Figure 8A,C,E). After 96 hours of differentiation the attained cells were positively stained for glial protein GFAP (Figure 8B) and for the growth-associated protein GAP-43 (Figure 8D). All nucleus of neural-like cells were also labeled with the neuron specific nucle‐ ar protein called NeuN (Figure 8F) showing that differentiation of MSCs in neural-like cells were successfully achieved [55].

and stained by Toluidine blue. Stereology is carried out on a DM4000B microscope equip‐ ped with a DFC320 digital camera and an IM50 image manager system (Leica Microsystems, Wetzlar, Germany). Systematic random sampling and D-disector is always adopted using a protocol previously described [115, 116]. Fiber density and total number of myelinated fi‐

In our experimental studies we expanded undifferentiated MSCs from human umbilical cord Wharton's jelly that exhibited a normal star-like shape with a flat morphology in cul‐ ture (Figures 4 and 5). To prevent the possibility of eventual mutations due to expansion ar‐ tifacts, a total of 20 Giemsa-stained metaphases of these cells, were analyzed for numerical aberrations. Sporadic, non-clonal aneuploidy was found in 3 cells (41-45 chromosomes). The other 17 metaphases had 46 chromosomes (Figure 7). The karyotype was determined in a completely analyzed G-banding metaphase. No structural alterations were found. The kar‐ yotype analysis to the MSCs cell line derived from Human Wharton jelly demonstrated that this cell line has not neoplastic characteristics and is stable during the cell culture proce‐

dures in terms of number and structure of the somatic and sexual chromosomes [55].

**Figure 7.** Selected metaphases from undifferentiated MSC cells isolated from Wharton's jelly, showing the normal

We differentiated MSC from Wharton's Jelly into neuroglial-like cells. After 96 hours of in‐ cubation in neurogenic medium, we observed a morphological change. The cells became ex‐ ceedingly long and there was a formation of typical neural-like cells with multi-branches and secondary branches (Figure 6). The differentiation was tested based on the expression of typical neuronal markers such as GFAP, GAP-43 and NeuN by neural-like cells attained

number of chromosomes (46, XY). Magnification: 1000X.

bers is estimated together with fiber and axon diameter and myelin thickness.

*3.3.1. Differentiation and metabolism of MSCs from Wharton's jelly*

484 Advances in Biomaterials Science and Biomedical Applications

**3.3. Results**

**Figure 8.** Undifferentiated MSC cells from the Wharton's jelly presenting a negative staining for: (A) GFAP which is a glial cell marker; (C) GAP-43 which is related with axonal outgrowth and (E) NeuN which is a marker for nucleus of neurons. Neuroglial-like cells obtained from HMSCs in vitro differentiated with neurogenic medium exhibiting a posi‐ tive staining for: (B) GFAP; (D) GAP-43 and (F) NeuN. Magnification: 200x [55].

The *in vitro* expansion and differentiation of MSCs for clinical cell-based therapy is a very expensive and long process that needs standardization. Although pre-clinical and clinical data demonstrated the safety and effectiveness of MSCs therapy in some pathologies such as neurological, there are still questions surrounding the mechanism of action. In our research work we aimed to disclose the possible role of metabolism not only in the MSCs mainte‐ nance and expansion but also during the differentiation in neural-like cells [55]. MSCs main‐ tenance and differentiation, to neural-like cells, depends on metabolic modulation. *In vitro*, glucose is the most widely used substrate for the generation ATP which is essential for cell growth and maintenance. It has been proposed that cells undergoing high proliferation rates depend on glycolysis to generate ATP, known as Warburg effect, although this pathway is less effective than the oxidative phosphorylation in terms of ATP production [117]. Our re‐ sults showed that during expansion, the undifferentiated MSCs consume glucose and pro‐ duce high concentration of lactate as a metabolic sub product which is consistent with the 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‐ tive metabolism [55].

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

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.
