Hydrogel based 3D carriers in the application of stem cell therapy by direct injection

Chengxin Luan; Ping Liu; Runzhe Chen; Baoan Chen

doi:10.1515/ntrev-2017-0115

Article Open Access

Hydrogel based 3D carriers in the application of stem cell therapy by direct injection

Chengxin Luan
Chengxin Luan received his BSc degree at medical school of Southeast University. He is a PhD candidate at Medical School and is studying at State Key Laboratory of Bioelectronics School of Biological Science and Medical Engineering of Southeast University. He is working on photonic crystal techniques and biological materials for medical application.
, Ping Liu
Ping Liu is a PhD candidate at Medical School of Southeast University and is studying at the hematology and oncology department of Zhongda Hospital. She is working on reversal of multidrug resistance of malignancies.
, Runzhe Chen
Runzhe Chen obtained her medical degree from Southeast University. She has published more than 10 SCI papers as first author. Presently, she is the joint PhD student in Southeast University and University of Texas MD Anderson Cancer Center. She is serving as the editor of Advances in Clinical Toxicology and the assistant editor of the Cancer Translational Medicine Journal. She is also the academic and section editor and peer reviewer of several international journals.
and Baoan Chen
Prof. Baoan Chen is a renowned hematologist and oncologist in China. He is the director of Faculty of Oncology of Southeast University, leader of Department of Hematology of the Affiliated Zhongda Hospital of Southeast University, and head of MDS Research Institute. He has rich experience in hematological malignancies’ diagnosis and treatment.

Published/Copyright: April 13, 2017

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From the journal Nanotechnology Reviews Volume 6 Issue 5

Abstract

Compared with systematic administration such as peripheral intravenous infusion, stem cell therapy by direct injection is theoretically more effective, but some technical barriers such as low stem cell retention rate and low engraftment rate still need to be overcome before its application in humans. Stem cell therapy supported by hydrogel carriers has been increasingly studied in recent years. These hydrogels with properties similar to natural tissues are able to fabricate various forms of carriers, which include in situ forming hydrogels, ex situ forming hydrogels, surface immobilization carriers, microencapsules, and microgels. Some of them are 3D carriers and promise to overcome the technical barriers of stem cell therapy by direct injection. They have different characteristics, application, and prospect in the application of stem cell therapy by direct injection, which is summarized by this review.

Keywords: 3D microcarriers; direct injection; hydrogel; stem cell therapy

1 Introduction

Stem cell therapy utilizes the pluripotency of stem cells to repair injuries or to treat various diseases [1], [2], [3], [4]. Compared with systematic administration such as peripheral intravenous infusion, in which most stem cells are stranded at the lungs or blocked by physiologic barriers such as blood-brain barrier and only few or even none of them arrive at target sites such as bone marrow through homing [5], [6], [7], stem cell therapy by direct injection is theoretically more effective [8], and the techniques of direct injection are safe and mature, for example, drug administration by intra-bone marrow injection (commonly used for emergency treatment) and transcatheter arterial chemoembolization of hepatic hepatocellular carcinoma [9], [10], [11]. Moreover, stem cell therapy by direct injection has the advantages of lesser graft versus host disease and faster engraftment [12]. While proven by animal experiments, evidence of stem cell therapy by direct injection in humans is scarce [13]. Besides extra pain to patients and risk of secondary infection, some technical barriers need to be overcome before its regular application in humans. Low cell retention rate can be caused by migration of stem cells from the target site to remote tissues or damage of stem cell membrane by mechanical shear forces during injection [5]. Low engraftment rate may result from a lack of protecting structure to enhance viability and proliferation of the injected stem cells [14]. Generally in practice, to enhance the therapeutic efficacy, a large dose of cells or cellular aggregates is needed to ensure sufficient number of stem cells at the target sites, which not only increases expenses and time but also brings side effects including unexpected and uncontrolled safety events because of nonspecific cell incorporation and undesired influence on the normal tissues [15]. Biological material-assisted drug delivery and cell therapy have been increasingly studied in recent years [16]. A competent carrier can not only protect stem cells from mechanical insults and prevent migration out of target sites, but also provide them favorable growth conditions such as essential growth factor, metabolic waste excretion, and exchange of nutrient substance and oxygen. These stem cell carriers are almost hydrogel based with the properties very similar to natural tissue, and many of them are injectable, therefore, hydrogels are promising to fabricate delivery vehicles for stem cell therapy by direct injection. Hydrogel based carriers are varied according to their configurations and application purpose, and in particular, conflicting terms often exist to define them. In the present review, after referring to the classification of injectable cell-based systems by Bidarra et al. [17], we propose to divide them into three categories: (1) surface immobilization, (2) microencapsulation, and (3) matrix entrapment. As shown in Figure 1, category 1 is not true 3D because the cells are on the surface of the carriers. The remaining, namely, categories 2 and 3, can be real 3D, and in the present review we collectively name them hydrogel based 3D carriers.

Figure 1:

The classification of hydrogel based carriers. 1. Surface immobilization. 2. Microencapsulation. 3 Matrix entrapment: (1) Microgel or other particles. (2) In situ forming hydrogel. A. Before injection. B. After injection. C. In situ forming hydrogel in solution form. D. In situ forming hydrogel in gel form. E. Component chains of in situ forming hydrogel, before cross-linking, they are independent and after they crosslink uniformly with each other. Adapted from Ref. [17].

Of the three categories, in situ forming hydrogels fabricated by natural polymers, synthetic polymers, or combined natural-synthetic polymers are the most studied to fabricate 3D carriers because of their easy administration, facile loading of stem cells, minimal invasion, and high contour adaptability [18], [19], [20], [21]. However, the technical barriers mentioned at the beginning paragraph have not been conquered by the existing hydrogel based carriers, and they seem to meet technical bottlenecks. Ex situ forming hydrogels can partly improve the drawback of in situ forming hydrogels, but they have new problems themselves as a competent candidate. A detailed review of them is in Section 2.

Novel designs of in situ or ex situ forming hydrogel based carriers such as one fabricated by Michael addition reaction are still being widely explored and reported [22], [23], and improvement may be acquired in the near future. These hydrogels are given high expectations, but their inherent defects can only be improved but not overcome. New carriers with innovative design ideas are continuously surfacing. With the development of technologies such as microarray chip and microfluidics [24], 3D microcarriers fabricated with hydrogels are emerging with potential to overcome the aforementioned technical barriers. However, many challenges are still faced by these new carriers to realize 3D microenvironment or stem cell niche. It is time to make summary and perspective of them.

2 Review of the in situ and ex situ forming hydrogel based 3D carriers

Many reviews have summarized these carriers and have high expectation of them; they can be real 3D carriers with many advantages such as easy administration, facile encapsulation of stem cells and factors, minimal invasion, high contour adaptability, and biocompatibility. In this section, we do not retell them, and interested readers can refer to a number of excellent published review articles [23], [25], [26], [27], [28]. We just briefly present them critically.

2.1 In situ forming hydrogel based 3D carriers

In brief, for syringeability, minimum invasion, and scaffold effect in vivo, in situ forming hydrogels undergo a sol-gel transition. Before injection, they are in liquid form so that stem cells are suspended within; after injection, they crosslink to the gel and anchor in the target site. To trigger the sol-gel transition, stimuli such as pH, light, temperature, kinetics, or enzymes of the tissue context are needed [22], [29], [30], [31]. Some of the trigger stimuli such as pH and enzymes are difficult to apply in vivo due to safety concerns, and that is why thermosensitive in situ cross-linkable hydrogels are mostly applied. Clearly, these in situ forming hydrogels must have at least two functional components: part A is to sense stimulus and trigger sol-gel transition, which are usually synthetic materials for their plasticity and sensitivity; part B and other parts are used to improve biocompatibility or provide other additional functions, and they are usually nature sourced or natural analogues [32], [33], [34]. Therefore, their compositions are usually complex especially the stimuli-sensing part, which leads to complex synthesis steps and unsatisfactory biocompatibility. Cross-linking mechanism may be physical or chemical, but they all fail to provide proper encapsulation time which may lead to migration or leakage of stem cells from target site to remote organ and can bring side effects [18], and their contingency ability is poor due to narrow range of stimuli for physical sol-gel transformation or safety concern for chemical sol-gel transformation in vivo [23]. During injection, mechanical insult to stem cells is inevitable due to poor mechanical strength of liquid hydrogels. The sol-gel transformation process in vivo is hard to control and may lead to bulk hydrogel, which easily puts the stem cell in a context of low nutrient substance, low oxygen, high toxic metabolites, and deficient interaction with matrix cells [15].

2.2 Ex situ forming hydrogel based 3D carriers

Ex situ forming hydrogels polymerize in vitro and can be applied at some tissues in which it is difficult to trigger polymerization due to lack of corresponding stimuli. These hydrogel based carriers can greatly reduce the mechanical insult to stem cells during injection, and their components are usually simple compared with in situ forming injectable hydrogels because of their greatly wider cross-linkable condition out of the body. For example, Aguado et al. [35] proved that cross-linked alginate hydrogel by Ca²⁺in vitro with certain modulus yielded high stem cell viability, 88.9±5.0%, while the corresponding not cross-linked alginate solutions which cross-linked in vivo just resulted in viability of 66.9–11.0%, not significantly higher than the phosphate-buffered saline (PBS) control group, which suggested that extensional flow force through the syringe needle was the main cause of stem cell death and could be prevented by ex situ forming hydrogel with stronger mechanical strength. Though different design philosophy was facilitated, these ex situ forming hydrogels impair their injectability and contour adaptability to the irregular tissue, which greatly limits their application.

3 Hydrogel based 3D injectable microcarriers

As illustrated in Section 1, microencapsulation and matrix entrapment can provide stem cells real 3D microenvironment. Generally, except for in situ and ex situ forming hydrogel carriers, the remainder are microencapsules and microgels (Figure 1), the microgel is a cross-linked gel particle, and the microcapsule is constituted by a shell with microscale diameter [36]. As illustrated in Section 2, neither in situ nor ex situ forming hydrogel based carriers are able to meet the requirements of competent carriers for stem cell therapy by direct injection. Microencapsule or microgel based carriers can combine the advantages of in situ and ex situ forming hydrogels. They are usually mixed, they are both 3D microcarriers [37], and we generally name them hydrogel based 3D microcarriers in the present review. Hydrogel based 3D microcarriers can encapsulate both stem cells and cellular niche components such as growth factors. They maintain a 3D microenvironment with their large surface area which improves cell-matrix interactions, nutrient and waste transfer, and oxygen and carbon dioxide exchange in vivo or ex vivo. They are able to prime stem cells ex vivo which may be important for stem cells to adapt in the in vivo environment. They also preserve the scaffold’s injectability as well as protect stem cells from mechanical insult, which cannot be achieved simultaneously by in situ forming hydrogels or other aforementioned hydrogels. Moreover, with the development new technologies such as microarray chip and microfluidics, monodisperse and uniform hydrogel based microcarriers can be conveniently fabricated with simple steps. Their comparison is briefly summarized in Table 1.

Table 1:

The comparison of hydrogel based 3D carriers.

3D carrier type	Advantages	Disadvantages
In situ forming hydrogel	Easy administration; facile loading of stem cells and related factors; minimal invasion; high contour adaptability	Poor mechanical properties; bulk hydrogel is prone to inefficient substance exchange; uncontrollable stem cell release; complex compositions due to stimuli sensing parts; limited and narrow range cross-linking stimuli; uncontrollable cross-linking process; and safety concern due to cross-linking in vivo
Ex situ forming hydrogel	Good mechanical properties; simple compositions and wider cross-linking condition out of body	Low contour adaptability and injectability
Microcarriers	Easy administration and injectability; facile loading of stem cells and related factors; preconditioning stem cell; minimal invasion; high contour adaptability; good mechanical properties; controllable stem cell loading, release and stem cell migration prevention; efficient substance exchange; low or no immune stimulation; sophisticated structure to mimic stem cell niche	Complex fabrication process; may be hard for batch production

3.1 Materials to fabricate hydrogel based 3D microcarriers

Similar to other gels, the materials to fabricate hydrogel based 3D microcarriers can be classified by their sources: natural polymers, synthetic polymers, and hybrid polymers, which are shown in Table 2 [17], [38], [39], [55], [56]. Compared with in situ forming hydrogels, their compositions are relatively simple, which benefits from removal of stimuli sensitivity part. Natural materials especially the extracellular matrix (ECM) components are the most used because of their high biocompatibility, low or no immune stimulation, and potential tissue specificity. However, the pure natural materials are difficult to self-assemble due to lack of efficient cross-linkable sites and their poor mechanical property; therefore, modification is usually needed. For example, gelatin is a denatured form of collagen from ECM protein, possessing bioactive or adhesive sequences for stem cell survival, proliferation, and differentiation. By substituting amines in gelatin with methacrylamide and addition of photoinitiator, photocross-linkable gelatin precursor was prepared, and the generated 3D microsphere was competent to apply in bone marrow-derived mesenchymal stem cells (BMSCs) injection for bone regeneration. It supported cell spreading inside the microspheres and enhanced cell proliferation [38]. Natural non-ECM sourced materials or synthetic polymers were also reported to fabricate these carriers. However, because of potential immune stimulation, lack of cell responsive anchorage point, and poor biodegradable property, the hydrogels merely based on them are limited to fabricate 3D microcarriers for stem cell therapy. Therefore, they are usually combined with natural materials to improve their biocompatibility. For the reasons above, hybrid polymers that combine the advantageous properties of synthetic polymers and natural polymers are increasingly studied and promising in stem cell therapy by direct injection [57].

Table 2:

The materials to fabricate hydrogel based 3D microcarriers.

Category	Polymer	3D microcarriers	Stem cell type	Tissue	References
Nature polymers	GelMA	Yes	hMSC	Bone	Zhao et al. [38]
	Dextran-HA	Yes	ADSC	In vitro	Kim et al. [39]
	Alginate	Yes	hMSC	Myocardium	Yu et al. [40]
	Alginate	Yes	DPSC	Bone	Kanafi et al. [41]
	HA-alginate	No	hAdMSC	Vocal fold wound healing	Kim et al. [42]
	Fibrion	Yes	ADSC	Intrinsic sphincter	Shi et al. [43]
	Alginate-fibrin	Yes	hUCMSC	Bone	Zhou and Xu [44]
	Collagen-chitosan	No	BMSC	Bone	Sun et al. [45]
	Pullulan-collagen	No	MSC	Skin	Wong et al. [46]
	Gelatin-collagen	Yes	ADSC	Bone	Kodali et al. [47]
Hybrid polymers (synthetic material, natural polymers)	PEG-HA	No	hADSC	Wound healing	Hassan et al. [22]
	PEG-peptide	Yes	hMSC	Bone and cartilage	Gao et al. [48]
	CNT-GelMA	Yes	hMSC	Skeletal muscle and neural lineage	Shin et al. [49]
	PLGA-alginate	Yes	NPC	In vitro	Ashton et al. [50]
	GNP-GelMA	No	ADSC	Bone	Heo et al. [51]
	PEGDA-collagen	No	hMSC	Cornea	Cho et al. [52]
	PEGDA-collagen	Yes	hMSC	Angiogenesis	Dilip et al. [53]
	PEG-fibrin	No	Dental stem cell	Dental tissue	Galler et al. [54]
	PEG-agarose-carbomer	No	hMSC	Spinal cord injury	Caron et al. [7]

GelMA, gelatin methacrylate; hMSC, human mesenchymal stem cell; HA, hyaluronic acid; ADSC, human adipose-derived stem cell; DPSC, dental pulp stem cell; hAdMSC, human adipose-derived mesenchymal stem cell; hUCMSC, umbilical cord mesenchymal stem cell; BMSC, bone marrow mesenchymal stem cell; PEG, poly(ethylene glycol); CNT, carbon nanotube; PLGA, poly(lactide-co-glycolide); NPC, neural progenitor cell; GNP, gold nanoparticle; PEGDA, poly(ethylene glycol) diacrylate. Synthetic materials are in italic.

Table 2 lists some reported materials used to synthesize hydrogel based 3D carriers for stem cell therapy [7], [22], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54].

3.2 Preparation method of hydrogel based 3D microcarriers

The shapes or configurations of microcarriers depend on the purpose of their application and types of the laden stem cells. Microspheres and microencapsules are the most reported structures to load stem cells because they are streamline which is benefit for stem cell loading and injection, while other shapes are also reported. For example, micro-cylinder-like microcarriers based on biodegradable gelatin microcryogels were fabricated by Li et al. [58], which enabled human adipose-derived mesenchymal stem cells (hMSCs) to prime within and resulted in tissue-like ensembles with enriched ECMs, enhanced cell-cell interactions, and increased stem cell delivery success rate to critical limb ischemia mouse model. Their diameters must be appropriate to carry certain numbers of stem cells per unit and maintain injectability through a syringe head. They must load stem cell easily, polymerize homogeneously, and possess enough mechanical strength to protect stem cell from forces insult during or after injection [35]. Therefore, competent preparation methods with custom-made device and polymerization condition as well as simple steps, efficiency, and low price are required.

Techniques to fabricate hydrogel based 3D injectable microcarriers for cell therapy include chemical methods, physical methods, and chemical-physical methods. According the fabrication course, they can be classified into two categories: formation pre-cell loading microcarriers and formation co-cell loading microcarriers, which is shown in Figure 2. For the former, firstly 3D microcarriers with desired shapes are fabricated through many ways such as electrospinning, phase separation, freeze-drying and solvent evaporation technique [59], [60], [61], [62], [63], [64]. Then cells are seeded on the microcarriers and expected to populate and create their own ECM. Lastly, microcarriers with stem cells dwelling in them are collected and injected to target sites through a syringe. For the latter, firstly cells are suspended homogeneously into the pre-gel. Then formation process is triggered, and the microcarriers loaded with stem cells are collected and injected to target sites through a syringe. For the former, steps to fabricate these scaffolds maybe complex or cytotoxic as long as the finished products are competent to culture stem cells without toxicity, while for the latter, complicated steps or cytotoxic organic solvents or harsh formation conditions are difficult to implement because of their cell vitality impact, and that is why acoustic or photic technique is most used for their noninvasive and facile handling of stem cells and particles [65]. These microcarriers fabrication techniques are various and widely reported [60], [61], [66], [67], [68], here we focused on two emerging and promising techniques.

Figure 2:

Fabrication course of microcarriers. 1. Formation pre-cell loading microcarriers. 2. Formation co-cell loading microcarriers. A. Formation process. B. Stem cell and other factors loading process. C. Certain time for stem cell growth and migration from the surface to inner mesh of the microcarriers. D. Injection process.

Microfluidic technology is a multidisciplinary field that precisely controls and manipulates fluids from milliliters to nanoliters and even to femtoliters and has broad application areas in inkjet, particles manufacture or manipulation, biochip, etc. [69], [70], [71], [72], [73]. As a kind of microfluidic technology, droplet-based microfluidics are gaining popularity in stem cell technology due to their ability to generate hydrogel microparticles with accurately controlled composition, monodisperse dimensions and shape, and simple operation and volume production capacity, which is difficult for other microcarriers fabrication techniques [74]. Droplet-based microfluidics mainly relies on the principle of emulsification process in which an aqueous pre-gel disperses into an immiscible nonpolar solution such as oil, and airjet droplet systems and dielectrophoresis-based systems have also been studied [75], [76], [77]. By fine control of the size of the orifice of the microfluidic channel, hydrophilicity or hydrophobicity of the channel surface, viscosity of the immiscible phases, velocity ratio of the continuous phase to discrete phase, and the size, morphology, and production rate of microcarriers can be uniformly formed as desired (shown in Figure 3A) [78]. Additionally, superior 3D microcarriers with more sophisticated structure that further mimics stem cell microenvironment can be fabricated by microfluidics. For example, by imitating the microstructure of the stem cell niche, Wang et al. [79] presented an ECM hydrogel based porous microcarrier with external-internal connected scaffold structures and biopolymer matrix fillers, in which the scaffold structure avoids shear forces, and the biopolymers can provide cells an ECM microenvironment to promote the formation of multicellular spheroid aggregates.

Figure 3:

Schematic diagram of microcarrier fabrication method. A. Droplet-based microfluidics method. (1) Schematic diagram of the microfluidic flow-focusing device. (2) The harvested microsphere. B. Photolithography methods. (1) A photomask with designed holes, which light passes to trigger polymerization. (2) After peeling, the harvested microgels. C. Mold method (see the description in Section 3.2).

Microgel array chip integrates microfabrication technology with gel preparation techniques, and flexibly fabricates injectable microcarriers with predefined sizes and shapes. Microgel array chip can be fabricated by photolithography methods or by a mold with many microunits (Figure 3B and C). After loading with liquid pre-gel, polymerization process is triggered and then the microcarriers with desired shapes are stripped with a matched ejector chip or other techniques [68], [80], [81]. Though low yield and cumbersome steps of microgel array chips may limit their application for now, the flexibility to provide microcarriers with unconstrained shapes is appealing compared with other techniques. For example, a microstencil chip was fabricated by poly(methymethacrylate) as a mold for the generation of poly(ethylene glycol) diacrylate (PEGDA) microgel array chips. After the PEGDA precursor solution was loaded in the microstencil chip, polymerization process was triggered, and then mesenchymal stem cells (MSCs) and other cell niche components were laden for a certain time. The matched ejector chip fabricated by polydimethylsiloxane was used to push out the microgels with uniform shapes. Then the harvested 3D stem cell-laden microgels that mimic ECM were injected subcutaneously to mice ischemic disease model and exhibited concentrated localization and enhanced stem cell retention at the injection site, which increased stem cell vitality and their treatment effect [15].

3.3 Characterization

After fabrication by proper techniques, microcarriers are evaluated to decide their usability. Besides uniform size, stable structure, biocompatibility, proper ratio of stem cells to a unit of a microcarrier, and injectability, other parameters must be fit with their corresponding stem cells. Elastic modulus is a parameter to measure the stiffness of 3D microcarriers, which must be strong enough to protect stem cells from shear forces experienced upon injection or any other mechanical stresses during their application; it also influences the differentiation of stem cells. The elastic modulus of a microcarrier is calculated as the slope of its stress-strain curve in the elastic deformation region which can be measured with nanoindentation technique [38]. Degradation behavior is critical to provide the mechanical properties and avoid immune reaction. Degradation rate of microcarriers should be controllable, which can be investigated by mass-loss analysis or surface morphology study [38], [64], [82]. Viability of stem cells in microcarriers whether in vitro or in vivo is also a crucial indicator for their usage, which is measured by various live/dead cell viability assays [38], [58], [83], [84].

4 Overview of the application of hydrogel based 3D carriers in stem cell therapy by direct injection

Hydrogel based 3D carriers have been applied in stem cell therapy by direct injection for many diseases with varying degrees. In this section, we briefly summarize and comment on some of the most reported or related diseases supported by hydrogel based 3D carriers in the application of stem cell therapy by direct injection including hemopathy, diabetes mellitus, myocardial regeneration, and wound healing. Meanwhile, their application is much broader such as in bone regeneration [38], cartilage regeneration [85], nervous diseases [86], and cornea regeneration [52] and show good application prospects.

Stem cell transplantation is a way that can cure many blood diseases. Stem cell transplantation by intra-bone marrow injection has been proposed as a strategy to bypass homing inefficiencies of stem cell transplantation by intravenous infusion, and some encouraging tests or trials have been reported, while low stem cell retention in the injected hematopoietic organs was still regarded as the main limiting factor for its translation from bench to bedside [12], [87], [88]. Though stem cell therapy assisted by hydrogel based 3D carriers is increasingly studied, the studies for stem cell transplantation by direct injection are rare [89]. For example, using mice model, a thermosensitive in situ forming collagen hydrogel was utilized as carriers for bone marrow stem cell transplantation by direct injection, which could achieve efficient retention of the injected bone marrow cells and show superior regeneration of hemopoietic cells than control group within PBS [90]. This seemed to be encouraging, but further studies are needed. Because microenvironment is very important for earlier regeneration of hemopoietic cells [91], [92], competent hydrogel based 3D microcarriers show great potential to realize stem cell niche and promote the application of stem cell transplantation by direct injection.

Islet transplantation combined with MSCs is a promising therapy for treatment of diabetes mellitus. MSCs can regulate neighbor cells by paracrine signaling, which makes them excellent candidates for improving the survival of islet cells [93], [94]. However, loss and poor engraftment of islets or MSCs and immunological rejection are major obstacles for islet therapy. Microencapsulation of islet cells and MSCs emerged as an immune-isolation strategy and showed advantages in nutrients and oxygen exchange, which enhanced the engraftment of islets and MSCs [37], [95], [96]. For example, using an isolated-graft model, Kerby et al. [97] suggested by co-transplantation of islets with MSCs in alginate microencapsules, insulin secretion and graft outcome could be improved, and the average blood glucose level of the transplanted mice was significantly lower. Though great improvement has been achieved, certain challenges such as hypoxia, massive death of β cells, and inflammatory response still need to be solved before its full availability in clinics, and microencapsulation technology combined with stem cells may further promote islet transplantation.

Currently, the heart is accepted to be able to renew its cardiomyocytes physiologically because of its stem cells [98], [99], which can be utilized to facilitate myocardial regeneration despite limited proofs from clinical trials and unsolved hurdles [100], [101]. The heart is a harsh organ for stem cells with high pressure from heartbeats, wall stress, and complicated blood vessel and nerve network, therefore, stem cell therapy is relatively hard to apply, and stem cells in thin tissue matrix are prone to death. Hydrogel based 3D carriers can protect stem cells from mechanical insult both from injection process and heartbeats, therefore, they show promise to improve stem cells vitality and enhance engraftment rate in the heart [102], [103]. These hydrogels are mostly in situ forming and have been summarized by Ye et al. [104], but other hydrogels with reliable mechanical properties such as alginate-poly-l-lysine-alginate microsphere [105] have also been studied [106].

Though wound healing outcomes have been greatly improved by surgical refinements, skin grafting, tissue engineering, and other techniques, the resulting tissues are still not fully recovered, with scars and/or without the full function of the normal tissue [107], [108]. A dressing is a usual method applied directly to the wound to promote healing by a sterile pad or gauze or hydrogel, which is broadly applied in clinic. Dressings with 3D carriers of hydrogels with stem cells entrapped inside can greatly enhance stem cell engraftment, in which stem cells differentiate and secrete various factors which are essential for wound healing. These injectable hydrogels are mostly in situ forming based [22], [109] besides their limit mechanical strengthen to reduce mechanical insult to stem cell during or after injection, they may not easily reach the deeper layer of the targeted lesion, and the bulk hydrogels might hinder the substance exchange, cell migration, and cell secretion and diffusion [38]. Other 3D carriers such as microgels or microspheres are studied and show promise to overcome the drawback of the in situ forming 3D carriers [110], [111].

5 Discussion and perspective

Stem cells with their ability to differentiate into specific lineages and secrete corresponding factors can contribute to the formation of new tissues, which can be used to treat various diseases or repair injuries. Stem cells are varied and different in differentiation capacity and renewability or paracrine effect, and therefore, their application scopes vary. For example, hematopoietic stem cells are used for treating leukemia because of their differentiation capacity, and MSCs are used for wound dressing because of their paracrine effect. Direct injection to the target sites greatly decreases stem cell loss compared to systematic administration, while a favorable microenvironment is indispensable for their vitality and function, which can be provided by hydrogel based 3D carriers (Take stem cell transplantation as example, shown in Figure 4). The requirements of ideal carriers for stem cell therapy are summarized as follows: (1) Nontoxic or very low toxicity to stem cells. (2) Efficient loading of stem cells and tailored cellular niche components such as bioactive factors. (3) Simple to manufacture on a large scale. (4) Flexible and tunable structure to offer optimal performance for delivering different sources of stem cells. (5) Protection of cells from mechanical insult or biochemical injury during loading and injection. (6) Good site-specificity without stem cell migration or leakage from the targeted site. (7) Long-term stem cell retention at the targeted site. (8) Maximum imitation of stem cell niche and provision of context that allows stem cells to survive, proliferate, differentiate, maintain the desired phenotype, and ultimately function as desired. (9) Biodegradable, adhesive or non-adhesive depending on therapeutic aim, and will not induce a severe and chronic inflammatory or immune response [15], [17], [112], [113], [114], [115]. In conclusion, competent 3D carriers must realize 3D microenvironment, namely, stem cell niche, as much as possible as well as protect stem cells from mechanism insult during and after injection.

Figure 4:

Symbolic schematic of three techniques to carry out stem cell transplantation. 1. Systematic administration by peripheral intravenous infusion. The size of the red arrow stands for the number of stem cells at the site they are arriving. After being consumed by mechanical insult during injection process (A), stranding at lung (B), liver and spleen (C), or other non-target site, only a small part of stem cells arrive at target site (D). 2. Direct injection of stem cells without assistance by competent 3 carriers. Without support of competent three carriers, they are prone to mechanical insult (A) and easily migrate out of the target sites (E1). 3. Direct injection of stem cells with assistance by competent 3D microcarriers, in which little or no mechanical insult is inflicted on stem cells and few stem cells migrate out of the target sites (E2).

However, the existing 3D carriers do not meet all the aforementioned requirements. Some studies focus on mechanism strength or the morphology of the 3D carriers, and some studies are modest to mimic a stem cell niche by just using a single or a few kinds of molecules of ECM or materials at a time, while ECM is a complex system structurally and functionally. Furthermore, the existing studies failed to realize the differences between stem cells and other cells or among the different types, sources, or stages of stem cells themselves. Sources and types of stem cells are various, and different stages of the same stem cell are different in characteristic and need special niche to grow, develop, and function. Obviously, the existing studies only basically form the 3D carriers with general fabrication and characterization methods, limited imitation to stem cell niche, and limited adaptability and specificity.

In spite of all the challenges, as research continues, new types of hydrogel based 3D carriers with better designs and properties can further mimic the ECM of the corresponding stem cells with adaptability and specificity. Hydrogels containing ECM analogous materials such as GelMA show low possibility in immune reaction and natural biodegradation behavior; moreover, the presence of peptide motifs can provide stem cells adherence, allowing stem cells to proliferate and spread within [67], [116]. By combining agarose/carbomer based hydrogel with arginine-glycine-aspartic acid tripeptide and ECM deposition, plus a new loading procedure on a lyophilized scaffold, Caron et al. [7] created a more optimized niche able to better sustain hMSCs viability. Stem cell niche is not a homogeneous microenvironment [117]. Inspired by that, gradient materials that can present a continuous series of microenvironments and mimic anatomical gradients are particularly attractive such as a gradient microspheres collagen hydrogel generated by microfluidics [118]. Promoting vascular reconstruction and oxygen diffusion are regarded as key factors in stem cell vitality especially for big areas of defect sites [119], [120], which is also the consideration of the new types of hydrogel based 3D carriers. In addition to promoting viability and proliferation of the stem cells, hydrogel with more sophisticated design can regulate stem cell differentiation. Inspired by biophysical and compositional properties of bone marrow, Ji and Harley [121] used ECM ligand-coated polyacrylamide ex situ forming hydrogel with defined stiffness and matrix ligand cues to regulate stem cell differentiation. They suggested that the interaction between stem cells and surrounding matrix ligands and matrix stiffness could decide lineage specification of stem cells by modulating integrin activation and actomyosin contractility within 24 h ex vivo. In brief, they reported that stem cells tended to primitive myeloid progenitors when they were in hydrogel substrates resembling the endosteal region, and they tended to erythroid lineages in vascular region resembling hydrogel substrates. Similar studies reported that substrate elasticity or stiffness, ligand types and concentration, dimensionality, etc. had different roles on the fate of hematopoietic stem cells. Though many of them were just applied ex vivo, findings emphasized the importance of selectively mimicking all aspects of specific stem cell niche with thorough consideration [122], [123].

In conclusion, hydrogel based 3D carriers are almost in situ forming based with limit cell retention rate and engraftment rate, though in situ forming hydrogel based 3D carriers may have advantages over some certain application areas and may be constantly improved by new techniques. Hydrogel based 3D microcarriers with superior design philosophy are gradually emerging to provide a more optimized stem cell niche. Although at this stage the existing 3D carriers cannot meet all the requirements of an ideal carrier, with further researches especially the development of 3D microcarriers, 3D carriers are promising to further meet the requirements of ideal 3D carriers for stem cell therapy by direct injection in the future.

Corresponding author: Professor Baoan Chen, MD/PhD, Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Dingjiaqiao 87, Gulou District, Nanjing 210009, Jiangsu Province, P.R. China, Phone: +86 25 83272006, Fax: +86 25 83272011

About the authors

Chengxin Luan

Chengxin Luan received his BSc degree at medical school of Southeast University. He is a PhD candidate at Medical School and is studying at State Key Laboratory of Bioelectronics School of Biological Science and Medical Engineering of Southeast University. He is working on photonic crystal techniques and biological materials for medical application.

Ping Liu

Ping Liu is a PhD candidate at Medical School of Southeast University and is studying at the hematology and oncology department of Zhongda Hospital. She is working on reversal of multidrug resistance of malignancies.

Runzhe Chen

Runzhe Chen obtained her medical degree from Southeast University. She has published more than 10 SCI papers as first author. Presently, she is the joint PhD student in Southeast University and University of Texas MD Anderson Cancer Center. She is serving as the editor of Advances in Clinical Toxicology and the assistant editor of the Cancer Translational Medicine Journal. She is also the academic and section editor and peer reviewer of several international journals.

Baoan Chen

Prof. Baoan Chen is a renowned hematologist and oncologist in China. He is the director of Faculty of Oncology of Southeast University, leader of Department of Hematology of the Affiliated Zhongda Hospital of Southeast University, and head of MDS Research Institute. He has rich experience in hematological malignancies’ diagnosis and treatment.

Acknowledgements

This work was supported by the National Natural Science Foundation of P.R. China (Grant Nos. 81170492 and 81370673), National High Technology Research and Development Program 863 of P.R. China (Grant No. 2012AA022703), National Key Basic Research Program 973 of P.R. China (Grant No. 2010CB732404), Key Medical Projects of Jiangsu Province (Grant No. BL2014078), and Key Discipline of Jiangsu Province (2011–2015).

Disclosure: The authors declare no conflicts of interest in this work.

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Received: 2017-1-9

Accepted: 2017-3-7

Published Online: 2017-4-13

Published in Print: 2017-10-26

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Articles in the same Issue

https://doi.org/10.1515/ntrev-2017-0115

Keywords for this article

3D microcarriers; direct injection; hydrogel; stem cell therapy

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