**3. The use of alginate hydrogels and MSCs in tissue engineering and regenerative medicine applications**

Due to the lack of bioactive molecules on the alginate structure, alginate hydrogels used for cell-based applications require functionalization with molecules which can aid cell adhesion, increase cellular proliferation, and/or guide stem cell differentiation toward the desired cell lineages. In an attempt to increase cell adhesion on alginate hydrogels, a wide variety of extracellular matrix proteins or protein fragments have been employed. The most commonly used molecules include collagen, gelatin (product of collagen hydrolysis), and arginylglycylaspartic acid (RGD) peptide, which is the functional adhesion sequence in several extracellular matrix (ECM) proteins. Gelatin has been widely utilized for the enhancement of cell adhesion and differentiation in alginate hydrogels [24] either mixed [25] or crosslinked with alginate [26]. It has also been shown that crosslinking of alginate with gelatin reduces gelatin leak over prolonged culture while enhancing cell adhesion and vascular endothelial growth factor (VEGF) secretion compared to natural alginate and RGD-alginate [27].

Oxidized alginate has been widely used for tissue engineering purposes. Alginate can be oxidized with the use of agents including sodium permanganate (KMnO4) and periodate, to produce two free aldehyde groups on the alginate backbone, offering enhanced in vitro and in vivo. Alginate oxidation is necessitated by the lack of natural alginate degrading enzymes in mammals, which is translated to a slower biodegradation of alginate hydrogels [28, 29]. Additionally, free aldehyde groups offer sites for possible crosslinking with amine group-containing molecules, which can be used for the robust functionalization of hydrogels used for tissue engineering [29, 30]. Similarly to natural alginate, a wide range of biomolecules have been used for the functionalization of oxidized alginate. The most commonly used are gelatin and RGD, which have been shown promote cell adhesion and viability [31] .

#### **3.1 In vitro paradigms of alginate/MSC constructs**

Culture of MSCs in alginate hydrogels has been attempted for applications ranging from the regeneration of bone, cartilage, and tendon to the repair of damaged myocardium and trachea. Most of the initial data on the use of alginate/MSC constructs have been obtained in vitro (**Table 1**).

*Alginates - Recent Uses of This Natural Polymer*

**2. Mesenchymal stem cells (MSCs) for tissue engineering**

MSCs are multipotent stem cells with the ability to proliferate and differentiate into a variety of mature cells, mainly osteocytes, chondrocytes, and adipocytes [5]. MSCs can be isolated from a multitude of adult and fetal tissues including but not limited to the bone marrow, adipose tissue, peripheral blood, synovial tissue, placenta, Wharton's jelly, and umbilical cord blood. Importantly, it has been shown that MSCs isolated from different tissue sources possess differential proliferation and differentiation capacity toward various lineages [6] (**Figure 1**). Since their description by Friedenstein et al. [7], MSCs have been evolved as the stem cell type with the most regenerative medicine applications and the only stem cell type used in clinic to date. MSCs represent attractive stem cell candidates for the use in tissue engineering and regenerative medicine applications for a variety of reasons. Firstly, they have the ability to proliferate and differentiate producing tissues, which are clinically relevant for regenerative medicine purposes such as musculoskeletal and neural tissues. In addition, they offer the possibility of autologous use, which can avoid adverse immune responses to allogeneic cells while also possessing an immunomodulatory capacity being able to regulate the immune environment even when implanted in an allogeneic fashion. Finally, the use of MSCs avoids the ethical shortcomings of embryonic stem cell (ESC) use and is not associated with the formation of teratomas which is a characteristic of pluripotent stem cell implantation (ESCs and induced pluripotent stem cells—iPSCs) [8–10]. Recently, protocols for the derivation of MSCs from iPSCs have also enabled the production of unlimited MSC

**66**

**Figure 1.**

*Tissue sources and properties of mesenchymal stem cells.*

#### *Alginates - Recent Uses of This Natural Polymer*

Functionalization of alginate has been achieved with molecules mimicking the ECM, the most commonly used of which are RGD and gelatin. RGD has been used to increase adhesion in photo cross-linked alginate hydrogels which were found to maintain viability and promote proliferation of bone marrow MSCs [32] and muscle differentiation of umbilical cord MSCs in alginate-fibrin hydrogels [33]. Tyramine has been also cross-linked to alginate to increase MSC adhesion [34]. In hydrogels functionalized with RGD, it has been shown that high cell density favors cell-cell contact and promotes osteogenic differentiation [35] as well as increasing survival and VEGF secretion from MSC spheroids [36]. The combination of RGD with a matrix metalloproteinase cleavable peptide (proline-valine-glycine-leucine-isoleucine-glycine) in alginate has been shown to promote adhesion and allow better


**69**

*The Use of Alginate Hydrogels for the Culture of Mesenchymal Stem Cells (MSCs): In Vitro and…*

elongation of MSCs than RGD alginate [37]. Increased chondrogenesis has been also demonstrated with the incorporation of fucoidan (a heparan sulfate analogue) in alginate hydrogels seeded with bone marrow MSCs [38]. Glycine-histidine-lysine (GHK), a tripeptide fragment 0f osteonectin (a bone ECM protein), has been cross-linked with natural alginate and oxidized alginate achieving enhanced VEGF secretion from bone marrow MSCs [39] and increased osteogenic differentiation of umbilical cord blood MSCs compared to oxidized alginate with gelatin [20]. Finally, functionalization of alginate with RGD has been shown to promote adipose tissue MSC chondrogenesis via integrin-dependent transforming growth factor (TGF)-β3

One of the most common applications of alginate/MSC constructs is for cartilage tissue engineering. It has been shown that cells differentiated to chondroblasts in alginate hydrogels produce more collagen type II than in monolayer where they predominantly produce collagen type I [41]. In addition, photocrosslinked alginate/ hyaluronic acid injectable hydrogels have been shown to support the chondrogenic differentiation of bone marrow MSCs for cartilage tissue engineering [42]. Alginate hydrogels have been also combined with synovial MSCs showing chondrogenic gene expression and collagen type II deposition under the effect of bone morphogenetic protein-2 (BMP-2). However, the authors noted that full progression of chondrogenesis was not feasible [43]. Interestingly enough when applied to bone marrow MSCs in RGD-alginate hydrogels, BMP-2 has promoted osteogenic differentiation

Several studies have demonstrated the suitability of alginate hydrogels in combination with MSCs for bone tissue engineering. Sodium alginate (SA)/

hydroxyethylcellulose (HEC)/hydroxyapatite (HA) hydrogels have been combined with bone marrow MSCs for bone tissue engineering maintaining high cell viability and proliferation [45]. Alginate-gelatin-hydroxyapatite [46] and oxidized alginategelatin-N-succinyl chitosan hydrogels [47] have been shown to promote the osteogenic differentiation of bone marrow MSCs. Injectable hydrogels have been also tested for the repair of bone defects such as alginate-calcium phosphate [48] and alginate-fibrin hydrogels [49] combined with umbilical cord MSCs. Such materials enable the direct injection of the hydrogel paste in a bone defect and have been shown to promote osteogenic differentiation of MSCs facilitating fracture healing. Hydroxyapatite (calcium-deficient) and RGD have also been combined with alginate for cartilage regeneration showing that placental MSCs could perform better chondrogenesis than bone marrow MSCs [50]. However, RGD-functionalized alginate has been also shown to enhance osteogenic differentiation, mineralization, and viability [51, 52]. Oxidized alginate hydrogels have been also widely utilized for bone tissue engineering. It has been cross-linked with fibrin achieving high cell viability and osteogenic differentiation of Wharton's jelly MSCs compared to plain natural and oxidized alginate [49]. Sarker and co-workers have described the crosslinking of oxidized alginate with gelatin hydrogels for bone regeneration, demonstrating enhanced osteogenesis of adipose tissue and increase of VEGF secretion from MG-63 osteosarcoma cells compared to plain alginate and RGDfunctionalized alginate [27, 53]. Other groups have also confirmed the suitability of oxidized alginate for the osteogenic differentiation of adipose-derived MSCs [53]

Apart from bone and cartilage regeneration, alginate hydrogels have found a limited number of other applications such as the regeneration of nucleus pulposus of the intervertebral disk, the cryopreservation of MSCs, and the three-dimensional printing of cellularized structures. Specifically, alginate hydrogels outperform chitosan hydrogels in glycosaminoglycan deposition and the production of collagen type II for nucleus pulposus engineering [55]. In addition, they have been

*DOI: http://dx.doi.org/10.5772/intechopen.88020*

showing that it favors osteogenic differentiation [44].

and muscle differentiation of Wharton's jelly MSCs [54].

activation [40].

#### **Table 1.**

*Representative in vitro studies combining alginate-based hydrogels with MSCs.*

#### *The Use of Alginate Hydrogels for the Culture of Mesenchymal Stem Cells (MSCs): In Vitro and… DOI: http://dx.doi.org/10.5772/intechopen.88020*

elongation of MSCs than RGD alginate [37]. Increased chondrogenesis has been also demonstrated with the incorporation of fucoidan (a heparan sulfate analogue) in alginate hydrogels seeded with bone marrow MSCs [38]. Glycine-histidine-lysine (GHK), a tripeptide fragment 0f osteonectin (a bone ECM protein), has been cross-linked with natural alginate and oxidized alginate achieving enhanced VEGF secretion from bone marrow MSCs [39] and increased osteogenic differentiation of umbilical cord blood MSCs compared to oxidized alginate with gelatin [20]. Finally, functionalization of alginate with RGD has been shown to promote adipose tissue MSC chondrogenesis via integrin-dependent transforming growth factor (TGF)-β3 activation [40].

One of the most common applications of alginate/MSC constructs is for cartilage tissue engineering. It has been shown that cells differentiated to chondroblasts in alginate hydrogels produce more collagen type II than in monolayer where they predominantly produce collagen type I [41]. In addition, photocrosslinked alginate/ hyaluronic acid injectable hydrogels have been shown to support the chondrogenic differentiation of bone marrow MSCs for cartilage tissue engineering [42]. Alginate hydrogels have been also combined with synovial MSCs showing chondrogenic gene expression and collagen type II deposition under the effect of bone morphogenetic protein-2 (BMP-2). However, the authors noted that full progression of chondrogenesis was not feasible [43]. Interestingly enough when applied to bone marrow MSCs in RGD-alginate hydrogels, BMP-2 has promoted osteogenic differentiation showing that it favors osteogenic differentiation [44].

Several studies have demonstrated the suitability of alginate hydrogels in combination with MSCs for bone tissue engineering. Sodium alginate (SA)/ hydroxyethylcellulose (HEC)/hydroxyapatite (HA) hydrogels have been combined with bone marrow MSCs for bone tissue engineering maintaining high cell viability and proliferation [45]. Alginate-gelatin-hydroxyapatite [46] and oxidized alginategelatin-N-succinyl chitosan hydrogels [47] have been shown to promote the osteogenic differentiation of bone marrow MSCs. Injectable hydrogels have been also tested for the repair of bone defects such as alginate-calcium phosphate [48] and alginate-fibrin hydrogels [49] combined with umbilical cord MSCs. Such materials enable the direct injection of the hydrogel paste in a bone defect and have been shown to promote osteogenic differentiation of MSCs facilitating fracture healing. Hydroxyapatite (calcium-deficient) and RGD have also been combined with alginate for cartilage regeneration showing that placental MSCs could perform better chondrogenesis than bone marrow MSCs [50]. However, RGD-functionalized alginate has been also shown to enhance osteogenic differentiation, mineralization, and viability [51, 52]. Oxidized alginate hydrogels have been also widely utilized for bone tissue engineering. It has been cross-linked with fibrin achieving high cell viability and osteogenic differentiation of Wharton's jelly MSCs compared to plain natural and oxidized alginate [49]. Sarker and co-workers have described the crosslinking of oxidized alginate with gelatin hydrogels for bone regeneration, demonstrating enhanced osteogenesis of adipose tissue and increase of VEGF secretion from MG-63 osteosarcoma cells compared to plain alginate and RGDfunctionalized alginate [27, 53]. Other groups have also confirmed the suitability of oxidized alginate for the osteogenic differentiation of adipose-derived MSCs [53] and muscle differentiation of Wharton's jelly MSCs [54].

Apart from bone and cartilage regeneration, alginate hydrogels have found a limited number of other applications such as the regeneration of nucleus pulposus of the intervertebral disk, the cryopreservation of MSCs, and the three-dimensional printing of cellularized structures. Specifically, alginate hydrogels outperform chitosan hydrogels in glycosaminoglycan deposition and the production of collagen type II for nucleus pulposus engineering [55]. In addition, they have been

*Alginates - Recent Uses of This Natural Polymer*

**First author [reference]**

Tohamy KM [45]

Weber M [41]\*\*\*

Kolambkar Y [64]

Karunanithi P [38]

Klontzas ME [20]

Bernhardt A [46]

Functionalization of alginate has been achieved with molecules mimicking the ECM, the most commonly used of which are RGD and gelatin. RGD has been used to increase adhesion in photo cross-linked alginate hydrogels which were found to maintain viability and promote proliferation of bone marrow MSCs [32] and muscle differentiation of umbilical cord MSCs in alginate-fibrin hydrogels [33]. Tyramine has been also cross-linked to alginate to increase MSC adhesion [34]. In hydrogels functionalized with RGD, it has been shown that high cell density favors cell-cell contact and promotes osteogenic differentiation [35] as well as increasing survival and VEGF secretion from MSC spheroids [36]. The combination of RGD with a matrix metalloproteinase cleavable peptide (proline-valine-glycine-leucine-isoleucine-glycine) in alginate has been shown to promote adhesion and allow better

**Year Type of hydrogel MSC type**

2002 Alginate C3H10T1/2 MSC cell

2007 Alginate Amniotic fluid MSCs

2016 Alginate-fucoidan Bone marrow MSCs

2019 Oxidized alginate-GHK Umbilical cord blood

2009 Alginate-gelatin-HA Bone marrow MSCs

Bone marrow MSCs

line

Placental MSCs and bone marrow MSCs

adipose MSCs

MSCs

Park Y [43] 2005 Alginate Synovial MSCs Coates EE [42] 2013 Methacrylated alginate-HA Bone marrow MSCs

(HEC)/hydroxyapatite (HA)

hydroxyapatite/RGD

Schütz K [58] 2017 Alginate/methylcellulose Bone marrow MSCs

Liu J [33] 2012 Alginate-fibrin-RGD Umbilical cord MSCs Du W-J [65] 2016 Alginate-HA Bone marrow and

Straccia M [66] 2015 Alginate-chitosan Bone marrow MSCs Maia F [35] 2014 Alginate-RGD Bone marrow MSCs Huang J [59] 2016 Alginate-gelatin-carboxymethyl chitosan Bone marrow MSCs

Jose S [39] 2014 Alginate-GHK Bone marrow MSCss Sarker B [53] 2017 Oxidized alginate-gelatin Adipose tissue MSCs

Wang Y [47] 2014 Oxidized alginate-gelatin-N-succinyl chitosan Bone marrow MSCs Zhao L [48] 2010 Alginate-calcium phosphate Umbilical cord MSCs Zhou H [49] 2011 Alginate-fibrin Umbilical cord MSCs

*Representative in vitro studies combining alginate-based hydrogels with MSCs.*

Yeatts A [60] 2011 Alginate Bone marrow MSCs Wang M [61] 2016 Alginate-HA Bone marrow MSCs Chen B [56] 2013 Strontium crosslinked alginate Bone marrow MSCs

2018 Sodium alginate (SA)/hydroxyethylcellulose

Hsu S [50] 2011 Alginate/nano-sized calcium-deficient

**68**

**Table 1.**

used for the cryopreservation of MSCs avoiding minimizing the effects of freezing and thawing on stem cell viability [56], and various formulations of alginate such as oxidized alginate-gelatin [57], alginate/methylcellulose [58], and alginategelatin-carboxymethyl chitosan [59] have been found to be suitable for 3D printing applications.

Finally, it should be mentioned that there is a constantly increasing use of dynamic bioreactor cultures for the cultivation of alginate/MSC constructs. For example, dynamic perfusion bioreactor cultures of bone marrow MSCs in alginate hydrogels have been shown to enhance early in vitro osteogenic commitment and late osteogenesis [60, 61], and dynamic cultures incorporating compression forces have been used for chondrogenic differentiation purposes [62].

Despite the encouraging in vitro results, it needs to be noted that in vitro data do not necessarily correlate to the efficiency of hydrogels in vivo. As shown by Yang et al. who performed a direct in vitro*-*in vivo comparison of differentiation in alginate-gelatin hydrogels with MSCs, subcutaneous implantation in mice inhibits tri-lineage differentiation despite the efficient in vitro differentiation [63]. These results highlight the fact that caution is needed when extrapolating in vitro results to the in vivo setting.

#### **3.2 In vivo paradigms of alginate/MSC constructs**

Various types of alginate hydrogels have been shown to promote bone healing in animal models (**Table 2**). Injectable materials such as chitosan-alginate-BMP-2 and alginate-hydroxyapatite (HA)-mineralized microsphere combinations have been used in conjunction with MSCs to promote bone healing in vivo, demonstrating the efficient formation of trabecular bone [67, 68]. When used for bone tissue engineering, alginate hydrogels are usually seeded with MSCs and are allowed to gradually obtain higher mechanical stability as a result of ECM deposition and mineralization. However, tough alginate hydrogels have been also developed in order to achieve high mechanical stability which has been shown to promote bone healing [69]. Additionally, animal experiments have shown that when RGD is used for alginate modification, faster stress relaxation of alginate hydrogels [70] and high peptide density are linked to more efficient osteogenic differentiation than low peptide density which was linked to cell migration [71]. This correlates with results showing that increasing RGD concentrations inhibit chondrogenic differentiation in vitro [72]. Rottensteiner et al. utilized oxidized alginate-gelatin-nano-Bioglass hydrogels for bone regeneration identifying evidence of in vivo vascularization without adverse reactions, despite the cytotoxic action of Bioglass in vitro [73]. Additionally, Paul et al. successfully treated critical size calvarial defects with serum-loaded oxidized alginate-gelatin-biphasic calcium phosphate hydrogels with rat BM MSCs [74]. Importantly, encapsulation of MSCs in oxidized and natural alginate hydrogels increases vascularization which is of utmost importance in bone tissue engineering and the repair of vascular lesions [75] such as hind limb ischemia [76].

The ability of alginate hydrogels with MSCs to repair cartilage defects in animal models has been demonstrated in a variety of studies with various MSC types and hydrogel formulations. Chung et al. have compared a variety of hydrogel formulations including alginate, HA, chitosan, pluronic, and combinations of them seeded with umbilical cord blood MSCs. Their results demonstrated that even though alginate mixed with pluronic and chitosan achieved a certain degree of healing in rat knee cartilage defects, it was 4% hyaluronic acid which resulted in the optimal cartilage repair with macroscopic and microscopic appearance of adjacent healthy cartilage [77]. High-quality repair of in vivo rabbit cartilage defects has been shown with the use of

**71**

*The Use of Alginate Hydrogels for the Culture of Mesenchymal Stem Cells (MSCs): In Vitro and…*

Yu J [85] 2010 Alginate-RGD Bone marrow MSCs Myocardial

Leijs M [91] 2017 Alginate Bone marrow MSCs Inflammatory

Wang S [90] 2016 Alginate Umbilical cord MSCs Skin wound healing

Re'em T [84] 2012 Alginate with TGF-β1 Bone marrow MSCs Cartilage

Schon LC [88] 2014 Alginate Bone marrow MSCs Tendon

2012 Alginate Adipose MSCs and

2018 Alginate-cyclic RGD Bone marrow MSCs Cardiac

chondrocytes

Gingival and periodontal MSCs

Zhang F [81] 2012 Alginate Co-culture of synovial

porous scaffolds

alginate-gelatin

with pluronic, HA, and chitosan

BMP-2

methacrylated alginate-RGD

TGF-β3

mineralized microspheres

2014 RGD-alginate with

*Representative in vivo studies combining alginate-based hydrogels with MSCs.*

2014 Oxidized alginate with nano-Bioglass®

**Year Type of hydrogel MSC type Application**

MSCs with transgenic chondrocytes

Cartilage regeneration

regeneration

multiple tissues

diseases

regeneration

regeneration

regeneration

regeneration

Tracheal repair

Tendon regeneration

Bone marrow MSCs Regeneration of

Bone marrow MSCs Vascularization

Bone marrow MSCs Bone regeneration

Bone marrow MSCs Bone regeneration

Bone marrow MSCs Bone regeneration

Bone marrow MSCs Bone regeneration

Umbilical cord blood Cartilage

bone marrow MSCs and natural alginate [78]. Alginate has been also combined with polylactic acid to promote in vivo cartilage repair with bone marrow MSCs [79]. In vivo cartilage differentiation in alginate hydrogels has been also attempted for the repair of tracheal tissue with the combination of adipose tissue MSCs and chondrocytes [80]. Synovial MSCs have also been co-cultured with chondrocytes transgenic for TGF-β3 in alginate hydrogels, demonstrating that TGF-β3 release can induce synovial MSC chondrogenesis [81]. The simultaneous activation of TGF-β3 and BMP-2 genes in MSC laden alginate hydrogels showed superior chondrogenesis compared to the isolated delivery of each one of the factors where cells progressed to endochondral osteogenesis instead of chondrogenesis [82]. Interestingly, alginate was found more capable in promoting endochondral osteogenesis than chondrogenesis when compared to chitosan [83].

*DOI: http://dx.doi.org/10.5772/intechopen.88020*

Yang C [63] 2009 Alginate-gelatin

Steiner D [92] 2018 Oxidized

Chung J [77] 2014 Alginate combined

Park D [67] 2005 Alginate-chitosan-

Ho SS [93] 2016 Oxidized

Ingavle GC [68] 2019 Alginate-HA-

**First author [reference]**

Rottensteiner [73]

Sondermeijer H

Hashemibeni B

Moshaverinia A [89]

[86]

[80]

**Table 2.**


*The Use of Alginate Hydrogels for the Culture of Mesenchymal Stem Cells (MSCs): In Vitro and… DOI: http://dx.doi.org/10.5772/intechopen.88020*

#### **Table 2.**

*Alginates - Recent Uses of This Natural Polymer*

applications.

to the in vivo setting.

used for the cryopreservation of MSCs avoiding minimizing the effects of freezing and thawing on stem cell viability [56], and various formulations of alginate such as oxidized alginate-gelatin [57], alginate/methylcellulose [58], and alginategelatin-carboxymethyl chitosan [59] have been found to be suitable for 3D printing

Finally, it should be mentioned that there is a constantly increasing use of dynamic bioreactor cultures for the cultivation of alginate/MSC constructs. For example, dynamic perfusion bioreactor cultures of bone marrow MSCs in alginate hydrogels have been shown to enhance early in vitro osteogenic commitment and late osteogenesis [60, 61], and dynamic cultures incorporating compression forces

Despite the encouraging in vitro results, it needs to be noted that in vitro data do not necessarily correlate to the efficiency of hydrogels in vivo. As shown by Yang et al. who performed a direct in vitro*-*in vivo comparison of differentiation in alginate-gelatin hydrogels with MSCs, subcutaneous implantation in mice inhibits tri-lineage differentiation despite the efficient in vitro differentiation [63]. These results highlight the fact that caution is needed when extrapolating in vitro results

Various types of alginate hydrogels have been shown to promote bone healing in animal models (**Table 2**). Injectable materials such as chitosan-alginate-BMP-2 and alginate-hydroxyapatite (HA)-mineralized microsphere combinations have been used in conjunction with MSCs to promote bone healing in vivo, demonstrating the efficient formation of trabecular bone [67, 68]. When used for bone tissue engineering, alginate hydrogels are usually seeded with MSCs and are allowed to gradually obtain higher mechanical stability as a result of ECM deposition and mineralization. However, tough alginate hydrogels have been also developed in order to achieve high mechanical stability which has been shown to promote bone healing [69]. Additionally, animal experiments have shown that when RGD is used for alginate modification, faster stress relaxation of alginate hydrogels [70] and high peptide density are linked to more efficient osteogenic differentiation than low peptide density which was linked to cell migration [71]. This correlates with results showing that increasing RGD concentrations inhibit chondrogenic differentiation in vitro [72]. Rottensteiner et al. utilized oxidized alginate-gelatin-nano-Bioglass hydrogels for bone regeneration identifying evidence of in vivo vascularization without adverse reactions, despite the cytotoxic action of Bioglass in vitro [73]. Additionally, Paul et al. successfully treated critical size calvarial defects with serum-loaded oxidized alginate-gelatin-biphasic calcium phosphate hydrogels with rat BM MSCs [74]. Importantly, encapsulation of MSCs in oxidized and natural alginate hydrogels increases vascularization which is of utmost importance in bone tissue engineering and the repair of vascular lesions

The ability of alginate hydrogels with MSCs to repair cartilage defects in animal models has been demonstrated in a variety of studies with various MSC types and hydrogel formulations. Chung et al. have compared a variety of hydrogel formulations including alginate, HA, chitosan, pluronic, and combinations of them seeded with umbilical cord blood MSCs. Their results demonstrated that even though alginate mixed with pluronic and chitosan achieved a certain degree of healing in rat knee cartilage defects, it was 4% hyaluronic acid which resulted in the optimal cartilage repair with macroscopic and microscopic appearance of adjacent healthy cartilage [77]. High-quality repair of in vivo rabbit cartilage defects has been shown with the use of

have been used for chondrogenic differentiation purposes [62].

**3.2 In vivo paradigms of alginate/MSC constructs**

[75] such as hind limb ischemia [76].

**70**

*Representative in vivo studies combining alginate-based hydrogels with MSCs.*

bone marrow MSCs and natural alginate [78]. Alginate has been also combined with polylactic acid to promote in vivo cartilage repair with bone marrow MSCs [79]. In vivo cartilage differentiation in alginate hydrogels has been also attempted for the repair of tracheal tissue with the combination of adipose tissue MSCs and chondrocytes [80]. Synovial MSCs have also been co-cultured with chondrocytes transgenic for TGF-β3 in alginate hydrogels, demonstrating that TGF-β3 release can induce synovial MSC chondrogenesis [81]. The simultaneous activation of TGF-β3 and BMP-2 genes in MSC laden alginate hydrogels showed superior chondrogenesis compared to the isolated delivery of each one of the factors where cells progressed to endochondral osteogenesis instead of chondrogenesis [82]. Interestingly, alginate was found more capable in promoting endochondral osteogenesis than chondrogenesis when compared to chitosan [83].

Finally, TGF-β1-releasing alginate hydrogels have been used to promote chondrogenesis of MSCs, demonstrating in vitro increase of chondrogenic markers and healing of articular cartilage defects in mice [84].

Another important application of alginate/MSC constructs is the treatment of myocardial lesions. Yu et al. combined RGD-functionalized alginate hydrogels with human bone marrow MSCs showing that they could improve left ventricular function after myocardial infarction in a rat acute myocardial infarction model [85]. Cyclic RGD in alginate hydrogels has been also shown to promote neoangiogenesis and cardiac neovascularization, improving cardiac function in animals postmyocardial infarction [86]. Finally, when alginate hydrogels are used for cardiac regeneration, it has been shown that G-type alginates possess properties suited for the regeneration of cardiac tissue [87].

MSCs have been combined with alginate hydrogels for tendon repair purposes in animal model of tendon tears. For example, rat Achilles tendon lesions have been treated with hydrogels loaded with MSCs [88] showing healing of higher quality than surgical meshes and sutures. In addition, RGD-functionalized hydrogels loaded with TGF-β3 and loaded with periodontal and gingival MSCs were found to efficiently produce tendon tissue when implanted subcutaneously in mice [89].

Alginate hydrogels have also been widely utilized as wound dressings either alone or in combination with MSCs. For this application, various types of MSCs have been used including umbilical cord MSCs [90] and bone marrow MSCs in alginate-chitosan hydrogels with antibacterial properties [66].

Finally, alginate hydrogels have been used to protect MSCs from the local immune response elicited when allogeneic cells are implanted in vivo. They have been shown to provide protection from the immune system increasing the survival of MSCs in the hostile environment of the host-releasing immunomodulatory factors [91].
