Mesenchymal Stem/Stromal Cells and Hydrogel Scaffolds for Tissue Engineering

Hydrogels are splendid biomaterials and play a critical role in multiple applications for disease management via offering a microenvironment for drug metabolism and exerting the bonding effect attribute to the preferable physical and chemical properties. State-of-the-art renewal has indicated the combination of hydrogels with mesenchymal stem/stromal cells (MSCs), which are heterogeneous populations with unique hematopoietic-supporting and immunoregulatory properties. For decades, we and other investigators have demonstrated the promising prospects of MSCs in regenerative medicine, and in particular, for the administration of recurrent and refractory disease. Very recently, we took advantage of the hydrogel/MSC composite for the applications in osteoarthritis, burn wounds, and refractory wounds associated with diabetic foot as well. Strikingly, the composite showed superiority in continuous improvement of the biological functions of the injured areas over hydrogels or MSCs, respectively. Collectively, hydrogel-based biomaterials are of importance for disease treatment and the accompanied regenerative medicine. Therefore, in this chapter, we will summarize the latest updates of hydrogel/MSCs composite in tissue engineering and put forward the direction of hotspot issues in the future including hydrogel/MSC and hydrogel/MSC-exosome in preclinical and clinical studies.


Introduction
Mesenchymal stem/stromal cells (MSCs) have been acknowledged as medicinal signaling cells as well as the most important niche cells in the microenvironment, and possess advantaged properties such as immunomodulatory capacity, hematopoietic-supporting effect and multi-lineage differentiation potential towards adipocytes, osteoblasts and chondrocytes, which thus hold promising prospects for tissue engineering and regenerative medicine [1][2][3].MSCs were first isolated from bone marrow in the 1960s, and followed by various stromal fractions of adult tissues [4,5], perinatal tissues [6][7][8], and even derived from stem cells [9][10][11].For decades, due to the limitation of unique biomarkers and the wide range of cell sources, MSCs are recognized as heterogeneous populations with great heterogeneity in cellular phenotypes and transcriptome characteristics [12][13][14].Generally, MSCs with diverse origins mainly function via direct-or trans-differentiation, paracrine or autocrine, homing, dual immunomodulation, neovascularization, and constitutive microenvironment [4,15,16].To date, more than 1340 MSC-based clinical trials have been registered for various disease treatment according to the ClinicalTrials.gov website.For instance, we and other investigators have indicated the therapeutic effects of MSCs upon multiple refractory and recurrent disorders including acute graft-versus-host diseases (aGVHD) [17], aplastic anemia [18,19], osteoarthritis [11,20], critical limb ischemia (CLI) [9], acute-on-chronic liver failure (ACLF) [21], Parkinson's syndrome [22], acute myocardial infarction (AMI) [23], rheumatoid arthritis (RA) [24], and coronavirus disease 2019 (COVID-19) [15,25].It's noteworthy that the variation of the therapeutic efficacy of MSCs upon acute liver failure and aGVHD has also been respectively observed by Zhang et al. and us, which further indicated the necessity and urgency of developing tissue engineering including biomaterials, three-dimensional (3D) printing, and MSC-based gene therapy [26,27].
Simultaneously, state-of-the-art updates have further suggested the preferable application of biomaterial/MSC composite as well [11,28,29].Of the current natural extracellular matrices (ECMs), hydrogels have been regarded as the most promising alternative biomaterials attribute to their excellent swelling property and the resemblance to soft tissues [11,30].In particular, synthetic biomimetic hydrogels with appropriate mechanical behavior and predictable biodegradation property can be easily synthesized and modulated for facilitating the biological phenotypes and bioapplications of the encapsulated MSCs such as adhesion, migration, differentiation, proliferation, and apoptosis [30].For instance, Gwon et al. and Huang et al. reported the influence of heparin-hyaluronic acid (HA) hydrogel upon cellular activity and hydrogel scaffolds for the differentiation of adipose-derived stem cells, respectively [30,31].Very recently, we took advantage of the HA hydrogel/MSC composite and demonstrated the reinforced cell vitality of human pluripotent stem cell-derived MSCs (hPSC-MSCs) over monolayer-cultured MSCs for chondrogenesis and the management of osteoarthritis rabbits [11].
Herein, we summarize the current progress in MSCs or MSC-derived exosomes and hydrogel scaffold for tissue engineering, and in particular, the potentially reinforcing or attenuating effects of hydrogel scaffold with unique biochemical and biophysical properties upon the MSC-based cytotherapy for regenerative medicine.

The cell sources of MSCs for tissue engineering
Not until 2006, the International Society for Cellular Therapy (ISCT) defined the preliminary criteria of defining multipotent MSCs including adherent property, multi-lineage differentiation capacities in vitro towards adipocytes, osteoblasts, and chondrocytes, together with high-levels of mesenchymal biomarker expression (CD73, CD90, and CD105) whereas minimal expression of hematopoietic or endothelial markers (CD31, CD34, and CD45) [32].After that, numerous studies aiming at dissecting the similarities and differences in biological phenotypes and biofunctions as well as transcriptome characteristics of MSCs derived from adult tissue, perinatal tissue, and PSCs have been extensively conducted (Table 1).

Adult tissue-derived MSCs
As mentioned above, adult tissue-derived MSCs hold vast prospect in tissue repairing and organ reconstruction [3].To date, massive literatures have reported the isolation and identification of MSCs from various adult tissues such as adipose tissue, bone marrow, synovium, dental pulp, peripheral blood, muscle tendon, and menstrual blood [40,41].According to the ClinicalTrials.govwebsite, a total of 1096 trials have been registered worldwide against disorders such as acute respiratory distress syndrome (ARDS), CLI, AMI, anoxic or hypoxic brain injury, moderate-to-severe Crohn's disease, idiopathic pulmonary fibrosis (IPF), and COVID-19.For example, a phase I interventional trial was led by Dr. Jesus JV Vaquero Crespo in Puerta de Hierro University Hospital was aiming to evaluate the security of local administration of autologous bone marrow-derived MSCs (BM-MSCs) in traumatic injuries of the spinal cord (NCT01909154), which was consistent with another study by Geffner and their colleagues [42].In details, 12 participants received 1 × 10 8 BM-MSCs by intrathecal injection (subarachnoid and intramedullary), and another 3 × 10 7 BM-MSCs by subarachnoid administration after 3 months depending on centromedullary post-traumatic injury.The safety outcomes of the patients were evaluated according to vital signs (ECG, blood pressure, and heart rate) and possibility of adverse reaction (headache, meningeal irritation, and infectious complications).The secondary outcomes were quantized from the view of sensitivity recovery (e.g., surface sensitivity and pain sensitivity), level of chronic pain, neurophysiological parameters, maximum cystometric capacity, and the decrease in volume and hyperintensity of intramedullary lesions.Very recently, Oraee-Yazdani et al. further verified that BM-MSCs in combination with autologous Schwann cell co-transplantation was safe and effective for treating 11 patients of spinal cord injury (SCI), and in particular for spinal cord regeneration during subacute period [43].
Notably, cutting-edge advances have also put forward the variations and limitations of adult tissue-derived MSCs in both preclinical and clinical studies [1,14,44].For example, among the indicated adult tissue-derived MSCs, BM-MSCs are recognized as the widest application in clinical practices whereas with inherent disadvantages such as ethical risk, pathogenic risk, invasive pain, replicative senescence and individual diversity for cell source, and in particular, the limitation in healthy donors and declined long-term ex vivo amplification further restrict the large-scale application in future [11,44].Interestingly, despite the variations in signatures and functions, we recently verified the potential conservative properties in adipose tissue-derived stem cells (AD-MSCs) from type 2 diabetics and healthy donors [4].However, multifaceted diversity among BM-MSCs, AD-MSCs, dental pulp stem cells (DPSCs), and supernumerary teeth-derived apical papillary stem cells (SCAP-Ss) were observed by investigators in the field [16,[45][46][47].

Perinatal tissue-derived MSCs
Perinatal tissues are abundant sources of MSCs and extracellular matrix with a wide range of therapeutic purposes in tissue engineering, which thus act as particularly interesting candidates for regenerative medicine [48,49].To date, a variety of perinatal tissue-derived MSCs have been identified such as placental-derived MSCs (P-MSCs), umbilical cord-derived MSCs (UC-MSCs), cord blood-derived MSCs (CB-MSCs) [49,50], amniotic-derived MSCs (A-MSCs) [51], amniotic fluid-derived MSCs (AF-MSCs) [52], decidua-derived MSCs (D-MSCs) [53], and chorionic villiderive MSCs (CV-MSCs) [54].Of the aforementioned perinatal tissue-derived MSCs, UC-MSCs are promising sources with preferable properties in long-term proliferation in vitro and immunoregulation, and most of all, without ethical risks and limitation in supply, and thus hold great prospect for large-scale clinical investigation and investigational new drug (IND) purposes [18,44].Up to November 11th of 2021, a total of 317 interventional clinical trials have been registered for the administration of numerous refractory diseases by UC-MSC infusion such as diabetic nephropathy, heart failure, perianal fistulas with Crohn's disease, lumbar discogenic pain, chronic obstructive pulmonary disease (COPD), Duchenne muscular dystrophy (DMD), and cerebral hemorrhage sequela (CHS) according to ClinicalTrials.govwebsite.
Similarly, other types of MSC sources are of equal importance in offering "seeds" for tissue engineering and regenerative medicine (e.g., umbilical cord, placenta, amniotic membrane, and amniotic fluid).For instance, Liu and the colleagues took advantage of A-MSCs and conducted intragastric administration and intraperitoneal injection for the management of hydrogen peroxide-induced premature ovarian failure (POF) model.As expected, POF mice with A-MSC transfusion in bilateral ovaries revealed increased estrogen levels, decreased follicle-stimulating hormone level, and evaluated ovarian index and fertility rate, which collectively suggested the ameliorative effects of MSCs in improving the follicular microenvironment and recovering ovarian function in POF [55].

Hydrogels/MSCs scaffolds for tissue engineering
The therapeutic effects of MSC transplantation largely attributed to the paracrine including soluble factors and extracellular vehicles (e.g., exosome and secretome), which could be rapidly sequestered and cleared [80].Hydrogels are ideally suited for MSCs cultivation and are adequate to offer splendid delivery platforms, enhance vehicles retention rates and thus enhance immunomodulation and tissue regeneration after in vivo transplantation (Figure 1, Table 1) [80][81][82].

Hydrogels/MSCs scaffolds for wound healing
Wound healings are regulated by series of events with overlapping phases, which represent an intractable issue in clinical practice [83,84].State-of-the-art renewal has indicated the prospective applications of hydrogel in combination with MSCs or MSC-derived exosomes in skin wound healing, and in particular, the recurrent and refractory cutaneous types (Table 1) [83,85].Of them, chronic refractory wounds are disorders attribute to multifactorial comorbidity with characteristics of inflammation and impaired vascular networks, which eventually result in unfavorable prognosis due to the lack of effective treatments [36,86].Recently, Yang and the colleagues topically applied UC-MSC-derived exosomes encapsulated into the thermosensitive PF-127 hydrogel (hydrogel/MSC-exo) and demonstrated that hydrogel/MSC-exo scaffold significantly upregulated expression of multiple cytokines (e.g., VEGF and TGFβ-1), enhanced regeneration of granulation tissue and accelerated wound closure rate in a streptozotocin-induced diabetic rat model, which was further verified by another study in a streptozotocin-induced diabetic model with hydrogel/AD-MSC composite [33,36] [88].Meanwhile, a full sheet consisting of A-MSCs on thermoresponsive polymers have been considered as advantaged skin substitute for the management of burn wounds [89].Collectively, these studies suggested that hydrogel-based MSC/MSC-exo therapy represent a novel therapeutic approach for refractory cutaneous regeneration of chronic wounds.

Hydrogels/MSCs scaffolds for osteoarticular diseases
Despite the dramatic progress in bone reconstruction, the osteoarticular diseases and bone regeneration in clinical practices are still challenging [82].Hydrogels have been extensively investigated in numerous osteoarticular diseases (e.g., osteoarthritis) and bone regeneration (e.g., craniofacial bone tissue) largely attribute to the high cell compatibility [34].For example, Ji et al. recently combined MSCs with a newly synthesized hybrid scaffold consisting of thermosensitive hydroxypropyl chitin hydrogel (HPCH) and 3D-printed nano-hydroxyapatite (nHA)/poly (ε-caprolactone) (PCL) for bone regeneration.Strikingly, they found the vascularization and osteogenesis and immunomodulation of encapsulated MSCs as well as cytokine secretion of macrophages were collectively orchestrated in bone defect mice model [35].
Osteoarthritis (OA) is recognized as the most prevalent chronic joint disease, which increases in prevalence with age and resultant in functional loss or decline in quality of life, and in particular, act as a major socioeconomic cost worldwide and a leading musculoskeletal cause of impaired mobility in individuals over 65-year-old [90,91].Despite joint replacement is an effective strategy for symptomatic end-stage disease, yet most of the functional outcomes are poor and the lifespans of prostheses are largely limited [91,92].Current studies have shown that MSC-based cytotherapy are promising for osteoarticular disease administration.For example, Portron et al. and Merceron et al. found that the in vivo chondrogenic potential of AD-MSCs encapsulated in a cellulose-based self-setting hydrogel (Si-HPMC) preconditioned by hypoxia (5% oxygen) was significantly enhanced compare to that in the control (20% oxygen) group, which was confirmed by subcutaneous transplantation of AD-MSCs with an injectable hydrogel in rabbits [93,94].Very recently, our group also found that the application of hyaluronic acid (HA) hydrogel/PSC-MSCs and HA hydrogel/ hydroxyapatite/UC-MSC (HA/HAP/UC-MSC) composite with reinforced efficacy upon OA rabbits and mice, respectively (Table 1) [11].It's noteworthy that Chung and their colleagues have systematically explored and detailed dissected the efficacy of articular cartilage repair in vivo by combining UC-MSCs with various hydrogels such as alginate, pluronic, HA, and chitosan.They finally concluded that HA hydrogel/UC-MSC composites resulted in preferable cartilage repair and collagen organization pattern, which were similar to adjacent uninjured articular cartilage [95].Additionally, the gingival MSC-laden photocrosslinkable hydrogels were also confirmed with preferable biocompatibility, biodegradability, and osteoconductivity for craniofacial bone tissue engineering in rat peri-implantitis model as well [34].Taken together, the biodegradable and biocompatible hydrogels can serve as advantaged scaffolds and supply structural integrity for cellular organization and morphogenic guidance of hydrogel scaffold-laden MSCs [88].

Hydrogels/MSCs scaffolds for reproductive diseases
Premature ovarian failure (POF) is a refractory disorder with declined fertility in females [96,97].In 2019, Yang and their colleagues took advantage of collagen scaffold loaded with UC-MSCs (collagen/UC-MSCs) and verified the efficacy in POF mice via increasing estrogen (E2) and ovarian volume, and promoting granulosa cell proliferation and ovarian angiogenesis [96].Similarly, Ding et al. reported the rescue of E2 concentrations and activation of follicles in the dormant ovaries of premature ovarian failure (POF) patients with long history of infertility after transplantation of collagen/UC-MSC scaffold (Table 1) [38].
As to premature ovarian insufficiency (POI), an intractable endocrine disease that severely restricts the reproductive and physiological function of females and resultant in menopausal symptoms, a series of literatures have suggested the ameliorative effect of hydrogel/MSC composite or hydrogel/MSC-derived microvesicles/secretomes via facilitating angiogenesis, enhancing granulosa cell generation and steroidogenesis, and accelerating follicular regeneration [98][99][100][101].Notably, Li et al. have summarized the current renewal of the therapeutic effects and molecular mechanisms of MSC-based cytotherapy in both preclinical research and clinical trials [102].

Hydrogels/MSCs scaffolds for vascular diseases
Peripheral arterial diseases (PAD) are severe medical conditions, which are characterized by blood vascular blockage and low limb Doppler signals and commonly associated with hind-limb ischemia or critical limb ischemia (CLI) [103].For decades, we and other investigators have primarily suggested the therapeutic of MSCs or MSCderived exosomes in hind limb ischemia models by alleviating the severity, promoting angiogenesis, and enhancing immunomodulation [9,104,105].In recently years, a certain number of outstanding researchers turned to injectable hydrogels such as self-assembled Nap-GFFYK-Thiol hydrogel, nitric oxide-releasing hydrogels, and the novel hydrogel composed of pooled platelet lysate (PL) to enhance the efficacy of MSCs or derivations upon peripheral artery diseases (PADs) [37,[106][107][108].For example, Lee et al. found that fucoidan was adequate to improve the bioactivity and vasculogenic potential of MSCs in hind limb ischemia murine with chronic kidney disease (CKD) whereas Nammian et al. further compared the variations of efficacy between BM-MSCs and AD-MSCs for CLI [109,110].Notably, Ding and their colleagues systematically dissected an injectable nanocomposite hydrogel consisting of chitosan, gelatin, β-glycerophosphate and Arg-Gly-Asp (RGD) peptide for potential applications of facilitating vascularization and tissue engineering [111].Collectively, the aforementioned studies suggest that hydrogel/MSC-based composites occupy a greater angiogenic potential over single hydrogel-or MSC-based treatment for PADs.

Hydrogels/MSCs scaffolds for digestive diseases
Gastroparesis is characterized by pyloric dysfunction, vomiting, severe nausea, delayed gastric emptying and impaired fundamental structures, which is related with consume of enteric neurons and interstitial cells of Cajal [112].Meanwhile, stem cell therapy has also been extensively explored in inflammatory bowel diseases (IBDs) including ulcerative colitis (UC) and Crohn's disease (CD) in both preclinical studies and clinical trials [113][114][115].State-of-the-art updates have indicated the mitigatory effects of MSCs in gastrointestinal diseases such as acute ulcerative colitis and perianal CD [8,10,68,116].For example, we recently reported the spatio-temporal metabolokinetics and efficacy of placenta-derived MSCs (P-MSCs) on intractable CD with enterocutaneous fistula in mice via simultaneously accelerating neovascularization and downregulating reactive oxygen species (ROS) [8].Interestingly, Joddar et al. conducted delivery of the MSC-alginate/gelatin/poly-l-lysine hydrogel atop stomach grafts facing the luminal side, and confirmed the significant advance towards the entire tissueengineered "microgels" or "gastric patch" [112].Of note, the therapeutic effects of MSCs via systemic administration are still contradictory largely due to the localization in the lungs, which is confirmed by the outcomes of two clinical trials with BM-MSC transplantation [117,118].Therefore, the local administration of hydrogel/MSC or hydrogel/ MSC-exosomes are promising alternatives for resolving refractory digestive diseases.

Conclusions
Tissue engineering is an inveterate and promising area in the field of regenerative medicine, which also has long-lasting limitations in engineering and regenerating tissues.MSCs of different origins are splendid "seeds" for the efficient administration of various refractory and recurrent diseases.As mentioned above, MSCs as well as the Mesenchymal Stem/Stromal Cells and Hydrogel Scaffolds for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.101793released extracellular vesicles (EVs) reveal substantial therapeutic effect in numerous pathophysiological conditions and potentially reconstructing an extensive range of diseased or damaged tissues and organs in tissue regeneration engineering, which have been predominantly demonstrated from pre-clinical or clinical in vitro and in vivo studies.MSC-encapsulated hydrogel scaffolds demonstrate enhanced cell vitality and committed differentiation, prolonged fundamental and operational consistency, which thus hold promising prospects for tissue engineering and the resultant regenerative medicine.Meanwhile, the exosomes and other nano-scale secretions released from the multivesicular MSCs encapsulated into appropriative hydrogel formulations (e.g., HA, nHAP, PLGA, pDA, and FHE) have manifested higher therapeutic potential in both fundamental research and clinical application.Overall, the current progress of regenerative medicine will extensively benefit from the "advantaged" artificial hydrogel/MSC-based cytotherapy in the near future.

CLI
. Marusina et al. and Xin et al. reported the influence of tunable bio-inert poly (ethylene glycol)-based hydrogels and microporous annealed particle hydrogels on MSCs and the optimization of cell-degradable hydrogels/MSCs delivery for wound re-epithelialization [81, 87].Zhang et al. took advantage of the bioluminescence imaging (BLI) technology and further demonstrated the therapeutic effects of prostaglandin E2 (PGE2) and chitosan (CS) hydrogel (PGE2 + CS hydrogel) in a murine wound healing model via modulating the M1 and M2 paradigms of macrophage activation

Table 1 .
Representative applications of HA/MSC-based scaffold in tissue engineering.