Stem cells are the foundation cells for every organ, tissue, and cell in the body [6, 7]. They may be thought of as a blank microchip that can ultimately be programmed to perform any number of specialized tasks. This role is justified based on two key properties: (1) the ability to self-renew, dividing in a way to make copies of themselves and (2) the ability to differentiate,

The stem cells can be generally divided into three groups: totipotent, pluripotent, and multipotent stem cells. Totipotent stem cells originate from the fertilized egg and give rise to the whole organism. These cells, through the process of proliferation and differentiation, become *pluripotent embryonic stem cells* that form three germ layers: ectoderm, mesoderm, and endoderm [8]. These three germ layers are the embryonic source of all cells of the body (adult organism consists of 200 different cells types). During embryonic development, stem cells

giving rise to the mature types of the cells that make up our organs and tissues [6, 7].

The MSC cells are multipotent—self-renewing cells found in adult tissues, which can be *in vitro* differentiated and form adipocytes, fibroblast, osteocyte, and chondrocytes lineage [13, 14]. These cells had been primarily isolated in the early 1970s when Friedenstein et al. discovered that a specific number of fibroblastic cells isolated from bone marrow have the capacity to form colonies *in vitro* and under appropriate stimulating environmental conditions, small aggregates of bone, and cartilage [15, 16]. Over the years, it has become clear that MSC are not an exclusive feature of the bone marrow [17–19], but can also be isolated from other organs and tissues such as fat [20–22], skeletal muscles [23, 24], and synovium [25].

The isolation and characterization of MSC among the other cell types are based on their properties to adhere and grow on plastic, phenotype characteristics, and differentiation potential [26]. Over the last decades of research, significant effort has been made to establish phenotypic characterization of MSC. Despite all the effort, to date, there is no specific marker or combination of markers which will allow isolation of the homogeneous MSCs pool [27].

Nevertheless, it has been generally agreed that MSCs express specific surface antigens which involve: CD105 (endoglin—type I glycoprotein), CD73 (ecto-5′-nucleotidase), CD44 (HCAM—homing cell adhesion molecule), CD90 (cluster of differentiation 90 [Thy 1]), CD71 (cluster of differentiation 71) and Stro-1 as well as the adhesion molecules CD106 (vascular cell adhesion molecule [VCAM]-1), CD166 (activated leucocyte cell adhesion molecule [ALCAM]), intercellular adhesion molecule (ICAM)-1, neurogenic locus notch homolog protein 3 (NOTCH3), integrin alpha-11 (ITGA11), and CD29 [17, 26, 28–31]. However, they do not express the hematopoietic-specific markers CD79a, CD45, CD11, CD34, CD19, or CD14 and co-stimulatory molecules CD80, CD40, CD86, or the adhesion molecules CD31 (platelet/ endothelial cell adhesion molecule [PECAM]-1), CD18 (leucocyte function-associated antigen-1 [LFA-1]), or CD56 (neuronal cell adhesion molecule-1) [26].

Hence, to confirm the presence of MSC and extract them among the other cell types, researchers use the different combinations of these markers.

Another way to identify supposed MSC population is by their differentiation capacity to bone, cartilage, and adipocyte tissue. Herein, MSC has to be cultured in the specific medium composed of the substituent known to stimulate and control these differentiations *in vivo*. These are mostly specific growth factors such as BMPs for osteocytes [32–34] and TGFs, BMPs, and FGFs for chondrocytes [35–38]. To optimize MSC differentiation, cells need to be put under the *in vivo*-like environment. Then MSC aimed to become osteocytes or chondrocytes will be cultured in 3D pellets [32–38] while differentiation to adipocytes will be performed in monolayer.

The fact that MSCs can be differentiated into several different cells types *in vitro* clearly makes MSC and MSC-like cells (progenitors) a promising cell source for tissue repair and regeneration. Moreover, MSCs are known to secrete a large number of growth factors (GFs), cytokines, and chemokines for mediating various functions including anti-inflammatory, anti-apoptotic, anti-fibrotic, angiogenic, mitogenic, and wound-healing through paracrine activity [27, 39, 40]. All these features are highly desired and support their candidature for therapeutic purpose.

#### **2.3. MSC potential for cartilage repair**

Current research into cartilage tissue engineering focuses on the use of adult MSCs as an alternative to autologous chondrocytes [41]. The advantage of MSC over chondrocytes is their ability to self-renew without loss of differentiation capacity. Likewise, MSCs may retain immunomodulatory activity in recipient tissue due to lack of human leucocyte antigen (HLA) class II expression [42, 43]. These properties make MSC promising for a diversity of clinical applications including *in vitro* development of the cartilage tissue and its transplantation into the joint defect.

To date, research has demonstrated that bone marrow, adipose, and synovial-derived MSCs are mostly relevant as MSC sources for cartilage repair [8].

#### *2.3.1. Bone marrow-derived MSC in cartilage repair*

#### *<IP_ADDRESS>. In vitro studies*

Since the Friedenstein study in the early 1970s to date, numerous reports confirmed the multipotency of MSC isolated from bone marrow (BMSC) [16, 44–48]. Although, they represent a minor fraction of the total nucleated cell population (1 MSC/5 ×103 mononuclear cells), they could significantly increase their number through *in vitro* expansion [44, 49–51]. Sakaguchi et al. confirmed that BMSC potential to divide persists even after 10 *in vitro* passages [49]. This is a significant achievement as the high cells number is required to fill the cartilage defects. Note that, as opposed to chondrocytes, MSC retain chondrogenetic potential even after long monolayer expansion [46, 52]. When a sufficient cell number is reached, cells are placed in the differentiation-specific medium. The quality of BMSC-derived chondrocytes and the formed cartilage tissue is then estimated [46, 52].

The obtained tissue exhibited high positive staining for cartilage ECM components: glycosaminoglycans, collagen II, and lubricin [45–48]. Note that, however, positive staining was also obtained for the collagen X, which is well-known as a marker of hypertrophic chondrocytes and produces calcified cartilage [45].

In a comparative study of MSC isolated from versus tissues, BMSC showed greater chondrogenetic potential over the fetal lung MSC or placenta MSC [45, 46]. Nevertheless, BMSCderived cartilage pellets exhibited significantly higher expression of collagen X than those derived from the two other sources [46]. Moreover, the capacity of BMSC to differentiate into chondrocytes was reduced by passaging of the cells [46]. This has been recently confirmed on the animal model [53]. The results showed that proliferative, differentiation, and metabolism profile of BMSC significantly decreases by age increase [53]. In the other comparative study from 2016, authors did not observe any preference in *in vitro* chondrogenesis among MSC derived from bone marrow, adipose tissue, and umbilical cord [54].

#### *<IP_ADDRESS>. Pre-clinical studies in animal model*

Hence, to confirm the presence of MSC and extract them among the other cell types, research-

Another way to identify supposed MSC population is by their differentiation capacity to bone, cartilage, and adipocyte tissue. Herein, MSC has to be cultured in the specific medium composed of the substituent known to stimulate and control these differentiations *in vivo*. These are mostly specific growth factors such as BMPs for osteocytes [32–34] and TGFs, BMPs, and FGFs for chondrocytes [35–38]. To optimize MSC differentiation, cells need to be put under the *in vivo*-like environment. Then MSC aimed to become osteocytes or chondrocytes will be cultured in 3D pellets [32–38] while differentiation to adipocytes will be performed in

The fact that MSCs can be differentiated into several different cells types *in vitro* clearly makes MSC and MSC-like cells (progenitors) a promising cell source for tissue repair and regeneration. Moreover, MSCs are known to secrete a large number of growth factors (GFs), cytokines, and chemokines for mediating various functions including anti-inflammatory, anti-apoptotic, anti-fibrotic, angiogenic, mitogenic, and wound-healing through paracrine activity [27, 39, 40]. All these features are highly desired and support their candidature for therapeutic purpose.

Current research into cartilage tissue engineering focuses on the use of adult MSCs as an alternative to autologous chondrocytes [41]. The advantage of MSC over chondrocytes is their ability to self-renew without loss of differentiation capacity. Likewise, MSCs may retain immunomodulatory activity in recipient tissue due to lack of human leucocyte antigen (HLA) class II expression [42, 43]. These properties make MSC promising for a diversity of clinical applications including *in vitro* development of the cartilage tissue and its transplantation into

To date, research has demonstrated that bone marrow, adipose, and synovial-derived MSCs

Since the Friedenstein study in the early 1970s to date, numerous reports confirmed the multipotency of MSC isolated from bone marrow (BMSC) [16, 44–48]. Although, they represent a

could significantly increase their number through *in vitro* expansion [44, 49–51]. Sakaguchi et al. confirmed that BMSC potential to divide persists even after 10 *in vitro* passages [49]. This is a significant achievement as the high cells number is required to fill the cartilage defects. Note that, as opposed to chondrocytes, MSC retain chondrogenetic potential even after long monolayer expansion [46, 52]. When a sufficient cell number is reached, cells are placed in the differentiation-specific medium. The quality of BMSC-derived chondrocytes and the formed

mononuclear cells), they

ers use the different combinations of these markers.

208 Mesenchymal Stem Cells - Isolation, Characterization and Applications

**2.3. MSC potential for cartilage repair**

are mostly relevant as MSC sources for cartilage repair [8].

minor fraction of the total nucleated cell population (1 MSC/5 ×103

*2.3.1. Bone marrow-derived MSC in cartilage repair*

cartilage tissue is then estimated [46, 52].

monolayer.

the joint defect.

*<IP_ADDRESS>. In vitro studies*

To investigate cartilage repair by MSC *in vivo*, most of these pre-clinical studies have been performed in rabbit models treated with MSCs combined with appropriated scaffold materials and environmental factors [55–57]. The histological outcomes confirmed formation of the hyaline cartilage-like tissue expressing collagen type II [55, 56, 58, 59] as well as collagen type I [55, 56, 58]. Note that, the latter is a marker of fibrocartilaginous tissue. However, compared to the traditional ACI, the MSC therapy of cartilage defect resulted in regenerated hyaline cartilage-like tissue and restored a smooth cartilage surface, while the chondrocyte-seeded constructs produced mostly fibrocartilage-like tissue with a discontinuous superficial cartilage contour [60].

This finding has been further tested in large animal models. The study on swine model confirmed the beneficial effect of MSC over the ACI [61, 62]. Moreover, ovine MSCs have been isolated from bone marrow, expanded, characterized, and injected with transforming growth factor (TGF) b3 in a fibrin clot [63]. Two months after implantation, histological analysis revealed chondrocyte-like cells surrounded by a hyaline-like cartilaginous matrix that was integrated to host cartilage [63, 64]. Similar findings had been observed in the *Cynomolgus macaque* OA-model. The 2 months postoperative evaluation confirmed regular surface integration with neighboring native cartilage, and reconstruction of trabecular subchondral bone in the BMSC filled defects [65].

Taken together, animal studies indicated that MSC may be a promising approach for cartilage repair. However, animal models could not completely mimic OA pathogenesis in humans. In human primary OA, disease generally develops as a result of disturbed cell homeostasis, which leads to misbalance in synthesis and degradation of cartilage and subchondral bone matrix. These pathological changes are widely spread in OA cartilage at advanced stages when OA is generally diagnosed. Unfortunately, at this stage of the disease, there is only a slight amount of normal cartilage left. In contrast, experimental OA induced by mechanical trauma represents cartilage lesion surrounded by healthy tissue. The implanted cartilage construct may interact differently with healthy tissue than with a damaged surrounding tissue. Thus, repair techniques performed on the OA experimental model may not be sufficient to predict outcomes of this technique in humans.

#### *<IP_ADDRESS>. Clinical studies*

The clinical reports of cartilage defects treated by bone marrow MSC did show promising results. The symptoms improvement was mostly expressed through the pain relief and progress in physical mobility [66, 67]. However, quality of regenerated tissue evaluated by MRI and histology vary with respect to the time elapsed since surgery [68–72].

Autologous BMSC embedded in a collagen gel were transplanted into articular cartilage defects and covered with autologous periosteum [68–71]. Six weeks follow-up revealed better arthroscopic and histological scores in the cell-transplanted compared to the cell-free control group [68]. The repaired defects were filled with hyaline-like cartilage tissue confirmed by positive Safranin O staining [71]. Moreover, pain and walking abilities have been improved significantly [69]. Nevertheless, 1-year follow-up analysis detected formation of fibrocartilaginous tissue instead of hyaline cartilage tissue in the repaired lesions [57, 70]. This has been further confirmed by a 5-year follow-up study, where in the first 6 months after surgery pain, walking, stairs climbing, patella crepitus, and flection contractures were all improved. However, after the 6 months, they started gradually to deteriorate [73].

In the comparative study of autologous BMSC and autologous chondrocyte implantation (ACI), it has been shown that older patients showed significantly lower improvement compared to the younger in the ACI group. Nevertheless, age did not make any difference for the patients treated by autologous BMSC [74]. This finding may indicate that cellular senescence downgraded chondrocytes molecular pathways that are involved in regulation of cell activity, which affected their ability to form functional cartilage tissue [75].

Yet, these results did not confirm significant improvement between ACI and MSC therapies [74, 76]. Moreover, the same as for ACI, being overweight and large lesion size are significant predictors of poor clinical and arthroscopic outcomes after MSC therapy [77, 78].

#### *2.3.2. Adipose tissue-derived MSC in cartilage repair*

#### *<IP_ADDRESS>. In vitro studies*

Even the BMSC were commonly investigated and used in treatment of cartilage defects, the harvesting of bone marrow is painful and followed by risk of wound infection. Moreover, the BMSC number in bone marrow is very low which requires extended *in vitro* expansion and may cause loss of cells regenerative potential [8]. Given that, the adipose tissue became a novel source of adult stem cells due to easier harvesting procedure from the wasted tissue after the liposuction treatment.

Moreover, the proportion of the AMSCs in adipose tissue is several times higher than of MSCs in bone marrow. Results have confirmed their potential for chondrogenesis, osteogenesis, adipogenesis, myogenesis, and some aspects of neurogenesis [79, 80].

Chondrogenesis of human AMSCs has shown significantly higher expression of chondrogenic markers after 1 week under appropriate conditions [81]. However, a significantly elevated expression of collagen type X was observed after 3 weeks of chondrogenic induction [41, 81]. The tendency of the AMSCs to differentiate in hypertrophic chondrocytes had been further confirmed by the other studies. These studies showed positive staining of the collagen I and X in newly formed tissue even after the stimulation with chondrogenic growth factors [82–84]. This indicates that the regulation of cellular activity by growth factors, scaffolds, and even gene therapy merits further investigation.

Compared to the BMSC, cartilage obtained from the adipose-derived MSC did not express significantly higher levels of hypertrophic markers: collagen X and MMP-13 [41]. The recent study from 2016 has emphasized that MSCs from bone marrow, adipose tissue, and umbilical cord share similar biological properties and that their chondrogenic potential does not vary [54].

Based on the *in vitro* studies, it is not clear if the AMSCs are the best choice for the cartilage repair. Even though their chondrogenic potential had been clearly justified, their predisposition to form hypertrophic and fibrous tissue should not be neglected.

#### *<IP_ADDRESS>. Pre-clinical studies*

construct may interact differently with healthy tissue than with a damaged surrounding tissue. Thus, repair techniques performed on the OA experimental model may not be sufficient

The clinical reports of cartilage defects treated by bone marrow MSC did show promising results. The symptoms improvement was mostly expressed through the pain relief and progress in physical mobility [66, 67]. However, quality of regenerated tissue evaluated by MRI

Autologous BMSC embedded in a collagen gel were transplanted into articular cartilage defects and covered with autologous periosteum [68–71]. Six weeks follow-up revealed better arthroscopic and histological scores in the cell-transplanted compared to the cell-free control group [68]. The repaired defects were filled with hyaline-like cartilage tissue confirmed by positive Safranin O staining [71]. Moreover, pain and walking abilities have been improved significantly [69]. Nevertheless, 1-year follow-up analysis detected formation of fibrocartilaginous tissue instead of hyaline cartilage tissue in the repaired lesions [57, 70]. This has been further confirmed by a 5-year follow-up study, where in the first 6 months after surgery pain, walking, stairs climbing, patella crepitus, and flection contractures were all improved.

In the comparative study of autologous BMSC and autologous chondrocyte implantation (ACI), it has been shown that older patients showed significantly lower improvement compared to the younger in the ACI group. Nevertheless, age did not make any difference for the patients treated by autologous BMSC [74]. This finding may indicate that cellular senescence downgraded chondrocytes molecular pathways that are involved in regulation of cell activ-

Yet, these results did not confirm significant improvement between ACI and MSC therapies [74, 76]. Moreover, the same as for ACI, being overweight and large lesion size are significant

Even the BMSC were commonly investigated and used in treatment of cartilage defects, the harvesting of bone marrow is painful and followed by risk of wound infection. Moreover, the BMSC number in bone marrow is very low which requires extended *in vitro* expansion and may cause loss of cells regenerative potential [8]. Given that, the adipose tissue became a novel source of adult stem cells due to easier harvesting procedure from the wasted tissue

Moreover, the proportion of the AMSCs in adipose tissue is several times higher than of MSCs in bone marrow. Results have confirmed their potential for chondrogenesis, osteogenesis,

predictors of poor clinical and arthroscopic outcomes after MSC therapy [77, 78].

and histology vary with respect to the time elapsed since surgery [68–72].

However, after the 6 months, they started gradually to deteriorate [73].

ity, which affected their ability to form functional cartilage tissue [75].

adipogenesis, myogenesis, and some aspects of neurogenesis [79, 80].

*2.3.2. Adipose tissue-derived MSC in cartilage repair*

*<IP_ADDRESS>. In vitro studies*

after the liposuction treatment.

to predict outcomes of this technique in humans.

210 Mesenchymal Stem Cells - Isolation, Characterization and Applications

*<IP_ADDRESS>. Clinical studies*

*In vitro* studies on animal models demonstrated that adipocyte-derived MSCs were able to restore symptoms of OA-induced cartilage. The improvement had been observed macroscopically where cartilage lesion had been covered by repaired tissue and the surface was relatively smooth. The histological assessment revealed only a few fissures, few cracks, and an almost continuous superficial zone [85]. Another study showed that injected AMSC migrated to the synovial membrane and meniscus, however not in cartilage. Nevertheless, reduced OA progression had been observed [86]. The benefits obtained by AMSCs treatment could be due to a trophic mechanism of action by the release of growth factors and cytokines [86]. Taken together, these few pre-clinical studies are in favor of AMSCs-based cartilage repair.

#### *2.3.3. Synovium-derived MSC in cartilage repair*

#### *<IP_ADDRESS>. In vitro studies*

Another source of adult stem cells is synovium (synovium-derived stem cells (SDSC)). The comparative study of stem cells from five different sources (bone marrow, synovium, skeletal muscle, periosteum, and adipose tissue) confirmed that SDSC have proliferation and differentiation capacity similar to BMSC [49]. Moreover, the pellets derived from synovium were heavier than those from other tissues, because of their higher secretion of cartilage matrix [87–89]. This makes synovium-derived MSC potentially superior to bone marrow-derived MSC.

#### *<IP_ADDRESS>. In vivo studies*

The transplantation of the implant composed of MSC from different sources into the full-thickness cartilage defects of rabbits showed that synovium and bone marrow MSCs had greater *in vivo* chondrogenic potential than adipose and muscle MSCs [89]. Moreover, synovium MSCs had the advantage of the highest proliferation potential [90]. This study also noted that cartilage repair by synovium-derived MSC requires injection of a high number of these cells into the defect [90]. By contrast, another group reported that the aggregates with a high density of synovium-derived MSCs failed to regenerate cartilage due to cell death and nutrient deprivation into the core of the aggregates. Though, aggregates with relatively low-cartilage density successfully regenerated damaged tissue [91]. When compared to the healthy cartilage, tissue regenerated by constructs composed of the synovium-derived MSCs showed more fibrocartilage-like characteristics mostly in the superficial zone of the repair tissue [92].

This finding needs to be further confirmed by more *in vitro* and *in vivo* studies before introducing these cell types in clinical trials.

#### **2.4. Regulation of the MSC chondrogenesis**

It has been proposed that *in vitro* chondrogenic differentiation of MSCs mimics *in vivo* embryonic cartilage development. Hence, *in vitr*o MSC expansion phase may correspond to the initial proliferation of mesenchymal cells before condensation. Switching over to the highdensity MSC pellet cultures mimics the *in vivo* MSCs condensation steps and early stage chondrogenesis during embryonic development [93]. It has been shown that mechanical forces employed on the cell mass during chondrogenesis may promote the cells differentiation and secretion of the matrix-specific molecule. These biomechanical applications mimic the natural articular cartilage *in vivo* conditions [94, 95].

#### *2.4.1. MSC isolation and in vitro culturing conditions*

The MSC to be subjected to the cartilage formation first need to be isolated from their native tissue. To date, bone marrow, fat, and synovium tissue presents the most suitable sources of adult stem cells [8] with each tissue necessitating a specific isolation procedure [6]. BMSC are aspired by syringe from bone shafts, while ADMS are released and collected due to enzymatic digestion of the tissue [6]. Subsequently, these cells are *in vitro* expanded in order to obtain sufficient cell numbers for the following experimental procedures [6]. After the proliferation step, expanded cells need to be cultured under the 3D conditions in order to stimulate chondrogenesis. Thus, they are cultivated in micromass (pellets) or in scaffold materials, such as polymers, alginate beads, collagen sponges or hydrogels, and microspheres for 2–3 weeks in special chondrogenic medium enriched by growth factors [96]. Growth factors enhance expression of chondrocyte markers and support formation of cartilage tissue [35, 44, 97–99]. Moreover, hypoxic conditions seem to be the logical choice to stimulate chondrogenesis as it is present in *in vivo* articular tissue [100–104]. It has been shown that hypoxia induces expression of crucial genes for cartilage formation like SOX9, SOX6, and SOX5 as well as secretion of ECM molecules typical for hyaline cartilage [44, 100–104].

Reported *in vitro* conditions provide MSC differentiation to chondrocytes, nevertheless, do not stop chondrogenesis at the pre-hypertrophic stage, while cells undergo terminal differentiation to hypertrophic chondrocytes. These cells produce calcified instead of hyaline cartilage [105]. This remains crucial, a limitation in the formation of functional articular cartilage, as calcified cartilage has different biomechanical characteristics compared to hyaline cartilage [105, 106].

#### *2.4.2. Role of growth factors in cartilage repair*

had the advantage of the highest proliferation potential [90]. This study also noted that cartilage repair by synovium-derived MSC requires injection of a high number of these cells into the defect [90]. By contrast, another group reported that the aggregates with a high density of synovium-derived MSCs failed to regenerate cartilage due to cell death and nutrient deprivation into the core of the aggregates. Though, aggregates with relatively low-cartilage density successfully regenerated damaged tissue [91]. When compared to the healthy cartilage, tissue regenerated by constructs composed of the synovium-derived MSCs showed more fibrocarti-

This finding needs to be further confirmed by more *in vitro* and *in vivo* studies before intro-

It has been proposed that *in vitro* chondrogenic differentiation of MSCs mimics *in vivo* embryonic cartilage development. Hence, *in vitr*o MSC expansion phase may correspond to the initial proliferation of mesenchymal cells before condensation. Switching over to the highdensity MSC pellet cultures mimics the *in vivo* MSCs condensation steps and early stage chondrogenesis during embryonic development [93]. It has been shown that mechanical forces employed on the cell mass during chondrogenesis may promote the cells differentiation and secretion of the matrix-specific molecule. These biomechanical applications mimic the natural

The MSC to be subjected to the cartilage formation first need to be isolated from their native tissue. To date, bone marrow, fat, and synovium tissue presents the most suitable sources of adult stem cells [8] with each tissue necessitating a specific isolation procedure [6]. BMSC are aspired by syringe from bone shafts, while ADMS are released and collected due to enzymatic digestion of the tissue [6]. Subsequently, these cells are *in vitro* expanded in order to obtain sufficient cell numbers for the following experimental procedures [6]. After the proliferation step, expanded cells need to be cultured under the 3D conditions in order to stimulate chondrogenesis. Thus, they are cultivated in micromass (pellets) or in scaffold materials, such as polymers, alginate beads, collagen sponges or hydrogels, and microspheres for 2–3 weeks in special chondrogenic medium enriched by growth factors [96]. Growth factors enhance expression of chondrocyte markers and support formation of cartilage tissue [35, 44, 97–99]. Moreover, hypoxic conditions seem to be the logical choice to stimulate chondrogenesis as it is present in *in vivo* articular tissue [100–104]. It has been shown that hypoxia induces expression of crucial genes for cartilage formation like SOX9, SOX6, and SOX5 as well as secretion of

Reported *in vitro* conditions provide MSC differentiation to chondrocytes, nevertheless, do not stop chondrogenesis at the pre-hypertrophic stage, while cells undergo terminal differentiation to hypertrophic chondrocytes. These cells produce calcified instead of hyaline cartilage [105]. This remains crucial, a limitation in the formation of functional articular cartilage, as calcified cartilage has different biomechanical characteristics compared to hyaline cartilage [105, 106].

lage-like characteristics mostly in the superficial zone of the repair tissue [92].

ducing these cell types in clinical trials.

**2.4. Regulation of the MSC chondrogenesis**

212 Mesenchymal Stem Cells - Isolation, Characterization and Applications

articular cartilage *in vivo* conditions [94, 95].

*2.4.1. MSC isolation and in vitro culturing conditions*

ECM molecules typical for hyaline cartilage [44, 100–104].

Chondrogenic differentiation of MSCs is induced by various intrinsic and extrinsic factors [107]. Growth factors play the most important role in this process [107]. The importance of growth factors in the maintenance and production of cartilage *in vivo* had been explained previously. Hence, introduction of these factors in *in vitro* controlled chondrogenesis was the logical choice. Below are listed studies that clarified the importance of growth factors in treatment of cartilage defects with MSC. Keep in mind that TGF-β superfamily (TGF-β 1 & 2 and bone morphogenic proteins—BMPs), insulin-like growth factors (IGFs), and fibroblast growth factors (FGFs) are the major factors regulating chondrogenesis and synthesis of cartilage matrix.

Porcine MSCs encapsulated in agarose hydrogels after treatment with TGF-b3 increase the sulfated glycosaminoglycans in surrounding culture media, highlighting their role in cartilage ECM anabolism [35]. Moreover, the expression of BMP4 in transgenic MSC enhances their chondrogenesis in rat model through the positive regulation of main cartilage component, collagen type II [108]. Moreover, after 24 weeks, animals treated with BMP-4 showed significantly better cartilage repair than untreated animals [108]. Nevertheless, better results were obtained in chondrogenesis of MSC when TGF-b1, IGF-1, BMP-2, and BMP-7 were combined [36]. Also, intra-articular application of another growth factor, FGF-18-induced dosedependent, increases the cartilage thickness of tibial plateau in rat OA model [37]. Similar effect to FGF-18 has FGF-2 which stimulates [38, 109] increase in glycosaminoglycan and collagen type II after its application on MSC culture in chondrogenic medium [38]. Overall, growth factors appear to be one of the main components in improving clinical cartilage regeneration, but they must be precisely combined and loaded on appropriate scaffold materials to simulate the conditions and three-dimensional (3D) structure most similar to the *in vivo* condition.

#### **3. Chondroprogenitors in cartilage**

#### **3.1. Chondrogenesis**

Chondrogenesis is a complex process that is initiated by MSC crowding and condensing on the bone-forming site, followed by maturation into terminally differentiated chondrocytes [110, 111]. This pathway is accompanied by stage-specific ECM production, synchronized by cellular interactions with the matrix, growth, and differentiation factors [110]. The latter initiate or suppress cellular signaling pathways and transcription of specific genes in a spatial-temporal manner [110, 111]. The anti-inflammatory and immunosuppressive properties of BMSCs suggest that these cells reduce inflammation in the joint. Moreover, BMSCs may initiate the repair process by differentiating into chondrocytes or by inducing proliferation and differentiation of the remaining healthy chondroprogenitor into mature chondrocytes or both. In addition, other features such as transcription factors, biological modulators, and extracellular matrix proteins expressed or produced by BMSCs may play an important role in enhancing cartilage formation.

Initially, MSCs express adhesion molecules including N-cadherin, N-CAM (Ncam1), tenascin-C (Tnc), and versican, which are involved in the compaction and condensation of MSCs regulated by different BMP factors [112]. Through progression of the condensation process, MSCs begin to express early cartilage markers collagen type II, aggrecan, and FGF receptor leading to chondrocytes progenitors stage of chondrogenesis [113]. Process of MSC condensation and chondrogenesis is triggered and positively regulated by major transcriptional factor, Sox 9. It is highly expressed in MSC before condensation and remains highly expressed in all stages of chondrogenesis through prechondrocytes to mature chondrocytes, while it is switched off when cells undergo hypertrophy [113, 114]. The formation of chondrocytes over osteocytes is regulated by combined action of Sox 9 and other transcriptional factors Pax/Nkx/ Barx2, Sox 9 through inhibition of Runx2 (Cbfa1) as a domain transcriptional factor required for osteoblast differentiation [113, 115]. Moreover, Sox 9 positively regulates two other Sox family members Sox 5 and Sox 6, which play a significant role in activation of cartilage-specific genes: type II, IX, and XI collagen, aggrecan, and cartilage oligomeric matrix protein [114, 116, 117]. The role and spatio-temporal expression of Sox 5 and Sox 6 in chondrogenesis has been studied through single and double null mutations in mice model. Single gene deletion resulted in moderate skeletal abnormalities; however, double mutation induced animal death caused by systemic chondrodysplasia and skeletal deformity. These results indicate simultaneous action of these two transcription factors in formation of functional skeletal system. Nevertheless, in the double mutant low level of cartilage, specific extracellular matrix component was sustained by normal Sox 9 expression, but it was insufficient to support proper MSC differentiation and formation of cartilage [116]. This implies that synchronized action of Sox 5, 6, and 9 trios is required to maintain sufficient ECM component expression and normal matrix composition. Furthermore, these three genes promote the chondrogenesis by inhibition of hypertrophic and osteogenic differentiation [113]. Chondrocytes maturation to hypertrophic chondrocytes is repressed by Sox 9 modulation of the Wnt/beta-catenin signaling pathway with beta-catenin degradation or inhibition of beta-catenin transcriptional activity without affecting its stability [118]. In addition, Sox 5 and Sox 6 delay chondrocyte hypertrophy by down-regulating Ihh signaling, FGFR3, and Runx2 and up-regulating BMP6 [115].

Further maturation of chondrocytes is essential for the final remodeling of the cartilage into bone. Terminal chondrocytes differentiation into the hypertrophic chondrocytes is promoted by upregulation of Runx 2 and calcified cartilage markers collagen X and MMP13 [113, 117]. Later, hypertrophic and terminal chondrocytes express angiogenic factors, including VEGF, which provide the genesis for vascularization and formation of primary ossification centers within osteoblasts, osteocytes, and hematopoietic cells [119]. Equally, terminal chondrocytes undergo apoptosis by release of collagen types X and I and mineralization of the ECM [117]. Contrary to growth plate chondrogenesis, normal articular chondrocytes never undergo hypertrophic differentiation, except at the tidemark [113].

#### **3.2. Chondroprogenitors potential in cartilage repair**

Recent research reported the presence of MSC and their progenitors in cartilage itself [104]. These cells possess characteristics similar to stem cells isolated from other adult tissues involving proliferation and differentiation potential under appropriate *in vitro* conditions [120–123]. They were subjected to the process of isolation, expansion, and identification in order to confirm their stem cells phenotype previously established on MSC from other adult tissues [121–124]. To date, studies investigated the presence of these cells in normal and OA cartilage. Interestingly, several authors observed that OA cartilage contains higher number of mesenchymal progenitors compared to normal [122, 125–129].

Subpopulation of cells determined as cartilage progenitor cells (ACPCs) possess high-colony forming efficiency and express surface antigens specific to MSC (Notch 1, CD 105 & CD 166) [121–123]. Moreover, after the cultivation in specific chondrogenic medium, they showed capacity to differentiate into cartilage in 3D pellet cultures [130]. The expression of MSC markers and differentiation potential confirmed presence of multipotential mesenchymal progenitor cells in articular cartilage [122]. Comparative study of ACPCs and BMSCs revealed positive expression of adult stem cells markers (Notch 1, Stro 1, CD105, and CD 166) on both cell types. Nevertheless, chondrogenesis of BMSCs resulted in hypertrophic cartilage tissue confirmed by positive staining of collagen X, while this marker was not detected in tissue obtained from ACPCs [124]. Similar was reported by Alsalameh et al. where CD105+ and CD166+ cells showed no signs of hypertrophic chondrocytes and osteogenesis in chondrogenic micromass cultures after 3 weeks [128].

Likewise, cells positive for other markers that have been identified in MSC CD9+ /CD90+ /CD166+ [131], CD105+ /CD166+ [128], and Notch-1+ /Stro-1+ [125] were capable of differentiating in chondrocytes and formed cartilage tissue *in vitro*. MCS differentiation into hypertrophic cartilage is the major limitation in hyaline functional cartilage production [105]. ACPCs may therefore be considered superior to MSCs from other tissues in cartilage repair [124, 125, 128, 129].

These results indicate the opportunity for using OA cartilage as a potential source of cells with cartilage-forming potential. Yet, further investigations are required to explore chondrogenesis regulation *in vitro*.

#### **4. Conclusion**

Initially, MSCs express adhesion molecules including N-cadherin, N-CAM (Ncam1), tenascin-C (Tnc), and versican, which are involved in the compaction and condensation of MSCs regulated by different BMP factors [112]. Through progression of the condensation process, MSCs begin to express early cartilage markers collagen type II, aggrecan, and FGF receptor leading to chondrocytes progenitors stage of chondrogenesis [113]. Process of MSC condensation and chondrogenesis is triggered and positively regulated by major transcriptional factor, Sox 9. It is highly expressed in MSC before condensation and remains highly expressed in all stages of chondrogenesis through prechondrocytes to mature chondrocytes, while it is switched off when cells undergo hypertrophy [113, 114]. The formation of chondrocytes over osteocytes is regulated by combined action of Sox 9 and other transcriptional factors Pax/Nkx/ Barx2, Sox 9 through inhibition of Runx2 (Cbfa1) as a domain transcriptional factor required for osteoblast differentiation [113, 115]. Moreover, Sox 9 positively regulates two other Sox family members Sox 5 and Sox 6, which play a significant role in activation of cartilage-specific genes: type II, IX, and XI collagen, aggrecan, and cartilage oligomeric matrix protein [114, 116, 117]. The role and spatio-temporal expression of Sox 5 and Sox 6 in chondrogenesis has been studied through single and double null mutations in mice model. Single gene deletion resulted in moderate skeletal abnormalities; however, double mutation induced animal death caused by systemic chondrodysplasia and skeletal deformity. These results indicate simultaneous action of these two transcription factors in formation of functional skeletal system. Nevertheless, in the double mutant low level of cartilage, specific extracellular matrix component was sustained by normal Sox 9 expression, but it was insufficient to support proper MSC differentiation and formation of cartilage [116]. This implies that synchronized action of Sox 5, 6, and 9 trios is required to maintain sufficient ECM component expression and normal matrix composition. Furthermore, these three genes promote the chondrogenesis by inhibition of hypertrophic and osteogenic differentiation [113]. Chondrocytes maturation to hypertrophic chondrocytes is repressed by Sox 9 modulation of the Wnt/beta-catenin signaling pathway with beta-catenin degradation or inhibition of beta-catenin transcriptional activity without affecting its stability [118]. In addition, Sox 5 and Sox 6 delay chondrocyte hypertrophy by

214 Mesenchymal Stem Cells - Isolation, Characterization and Applications

down-regulating Ihh signaling, FGFR3, and Runx2 and up-regulating BMP6 [115].

hypertrophic differentiation, except at the tidemark [113].

**3.2. Chondroprogenitors potential in cartilage repair**

Further maturation of chondrocytes is essential for the final remodeling of the cartilage into bone. Terminal chondrocytes differentiation into the hypertrophic chondrocytes is promoted by upregulation of Runx 2 and calcified cartilage markers collagen X and MMP13 [113, 117]. Later, hypertrophic and terminal chondrocytes express angiogenic factors, including VEGF, which provide the genesis for vascularization and formation of primary ossification centers within osteoblasts, osteocytes, and hematopoietic cells [119]. Equally, terminal chondrocytes undergo apoptosis by release of collagen types X and I and mineralization of the ECM [117]. Contrary to growth plate chondrogenesis, normal articular chondrocytes never undergo

Recent research reported the presence of MSC and their progenitors in cartilage itself [104]. These cells possess characteristics similar to stem cells isolated from other adult tissues Based on self-repair and multilineage potentials, MCS provide hyaline cartilage regeneration opportunities. Studies on cartilage regeneration with adult mesenchymal stem cells have shown that BMSC are the most commonly used cell types to address cartilage regeneration. However, although short-term results appear satisfactory, hypertrophic chondrocyte and fibrocartilage formation emerge thereafter with hypertrophically differentiated MSC. Note that fibrocartilage provides a molecular pattern secreted by hypertrophic chondrocytes, leading to different biomechanical characteristics compared with hyaline cartilage.

Furthermore, harvesting bone marrow is a painful procedure with donor-site morbidity and risk of wound infection and sepsis. Hence, both AMSCs and synovium-derived stem cells have been considered as alternatives. However, results using these two cell lines have been similar to those obtained employing the bone marrow approach. In fact, although a high expression of chondrogenic markers was initially obtained, they appear to be expressed as collagen type X confirming the presence of hypertrophy.

Therefore, further investigations regarding the regulation of cellular activity by growth factors, scaffolds and even gene therapy remain viable options. Recently, one more potential source of MSC and progenitors for cartilage repair engineering from the cartilage itself has been tested. Cells isolated from the surface zone of articular cartilage have the capacity to differentiate into cartilage in 3D pellet culture. Moreover, no signs of hypertrophic chondrocytes and osteogenesis were observed. Thus, ACPCs could be considered more adequate than MSC in cartilage repair.

#### **Abbreviations**

#### **Author details**

expression of chondrogenic markers was initially obtained, they appear to be expressed as

Therefore, further investigations regarding the regulation of cellular activity by growth factors, scaffolds and even gene therapy remain viable options. Recently, one more potential source of MSC and progenitors for cartilage repair engineering from the cartilage itself has been tested. Cells isolated from the surface zone of articular cartilage have the capacity to differentiate into cartilage in 3D pellet culture. Moreover, no signs of hypertrophic chondrocytes and osteogenesis were observed. Thus, ACPCs could be considered more adequate than MSC

collagen type X confirming the presence of hypertrophy.

216 Mesenchymal Stem Cells - Isolation, Characterization and Applications

OA Osteoarthritis AC Articular cartilage PRP Platelet-rich plasma ECM Extra-cellular matrix MSC Mesenchymal stem cells BMSCs Bone marrow stromal cells

ACI Autologous chondrocytes implantation COMP Cartilage oligometric matrix protein

IGFs Insulin-like growth factors FGFs Fibroblast growth factors BMPs Bone morphogenetic proteins ALK Activin receptor like-kinase IHH Indian hedgehog protein

IRS Insulin receptor-substrate family

FGFR Fibroblast growth factor receptor CD105 Endoglin-type I glycoprotein

FGF Fibroblast growth factors

CD73 Ecto-5′-nucleotidase

ITGA11 Integrin alpha-11

CD90 (Thy) Cluster of differentiation 90 CD106 (VCAM-1) Vascular cell adhesion molecule-1

CD166 (ALCAM) Activated leucocyte cell adhesion molecule

NOTCH Neurogenic locus notch homolog protein

CD106 (ICAM-1) Intercellular adhesion molecule-1

TGF-β Transforming growth factors-beta superfamily

in cartilage repair.

**Abbreviations**

Hechmi Toumi\*, Eric Lespessailles and Marija Mazor

\*Address all correspondence to<EMAIL_ADDRESS>
Service de Rhumatologie, Centre Hospitalier Régional d'Orléans, Collegium ST, University of Orleans, Orleans, France

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224 Mesenchymal Stem Cells - Isolation, Characterization and Applications

#### **Chapter 11**

**Provisional chapter**

#### **Prerequisites for Mesenchymal Stem Cell Transplantation in Spinal Cord Injury Transplantation in Spinal Cord Injury**

**Prerequisites for Mesenchymal Stem Cell**

DOI: 10.5772/intechopen.69554

Sherif M. Amr Sherif M. Amr Additional information is available at the end of the chapter

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69554

#### **Abstract**

We have aimed at distinguishing obligatory prerequisites for mesenchymal stem cell transplantation in spinal cord injury from those prerequisites which are unnecessary or are prerequisites that have to be further investigated. Obligatory prerequisites include the following. First, the site of injury is extensively gliotic, constituting an unsuitable medium for stem cell transplantation. It has to be dissolved by neurolyzing agents, chondroitinase ABC as an example. Second, stem cells need a suitable biomaterial scaffold for their proper integration. Third, the biomaterial scaffold necessitates a tissue filler harboring stem cells, other cells and neurotrophic factors in a combinatorial approach. Fourth, the efficiency of mesenchymal stem cells themselves has to be increased (by reducing oxidative stress-induced apoptosis, by hypoxic preconditioning, by modulating the extracellular matrix and by other measures). Prerequisites that have to be further investigated include the ideal source, mode, quantity, time point and number of injections of mesenchymal stem cells; which growth factors and cells to be used in the combinatorial approach; transforming mesenchymal stem cells into motor neuron-like cells or Schwann cells; increasing the homing effect of stem cells and how to establish a continuous drug and cell delivery system.

**Keywords:** spinal cord injury, mesenchymal stem cells, scaffolds, nerve grafting, neurotrophic factors, chondroitinase ABC, continuous drug delivery systems

#### **1. Introduction**

Traumatic spinal cord injury results usually from cervical and lumbar fractures; it may be associated with complete paraplegia. Regeneration after such an injury is fairly limited mainly due to the inhibitory milieu (the gliosis) within the spinal cord. Cellular therapeutic strategies may overcome this milieu by neuroprotection, immunomodulation, axon regeneration,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

neuronal relay formation and myelin regeneration [1]. Clinically, in a meta-analysis on cellular therapy in traumatic spinal cord injury in humans published in 2012 [2], the authors reviewed eight bone marrow mesenchymal and hematopoietic stem cell studies, two olfactory ensheathing cell studies, one Schwann cell study and one fetal neurogenic tissue study. Three of these were Grade III and nine Grade IV level of evidence. It was concluded that improved preclinical studies and prospective, controlled clinical trials were needed. Nevertheless, ever since, the number of clinical trials have been increased. Mesenchymal stem cells, in particular, are easy to isolate, can be rapidly expanded in culture and can be cryopreserved without loss of potency [3, 4]. Clinical reports on their use have varied, starting from documenting their safety [5, 6] up to limited clinical efficacy [7], even partial or complete efficacy [8–11].

The aim of this review is to distinguish necessary prerequisites for effective mesenchymal stem cell transplantation in spinal cord injuries from those prerequisites which are unnecessary or are prerequisites that have to be further investigated.

#### **2. Establishing a suitable niche**

#### **2.1. Dissolving the gliosis**

Axonal regeneration following spinal cord injury is limited not only because central nervous system neurons have a poor intrinsic capacity for growth but also because injured axons encounter a series of inhibitory factors that are non-permissive for growth. These include myelin inhibitors [Nogo-A, MAG108 (myelin-associated glycoprotein) and OMgp109 (oligodendrocyte myelin glycoprotein)]; chondroitin sulfate proteoglycans (neurocan, versican, aggrecan, brevican, phosphacan and NG2); semaphorins and ephrins. In the central nervous system, laminin is replaced by netrins [12–15].

#### *2.1.1. Chondroitinase ABC*

Chondroitinase ABC [16–18] has improved recovery of function in synergy with mesenchymal stromal cells without [19] or with the addition of an acellular nerve allograft [20] or in synergy with brain-derived neurotrophic factor (BDNF) secreting mesenchymal stem cells [21]. Chondroitinase ABC should be thermostabilized with the sugar trehalose to reduce its temperature-dependent loss of activity [22]; it should be injected in high doses (50 or 100 IUs) [23–25], at multiple times [26–29] and be combined with cell transplantation and growth factor infusion [30, 31].

#### *2.1.2. Other measures to overcome the gliosis*

In a rat model of spinal cord contusion injury [32], infused *sialidase* has acted robustly throughout the spinal cord gray and white matter, whereas chondroitinase ABC activity has been more intense superficially, thus raising the possible consideration that it might be superior to chondroitinase ABC. Blocking myelin-associated inhibitors with *Nogo-A monoclonal antibodies or with Nogoreceptor competitive agonist peptide (NEP1-40)* has been shown to increase axonal regeneration [33]. Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing have been used for repair of spinal cord injury [34]. *Blocking Rho-A with Rho inhibitor 'cethrin'* might overcome its effect; a synthetic membrane-permeable peptide mimetic of the protein tyrosine phosphatase σ, wedge domain can bind to tyrosine phosphatase σ and relieve chondroitin sulfate proteoglycan-mediated inhibition [35]. Chondroitin sulfate proteoglycans inhibition of phosphoinositide 3-kinase (PI3K) signaling is reversed by *cell permeable phosphopeptide (PI3Kpep)* [36]; *rolipram,* a phosphodiesterase4 inhibitor, can increase intracellular cAMP levels [33]; *taxol*, a microtubule-stabilizing agent, increases neurite outgrowth [37, 38].

#### *2.1.3. Emerging role of heparin in lysing the gliosis*

neuronal relay formation and myelin regeneration [1]. Clinically, in a meta-analysis on cellular therapy in traumatic spinal cord injury in humans published in 2012 [2], the authors reviewed eight bone marrow mesenchymal and hematopoietic stem cell studies, two olfactory ensheathing cell studies, one Schwann cell study and one fetal neurogenic tissue study. Three of these were Grade III and nine Grade IV level of evidence. It was concluded that improved preclinical studies and prospective, controlled clinical trials were needed. Nevertheless, ever since, the number of clinical trials have been increased. Mesenchymal stem cells, in particular, are easy to isolate, can be rapidly expanded in culture and can be cryopreserved without loss of potency [3, 4]. Clinical reports on their use have varied, starting from documenting their

safety [5, 6] up to limited clinical efficacy [7], even partial or complete efficacy [8–11].

are prerequisites that have to be further investigated.

228 Mesenchymal Stem Cells - Isolation, Characterization and Applications

**2. Establishing a suitable niche**

**2.1. Dissolving the gliosis**

*2.1.1. Chondroitinase ABC*

factor infusion [30, 31].

*2.1.2. Other measures to overcome the gliosis*

The aim of this review is to distinguish necessary prerequisites for effective mesenchymal stem cell transplantation in spinal cord injuries from those prerequisites which are unnecessary or

Axonal regeneration following spinal cord injury is limited not only because central nervous system neurons have a poor intrinsic capacity for growth but also because injured axons encounter a series of inhibitory factors that are non-permissive for growth. These include myelin inhibitors [Nogo-A, MAG108 (myelin-associated glycoprotein) and OMgp109 (oligodendrocyte myelin glycoprotein)]; chondroitin sulfate proteoglycans (neurocan, versican, aggrecan, brevican, phosphacan and NG2); semaphorins and ephrins.

Chondroitinase ABC [16–18] has improved recovery of function in synergy with mesenchymal stromal cells without [19] or with the addition of an acellular nerve allograft [20] or in synergy with brain-derived neurotrophic factor (BDNF) secreting mesenchymal stem cells [21]. Chondroitinase ABC should be thermostabilized with the sugar trehalose to reduce its temperature-dependent loss of activity [22]; it should be injected in high doses (50 or 100 IUs) [23–25], at multiple times [26–29] and be combined with cell transplantation and growth

In a rat model of spinal cord contusion injury [32], infused *sialidase* has acted robustly throughout the spinal cord gray and white matter, whereas chondroitinase ABC activity has been more intense superficially, thus raising the possible consideration that it might be superior to chondroitinase ABC. Blocking myelin-associated inhibitors with *Nogo-A monoclonal antibodies or with Nogoreceptor competitive agonist peptide (NEP1-40)* has been shown to increase axonal regeneration [33]. Bone marrow mesenchymal stem cells with Nogo-66

In the central nervous system, laminin is replaced by netrins [12–15].

There is an emerging role of heparin in lysing of the gliosis, as reviewed elsewhere [39]. Both unfractionated and low molecular weight heparins have a fibrolytic (gliolytic) effect, can modulate astrocyte function and are used as lumen fillers. Astrocytes release a variety of trophic factors. These trophic factors include nerve growth factor, basic fibroblast growth factor, transforming growth factor-β, platelet-derived growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor and others. Astrocyte stress response and trophic effects are mediated by the fibroblastic growth factor family member, on which heparin exerts a profound influence [40–42].

#### **2.2. Providing a suitable scaffold, both to bridge the gap and to harbor the cells**

#### *2.2.1. Biomaterial scaffolds in spinal cord injury*

Biomaterial scaffolds in spinal cord injury have been reviewed elsewhere [43, 44]. Mesenchymal stromal cells have been grown onto *fibrin* scaffolds [45, 46]. The survival and neural differentiation of human bone marrow stromal cells have been tested on fibrin versus fibrin platelet-rich plasma scaffolds. The results have shown a clear superiority of platelet-rich plasma scaffolds, mainly after BDNF administration [47]. Mesenchymal stem cells have also been grown onto *collagen* scaffolds [48]. Rat adipose-derived stem cells have differentiated into olfactory ensheathing cell-like cells on collagen scaffolds by co-culturing with olfactory ensheathing cells [49]. *Acellular* spinal cord scaffolds [50, 51] and acellular muscle bioscaffolds [52] seeded with bone marrow stromal cells have promoted functional recovery in spinal cord-injured rats. Electroacupuncture has been found to promote the survival and differentiation of transplanted bone marrow mesenchymal stem cells pre-induced with neurotrophin-3 and retinoic acid in *gelatin* sponge scaffold after rat spinal cord transaction [53]. Human bone marrow mesenchymal stem cells and endometrial stem cells have been found to differentiate better into motor neurons on electrospun *poly(ε-caprolactone)* scaffolds [54]. Nogo-66 receptor gene-silenced cells have been transplanted in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury [55]. Bone marrow mesenchymal stem cells seeded in *chitosan*-alginate scaffolds [56] and biodegradable chitin conduit tubulation combined with bone marrow mesenchymal stem cell transplantation have reduced glial scar and cavity formation in spinal cord injury [57]. In a comparative study investigating the efficacy of allogeneic mesenchymal stem cell transplantation via simple intralesional injection versus the use of a poly (lactic-co-glycolic acid) scaffold or a chitosan scaffold, higher mesenchymal stem cell engraftment rates have been reported in the scaffold groups, particularly, in the chitosan scaffold group [58].

Injectable extracellular matrix *hydrogels* have been used as scaffolds for spinal cord injury repair [59]. Matrix metalloproteinase-sensitive, hyaluronic acid-based biomimetic hydrogel scaffolds containing brain-derived neurotrophic factor have been implanted [60]. Cell-seeded alginate hydrogel scaffolds have promoted directed linear axonal regeneration in the injured rat spinal cord [61]. Multichannel polymer scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] hydrogel and loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats have been successfully implanted into rat spinal cords following T9 complete transection [62]. Highly superporous poly(2-hydroxyethyl methacrylate) scaffolds with oriented pores [63] and highly superporous cholesterol-modified poly(2-hydroxyethyl methacrylate) scaffolds have been developed for spinal cord injury repair [64].

*Three-dimensional* culture can mimic the stem cell niche compared to conventional twodimensional culture. Bone marrow-derived mesenchymal stem cells cultured in threedimensional collagen scaffold have exhibited distinctive features including significantly enhancing neurotrophic factor secretions and reducing macrophage activations challenged by lipopolysaccharide [65]. A polyhydroxybutaryl-hydroxyvinyl-based three-dimensional scaffold for a tissue engineering and cell-therapy combinatorial approach for spinal cord injury regeneration has been developed [66]. A three-dimensional biomimetic hydrogel has been implemented to deliver factors secreted by human mesenchymal stem cells in spinal cord injury [67]. Bone marrow mesenchymal stem cells in a three-dimensional gelatin sponge scaffold have attenuated inflammation, have promoted angiogenesis and have reduced cavity formation in experimental spinal cord injury [68].

#### *2.2.2. Prerequisites for the use of biomaterial scaffolds in spinal cord injury*

*Biomaterial scaffolds* should be biocompatible, non-toxic, chemically stable, of known absorption and degradation kinetics matching the degree of in vivo cell/tissue growth and should have adequate surface for cell access, proliferation and cell differentiation [69, 70]. They *should meet macroengineering requirements* being of proper form [71, 72], design (shape) [73] and size (diameter) [74]. They should be supplied with macrogrooves [43, 75, 76] and have a wall thickness of 0.6 mm, a porosity of 80% and a pore size range of 10–40 μm [77–79]. They *should meet microengineering requirements*, microgrooves directing axonal growth [80–87]. Prestretchinduced surface anisotropy has been beneficial in enhancing axon alignment, growth and myelination [88]. Also, filament inclusion has been more effective for bridging long nerve defect gaps [43, 89, 90]; Schwann cell migration over gaps exceeding 18 mm is superior in the presence of filaments. Yoshii et al. [91, 92] have tested collagen microfilaments with diameters of 20 μm to repair long gaps (20 or 30 mm) in the rat sciatic nerve. Increasing fiber number (4000 versus 2000 filaments) has enhanced nerve regeneration. Thus, increasing the whole filament surface area by increasing their number and reducing their diameter (increased surface area-to-volume ratio) is also critical [89, 93, 94].

*Scaffolds should fulfill nearly the same mechanical conditions of the recipient spinal cord, exerting incremental tensile forces on intact cord segments to promote axonal regeneration while unloading gliotic segments to reduce gliosis and harbor cellular transplants* (**Figure 1a and b**)*.* A scaffold should Prerequisites for Mesenchymal Stem Cell Transplantation in Spinal Cord Injury http://dx.doi.org/10.5772/intechopen.69554 231

Injectable extracellular matrix *hydrogels* have been used as scaffolds for spinal cord injury repair [59]. Matrix metalloproteinase-sensitive, hyaluronic acid-based biomimetic hydrogel scaffolds containing brain-derived neurotrophic factor have been implanted [60]. Cell-seeded alginate hydrogel scaffolds have promoted directed linear axonal regeneration in the injured rat spinal cord [61]. Multichannel polymer scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] hydrogel and loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats have been successfully implanted into rat spinal cords following T9 complete transection [62]. Highly superporous poly(2-hydroxyethyl methacrylate) scaffolds with oriented pores [63] and highly superporous cholesterol-modified poly(2-hydroxyethyl methacrylate) scaffolds have been developed for

*Three-dimensional* culture can mimic the stem cell niche compared to conventional twodimensional culture. Bone marrow-derived mesenchymal stem cells cultured in threedimensional collagen scaffold have exhibited distinctive features including significantly enhancing neurotrophic factor secretions and reducing macrophage activations challenged by lipopolysaccharide [65]. A polyhydroxybutaryl-hydroxyvinyl-based three-dimensional scaffold for a tissue engineering and cell-therapy combinatorial approach for spinal cord injury regeneration has been developed [66]. A three-dimensional biomimetic hydrogel has been implemented to deliver factors secreted by human mesenchymal stem cells in spinal cord injury [67]. Bone marrow mesenchymal stem cells in a three-dimensional gelatin sponge scaffold have attenuated inflammation, have promoted angiogenesis and have reduced cav-

*Biomaterial scaffolds* should be biocompatible, non-toxic, chemically stable, of known absorption and degradation kinetics matching the degree of in vivo cell/tissue growth and should have adequate surface for cell access, proliferation and cell differentiation [69, 70]. They *should meet macroengineering requirements* being of proper form [71, 72], design (shape) [73] and size (diameter) [74]. They should be supplied with macrogrooves [43, 75, 76] and have a wall thickness of 0.6 mm, a porosity of 80% and a pore size range of 10–40 μm [77–79]. They *should meet microengineering requirements*, microgrooves directing axonal growth [80–87]. Prestretchinduced surface anisotropy has been beneficial in enhancing axon alignment, growth and myelination [88]. Also, filament inclusion has been more effective for bridging long nerve defect gaps [43, 89, 90]; Schwann cell migration over gaps exceeding 18 mm is superior in the presence of filaments. Yoshii et al. [91, 92] have tested collagen microfilaments with diameters of 20 μm to repair long gaps (20 or 30 mm) in the rat sciatic nerve. Increasing fiber number (4000 versus 2000 filaments) has enhanced nerve regeneration. Thus, increasing the whole filament surface area by increasing their number and reducing their diameter (increased sur-

*Scaffolds should fulfill nearly the same mechanical conditions of the recipient spinal cord, exerting incremental tensile forces on intact cord segments to promote axonal regeneration while unloading gliotic segments to reduce gliosis and harbor cellular transplants* (**Figure 1a and b**)*.* A scaffold should

spinal cord injury repair [64].

ity formation in experimental spinal cord injury [68].

230 Mesenchymal Stem Cells - Isolation, Characterization and Applications

face area-to-volume ratio) is also critical [89, 93, 94].

*2.2.2. Prerequisites for the use of biomaterial scaffolds in spinal cord injury*

**Figure 1.** (a) How a spinal cord lesion looks like; (1) cranial spinal cord; (2) rostral spinal cord and (3) the gliotic segment. (b) A biomaterial scaffold (4) should fulfill nearly the same mechanical conditions of the recipient spinal cord, exerting incremental tensile forces (5—arrows) on intact cord segments to promote axonal regeneration while unloading gliotic segments (6—arrows) to reduce gliosis and harbor cellular transplants. In addition, it should meet macro- and microengineering requirements; it should provide adequate space for the interplay and manipulation of the different molecular pathways for axonal regeneration through lumen filling technology and it should meet requirements based on spatial distribution of neurotrophic factor gradients. Lumen filling technology allows for the incorporation and gradual local release of stem cells (7), accessory cells (8), molecular growth factors (e.g. BDNF, neurotrophin-3, etc.) (9) and neurolyzing agents (e.g. chondroitinase ABC) (10), either by combining them with a growth-supporting matrix in the lumen (11), by crosslinking (12) them to nerve conduit walls or by using microspheres (13) to deliver them. Growthsupporting matrices (11) in the lumen include hydrogel-forming collagen, fibrin, laminin, alginate, heparin and heparin sulfate. A natural and low-toxicity crosslinking agent (12), genipin, is commonly used.

possess sufficient toughness to resist compression or collapse, yet still be flexible and suturable [95]. A brittle scaffold that sustains little or no plastic deformation before fracture might break hampering axonal progression.

A scaffold should have an elastic modulus comparable with that of the recipient spinal cord. To approach appropriate mechanical properties, one strategy has been to form polymer composites with biopolymers such as chitosan [96], a polymer which has been established as being "softer" and biocompatible. The role of mechanical compliance in directing cell fate and function is a critical issue in material design [97–99]. A low elasticity and hierarchically aligned fibrillar fibrin hydrogel fabricated through electrospinning and concurrent molecular selfassembly process has been tested. Matrix stiffness and aligned topography have instructed stem cell neurogenic differentiation and rapid neurite outgrowth [100].

*Scaffolds should provide adequate space for the interplay and manipulation of the different molecular pathways for axonal regeneration* [80, 81, 101–103].

To provide adequate space and adherence for cells and molecules, biomaterial polymer nerve scaffolds should be porous [43]. Currently, ideal scaffolding should have 80–90% porosity with a pore size of 50–250 μm. Its pores should be interconnected so as to provide physical support to cells and guide their proliferation and differentiation, also facilitating neovascularization [69, 104]. The porous structure can be stabilized by adding glutaraldehyde, polyethylene glycol, heparin or collagen, allowing the structure to become more resistant and to maintain elasticity. A natural and low-toxicity cross-linking agent, genipin, has been used to immobilize nerve growth factor, a neurotrophic factor, onto chitosan-based neural scaffolds to generate a novel nerve graft, which has been beneficial for peripheral nerve repair [105]. A novel method has been introduced for standardized microcomputed tomography-guided evaluation of scaffold properties in bone and tissue research [106].

*Scaffolds should provide adequate space for lumen fillers* Methods of lumen filling allow for incorporation of cells and molecular factors either by combining them with a *growth-supporting matrix* in the lumen, by crosslinking them to nerve conduit walls or by using microspheres to deliver them [107]. *Growth-supporting matrices in the lumen* include hydrogel-forming collagen, fibrin, laminin, alginate, heparin, and heparin sulfate.

#### *Scaffolds should meet requirements based on spatial distribution of neurotrophic factor gradients*.

Spatial molecular concentration gradients of nerve growth factor [108] and laminin [43, 109, 110] promote axonal sprouting. Thus, axonal growth can be hypothetically made to bridge the whole length of the neural gap by seeding the scaffolds with multiple nerve growth factor/laminin spatial concentration gradients [111].

#### **3. Optimizing the therapeutic effect of mesenchymal stem cell transplantation**

#### **3.1. The ideal source for mesenchymal stem cells**

Mesenchymal stem cells reside not only in various tissues of mesenchymal origin (e.g. bone marrow, adipose tissue, skin and peripheral blood) but also in perinatal sources (e.g. umbilical cord blood, umbilical cord matrix or Wharton's jelly, amniotic fluid and placenta) [112].

In a comparative study using mesenchymal stem cells extracted from both bone marrow and adipose tissue for spinal cord injury, animals receiving adipose tissue cells have presented higher levels of tissue brain-derived neurotrophic factor, increased angiogenesis, higher number of preserved axons and a decrease in the number of macrophages, suggesting the superiority of mesenchymal stem cells extracted from adipose tissue [113]. In another study, however, no difference has been found between animals receiving mesenchymal stem cells derived from bone marrow or adipose tissue, whether in terms of axonal regeneration, neuroprotection or functional recovery [114].

Mesenchymal stem cells obtained from perinatal sources can proliferate more rapidly and extensively than adult mesenchymal stem cells and are easily obtained after normal and cesarean births, with low risk of viral contamination. They may be used for allogenic transplantation because they act by suppressing immune response and are, therefore, considered non-immunogenic cells [112].

In a study comparing mesenchymal stem cells derived from fat, bone marrow, Wharton's jelly and umbilical cord blood for treating spinal cord injuries, dogs have been treated with only matrigel or matrigel mixed with each type of mesenchymal stem cells. Although there have been no significant differences in functional recovery among the mesenchymal stem cell groups, application of umbilical cord stem cells has led to more nerve regeneration, neuroprotection and less inflammation compared to other mesenchymal stem cells [115].

Central nervous system pericytes (perivascular stromal cells) have recently gained significant attention. These cells not only display a mesenchymal stem cell phenotype in vitro but also have similar in vivo immunomodulatory effects after spinal cord injury that are more potent than those of non-central nervous system tissue-derived cells [116].

#### **3.2. Increasing the efficiency of mesenchymal stem cells and their influence on spinal cord regeneration**

#### *3.2.1. Influence of mesenchymal stem cells on spinal cord regeneration in general*

with a pore size of 50–250 μm. Its pores should be interconnected so as to provide physical support to cells and guide their proliferation and differentiation, also facilitating neovascularization [69, 104]. The porous structure can be stabilized by adding glutaraldehyde, polyethylene glycol, heparin or collagen, allowing the structure to become more resistant and to maintain elasticity. A natural and low-toxicity cross-linking agent, genipin, has been used to immobilize nerve growth factor, a neurotrophic factor, onto chitosan-based neural scaffolds to generate a novel nerve graft, which has been beneficial for peripheral nerve repair [105]. A novel method has been introduced for standardized microcomputed tomography-guided

*Scaffolds should provide adequate space for lumen fillers* Methods of lumen filling allow for incorporation of cells and molecular factors either by combining them with a *growth-supporting matrix* in the lumen, by crosslinking them to nerve conduit walls or by using microspheres to deliver them [107]. *Growth-supporting matrices in the lumen* include hydrogel-forming collagen,

*Scaffolds should meet requirements based on spatial distribution of neurotrophic factor gradients*.

**3. Optimizing the therapeutic effect of mesenchymal stem cell**

Spatial molecular concentration gradients of nerve growth factor [108] and laminin [43, 109, 110] promote axonal sprouting. Thus, axonal growth can be hypothetically made to bridge the whole length of the neural gap by seeding the scaffolds with multiple nerve growth factor/laminin

Mesenchymal stem cells reside not only in various tissues of mesenchymal origin (e.g. bone marrow, adipose tissue, skin and peripheral blood) but also in perinatal sources (e.g. umbilical cord blood, umbilical cord matrix or Wharton's jelly, amniotic fluid and placenta) [112].

In a comparative study using mesenchymal stem cells extracted from both bone marrow and adipose tissue for spinal cord injury, animals receiving adipose tissue cells have presented higher levels of tissue brain-derived neurotrophic factor, increased angiogenesis, higher number of preserved axons and a decrease in the number of macrophages, suggesting the superiority of mesenchymal stem cells extracted from adipose tissue [113]. In another study, however, no difference has been found between animals receiving mesenchymal stem cells derived from bone marrow or adipose tissue, whether in terms of axonal regeneration, neuro-

Mesenchymal stem cells obtained from perinatal sources can proliferate more rapidly and extensively than adult mesenchymal stem cells and are easily obtained after normal and cesarean births, with low risk of viral contamination. They may be used for allogenic transplantation because they act by suppressing immune response and are, therefore, considered

evaluation of scaffold properties in bone and tissue research [106].

fibrin, laminin, alginate, heparin, and heparin sulfate.

232 Mesenchymal Stem Cells - Isolation, Characterization and Applications

**3.1. The ideal source for mesenchymal stem cells**

protection or functional recovery [114].

non-immunogenic cells [112].

spatial concentration gradients [111].

**transplantation**

Present around blood vessels, mesenchymal stem cells respond more readily to tissue damage [3]. The transdifferentiation capacity of mesenchymal stem cells into neuronal and glial lineages has been debated; transplanted mesenchymal stem cells do not differentiate into a neuronal fate, even if they display weak expression of NeuN (a neuronal marker) [3]. Mesenchymal stem cell-based cell therapy, even when applied during the chronic phase of spinal cord injury, leads to changes in a number of structural and functional parameters, all of which indicate improved recovery [117]. Mesenchymal stem cells promote repair in the injured cord by secreting growth factors that overcome the inhibitory environment of the lesion. These cells have anti-inflammatory, immunomodulatory, vascular promoting oxidative stress reducing and neuroprotective effects. They can secrete trophic factors thus exerting a paracrine effect that can stimulate axon regeneration contributing to functional recovery enhancement [112, 118]. Human mesenchymal stem/stromal cells suppress spinal inflammation in mice with contribution of pituitary adenylate cyclase-activating polypeptide [119]. Intrathecal transplantation of mesenchymal stem cells activates extracellular adjusting protein kinase1 and 2 in the spinal cord following ischemia reperfusion injury, partially improving spinal cord function and inhibiting apoptosis in rats [120].

Measures to increase the *efficiency of mesenchymal stem cells* include the following. *Replacing fetal bovine serum* has been proposed as a gold standard for human cell propagation [121]. *Mechanical fibrinogen-depletion* has been found to support heparin-free mesenchymal stem cell propagation in human platelet lysate [122]. A combination of *electroacupuncture* and grafted mesenchymal stem cells overexpressing tyrosine kinase C has been found to improve remyelination and function in demyelinated spinal cord of rats [123]. Arginine decarboxylase is a rate-limiting enzyme of agmatine synthesis and is known to exist in the central nervous system of mammals. Arginine decarboxylase-secreting human mesenchymal stem cells have been found to be more suitable candidates than human mesenchymal stem cell for stem cell therapy after spinal cord injury [124]. Heme oxygenase-1 is a stress-responsive enzyme that modulates immune response and oxidative stress associated with spinal cord injury. Functional recovery after spinal cord injury has been promoted by transplantation of mesenchymal stem cells overexpressing heme oxygenase-1 [125]. *Hypothermia* is known to improve the microenvironment of the injured spinal cord in a number of ways. Neural cell transplantation has promoted the recovery of hind limb function in rats, and a combination treatment with hypothermia has produced synergistic effects [126]. *Extracorporeal shock wave* can introduce alteration of microenvironment in cell therapy for chronic spinal cord injury [127].

#### *3.2.2. Peculiarities of bone marrow stromal cells in spinal cord regeneration*

Bone marrow stromal cell transplantation has been shown to overcome the gliosis [3]. They have been reported to enhance neuronal protection and cellular preservation *via* reduction in injury-induced sensitivity to mechanical trauma. They can attenuate astrocyte reactivity and chronic microglia/macrophage activation. They have been found to infiltrate primarily into the ventrolateral white matter tracts, spreading to adjacent segments rostrocaudal to the injury epicenter. However, bone marrow stromal cell transplantation present certain issues. Migration beyond the injection site after intraspinal delivery is limited and inter-donor variability in efficacy and immunomodulatory potency might affect clinical outcome [4].

Measures to increase the *efficiency of bone marrow mesenchymal stem cells* include mainly measures to reduce oxidative stress-induced apoptosis, hypoxic preconditioning, measures to modulate the extracellular matrix and other measures.

Studies have demonstrated that the inhibition of the Notch1 pathway in bone marrow mesenchymal stem cells contributes to the differentiation of these cells. Research findings that certain antioxidants induce bone marrow mesenchymal stem cells to differentiate into neuronal cells suggest that bone marrow mesenchymal stem cell differentiation is related to the level of reactive oxygen species in cells. After bone marrow mesenchymal stem cell induction with the antioxidant β-mercaptoethanol, Western blotting and immunofluorescence have revealed gradual increases in the expression of Nestin (a neural stem cell-specific protein) and neuron-specific enolase but decreases in Notch1 expression. The decreased expression levels of Notch1 have correlated positively with changes in reactive oxygen species [128]. The effects of a calpain inhibitor (MDL28170) on increasing survival of bone marrow mesenchymal stem cells transplanted into the injured rat spinal cord have been investigated. The protective effects of MDL28170 on survival of bone marrow mesenchymal stem cells have inhibited the activation of calpain and stress-induced apoptosis [129]. Treatment with bone marrow mesenchymal stem cells combined with plumbagin may alleviate spinal cord injury by affecting oxidative stress, inflammation, apoptosis and the activation of the Nrf2 pathway [130]. Polydatin, a glucoside of resveratrol, has been reported to possess potent antioxidative effects and can used in combination with bone marrow mesenchymal stem cell for the treatment of spinal cord injury. Polydatin significantly protects bone marrow mesenchymal stem cell against apoptosis due to its antioxidative effects and the regulation of Nrf 2/ARE pathway [131]. Carvedilol, a nonselective β-adrenergic receptor blocker, has been reported to exert potent anti-oxidative activities. It has been shown that carvedilol protects cell death of H2O2 induced bone marrow mesenchymal stem cells partly through PI3K-Akt pathway, suggesting its use in combination with bone marrow mesenchymal stem cells to improve cell survival in oxidative stress microenvironments [132].

Hypoxic preconditioning effectively increases the survival rate of bone marrow mesenchymal stem cells following transplantation and increases their protective effect on injured tissues. Hypoxic preconditioning has upregulated the expression of hypoxia-inducible factor 1α in spinal cord tissues [133].

overexpressing heme oxygenase-1 [125]. *Hypothermia* is known to improve the microenvironment of the injured spinal cord in a number of ways. Neural cell transplantation has promoted the recovery of hind limb function in rats, and a combination treatment with hypothermia has produced synergistic effects [126]. *Extracorporeal shock wave* can introduce alteration of micro-

Bone marrow stromal cell transplantation has been shown to overcome the gliosis [3]. They have been reported to enhance neuronal protection and cellular preservation *via* reduction in injury-induced sensitivity to mechanical trauma. They can attenuate astrocyte reactivity and chronic microglia/macrophage activation. They have been found to infiltrate primarily into the ventrolateral white matter tracts, spreading to adjacent segments rostrocaudal to the injury epicenter. However, bone marrow stromal cell transplantation present certain issues. Migration beyond the injection site after intraspinal delivery is limited and inter-donor vari-

ability in efficacy and immunomodulatory potency might affect clinical outcome [4].

Measures to increase the *efficiency of bone marrow mesenchymal stem cells* include mainly measures to reduce oxidative stress-induced apoptosis, hypoxic preconditioning, measures to

Studies have demonstrated that the inhibition of the Notch1 pathway in bone marrow mesenchymal stem cells contributes to the differentiation of these cells. Research findings that certain antioxidants induce bone marrow mesenchymal stem cells to differentiate into neuronal cells suggest that bone marrow mesenchymal stem cell differentiation is related to the level of reactive oxygen species in cells. After bone marrow mesenchymal stem cell induction with the antioxidant β-mercaptoethanol, Western blotting and immunofluorescence have revealed gradual increases in the expression of Nestin (a neural stem cell-specific protein) and neuron-specific enolase but decreases in Notch1 expression. The decreased expression levels of Notch1 have correlated positively with changes in reactive oxygen species [128]. The effects of a calpain inhibitor (MDL28170) on increasing survival of bone marrow mesenchymal stem cells transplanted into the injured rat spinal cord have been investigated. The protective effects of MDL28170 on survival of bone marrow mesenchymal stem cells have inhibited the activation of calpain and stress-induced apoptosis [129]. Treatment with bone marrow mesenchymal stem cells combined with plumbagin may alleviate spinal cord injury by affecting oxidative stress, inflammation, apoptosis and the activation of the Nrf2 pathway [130]. Polydatin, a glucoside of resveratrol, has been reported to possess potent antioxidative effects and can used in combination with bone marrow mesenchymal stem cell for the treatment of spinal cord injury. Polydatin significantly protects bone marrow mesenchymal stem cell against apoptosis due to its antioxidative effects and the regulation of Nrf 2/ARE pathway [131]. Carvedilol, a nonselective β-adrenergic receptor blocker, has been reported to exert potent anti-oxidative activities. It has been shown that carvedilol protects cell death of H2O2 induced bone marrow mesenchymal stem cells partly through PI3K-Akt pathway, suggesting its use in combination with bone marrow mesenchymal stem cells to improve cell survival in

environment in cell therapy for chronic spinal cord injury [127].

234 Mesenchymal Stem Cells - Isolation, Characterization and Applications

modulate the extracellular matrix and other measures.

oxidative stress microenvironments [132].

*3.2.2. Peculiarities of bone marrow stromal cells in spinal cord regeneration*

Cytokines and extracellular matrix can trigger various types of neural differentiation. To highlight the current understanding of their effects on neural differentiation of human bone marrow-derived multipotent progenitor cells, extracellular matrix proteins, tenascin-cytotactin, tenascin-restrictin and chondroitin sulfate, with the cytokines, nerve growth factor/brainderived neurotrophic factor/retinoic acid, have been incorporated to induce transdifferentiation of human bone marrow-derived multipotent progenitor cells. Greater amounts of neuronal morphology have appeared in cultures incorporated with tenascin-cytotactin and tenascin-restrictin than those with chondroitin sulfate. It has been suggested that the combined use of tenascincytotactin, nerve growth factor /brain-derived neurotrophic factor/retinoic acid and human bone marrow-derived multipotent progenitor cells offers a new feasible method for nerve repair [134]. Fibronectin secreted by mesenchymal stem cells in the early stage has been found to accumulate on gelatin sponge scaffolds and promote neurite elongation of neuronal differentiating mesenchymal stem cells as well as nerve fiber regeneration after spinal cord injury [135].

Transplanted bone mesenchymal stem cells can be mobilized by erythropoietin toward lesion sites following spinal cord injury [136]. Propofol injection combined with bone marrow mesenchymal stem cell transplantation has improved electrophysiological function in the hindlimb of rats with spinal cord injury than monotherapy [137]. Combining bone marrow stromal cells with green tea polyphenols has attenuated the blood-spinal cord barrier permeability in rats with compression spinal cord injury [138]. Bone marrow stromal cells transplantation combined with ultrashortwave therapy has promoted functional recovery in spinal cord injury in rats [139].

Microtubule-associated protein 1B plays an important role in axon guidance and neuronal migration. Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 in bone marrow mesenchymal stem cells have been found to modulate the phosphorylation of microtubule-associated protein 1B via a cross-signaling network and have affected the migratory efficiency of bone marrow mesenchymal stem cells towards injured spinal cord [140]. Administration of valproic acid potentiates the therapeutic effect of mesenchymal stem cell therapy [141]. Interleukin-8 enhances the angiogenic potential of human bone marrow mesenchymal stem cells by increasing vascular endothelial growth factor production [142].

#### *3.2.3. Peculiarities of adipose-derived stem cells in spinal cord regeneration*

Human mesenchymal cells from adipose tissue have deposited laminin and have promoted regeneration of injured spinal cord in rats [143–146]. Transplanted during the acute and subacute phases after spinal cord injury, they have enabled the remodulation and regeneration of the lesion site, decreasing the importance of transplantation time in the treatment of spinal cord injury [145]. Chondroitinase ABC-adipose-derived stem cells constructed using lentiviral vector transfection have stably expressed chondroitinase ABC, and chondroitinase ABC expression has significantly enhanced their migratory capacity [146]. Cytoplasmic extracts prepared from adipose tissue stromal cells have *inhibited H2 O2 -mediated apoptosis* of cultured spinal cord-derived neural progenitor cells and have improved cell survival. *Predifferentiation* of adipose tissuederived stromal cells has promoted the protection of denuded axons and cellular repair. Such predifferentiated cells and hematopoietic stem cells have been successfully infused intrathecally [143]. Nevertheless, no evidence points to the superiority of neural differentiated adipose tissuederived stromal over undifferentiated ones. *Allogenic* adipose-derived stem cells have improved neurological function in a canine model. All of the former evidence, however, is contradicted by a study in a rat C3–C4 hemisection in which adipose tissue-derived stromal cell transplantation has significantly reduced sprouting of the descending serotonergic fibers at the injured site [147].

*Hypoxic preconditioning* of adipose tissue-derived mesenchymal stem cells has increased their survival. Cotransplantation of such cells with engineered neural stem cells has improved both cell survival and gene expression of the engineered neural stem cells [4].

#### *3.2.4. Peculiarities of human umbilical cord blood-derived mesenchymal stem cells in spinal cord regeneration*

Human umbilical cord blood-derived mesenchymal stem cells (whether Wharton's jelly mesenchymal stem cells or human umbilical cord perivascular cells) may reverse spinal cord injury pathophysiology by *downregulating apoptotic genes and secreting neurotrophic factors* in few days; they may *transdifferentiate* toward neuronal and oligodendroglial phenotypes [3]. Intrathecal transplantation of human amniotic mesenchymal stem cells has promoted functional recovery in a rat model of traumatic spinal cord injury [148] and in a chronic constrictive nerve injury model [149]. Placental mesenchymal stromal cells have rescued ambulation in ovine myelomeningocele [150]. Umbilical cord-derived mesenchymal stem cell therapy for neurological disorders may act via inhibition of mitogen-activated protein kinase pathwaymediated apoptosis [115]. Through the effect on glial cells(suppression of activated astrocytes and microglia), proinflammatory (Interleukin-1β and Interleukin-17A) and anti-inflammatory cytokines (anti-inflammatory cytokine Interleukin-10), intrathecal injection of human umbilical cord-derived mesenchymal stem cells has ameliorated neuropathic pain in rats [151]. Also, neurotrophic factors have been expressed in the injured spinal cord after transplantation of human-umbilical cord blood stem cells in rats [152].

Preconditioning of umbilical cord mesenchymal stem cells in physioxic environment can enhance the regenerative properties of these cells in the treatment of rat spinal cord injury. In a study on umbilical cord, mesenchymal stem cells pretreated with either atmospheric normoxia (21% O2 ) or physioxia (5% O2 ) have grown faster, whereas physioxia has upregulated the expression of trophic and growth factors, including hepatocyte growth factor, brainderived neurotrophic factor and vascular endothelial growth factor. This has been associated with a significant increase in axonal preservation and a decrease in the number of caspase-3+ cells and ED-1+ macrophages [153].

Calcitonin gene-related peptide, a neural peptide synthesized in spinal cord, contributes to homing of human umbilical cord mesenchymal stem cells. The PI3K/Akt and p38MAPK signaling pathways have played a critical role in the calcitonin gene-related peptide-induced chemotactic migration of human umbilical mesenchymal stem cells [154].

has significantly enhanced their migratory capacity [146]. Cytoplasmic extracts prepared from

neural progenitor cells and have improved cell survival. *Predifferentiation* of adipose tissuederived stromal cells has promoted the protection of denuded axons and cellular repair. Such predifferentiated cells and hematopoietic stem cells have been successfully infused intrathecally [143]. Nevertheless, no evidence points to the superiority of neural differentiated adipose tissuederived stromal over undifferentiated ones. *Allogenic* adipose-derived stem cells have improved neurological function in a canine model. All of the former evidence, however, is contradicted by a study in a rat C3–C4 hemisection in which adipose tissue-derived stromal cell transplantation has significantly reduced sprouting of the descending serotonergic fibers at the injured site [147]. *Hypoxic preconditioning* of adipose tissue-derived mesenchymal stem cells has increased their survival. Cotransplantation of such cells with engineered neural stem cells has improved both

*-mediated apoptosis* of cultured spinal cord-derived

*O2*

*3.2.4. Peculiarities of human umbilical cord blood-derived mesenchymal stem cells in spinal cord*

Human umbilical cord blood-derived mesenchymal stem cells (whether Wharton's jelly mesenchymal stem cells or human umbilical cord perivascular cells) may reverse spinal cord injury pathophysiology by *downregulating apoptotic genes and secreting neurotrophic factors* in few days; they may *transdifferentiate* toward neuronal and oligodendroglial phenotypes [3]. Intrathecal transplantation of human amniotic mesenchymal stem cells has promoted functional recovery in a rat model of traumatic spinal cord injury [148] and in a chronic constrictive nerve injury model [149]. Placental mesenchymal stromal cells have rescued ambulation in ovine myelomeningocele [150]. Umbilical cord-derived mesenchymal stem cell therapy for neurological disorders may act via inhibition of mitogen-activated protein kinase pathwaymediated apoptosis [115]. Through the effect on glial cells(suppression of activated astrocytes and microglia), proinflammatory (Interleukin-1β and Interleukin-17A) and anti-inflammatory cytokines (anti-inflammatory cytokine Interleukin-10), intrathecal injection of human umbilical cord-derived mesenchymal stem cells has ameliorated neuropathic pain in rats [151]. Also, neurotrophic factors have been expressed in the injured spinal cord after transplantation of

Preconditioning of umbilical cord mesenchymal stem cells in physioxic environment can enhance the regenerative properties of these cells in the treatment of rat spinal cord injury. In a study on umbilical cord, mesenchymal stem cells pretreated with either atmospheric

lated the expression of trophic and growth factors, including hepatocyte growth factor, brainderived neurotrophic factor and vascular endothelial growth factor. This has been associated with a significant increase in axonal preservation and a decrease in the number of caspase-3+

Calcitonin gene-related peptide, a neural peptide synthesized in spinal cord, contributes to homing of human umbilical cord mesenchymal stem cells. The PI3K/Akt and p38MAPK signaling

) have grown faster, whereas physioxia has upregu-

cell survival and gene expression of the engineered neural stem cells [4].

human-umbilical cord blood stem cells in rats [152].

) or physioxia (5% O2

adipose tissue stromal cells have *inhibited H2*

236 Mesenchymal Stem Cells - Isolation, Characterization and Applications

*regeneration*

normoxia (21% O2

cells and ED-1+ macrophages [153].

Lavandula angustifolia has neuroprotective effects; it has potentiated the functional and cellular recovery with human umbilical mesenchymal stem cell treatment in rats after spinal cord injury [155]. The combined treatment with methylprednisolone and amniotic membrane mesenchymal stem cells after spinal cord injury in rats has potentiated the anti-inflammatory and anti-apoptotic effect of mesenchymal stem cell transplantation [156]. The neuroprotective effects of conditioned medium from cultured human CD34(+) cells have been similar to those of human CD34(+) cells and the conditioned medium has been found to enhance the neuroprotective effects of 17β-estradiol in rat spinal cord injury [157].

#### **3.3. Inducing the transformation of mesenchymal stem cells into motor neuron-like cells or Schwann cells**

A third method for optimizing the therapeutic effect of mesenchymal stem cell transplantation is inducing their transformation into motor neuron-like cells or Schwann cells [158–169]. Their differentiation into *motor neuron-like cells* has been induced through a pre-induction step using β-mercaptoethanol followed by 4 days of induction with retinoic acid and sonic hedgehog [158]. Motor neuron axonal sprouting has been induced by adding different concentrations of a nerve growth factor to the differentiation media. In another study [159], such cells have been tested for 2′,3′-cyclic-nucleotide-3′-phosphodiesterase and microtubule-associated protein 2, as well as to glial fibrillary acidic protein and beta III tubulin. Cells have been injected percutaneously into the spinal cord of paraplegic dogs for two times separated by a 21-day interval. *Optimal culture conditions* have been investigated as to the production of neural cells and neural stem cells [160]. β-Mercaptoethanol has been used as the main inducer of the neurogenesis pathway. Three types of neural markers have been used: nestin as the immaturation stage marker, neurofilament light chain as the early neural marker, and microtubule-associated protein 2 as the maturation marker. Results have shown that the best exposure time for the production of neural stem cells is 6 hours. It has also been demonstrated that LY294002, a small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, can promote neuronal differentiation of mesenchymal stem cells cultured on polycaprolactone/ collagen scaffolds [161]. Similarly, microRNA-124 has promoted bone marrow mesenchymal stem cell differentiation into neurogenic cells for accelerating recovery in the spinal cord injury [166, 169]. *Such induced motor neuron-like cells* have promoted axonal regeneration into the injured spinal cord, whether derived from bone marrow [162, 163, 168], human chorion [164] and placenta [167]. Their in vivo tracking by magnetic resonance has been possible in rabbit models of spinal cord injury [169].

#### **3.4. Mode, quantity and number of injections; time point for injection age and donor variation; allo- and xenotransplantation**

The mode, quantity and number of injections may influence the therapeutic effect of mesenchymal stem cell transplantation

#### *3.4.1. Mode of injection*

All methods for stem cell transplantation (intravenous, intrathecal, intramedullary, intranasal or skeletal muscle injection ) are based on the homing effect, the ability of implanted stem cells to move to the injured area [170–180]. Mesenchymal progenitor cells have been injected *intravenously* in two models of cervical spinal cord injury, unilateral C5 contusion and complete unilateral C5 hemisection. Cells have been isolated from green fluorescence proteinluciferase transgenic mice and have been injected via the tail vein at D1, D3, D7, D10, or D14. Transplanted cells have been tracked via postmortem bioluminescence imaging. Cells have been found to accumulate in the lungs, irrespective of the time of injection or injury model. It has been proposed that they modulate the immune system via the lungs through secreted immune mediators [173]. The antioxidant and anti-inflammatory effects of intravenously injected adipose-derived mesenchymal stem cells have been proven in dogs with acute spinal cord injury [174]. Diffuse and persistent blood-spinal cord barrier disruption after contusive spinal cord injury has recovered following intravenous infusion of bone marrow mesenchymal stem cells [177]. Intravenous mesenchymal stem cell therapy has been effective after recurrent laryngeal nerve injury [179]. In a meta-analysis, the efficacy of intravenous bone marrow mesenchymal stem cell transplantation in spinal cord injury has been investigated. It has been concluded that the therapeutic window of intravenous bone marrow mesenchymal stem cell transplantation is wide [180]. The feasibility and safety of *intrathecal* transplantation of autologous bone marrow mesenchymal stem cells have been investigated in horses [175]. The *intranasal* delivery of bone marrow stromal cells to spinal cord lesions has been successfully tried out [176]. Stem cell injection in the *hindlimb* skeletal muscle has enhanced neurorepair in mice with spinal cord injury [178].

Although intrathecal is more effective than intravenous injection, it needs large stem cell numbers. Subarachnoid adhesions may prevent the cells from reaching the target site. The homing effect is absent in the chronic stage of spinal cord injury. Therefore, direct intramedullary injection into the injured site is the most effective method for delivering stem cells. Intramedullary injection proximal to the injured area is ideal for stem cell survival, but is hampered by volume effects caused by high tissue pressure and subsequent normal spinal cord damage. On the contrary, large volumes can be injected into the cavity area at the injured level. Injecting into the contused cavity may lead to resolution of the glial scar and may bridge for axonal regeneration. Therefore, Park et al. [171, 172] have injected into both the normal proximal spinal cord and the injured area. In addition, subdural stem cells have been applied in the hope the homing effect has been reinduced because of intramedullary injection.

#### *3.4.2. Quantity, number and time point for mesenchymal stem cell transplantation*

#### *<IP_ADDRESS>. Quantity and number*

Diversity of lesion models, animal types and route of cell administration influence the quantity of mesenchymal stem cells administered. Cell survival and enhancement in locomotor performance have been observed both after intravenous injection of one million cells in a volume of 0.5 mL of DMEM in a model of balloon compressive injury in rats and after transplantation of 600,000 cells in a volume of 6 μL directly into the injury site after contusive injury in rats [112]. Other studies have advocated intrathecal administration from 100 × 106 up to 230 × 106 cells followed by an additional 30 × 106 cell administration at 3 months [5], or the administration of two or three intrathecal injections with a median of 1.2 × 106 mesenchymal stem cells/kg body weight [6]. In a phase III clinical trial, limited efficacy has been proven after injecting 1.6 × 10 autologous mesenchymal stem cells into the intramedullary area at the injured level and 3.2 × 10 autologous mesenchymal stem cells into the subdural space. Single mesenchymal stem cell application to intramedullary and intradural space has had a very weak therapeutic effect compared to multiple injections [7]; partial efficacy has been demonstrated in other trials [8–11]. Continuous improvement after multiple mesenchymal stem cell transplantations has been observed in a patient with complete spinal cord injury [181]. Multiple injections of human umbilical cord-derived mesenchymal stromal cells through the tail vein have improved microcirculation and the microenvironment in a rat model of radiation myelopathy [182].

#### *<IP_ADDRESS>. Time point*

*3.4.1. Mode of injection*

238 Mesenchymal Stem Cells - Isolation, Characterization and Applications

pair in mice with spinal cord injury [178].

injection.

*<IP_ADDRESS>. Quantity and number*

All methods for stem cell transplantation (intravenous, intrathecal, intramedullary, intranasal or skeletal muscle injection ) are based on the homing effect, the ability of implanted stem cells to move to the injured area [170–180]. Mesenchymal progenitor cells have been injected *intravenously* in two models of cervical spinal cord injury, unilateral C5 contusion and complete unilateral C5 hemisection. Cells have been isolated from green fluorescence proteinluciferase transgenic mice and have been injected via the tail vein at D1, D3, D7, D10, or D14. Transplanted cells have been tracked via postmortem bioluminescence imaging. Cells have been found to accumulate in the lungs, irrespective of the time of injection or injury model. It has been proposed that they modulate the immune system via the lungs through secreted immune mediators [173]. The antioxidant and anti-inflammatory effects of intravenously injected adipose-derived mesenchymal stem cells have been proven in dogs with acute spinal cord injury [174]. Diffuse and persistent blood-spinal cord barrier disruption after contusive spinal cord injury has recovered following intravenous infusion of bone marrow mesenchymal stem cells [177]. Intravenous mesenchymal stem cell therapy has been effective after recurrent laryngeal nerve injury [179]. In a meta-analysis, the efficacy of intravenous bone marrow mesenchymal stem cell transplantation in spinal cord injury has been investigated. It has been concluded that the therapeutic window of intravenous bone marrow mesenchymal stem cell transplantation is wide [180]. The feasibility and safety of *intrathecal* transplantation of autologous bone marrow mesenchymal stem cells have been investigated in horses [175]. The *intranasal* delivery of bone marrow stromal cells to spinal cord lesions has been successfully tried out [176]. Stem cell injection in the *hindlimb* skeletal muscle has enhanced neurore-

Although intrathecal is more effective than intravenous injection, it needs large stem cell numbers. Subarachnoid adhesions may prevent the cells from reaching the target site. The homing effect is absent in the chronic stage of spinal cord injury. Therefore, direct intramedullary injection into the injured site is the most effective method for delivering stem cells. Intramedullary injection proximal to the injured area is ideal for stem cell survival, but is hampered by volume effects caused by high tissue pressure and subsequent normal spinal cord damage. On the contrary, large volumes can be injected into the cavity area at the injured level. Injecting into the contused cavity may lead to resolution of the glial scar and may bridge for axonal regeneration. Therefore, Park et al. [171, 172] have injected into both the normal proximal spinal cord and the injured area. In addition, subdural stem cells have been applied in the hope the homing effect has been reinduced because of intramedullary

Diversity of lesion models, animal types and route of cell administration influence the quantity of mesenchymal stem cells administered. Cell survival and enhancement in locomotor performance have been observed both after intravenous injection of one million cells in a

*3.4.2. Quantity, number and time point for mesenchymal stem cell transplantation*

Acute phase is defined as the first three days after spinal cord injury and chronic phase is defined as more than 12 months after spinal cord injury. Subacute phase is defined as the period between acute and chronic phase. In the acute phase, reactive oxygen-free radicals, excitatory transmitters, inflammatory molecules and hypoxia caused by hypoperfusion are cytotoxic to implanted stem cells. In the chronic phase, glial scar tissue acts as a physical barrier to axonal regrowth. Thus, it is difficult for implanted stem cells to survive in chronic spinal cord injury. In contrast, in the subacute phase, the inflammatory response is reduced and the glial scar formation has not formed. Therefore, the subacute phase seems to be an optimal phase in the respect of timing of stem cell application [170]. Experimentally, bone marrow-derived stem cells have been infused intravenously 10 weeks after spinal cord injury [183].

#### *3.4.3. Age and donor variation, allo- and xenotransplantation*

#### *<IP_ADDRESS>. Age and donor variation*

The potency of mesenchymal stem cells exhibits significant age and donor variation [3, 184–186]. A robust potency assay has been established based on pooling responder leukocytes to minimize individual immune response variability. It has highlighted significant donor variation of human mesenchymal stem/progenitor cell immune modulatory capacity and extended radioresistance [184, 185].

#### *<IP_ADDRESS>. Allo- and xenotransplantation*

The neuroprotective and immunomodulatory effects of *xenotransplantation* of adipose tissue mesenchymal stem cells in Lewis rats after lumbar ventral root avulsion have been proven [187]. The therapeutic effects of autologous and *allogenic* bone marrow-derived mesenchymal stem cell transplantation have been established in canine spinal cord injury [188]. Immunosuppression of allogenic mesenchymal stem cells transplantation after spinal cord injury may improve graft survival [189].

#### *3.4.4. Evaluating the therapeutic effect of mesenchymal stem cell transplantation*

Although neurological evaluation of the spinal cord injured patient is usually conducted according to the International Standards for Neurological Classification of Spinal Cord Injury recommended by the American Spinal Cord Injury Association, it should be confirmed by electrophysiological studies (somatosensory evoked potentials and motor evoked potentials) and magnetic resonance imaging studies. Magnetic resonance imaging findings after stem cell therapy include widening of cord diameter, blurring of intramedullary cavity margin and appearance of fiber-like streak pattern in the injured spinal cord. Diffusion tensor imaging can perform accurate visualization and assessment of white matter tracts and is useful for the prediction of neurological recovery in spinal cord injury patients. Fiber continuity on diffusion tensor imaging not seen before stem cell therapy may be an indicator of axonal regeneration in stem cell therapy. Cell labeling techniques for *in vivo* visualization using biological indicators or contrast agents have helped monitoring the status of the transplanted stem cells in the body (survival, migration and exact location of implanted stem cells). Typical examples are supermagnetic iron oxide particle monitoring using magnetic resonance imaging and radionuclide monitoring using positron emission tomography or single-photon emission computed tomography [170, 190, 191].

#### **4. Supplying neurotrophic factors and accessory cells**

A combinatorial approach has been agreed upon for effective treatment of spinal cord injury [192–208].

The combination of *neurotrophic factors* such as BDNF and neurotrophin-3 has enhanced axonal regeneration and myelination [193]. Cyclic adenosine monophosphate (a neuronal stimulator) and neurotrophin-3 (neurotrophic factor) have been injected 5 days prior to a C4 transection at L4 to precondition the dorsal root ganglion soma. Bone marrow mesenchymal stem cells have been transplanted 7 days post injury. The effect of bone marrow mesenchymal stem cells on spinal cord regeneration has been augmented by modifying them to either express human brain-derived neurotrophic factor (BDNF) in an acute injury or neurotrophin-3 in a chronic injury model, by prestimulating them to secrete neurotrophic factors, e.g. by pretreating them with Schwann cell differentiating factors [3]. In an attempt to generate mesenchymal-derived differentiated neural cells expressing nerve growth factor or neurotrophin-3, mesenchymal stem cells have been infected with recombinant lentiviruses that express nerve growth factor both to induce their neural lineage genes and as a combinatorial approach [194]. Magnetic targeting of neurotrophin-3 gene-transfected bone marrow mesenchymal stem cells via lumbar puncture has enhanced their delivery to the site of injury and has significantly improved functional recovery and nerve regeneration compared to transplanting neurotrophin-3 gene-transfected bone marrow mesenchymal stem cells without magnetic targeting system [195, 196]. Pulsed electromagnetic field exposure near the injured site and for 8 hours per day over 4 weeks has been suggested as a suitable protocol for directing the cells to the site of injury [197]. Electro-acupuncture has promoted the survival and differentiation of transplanted bone marrow mesenchymal stem cells pre-induced with neurotrophin-3 and retinoic acid in gelatin sponge scaffold after rat spinal cord transection [53, 198].

stem cell transplantation have been established in canine spinal cord injury [188]. Immunosuppression of allogenic mesenchymal stem cells transplantation after spinal cord injury may