Paracrine Effects of Mesenchymal Stromal Cells Cultured in Three

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Paracrine Effects of Mesenchymal Stromal Cells Cultured in Three-Dimensional Settings on Tissue Repair Holly M. Wobma, David Liu, and Gordana Vunjak-Novakovic ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 28 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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ACS Biomaterials Science & Engineering

Paracrine Effects of Mesenchymal Stromal Cells Cultured in Three-Dimensional Settings on Tissue Repair

Holly M. Wobma, BS1, David Liu, BS1, Gordana Vunjak-Novakovic, PhD1,2, # 1

Department of Biomedical Engineering, and 2Department of Medicine

Columbia University, 622 west 168th Street, VC12-234, New York, NY 10032

#

Please address correspondence to Gordana Vunjak-Novakovic, [email protected]

Submitted to the special issue on Regenerative Biomaterials, by invitation of Prof. Kara Spiller Keywords: Mesenchymal stromal cells; regeneration; wound healing; biomaterials; immunosuppression

ABSTRACT Mesenchymal stromal cells (MSCs) are a promising cell source for promoting tissue repair, due to their ability to release growth, angiogenic, and immunomodulatory factors. However, when injected as a suspension, these cells suffer from poor survival and localization, and suboptimal release of paracrine factors. While there have been attempts to overcome these limitations by modifying MSCs themselves, a more versatile solution is to grow them in three dimensions (3D), as aggregates or embedded into biomaterials. Here we review the mechanisms by which 3D culture can influence the regenerative capacity of undifferentiated MSCs, focusing on recent examples from the literature. We further discuss how knowledge of these mechanisms can lead to strategic design of MSC therapies that overcome some of the challenges to their effective translation.

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INTRODUCTION In 2010, Arnold Caplan suggested the term “mesenchymal stem cell” (MSCs), which he first coined in the 1980s, should be rebranded as “medicinal signaling cell”1. While tri-lineage mesenchymal differentiation is still a standard method to demonstrate this population in vitro, there is a growing appreciation that their normal physiological role may have more to do with their paracrine effects2,3. This is based on in vivo observations that MSC delivery without differentiation and engraftment into injured tissue still leads to regeneration via alternative therapeutic mechanisms4–7. Specifically, MSCs can release numerous growth factors (GFs) (e.g. EGF, FGF) and angiogenic factors (e.g. VEGF, PDGF), as well as express immunomodulatory proteins and lipids capable of subduing both the innate and adaptive immune responses (e.g. IDO, PGE-2, HGF)8,9. This pßaracrine repertoire makes MSCs particularly attractive for wound healing applications10. Injured tissues in the body share a number of common responses. First, there is a transient period of hypoxia from the clotting process, and damage signals are released from dying cells. Inflammatory cells infiltrate the wound and contribute to dead tissue clearance and extracellular matrix (ECM) remodeling. New blood vessels form, and neighboring cells – including tissue resident MSC-like cells – may enter to promote healing3,10. Ideal injury resolution is fast with minimal scar formation. While healthy individuals can generally achieve this outcome after a mild injury, severe injuries overwhelm the capacity for healing. Furthermore, there are several states in which even the repair of less severe injuries can fail: older age, poor perfusion (e.g. patients with diabetes or vascular disease), malnutrition, obesity, burn damage, and immunodeficiency10. It is in these situations that MSCs may enable improved outcomes because of their ability to facilitate ECM remodeling, neovascularization, and an anti-inflammatory response8,10. Over the last decade, there have been over 800 completed and ongoing clinical trials (clinicaltrials.gov) using MSCs to treat a wide variety of pathologies. One of the earliest and most extensively explored applications has been for treatment of heart disease. Initial preclinical studies in large animal models (porcine, canine, ovine) showed promising data for treatment of acute myocardial

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infarction (MI)11,12, subacute MI13,14, and chronic ischemia15,16, using standard benchmarks of cardiac health such as ejection fraction, left ventricular end-diastolic volume, contractility, scarring, and vascular density. Similar studies were then conducted in human patients such as the phase I/II POSEIDON trial, which showed improved ejection fraction, fewer arrhythmic events, and reduced cardiac hypertrophy in patients after acute MI17. Results for chronic ischemia followed the same trends, as the PROMETHEUS and TAC-HFT trials all showed significant decrease in scar size after injection of MSCs, leading to better perfusion and contractility18,19 . Based on these and other promising phase I/II data, phase III trials followed. However, these results were less encouraging. For example, the BOOST trial showed that intracoronary delivery of MSCs improved left ventricle ejection fraction in acute infarction after 6 months relative to control, but this difference was no longer significant after 18 months20. This questionable efficacy was not unique to cardiac studies. Large-scale studies by Osiris investigating the effect of MSC therapy in treatment of graft-versus-host disease did not corroborate their earlier phase II success, as the therapy failed to meet its primary endpoints in two phase III trials. In other indications, Osiris’s testing did not fare much better: a phase III trial geared toward Crohn’s disease was discontinued, while treatment of chronic obstructive pulmonary disease also failed to meet its efficacy endpoint21,22. These mixed results are confounded by the fact that it is difficult to elucidate the precise action and fate of MSCs once they are delivered. While it seems like they may have some useful trophic effects (at least temporarily), there is still much room for optimization of the cell therapy itself, particularly in the area of delivery. Specifically, although conventional intravascular delivery strategies may be an intuitive and facile first step towards translational MSC therapies, there are several challenges to this methodology (Figure 1). For example, as apparent from many animal studies and some human trials, the MSCs are at least temporarily retained in lung microvasculature (more significant in rodent models) if delivered systemically (“first pass effect”) and leave the site of administration if delivered locally 8,23–25. Additionally, many studies suggest the MSCs have a short-life span, which is attributed to the loss of ECM adherence (anoikis), as well as metabolic stress after going from a high serum, high glucose,

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and 21% O2 laboratory culture environment to an in vivo environment that is less flush with these nutrients and may present further insults such as oxidative stress (e.g. in ischemic disease)26,27. Finally, while MSCs expanded in regular culture media

can

release

angiogenic

and

anti-

inflammatory factors, there is much evidence to suggest that they would be far more effective if Figure 1 Environmental conditions that limit the

exposed to instructive cues such as hypoxia, pro- efficacy of MSC therapies in wound healing. inflammatory cytokines, and/or toll-like receptor (TLR) agonists, before being administered24,28,29. One broad strategy to overcome these challenges is to alter the cells, themselves, through genetic engineering, cell coating, or by altering culture conditions. To enhance localization and targeting, MSCs have been transfected to overexpress CXCR430. This encodes a chemokine receptor thought to be an important player in the cells’ chemotactic response to SDF-1α gradients that are created at pathological sites30. MSCs have also been coated with antibodies to integrins or ligands for selectins to improve adhesion to endothelial cells in inflamed regions30. To combat rapid cell death, MSCs have been engineered to express pro-survival factors, such as Bcl-2, HO-1, and Akt30. Overexpression of human leukocite antigen-G (HLA-G)31 or cell surface “painting” with protein H32 have also been used to confer protection against immune cell attack and complement binding, respectively. Finally, there is evidence that initial culture of MSCs with pro-inflammatory cytokines or hypoxia can improve their antiinflammatory/immunosuppressive capacity24,28,29 and that hypoxia preconditioning can induce resilience against ischemic damage when MSCs are implanted in animal models of stroke and myocardial infarction33–35. These strategies seem promising, particularly for treating systemic diseases in which MSC delivery into circulation is needed for them to impact multiple tissue sites; however, for treating a single, localized defect, many of the same benefits can be achieved by growing the MSCs as aggregates or using

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biomaterials as cell delivery agents. Here we review how 3D culture may influence and enhance the behavior of MSCs for regenerative applications, using studies from the last 5-10 years to highlight various concepts. Although MSCs can differentiate into multiple cell types, our discussion will be focused on their paracrine effects in their undifferentiated state.

DESIGN CRITERIA 3D culture can influence MSC behavior through intrinsic chemical and physical cues as well as by purposeful addition of cell instructive factors. While aggregate formation has few modifiable parameters (e.g. method, size), there is great flexibility in choosing a biomaterial. To this end, the following minimal design criteria may be taken into consideration: (i) previous FDA approval for similar applications, (ii) biocompatibility, (iii) promotion of MSC “stemness”, (iv) tunable properties, such as porosity, degradation rate, or ability to load with bioactive cues, and (v) permissive of MSC paracrine effects. Given these considerations, it is not surprising that the evaluation of MSC-biomaterial interactions often includes measures of cell viability, stemness, migration36, paracrine activity

37–40

, and capacity for tissue

repair.

THE PHYSICAL ENVIRONMENT By default, three-dimensional (3D) formulation of MSCs mitigates cell death from loss of attachment “anoikis”. However, cell–cell and cell-ECM interactions also provide important signaling mechanisms that can affect the regenerative function of MSCs. This is, perhaps, best exemplified by the simple scenario of growing MSCs as spheroids/aggregates. MSCs have an innate ability to self-assemble into aggregates (103-105 cells) when cultured on non-adhesive surfaces41. This morphology can also be promoted via other methods such as the hanging drop method, by coating of plates with charged materials (e.g. chitosan)42, or by forced aggregation (e.g. Aggrewell plates)41. MSCs experience a much smaller elastic modulus from the cell-cell interactions in spheroids than in 2D culture (~0.1kPa vs GPa)43, although these interactions are not perfectly homogenous throughout the aggregate. Cells on the surface

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are more elongated and can interact with cells and ECM in their implanted/injected environment, whereas interior cells are more rounded and only have cell-cell interactions with each other. Interestingly, spheroid formulation has recently been shown to promote a more “pluripotent-like” state of MSCs, with enhanced expression of pluripotency markers and the ability to transdifferentiate into cells types beyond mesenchymal origin (e.g. neuron and hepatocyte-like cells)42,44,45. At least one hypothesis for this behavior is that aggregation causes mitochondrial reorganization and glycolytic metabolism that promotes the more primitive phenotype44,45. Spheroid formulation also alters the survival and activity of MSCs in their undifferentiated state. MSCs upregulate CXCR4 and superoxide dismutase, enhancing cell homing and survival, respectively41. Furthermore, a hypoxic core and metabolic signals (e.g. lactate) have been suggested to increase HIF-1α expression and the release of pro-angiogenic and immunomodulatory factors (e.g. prostaglandin E2 (PGE-2)41, although the full signaling pathways are not well understood. MSCs may also upregulate immunomodulatory factors in response to differences in the physical stress they experience. Indeed, some initial evidence of this was accidentally discovered when MSCs were used in a mouse model of myocardial infarction7. Surprisingly, when the relatively large MSCs got trapped in lung microvasculature after tail vein injection, they were still able to quell ischemic damage. This was attributed to the clustering of entrapped MSCs and subsequent secretion of TNF-stimulated gene 6 protein (TSG-6), which is minimally expressed in 2D culture46. Even though the MSCs were trapped, the circulation of this antiinflammatory glycoprotein enabled them to provide cardioprotective effects from a distance. Since then, MSCs have been shown to upregulate other secreted factors such as PGE-2 and hepatocyte growth factor (HGF) when grown as spheroids and also when encapsulated in biomaterials46. These paracrine factors have a direct influence on nearby immune cell populations in vivo. For example, in their recent paper, Vallés et al. explored the cross-talk between MSCs and macrophages in both 2D and 3D culture (highly porous synthetic scaffold). Monocytes exposed to MSCs cultured in 3D were less likely to be recruited than when cultured with MSCs in 2D due to a decrease in the local levels of IL-6 and MCP-1. Furthermore, the 3D MSC environment led to macrophages that expressed more of the anti-

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inflammatory cytokine IL-10 and less of the pro-inflammatory TNF-α.47 This is consistent with other reports that MSCs encapsulated in a biomaterial or cultured in spheroids induce an M2/anti-inflammatory macrophage phenotype37,40,48. Such studies highlight the role of physical environment in shaping the suppressive response of MSCs. Curiously, while studies of MSCs cultured in 2D on tissue culture plastic (TCP) suggest that both contact dependent and independent mechanisms are important for MSC-based immunosuppresion28, the former are limited in biomaterial encapsulated MSCs (depends on porosity and degradation rate) and only possible for the outer layer of cells in MSC spheroids, since interior cells are not available for interaction at the surface. Nevertheless, MSCs grown in 3D have been found to exhibit equal or greater immune cell suppression than those in 2D, presumably due to the compensatory boost in secreted suppressive factors46. The mechanisms by which the 3D physical environment elicits changes in paracrine factor secretion are still poorly understood. The hypoxic core of spheroidal MSCs has been associated with upregulation of PGE-2, HGF, and other suppressive factors46; however, biomaterials are often far more porous and do not consistently form the same O2 gradients. In the study by Valles et al., even when MSCs were cultured in a scaffold of 90% porosity, they still demonstrated enhanced secretion of PGE-2 and TSG-6, which is unlikely due to a hypoxic gradient. It is attractive to think that mechanotransduction pathways could influence MSC immunomodulation. MSCs grown on in 2D were shown to have a forced polarization with extensive interactions between the basal surface of each cell and an extremely stiff plate (Young’s modulus on the order of GPa)46,49. The setting could not be more different in 3D culture. Cells in spheroids experience mild strain (Young’s modulus ~60 Pa)46 on all sides from extensive cell-cell interactions, and in biomaterials, the cell-matrix interactions are similarly of a much smaller modulus and in all directions. MSCs are acutely sensitive to variations in stiffness, which can influence their differentiation status50. For example, MSCs in soft substrates are more likely to exhibit adipogenic differentiation, whereas those in a stiff matrix are more likely to start down an osteogenic pathway50. A key mechanotransduction pathway implicated in these responses involves the transcriptional regulators yes-

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Figure 2 Substrate stiffness and dimensionality affect MSC morphology and spread area. MSCs seeded atop hydrogels of low (1 kPa), medium (5 kPa), and high (20 kPa) stiffness measured for representative F-actin (red) and nuclear (blue) staining. (Reused with permission from ref [51]. Copyright [2016] [Elsevier].

associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Recently, Caliari et al. explored the YAP/TAZ pathway in MSCs51 and compared norbornene-functionalized hyaluronic acid (HA) hydrogels that had been crosslinked to modulate stiffness and capacity for degradation. MSCs were grown either on top of or within the gel. The study showed that cell morphology and polarization in response to differences in substrate stiffness were different for 2D and 3D culture (Figure 2), and these differences correlated with opposite trends in YAP/TAZ nuclear translocation. Clearly, YAP/TAZ signaling is important for MSCs in terms of sensing 2D vs. 3D environment. While the functional outcome of this pathway has been explored in the setting of organ development and tumors, there have been no studies exploring the direct contribution to immunomodulation. Such studies would make a fascinating new avenue for pursuit. Of course, the YAP/TAZ pathway could indirectly play a role by influencing MSC stemness through cell shape and polarity52. There have been contradictory reports on

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how MSC immunomodulation is affected by differentiation status, with suggested decreases in immunosuppressive capacity53–56. Regardless, Caliari et al.51 make a strong case for the context-specific nature of mechanotransduction, which must be recognized when moving MSCs from 2D to 3D culture. Another important physical parameter is porosity. In general, material interconnectedness is necessary for oxygen and nutrient supply and waste elimination: too small pore sizes limit nutrient transport and cell migration, while too large pores limit cell adhesion57. There are also signs that pore size can influence the interplay between encapsulated MSCs and host factors and cells. This was shown nicely by Moshaverinia et al.58. Following the observation that pro-inflammatory cells and cytokines can inhibit osteogenic differentiation of MSCs, they sought to test whether diffusion of these cytokines into an MSCloaded scaffold can be limited by using alginate hydrogels of intermediate stiffness and low diffusion capacity. Compared to absorbable collagen sponges (ACS), the penetration of TNF- α and IL-17 into their scaffolds was greatly reduced, and this feature was not unique to alginate and could be recapitulated in PEG-DMA, a synthetic material with similar pore sizes (0.5 – 1µm). This study demonstrates how a material can shield cells from experiencing in vivo cues. However, the objective of the investigators should not be overlooked, as their intention was to promote bone regeneration by blocking exposure of MSCs to pro-inflammatory cytokines. For tissue repair, the opposite may be desirable, as pro-inflammatory cytokines are known to induce a more suppressive MSC phenotype. Thus, one could extrapolate that using materials with such small pores would not be conducive to MSCs receiving the right signals, unless these signals were packaged within the material. However, even in that case, it would be questionable as to whether the regenerative paracrine factors that MSCs secrete could leave the scaffold, given that cytokines had poor capacity to enter.

THE EFFECT OF THE MATERIAL Beyond the physical 3D environment, different biomaterials have been shown to influence the regenerative capacity of MSCs. These include natural matrix proteins (e.g. fibrin, collagen), decellularized tissue ECM, and synthetic polymers. Of course, these materials should not be regarded as

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inert vehicles for MSC delivery. Indeed, decades of research have been devoted towards evaluating their potential for facilitating healing. While most preclinical and clinical trials involving MSC therapies have utilized IV and direct injection methods59, the potential of biomaterials has been widely explored, especially in the osteochondral and skin fields. A non-exhaustive summary of the methods and outcomes of preclinical and clinical studies utilizing biomaterials in conjunction with MSCs is presented in Table 1. We included examples of preclinical and clinical trials with n > 5, sorted by material and year. Note also that this table is limited to studies with MSCs, but numerous other trials have also been conducted using bone marrow-derived cells, which include not only MSCs and precursor cells, but also accessory cells that support angiogenesis and vasculogenesis by producing additional growth factors.60–63. The materials listed in Table 1 mediate their effects through interactions with cell populations within the damaged tissue (fibroblasts, epithelial cells, tissue-resident multipotent progenitor cells, macrophages), influencing processes such as inflammation and angiogenesis3. For example, Spiller et al. examined how materials can affect macrophage populations by comparing subcutaneous implantation of unmodified collagen sponges with those either coated with lipopolysaccharide (LPS) or cross-linked with

Table 1 Representative preclinical and clinical studies utilizing biomaterials and MSCs

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BIOMATERIAL SUBJECT/INJURY

METHOD

RESULTS

Collagen Zhang et al.64

Minipigs (20)

MSC source: Pig BM-derived. Seeded in: Pig collagen II gel.

Osteochondral

1 defect per lateral femoral trochlea, 6 mm diameter (n = 40 total).

Glossy regeneration in both cell-treated groups, complete filling w/ hyaline-like cartilage at 8 wk. Defects remained in cell-free animals. O’Driscoll score higher in cell-treated groups (collagen-only gel also higher than control).

Maureira et al.65

Rat (20 M)

Cardiac

Induced infarct by coronary ligation of LAD artery

Koga et al.66

Rabbit (24?) 2.8 – 3.5 kg

Osteochondral Full-thickness defect in femur trochlear groove, 5 x 5 mm (n = 24 total). Yoshikawa al.67 Skin

et

Human (20: 9 M, 11 F)

4 groups: BMSC/gel; BNC/gel; only gel; control (n = 5 each). 2 time points: 4, 8 weeks. MSC source: Autologous BM-derived. Seeded in: Collagen I patch. 2 groups: MSC-patch engraftment (n = 10); untreated control (n = 10) 1 month follow-up. Compared MSC chondrogenic potential from 4 autologous sources: bone marrow; synovium; adipose tissue; muscle. Seeded in: Collagen I. Implanted w/ periosteum patch (n = 3 each). 2 time points: 4, 12 weeks. MSC source: Human BM-derived. Seeded in: Collagen sponge, applied topically.

MSC transplant effective in all patients. Wound healed or mostly healed in 18/20 patients within 3-8 wk; other 2 died of unrelated causes. Histology confirmed regeneration of subcutaneous tissue.

Burns or chronic wounds > 2 months follow-up.

Collagen + other Tong et al.68 Skin

Rustad et al.69 Skin

Rat (60? M) Induced diabetes, followed by left femoral artery ligation to induce ischemia. 1 wk later induced 1 cm diameter wounds. Mouse (24 F) 2 full-thickness dorsal wounds, 6 mm diameter (n = 48 total).

MSC source: Rat BM-derived Seeded in: Collagen-chitosan sponges (CCSS). Examined hypoxia pretreatment on CCSS scaffold. 4 groups: untreated control; CCSS-only; CCSS/MSC normoxic; CCSS/MSC hypoxic (n = 5? each). 3 time points: 7, 14, 21 days. MSC source: CAG-luc-eGFP transgenic mice BMderived Seeded in: Collagen-pullulan gel. 4 groups: untreated control; gel-only; injection; MSC/gel treatment (n = 12 each). 14 days follow-up.

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In-vitro patch analysis showed collagen slows MSC growth but not phenotype or potential. SPECT imaging showed significant improvement in perfusion and infarction area in patch group vs control. LV dp/dtmax and dp/dtmin were improved. Histology showed significant increase in ventricular wall thickness. Synovium and BM MSCs produced much more cartilage matrix at 4 wk by staining and histology. Synovium-derived cells showed further improved histologic scores at 12 wk. Synovium-MSCs showed positive correlation w/ transplant cell density and amount of cartilage matrix. Absence of periosteal patch negatively affected matrix production.

MSC


Hypoxic group showed accelerated wound closure at 7, 14d. Normoxic showed some benefit vs CCSS-only and control, but less. Staining of hypoxic group showed near-normal strata structure and reepithelialization. qPCR showed reduction in IL-6, TNF-α levels in hypoxic group, as well as increase in IL-10 levels. Showed angiogenesis promotion via increases in p-AKT and VEGF expression. Wounds healed significantly faster in MSC/gel group, w/ more hair follicles and sebaceous glands. Imaging on MSCs expressing luciferase 1hr post-wound showed decrease in MSC viability in injection group vs those in scaffold. Fewer GFP+ cells found in injected group. MSC/gel wounds had more vascularization and higher expression of pro-angiogenic cytokines like VEGF.


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Xiang et al.70

Rat (40 F at start)

Cardiac

Induced infarct by coronary ligation of left marginal artery.

Fibrin Yang et al.71 Skin

Rat (15) 2 scald wounds on back per rat (n = 30 total).

Bekkers et al.72

Goat (8 F)

Osteochondral

1 defect per medial femoral condyle, 5 mm diameter (n = 16 total).

Falanga et al.73

Human (13)

Skin

Acute wounds from skin cancer surgery (n = 5), and chronic wounds (n = 8). Mouse (? F)

MSC source: Allogeneic. Seeded in: Dehydrothermally (DHT) crosslinked collagen I-GAG scaffolds. 4 groups: sham control; DHT scaffold w/o cells; DHT+carbodiimide w/o cells; DHT w/ MSCs (n = 4, 10, 8, 9, respectively). 3 weeks follow-up. MSC source: Allogeneic BM-derived Seeded in: Fibrin glue. 3 groups: MSC/fibrin; fibrin-only; untreated control (n = 10 each). 30 days follow-up. MSC source: Goat BM-derived. Seeded in: Fibrin glue, cocultured w/ chondrons (Beriplast). Compared w/ microfracture (MX). 2 groups: 1 knee treated w/ MSC/chondron-fibrin glue; and 1 knee w/ MX per goat. 6 months follow-up. MSC source: Autologous BM-derived. Seeded in: Fibrin polymer spray, applied 3x. 3 subjects excluded for various reasons. 4 – 12 months follow-up. MSC source: Mouse BM-derived. Seeded in: Fibrin polymer spray.

Full-thickness tail wounds. 2 groups: diabetic (db/db); control (db/+). 25 days follow-up. Silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel Mathieu et al.74 Rat (62 F at start) MSC source: Allogeneic BM-derived. Seeded in: Si-HPMC gels (cellulose-derived). Cardiac Induced MI by ligation of the LAD coronary artery 4 groups: PBS control; gel-only; inject MSCs; MSC/gel (n = 6, 7, 8, 9, respectively). 19/62 rats died 24 hr post- 56 days follow-up. infarct. (13/43 post-infarct rats didn’t meet MI standards of LVEF < 70%, excluded.) Plasma-polymerized acrylic acid (ppAAc) carriers

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Histologically, most of the scaffolds in the MSC-seeded group were degraded, macrophages were seldom seen, there was significant vascularization, and no noticeable scaffoldtissue boundary, in contrast w/ the carbodiimide group. Staining showed good cell distribution. (6 rats died during infarct, 3 died during or right after scaffold implantation.) MSC/fibrin group showed greater wound contraction by 7d vs fibrin-only and control, and continued to 21d, when all but 1 wound healed. Other 2 groups did not show all wounds healing till 30d. Histology showed thinner epidermal layer and sebaceous gland proliferation in MSC/fibrin group, consistent w/ normal skin. Imagining and ICRS score showed more complete defect fill in MSC/chondron group. O’Driscoll histologic score, GAG/wt, and GAG/DNA also significantly higher. Note: group also reported mice study where subcutaneous addition of MSCs to chondrons showed higher GAG/DNA production vs only chondrons, pointing to a stimulating effect of MSCs on matrix production. Acute: Healing complete by 8 wk. 1 much larger wound healed faster than smaller wounds, suggesting MSCs could accelerate resurfacing. Chronic: ulcer size decrease or closure by 16-20 wk, except one patient. Histology suggest cells migrated from fibrin matrix in both acute and chronic cases. By 20d application of MSC/fibrin led to significantly better results in both control and db/db mice vs fibrin alone. Histology showed that applied cells may not persist longterm, but may play a stimulatory role.

MSC/gel group saw significant increase in LVEF compared to PBS after 7d; also higher vs gel-only and MSC-only groups. Infarct size significantly smaller in MSC-only and MSC/gel groups vs other two. Data suggested a short-term effect of the gel and a delayed effect of MSC injection, pointing toward a benefit in combined use.

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Jiang et al.75

Mouse (30 F)

Skin

2 dorsal excision wounds per mouse, 6 mm diameter (n = 60 total).

Poly(lactide-co-ε-caprolactone) (PLCL) Jin et al.76 Rat (32 M) Cardiac

Induced infarct by cryoinjury w/ cooled metal probe.

Pullulan hydrogel Wong et al.77 Mouse (F) Skin

Excisional Study: Dorsal wounds on mouse, 6 mm diameter (n = 8 total). High ROS study: Wounds in distal third of ischemic flap (highest ROS levels), 3 mm diameter (n = 8 total). Aloe vera-polycaprolactone (AV/PCL) Tam et al.78 Mouse

Skin Excisional: SCID mice (54 F) 2 dorsal wounds per mouse, 8 mm diameter (n = 108 total). Diabetic: Diabetic mice (36) 2 dorsal wounds per mouse, 6 mm diameter (n = 72 total).

MSC source: Human adipose-derived. Seeded in: acrylic acid on silicone carriers. 4 groups: ppAAc/MSCs; empty carriers; MSC injections; PBS sham injections (n = 20 each). Carriers removed on day 3, wounds analyzed on days 0, 3, 5, 7, 9. MSC source: Rat BM-derived. Seeded in: PLCL scaffolds. 4 groups: PLCL/MSCs; PLCL w/o cells; MSC-only; saline control (n = 8 each). 4 weeks follow-up.

Deposited MSCs remained viable for at least 5 days. ppAAC/MSC group saw accelerated healing compared to all groups. ppAAc/MSC and MSC injection groups saw reduced TNF-α levels yet more macrophages, but these were likely M2 macrophages. These 2 groups also saw higher conc. of TGF-β1, and enhanced levels of PECAM-1, VEGF-A, and αSMA. PLCL/MSC and MSC-only groups improved cardiac function. LVEDD and LVESD lower, LVEF higher in MSC groups than in PLCL and control. PLCL/MSC group showed more myogenesis. Infarct area significantly reduced in PLCL/MSC group vs all other groups.

MSC source: Allogeneic BM-derived, expressing luciferase. Seeded in: Pullulan polymer w/ 5 wt% collagen I. 2 groups: MSC/gel; MSC injection (n = 4 each). MSC/gel delivery significantly prolonged cell survival up to 30 days follow-up. 30 d compared to injection alone, suggesting acute inflammatory responses impair cell survival after delivery. 2 groups: MSC/gel; MSC injection (n = 4 each). MSC/gel delivery significantly slowed decrease in cell signal 10 days follow-up. vs injection at day 4, and had a greater signal at day 7, though nonsignificant. Note: in vitro tests w/ H2O2 confirmed gel protects MSCs from ROS damage; better protection vs acellular dermis. MSC source: Human Wharton’s jelly from umbilical cords (hWJSCs). Human foreskin fibroblasts (CCD) taken as controls. Both transduced for GFP and grown in media, which was retrieved as conditioned media (CM). Seeded in: AV/PCL electrospun nanoscaffolds. 6 groups: GFP-hWJSCs + AV/PCL; hWJSC-CM + Rapid wound closure by 14d in hWJSCs + AV/PCL and AV/PCL (scaffold soaked in CM); GFP-CCDs + hWJSC-CM + AV/PCL treatment groups vs controls, w/ the AV/PCL; CCD-CM + AV/PCL; PBS + AV/PCL; former significantly greater than all other groups. Histology untreated control (n = 18 each). showed reepithelialization, sebaceous glands, and hair follicles by 3d for hWJSCs. By 7 and 14d, treatment groups 3 mice per group analyzed on days 3, 7, 14 showed greater cellularity and vascularity. ICAM-1 mRNA levels significantly higher in hWJSCs group vs controls. 3 groups: GFP-hWJSCs + AV/PCL; hWJSC-CM + Treatment groups showed complete wound closure by 28d. AV/PCL; unconditioned media (UCM) + AV/PCL hWJSCs + AV/PCL group healed faster than other 2 groups, control (n = 24 each). TIMP-1 and VEGF-A expression also higher. Histology followed similar trend as excisional data, but w/ more 4 mice per group taken on days 7, 14, 28 for extended timeline. Staining for cytokeratin, involucrin, and analysis. filaggrin corroborated trends.

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glutaraldehyde. While unmodified sponges led to the formation of a fibrous capsule that was coincident with M2 (alternatively activated) macrophages on histology, LPS coating led to scaffold degradation by M1 (classically activated) macrophages. Curiously, glutaraldehyde crosslinked scaffolds, which were expected to cause a milder inflammatory stimulus than LPS, led to extensive angiogenesis, with evidence of sequential contributions of both M1 and M2 macrophages79. A conclusion was that sequential involvement of both types of macrophages is needed for a pro-angiogenic response to materials. Implanted biomaterials can also recruit endogenous “MSC-like cells”, which are thought to reside in all tissues3,80. A careful selection of a scaffold material can enhance recruitment of these endogenous populations. An instructive example comes from a study of dehydrated human amnion/chorion allografts (EpiFix®)39. These were shown to release numerous GFs and cytokines into solution and were able to recruit MSCs in vitro and in vivo when implanted subcutaneously in wild type mice (endogenous MSCs). The regenerative property of dehydrated amnion and chorion scaffolds is consistent with their role in promoting human development and preventing rejection at the maternal-fetal interface. Since MSCs are also supposed to promote secretion of GFs and immunosuppressive moieties, it is not surprising that scaffolds temporarily exposed to MSCs have been shown to contain many bioactive molecules that enhance tissue regeneration. In their study from 2014, Navone et al. cultured MSCs on electrospun silk fibroin (SF) scaffolds for seven days and then removed the cells from one group of scaffolds via incubation in pure H2O.

The cellularized and decellularized SF scaffolds were then

compared for their ability to treat diabetic db/db mice that had underwent a 5mm skin lesion via punch biopsy81. Whereas SF scaffolds alone conferred no improvement over lack of treatment in terms of wound closure at day 10 (~50% closure), both cellularized and decellularized SF scaffolds facilitated wound closure and to similar extents (99.84 % ± 1.9 vs 94 % ± 2.9, respectively; Figure 3). Conditioned medium from both situations could significantly enhance endothelial cell migration, but only cellular grafts enhanced the presence of VEGF in culture media (curiously, decellularized scaffolds led to the most TGF-β). The above studies reinforce that scaffolds can have a significant influence on regeneration and

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endogenous cell migration, even in the absence of live MSCs. Thus, when MSCs are brought into the mix, it is important to bear in mind that they act on the surrounding tissue environment in concert with a biomaterial.

Intrinsic bioactive cues In addition to directly promoting regeneration, the biochemical composition of a material may also directly impact the behavior of MSCs. Admittedly, Figure 3 Comparison of wound healing in diabetic

it can be difficult to parse out the contribution of a mice. Electrospun nanofibrous silk fibroin (SF)

patches with human adipose-derived MSCs (Ad-MSCsand D-Ad-MSCs-SF, respectively) showed significantly improved healing by wound closure structure, as most investigators compare an measurement and qualitative assessment compared to just SF and no graft control. Reused with permission individual biomaterial to a TCP control (changing from reference [81]. Copyright [2014] Navone et al.; licensee BioMed Central Ltd.

material’s biochemical cues vs. its physical SF

both substrate and mechanical properties). For example, in a study exploring the healing capacity of MSCs for treating brain injury, hippocampal slices were exposed to LPS to induce inflammation and then treated with MSCs from TCP or microencapsulated in alginate. When the hippocampal tissue had inflammatory signals from the LPS, MSCs in both 2D and 3D culture were primed towards an immunosuppressive phenotype and were able to produce PGE-2 and reduce TNF-α levels in the media82. However, MSCs encapsulated in alginate did not even need to be exposed to the inflammation in order to produce PGE-2. It would be ideal to be able to conclude whether alginate, as a material, had any role in this outcome; however, it seems far more likely that the MSC behavior was simply the consequence of physical 3D encapsulation. Another recent example involves the creation of a hydrogel delivery system for MSCs using a PEG-based copolymer combined with thiolated HA. Compared to freely injected MSCs, embedded cells showed increased secretion of pro-angiogenic factors like PGF, VEGF, and TGF-β, while producing low

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levels of pro-inflammatory cytokines like IL-2 and IFN-γ83. These paracrine secretions could suggest a possible positive impact of the HA-PEG system on cell behavior. However, as with the previous study, this could also be due to changes in physical environment. There are many other studies in this area, but most of them seem oriented towards simply showing that MSCs + biomaterial have a positive outcome without insights into the underlying reasons. Wahl et al. performed a rare side-by-side comparison of four different MSC-loaded biomaterials, focusing on those materials already in use or under development for wound healing, including BioPiel (chitosan), Smart Matrix (fibrin-alginate composite), Integra Dermal Regenerative Template (DRT; bilayer with collagen/chondroitin-6-sulfate and a thin layer of silicon), and Strattice (decellularized dermis)25. The various scaffolds led to different MSC seeding distributions (Figure 4A) and levels of cytotoxicity. Supernatants from cultures of MSCs on various matrices were added to a chicken chorioallantoic membrane to assess their pro-angiogenic capacity. Curiously, differences seen did not relate to the trends in VEGF from the various supernatants. Even if there were a clear narrative as to the biomaterial-MSC combination that led to the best survival and angiogenic behavior, it could not be clearly attributed to the intrinsic material biology. The materials tested had widely disparate dry weights and thicknesses, as well as different porosities/fluid capacities (Figure 4B). This study exemplifies an ongoing challenge in comparing biomaterials, which often differ in numerous properties simultaneously. One way to separate cues from physical architecture and surface chemistry could be to compare MSC behavior on thin polymer films. Levato et al. conducted such a study by comparing polylactic acid microcarriers coated with either collagen or RGD peptides, covalently or via physisorption (4 conditions total)84. Their studies focused mainly on the influence of these surface coatings on adhesion, proliferation, and MSC chemotaxis. One of the most interesting findings was related to surface expression of the chemokine receptor CXCR4. Damaged tissues release SDF-1α, which MSCs migrate towards via their expression of CXCR4. Consistent with previous reports, Levato et al. found the majority of CXCR4 is internalized in 2D TCP culture. However, surface expression was enhanced via culture on microcarriers, with the greatest increases seen from both physically absorbed and covalently bound collagen. Since

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Figure 4 Comparison of four MSC-loaded biomaterials side-by-side. Data are shown for chitosan, fibrinalginate composite, DRT (bilayer with collagen/chondroitin-6-sulfate and a thin layer of silicon), and decellularized dermis. Different scaffolds led to different MSC seeding distributions and levels of cytotoxicity (A). Scaffolds were significantly non-uniform in dimensions (B). Reused with permission from reference [25]. Copyright [2015] Elizabeth A. Wahl et al.

return to TCP led to swift internalization of the surface CXCR4, this suggested that something about celladhesion to ECM, particularly collagen, influenced CXCR4 localization and thus the capacity for migration to damaged tissue.

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Added bioactive cues Just as the physical characteristics of materials can be tuned via adjusting polymer mixtures or altering cross-linking agents, bioactive cues can also be added to a material. As with intrinsic material properties, these factors can influence both the damaged tissue niche into which the loaded biomaterial is introduced, or they can be used to influence MSC behavior. Scaffolds made from ECM proteins like collagen have also been shown to be widely versatile in delivering a variety of bioactive molecules, including hormones like human growth hormone proteins like EGF85, vitamins, glucose86, and cytokines, all of which can promote tissue repair. Others have used scaffolds to incorporate a local reservoir of antimicrobial agents, including drugs like vancomycin87 or compounds like silver sulfadiazine88. In addition to promoting healing via other cell types, added factors can also be used to bolster the efficacy of MSCs by improving survival (e.g. tenascin-C)89, homing (e.g. SDF-1α)90, and paracrine factor secretion (GFs and immunomodulatory factors)91. To parse out the influence of these factors on MSCs, the best laid out studies will have control groups that include added factors in the absence of MSCs and vice versa. An example of such a study was recently published by Pumberger et al.91. Their aim was to locally apply MSCs to regions of muscle damage to promote regeneration via release of paracrine factors. To enhance release, they investigated adding VEGF and IGF to MSC culture. Media alone, media with GFs, conditioned media from MSCs, and conditioned media from MSCs exposed to GFs were added to C2C12 myoblasts (Figure 5). Both conditioned medias (from MSC alone and MSC + GF) promoted myoblast proliferation, survival, and migration, while inhibiting premature differentiation and myotube formation (thought to be desirable), but the degree of these effects was greater for conditioned media from MSCs exposed to IGF and VEGF.

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Figure 5 Growth factor (VEGF, IGF) loaded alginate scaffold provides MSCs with the physical and chemical environment for optimal paracrine secretion toward wound healing. Reused with permission from reference [91]. Copyright [2016] [Elsevier].

To further demonstrate that these GFs were acting through the MSCs and not simply providing an additive effect, the investigators added blocking antibodies. As expected, such blocking antibodies reduced the beneficial effect of GFs spiked into regular media. However, they did not reverse the added benefits of conditioned media of MSCs cultured with GFs. This suggests that the GFs influenced MSC behavior to enhance paracrine secretion and had a relatively minor therapeutic contribution in that context. The investigators then created implantable “stem cell niches” by formulating the MSCs or MSCs + GFs in alginate cryogels, and tested them in a rat soleus muscle crush injury. As with their in vitro findings, both MSCs and MSC + GFs (now all in alginate) had positive benefits, but the latter were more successful at reducing scarring and increasing muscle density and force compared with alginate or alginate + MSCs alone.

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The Pumberger et al. study provided elegant insight towards using extrinsic GFs to boost and broaden paracrine secretion by MSCs. While such factors play an important role in facilitating wound resolution, another important behavior of MSCs is to reduce inflammation. In that regard, we have already described how this effect can be bolstered via 3D culture; however, priming MSCs towards a more immunosuppressive phenotype through inflammatory cues could enhance this behavior further. Zimmerman et al. pursued this objective by using biomaterials to provide sustained priming cues to promote and prolong an immunosuppressive MSC phenotype92. Heparin microparticles were loaded with IFN-γ, since heparin is known to have a high affinity for and prevent proteolytic degradation of IFN-γ. The particles demonstrated sustained release of IFN-γ for over a week, after an initial burst release of 130% of loaded IFN-γ (depending on initial loading dose). The loaded microparticles were then incorporated into MSC spheroids. Compared to MSCs spheroids simply pretreated with IFN-γ, formulating the IFN-γ within the embedded microparticles enabled sustained exposure of MSCs to the IFN-γ, resulting in maintained expression of the immunosuppressive enzyme IDO. In vitro assays demonstrated enhanced suppression of the T-cell fraction of activated peripheral blood mononuclear cells, which was attributed to enhanced IDO expression but also to the monocyte fraction with the PBMCs adopting a more anti-inflammatory phenotype. The latter relates to the ability for immunosuppressive MSCs to promote anti-inflammatory macrophage phenotypes, Therefore, by providing an ongoing IFN-γ (and 3D) “instruction” for the MSCs to be immunosuppressive, they can inhibit T-cell division and polarize monocytes away from an inflammatory phenotype.

DISCUSSION Our exploration of materials as MSC-delivery agents was motivated by the two challenges of MSC cell therapies - poor survival and localization of cells, and an untapped boost in the paracrine activity of MSCs when cultured in basic media on TCP. Formulating MSCs in a biomaterial automatically aids in cell localization and survival, assuming that a non-toxic, biocompatible material is used, of which there are many already approved for human therapies.

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Given that most materials will provide some level of survival and localization, the choice in material should be further guided by intrinsic capacity to promote tissue regeneration and the influence on MSC behavior. Indeed, while MSCs alone are the therapeutic agents in free cell therapies, it is, in fact, the pairing of material and MSC that together make up a therapy for localized applications. Admittedly, GFs and other useful molecules can be added or already present in the biomaterial (e.g. in the situation of Navone et al.81), and these bioactive scaffolds can be therapeutic. This raises the question as to whether MSCs are even necessary to add, especially since these cells will likely have only a transient lifespan/role as a source of paracrine signals. While live MSCs may not be absolutely necessary, they can enhance a therapy in multiple ways. An excellent example of the latter is for patients who have poor intrinsic regenerative capacity (e.g. aging, vascular disease, burn victim), where an affected person’s endogenous MSCs and other cell types may be dysfunctional and/or less capable of homing to a GF-loaded scaffold than in healthy individuals, and receptor expression for many growth factors may be limited93. In this scenario, providing a source of pro-angiogenic and anti-inflammatory cells could prevent a negative outcome (scarring or chronic wound/lack of closure). Furthermore, as shown by Pumberger et al.91, Zimmermann et al.92 and many others, scaffolds loaded with factors can serve as instructive cues to bolster the MSC therapeutic activity in addition to providing their own healing benefit. This approach can have far reaching effects, as it may also help recruit endogenous MSCs and convert macrophages to a more beneficial phenotype. Lastly, as has been unequivocally shown by numerous studies, 3D formulation of MSCs also enhances their behavior through upregulation of factors such as HGF and PGE-2. It is unlikely that one could achieve the same diversity and duration of beneficial paracrine factor secretion in the absence of incorporating MSCs, which makes a composite therapy – both cell and material – particularly suitable for localized tissue repair applications. Unfortunately, we still do not know the “best” combination of material and regulatory factors. Indeed, there seem to be numerous limitations with the current state of the art. Due to a paucity of strictly controlled studies, we do not know which materials are superior and why. It is difficult to parse out the contribution of the physical versus the biochemical cues. Understandably, many groups are more

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interested in showing a beneficial MSC + material outcome than exploring the mechanisms. There should at least always be “material-only” and “MSC-only” controls along with the cell loaded scaffold, yet these two controls are rarely both present in the same study. A further consideration is that different tissue injuries in different animal models may be difficult to compare, and immunocompromised species are not ideal for modeling diseases in which inflammation and counteracting inflammation are fundamental to the pathology and its treatment. In light of the use of minimal sets of control groups and challenges with making comparisons across different studies, the best we can do is to observe how general principles like angiogenesis and immunosuppression are combatted in each approach to try to make generalizations. As a final note, while a variety of approaches were featured in this review, one need not feel limited to a single strategy. For example, as demonstrated by Zimmermann et al.92, spheroids can be put into biomaterials and then given instructive cues. If we consider beneficial features of growth factors, inflammatory priming cues, 3D culture, and hypoxia, then it seems reasonable to try to combine all of these factors to make a superior therapy. All that remains is the choice of material and delivery method, which may be tailored to treating a particular type of injury. Further insights towards the most successful strategies are bound to come in the near future, due to the widespread recognition of the remarkable abilities for MSCs to promote regeneration.

AUTHOR INFORMATION Corresponding author: Professor Gordana Vunjak-Novakovic [email protected] Competing interests: The authors declare no competing financial interests

ACKNOWLEDGMENTS The authors gratefully acknowledge the funding support of NIH (EB002520).

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