Biomaterial based strategies for engineering new lymphatic vasculature

Abstract

The lymphatic system is essential for tissue regeneration and repair due to its pivotal role in resolving inflammation, immune cell surveillance, lipid transport and maintaining tissue homeostasis. Loss of functional lymphatic vasculature has been directly implicated in a variety of diseases, including lymphedema, obesity and the progression of cardiovascular diseases. Strategies that stimulate the formation of new lymphatic vessels (lymphangiogenesis) could provide an appealing new approach to reverse the progression of these diseases. However, lymphangiogenesis has been relatively understudied and stimulating therapeutic lymphangiogenesis faces challenges in precise control of lymphatic vessel formation. Biomaterial delivery systems could be used to unleash the therapeutic potential of lymphangiogenesis for a variety of tissue regenerative applications due to their ability to achieve precise spatial and temporal control of multiple therapeutics, direct tissue regeneration and improve the survival of delivered cells. In this review, we begin by introducing therapeutic lymphangiogenesis as a target for tissue regeneration, then an overview of lymphatic vasculature will be presented followed by a description of the mechanisms responsible for promoting new lymphatic vessels. Importantly, this work will review and discuss current biomaterial applications for stimulating lymphangiogenesis. Finally, we consider challenges and future directions for utilizing biomaterials for lymphangiogenic based treatments.

Graphical Abstract

Stimulating new lymphatic vasculature (lymphangiogenesis) has the potential to drive various tissue regeneration applications due to their role in tissue homeostasis. Hydrogel systems are a promising strategy to promote lymphangiogenesis due to their ability to 1) provide spatiotemporal control of factors, 2) direct and stimulate surrounding tissue growth and 3) provide a controllable microenvironment to deliver cell based therapies.

Introduction

Engineering new lymphatic vasculature represents a largely unexplored therapeutic strategy to stimulate new tissue regeneration. The human body is composed of both blood and lymphatic vasculature, which are both responsible for maintaining tissue homeostasis.^{[1, 2]} Specifically, lymphatic vessels have an important role in resolving inflammation, maintaining interstitial fluids and transporting cholesterol from tissues.^[1-3] Unsurprisingly, loss of lymphatic function has been implicated in a wide range of diseases including reduced immune response, impaired metabolic function and the formation of localized swelling (lymphedema).^[4-7] Lymphedema is estimated to affect approximately 200 million patients globally and one in seven persons treated for cancer.^[8-11] Additionally, lymphatic dysfunction has also been associated in the pathogenesis of cardiovascular disorders, which are the leading cause of death globally and accounted for 17.3 million deaths per year in 2016 and are expected to increase to 23.6 million in 2030.^[12] Specifically, lymphatic dysfunction has been associated with the progression of inflammation, obesity, atherosclerosis, hypertension and myocardial infarction.^{[3, 7, 13-20]} Stimulating new lymphatic vessels could provide a promising strategy to help reverse the progression of these diseases.^[21] However, the process of new lymphatic vessel formation (lymphangiogenesis) has been relatively understudied. Lymphatic vessels, though having blood vascular origins, have received significantly less attention than their blood counterparts. According to a search in PubMed, there are many more publications for “angiogenesis” than “lymphangiogenesis” (Figure 1A). Clinical trials have also focused predominantly on angiogenesis, with nearly 180 times more trials for therapeutic angiogenesis than lymphangiogenesis as of the end of 2019 (Figure 1B). Thus, localized stimulation of new lymphatic vasculature could provide a novel and appealing strategy for promoting tissue regeneration and reversing the progression of a variety of diseases.

Figure 1. Lymphangiogenesis is Significantly Less Studied than Angiogenesis.

Although both lymphatic and blood vasculature have an important role in homeostasis, there is significantly more research focusing on blood vasculature formation. According to PubMed, there are currently over one hundred thousand more publications for “angiogenesis” than “lymphangiogenesis” (A). Clinical therapies have also primarily focused on therapeutic angiogenesis, with over two orders of magnitude more clinical trials for angiogenesis than lymphangiogenesis as of the end of 2019 (B).

Limitations with conventional administration of drug and cell-based therapies represents a challenge for future lymphangiogenic applications. Therapies delivered into the body without control of the location or rate of delivery frequently encounter challenges due to low efficacy and undesirable side effects. Conventional therapeutic administration often necessitates either multiple administrations or large dosages, which not only are wasteful, but can also result in high systemic concentrations, decreased patient compliance and toxic side effects (Figure 2A).^[22-26] Oral administration is the most common approach for delivering therapeutics, though limited half-lives of protein and peptide based drugs (minutes to hours ^[27]), systemic delivery and short circulation times (<12 hours) frequently limit their applications.^[28] Alternatively, cell-based therapies, which are commonly delivered via bolus and intravenous injections, can result in poor cell survival and limited homing to desired tissues.^{[29, 30]} This has sparked interest over the last few decades into designing biomaterial delivery systems that can control therapeutic availability to local tissue and to improve the efficacy of cell-based therapies (Figure 2B).

Figure 2. Comparing Enteral, Bolus and Intravenous Administration Versus Hydrogel Delivery.

Therapies delivered into the body without control of location or rate of delivery often have low efficacy and undesirable side effects (A). Therapeutics delivered orally (enterally) typically require large or multiple doses, experience systemic concentrations and can be associated with potential toxic or undesirable side effects. Cell delivery via bolus or intravenous injections often face challenges with maintaining cell viability and localizing the cells to desired tissue, thereby limiting the efficacy of these therapies. Biomaterial systems have the advantage of overcoming some of these limitations by providing spatial and temporal control of delivered therapeutics and improving cell viability and localization, thus minimizing required doses and improving potential therapeutic benefit (B).

The use of biomaterial delivery systems is a particularly appealing strategy to promote lymphatic regeneration that applies engineering principles to provide spatiotemporal control over therapeutics and improve the efficacy of cell and tissue based therapies. Biomaterial systems deliver therapies that are localized to a desired tissue through using material carriers that can be designed to control therapeutic presentation and stimulate exogenous or endogenous cells. For example, hydrogels are a specific class of biomaterials that are mostly liquid by content, consist of crosslinked polymers and are characterized as viscoelastic or “solid-like material”.^{[31, 32]} These hydrogels have the ability to encapsulate a wide range of therapeutic agents to provide spatiotemporal control over therapeutic release and protect various therapeutic agents within its mesh structure from native enzymes via controlling hydrogel mechanical properties including porosity, crosslinking density, polymer alignment, stiffness and degradation rate. ^{[25, 33]} Furthermore, hydrogels can also mimic properties of the extracellular matrix (ECM) to improve cell adhesion, survival and functionality.^{[30, 34, 35]} This has led to the development of many hydrogel systems for blood revascularization applications, including delivering small molecules, proteins, endothelial cells and stem cells.^[36-42]

The properties of hydrogel systems are essential for their ability to target, protect and provide control over locally delivered therapies to stimulate lymphatic vasculature. This review will begin by introducing lymphatic vasculature and the process of lymphatic vessel development. Current hydrogel strategies to provide controlled release over therapeutic cargo, including diffusion and degradation mediated release, are introduced and examples are provided. We then discuss hydrogel applications to direct lymphatic regeneration via controlling hydrogel mechanical properties and through delivering exogenous cells. Furthermore, we provide considerations to help determine the choice of polymer and therapeutic mechanism depending on the desired lymphangiogenic application. Finally, we will discuss challenges and future directions for utilizing hydrogel delivery systems to promote lymphangiogenesis.

Lymphatic Vasculature Structure, Function and Development

Lymphatic vasculature has attracted attention as a target for tissue engineering and regenerative medicine applications, though the process of restoring lymphatic function in diseased tissue remains challenging. These challenges have prompted interest in developing therapeutic strategies to promote lymphangiogenesis. Here, we provide an introduction to lymphatic vasculature and the process of lymphatic vessel formation to identify potential therapeutic mechanisms to stimulate lymphangiogenesis.

Overview of Lymphatic Vasculature

Blood and lymphatic vessels were first described in the 17^th century and both types of vasculature have an essential role in maintaining tissue homeostasis.^{[2, 43, 44]} Blood vessels are essentially responsible for the delivery of oxygen, nutrients and soluble factors to the body, with capillaries providing direct exchange of these molecules with tissues (Figure 3A). Components of blood that enters the interstitial space of tissue from blood capillaries is recycled and regulated by the lymphatic system (Figure 3B).^{[2, 45]} Interstitial fluid is taken up by lymphatic capillaries, which are blind ended structures composed of single layered lymphatic endothelial cells (LECs) that are permeable to interstitial fluids.^[1] When designing biomaterial systems specific for promoting lymphangiogenesis, it is important to recognize the critical differences between lymphatic and blood capillaries. Foremost, lymphatic capillaries have a wide and irregular lumen, numerous cytoplasmic vesicles, multiple intercellular junctions and lack pericytes, which are connective tissue cells found on blood capillaries.^[46-49] Additionally, lymphatic capillaries contain a discontinuous basement membrane that forms overlapping LEC edges ^[50-52] and have anchoring filaments attaching the vessel to the surrounding tissue ^[53] to provide access for interstitial fluids and cells, including leukocytes, to enter lymphatic vessels.^{[48, 49, 54]} This is in sharp contrast to blood capillaries, which have a developed basal lamina and more interconnecting tight junctions.^{[1, 55, 56]}

Figure 3. The Structure and Function of Blood and Lymphatic Vasculature.

Blood vasculature is composed of arteries and veins that are responsible for exchanging gas and nutrients with tissue through capillary beds. Blood endothelial cells in these vessels have continuous membranes and are surrounded by pericytes (A). In contrast, lymphatic vasculature consists of interconnected, highly permeable and blind ended capillaries that are responsible for regulating interstitial fluids and immune cell trafficking. LECs are characterized via wide and irregular lumen, lack pericytes and have a discontinuous membrane which supports the entrance of cells into lymphatic vessels (B).

Fluids traveling through lymphatic vessels, also known as lymph, will then travel through capillaries and enter collecting lymphatic vessels.^[51] Collecting lymphatic vessels contain continuous zipper-like junctions, intraluminal valves and are surrounded by a layer of lymphatic muscle cells.^[57-62] The transportation of lymph through collecting lymphatic vessels relies heavily on the lymphatic muscle cells surrounding the collectors. ^{[57, 59, 60, 63]} The contraction of lymphatic muscle cells are driven by changes in the muscle membrane potential.^{[64, 65]} Specifically, the depolarization and action potential will spread along the lymphatic muscle cells leading to increases in intracellular calcium via the L-type Ca²⁺ channel and correspondingly promotes contraction.^{[57, 60-62, 66]} Interestingly, lymphatic muscle cells have recently been identified to have both striated and smooth muscle contractile elements which differ greatly from their counterparts in blood vasculature.^{[58, 60]} The contractions provided by the lymphatic muscle cells in combination with the system of one-way valves found in collecting vessels provide the active pumping of lymphatic flow in the collecting vessels.^{[59, 63, 67]} Additionally, lymph is also transported through the lymphatic collecting vessels via various systemic forces including interstitial fluid pressure, skeletal muscle contractions and blood pressure.^[68-71]

Lymph is then transported from the collecting vessels to lymphatic trunks and finally lymphatic ducts.^[69] Prior to being transported back into venous circulation, the lymph is filtered for debris and pathogens in lymph nodes. These lymph nodes contain antigen producing cells that respond to foreign particles and initiate the specific immune response. The process of lymph circulation through lymphatic vasculature allows the lymphatic system to both regulate interstitial fluids while also mediating the immune response.^[72] Another important role of the lymphatic system is in transporting lipids in the digestive tract.^{[73, 74]} Specialized lymphatics known as lacteals are found in the villi of the intestines and are responsible for transporting triglyceride-loaded particles (chylomicrons) containing dietary lipids from the gut to venous circulation.^[75] Interestingly, lacteal dysfunction in animal models have contributed to a number of diseases including obesity ^[7] and abnormal lipid metabolism ^{[76, 77]} further demonstrating the critical role lymphatics have in dietary functions. In summary, both types of vasculature have a complimentary role in maintaining tissue homeostasis, with the lymphatic system responsible for regulating interstitial fluids, immune function and dietary fat adsorption.

Lymphangiogenesis

Developmental biology is one of the main sources of inspiration for new tissue engineering therapies. These developmental mechanisms can serve as templates to design new biomaterial based strategies to promote lymphatic vessels. In mammals, the majority of LECs are believed to originate from venous endothelial cells.^{[78, 79]} The exact molecular mechanisms that govern these events are still uncertain, however venous endothelial cells in the posterior cardinal vein are believed to transition into LECs and form primary lymph sacs around E42 to E49 in humans.^{[2, 80]} This transition is regulated by several genes. The prospero homeobox protein 1 (Prox-1) gene is typically recognized as the master regulator gene responsible for transitioning venous endothelial cells into LECs.^[81] Indeed, the upregulation of Prox-1 drives the formation of specific LEC markers including vascular endothelial growth factor receptor 3 (VEGFR-3) and podoplanin.^{[81, 82]} Prox-1 is also responsible for suppressing about 40% of blood vascular specific genes in LECs, including those responsible for the upregulation of integrin α5, ICAM 2 and CD44.^{[81, 82]} The newly established lymphatic vessels will begin to sprout and migrate from the primary lymph sacs under the influence of specific molecular and microenvironmental cues including VEGFR-3 stimulation.^[83-85] Interestingly, VEGFR-3 is expressed in all endothelia during development, but later becomes primarily restricted to lymphatic cells with notable exceptions including fenestrated blood vessels and tip cells in sprouts during angiogenesis.^{[86, 87]} Fully mature VEGF-C and VEGF-D are the only known ligands for VEGFR-3, though only VEGF-C is essential during development.^{[84, 88]} VEGF-C and VEGF-D are secreted from a wide variety of cells including macrophages, neutrophils, dendritic cells, mast cells and fibroblasts.^[89-92] LECs will then form into capillaries , precollecting ducts and collecting ducts, with collecting vessels recruiting lymphatic muscle cells ^[59] and developing valves with the fork head transcription factor FOXC2 having a critical role in valve formation.^[93] Podoplanin and the hematopoietic signaling proteins SLP-76 and Syk also have an important role in lymphatic development through helping regulate separation of lymphatic vasculature from blood vasculature.^{[94, 95]} Recent work has also identified that many organs may have lymphatic vasculature from a diverse population of LEC origins, including LEC progenitors and precursors, which is reviewed in detail elsewhere.^{[96, 97]}

Hydrogels for Therapeutic Lymphangiogenic Applications

Utilizing hydrogel systems to deliver lymphangiogenic therapies are an appealing strategy to restore lost lymphatic function through providing controlled and localized therapeutic stimulation to diseased tissue. Hydrogels consist of cross-linked polymer chains that swell in aqueous solutions and, as a result, can recapitulate many tissue-like characteristics.^[25] In general, hydrogels can be placed into one of three mechanistic categories, depending on whether the potential therapeutic effect is achieved by 1) providing controllable release of therapeutic cargo, 2) supporting or directing tissue growth, or 3) encapsulating exogenous cells to incorporate into native tissue. Homing specific cells to the hydrogel, typically triggered by mechanical or antigen presentation, can also provide another therapeutic mechanism via promoting an endogenous source of cells. In practice, these biomaterial delivery systems often combine these mechanisms to achieve their therapeutic effect. The utility of each of these strategies largely depends on the applied polymer. Different types of polymers have been used for lymphatic revascularization, each with unique properties for their proposed therapeutic mechanisms (Table 1). In what follows, we provide a brief description of the various biomaterial delivery strategies, polymers used to promote lymphatic vessels and their current applications for lymphangiogenesis.

Table 1:

Properties of polymers and hydrogels commonly used for lymphatic revascularization strategies

Polymer	Mesh Size (nm)	Isoelectric Point/Charge	Biodegradable	FDA Approvals
Alginate	5-100 [98-100]	2.0-3.0, Anionic [101]	Nondegradable without Modification: Hydrolysis [102] Enzymatic [98, 103-105]	Food Products [106] Dental Applications [107] Wound Dressing [108]
Collagen	0.5-1.5 x 104 [109, 110]	7.0-8.0, Neutral	Biodegradable via Collagenases [111]	Cosmetics [112] Dental Applications [107] Skin Constructs [113] Repairing Cartilage Defects [114] Absorbable Sutures [115] Vascular Grafts [116]
Fibrin	1.0-5.0 x 102 [117]	5.5-5.6, Anionic	Biodegradable via Plasmin [118]	Sealant [119]
Gelatin	5-100 [120, 121]	Type A: 7.0-9.0, Neutral/Cationic Type B: 4.7-5.2, Anionic	Biodegradable via Collagenases [102], Thermally [122]	Food Products [123] Cosmetics [124] Drug Capsules [125] Surgical Hemostasis Sponge [126] Ophthalmic Demulcents [127] Partial Ossicular Replacements Prosthesis [128]
PLGA	5-100 [129]	3.0, Anionic [130]	Biodegradable via Krebs Cycle/Respiration [131]	Surgical Sutures [115] Implants [132] Drug Delivery [132]

Controlled Release Strategies

Therapeutic cargo release from hydrogels is generally mediated by the diffusion of the cargo within the polymer system and degradation of the biomaterial carrier (Figure 4). In the case where the therapeutic cargo (r_cargo) is significantly smaller than the average space between polymer crosslinks (mesh size) (r_mesh), therapeutic release is primarily mediated by diffusion (r_mesh/r_cargo>1).^[25] In diffusion based systems, the effective diffusion coefficient depends on the steric interactions between the polymer and the therapeutic cargo. Numerous mathematical equations have been developed to predict the diffusion coefficient of therapeutics within a hydrogel.^[133-136] In general, these models are dependent on the ratio of the hydrodynamic radius of the therapeutic to the hydrogel mesh size, with larger ratios resulting in more hindered diffusion. Additional interactions between the polymers and therapeutics can also influence diffusion mediated release, including electrostatic properties, van der Waals forces, hydrophobic effects, hydrogen bonding and other reversible interactions between the therapeutic and polymer system.^[137] In cases where the therapeutic cargo is significantly larger than the average mesh size or when the therapeutic has strong affinity to the polymer, degradation primarily mediates release (r_mesh/r_cargo<1).^[25] Factors influencing hydrogel degradation include the degree of susceptibility of the polymer to enzymatic, hydrolytic or thermal degradation. Degradation also typically occurs on the surface or throughout the hydrogel and results in loss of material from the polymer system.^[23] These parameters can help guide selection of polymer materials to deliver lymphangiogenic therapeutics. Here we consider different properties of hydrogel applications utilizing controlled therapeutic release strategies to promote lymphangiogenesis (Table 2).

Figure 4. Hydrogel Strategies to Provide Spatiotemporal Control over Therapeutic Release.

Therapeutic release from hydrogels is typically mediated via diffusion and degradation. Diffusion primarily mediates release when the therapeutic is significantly smaller than the mesh size of the hydrogel and lacks strong affinity to the polymer (A). Degradation is responsible for release when therapeutics are larger than the mesh size of the hydrogel or when tissue ingrowth is required to cleave bonds between the therapeutic and polymer (B).

Table 2:

Selected examples and properties of therapeutics delivered with biomaterials to promote lymphangiogenesis

Therapeutic	Biomaterial Carrier	Therapeutic Concentrations (μg/ml)	Isoelectric Point	Mass (kDa)	Hydrodyanmic Radius (nm) [138]
ANG-2	Hyaluran/ Methylcellulose	1.0-3.0 [139]	6.7 [140]	1.0	0.7
SDF-1α	PLGA	0.4 [141]	9.8 [142]	7.0-8.0	1.5-1.6
VEGF-C	Alginate	0.5-2.0 [143] 7.5 x 103 [18]	8.3 [144]	15.5-21.0	2.0-2.3
	Fibrin/Collagen	5.0-40.0 [145]
	Gelatin	1.0 x 103 [146, 147]
	Hyaluran/ Methylcellulose	0.2 [148] 1.0-3.0 [139]
VEGF-D	Alginate	0.5-2.0 [143]	6.2	12.0-20.0	1.8-2.2

Polymer systems that support a large range of mesh sizes and have affinity to the delivered therapeutic cargo have applications for delivering lymphangiogenic growth factors. An example of one such polymer is alginate, which is a naturally occurring anionic polysaccharide that has Food and Drug Administration (FDA) approval for food products, dental applications and wound dressings and has a long history of supporting biomedical therapies.^[106-108] Although alginate is biologically inert in mammals, alginate can be partially oxidized to allow for the polymer backbone to become susceptible to hydrolysis ^[149] and can be enzymatically degraded through the incorporation of exogenous alginate lyases ^[98] or polymer modification.^[103-105] Alginate has many applications for delivering VEGF members due to these hydrogels being nanoporous ^{[99, 102, 150]} and having affinity for heparin binding proteins including basic fibroblast growth factor and VEGF-A.^[151] Work applying alginate hydrogels to deliver VEGF-C and VEGF-D found that both growth factors experienced sustained release that could promote LEC sprouts in vitro and new blood vasculature in vivo via a chick chorioallantoic membrane (CAM) assay.^[143] Additionally, albumin-alginate microspheres loaded with VEGF-C_C152S, a mutated form of VEGF-C specific for rat VEGFR-3, were delivered into rat myocardium’s after inducing myocardial infarction where they led to an increase in lymphatic vessel density, decreased fibrosis and increased cardiac perfusion.^[18] Hydrogels can also be prepared from peptides, which can support nanoporous mesh sizes and can bind/stabilize delivered proteins depending on the utilized peptides.^{[152, 153]} Recent work using custom synthesized peptide hydrogels delivering VEGF-C provided sustained release of VEGF-C in vitro and was able to reduce edema and improve cardiac function when injected into the heart of a rat myocardial infraction model.^[152]

Another polymer that has applications for providing spatiotemporal control over VEGF-C delivery is gelatin. Gelatin is extracted from collagen and has FDA approval for a variety of applications including as a food product, cosmetic applications, drug capsules, surgical hemostasis sponges, ophthalmic demulcents and partial ossicular replacement prostheses.^[123-128] Gelatin hydrogels support nanoscale mesh sizes and can be either positively or negatively charged depending on the concentration and type of gelatin utilized.^{[120, 121, 154]} A study delivering VEGF-C found that decreasing the crosslinking density of gelatin hydrogels could lead to more rapid VEGF-C release and that gelatin hydrogels delivering VEGF-C promoted an increase in lymphatic vessels in a lymphedema mouse model.^[146] Another study delivering VEGF-C with gelatin hydrogels in combination with extracorporeal shock therapy induced lymphatic revascularization of the mid-thigh in the lymphedema mouse model.^[147] Although hydrogels with VEGF-C were found to promote lymphangiogenesis and reduce edema, the greatest effect was observed when combining both therapies.^[147]

Hydrogel delivery systems can be designed to release therapeutics as a function of hydrogel degradation. For instance, fibrin is a very commonly used natural biomaterial involved in blood clotting and is the product of the enzymatic cleavage of fibrinogen. Fibrinogen is a very large glycoprotein (350 kDa) that is produced by hepatocytes in the liver ^{[155, 156]}, has FDA approval as a sealant and is naturally degraded by plasmin following tissue regeneration.^{[119, 157, 158]} Although fibrin hydrogels typically have relatively larger mesh sizes compared to some of the other previously described polymer systems ^[99], fibrin contains many active sites for protein binding that can be utilized to mediate controlled release of therapeutics.^{[117, 159]} A recent study engineered a fibrin binding VEGF-C (FB-VEGF-C) variant with enzymatically degradable sequences to control FB-VEGF-C release from fibrin hydrogels.^[145] FB-VEGF-C was released from fibrin hydrogels as a function of fibrin degradation and experienced prolonged release compared to unmodified VEGF-C in vivo.^[145] Furthermore, fibrin hydrogels delivering FB-VEGF-C promoted lymphangiogenesis in a dose dependent manner in the ear of mice and stimulated increased wound healing in a diabetic mouse wound.^[145]

Other hydrogel delivery applications, including homing and dual factor release strategies, have also been used to promote new lymphatic vasculature. Poly(lactic-co-glycolic acid) (PLGA) is a anionic synthetic polymer that boasts many therapeutic applications and is comprised of both lactic and glycolic acid in varying ratios. PLGA is nonimmunogenic, has acceptable toxicity and has been approved by the FDA for a variety of clinical uses including surgical sutures, implants and drug delivery applications.^{[115, 132, 160]} Additionally, PLGA hydrogels typically are nanoporous and have easily adjustable mechanical properties via modifying the ratio of lactic to glycolic acid, with glycolic acid having a relatively faster degradation rate.^[161] Herein, any biomaterial application containing any ratio of glycolic to lactic acid will be referred in an over simplistic way as PLGA, including specific references to polyglycolic acid (PLG) and polylactic acid (PLA/PLLA). Work using PLGA scaffolds loaded with SDF-1α were implanted into the subcutaneous cavity of mice and substantially increased the number of mesenchymal stem cells (MSCs) at the implantation site.^[141] This homing strategy was also able to recruit hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs) when an osmotic pump delivered continuous SDF-1α, which promoted lymphangiogenesis and increased local proinflammatory cytokines associated with lymphatic vascularization.^[141] Other polymers used to deliver lymphangiogenic factors include methylcellulose and hyaluronan, which are polymers derived from constituents found in both plant and animal cells. Hydrogels incorporating methylcellulose and hyaluronan have been used to deliver VEGF-C or VEGF-C_156S via a transcranial injection to promote meningeal lymphatics as a potential strategy for treating neurological diseases.^[148] Another study also used methylcellulose and hyaluronan hydrogels to deliver VEGF-C_156S and angiotensin II (ANG-2).^[139] These hydrogels were found to protect the bioactivity of both factors, promote lymphangiogenesis and reduce edema in sheep after removing a lymph node.^[139]

Directing Tissue Growth

In certain situations, it may be desirable to provide very localized signals to specific cells that are in contact with the hydrogel while simultaneously minimizing the amount of stimulation to the surrounding tissue. These hydrogels often employ immobilized factors or control of mechanical properties including stiffness, crosslinking density, polymer orientation or cell adhesion sites to direct cell or tissue growth (Figure 5).^{[98, 162-165]} In general, these hydrogel applications can allow for long term therapeutic stimulation via protecting immobilized factors from internalization or adjusting degradation rates to maintain desirable mechanical properties.^{[98, 166, 167]} Hydrogels utilizing this strategy have applications for aligning, orienting and directing cell migration and driving stem cell differentiation to restore tissue function.^{[41, 168]} Here we describe different biomaterial applications for directing tissue growth for the purpose of promoting lymphangiogenesis (Table 3).

Figure 5. Designing Hydrogels to Direct Tissue Growth and Function.

Hydrogels can provide localized and long-term regeneration signals to tissue in contact with the biomaterial while simultaneously minimizing stimulation to surrounding tissue. These strategies typically employ immobilized factors to provide prolonged stimulation to native tissue (A) or alter local mechanical properties of the hydrogel to direct and align cells (B).

Table 3:

Selected examples of biomaterials designed to support lymphangiogenesis through promoting tissue ingrowth

Biomaterial Carrier	Targeted Cells	Predicted Biomaterial Mechanism	Intended Purpose	Reference
Collagen	LECs	Supports Sprout Ingrowth	Biological studies/Therapeutic screening	[169-172]
	LECs	Supports 3D microscale lymphatic vessels	Biological studies/Therapeutic screening	[173, 174]
	ECs	Interstitial flow through hydrogels	Biological studies	[175]
	LECs Caco-2	Supports coculture and interactions between both cell types	Biological studies/therapeutic screening of intestinal lymphatics	[176]
	LECs	PLGA nanofibers directed LEC migration	Directed lymphangiogenesis	[177]
	LECs	Promotes tissue regeneration and prevents scar formation	Topical dressing to promote lymphatic revascularization	[178, 179]
	LECs	Aligned nanofibers promotes LEC alignment/migration	Stimulate lymphatic regeneration	[180]
Fibrin	LECs	Supporting Sprout Ingrowth	Therapeutic screening	[143, 171, 181]
	ECs	Incorporates matrix bound VEGF and interstitial flow	Biological studies/Therapeutic screening	[182, 183]
	ECs	Varying hydrogel mechanical properties effect sprouting	Designing in vitro vascularized tissue	[184]
	ECs	Developing hydrogel channels to replicate lymphatic drainage	Designing engineered tissues	[185]
PLGA	LECs	Aligning polymers to direct LEC behavior	Repairing severed lymphatic vessels	[186]

Hydrogel systems that direct tissue growth should support interactions between the polymers and cells within native tissue. Fibrin is an example of a polymer that has many applications for supporting cell growth due to its natural role in tissue repair.^[187] Some of these advantages include having chemical and physical properties that can be altered by adjusting the ionic strength, pH and fibrinogen concentration as well as possessing many sites to promote cell adhesion.^[188] Studies seeding LECs on cytodex carrier beads and encapsulating the cell laden beads in fibrin gels found that fibrin supported LEC sprouting for potential therapeutic screening applications.^{[143, 171, 181]} Fibrin also has applications for immobilizing factors to provide long term stimulation to cells. Work utilizing a fibrin binding variant of VEGF-A₁₂₁ (FB-VEGF-A₁₂₁), an isoform of VEGF-A involved in promoting endothelial cell migration ^[189], found that LECs formed lymphatic capillaries to a much greater extent in fibrin gels with FB-VEGF-A₁₂₁.^{[182, 183]} Additional work applying either lymphatic or blood endothelial cells in fibrin hydrogels with FB-VEGF-A₁₂₁ found that LECs produced capillaries with fine lumens under the presence of interstitial flow.^[184] Interestingly, LECs preferred fibrin hydrogels with low hydraulic permeability and formed slender and overlapping networks, while blood endothelial cells preferred hydrogels with higher permeability and formed thick and branched vessels.^[184] Another study investigated controlling fibrin mechanical properties, including porosity, to design scaffolds to replicate lymphatic drainage for potential tissue engineering applications.^[185]

Another polymer with applications for directing lymphatic revascularization is collagen. Type I collagen consist of three polypeptide chains wrapped around each other and held together through hydrogen and covalent bonds. Collagen has FDA approval for a variety of uses including cosmetics, dental bone grafts, absorbable sutures and vascular grafts.^{[107, 112-116]} Collagen has many advantages as a biomaterial due to having cell adhesion sites, attunable mechanical properties with the addition of chemical or physical crosslinking methods ^[190] and is biodegradable due to its susceptibility to metalloproteases including collagenase.^[111] Studies found that LECs could invade into collagen hydrogels and form lumenized lymphatic capillaries, which could be enhanced in the presence of various factors including basic fibroblast growth factor, VEGF-A and heparin.^{[169, 172]} A more recent study showed that interstitial flow through collagen hydrogels could stimulate LEC microvascular organization.^[175] Other work has also shown that collagen hydrogels can support LECs to model sprout formation ^{[170, 171]}, microscale lymphatic vessels ^{[173, 174]} and the intestinal microenvironment ^[176] for potential therapeutic screening applications. Additional work utilizing collagen hydrogels to promote wound repair found that collagen hydrogels reduced scarring and fibrosis when applied in a mouse tail excision model, which resulted in decreased edema and increased local lymphatic vasculature in the tail.^{[178, 179]}

Hydrogels can also control LEC behavior through applying tight control of polymer alignment. For instance, parallel aligned collagen fibrils were found to preserve endothelial cell phenotype, provide LEC alignment and promote LEC migration along the fibrils.^[180] These parallel aligned collagen fibers significantly increased both blood and lymphatic vasculature density, led to an increase in lymphatic collector vessels around the scaffold and reduced bioimpedance in a porcine model of secondary lymphedema.^[180] Another polymer with applications for directing LEC migration is PLGA. PLGA hydrogels support a wide range of mechanical properties via varying the ratio and molecular weight of glycolic and lactic acid.^{[191, 192]} Work applying electrospun PLGA found that LECs could adhere and migrate along the fibers.^[186] The aligned PLGA fibers were determined to promote LEC migration, while randomly oriented polymers were found to inhibit migration.^[186] Combinations of PLGA fibers on the surface of collagen gels found that fibril diameter, orientation and cell adhesion properties can direct LEC growth and migration on the surface of the hydrogel.^[177]

Cell-based Therapies

Hydrogels have many applications for delivering cells to promote regeneration on the tissue level. Generally, cell-based strategies for lymphatic revascularization include delivering cells which directly incorporate into native vasculature or can exert their therapeutic benefit indirectly through paracrine signaling or tissue remodeling (Figure 6). Regardless of the therapeutic strategies, hydrogels should provide appropriate matrix-related signals necessary for cell adhesion, survival and function.^{[30, 34, 35]} This has led to many hydrogel applications with polymers that mimic the ECM or are naturally involved in wound healing. Additionally, hydrogel carriers should deliver a large number of cells while providing mechanical and chemical cues to maximize cell viability, direct cells towards the desired phenotype, promote encapsulated cell migration and allow for host tissue ingrowth.^[102] This has led to many applications of porous or pore forming hydrogels that have properties conducive to supporting cell survival, growth, differentiation and migration into native tissue. Here we present some hydrogel strategies to deliver cell-based therapies for lymphangiogenic applications (Table 4).

Figure 6. Hydrogels to Deliver Cell-based Therapies for Lymphatic Revascularization Applications.

Many different cellular based therapies have been delivered with biomaterials to promote lymphangiogenesis. These strategies include delivering LECs, which can directly interact and incorporate with native lymphatic vasculature. Hydrogels can also deliver cells which can support lymphatic revascularization through secreting paracrine signals, assisting in tissue remodeling and differentiating into mature LECs.

Table 4:

Selected examples of cell-based biomaterial strategies for lymphangiogenesis

Biomaterial Carrier	Delivered Cell Type	Therapeutic Concentrations (Cells/ml Hydrogel)	Predicted Lymphangiogenic Mechanism	Intended Purpose	Reference
Collagen	ECs	Not specified	Formed lymphatic vessels that support drainage	Vascularized scaffolds for tissue engineering applications	[193-195]
	Thoracic duct fragments	Not specified	3D matrix to Support Lumen Forming Capillaries	Identify Lymphangiogenesis Regulators	[196]
	Lymph node fragment	Not specified	Stimulate lymphatic regeneration	Direct lymphangiogenesis for secondary lymphedema	[180]
	Adipose tissue-derived microvascular fragments	Not specified	Rapidly assemble into vasculature networks	Implant for full-thickness skin defects	[197, 198]
	LECs Fibroblasts	6.0 x 1044.0 x 104	Creating prevascularized hydrogels with blood and lymphatic vessel	Skin grafts for severe skin defects	[199]
Fibrin	ECs Adipose-Derived Stromal Cells	Not specified	Creating prevascularized hydrogels with blood and lymphatic vessel	Drug screening and tissue engineering applications	[200]
	LECs Fibroblasts Keratinocytes	4.0-6.0 x 104 4.0 x 1041.0 x 106	Supports blood and lymphatic vessel that anastomosis in vivo	Skin grafts for severe skin defects	[199]
	LECs Fibroblasts	8.0 x 1061.0 x 107	Provides interstitial flow to promote lymphatic sprouting	Drug screening and biological studies application	[201]
Gelatin	Adipose-Derived Stem Cells	1.0 x 104	Paracrine signaling and lymphatic endothelial differentiation	Lymphangiogenesis for lymphedema treatment	[146]
Gelatin/PCL	MSCs	Not specified	Enhancing host cell survival and providing paracrine signaling	Therapy for myocardial infarction	[202]
Peptide	EPCs	1.7 x 107	Supporting EPC survival in hypoxia and incorporating into lymphatic vascular	Therapy for myocardial infarction	[152]
PLGA	Aortic Endothelial Cells Smooth Muscle Cells Skeletal Muscle Cells	Not specified	Supporting new vasculature	Promote lymphatic structures	[203]
	LECs	2.4 x 108	Allow for cell adhesion	Structure for new lymphatic vessels	[204]
	Intestinal Organoids	Not specified	Increase VEGFR-3 positive cells at scaffold	Developing lymphatic vessels in the neointestine	[205]

One appealing strategy to promote lymphatic regeneration is to develop hydrogel systems which can utilize ECM related signals to support cell-based therapies and direct new vasculature formation. This has resulted in many hydrogels that have polymers which are naturally derived from plant, animal or human tissue to recapitulate properties of the ECM and subsequently mimic key characteristics of native tissue.^[41] For instance, hydrogels made from collagen, which is the primary component of the ECM, supports cell adhesion, promotes cell survival, is readily remodeled by delivered cells or surrounding tissue and can form hydrogels with a large mesh size to support cell-based therapies.^{[109, 110, 193, 206]} Previous work has shown that collagen hydrogels can promote lymphatic vessels that can support drainage of fluid in vitro for potential regenerative tissue engineering applications.^[193-195] Another study seeded LECs and dermal fibroblasts into collagen hydrogels where the cells were able to form lumen-forming lymphatic capillaries within the hydrogels.^[199] Additionally, collagen hydrogels encapsulating a section of isolated mouse thoracic duct supported the formation of lumenized lymphatic capillaries in vitro and have applications for identifying new therapeutic and genetic lymphangiogenic regulators.^[196] Lymph node fragments have also been delivered with collagen to promote therapeutic lymphangiogenesis in a porcine model of secondary lymphedema.^[180] Furthermore, work using collagen scaffolds seeding adipose tissue-derived microvasculature fragments were able to form lymphatic networks when implanted into the dorsal skinfold ^[198] or full thickness skin defect model in mice.^[197] Gelatin also has many advantages as a biomaterial for cell delivery as gelatin shares many similar properties to collagen including containing peptides to support cell adhesion and allowing for chemical modification due to having diverse and accessible functional groups.^[207] Gelatin scaffolds delivering either ADSCs or VEGF-C were found to promote lymphangiogenesis in the hindlimb mouse model of lymphedema, though the combination of both led to the most increase in lymphatic vessel density and decrease in edema in the footpad.^[146] Furthermore, the ADSCs were speculated to provide paracrine signaling to promote lymphangiogenesis and were found to differentiate into a lymphatic lineage.^[146]

Other naturally derived polymers have also been used to design hydrogel systems to deliver cells to stimulate lymphatic vasculature. Fibrin hydrogels have many cell delivery applications due to it being biodegradable and containing ample adhesion sites for cell anchoring and proliferation, including the RGD amino acid sequences.^{[187, 208]} Additionally, neutrophils, fibroblasts and macrophages can infiltrate through fibrin to secrete ECM components including collagen and fibronectin, which can allow for natural ECM production and provide additional cellular adhesion sites.^[209] Previous work has found that LECs readily formed microvascular networks in fibrin, which could be enhanced by coculturing with adipose-derived stromal cells or the addition of exogenous VEGF-C.^[200] Other work delivering LECs with fibroblasts and keratinocytes showed that lumenized lymphatic capillaries formed within the fibrin hydrogels.^[199] Fibroblasts were believed to support lymphatic capillary formation through depositing ECM and insoluble factors.^[199] Additionally, these prevascularized fibrin hydrogels were also found to anastomose with native vasculature and support lymphatic drainage when implanted in a back wound in rats.^[199] Other work encapsulating fibroblasts in fibrin hydrogels within microfluidic devices found that LECs could form sprouts in vitro in the presence of lymphangiogenic factors and that LEC sprouting increased with interstitial flow.^[201] This microfluidic device also supported the incorporation of various factors for potential lymphangiogenic drug screening applications.^[201] Peptide based hydrogels have also been used to deliver cells to promote lymphatic vasculature as the peptides can be designed to promote cell adhesion, survival and allow for control over a variety of mechanical properties including degradation.^[153] One study used a peptide based hydrogel system to deliver EPCs in the heart of rats following myocardial infarction, were the EPCs incorporated into lymphatic vasculature and improve cardiac function.^[152] Interestingly, hydrogels co-delivering VEGF-C and EPCs resulted in the most lymphangiogenesis and recovery of cardiac function.^[152]

Synthetic hydrogels also have many applications for cell-based therapies to promote lymphatic vasculature due to having reproducible and readily controllable properties.^[41] One example of a synthetic polymer used to promote lymphangiogenesis is PLGA, which supports a wide range of controllable degradation rates, can be modified to attenuate cell adhesion ^[210] and is naturally removed from the body via the Kreb’s cycle/respiration.^{[160, 211, 212]} Some of the earliest work using PLGA scaffolds to transplant cells for revascularization applications were observed to support new lymphatics. Aortic endothelial cells, skeletal muscle cells or aortic smooth muscle cells were seeded into PLGA scaffolds and were implanted in the subcutaneous space in rats, where all three cell types led to an increase in lymphatic structures.^[203] More recent work has used PLGA scaffolds to transplant LECs, which allowed for the formation of a vessel like structure that expressed lymphatic genes when delivered into the caudal area of mice.^[204] PLGA and collagen scaffolds encapsulating intestinal organoid units consisting of mesenchymal cores and polarized epithelium were implanted into the omenta of rats.^[205] These transplanted cells led to an increase in VEGFR-3 positive vessels believed to be lymphatic at the site of the scaffold.^[205] Polycaprolactone (PCL) is another synthetic polymer that has been used to deliver stem cell therapies to promote lymphangiogenesis. PCL is synthesized from the ring opening of caprolactone or condensation of 6-hydroxycaproic (6-hydroxyhexanoic) acid and has high mechanical strength and slow degradation.^[213] Work delivering mesenchymal stem cells (MSCs) in patches with PCL and gelatin found that the delivered MSCs could promote new lymphatic vessels in a rat myocardial infarction model.^[202]

Conclusions and Future Directions

Therapeutic lymphangiogenesis holds significant potential to promote tissue regeneration due to the role lymphatics have in maintaining tissue homeostasis. This has led to interest in designing biomaterial delivery systems to stimulate lymphatic revascularization. In particular, hydrogels are an appealing therapeutic delivery vehicle due to their ability to incorporate a large range of biological agents, provide spatial and temporal control over therapeutic release and allow for a wide range of mechanical properties. However, despite the major advances in designing hydrogels that can provide sustained release of therapeutics, the ability to provide long term release of desired lymphangiogenic factors at physiologically relevant concentrations can still be challenging. One solution involves delivering gene therapies, which can provide continuous expression of therapeutics at a specifically targeted cell type or tissue to promote revascularization.^[214] An appealing gene therapy strategy involves direct gene delivery in vivo.^[215] This direct approach avoids the technical challenges with ex vivo manipulation, but at the loss of control over cell targeting and dosages.^[215] Applying hydrogel delivery systems for gene therapy can overcome some of these limitations by allowing for spatio-temporal control of gene delivery, maintaining sustained gene expression and minimizing copies of genes inserted into native cells.^[215-219] There are many studies utilizing genes encoding lymphangiogenic factors ^[220-226], however, to our knowledge, there has not been any research in utilizing hydrogels for the controlled delivery of lymphangiogenic gene therapies. Applying hydrogel systems to control the delivery of lymphangiogenic gene therapies could offer a safe and efficient strategy for providing sustained and controlled expression of lymphangiogenic factors in diseased tissue.

Biomaterials that mimic the extracellular environment have many applications for promoting tissue regeneration, but there are currently limited strategies to recapitulate the dynamic signals present in diseased tissue. The natural ECM provides adhesion signals that, in synergy with cellular and growth factor stimulus, influences endothelial cell behavior.^[227-232] Hydrogels that can simulate and control these ECM cues have many applications for driving tissue regeneration. However, there is dynamic regulation of the ECM in disease and healthy conditions that have an intrinsic role in stimulating tissue repair. Following injury, proteases will degrade the basement membrane, endothelial cells will migrate and respond to varying concentrations of both soluble and immobilized factors and support cells will assist in revascularization and remodeling the ECM.^[233] This has led to interest in developing biomaterials that can replicate this process by simulating ECM mechanical cues, providing a source of diffusible and immobile factors and supporting tissue remodeling for potential revasculature applications.^[233] Furthermore, developing biomaterial strategies that can replicate conditions present during development could also have applications for promoting more complex lymphatic vasculature, including lymphatic collecting vessels, which could have many important therapeutic applications for restoring lymphatic function in diseased tissue. Hydrogel strategies utilizing a combination of cell therapies, lymphangiogenic factors and dynamically regulated mechanical cues could provide a potential strategy to recapitulate the regenerative process of diseased tissue to promote lymphatic revascularization.

Biography

Kevin Campbell graduated from the University of California, Berkeley in 2013 and majored in Chemical Biology and minored in Bioengineering. He then attended California Polytechnic State University for a master’s in Biomedical Engineering where he did research on new therapies for skin cancer and computational modeling of therapeutic diffusion through the dermal microenvironment. In 2015, Kevin joined the PhD program in Biomedical Engineering in the University of California, Davis under the supervision of Prof. Eduardo Silva. His current research utilizes multiple disciplines from his background to model and design lymphatic based biomaterial delivery strategies for potential tissue regeneration applications.

Eduardo Silva is an associate professor at the department of Biomedical Engineering at the University of California, Davis. He received his PhD in Engineering Sciences: Bioengineering in 2008 while working in the laboratory of Prof. David Mooney, both at the University of Michigan and at Harvard University. After finishing his PhD, he was a postdoctoral fellow at Harvard University and was awarded a Harvard Wyss Technology Development fellowship. His research focuses on developing polymer systems to obtain control over multiple biological cues, including proteins, viral vectors and cells for the revascularization of ischemic tissues and other regenerative applications in the body.