*5.2.2 MSC culturing protocols*

Traditionally, BM-MSCs have been separated from other BM cells following strict laboratory cell processing protocols. These cell processing techniques are lengthy procedures, as they cannot be performed at POC, as a same-day procedure. In many parts of the world, clinicians are allowed to use autologous, fresh, and non-cultured BMA and BMC products that are prepared at POC. In the USA, regenerative medicine biological procedures demand the use of the so-called 510-K FDA-approved devices. The use of MSCs following laboratory expansion techniques is facing considerable legislative barriers. Furthermore, the literature has cited potential risks associated with laboratory MSC cell processing techniques, like tumorigenicity [63], genetic instability [64], and immunogenicity [65]. Others raise concern regarding the efficacy and function of cultured MSCs by in vitro culture conditions during the cell passages for cell expansion. Karp and Teo reported on the loss of specific MSC surface receptors functions, negatively affecting chemotaxis [66]. Others have informed on impaired homing abilities and disappearing CXCR4 receptors following cell culturing, when compared to non-cultured BMA, in which high CXCR4 concentrations were measured [67]. Last but not least, laboratory MSC cell culturing methods for regenerative medicine practices require the availability of a specialized and dedicated facility, using strict regulatory protocols which will increase costs [68].

### *5.2.3 Characterization of bone marrow mesenchymal stem cells*

In order to better understand the specifics of MSC cell concentrations, counts, and quality, it's important to understand the differences between laboratory techniques analyzing HSCs and MSCs, as they differ with regard to the specificity

**17**

CFU need to be defined.

**5.3 MSC capacities**

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use…*

and relevance of the different BM cell types, possibly effecting regenerative

The International Society of Cellular Therapy (ISCT) has developed criteria in order to outline human MSCs for both laboratory-based scientific investigations and for preclinical studies [69]. MSCs are defined as those cells that are able to adhere to plastic and express a number of cell surface markers while undergoing

It has been difficult to determine what type of cells is neighboring both MSCs and HSCs and contributes to the regulation of the functional continuation of stem cell, as immunostaining methods are complex procedures to perform. Flow cytometry is a laboratory technique used to detect and measure characteristics of cell/particle populations by measuring their physical and chemical properties. A specific protocol for the identification of dissimilar cell surface molecules is called cluster of differentiation (CD) where monoclonal antibodies (markers) are used to establish positive and negative staining for certain cell types. Specifically for MSC and HSC, explicit CD markers are established to validate BM cellular content, as it is widely accepted that MSC cultures are a heterogenous source of cells with varying self-renewal and differentiation properties [71]. This indicates that there is no single unique indicator for identification. Hence, a panel of both positive and negative protein markers is used to identify the cell surface markers that are expressed by MSC populations, like CD29, VD44, CD51, CD73, CD90, CD105, CD166, and Stro1. While they must be negative for hematopoietic stem cell markers like CD14, CD34, and CD45 [72], some of these markers are included in the minimum ISCT criteria.

In the initial BM monolayer, several hematopoietic oriented cells (macrophages, endothelial cells, and lymphocytes) adhere to plastic [7]. Nevertheless, in culturing conditions only fibroblast-like spindle-shaped cells proliferate and form ultimately CFU-F colonies. These cells are representative of the more highly proliferative cells in MSCs [73]. The CFU-F assay is a different method used to determine the MSC presence in a vial of BM tissue. Unlike a complete blood count test, which is a quantitative blood cell analysis, the CFU-F assay is a laboratory assay in particular for stem and progenitor cell determination (**Figure 9**) [74]. The CFU-F assay is a qualitative indicator of the proliferative and differentiation capability of individual MSC cells within a BMA or BMC sample. The cells are seeded and cultured in a growth medium where they have to adhere to plastic, at 37°C. After 14 days the cultures are evaluated, and CFU-Fs are counted, whereby a minimum of 50 cells per

MSCs are multipotent stem cells which can be obtained from various adult tissues, like the BM stroma, adipose tissue, synovium, periosteum, and trabecular bone. Typical features are their ability for self-renewal, defined as sustaining

*DOI: http://dx.doi.org/10.5772/intechopen.91310*

medicine therapy outcomes.

multilineage differentiation [70].

*5.2.3.2 Flow cytometry and CD markers*

*5.2.3.3 Colony-forming unit fibroblast assay*

*5.2.3.1 ISCT criteria*

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use… DOI: http://dx.doi.org/10.5772/intechopen.91310*

and relevance of the different BM cell types, possibly effecting regenerative medicine therapy outcomes.

#### *5.2.3.1 ISCT criteria*

*Regenerative Medicine*

*5.2.2 MSC culturing protocols*

*individual marrow components.*

**Figure 8.**

Traditionally, BM-MSCs have been separated from other BM cells following strict laboratory cell processing protocols. These cell processing techniques are lengthy procedures, as they cannot be performed at POC, as a same-day procedure. In many parts of the world, clinicians are allowed to use autologous, fresh, and non-cultured BMA and BMC products that are prepared at POC. In the USA, regenerative medicine biological procedures demand the use of the so-called 510-K FDA-approved devices. The use of MSCs following laboratory expansion techniques is facing considerable legislative barriers. Furthermore, the literature has cited potential risks associated with laboratory MSC cell processing techniques, like tumorigenicity [63], genetic instability [64], and immunogenicity [65]. Others raise concern regarding the efficacy and function of cultured MSCs by in vitro culture conditions during the cell passages for cell expansion. Karp and Teo reported on the loss of specific MSC surface receptors functions, negatively affecting chemotaxis [66]. Others have informed on impaired homing abilities and disappearing CXCR4 receptors following cell culturing, when compared to non-cultured BMA, in which high CXCR4 concentrations were measured [67]. Last but not least, laboratory MSC cell culturing methods for regenerative medicine practices require the availability of a specialized and dedicated facility, using strict regulatory protocols which will

*Bone marrow gravity separation following centrifugation. (A) Bone marrow aspirate in concentration device before centrifugation. In (B), the bone marrow aspirate is concentrated, with a view on the buffy coat stratum (gray layer on top of the RBC layer), referenced by the two black lines. Following a two-step centrifugation protocol, the centrifugal forces achieve density marrow cell separation due to the specific gravities of the* 

In order to better understand the specifics of MSC cell concentrations, counts, and quality, it's important to understand the differences between laboratory techniques analyzing HSCs and MSCs, as they differ with regard to the specificity

**16**

increase costs [68].

*5.2.3 Characterization of bone marrow mesenchymal stem cells*

The International Society of Cellular Therapy (ISCT) has developed criteria in order to outline human MSCs for both laboratory-based scientific investigations and for preclinical studies [69]. MSCs are defined as those cells that are able to adhere to plastic and express a number of cell surface markers while undergoing multilineage differentiation [70].

#### *5.2.3.2 Flow cytometry and CD markers*

It has been difficult to determine what type of cells is neighboring both MSCs and HSCs and contributes to the regulation of the functional continuation of stem cell, as immunostaining methods are complex procedures to perform. Flow cytometry is a laboratory technique used to detect and measure characteristics of cell/particle populations by measuring their physical and chemical properties. A specific protocol for the identification of dissimilar cell surface molecules is called cluster of differentiation (CD) where monoclonal antibodies (markers) are used to establish positive and negative staining for certain cell types. Specifically for MSC and HSC, explicit CD markers are established to validate BM cellular content, as it is widely accepted that MSC cultures are a heterogenous source of cells with varying self-renewal and differentiation properties [71]. This indicates that there is no single unique indicator for identification. Hence, a panel of both positive and negative protein markers is used to identify the cell surface markers that are expressed by MSC populations, like CD29, VD44, CD51, CD73, CD90, CD105, CD166, and Stro1. While they must be negative for hematopoietic stem cell markers like CD14, CD34, and CD45 [72], some of these markers are included in the minimum ISCT criteria.

#### *5.2.3.3 Colony-forming unit fibroblast assay*

In the initial BM monolayer, several hematopoietic oriented cells (macrophages, endothelial cells, and lymphocytes) adhere to plastic [7]. Nevertheless, in culturing conditions only fibroblast-like spindle-shaped cells proliferate and form ultimately CFU-F colonies. These cells are representative of the more highly proliferative cells in MSCs [73]. The CFU-F assay is a different method used to determine the MSC presence in a vial of BM tissue. Unlike a complete blood count test, which is a quantitative blood cell analysis, the CFU-F assay is a laboratory assay in particular for stem and progenitor cell determination (**Figure 9**) [74]. The CFU-F assay is a qualitative indicator of the proliferative and differentiation capability of individual MSC cells within a BMA or BMC sample. The cells are seeded and cultured in a growth medium where they have to adhere to plastic, at 37°C. After 14 days the cultures are evaluated, and CFU-Fs are counted, whereby a minimum of 50 cells per CFU need to be defined.

#### **5.3 MSC capacities**

MSCs are multipotent stem cells which can be obtained from various adult tissues, like the BM stroma, adipose tissue, synovium, periosteum, and trabecular bone. Typical features are their ability for self-renewal, defined as sustaining

#### **Figure 9.**

*MSC culturing. A picture of a flask cultured with stained human MSCs. The zoomed-in area is a light micrograph showing the morphology of a MSC colony in a patient treated with BMC. After culturing for 14 days, the MSC count in this example was 1065/mL (picture courtesy of BioSciences Research Associates, Cambridge, MA, USA).*

biological pathways and mechanisms to preserve the undifferentiated stem state, and the regulation of lineage-specific differentiation [39]. Although the number of MSCs represents only a small fraction of non-hematopoietic, multipotent cells of the bone marrow (0.001–0.01%), understanding these unique cells has taken great strides forward. Generally, MSCs have developed a great attractiveness for regenerative medicine autologous therapeutic applications and tissue engineering opportunities, because of their multipotentiality and relative ease of isolation from numerous tissues, like BM [75]. MSCs can be also identified as specialized populations of mural cells or pericytes, sharing a niche with HSCs. Under appropriate conditions and an optimal microenvironment, MSCs can differentiate into various mesodermal lineages like osteoblasts, chondrocytes, endothelial cells, adipose tissue, and smooth muscle cells (**Figure 10**) [76]. These MSC proficiencies have led to the use of MSC as a potential strategy for treating various diseases, since they encourage biological processes, for example angiogenesis, cell proliferation, and cell differentiation [77]. Furthermore, they synthesize cytokines and trophic mediators which participate in tissue repair processes, immune modulation, and the regulation of inflammatory processes [78]. Caplan also suggested that the modulation of inflammation is instigated by the suppression of inflammatory T-cell proliferation and inhibition of monocyte and myeloid cell maturation [79]. Based on the above characteristics, it can be assumed that MSCs are capable to institute a regenerative microenvironment at the site of release and improve the various cell recruitment, cell-signaling, and differentiation of endogenous stem cells, with the potential to instigate tissue repair in a variety of disease states.

#### **5.4 MSC immunomodulatory effects**

In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contact, secretion of cytokines, and/or by a combination of both mechanisms. MSCs have been shown to exert profound anti-inflammatory and immunomodulatory effects on almost all the cells of the innate and adaptive immune systems via a variety of mechanisms, notably cytokine and chemokine secretion [80]. The immunosuppressive capabilities of MSCs are only exploited when they are exposed to sufficiently high concentrations of pro-inflammatory cytokines, like interferon-gamma (IFN-γ), tissue necrosis factor α, (TNF- α), and interleukins α or ß (IL-1α, IL ß).

**19**

**Figure 10.**

medicine applications hold great promise [87].

**5.5 MSC growth factor activity**

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use…*

In order for MSCs to become "immunosuppressants," they need to be triggered by these inflammatory cytokines, and the inflammatory environment is then a crucial factor for MSCs to exert their immunomodulatory effects. These are wielded by blocking apoptosis of native and activated neutrophils, aside from decreasing neutrophils from binding to vascular endothelial cells and the mobilization of neutrophils to the area of damage [81]. Furthermore, MSCs constrain the complementmediated effects of peripheral blood mononuclear cell proliferation [82], and they limit mast cell degranulation and the secretion of pro-inflammatory cytokines, while at the same time MSCs migrate towards CXCL12 and other chemotactic factors [83]. In **Figure 11** the MSC cell-dependent trophic support mechanisms are shown. Data from Jiang and others suggested that MSCs can block the differentiation of CD34+ cells from BM or blood monocytes into mature dendritic cells by direct contact as well as by secreted paracrine factors [84]. Under their influence, M1 (pro-inflammatory) macrophages are transformed into M2-type cells with an anti-inflammatory phenotype, and the interleukin-10 secreted by them inhibits T-cell proliferation. This immunosuppressive effect related to T-cell proliferation and decrease in cytokine production by MSCs was, among others, confirmed by Sato et al. [85]. However, the mechanisms by which MSCs are mobilized and recruited to damaged sites are not known. In addition, how they survive and differentiate into distinct cell types is still not clear. Once MSCs have been applied to the microenvironment of injured or degenerated tissues, many factors stimulate the release of many growth factors by MSCs; a detailed growth and trophic factor overview is shown in **Table 3**. These growth factors stimulate the development of fibroblasts, endothelial cells, and tissue progenitor cells [86]. It is credible to state that the use of MSCs and their potential in immunomodulation in regenerative

*MSC differentiation potential. MSC differentiation potential into endodermal, ectodermal, and mesodermal lineages. The mesodermal lineage differentiation has been recognized as the most attractive differentiation lineages for regenerative medicine applications, executed at point of care, as these produce osteoblasts,* 

In order for MSCs to differentiate into several cell lineages, the action of specific

growth factors and chemical mediators are needed in these processes [88, 89].

*DOI: http://dx.doi.org/10.5772/intechopen.91310*

*chondrocytes, tenocytes, adipose tissues, and smooth muscle cells.*

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use… DOI: http://dx.doi.org/10.5772/intechopen.91310*

#### **Figure 10.**

*Regenerative Medicine*

**Figure 9.**

*Cambridge, MA, USA).*

biological pathways and mechanisms to preserve the undifferentiated stem state, and the regulation of lineage-specific differentiation [39]. Although the number of MSCs represents only a small fraction of non-hematopoietic, multipotent cells of the bone marrow (0.001–0.01%), understanding these unique cells has taken great strides forward. Generally, MSCs have developed a great attractiveness for regenerative medicine autologous therapeutic applications and tissue engineering opportunities, because of their multipotentiality and relative ease of isolation from numerous tissues, like BM [75]. MSCs can be also identified as specialized populations of mural cells or pericytes, sharing a niche with HSCs. Under appropriate conditions and an optimal microenvironment, MSCs can differentiate into various mesodermal lineages like osteoblasts, chondrocytes, endothelial cells, adipose tissue, and smooth muscle cells (**Figure 10**) [76]. These MSC proficiencies have led to the use of MSC as a potential strategy for treating various diseases, since they encourage biological processes, for example angiogenesis, cell proliferation, and cell differentiation [77]. Furthermore, they synthesize cytokines and trophic mediators which participate in tissue repair processes, immune modulation, and the regulation of inflammatory processes [78]. Caplan also suggested that the modulation of inflammation is instigated by the suppression of inflammatory T-cell proliferation and inhibition of monocyte and myeloid cell maturation [79]. Based on the above characteristics, it can be assumed that MSCs are capable to institute a regenerative microenvironment at the site of release and improve the various cell recruitment, cell-signaling, and differentiation of endogenous stem cells, with the potential to

*MSC culturing. A picture of a flask cultured with stained human MSCs. The zoomed-in area is a light micrograph showing the morphology of a MSC colony in a patient treated with BMC. After culturing for 14 days, the MSC count in this example was 1065/mL (picture courtesy of BioSciences Research Associates,* 

In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contact, secretion of cytokines, and/or by a combination of both mechanisms. MSCs have been shown to exert profound anti-inflammatory and immunomodulatory effects on almost all the cells of the innate and adaptive immune systems via a variety of mechanisms, notably cytokine and chemokine secretion [80]. The immunosuppressive capabilities of MSCs are only exploited when they are exposed to sufficiently high concentrations of pro-inflammatory cytokines, like interferon-gamma (IFN-γ), tissue necrosis factor α, (TNF- α), and interleukins α or ß (IL-1α, IL ß).

instigate tissue repair in a variety of disease states.

**5.4 MSC immunomodulatory effects**

**18**

*MSC differentiation potential. MSC differentiation potential into endodermal, ectodermal, and mesodermal lineages. The mesodermal lineage differentiation has been recognized as the most attractive differentiation lineages for regenerative medicine applications, executed at point of care, as these produce osteoblasts, chondrocytes, tenocytes, adipose tissues, and smooth muscle cells.*

In order for MSCs to become "immunosuppressants," they need to be triggered by these inflammatory cytokines, and the inflammatory environment is then a crucial factor for MSCs to exert their immunomodulatory effects. These are wielded by blocking apoptosis of native and activated neutrophils, aside from decreasing neutrophils from binding to vascular endothelial cells and the mobilization of neutrophils to the area of damage [81]. Furthermore, MSCs constrain the complementmediated effects of peripheral blood mononuclear cell proliferation [82], and they limit mast cell degranulation and the secretion of pro-inflammatory cytokines, while at the same time MSCs migrate towards CXCL12 and other chemotactic factors [83]. In **Figure 11** the MSC cell-dependent trophic support mechanisms are shown. Data from Jiang and others suggested that MSCs can block the differentiation of CD34+ cells from BM or blood monocytes into mature dendritic cells by direct contact as well as by secreted paracrine factors [84]. Under their influence, M1 (pro-inflammatory) macrophages are transformed into M2-type cells with an anti-inflammatory phenotype, and the interleukin-10 secreted by them inhibits T-cell proliferation. This immunosuppressive effect related to T-cell proliferation and decrease in cytokine production by MSCs was, among others, confirmed by Sato et al. [85]. However, the mechanisms by which MSCs are mobilized and recruited to damaged sites are not known. In addition, how they survive and differentiate into distinct cell types is still not clear. Once MSCs have been applied to the microenvironment of injured or degenerated tissues, many factors stimulate the release of many growth factors by MSCs; a detailed growth and trophic factor overview is shown in **Table 3**. These growth factors stimulate the development of fibroblasts, endothelial cells, and tissue progenitor cells [86]. It is credible to state that the use of MSCs and their potential in immunomodulation in regenerative medicine applications hold great promise [87].

#### **5.5 MSC growth factor activity**

In order for MSCs to differentiate into several cell lineages, the action of specific growth factors and chemical mediators are needed in these processes [88, 89].

#### **Figure 11.**

*MSC trophic mechanisms. After bone marrow cell injections, MSCs produce a variety of trophic factors impacting healing cascades by reducing cell apoptosis, fibrosis, and inflammation. Furthermore, by acting on cell proliferation cascades, they contribute to differentiation and mobilization of cells. MSC paracrine trophic factors are potentially important in maintaining endothelial integrity and promoting angiogenesis and the secretion of various growth factors and reparative cytokines.*


**21**

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use…*

Once MSCs are mobilized, or after BM tissue injections, they produce a number of trophic factors that impact healing responses. At a local tissue level, they act by reducing cell apoptosis, fibrosis, inflammation, and activation of cascades that lead to cell proliferation and differentiation, mobilization of cells, and an onset of angiogenesis via paracrine and autocrine pathways [90]. Crucial agents involved in these processes include a variety of growth factors. The MSC trophic effects are associated with the secretion of reparative cytokines and growth factors [91], which contribute finally to tissue repair of inflamed and degenerated tissues, retaining positive MSC paracrine effects [92]. Many of the MSC growth factors are generated on the principle of the cell regulating protein nuclear factor-κB activation, after an initial exposure to pro-inflammatory stimuli such as IFN-γ, TNF-α, and IL-1β or even hypoxia [93]. These factors most likely coexist in prepared MSC-containing BM vials and delivered at tissue injury sites. In this situation, MSC growth factors and other cell mediators may have the potential to exert their specific activities via molecular interplays and subsequently promote optimal MSC-associated therapeutic tissue healing, in particular in a highly concentrated environment [94]. The endothelial monolayer barrier function of tissue capillary beds is often disturbed under degenerative and inflamed conditions, allowing for the blood to release proteins and white blood cells, while MSCs produce and release growth factors that affect endothelial cell and subsequently promote the development of tissue progenitor cells and fibroblasts and support tissue regeneration and repair [95]. Some clinicians combine platelet-rich plasma concentrations [96] with BM products in order to have a more biologically active graft, projected to optimize regenerative medicine treatment outcomes. However, it is important to comprehend the detailed mechanisms underlying the inflammation-modulated production of growth factors by MSCs, as this will provide a better perspective for the clinical application of

*DOI: http://dx.doi.org/10.5772/intechopen.91310*

MSCs or their paracrine factors in tissue regeneration.

MSC paracrine trophic factors are potentially important in maintaining endothelial integrity and promoting angiogenesis through their ability to regulate endothelial cell proliferation and ECM production [97]. Furthermore, endothelial cell permeability is reduced, and MSCs inhibit interactions between leukocytes and endothelial cells [98]. Apart from MSC trophic factors, fibroblasts have fundamental functions in maintaining tissue integrity and promote tissue healing through their secretion of cytokines that support ECM building. These endothelial and angiogenetic capabilities have been demonstrated in clinical studies addressing chronic wound healing [99] and recovery from postmyocardial infarction [100].

An enduring problem in the field of cell-based regenerative medicine therapies is the factual delivery of the harvested and prepared cells to the site of injury, a process termed "homing" [101]. One of the major characteristics of MSCs after administration is that they are able to migrate to sites of inflammation and tissue damage, which are typically associated with cytokine outburst [102]. Homing mechanism to degenerated and injured tissue sites are influenced by factors like age, cell viability, the number of available cells (dosage), and the delivery method. Unlike the well-characterized phenomenon of leukocyte homing by de novo, or exogenously delivered (BM) MSCs, is still unclear. Evidently, an increase in leukocyte migration, with induced rolling response to inflamed tissue sites, has been noted by engineered MSCs [103]. For successful cell-based regenerative therapies,

**5.6 MSCs and angiogenesis**

**5.7 Homing and migration**

#### **Table 3.**

*Growth and trophic factors contributing to MSC tissue regenerative processes.*

*The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use… DOI: http://dx.doi.org/10.5772/intechopen.91310*

Once MSCs are mobilized, or after BM tissue injections, they produce a number of trophic factors that impact healing responses. At a local tissue level, they act by reducing cell apoptosis, fibrosis, inflammation, and activation of cascades that lead to cell proliferation and differentiation, mobilization of cells, and an onset of angiogenesis via paracrine and autocrine pathways [90]. Crucial agents involved in these processes include a variety of growth factors. The MSC trophic effects are associated with the secretion of reparative cytokines and growth factors [91], which contribute finally to tissue repair of inflamed and degenerated tissues, retaining positive MSC paracrine effects [92]. Many of the MSC growth factors are generated on the principle of the cell regulating protein nuclear factor-κB activation, after an initial exposure to pro-inflammatory stimuli such as IFN-γ, TNF-α, and IL-1β or even hypoxia [93]. These factors most likely coexist in prepared MSC-containing BM vials and delivered at tissue injury sites. In this situation, MSC growth factors and other cell mediators may have the potential to exert their specific activities via molecular interplays and subsequently promote optimal MSC-associated therapeutic tissue healing, in particular in a highly concentrated environment [94]. The endothelial monolayer barrier function of tissue capillary beds is often disturbed under degenerative and inflamed conditions, allowing for the blood to release proteins and white blood cells, while MSCs produce and release growth factors that affect endothelial cell and subsequently promote the development of tissue progenitor cells and fibroblasts and support tissue regeneration and repair [95]. Some clinicians combine platelet-rich plasma concentrations [96] with BM products in order to have a more biologically active graft, projected to optimize regenerative medicine treatment outcomes. However, it is important to comprehend the detailed mechanisms underlying the inflammation-modulated production of growth factors by MSCs, as this will provide a better perspective for the clinical application of MSCs or their paracrine factors in tissue regeneration.

#### **5.6 MSCs and angiogenesis**

*Regenerative Medicine*

**Figure 11.**

**Growth factor/cytokine Activity in MSC regenerative repair**

*MSC trophic mechanisms. After bone marrow cell injections, MSCs produce a variety of trophic factors impacting healing cascades by reducing cell apoptosis, fibrosis, and inflammation. Furthermore, by acting on cell proliferation cascades, they contribute to differentiation and mobilization of cells. MSC paracrine trophic factors are potentially important in maintaining endothelial integrity and promoting angiogenesis and the* 

Tissue regeneration

Neurogenesis

Wound healing

Tissue repair

Wound healing

Intrinsic stem cell survival Tissue regeneration Neurogenesis

Intrinsic neural cell regeneration

Epidermal growth factor Wound healing

*secretion of various growth factors and reparative cytokines.*

Fibroblast growth factor Tissue repair

Hepatocyte growth factor Vasculogenesis

Insulin-like growth factor Wound healing

Keratinocyte growth factor Wound healing Platelet-derived growth factor Tissue repair Transforming growth factor beta Wound healing Vascular endothelial growth factor Angiogenesis

Angiopoietin-1 Angiogenesis

Erythropoietin Angiogenesis Interleukin-8 Wound healing Stem cell-derived factor-1 Neuroprotective effect

*Growth and trophic factors contributing to MSC tissue regenerative processes.*

**20**

**Table 3.**

MSC paracrine trophic factors are potentially important in maintaining endothelial integrity and promoting angiogenesis through their ability to regulate endothelial cell proliferation and ECM production [97]. Furthermore, endothelial cell permeability is reduced, and MSCs inhibit interactions between leukocytes and endothelial cells [98]. Apart from MSC trophic factors, fibroblasts have fundamental functions in maintaining tissue integrity and promote tissue healing through their secretion of cytokines that support ECM building. These endothelial and angiogenetic capabilities have been demonstrated in clinical studies addressing chronic wound healing [99] and recovery from postmyocardial infarction [100].

#### **5.7 Homing and migration**

An enduring problem in the field of cell-based regenerative medicine therapies is the factual delivery of the harvested and prepared cells to the site of injury, a process termed "homing" [101]. One of the major characteristics of MSCs after administration is that they are able to migrate to sites of inflammation and tissue damage, which are typically associated with cytokine outburst [102]. Homing mechanism to degenerated and injured tissue sites are influenced by factors like age, cell viability, the number of available cells (dosage), and the delivery method. Unlike the well-characterized phenomenon of leukocyte homing by de novo, or exogenously delivered (BM) MSCs, is still unclear. Evidently, an increase in leukocyte migration, with induced rolling response to inflamed tissue sites, has been noted by engineered MSCs [103]. For successful cell-based regenerative therapies,

it is critically important for MSCs to control cell adhesion in the ECM of the treated tissue. This will occur through the expression of fibronectin and specific integrin and selectin protein adhesion molecules, which are binding to collagen and fibrin ECM components [102], initiating tissue healing and regeneration through cell adhesion, cell growth, migration, and differentiation [104]. The migration ability of MSCs is further controlled by a wide range of growth factors, acting under the receptor tyrosine kinase signaling principle [105], once more illustrating the importance and presence of platelets and their growth factors in the collected BM vial. Furthermore, the administration of MSCs via various delivery routes (intravenous, intraperitoneal, intra-arterial, in situ injections) seems to have an effect on MSC homing [66].
