Multi-parameter tunable synthetic matrix for engineering lymphatic vessels

Abstract

A hyaluronic acid based three dimensional bioengineered vessel includes living biological cells, and norbornene functionalized hyaluronic acid (Nor-HA) comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative, at least a portion of the plurality of norbornene moieties being crosslinked via a thiol linkage, respectively, to each of a thiol functionalized metalloproteinase (MMP) sensitive peptide, and a thiol functionalized RGD peptide to provide an HA-crosslinked hydrogel matrix that comprises HA- norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages, the matrix maintained in a culture medium comprising MV2 culture media and at least VEGF-C and Ang2. The three dimensional bioengineered vessel comprises one or more of individual tubes, branched tubes, lymphatic capillaries with button like junction, and lymphatic valve with zipper like junction, and can transport lymph fluid and immune cells.

Description

MULTI-PARAMETER TUNABLE SYNTHETIC MATRIX FOR ENGINEERING LYMPHATIC VESSELSSTATEMENT REGARDING FEDERAL FUNDING

[0001] This invention was made with government support under grants R35 GM143055 and R35 GM13798 awarded by the National Institutes of Health and grants 2047903 and 1944875 awarded by the National Science Foundation. The government has certain rights in the invention.

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 516,325, entitle MULTI-PARAMETER TUNABLE SYNTHETIC MATRIX, filed July 28, 2023, the entire contents of which is hereby incorporated by reference.

[0004] FIELD

[0005] The invention relates to formation of lymphatic vessels in a hyaluronic based hydrogel including peptide growth factors and culture media suitable to support cell growth and morphogenesis and vessel remodeling.

[0006] BACKGROUND

[0007] Lymphatic vasculature is responsible for extracellular fluid homeostasis, lipid transport, immune cell trafficking, and inflammatory responses. Consequently, the lymphatic system is associated with numerous conditions, including lymphedema, cardiovascular disease, neurodegenerative diseases, inflammation, and metabolic disorders. Despite the significant impact of the lymphatic system and the wide spectrum of diseases it is implicated with, current treatments remain non-existent or very transient. Compared to blood vasculature, the field of lymphatic biology is still very nascent, and many molecular mechanisms and regulators of lymphatic growth, maintenance, and disease remain unknown. Controlling the formation of new lymphatic vessels, whether in vivo or through engineering approaches, has been postulated as a novel long-term therapeutic strategy for treating various disease phenotypes including primary and secondary lymphedema, lymphatic malformations, cardiovascular disease, and metabolic diseases.

[0008] While there have been previous approaches to generate lymphatic vessels in vitro for various applications, they require the use of natural matrices and / or co-culture with other supporting cells. In the case of natural matrices, different mixtures of fibrin and collagen gels were used to investigate lymphatic vessel formation stimulated by vascular endothelial growth factor C(VEGF-C) and interstitial flow. Fibrin gels have also been used to generate dermal skin grafts with blood and lymphatic capillaries to support fluid drainage. Another approach utilized a microfluidic-based system with a fibrin hydrogel to show that the addition of interstitial flow, combined with a cocktail of growth factors, was able to develop in vivo-like capillaries to investigate lymphatic transport. While natural gels like collagen and fibrin can support lymphatic vessel formation, their inherent chemical and physical properties may limit the ability to systematically study the influence of matrix properties on lymphatic vessel formation. To control the matrix properties more fully, a poly(ethylene glycol) (PEG)-based hydrogel system was developed where parameters were independently tuned to engineer a synthetic environment that fostered sprouting of implanted segments of excised rat collecting lymphatic vessels. Another study utilized poly(lactide-co-glycolide) PLGA scaffolds to co-culture lymphatic endothelial cells (Lymphatic endothelial cells) and dental pulp stem cells to generate lymphatic vessel networks. Nonetheless, many of these synthetic hydrogels require co-culture of lymphatic endothelial cells with other supporting cells to promote lymphatic vessel formation.

[0009] Therefore, there is a need for tunable synthetic hydrogels that can generate lymphatic networks from endothelial cells alone, which can be used to decouple contributions of various cellular components, as well as chemical and mechanical regulators.

[0010] SUMMARY

[0011] Controlling the formation of new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Utilizing hyaluronic acid (HA)- hydrogels a viable lymphatic vessel transplant was developed wherein the HA-hydrogels were designed with the ability to spatially and temporally pattern specific peptides and substrate stiffnesses. A matrix metalloproteinase (MMPs)-sensitive crosslinker allowed cells to degrade and remodel their matrix and the incorporation of a RGD binding motif provided sites for cell attachment. The HA-hydrogels are demonstrated herein as versatile and unique matrices that preserve key lymphatic markers Proxl, LYVE-1, and Pdpn, and controlled matrix stiffness is shown to prime lymphatic tube formation and RGD is required for cell elongation and branching.

[0012] As shown in the examples, VEGF-C and Ang2 synergistically enhance lymphatic tube formation. By three days post-encapsulation, mature vessels with lumens can be observed and stable up to 3-weeks during in vitro culture. Upon transplantation into immunodeficient mice, the engineered lymphatic networks integrated with the host lymphatic system and continued to mature,as evidenced by fluorescent staining at two weeks post-implantation. Collectively, this disclosure provides a novel 3D synthetic matrix system that can be used for higher-throughput in vitro mechanistic studies as well as implanted in vivo for regenerative medicine applications such as vascularizing organoids and transplantation therapies for lymphatic dysfunction.

[0013] In various embodiments, the disclosure provides a method for forming hyaluronic acid based three dimensional bioengineered lymphatic vessels, the method including providing a biological matrix generative system for forming an hyaluronic acid hydrogel. The biological matrix generative system including norbornene functionalized hyaluronic acid (Nor-HA), the Nor- HA comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative, at least one peptide crosslinker selected from the group consisting of thiol functionalized metalloproteinase (MMP) sensitive peptides, at least one celladhesive peptide, wherein the at least one cell-adhesive peptide is thiol functionalized, and at least one photoinitiator for initiating photo-crosslinking. The method further including providing living biological cells comprising at least one cultured animal cell, mixing the components of the provided biological matrix generative system and the living biological cells and irradiating with UV light 320-390 nm to crosslink HA bound norbornene moieties of the Nor-HA with the peptide crosslinker to and to crosslink HA bound norbornene moieties of the Nor-HA with the celladhesive peptide to form an HA-crosslinked hydrogel matrix comprising HA-norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages. The method further including providing peptide growth factor comprising at least VEGF-C, and maintaining the combined HA-crosslinked hydrogel matrix in culture supplemented with the provided at least one peptide growth factor, whereby the hyaluronic acid based three dimensional bioengineered lymphatic vessels are formed.

[0014] In some embodiments, the living biological cells comprise mammalian cells selected from the group consisting of human lymphatic endothelial cells, lymphatic endothelial cells, lymphatic muscle cells, fibroblasts, endothelial progenitor cells, stem cell derived endothelial cells, and combinations thereof.

[0015] In some embodiments, mechanical properties of the bioengineered vessel are tuned or modulated by varying one or more parameters selected from the group consisting of: extent of HA binding site availability corresponding to degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA, concentration of the at least one peptide crosslinker, concentration of cell adhesive peptide binding sites on the Nor-HA corresponding to a concentration of the atleast one cell-adhesive peptide wherein the cell adhesive peptide is bound to the HA via a norbornene moiety, concentration of the living biological cells, one or more of concentration, frequency of addition, and combinations of these with respect to peptide growth factor supplementation, duration of time of exposure to UV radiation, concentration of the at least one peptide crosslinker, crosslinking chemistry of the at least one peptide crosslinker, and combinations thereof.

[0016] In some embodiments, the hyaluronic acid derivative comprises sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa, the at least one peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQGJ.IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase, the at least one cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6), the living biological cells comprise human lymphatic endothelial cells, and the at least one peptide growth factor comprises angiopoietin 2 (ang2), and optionally comprises at least one other peptide growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF- C156S, fibroblast growth factor-2 (FGF-2), angiopoietin- 1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1 -phosphate (SIP), and combinations thereof.

[0017] In some embodiments, the human lymphatic endothelial cells self-assemble into vessels in vitro within three (3) days, and wherein the presence of the degradable crosslinker allows the living biological cells to remodel the HA-crosslinked hydrogel matrix.

[0018] Also provided, in various embodiments, is a biological matrix generative system for forming a hyaluronic acid based three dimensional bioengineered lymphatic vessel, the system including norbornene functionalized hyaluronic acid (Nor-HA), the Nor-HA comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative, at least one peptide crosslinker selected from the group consisting of thiol functionalized metalloproteinase (MMP) sensitive peptides, at least one thiol functionalized cell-adhesive peptide, living biological cells comprising at least one cultured animal cell, at least one peptide growth factor comprising at least VEGF-C, and optionally at least one additional peptide growth factor selected from the group consisting of ang2, VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-C156S, fibroblast growth factor-2 (FGF-2), angiopoietin- 1, angiopoietin-3,endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1 -phosphate (SIP), and combinations thereof, and at least one photoinitiator for initiating photo-crosslinking, wherein the matrix generative system is employed to form an HA hydrogel matrix by means of UV photocrosslinking to crosslink HA bound norbornene moieties of the Nor-HA with the peptide crosslinker and to crosslink HA bound norbornene moieties of the Nor-HA with the cell adhesive peptide, and wherein the HA hydrogel matrix, when formed, comprises HA-norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages.

[0019] In some embodiments, the hyaluronic acid derivative is selected from the group consisting of sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa, hyaluronic acid derivatives compatible with norbornene chemistry, and combinations thereof.

[0020] In some embodiments, the at least one peptide crosslinker comprising an MMP sensitive peptide comprises an amino acid having susceptibility to cleavage by an MMP selected from the group consisting of MMP- 1, MMP-2, MMP- 14, and MMP-9.

[0021] In some embodiments, the at least one cell-adhesive peptide comprises an amino acid sequence selected from the group consisting of GCGYGRGDSPG (SEQ ID NO: 6), RGD(S) (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), YIGSR (SEQ ID NO: 9), PDSGR (SEQ ID NO: 10), RNIAEIIKDI (SEQ ID NO: 11), RYWLPR (SEQ ID NO: 12), POG (SEQ ID NO: 13), DGEA (SEQ ID NO: 14), GFOGER (SEQ ID NO: 15), VFDNFVLK (SEQ ID NO: 16), FPGERGVEGPGP (SEQ ID NO: 17), and HAV(DI) (SEQ ID NO: 18).

[0022] In some embodiments, the living biological cells comprise mammalian cells selected from the group consisting of human lymphatic endothelial cells, lymphatic endothelial cells, lymphatic muscle cells, fibroblasts, endothelial progenitor cells, stem cell derived endothelial cells, and combinations thereof.

[0023] In some embodiments, the at least one peptide growth factor comprises at least VEGF-C and ang2, and optionally comprises at least one other peptide growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-C156S, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain- containing protein 1 (CCBE1), Sphingosine 1 -phosphate (SIP), and combinations thereof.

[0024] In some embodiments, the hyaluronic acid derivative comprises sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa.

[0025] In some embodiments, the at least one peptide crosslinker comprises an MMP sensitive peptide comprising an amino acid sequence selected from the group consisting of GCRDGPQG / IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase, PLGL (SEQ ID NO: 2), GPQGIWCQ (SEQ ID NO: 3), PVGLIG (SEQ ID NO: 4), and GTAGLIGQ(SEQ ID NO: 5).

[0026] In some embodiments, the at least one peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQG / IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase.

[0027] In some embodiments, the at least one cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6).

[0028] In some embodiments, the at least one peptide growth factor comprises Ang2.

[0029] In some embodiments, the hyaluronic acid derivative comprises sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa, the at least one peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQG / IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase, the at least one cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6), the living biological cells comprise human lymphatic endothelial cells, and the at least one peptide growth factor comprises angiopoietin 2 (ang2), and optionally comprises at least one other peptide growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF- C156S, fibroblast growth factor-2 (FGF-2), angiopoietin- 1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1 -phosphate (SIP), and combinations thereof.

[0030] Also provided, in various embodiments, is an hyaluronic acid based three dimensional bioengineered vessel, including living biological cells, and norbornene functionalized hyaluronic acid (Nor-HA) comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative, at least a portion of the plurality of norbornene moieties being crosslinked via a thiol linkage to a thiol functionalized metalloproteinase (MMP) sensitive peptide, and at least a portion of the norbornene moieties being crosslinked via a thiollinkage to a thiol functionalized RGD peptide to provide a HA-crosslinked hydrogel matrix that comprises HA-norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages; and culture medium comprising MV2 culture media and at least VEGF-C and ang2, wherein the three dimensional bioengineered vessel comprises one or more of individual tubes, branched tubes, lymphatic capillaries with button like junction, and lymphatic valve with zipper like junction, and wherein the three dimensional bioengineered vessel can transport lymph fluid and immune cells.

[0031] In some embodiments, the hyaluronic acid based three dimensional bioengineered vessel includes human lymphatic endothelial cells.

[0032] In some embodiments, the Nor-HA comprises about 1% to about 3%, by weight of the HA hydrogel.

[0033] In some embodiments, a degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises from about 10% to about 75%.

[0034] In some embodiments, the degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises about 21%.

[0035] In some embodiments, the thiol functionalized RGD peptide is present in an amount from about 1 mM to about 10 mM, and the thiol functionalized MMP sensitive peptide comprises about 0.01% to about 0.1%, by weight of the HA hydrogel.

[0036] In some embodiments, the lymphatic vessels demonstrate genetic expression of lymphatic markers, Proxl, LYVE-1, and Pdpn, and can be maintained up to 22 days in culture in an endothelial culture medium.

[0037] In some embodiments, the culture medium does not include support cells comprising any of cells selected from fibroblasts, endothelial progenitor cells, dermal pulp stem cells, or other supporting cells.

[0038] DESCRIPTION OF THE DRAWINGS

[0039] Figure 1. (A) Norbornene groups were functionalized along the HA backbone in low, medium, or high frequency based on parameters during the synthesis process. (B) Lyophilized Nor-HA polymer was dissolved in complete cell media, along with Irgacure 2959, a RGD peptide (GCGYGRGDSPG), and an MMP-sensitive crosslinker (GCRDGPQGJ.IWGQDRCG; down arrow indicates the site of proteolytic cleavage). The pre-polymer solution was then cast in a cylindrical mold and crosslinked with UV light to yield a hydrogel with human lymphaticendothelial cells encapsulated throughout. (C) Several days after encapsulation, lymphatic endothelial cells self-assembled into vessels to varying extents based on the hydrogel conditions. These engineered in vitro constructs, created with a fully defined matrix and growth factor components along with only human lymphatic endothelial cells and no feeder layers or support cells, can be used in mechanistic and growth factor screenings, for disease modeling, and as a therapeutic transplant.

[0040] Figure 2. Hydrogels were lysed at day five (5) and RNA was purified for real-time qRT- PCR. lymphatic endothelial cells encapsulated in low modification (21% D.S.) gels showed increased expression of LYVE-1 (A), Proxl (B), and Podoplanin (C), compared to lymphatic endothelial cells cultured in high modification (63% D.S.) gels. Interestingly, while LYVE-1 expression increased, its homologue CD44 exhibited no change in expression (D). Encoding for VEGFR-3, Flt4 exhibited very minor differences in expression (E), as hypothesized due to the uniform mechanics across %D.S. conditions. A co-receptor of VEGFR-3, Nrp2 expression also exhibited very minor differences in expression (F). Staining lymphatic tubes within the hydrogels for DAPI and LYVE-1 demonstrated that 21% D.S. gels (G) fostered more sprouting than the 44% D.S. gels (H). Differential gene expressions as a result of using different %D.S. polymers was analyzed using unpaired t-tests. Significance levels were set at ns>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar: 100pm.

[0041] Figure 3. Hydrogel constructs were created with identical parameters, except for the amount of RGD peptide incorporated. Gels made with 1 mM RGD (A), 3 mM RGD (B), 5 mM RGD (C), and 7 mM RGD (D) were fixed at day five (5) for immunofluor escent imaging to observe the extent of tube formation and branching. Scale bar: 100pm. To investigate specific integrin subunits and their role during the kinetics of lymphatic tube formation, n=4 hydrogels were pooled, and RNA was isolated from gels at 24, 48, and 72hrs and expression of ITGA9 (E), ITGAv (F), ITGB1 (G), and ITGA5 (H) was measured by qRT-PCR. 5 mM RGD served as a scrambled, negative control.

[0042] Figure 4. Hydrogel constructs were fixed at day five (5) and immunofluorescently stained to visualize lymphatic tubes. Hydrogels were created with identical parameters except for the concentration of VEGF-C supplemented. When no VEGF-C was added (A), lymphatic endothelial cells only had 0.5ng / mL of VEGF-A from the complete media and only a few cells demonstrate short sprouts. The addition of lOng / mL VEGF-C (B) results in some lymphatic endothelial cells beginning to form tubes, but a large proportion of lymphatic endothelial cells remain unassembled.Upon addition of 50ng / mL VEGF-C (C), long and numerous lymphatic tubes can be observed. Supplementation of lOOng / mL VEGF-C (D) qualitatively yields a few additional sprouts, compared to 50ng / mL VEGF-C but significant differences between those two conditions are not observed. Scale bar: 100pm.

[0043] Figure 5. Hydrogels were fabricated with varied crosslinker concentrations to precisely tune the mechanical properties. (A) Crosslinker concentrations corresponding to 10%, 25%, 50%, and 75% of the total available norbornene arms (of low modification Nor-HA) were used and mechanical properties were measured, reporting bulk moduli (G’) of 103±18.0Pa, 548±158Pa, 868±75.9Pa, and 1800±789Pa, respectively. Linear regression analysis reported a high coefficient of determination (R2=0.96), indicating that the mechanical properties of this hydrogel system can be precisely tuned through modulation of the crosslinker concentration. Samples were immunofluorescently stained and imaged at day five (5) post-encapsulation to evaluate how matrix mechanics influence vessel formation, demonstrating that lymphatic endothelial cells assemble and start forming capillaries in (B) 25% crosslinked gels, while they remain dispersed and nonbranched in (C) 50% crosslinked and (D) 75% crosslinked gels. Initial screenings revealed that 10% crosslinking was too soft beyond day three (3) to support LEC branching. To examine the role of key matrix remodeling genes during the initial periods of LEC sprouting and branching, hydrogels were collected (n=4, pooled) and lysed at 24, 48, and 72hrs and compared against gels that were crosslinked with the non-degradable crosslinker DTT. qRT-PCR revealed that stiffer environments, higher crosslinking percentages, resulted in lymphatic endothelial cells upregulating expression of (E) MMP-2 and (F) MMP-14 in an initial burst, suggesting that lymphatic endothelial cells are sensing and responding to their environment and regulating expression of MMPs accordingly to attempt to compensate for the mechanical environment. Scale bar: 100pm.

[0044] Figure 6. Hydrogel constructs were fixed at day five (5) and immunofluorescently stained to visualize lymphatic tubes. Hydrogels were created with identical parameters except for the concentration of Ang2 and VEGF-C supplemented. When only Ang2 was added, albeit a very high concentration, no tube development is observed (A). When Ang2 is supplemented with 50ng / mL VEGF-C, increased sprouting is observed with 400ng / mL Ang2 (B) and even greater sprouting is observed with higher concentrations of Ang2 (C), indicating a synergistic signaling effect. Reference images for 50ng / mL VEGF-C without Ang2 are included (D) from Figure 4.4 for ease of comparison. Scale bar: 100pm.

[0045] Figure 7. (A) lymphatic endothelial cells were encapsulated during hydrogel fabrication and cultured in VEGF-C supplemented media for two (2) days to allow lymphatic endothelial cells to begin to elongate and self-assemble, whereafter they were (B) implanted in the pocket of mammary gland 4 in NOD / SCID female mice. Two weeks post-implantation, two lectin dyes to label endothelial cells (ECs) of each species were injected into the extracellular space of the hindfoot for uptake to the lymphatic circulatory system before tissue harvest. The fluorescein- conjugated Isolectin Griffonia simplicifolia (GS-IB 4 isolectin) stained mouse ECs and the rhodamine-conjugated Ulex Europaeus Agglutinin I (UEA-1 lectin) stained the human lymphatic endothelial cells. The hydrogel and surrounding tissue of the mammary gland were harvested, fixed, and imaged to assess integration. (C) Hydrogels were implanted in a pocket made in the #4 mammary gland where the vasculature is readily visible in a close proximity to the lymph node (LN), two (2) weeks post-implantation, then hydrogels and the surrounding tissue were harvested to visualize mouse and human ECs. Mouse ECs are stained in green with GS-IB4 isolectin and human lymphatic endothelial cells are stained in red with UEA-1 lectin. (D) A lOx overview of the hydrogel showing that the human lymphatic tubes (red) continue to grow once implanted, and mouse vasculature (green) can be seen invading the hydrogel region. Scale bar: 200pm. (E) Higher magnification imaging shows that the implanted vessels integrate with the host vasculature, as indicated by the arrows pointing to areas of fluorescent signal overlap which corresponds to both mouse and human ECs being present there. Imaris renderings of confocal z-stacks reveals that the vessels exhibit lumens (F), highlighted with the dashed circle, and that the two species of ECs wrap around each other and truly form integrated vessels (G and H), supporting the overlapping fluorescent signals observed in (E). Scale bars for E-H: 50pm.

[0046] Figure 8. (A) Two-step reaction to generate Nor-HA using an HA-TBA intermediate. (B) IH NMR of HA-TBA.

[0047] Figure 9. 1H NMR of Nor-HA with (A) 21% degree of substitution, (B) 44% degree of substitution, and (C) 63% degree of substitution.

[0048] Figure 10. To investigate how cell density affects lymphatic tube formation and to determine what cell density was a critical minimum for this system, hydrogels were fabricated with varying cell densities. At day 5, hydrogels were fixed and immunofluorescently stained to visualize tubes. A density of 8 million lymphatic endothelial cells / mL was determined to be the critical minimum.

[0049] Figure 11. To investigate the role of integrin binding site availability for lymphatic tube formation, hydrogels were fabricated with varied concentrations of RGD peptides incorporated. When no RGD was incorporated (A), no cell elongation or tube formation is observed, demonstrating that integrin binding sites are required for tube formation. When 5 mM of a scrambled peptide, RDG, was incorporated as a negative control (B), no tube formation was observed, indicating that the tube formation observed in Figure 4 is due to interaction with the specific RGD sequence. Scale bar: 100pm.

[0050] Figure 12. To investigate the influence of RGD (A) and MMP-sensitive peptide (B) concentrations on the expression of key lymphatic markers, hydrogels were fabricated with varied concentrations of the respective peptide. Hydrogels were lysed at day five (5) and RNA was purified for real-time qRT-PCR. Differential gene expression was analyzed using unpaired t-tests. Significance levels were set at ns>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0051] Figure 13. To investigate cell viability during hydrogel fabrication and lymphatic tube formation, hydrogels were fabricated with an embodiment of optimized conditions (21% D.S., 5 mM RGD, and 25% crosslinker). On days 1, 3, and 5, hydrogels were stained and imaged using a LIVE / DEAD assay to qualitatively assess cell death. While a substantial number of cells stained red (dead) on day 1, the amount of dead cells remained relatively similar across days, suggesting that the UV crosslinking process on day zero (0) contributes to a majority of the cell death but that cell viability is stabilized after that. Scale bars are 100 micro m.

[0052] Figure 14. Hydrogels fabricated with an embodiment of optimized parameters were cultured long-term and visually inspected daily to monitor how long they were stable for. Media was replaced daily. After 22 days of culture, vessels could still be observed and hydrogels were fixed for immunofluorescent staining. Staining for DAPI, f-actin, and VE-Cadherin outlined the vessels and demonstrated that vessels were still throughout the hydrogel after 3 weeks of in vitro culture. Scale bar: 100pm.

[0053] Figure 15. To assess perfusion of the engineered lymphatic networks, a p-Slide Chemotaxis (ibidi) chip (A) was used. The schematic of the chip was adapted from the user manual. The hydrogel pre-polymer solution was injected into the center channel and then UV crosslinked. (B) At day five (5) of culture, brightfield images show robust lymphatic networks formed in the center channel. (C) To assess perfusion of these networks, FluoSpheres Polystyrene Microspheres (1pm in diameter, red fluorescent) were added to one side channel. Confocalmicroscopy indicated that these fluorescent beads were able to travel into the networks, indicating that the networks are perfusable.

[0054] Figure 16. To investigate the specific role of VEGF-C concentration and to also investigate the difference of using VEGF-C versus VEGF-C156S, a lymphangiogenesis-specific form that only activates VEGFR3, hydrogels with identical parameters were fabricated and cultured in the respective growth factor supplemented media. After three (3) days of culture, hydrogels (n=3, pooled) were lysed and real-time qRT-PCR was run to examine key lymphatic genes including (A)LYVE-l, (B)Proxl, (C)Flt4, (D)Nrp2, (E)Ang2, and (F)CDH5. Unpaired t-tests were used to compare VEGF-C vs VEGF-C156S. Significance levels were set at ns>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0055] Figure 17: Table 1. Primers used in the studies.

[0056] Figure 18: Table 2. Immunohistochemical and Immunofluorescence Staining Antibodies used in the studies.

[0057] DESCRIPTION

[0058] The inventors have engineered multi-parameter tunable synthetic HA-hydrogel matrices which can be used to generate lymphatic vessels from lymphatic endothelial cells alone (i.e., without the need for any other cells) by tuning one or more individual components of the system. Since lymphatic endothelial cells uniquely express lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), hyaluronic acid was used as a template, which can be systematically modified with integrin binding motifs and matrix metalloproteinase (MMP)-sensitive crosslinkers. As shown in the examples herein, the inventors have shown report that matrix composition, growth factor concentration, and matrix mechanics all individually contribute to lymphatic vessel growth. The inventive system allows for independent modulation of parameters while keeping other factors fixed, highlighting the novelty and elegance of this system in which it is possible to directly investigate the effect of matrix composition, mechanics, and growth factors which is not achievable using natural biomaterials of mouse models where factors cannot be uncoupled. These engineered lymphatic networks made of only human lymphatic endothelial cells in a fully defined matrix can be cultured in vitro for at least 3 weeks and also demonstrate the ability to integrate with host vasculature upon implantation into immunodeficient mice.

[0059] The examples herein demonstrate that this novel HA-hydrogel system preserves key lymphatic markers, Proxl, LYVE-1, and Pdpn, and that HA binding site availability can be tuned to modulate expression of key lymphatic markers. As reported herein, RGD binding siteconcentration can be tuned to promote and increase lymphatic branching. Also reported herein, screening a range of matrix stiffnesses revealed that the mechanical environment primes lymphatic endothelial cells for tube formation, and that there is a critical maximum where beyond that, lymphatic endothelial cells will not form tubes. Supplementation of the pro-lymphangiogenic growth factor VEGF-C resulted in increased tube formation and branching, and the addition of Ang2 demonstrates a synergistic signaling effect between these two main pathways of lymphatic development.

[0060] Biological Matrix Generative System For Forming A Hyaluronic Acid Based Three Dimensional Bioengineered Lymphatic Vessel

[0061] In various embodiments, the disclosure provides a biological matrix generative system for forming a hyaluronic acid based three dimensional bioengineered lymphatic vessel, wherein the matrix generative system is employed to form an HA hydrogel matrix by means of UV photocrosslinking to crosslink HA bound norbornene moieties of the Nor-HA with the peptide crosslinker and to crosslink HA bound norbornene moieties of the Nor-HA with the cell adhesive peptide, and wherein the HA hydrogel matrix, when formed, comprises HA-norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages.

[0062] Norbornene functionalized hyaluronic acid (Nor-HA)

[0063] In various embodiments, the matrix generative system includes hyaluronic acid functionalized with norbornene moieties.

[0064] As used herein, “hyaluronic acid (HA)” refers to a polymer having a repeat unit represented by the following General Formula 1 unless otherwise indicated, and is used with a meaning encompassing a salt or derivative of hyaluronic acid.

[0065] General Formula 1

[0066] In General Formula 1, n may be an integer ranging from 50 to 10,000.

[0067] Hyaluronic acid is present in most animals and is a linear polysaccharide polymer with biodegradability, biocompatibility, and no immune responses, and thus may be safely applied to the human body. Since hyaluronic acid plays a number of different roles in the body depending on its molecular weight, it may be used for a variety of uses.

[0068] In the present invention, the “hyaluronic acid derivative” refers to all of the modified forms of hyaluronic acid based on the basic structure of hyaluronic acid of General Formula 1, into which a functional group is introduced.

[0069] In various embodiments, the hyaluronic acid includes hyaluronic acid and chemically attached moieties of norbornene, the synthesis of which is described in the examples and materials and methods herein.

[0070] In some embodiments, the hyaluronic acid derivative is selected from the group consisting of sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa, hyaluronic acid derivatives compatible with norbornene chemistry, and combinations thereof.

[0071] As used herein, the hyaluronic acid, salt of hyaluronic acid, or derivative of hyaluronic acid is not limited in configuration thereof, but the molecular weight thereof preferably ranges from 10,000 to 3,000,000 Dalton (Da).

[0072] In some embodiments, as exemplified herein, the hyaluronic acid is sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa.

[0073] Peptide Crosslinker

[0074] In various embodiments, the matrix generative system includes a peptide crosslinker selected from the group consisting of thiol functionalized metalloproteinase (MMP) sensitive peptides.

[0075] In some embodiments, the peptide crosslinker includes an MMP sensitive peptide comprises an amino acid having susceptibility to cleavage by an MMP selected from the group consisting of MMP- 1, MMP-2, and MMP-9.

[0076] In some embodiments, the at least one peptide crosslinker comprises an MMP sensitive peptide comprising an amino acid sequence selected from the group consisting of GCRDGPQG / IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase, PLGL (SEQ ID NO: 2), GPQGIWCQ (SEQ ID NO: 3), PVGLIG (SEQ ID NO: 4), and GTAGLIGQ(SEQ ID NO: 5).

[0077] In some embodiments the at least one peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQG^IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase.

[0078] As exemplified herein the peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQGJ.IWGQDRCG (SEQ ID NO: 1) is represented by the structure below Formula 2

[0079] Formula 2

[0080] Cell-Adhesive Peptide

[0081] In various embodiments, the matrix generative system includes at least one thiol functionalized cell-adhesive peptide.

[0082] In some embodiments the at least one cell-adhesive peptide comprises an amino acid sequence selected from the group consisting of GCGYGRGDSPG (SEQ ID NO: 6), RGD(S) (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), YIGSR (SEQ ID NO: 9), PDSGR (SEQ ID NO: 10), RNIAEIIKDI (SEQ ID NO: 11), RYWLPR (SEQ ID NO: 12), POG (SEQ ID NO: 13), DGEA (SEQ ID NO: 14), GFOGER (SEQ ID NO: 15), VFDNFVLK (SEQ ID NO: 16), FPGERGVEGPGP (SEQ ID NO: 17), and HAV(DI) (SEQ ID NO: 18).

[0083] In some embodiments the at least one cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6).

[0084] As exemplified herein the cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6) is represented by the structure below Formula 3.

[0085] Formula 3

[0086] Cultured Animal Cell (lymphatic origin)

[0087] In various embodiments, the matrix generative system includes at least one cultured animal cell, in particular of lymphatic origin.

[0088] In various embodiments, the living biological cells comprise mammalian cells selected from the group consisting of human lymphatic endothelial cells, lymphatic endothelial cells, lymphatic muscle cells, stem cell derived endothelial cells, fibroblasts, endothelial progenitor cells, and combinations thereof.

[0089] In some particular embodiments, the living biological cells comprise human lymphatic endothelial cells.

[0090] Peptide Growth Factor

[0091] In various embodiments, the matrix generative system includes at least one peptide growth factor comprising at least VEGF-C. In some embodiments, the matrix generative system includes at least one additional peptide growth factor comprising ang2.

[0092] In some embodiments, the matrix generative system includes at least one additional peptide growth factor selected from the group consisting of the at least one peptide growth factor comprises VEGF-C, and optionally comprises at least one other peptide growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-C156S, angiopoietin 2 (Ang2), angiopoietin-1 (Angl), angiopoietin-3 (Ang3), fibroblast growth factor-2 (FGF-2), endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1 -phosphate (SIP), and combinations thereof.

[0093] Method For Forming Hyaluronic Acid Based Three Dimensional Bioengineered Lymphatic Vessels

[0094] In various embodiments, the disclosure provides a method for forming hyaluronic acid based three dimensional bioengineered lymphatic vessels. The method includes providing the matrix generative system as described herein above, including at least one photoinitiator for initiating photo-crosslinking.

[0095] According to the method, the components of the provided biological matrix generative system are combined and subjected to UV irradiation generally in the range from 320 to about 390 nm to crosslink HA bound norbornene moieties of the Nor-HA with the peptide crosslinker to and to crosslink HA bound norbornene moieties of the Nor-HA with the cell-adhesive peptide. Theformed HA-crosslinked hydrogel matrix is a matrix, as depicted in Figure 1, which includes HA- norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages. The method further includes providing peptide growth factor comprising at least VEGF-C, and maintaining the combined HA-crosslinked hydrogel matrix in culture supplemented with the provided at least one peptide growth factor, for example according to the culture methods as described in the examples herein, whereby the hyaluronic acid based three dimensional bioengineered lymphatic vessels are formed.

[0096] Successful lymphatic vessel formation is demonstrated by assembly of the cells, for example human lymphatic endothelial cells as exemplified herein, into vessels in vitro within three (3) days after initial culture. Also as demonstrated herein, the vessels are characterized by biological remodeling owing to the presence of the MMP degradable crosslinker which is susceptible to MMP produced by the cells. Of course, it is possible to also utilize exogenously provided MMP to accomplish crosslinker degradation. According to the exemplified embodiments, exogenously provided MMP is not included in the method.

[0097] In various embodiments, the method includes deliberately modulating features of the biological matrix generative system and method to “tune” or vary mechanical and chemical properties of the novel matrix.

[0098] As more specifically illustrated in the examples herein, the features listed herein below may be modulated.

[0099] In various embodiments, the extent of HA binding site availability corresponding to degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA may be varied. Reference may be made to the specific Examples 1 , 2 and 5 herein, which demonstrate tunability of the novel matrix with respect to HA binding sites available for interaction with cells. As more Nor group added to the HA backbone, scope of modification like RGD binding site, MMP sensitive crosslinking increases but availability of interacting HA unit decreases. As various cell types have inclination towards different stiffness, RGD concentration and HA units this opens up possibility of using the platform to use on wide range of cell lines.

[0100] In some embodiments, the Nor-HA comprises about 1% to about 3%, by weight of the HA hydrogel. More generally, the Nor-HA may comprise from about 1% to about 7%, by weight of the HA hydrogel.

[0101] In some embodiments, the degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises about 21%.

[0102] More generally, in some embodiments a degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises from about 10% to about 75% based on the units of HA.

[0103] In some embodiments, the thiol functionalized RGD peptide is present in an amount from about 0.5 mM to about 10 mM.

[0104] In various embodiments, concentration of the at least one peptide crosslinker may be varied. Reference may be made to the specific Example 5 herein, which demonstrates tunability of the novel matrix mechanics and ability to remodel based on the nature and extent of crosslinked peptide crosslinker As exemplified herein, the inventors fabricated hydrogels with the previously optimized parameters of 21 % D.S. NorHA, 8 million / mL LECs, 5 mM RGD, and 50 ng / mL VEGF-C supplemented into the media, and then varied the concentration of crosslinker added in order to directly control the elastic modulus. A range of crosslinker concentrations corresponding to 10%, 25%, 50%, and 75% of the available norbornene groups being crosslinked to the MMP susceptible peptide crosslinker was used and the mechanical properties were evaluated (Figure 5A). Rheological measurements reported bulk moduli (G’) of 103±18.0Pa, 548±158Pa, 868±75.9Pa, and 1800±789Pa for 10, 25, 50, and 75% crosslinking, respectively. The high coefficient of determination (R2=0.96) indicates that this system can be precisely tuned through modulation of the crosslinker concentration, while importantly maintaining the same composition of the system.

[0105] In some embodiments, the thiol functionalized MMP sensitive peptide comprises about 10% to about 80% of the total available norbornene moieties. In some particular embodiments the thiol functionalized MMP sensitive peptide comprises about 25% to about 75% of the total available norbornene moieties.

[0106] Accordingly, in some embodiments, formed hydrogels demonstrate bulk modulus in a range from about 100 Pa to about 2000 Pa in a hydrogel having about 21% DS in which the amount of MMP crosslinker comprises about 10% to about 80% of the total available norbornene moieties. In some particular embodiments wherein the amount of MMP crosslinker comprises about 20% to about 25%, the bulk modulus is about 500 Pa.

[0107] In various embodiments, concentration of cell adhesive peptide binding sites on the Nor-HA corresponding to a concentration of the at least one cell-adhesive peptide may be varied. Reference may be made to the specific Example 3 herein, which demonstrates tunability of the novel matrix with respect to cell adhesive peptide binding sites. The RGD (Arg-Gly-Asp) peptideis an integrin recognition motif present in several ECM proteins, including fibronectin. The incorporation of fibronectin or RGD peptide has been previously demonstrated to promote lymphatic proliferation and tube formation, both in 2D cell culture and in 3D systems. To establish baseline morphology, gels were made without any RGD and after 5 days of culture, cells were observed to remain spherical and no elongation was observed. We also created gels with 5 mM of a scrambled RDG peptide, which serves as a negative control to samples with the proper RGD sequence that can be recognized by LECs. After 5 days of culture, gels with 5 mM RDG demonstrated a similar morphology to the 0 mM RGD samples. Upon addition of 1 mM RGD, short tubes with minimal branching can be observed, and with 3 mM RGD, tubes remain short but the extent of branching increases. With the incorporation of 5 mM RGD, longer tubes and branching from the wider tubes can be observed. The incorporation of 7 mM RGD resulted in shorter and more disconnected tubes compared to 5 mM RGD. So this flexibility of tuning RGD gives us the ability to have different up / downregulation of certain properties within the same cell line. On top of that as various cell types have different inclination towards different degree of RGD this platform opens up lot of opportunities going forward.

[0108] In various embodiments, the concentration of the living biological cells may be varied. In some embodiments, the density of cells in the HA hydrogel is in a range from at least about 2 million / mL to about 20 million / mL. In some particular embodiments, the density of cells in the HA hydrogel is in a range from at least about 8 million / mL to about 10 million / mL.

[0109] As referenced herein below, the inventors’ benchmarking studies indicated that 8 million / mL LECs was a critical density required to form lymphatic tubes (Supplementary Eigure 3). Increasing the cell density beyond 8 million / mL resulted in qualitatively slightly more connected lymphatic networks , accordingly, 8 million / mL LECs was determined to be a critical threshold at least for use of lymphatic endothelial cells, and was selected for use in the subsequent parameter screenings, both to limit the cells required per hydrogel construct and to allow for systematic investigation of individual parameters.

[0110] In various embodiments, with respect to peptide growth factor supplementation the frequency of addition and combinations of these factors may be varied. Reference may be made to the specific Examples 4, 6 and 7 herein, which demonstrate tunability of the novel matrix with respect to use of growth factors.

[0111] As shown in the examples, VEGF-C is essential to formation of the lymphatic vessels, and addition of ang2 is shown to be synergistic. Reference may be made to the specificExamples 6 and 7 herein, which demonstrate tunability of the novel matrix with respect to use of the combination of growth factors including VEGF-C and ang2.

[0112] In some embodiments, other growth factors such as fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain- containing protein 1 (CCBE1), Sphingosine 1 -phosphate (SIP), and combinations may be used.

[0113] In some embodiments, the concentration of VEGF-C in the culture medium is in a range from about 10 ng / mL to about 100 ng / mL.

[0114] In some embodiments, the concentration of Ang2 in the culture medium is in a range from about 400 ng / mL to about 1000 ng / mL.

[0115] In some embodiments, the concentration of FGF2 in the culture medium is in a range from about 2 ng / mL to about 50 ng / Ml

[0116] More generally, in various embodiments, one or a combination of growth factors may be used an a concentration ranging from about 1 ng / mL to about 1000 ng / mL.

[0117] In various embodiments, the duration of time of exposure to UV radiation may be varied - In this study 320-390nm UV light filter at 10mW / cm2 intensity for 1 minute was used. But changing the wavelength of the UV and exposure time (1 - 15 minutes) leaves room for tuning the gels initial stiffness and rheological property.

[0118] Three Dimensional Bioengineered Vessels

[0119] According to the method and use of the generative system, the disclosure provides and exemplifies hyaluronic acid based three dimensional bioengineered vessels that are viable in vitro in incubated culture with suitable medium, and are implantable and functional in vivo upon implantation.

[0120] In various embodiments, the bioengineered lymphatic vessels include living biological lymphatic cells in a norbornene functionalized hyaluronic acid matrix comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative, at least a portion of the plurality of norbornene moieties being crosslinked via a thiol linkage to a thiol functionalized metalloproteinase (MMP) sensitive peptide, and at least a portion of the norbornene moieties being crosslinked via a thiol linkage to a thiol functionalized RGD peptide. The provided HA-crosslinked hydrogel matrix comprises HA- norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesivepeptide-biological cell linkages. In culture, the medium includes MV2 culture media and at least VEGF-C and optionally ang2.

[0121] As shown in the examples, the lymphatic vessels demonstrate genetic expression of lymphatic markers, Proxl, LYVE-1, and Pdpn, and can be maintained up to 22 days in culture in an endothelial culture medium. Further, the culture medium does not include support cells comprising any of cells selected from fibroblasts, endothelial progenitor cells, dermal pulp stem cells, or other supporting cells.

[0122] In various embodiments, the method by which the three dimensional bioengineered vessel is formed includes a photoinitiator used in the crosslinking reaction in a range from about 0.01% to about 1%, or from about 0.01% to about 0.5% by weight of the reaction mixture. In some particular embodiments, the amount of photoinitiator used is about 0.05 wt%. In some embodiments, the photocrosslinker used is 12959. It will be appreciated that other chemical photoinitiator may be used such as for example 1184 and 1651, both from Irgacure. In some embodiments, the photoinitiator may be Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) with concentrations from 0.01 wt% to 0.5 wt% and the UV light employed is about 405 nm wavelength.

[0123] In some embodiments, the three dimensional bioengineered vessel comprises one or more of individual tubes, branched tubes, lymphatic capillaries with button like junction, and lymphatic valve with zipper like junction, wherein the three dimensional bioengineered vessel can transport lymph fluid and immune cells.

[0124] In some embodiments, the hyaluronic acid based three dimensional bioengineered vessel includes human lymphatic endothelial cells.

[0125] In some embodiments, the Nor-HA comprises about 1% to about 3%, by weight of the HA hydrogel. More generally, the Nor-HA may comprise from about 1% to about 7%, by weight of the HA hydrogel.

[0126] In some embodiments, the degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises about 21%.

[0127] More generally, in some embodiments a degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises from about 10% to about 75% based on the units of HA.

[0128] In some embodiments, the thiol functionalized RGD peptide is present in an amount from about 0.5 mM to about 10 mM.

[0129] In some embodiments, the HA hydrogel includes an amount of hyaluronic acid crosslinked with thiol functionalized RGD peptide in a range from about 0.5% to about 75%, or from about 1% to about 35%, or from about 3% to about 15% based on the degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA.

[0130] In some embodiments, the HA hydrogel includes an amount of hyaluronic acid crosslinked with thiol functionalized RGD peptide that is about 18% based on the degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA. In some embodiments, the amount of hyaluronic acid crosslinked with thiol functionalized RGD peptide that is about 2% based on the weight of the HA hydrogel.

[0131] In some embodiments, the thiol functionalized MMP sensitive peptide comprises about 10% to about 80% of the total available norbornene moieties. In some particular embodiments the thiol functionalized MMP sensitive peptide comprises about 25% to about 75% of the total available norbornene moieties.

[0132] In some embodiments, the density of cells in the HA hydrogel is in a range from about 2 million / mL to about 10 million / mL.

[0133] In some embodiments, the concentration of VEGF-C in the culture medium is in a range from about 10 ng / mL to about 100 ng / mL.

[0134] In some embodiments, the concentration of Ang2 in the culture medium is in a range from about 400 ng / mL to about 1000 ng / mL.

[0135] In some embodiments, the concentration of FGF2 in the culture medium is in a range from about 2 ng / mL to about 50 ng / mL

[0136] EXAMPLES

[0137] EXAMPLE 1: Nor-HA synthesis and hydrogel fabrication

[0138] HA was modified with norbornene groups to generate norbornene-modified hyaluronic acid (Nor-HA), which was first reported by Gramlich, et al, and is schematically depicted in Figure 1A and Figure 8. The extent of modification of the HA backbone can be controlled during the synthesis process to produce Nor-HA with varying abundances of norbornene groups, referred to as the degree of substitution (D.S.), as depicted in Figure 1A. The D.S. was calculated as the number of repeat units functionalized with norbornene per every 100 units and is reported as a percentage (% D.S.). We synthesized Nor-HA with three different D.S. 1values, low (21% D.S.), medium (44% D.S.), and high (63% D.S.), to use in subsequent characterization studies (Figure 9A-C).

[0139] Hydrogels were fabricated as described in the methods section and resulted in a covalently crosslinked hydrogel construct, shown in Figure IB, wherein human lymphatic endothelial cells were encapsulated throughout the gel and peptides could be selectively incorporated. Ratios of the components in the system were systematically varied throughout the following characterization studies, while maintaining a consistent 2 wt% of Nor-HA polymer, in order to engineer a system in which lymphatic endothelial cells would self-assemble into vessels, which will have applications for drug screening and mechanistic studies, disease modeling, and as a therapeutic transplant (Figure 1C) for lymphatic disorders.

[0140] EXAMPLE 2: HA binding site availability influences LEC phenotype and vessel formation

[0141] To directly investigate how HA binding site availability influences lymphatic endothelial cells, the inventors fabricated hydrogel constructs from the three different % D.S. options with otherwise identical parameters. Based on the final pre-crosslinked volume, 5 mM RGD peptide, 2.6 mM MMP-sensitive peptide, 0.05 wt% of 12959, and 8 million / mL lymphatic endothelial cells were added, and then hydrogels were crosslinked by UV light. Earlier benchmarking studies indicated that 8 million / mL lymphatic endothelial cells was a critical density required to form lymphatic tubes (Figure 10). While increasing the cell density beyond 8 million / mL resulted in qualitatively slightly more connected lymphatic networks, 8 million / mL lymphatic endothelial cells was determined to be a critical threshold and was selected for use in the subsequent parameter screenings, both to limit the cells required per hydrogel construct and to allow for systematic investigation of individual parameters.

[0142] After five (5) days of culture, individual gels were lysed with a tissue homogenizer to extract RNA or fixed for immunofluorescent analysis. Real-time qRT-PCR revealed that lymphatic endothelial cells encapsulated and cultured in low modification (21% D.S.) gels showed increased expression of LYVE-1, Prospero Homeobox 1 (Prox-1), and Podop lanin (Pdpn) compared to lymphatic endothelial cells cultured in high modification (63% D.S.) gels (Figure 2A- C). In earlier work not disclosed herein in detail inventors hereof reported that LYVE-1 and Prox- 1 expression increased with decreasing substrate stiffness, and now the inventors report a novel and unexpected method to increase the expression of these key lymphatic markers. The availability of HA binding sites represents another tunable parameter that can be controlled in this syntheticmatrix to probe the influence of HA on lymphatic vessel physiology and functions. Interestingly, while LYVE-1 expression increased, its homolog CD44 had no change in expression (Figure 2D). The expression of Flt4, the gene that encodes VEGFR-3, has been previously reported to be mechanosensitive, the inventors thus hypothesized that the expression would remain unchanged in the inventive system due to the mechanics remaining constant across varied % D.S. conditions. There was a statistically significant difference in Flt4 expression between conditions (Figure 2E), which could be explained by the differences in tube formation the inventors observed across % D.S. conditions. We also checked if expression of Neuropilin 2 (Nrp2), a co-receptor of VEGFR- 3, was altered by % D.S. but report no differences resulting from varied HA binding site availability (Figure 2F). Collectively, the data suggests that while the availability of HA influences the expression of Flt4 and Nrp2, it has a more significant impact on the expression of LYVE-1, Prox-1, and Pdpn.

[0143] Hydrogel constructs were observed daily for changes in morphology and sprouting, and a clear difference in morphology based on % D.S. was observed so samples were fixed at Day five (5) for immunofluorescent imaging, lymphatic tubes could readily be observed in 21% D.S. gels (Figure 2G), while minimal elongation and sprouting was observed in 44% D.S. gels (Figure 2H), and no elongation or sprouting was observed in 63% D.S. gels (data not shown). While the 44% D.S. polymer had minimally higher expression of lymphatic markers by real time qRT-PCR analysis compared to the 21% D.S. polymer, 21% D.S. constructs had substantially better tube formation, so the inventors selected 21% D.S. to be used in all of the subsequent characterizations in this study.

[0144] EXAMPLE 3: Fibronectin binding motif required for tube formation

[0145] The RGD (Arg-Gly-Asp) peptide is an integrin recognition motif present in several ECM proteins, including fibronectin. The incorporation of fibronectin or RGD peptide has been previously demonstrated to promote lymphatic proliferation and tube formation, both in 2D cell culture and in 3D systems. Here, the inventors have demonstrated how the incorporation of an RGD peptide can be used to promote lymphatic tube formation within the inventive 3D hydrogel system. The RGD peptide was modified with a thiol group on one end to covalently crosslink to the Nor-HA polymer during the UV-crosslinking process. Hydrogels were fabricated with identical parameters, except for the concentration of RGD peptide incorporated, to directly investigate the effect of integrin recognition site availability on lymphatic tube formation.

[0146] Based on previous 2D studies, 50 ng / mL VEGF-C was supplemented in the media and gels, and an embodiment of optimized parameters of 21% D.S. Nor-HA and 8 million / mL lymphatic endothelial cells were used. To establish baseline morphology, gels were made without any RGD and after five (5) days of culture, cells were observed to remain spherical, and no elongation was observed (Figure 11 A). We also created gels with 5 mM of a scrambled RDG peptide, which serves as a negative control to samples with the proper RGD sequence that can be recognized by lymphatic endothelial cells. After five (5) days of culture, gels with 5 mM RDG demonstrated a similar morphology to the 0 mM RGD samples (Figure 1 IB). Upon addition of 1 mM RGD (Figure 3 A), short tubes with minimal branching can be observed, and with 3 mM RGD (Figure 3B), tubes remain short but the extent of branching increases. With the incorporation of 5 mM RGD (Figure 3C), longer tubes and branching from the wider tubes can be observed. The incorporation of 7 mM RGD (Figure 3D) resulted in shorter and more disconnected tubes compared to 5 mM RGD. Analysis of key lymphatic markers by qRT-PCR at day five (5) demonstrated an inverse trend compared to the observed morphology, where increasing RGD peptide concentrations decreased the expression of CD44, Flt4, and Nrp2 (Figure 12A). Due to RNA being isolated at peak network formation, the inventors hypothesize that by day five (5), lymphatic endothelial cells in sub-optimal gel conditions are upregulating expression in an attempt to signal lymphangiogenesis, but it might not be translated. Future studies should more extensively investigate the kinetics of key lymphatic marker expression in correspondence with morphology.

[0147] In addition to evaluating tube morphology, the inventors have demonstrated the role of specific integrin subunits and their dynamic expression during lymphatic tube formation by collecting RNA at 24, 48, and 72 hrs post-encapsulation. Based on observed morphology, 1 mM, 3 mM, and 5 mM RGD were investigated and compared against 5 mM RDG as a baseline reference. Integrin subunit a9 is required for lymphatic valve morphogenesis, but it has also been shown to be involved in Proxl induced lymphatic endothelial differentiation, and 0,9(31 is capable of binding with VEGF-C22. In the case of ITGA9 (Figure 3E), a clear trend in expression increasing both over time and with RGD concentration was observed, correlating with changes in morphology as lymphatic endothelial cells self-assemble into tubes from days 1 to 3. We also investigated ITGAv (Figure 3F), ITGB1 (Figure 3G), and ITGA5 (Figure 3H), but saw only minimal differences in expression over time and RGD concentration. Based on tube morphology and trends in expression of ITGA9, the inventors concluded that 5 mMRGD peptide incorporation represents and embodiment of optimized conditions for the inventive system. Of course it will beappreciated that while the instant results reflect an embodiment of an optimal amount of RGD peptide given other parameters used, other amounts of the peptide may be appropriate when tuning the matrix system for other features such as D.S., quantity of lymphatic endothelial cells, added growth factors, etc., according to the teachings herein.

[0148] EXAMPLE 4: VEGF-C is required for and modulates lymphatic tube formation

[0149] Since its discovery as a key pro-lymphangiogenic growth factor, VEGF-C has been extensively reported to induce lymphangiogenesis both in vitro and in vivo and elevated concentrations of VEGF-C have been used in previous approaches to induce tube formation. Previously, the inventors used 50 ng / mL VEGF-C in a 2D system to promote the development of CLS, and other studies have used ranges of 10-100 ng / mL VEGF-C in their systems. Here, the inventors screened a range of VEGF-C concentrations to evaluate what concentration was needed to induce tube formation in the inventive 3D system. Based on embodiments of optimization of parameters discussed in previous sections, the inventors used 21% D.S. Nor-HA, 8 million / mL lymphatic endothelial cells, and 5 mM RGD peptide to fabricate the hydrogels.

[0150] When no VEGF-C is added to the system and the cells only have 0.5 ng / mL VEGF- A in the media, only a few cells are observed to be elongating (Figure 4A). With the addition of 10 ng / mL VEGF-C, lymphatic endothelial cells begin to sprout, and some short tubes can be observed by day five (5), but very few appear to be connected to each other (Figure 4B). Upon addition of 50 ng / mL VEGF-C, long and continuous tubes can be observed with some sprouting occurring off the wider tubes (Figure 4C). A similar phenotype is observed when 100 ng / mL VEGF-C is added to the system (Figure 4D). While 100 ng / mL VEGF-C supplementation qualitatively appears to yield a few additional spouts compared to 50 ng / mL VEGF-C supplementation, the effect is incremental and due to the possible adverse effects of higher levels of VEGF-C, 50 ng / mL VEGF-C supplementation was considered an embodiment of optimal conditions for subsequent experiments.

[0151] EXAMPLE 5: Matrix mechanics control and tune lymphatic tube formation

[0152] While many studies have historically focused on biochemical and genetic mechanisms of lymphatic development, studies on mechanical forces have been limited to flow and contractility, and mechanics of the environment have remained poorly studied. Several recent papers have highlighted the role of matrix mechanics in lymphatic development and there is a growing body of work looking into how microenvironment factors influence or regulate lymphaticdevelopment and function. Previously, the inventors showed that Flt4 expression, the gene that encodes for VEGFR3, was influenced by matrix mechanics and not by VEGF-C supplementation, the inventors hypothesized that matrix mechanics could be tuned to promote lymphatic tube formation in this 3D system.

[0153] To examine how matrix mechanics influence lymphatic tube formation, the inventors fabricated hydrogels with the previously described parameters of 21 % D.S. Nor-HA, 8 million / mL lymphatic endothelial cells, 5 mM RGD, and 50 ng / mL VEGF-C supplemented into the media, and then varied the concentration of crosslinker added in order to directly control the elastic modulus. A range of crosslinker concentrations corresponding to 10, 25, 50, and 75% of the available norbornene groups being crosslinked was used and the mechanical properties were first investigated (Figure 5A). Rheological measurements reported bulk moduli (G’) of 103±18.0Pa, 548±158Pa, 868±75.9Pa, and 1800±789Pa for 10, 25, 50, and 75% crosslinking, respectively. The high coefficient of determination (R2=0.96) indicates that this system can be precisely tuned through modulation of the crosslinker concentration, while importantly maintaining the same composition of the system.

[0154] Lymphatic endothelial cells were encapsulated in hydrogels of varied stiffnesses and observed daily for morphological changes, whereafter they were fixed at day five (5) for immunofluorescent imaging. In softer environments (25% crosslinked gels), self-assembled tubes were observed by day three (3) and a network of tubes was observed by day five (5) (Figure 5B). Moreover, lymphatic endothelial cells cultured in stiffer environments (50% and 75% crosslinked gels) failed to self-assemble into tubes (Figure 5C and 5D). Interestingly and contrary to results in a separate 2D hydrogel system where Flt4 expression increased with decreasing substrate stiffness, in this 3D system Flt4 expression increased with increasing matrix stiffness (Figure 12B), despite lymphatic endothelial cells failing to self-assemble in these stiffer environments. This data may suggest that lymphatic endothelial cells try to upregulate key genes to compensate for the higher crosslinking density and stiffer environment. In order to confirm that lymphatic endothelial cells could not self-assemble in stiffer environments, even given a longer time frame to remodel their environment, 75% crosslinked gels were cultured for up to 21 days but tubes failed to develop (data not shown), suggesting that Flt4 might not be translated into VEGF-C in these stiffer environments and is a point of investigation in future studies.

[0155] Daily visual observations of the hydrogels revealed that not only do matrix mechanics control whether tubes form or not, but they also control the kinetics of tube formation.When lymphatic endothelial cells were encapsulated in 10% crosslinked gels, primitive tubes could already be observed by day 1 or 2 (data not shown). However, structural integrity over time was a limitation as lymphatic endothelial cells degraded the MMP-sensitive crosslinker and by day three (3) collapsing tubes were observed due to the lack of structural support in the 10% crosslinked condition. The lack of tube formation at higher stiffnesses indicate there is a critical maximum that cannot be overcome, and collapsing tubes at the lower stiffness indicate a minimum threshold of crosslinking is required for longer-term culturing of these constructs.

[0156] A novel aspect of this hydrogel system is that cells were able to remodel their matrix through MMP secretion and subsequent cleaving of the MMP-sensitive crosslinker peptide, resulting in a dynamic mechanical environment for tube formation. To investigate how matrix stiffness influenced MMP expression, the inventors collected samples at 24, 48, and 72 hrs for real time qRT-PCR analysis. A burst response followed by a decline in MMP expression can be observed across conditions for MMP2 and MMP14 (Figure 5E and 5F), suggesting that cells secrete a higher amount of MMPs early on and then expression is reduced once tubes begin to form. These elevated levels of MMPs would enable cell migration which is a necessary step to create lymphatic tubes. Stiffer environments (75% crosslinked) demonstrated higher levels of MMP2 and MMP 14 expression, suggesting that lymphatic endothelial cells can sense and respond accordingly to their environment and this higher MMPs expression could be a method of trying to compensate for mechanical differences. To evaluate the benefit of using a degradable crosslinker to allow cells to remodel their matrix, the inventors also created hydrogels using dithiothreitol (DTT) which cannot be degraded by cells. Real time qRT-PCR analysis showed that MMP2 and MMP14 expression was the lowest in Nor-HA gels crosslinked with DTT compared to gels crosslinked with the MMP-sensitive peptide (Figure 5D and E), again suggesting that cells can sense and respond to their environment and highlights one of the unique features of the inventive synthetic hydrogel system.

[0157] EXAMPLE 6: Exemplary parameters for formation of perfusable lymphatic networks

[0158] The inventors show that Nor-HA %D.S., LEC encapsulation density, RGD motif concentration, VEGF-C concentration, and matrix stiffness, hydrogels with self-assembled lymphatic vessels throughout the thickness of the gel can be created (data not shown). Using a LIVE / DEAD stain across different time points of network self-assembly, the inventors observe that healthy lymphatic endothelial cells assemble into tubes while many of the cells that do notcontribute to tubes were dead, possibly as a result of the UV crosslinking process (Figure 13). After employing embodiments of optimized conditions, the inventors investigated the longevity of these constructs and report that they can be cultured for at least 3 weeks (Figure 14).

[0159] One of the main functions of lymphatic vasculatures is the ability to drain excess fluid. In order to evaluate this important function, lymphatic endothelial cells were encapsulated in Nor-HA hydrogels with an embodiment of optimized parameters within a perfusion chip, which has been previously used to assess lymphatic drainage function (Figure 15 A). After five (5) days of culture, robust lymphatic networks were formed within the middle chamber. To assess lymphatic drainage, 1 pm rhodamine-conjugated polystyrene beads were perfused on one side of the chamber (Figure 15B). Confocal microscopy demonstrates that these polystyrene beads can drain through the lymphatic networks (Figure 15C) and while this perfusion chip does not recapitulate the complete tissue physiology found in vivo, these findings suggest that these engineered lymphatic networks can be functional and perfusable.

[0160] EXAMPLE 7: Hydrogel platform allows for higher throughput growth factor screening

[0161] In order to explore the potential of using this hydrogel platform for additional mechanistic screenings and future disease modeling studies, the inventors investigated how the addition of specific growth factors impact lymphatic tube formation. While the activity of angiopoietin 2 (Ang2) is context dependent in blood vasculature, Ang2 has been reported to be an agonist of Tie2 in lymphatic vessels. In a recent study which revealed a mechanism of crosstalk between VEGF-C and Ang2 signaling through phosphoinositide 3-kinases (PI3K), Tiel and Ang2 were required for proper presentation of VEGFR3 on the surface of lymphatic endothelial cells in capillaries.

[0162] To probe this novel mechanism of lymphatic growth, the inventors fabricated hydrogels with identical parameters except for the growth factor composition they were cultured in. Ang2 alone has been reported to be a weak agonist, and even with 1,000 ng / mL Ang2 supplementation, no LEC elongation or tube formation was observed by day five (5) (Figure 6A). When encapsulated lymphatic endothelial cells were cultured with 50ng / mL VEGF-C and 400 ng / mL Ang2 (Figure 6B), increased lymphatic sprouting was observed, compared to 50 ng / mL VEGF-C alone (Figure 6D and shown in Figure 4C). The supplementation of 50 ng / mL VEGF-C and 1,000 ng / mL Ang2 resulted in even greater branching (Figure 6C), and importantly, suggestsa synergistic effect of Ang2 and VEGF-C where Ang2 alone had no effect, but when combined with VEGF-C, resulted in a significant increase in lymphatic sprouting.

[0163] In addition to screening VEGF-C and Ang2 supplementation in this system, the inventors also used this platform to investigate VEGF-C versus VEGF-C156S which is a lymphangiogenesis-specific, engineered mutant form of VEGF-C. While VEGF-C can activate both VEGFR2 and VEGFR3, VEGF-C156S is only able to activate VEGFR338. VEGFR2 has been shown to be required for lymphangiogenesis and in a previous growth factor therapy study conducted in pigs, VEGF-C 156S demonstrated a lower lymphangiogenic response. Hydrogels were fabricated with identical conditions except for the respective growth factor supplemented media that they were cultured in. Three days after encapsulation and culturing, hydrogels were lysed and examined using qRT-PCR to assess differential gene expression. Across all concentrations, VEGF-C resulted in higher expression of key lymphatic genes when compared to VEGF-C156S (Figure 16A-F), agreeing with previous reports of the necessity of VEGFR2 activation for lymphangiogenesis and demonstrating that this platform can be used for growth factor screening and future mechanistic studies in a high-throughput format.

[0164] EXAMPLE 8: Hydrogel constructs demonstrate in vivo integration and maturation

[0165] To investigate the functionality of the engineered human lymphatic vessels, the inventors used a murine mammary gland model, which has been previously used in the context of patient-derived tumor xenograft models of breast cancers. Hydrogel constructs were fabricated and cultured in vitro for two days to allow lymphatic endothelial cells to begin to elongate and sprout, and then they were implanted into a pocket created in the #4 mammary gland of NOD / SCID mice, shown in Figure 7A and 7B. The #4 mammary gland was selected for the implantation site due to it is ease of identification, proximity to a lymph node, being extensively used in the inventors’ previous studies, and to keep the hydrogel from shifting around the body. Implantation of the hydrogel in the mammary gland pocket and the neighboring vasculature can be observed in Figure 7C.

[0166] Notably, anastomosis was observed wherein on implantation, the engineered LV was not aligned with the native lymphatic vessels; subsequent growth resulted in alignment which is believed to occur via cellular signaling.

[0167] Two weeks post-implantation, hydrogels and the surrounding tissue were harvested to investigate how the implant integrated with the host vasculature. To assess lymphaticintegration, as well as to distinguish mouse vasculature from the implanted human LEC networks, two lectin dyes to label endothelial cells (ECs), 1 specific to mouse and 1 specific to human ECs, were injected into the extracellular space of the hindfoot for uptake to the lymphatic circulatory system before tissue harvest. The fluorescein-conjugated Isolectin Griffonia simplicifolia (GS-IB4 isolectin) stained mouse ECs, both blood and lymphatic, and the rhodamine-conjugated Ulex Europaeus Agglutinin I (UEA-1 lectin) stained the human lymphatic endothelial cells. While UEA-1 lectin can stain both blood and lymphatic human ECs, the entirety of the rhodamine signal in these explants can be attributed to the implanted human lymphatic endothelial cells due to using a monoculture hydrogel system.

[0168] In recent years, it has been shown that a tumor suppressor, Folliculin, is responsible for maintaining distinct blood and lymphatic vasculatures. Naturally present, Folliculin prevents vessel mismatching but when knocked out, anastomoses between blood and lymphatic vessels can be observed. Since NOD / SCID mice without altered Folliculin gene were used in this study, it is expected that any signal overlap between the two lectin dyes is from the interaction of lymphatic vessels between species and not a vasculature mismatch.

[0169] Confocal imaging of the hydrogel construct and surrounding tissue revealed that the human lymphatic networks (stained in red) continued to grow once implanted, evidenced by the long and aligned vessels observed, and that the mouse vasculature (green) could invade the hydrogel (Figure 7D). Higher magnification imaging revealed that the implanted human lymphatic endothelial cells integrated with the host lymphatic vasculature, indicated by overlapping fluorescent signals for several vessels (Figure 7E). Using Imaris imaging software, renderings of the confocal z-stacks were created for further visualization of the 3D structure and vessel integration. Renderings indicated that these implanted tubes (red) develop lumens (Figure 7F) and that the mouse and human vasculature truly integrate (Figure 7G and 7H) rather than existing as parallel planes. The host lymphatic vasculature is shown wrapped around the engineered human lymphatic vasculature, suggesting functional chimeric lymphatic vessels that can fully integrate with the host lymphatic vasculatures.

[0170] MATERIALS AND METHODS

[0171] Cell culture

[0172] Human lymphatic endothelial cells (Lymphatic endothelial cells) derived from the dermis of an adult donor (PromoCell, Heidelberg, Germany) were expanded and used for experiments between passages 5 and 9. Human lymphatic endothelial cells were grown inendothelial cell growth medium MV2 (EGM MV2; PromoCell) without antibiotics and incubated at 37 °C with 5% CO2. lymphatic endothelial cells were characterized for the positive expression of CD31 and Proxl throughout the experiments. All cell lines were routinely tested for mycoplasma contamination and were negative throughout this study.

[0173] Nor-HA synthesis and characterization

[0174] Nor-HA was synthesized in two steps as reported previously. First, HA was dissolved in DI water at 2 wt%, then Dowex 50w (H form, 3 g Dowex 50w per 1 g HA) was added and stirred at room temperature for 2 h. Then the mixture was filtered and neutralized with TBA- OH to a pH of 7.00. The resulting HA-TBA was freeze dried and stored at -20 °C. To synthesize Nor-HA, HA-TBA (1.96 g) was dissolved in anhydrous DMSO (2 wt%) at 45 °C. 5-norbornene- 2-carboxylic acid (mixture of endo and exo isomers, 910 pL, 3 equivalents to HA-TBA repeat units) and 4-(dimethylamino)pyridine (453.9 mg, 1.5 equivalents to HA-TBA repeat units) were added to the mixture. Di-tert-butyl dicarbonate (Boc2O, 455.2 pL, 0.8 equivalents to HA-TBA repeat units) was added to start the reaction, followed by stirring at 45 °C for 14 hours. Nor-HA was purified by dialysis against 1 wt% NaCl at 4 °C for three (3) days and against deionized water at 4 °C for two (2) days, lyophilized and stored at -20 °C until use. Nor-HA was characterized by 1H NMR in D2O for peaks at 1.97 ppm ((C=O)CH3 in HA) and 5.9-6.3 ppm (double bond in Nor). Representative NMR spectra for HA-TBA and Nor-HA are shown in Figure 8. The extent of modification of the HA backbone was controlled during the synthesis process to produce three variations of Nor-HA with differing degrees of substitution (D.S.). The D.S. was calculated as the number of repeat units functionalized with norbornene per every 100 units and is reported as a percentage. NMR spectra for low, medium, and high D.S. are shown in Figure 9A-C.

[0175] Peptide synthesis

[0176] We obtained the cell-adhesive peptide GCGYGRGDSPG (SEQ ID NO: 6) (molecular weight [MW]: 1025.1 Da, referred to as RGD peptide from here on), cell-nonadhesive peptide GCGYGRDGSPG (SEQ ID NO: 19) (MW: 1025.1 Da, referred to as RDG peptide from here on), and MMP-sensitive peptide crosslinker GCRDGPQGJ.IWGQDRCG (SEQ ID NO: 1) (MW: 1754.0 Da; down arrow indicates the site of proteolytic cleavage) from GenScript Corporation. All peptides had greater than 95% purity, as indicated by HPLC analysis and were dissolved in sterile IX DPBS.

[0177] Hydrogel fabrication

[0178] Hydrogels were made with 2 wt% Nor-HA lyophilized polymer, dissolved in MV2 cell culture media, in order to balance material usage, time required for the polymer to dissolve, and the amount of norbornene groups available for crosslinking. Irgacure 2959 (12959) served as a photo initiator and was added at 0.05 wt%. RGD and MMP-sensitive peptides were incorporated at varied concentrations based on the parameter conditions being evaluated, lymphatic endothelial cells were pelleted and then resuspended at high concentrations to add to the pre-crosslinked polymer solution immediately before gel fabrication. 30pL pre-crosslinked solution was pipetted into a 5 mM diameter mold and crosslinked with a 320-390nm UV light filter at 10mW / cm2 intensity for 1 minute.

[0179] Hydrogel culture

[0180] After hydrogels were casted and crosslinked, they were transferred to a 96-well plate and cultured in 200 pL complete cell media per well, plus any additional growth factors used in the media for specific conditions. Media was gently removed and replaced every 24 hours to support the high density of cells and to replenish growth factors. Hydrogel constructs were imaged daily using an inverted brightfield microscope to monitor cell elongation and tube formation.

[0181] Gene expression

[0182] Hydrogels were collected at specified time points and mechanically digested using a tissue homogenizer and then RNA was isolated and purified. RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher) according to the manufacturer’s protocol. cDNA was then used with the TaqMan Universal PCR Master Mix and gene expression assays for the selected genes. Each sample was prepared in triplicate, the relative expression was normalized to GAPDH, and results were analyzed using theAACt method. Gene expression assays used in this study are listed in Figure 17.

[0183] Brightfield and immunofluorescent imaging

[0184] To monitor lymphatic morphogenesis, hydrogels were imaged every 24hrs using brightfield microscopy to evaluate the progress of tube formation. Once tube formation had plateaued, hydrogels were fixed with 4% PFA for 1 hour and then immunofluorescently stained to visualize cell membranes, junctions, and nuclei. Fluorescent images were acquired with a Nikon A1X confocal microscope at 20x, and z-stacks were taken to visualize the 3D structure. Antibodies used for this study are listed in Figure 18.

[0185] Live dead assay

[0186] To analyze live and dead cell populations, cell viability assay (cat no. L3224) was used at day 1, 3 and 5. The gel constructs were washed and incubated with the cell viability staining for 30 minutes, then samples were imaged using confocal microscopy.

[0187] Perfusion assay

[0188] Hydrogels were prepared inside p-Slide Chemotaxis slides (Ibidi, cat no. 80326) and FluoSpheres Polystyrene Microspheres (Invitrogen Fl 3083) were used to assess the perfusability of the engineered lymphatic networks. On day five (5) after the mature lymphatic networks were formed, one of the reservoirs was filled with microbeads and the perfusion chips were imaged to assess bead transport through the lymphatic networks.

[0189] In vivo model

[0190] Female NOD / SCID mice were used in order to prevent immune rejection of the implanted human cell-laden hydrogel scaffolds. All mice used in this study were maintained in a pathogen-free environment and all animal experiments were conducted with approval from the University of Notre Dame Institutional Animal Care and Use Committee. 40pL hydrogel constructs were prepared as described earlier, and constructs were cultured in vitro for two days to allow lymphatic endothelial cells to begin to self-assemble. Constructs were maintained in a sterile environment and transferred to the animal facility for implantation into the mice. Under isoflurane anesthesia, an incision was made to access both the left and right #4 mammary gland. A pocket was then created with tweezers and the hydrogel construct was inserted into each pocket. Incisions were closed up with staples and mice were kept on a heating pad to recover from anesthesia, before being transferred back to their cage for daily monitoring.

[0191] Hydrogel implant and tissue collection and imaging

[0192] Two weeks post-implantation, two lectin dyes to label endothelial cells (ECs) of each species were injected into the extracellular space of the hindfoot for uptake to the lymphatic circulatory system before tissue harvest. The fluorescein- conjugated Isolectin Griffonia simplicifolia (GS-IB4 isolectin) stained mouse ECs and the rhodamine-conjugated Ulex Europaeus Agglutinin I (UEA-1 lectin) stained the human lymphatic endothelial cells. Hydrogels and the surrounding mammary gland tissue were then harvested. Tissues and hydrogels were arranged in cassettes and fixed with 4% PFA overnight, and then stored in sterile PBS. Due to low signal intensity and challenges with auto-fluorescence from the surrounding adipose tissue, explants were stained a second time with the lectin dyes after fixation.

[0193] Tissues and hydrogels were then imaged as a whole- mount sample in a coverslip chamber- well with a Nikon AXR confocal microscope. The entire tissue area was scanned at lOx to determine the regions of vessel integration, and then z-stacks at 20x were taken to visualize the 3D structure and spatial arrangement of the vasculature. Regions of interest included where the mouse vasculature invaded the hydrogel region and where the human lymphatic vessels within the hydrogel continued to grow and connect with the host vasculature. Regions of fluorescent signal overlap were deemed areas of host-implant integration.

[0194] Statistical analysis

[0195] Statistical analysis of gene expression was performed with GraphPad Prism (GraphPad Software Inc., La Jolla, CA). Each condition contained a minimum of n=3 biological replicates and qRT-PCR was performed in technical triplicate. Statistical comparisons were made using Student’s t-test for paired data. Significance levels were set at the following: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0196] Discussion

[0197] Lymphatic endothelial cells uniquely express the receptor LYVE-1 which can bind to HA and activate intracellular signaling to promote lymphangiogenesis. Due to this interaction, the inventors previously investigated and reported that culturing lymphatic endothelial cells on 2D HA-hydrogels increased expression of LYVE-1 and Prox-1, compared to traditional tissue culture methods. Advancing to the current and more complex 3D Nor-HA system, the inventors report an additional method for promoting the expression of key lymphatic markers through modulation of available HA binding sites within the synthetic matrix. .

[0198] While Prox-1 is known as the master lymphatic regulator, LYVE-1 and Pdpn are important lymphatic markers, responsible for leukocyte trafficking and LEC recognition, respectively. The ability to independently modify the amount of available HA binding sites, without altering mechanical properties or the abundance of ECM, allowed for decoupled investigation into the direct impact of matrix composition on lymphatic gene expression and vessel formation. Upregulation of key genes due to changes in HA binding sites highlights the importance of matrix composition and indicates that it should be a critical design consideration in future lymphatic vasculature tissue engineering approaches. These findings regarding the matrix composition are also consistent with previous studies which highlighted how matrix selection can influence differences in lymphatic vessel formation, specifically fibrin or collagen gels.

[0199] The role of integrin signaling in lymphatic development has been widely investigated, particularly Integrin alpha-9 and its role in the development of valve leaflets. Additionally, integrin signaling has been investigated in a synthetic matrix for its role in blood vasculature development, but in vitro insights have been limited to 2D studies for lymphatic development. As described and exemplified herein, the inventors demonstrate that the concentration of RGD binding sites can be tuned to promote lymphatic self-assembly and sprouting, showing that it is required for tube formation. Additionally, this hydrogel system allowed us to investigate the kinetics of integrin expression and how it corresponds to morphological changes observed. We report Itga expression increases both with RGD peptide concentration and as lymphatic tubes self-assemble, highlighting a kinetic relationship to further explore in future studies.

[0200] When varying concentrations of VEGF-C were investigated, 100 ng / mL VEGF-C supplementation qualitatively appeared to have resulted in slightly increased sprouting compared to 50 ng / mL VEGF-C. However, prolonged high levels of VEGF-C have been reported to have adverse effects in some cases, such as blood vessels becoming enlarged and leaky, and aggravation of lymphedema and increased vascular leakage. Due to the overall goal of engineering vessels capable of being implanted and used for regenerative medicine, 50 ng / mL VEGF-C was determined to be an optimal embodiment in order to minimize adverse effects of high levels of VEGF-C in future translational applications.

[0201] The ability to precisely control matrix stiffness and screen a range of stiffnesses, decoupled from other factors in this system, revealed that there is a critical maximum which cannot be exceeded, otherwise lymphatic cells will fail to migrate, sprout, and self-assemble. Previously the inventors reported that matrix mechanics tune cord-like structure formation and modulate gene expression. As shown herein, the inventors report in a 3D system that matrix mechanics dominate lymphatic tube formation, guided by VEGF-C. In softer environments, those that are representative of the mechanics found outside of the cardinal vein during embryonic development, lymphatic vessels could develop with as little as lOng / mL VEGF-C supplemented daily. Both vessel length and the extent of branching could be increased by increasing VEGF-C supplementation. Conversely, when lymphatic endothelial cells were cultured in environments which could still be considered “soft” in most applications but were three times the bulk modulus (G’) of the softer gels in this study, lymphatic endothelial cells failed to migrate and self-assemble into vessels, even when supplemented with high levels of VEGF-C. Despite higher levels of MMPs being secretedin these more rigid environments, and consequently more remodeling of the matrix through degradation of the MMP-sensitive crosslinker, lymphatic endothelial cells were not able to overcome the higher threshold. Collectively, these results indicate that while VEGF-C can be used to promote lymphatic tube formation, unsuitable matrix mechanics cannot be overcome through VEGF-C supplementation. As shown herein, the inventors demonstrate that matrix mechanics must be a primary consideration in future lymphatic tissue engineering approaches, and that supplementation of growth factors allows for modulation of the resulting vessels.

[0202] Most studies on matrix mechanics and lymphatic development, including this study, focused on embryonic development or de novo growth while the role of mechanics during post-natal lymphangiogenesis or fibrosis remain undiscovered. By using a UV-driven crosslinking system, this hydrogel platform allows for sequential cross-linking steps and could serve as a novel platform to investigate the effects of a stiffening matrix. For example, lymphatic endothelial cells could be encapsulated and allowed to self-assemble in a partially crosslinked (<100%) system, and then additional crosslinker could be swelled in to stiffen the matrix and to investigate genetic and morphological changes on developed vessels after matrix stiffening occurs. Recent advances in dynamic hydrogels can also be used to investigate the influence of viscoelastic properties on lymphatic tube formation.

[0203] The novelty and versatility of this system allowed for the systematic investigation of individual components without confounding influences, providing an advantage or complement to complex mouse models used for mechanistic investigations. Instead of needing a large cohort of mice to account for variability between animals for a growth factor or drug response study or needing to undergo the time and cost-intensive endeavor of generating a transgenic mouse model to investigate a gene, this hydrogel platform could provide an efficient alternative. Dozens of hydrogel replicates can easily be fabricated, and specific concentrations of growth factors or drugs can be incorporated to allow for the rapid investigation of its effect, or siRNA could be used to knock-down a gene of interest and the resulting genetic and morphological changes investigated in a shorter timeframe than animal models. This modular hydrogel platform can also be used within a perfusion chip to study the effect of various growth factors or drugs on lymphatic drainage using high-throughput imaging platforms. The recent discovery of PI3K linking the Ang / Tie and VEGF- C / VEGFR-3 pathways highlights the elaborate signaling involved in lymphatic development and provides numerous additional targets to investigate for therapeutic applications. The ability to screen VEGF-C, Ang2, and VEGF-C 156S in this hydrogel platform provides an early-stage proofof concept that this system could be used for future modeling studies. While this system is less complex than animal models and lacks certain features like an immune response, the rapid and simpler fashion of this system provides promise as a complement to in vivo models for higher- throughput studies in the future.

[0204] The ability of these hydrogel constructs to integrate with the host vasculature upon implantation provides promise for this technology to be used in regenerative medicine applications. By using only lymphatic endothelial cells, derived from humans, and a fully defined matrix, this disclosure represents an novel method, system and engineered 3D lymphatic vessels without the need for support cells. A few studies with synthetic gels have reported forming lymphatic tubes, but they have required a co-culture system with fibroblasts, endothelial progenitor cells, dermal pulp stem cells, or other supporting cells. The inventive tunable system is shown herein to promote lymphatic formation with only lymphatic endothelial cells. Application of the inventive tunable system provides the ability to systematically study LEC-material interactions while avoiding translational challenges of additional cell types and the possibility of off-target differentiation. As shown herein, after only two days of culture, engineered lymphatic tubes can be implanted and continue to grow in vivo, reporting a rapid method for engineering lymphatic vessels. Not only is the methodology timeline shorter than some other approaches, the inventors report that these lymphatic tubes can be cultured for up to 21 days in vitro and that they survive for at least two (2) weeks in vivo, demonstrating the novelty of this Nor-HA hydrogel system for both in vitro and in vivo applications of lymphatic vessel engineering. Upon implantation, the host lymphatic vasculatures are found to wrap around the engineered human lymphatic vasculatures suggesting a “wrapping and tapping” mechanism, which was first observed in blood vasculatures. The interaction between the engineered human lymphatic vessels and murine lymphatic vessels raises many interesting questions surrounding the fate of the engineered human cells, which could be further elucidated using genetic labeling and lineage tracing. While the uptake of speciesspecific lectin dyes suggest that the engineered lymphatic networks can function and integrate with the host lymphatic vasculatures, future studies should explore the prospect of the lymphatic engineered approaches to drain fluid and how they interact with the host lymphatic vessels in clinically relevant lymphedema models. The in vivo implantation was in a murine mammary gland model using female mice in order to mimic the clinical case of secondary lymphedema in breast cancer patients, but precludes sex as a biological variable. The results provide clear support use ofthe inventive HA-hydrogels for at least rapid screening, researching developmental lymphatic biology, and tissue and lymphatic network formation and regeneration applications.

[0205] DEFINITIONS

[0206] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001 or Singleton, etal., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N.Y. (1991). General laboratory techniques (DNA extraction, RNA extraction, cloning, PCR amplification, cell culturing, etc.) are known in the art and described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, 2012.

[0207] The terms “optimal” and “optimized” may be used herein in referenced to certain exemplified conditions, and it will be appreciated that while any results reflect an “optimal” or “optimized” amount or conditions in the context of any other given parameters used, other amounts or conditions may be appropriate and “optimal” or “optimized” when tuning the matrix system for other features according to the teachings herein. Accordingly, the terms “optimal” and “optimized” are not intended to be limiting on other possible conditions.

[0208] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0209] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further notedthat the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0210] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring.

[0211] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0212] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0213] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0214] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number 2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, ... , 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than number 10, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than number 10, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about,” whose meaning has been described above.

[0215] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The inventiontherefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0216] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0217] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein.

[0218] While specific embodiments are described below with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

[0219] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. A method for forming hyaluronic acid based three dimensional bioengineered lymphatic vessels, the method comprising: a. providing a biological matrix generative system for forming an hyaluronic acid hydrogel, the system comprising: i. norbornene functionalized hyaluronic acid (Nor-HA), the Nor-HA comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative; ii. at least one peptide crosslinker selected from the group consisting of thiol functionalized metalloproteinase (MMP) sensitive peptides; iii. at least one cell-adhesive peptide, wherein the at least one cell-adhesive peptide is thiol functionalized; and iv. at least one photoinitiator for initiating photo-crosslinking; and b. providing living biological cells comprising at least one cultured animal cell; c. mixing the components of the provided biological matrix generative system and the living biological cells and irradiating with UV light 320-390 nm to crosslink HA bound norbornene moieties of the Nor-HA with the peptide crosslinker to and to crosslink HA bound norbornene moieties of the Nor-HA with the cell-adhesive peptide to form an HA-crosslinked hydrogel matrix comprising HA-norbornene- MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages; d. providing peptide growth factor comprising at least Vascular Endothelial Growth Factor (VEGF)-C; e. maintaining the combined HA-crosslinked hydrogel matrix in culture supplemented with the provided at least one peptide growth factor, whereby the hyaluronic acid based three dimensional bioengineered lymphatic vessels are formed.

2. The method according to claim 1, wherein the living biological cells comprise mammalian cells selected from the group consisting of human lymphatic endothelialcells, lymphatic endothelial cells, lymphatic muscle cells, stem cell derived endothelial cells, fibroblasts, endothelial progenitor cells, and combinations thereof.

3. The method according to claim 1 wherein mechanical properties of the bioengineered vessel are tuned or modulated by varying one or more parameters selected from the group consisting of: a. extent of HA binding site availability corresponding to degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA, b. concentration of the at least one peptide crosslinker based on the degree of binding to available norbornene moieties, c. concentration of cell adhesive peptide binding sites on the Nor-HA corresponding to a molar concentration of the at least one cell-adhesive peptide, d. concentration of the living biological cells, e. one or more of concentration, frequency of addition, and combinations of peptide growth factor supplementation, f. duration of time of exposure to UV radiation, g. concentration of the at least one peptide crosslinker, h. crosslinking chemistry of the at least one peptide crosslinker, and i. combinations thereof.

4. The method according to claim 3, wherein: a. the hyaluronic acid derivative comprises sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa; b. the at least one peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQGJ.IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase; c. the at least one cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6); d. the living biological cells comprise human lymphatic endothelial cells; and e. the at least one peptide growth factor comprises angiopoietin 2 (ang2), and optionally comprises at least one other peptide growth factor selected from thegroup consisting of VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-C156S, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1- phosphate (SIP), and combinations thereof, wherein the human lymphatic endothelial cells self-assemble into vessels in vitro within three (3) days, and wherein the presence of the degradable crosslinker allows the living biological cells to remodel the HA-crosslinked hydrogel matrix.

5. A biological matrix generative system for forming a hyaluronic acid based three dimensional bioengineered lymphatic vessel, the system comprising: a. norbornene functionalized hyaluronic acid (Nor-HA), the Nor-HA comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative; b. at least one peptide crosslinker selected from the group consisting of thiol functionalized metalloproteinase (MMP) sensitive peptides; c. at least one thiol functionalized cell-adhesive peptide; d. living biological cells comprising at least one cultured animal cell; e. at least one peptide growth factor comprising at least VEGF-C, and optionally at least one additional peptide growth factor selected from the group consisting of ang2, VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-C156S, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1 -phosphate (SIP), and combinations thereof; and f. at least one photoinitiator for initiating photo-crosslinking, wherein the matrix generative system is employed to form an HA hydrogel matrix by means of UV photocrosslinking to crosslink HA bound norbornene moieties of the Nor-HA with the peptide crosslinker and to crosslink HA bound norbornene moieties of the Nor-HA with the cell adhesive peptide, and wherein the HA hydrogel matrix,when formed, comprises HA-norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages.

6. The biological matrix generative system of claim 5, wherein: a. the hyaluronic acid derivative is selected from the group consisting of sodium hyaluronic acid (NaHA) with a Mnof 90 kg / mol and a MW of 60-70 kDa, hyaluronic acid derivatives compatible with norbornene chemistry, and combinations thereof, b. the at least one peptide crosslinker comprising an MMP sensitive peptide comprises an amino acid having susceptibility to cleavage by an MMP selected from the group consisting of MMP- 1 , MMP-2, and MMP-9, and c. the at least one cell-adhesive peptide comprises an amino acid sequence selected from the group consisting of GCGYGRGDSPG (SEQ ID NO: 6), RGD(S) (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), YIGSR (SEQ ID NO: 9), PDSGR (SEQ ID NO: 10), RNIAEIIKDI (SEQ ID NO: 11), RYWLPR (SEQ ID NO: 12), POG (SEQ ID NO: 13), DGEA (SEQ ID NO: 14), GFOGER (SEQ ID NO: 15), VFDNFVLK (SEQ ID NO: 16), FPGERGVEGPGP (SEQ ID NO: 17), and HAV(DI) (SEQ ID NO: 18), d. the living biological cells comprise mammalian cells selected from the group consisting of human lymphatic endothelial cells, lymphatic endothelial cells, lymphatic muscle cells, stem cell derived endothelial cells, fibroblasts, endothelial progenitor cells, and combinations thereof, and e. the at least one peptide growth factor comprises at least VEGF-C and ang2, and optionally comprises at least one other peptide growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-C156S, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1- phosphate (SIP), and combinations thereof.

7. The biological matrix generative system of claim 5, wherein the hyaluronic acid derivative comprises sodium hyaluronic acid (NaHA) with a Mnof 90 kg / mol and a MW of 60-70 kDa.

8. The biological matrix generative system of claim 5, wherein the at least one peptide crosslinker comprises an MMP sensitive peptide comprising an amino acid sequence selected from the group consisting of GCRDGPQGJ.IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase, PLGL (SEQ ID NO: 2), GPQGIWCQ (SEQ ID NO: 3), PVGLIG (SEQ ID NO: 4), and GTAGLIGQ(SEQ ID NO: 5).

9. The biological matrix generative system of claim 5, wherein the at least one peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQG / IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase.

10. The biological matrix generative system of claim 5, wherein the at least one cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6).

11. The biological matrix generative system of claim 5, wherein the at least one peptide growth factor comprises Ang2.

12. The biological matrix generative system of claim 5, wherein: a. the hyaluronic acid derivative comprises sodium hyaluronic acid (NaHA) with a Mn of 90 kg / mol and a MW of 60-70 kDa; b. the at least one peptide crosslinker comprises an MMP sensitive peptide having an amino acid sequence comprising GCRDGPQG / IWGQDRCG (SEQ ID NO: 1) wherein J, represents a site of proteolytic cleavage by a metalloproteinase; c. the at least one cell-adhesive peptide comprises at least an RGD peptide having an amino acid sequence comprising GCGYGRGDSPG (SEQ ID NO: 6); d. the living biological cells comprise human lymphatic endothelial cells; and e. the at least one peptide growth factor comprises angiopoietin 2 (ang2), and optionally comprises at least one other peptide growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-C156S,fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1- phosphate (SIP), and combinations thereof.

13. An hyaluronic acid based three dimensional bioengineered vessel, comprising living biological cells, and norbornene functionalized hyaluronic acid (Nor-HA) comprising a hyaluronic acid derivative and a plurality of norbornene moieties covalently linked to the hyaluronic acid derivative, at least a portion of the plurality of norbornene moieties being crosslinked via a thiol linkage to a thiol functionalized metalloproteinase (MMP) sensitive peptide, and at least a portion of the norbornene moieties being crosslinked via a thiol linkage to a thiol functionalized RGD peptide to provide a HA-crosslinked hydrogel matrix that comprises HA-norbornene-MMP sensitive peptide-norbornene-HA linkages and HA-norbornene- cell adhesive peptide-biological cell linkages; and culture medium comprising MV2 culture media and at least VEGF-C and ang2, wherein the three dimensional bioengineered vessel comprises one or more of individual tubes, branched tubes, lymphatic capillaries with button like junction, and lymphatic valve with zipper like junction, and wherein the three dimensional bioengineered vessel can transport lymph fluid and immune cells.

14. The hyaluronic acid based three dimensional bioengineered vessel according to claim 13, comprising human lymphatic endothelial cells.

15. The hyaluronic acid based three dimensional bioengineered vessel according to claim 13, wherein the Nor-HA comprises about 1% to about 3%, by weight of the HA hydrogel.

16. The hyaluronic acid based three dimensional bioengineered vessel according to claim 13, wherein a degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises from about 10% to about 75%.

17. The hyaluronic acid based three dimensional bioengineered vessel according to claim 16, wherein the degree of substitution of norbornene moieties per every 100 units of HA in the Nor-HA comprises about 21%.

18. The hyaluronic acid based three dimensional bioengineered vessel according to claim 13, wherein the thiol functionalized RGD peptide is present in an amount from about 1 mM to about 10 mM, and the thiol functionalized MMP sensitive peptide comprises about 0.01% to about 0.1%, by weight of the HA hydrogel.

19. The hyaluronic acid based three dimensional bioengineered vessel according to claim 13, wherein the lymphatic vessels demonstrate genetic expression of lymphatic markers, Proxl, LYVE-1, and Pdpn, and can be maintained up to 22 days in culture in an endothelial culture medium.

20. The hyaluronic acid based three dimensional bioengineered vessel according to claim 13, wherein the culture medium does not include support cells comprising any of cells selected from fibroblasts, endothelial progenitor cells, dermal pulp stem cells, or other supporting cells.

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