Stem Cell Delivery in Regenerative Engineering and Medicine

Regenerate the Vascular Tissue

Blood vessels composed of endothelial cells and mural cells play key roles in tissue regeneration and repair by serving as conduits to deliver oxygen and nutrients to injured and newly-formed tissues.[88] Many CVDs such as coronary artery disease (CAD) and peripheral vascular disease (PAD) are resulted from the loss of vascular supply leading to organ failure. Regeneration of blood vessels can be generally divided into two major aspects: the neovascularization in ischemic tissues, and repairing of injured blood vessels.[89]

Insufficient neovascularization is the essential hurdle for treatment of ischemic diseases, especially coronary artery diseases (CADs), by regeneration of thick tissues (> 200 ??m) that cannot survival under nutrients diffusion supply alone. Delivery of endothelial cells (ECs) or related progenitor cells together with stem cells can effectively improve the neovascularization, which consequentially enhance the efficacy of stem cell therapy. For instance, a recent research reported by Zhang group encapsulated a combination of hiPSC-CMs, hiPSC-ECs, and hiPSC-derived smooth muscle cells (hiPSC-SMCs) in fibrinogen gel at ratio of 2:1:1 to fabricated human cardiac muscle patches (hCMPs) for MI treatment. Co-delivery of the hiPSC-ECs and hiPSC-SMCs with fibrinogen gel into the porcine model of MI leaded to angiogenesis factor-enriched exosome secretion that significantly improved CMs survival and maturation, and contributed to the engraftment and angiogenesis at periscar border zone, which improved functional recovery of myocardium as a consequence. Besides the co-delivery strategy, direct encapsulation of pro-survival peptides in biomaterials was also proved to improve angiogenesis (Figure 5).[90] Wu group conjugated bone morphogenetic protein-2 peptide analogue (BMP2), erythropoietin peptide analogue (EPO), and fibroblast growth factor-2 peptide analogue (FGF2) with collagen fibers (Col?— D?— Pep) via dendrimer linker to realize the slow release of the pro-survival factors to delivered BMMNCs. The peptide had similar biological effects as the full-length protein, but with improved stability, fewer side effects and better delivery. Due to the chemical conjugation, the pro-survival factors can be slowly released by degradation of the collagen hydrogel. The hydrogel delivery system improved the cell survival and retention. In vivo delivery of the BMMNCs with the Col?— D?— Pep hydrogel improved the blood perfusion in ischemic limb of both severe combined immune-deficient mice and immunocompetent mice.

Besides the paracrine effect, ECs derived from human pluripotent stem cells (hPSC, including ESCs and iPSCs) were reported to integrate with host tissue to from vascular structures.[91] The researchers from Yoon Group delivered the hPSC-ECs using a self-assembled peptide amphiphile conjugated with RGDS (PA-RGDS) nanomatrix gel into ischemic limb to reconstruct the vascular structure for CVDs treatment. The PA-RGDS molecule was consisted of a hydrophobic alkyl tail, and hydrophilic domain of cell adhesive ligand RGDS and biodegradable sequence of metalloprotease-2 (MMP-2). The RGD ligand promoted the adhesion and avoided anoikis of encapsulated hPSC-ECs in nanomatrix gel which is critical for cell survival during engraftment, while the MMP-2 site provided a controlled degradation of the gel which played key roles in cell integration with the host tissue.[92] Although the mechanism was not shown, the PA-RGDS nanomatrix preserved the cell viability from oxidative stress which is important cytoprotection as discussed. Meanwhile, the nanomatrix gel could enhance the retention of delivered cells that were still detectable up to 21 weeks. After 10 months, the transplanted ECs integrated into host tissues which contribute to the neovascularization in ischemic areas. Additionally, providing the structural guidance such as micro-channels would further accelerate the neovascularization process.[80b, 93]

Dysfunction of the narrowed arteries results in reduced blood supply to organs that will cause ischemic disease such as CAD, stroke and periphery artery disease (PAD). Current repairing strategy is bypass surgery using autologous grafts (e.g. patient vein) or synthetic polymer graft (e.g. Teflon). However, the availability of autologous grafts is patient-limited availability and the harvest process is invasive.[94] However, the usage of synthetic graft usually resulted in repeated revascularization procedures due to the acute and chronic occlusion caused by graft infection and other complication.[95] Recent development in biomaterials and cell therapy could shed light on the possible solution. For instance, Woo group manufactured the engineered vascular conduits (EVCs) using cell sheets from patient-derived smooth muscle cells (SMCs) and fibroblasts.[96] The EVCs was further perfused with human umbilical vein endothelial cells (HUVECs) for maturation. The artificial mature EVCs showed native artery comparable structure and mechanical strength, which leaded to completely recovery of blood perfusion in ligated hindlimb of rats. Besides the cannular vascular grafts, stem cell patch was also shown to be useful in repairing of ateries. Mayer group fabricated a hybrid scaffold using an elastomeric polymer of poly-4-hydroxybutyrate (P4HB) and methacrylated gelatin (GelMa) hydrogel (Figure 6).[97] A fibrous elastic scaffold was firstly prepared with dry spinning of P4HB in random and aligned manner which had uniform and anisotropic mechanical properties, respectively. Then GelMa was introduced into the fibrous P4HB scaffold to provide a hospitable environment for cell growth. Cells encapsulated in the hybrid scaffold showed more uniform distribution compared to the cells directly seeded onto P4HB scaffold. In vivo delivery of MSCs and endothelial progenitor cells (EPCs) using the hybrid scaffold on sheep pulmonary artery enabled the tissue formation throughout the scaffold and prevented the surface thrombosis. This was attributed to the compatible environment provided by the GelMa hydrogel encapsulation, as well as the mechanical support by P4HB fibrous scaffold under physiological pressure in vivo. Other potential strategies via decellularized vascular structures without pre-seeding of stem cells are also proved to be efficient to induce vascularization which is beyond the scope of this review.[98]

Despite the achievements in preclinical studies, few clinical trials were carried involving biomaterials delivery system for stem cells therapy (Table 2).[99] Therefore, more work is required to further evaluate and translate the developed functional biomaterials to provide new options for cardiovascular regeneration to fulfill the bench-to-bedside approach.

Biomaterials for Stem Cell Delivery in Neruoregeneration

Damages to the brain, the spinal cord and the peripheral nerves due to traumatic injuries, stroke, tumor, infection or neurodegenerative diseases are often permanent and incapacitating, presenting tremendous burden on the patients, their families and society. The adult nervous system, especially the central nervous system (CNS), has limited capability to regenerate to establish the correct axonal and dendritic connections.[100] Instead, a cascade of events happen as a consequence of damages, including the production of glial scar, which represents a molecular and physical barrier to regeneration, leading to neuronal degeneration and cell death.[101] Current clinical treatments are designed to pharmacologically improve the disease symptoms in combination with rehabilitation activities to restore physical function to some extent; however, no therapies are yet available to fully restore lost functions or slow ongoing neurodegeneration following the injury or disease.[102] It has been recognized that the failure of neurons to regenerate was not an intrinsic deficit of the neuron, but rather a characteristic feature of the damaged environment that either did not support or prevented regeneration.[101] Therefore, strategies aim to fully regenerate the damaged nerves should not only focus on supplying the cells lost due to injury or disease to re-establish the connections, but also improving the local environment to support regeneration and prevent further degeneration.

Stem cells have great promise as a source for introducing new neurons or glial cells to the damaged nervous system. For instance, neural stem/progenitor cells (NS/PCs) can be isolated from the mammalian neonatal CNS,[103] or derived from pluripotent stem cells such as ESCs and iPSCs,[104] which can in turn be differentiated into neurons and glia cells of the nervous system when delivered to the damaged regions. In addition, NS/PCs can also modulate immune and inflammatory responses, leading to neuroprotective effect to the surrounding tissues.[105] Clinical trials of allogenic NS/PCs transplantation to patients with ALS,[106] stroke,[107] and Pelizaeus-Merzbacher disease (PMD)[108] have found consistently favorable safety profile during early phase trials. Other cell types, such as mesenchymal stem cells (MSCs) and adipose-derived stem cells (ASCs) are also investigated by researchers preclinically[109] and clinically[110] for their ability to facilitate neuroregeneration via paracrine actions of neural protection, plasticity, antiinflammation, and angiogenesis, rather than direct transdifferentiating into neural cells.[111]

Advanced biomaterials can provide a structural platform to bridge the gap of damaged neural tissue, deliver stem cells to the site of injury, enable and augment the targeted delivery of therapeutic molecules, and help rebuild damaged circuits and repair damaged neuronal pathways.[102b, 112] In a 2016 meta-analysis of preclinical studies using NSPC transplantation for spinal cord injury treatment, it was found that scaffold use in NSPC transplantation effectively raise functional recovery comparing to scaffold-free cell suspension.[113] The ideal materials for neural regeneration should be biocompatible with low immunogenicity, mechano-compatible to provide structural support to the surrounding neural tissue, and encouraging stem cell survival, differentiation and integration.[102b, 114] In this section, we highlight some of our colleagues' recent work of neuroregeneration in the brain, the spinal cord and the peripheral nerves (Table 3) that utilized advanced biomaterial-assisted stem cell delivery strategies to regenerate neural tissues.

Regenerate the Brain Tissues

Stem or progenitor cell transplantation after brain damage due to ischemic stroke or traumatic injury was shown to promote neuro-regeneration in pre-clinical models.[115] Early-phase clinical studies to evaluate the intracerebral transplantation of allogenic human neural stem cells (NSCs)[107] and MSCs[116] have also demonstrated promising safety profiles in stroke patients. However, these studies are limited by poor survival of the transplant when administered as a suspended form into the damaged brain, most likely due to the ischemic and pro-inflammatory environment, the immunological attack and the abrupt withdrawal of growth factor and adhesive support.[114] To solve this problem, biomaterial scaffolds can serve as delivery vehicles for transplanted cells, facilitating neuronal outgrowth and connectivity between grafted and host cells. Moreover, the scaffolds can be modified to provide local and sustained delivery of proteins, and the surface can be topographically altered to form alignment, coated with ligands or modified to possess a surface charge to promote attachment, growth and differentiation of neural stem cells.[102b]

Some earlier works used porous biodegradable polymers such as polyglycolic acid (PGA) as cell-seeding scaffolds, which supported new brain tissue regeneration around the degenerating scaffold and formed a network of neurites with axonal regeneration.[117] However, the transplantation of cell seeded polymer scaffold requires invasive surgical procedures, which may cause further damage to the vulnerable brain tissue. Alternatively, hydrogels containing ECM proteins such as collagen, laminin and fibronectin could be injected with stem cells to allow minimally invasive cell delivery.[118] The ECM-based scaffolds were found to significantly improve cell survival rate comparing to cells in suspension by many groups.[119] However, the xenogeneic nature of ECM-based scaffolds and the potential immune response to the antigenic components of xenogeneic materials represent a critical barrier to the use of xenogeneic scaffolds in translational applications.[120] Synthetic ECM-derived peptides, such as laminin-derived IKVAV and fibronectin-derived RGD could be used to form ECM-memetic hydrogels by self-assembly[121] or incorporation with other biopolymer materials (e.g. hyaluronic acid[122] and alginate[123]) to allow effective delivery and differentiation of stem cells to the brain, without the use of xenogeneic materials.

Engineering strategies can further encourage the migration, survival and differentiation of NS/PCs by promoting a stem cell niche-like environment that helps rebuild a functional neuronal network. As an example, the Segura group has engineered a series of hyaluronic acid (HA) based hydrogels with various peptide modifications.[122, 124] Their initial delivery of iPS-NPCs to the stroke cavity within the HA based matrix modified with RGD and MMP sensitive peptide promoted the differentiation of transplanted cells; however, the material did not significantly promote stem cell survival.[122a] To improve stem cell survival, the group utilized the Design of Experiments (DOE) methodology to optimize the concentrations of three ECM ligands (RGD, YIGSR, IKVAV) in vitro for the survival and differentiation of NPCs by determining the individual and combinatorial effects of each factor on cell activity.[124] Further DOE optimization and modification was carried out combining mechanical, biochemical and trophic factors, and a later animal study using the systematically optimized HA hydrogel resulted in selective control of human neural stem cell survival and differentiation after transplantation in the stroke brain[122b] (Figure 7A). Moreover, the HA hydrogel can be tracked in vivo with MRI, enabling non-invasive tracking for material distribution and degradation. Recent work from the same group also focuses on cell-free approach of brain tissue regeneration after stroke by promoting vascularization and reducing inflammation, which is beyond the scope of this review.[125]

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