SYNTHESIS AND ASSEMBLY OF CLICKABLE MICROGELS INTO CELL-LADEN POROUS SCAFFOLDS

Abstract

This invention is in the field of medicinal chemistry. The present invention provides cell-laden hydrogels and hydrogel assemblies thereof for use in tissue engineering. The present invention provides methods of producing various hydrogels and hydrogel assemblies and pharmaceutical compositions thereof. The present invention provides for a microgel comprising an encapsulated population of live primary human cells in a hydrogel comprising a polymeric network.

Claims

1. A hydrogel network comprising assembled polymeric microgel particles with complementary clickable reactive surface groups.

2. The hydrogel network of claim 1, wherein said network is porous.

3. The hydrogel network of claim 1, wherein said hydrogel network comprises poly(ethylene glycol) microgel particles with dibenzocylcoctyne surface functionalities that have reacted with azide surface functionalities on poly(ethylene glycol) microgel particles.

4. The hydrogel network of claim 1, wherein live cells are encapsulated within the network.

5. The hydrogel network of claim 1, wherein said cells are primary cells.

6. The hydrogel network of claim 1, wherein said cells are human cells.

7. The hydrogel network of claim 6, wherein said human cells are mesenchymal stem cells (hMSCs).

8. The hydrogel network of claim 1, wherein said particles are composed of materials selected from the group consisting of poly(ethylene glycol), hyaluronic acid, gelatin, alginate, poly(vinyl alcohol), and polypeptides.

9. The hydrogel network of claim 1, wherein said groups react via click reactions.

10. The hydrogel network of claim 9, wherein said click reactions are selected from the group consisting of thiol-ene chemistry, Michael type additions, copper-click azide alkyne chemistries, strain-promoted alkyne-azide cycloadditions chemistry, and Diels Alder type reactions.

11. The hydrogel network of claim 3, wherein said poly(ethylene glycol) microgel particles with dibenzocylcoctyne surface functionalities comprise 20 kDa 8-arm poly(ethylene glycol) and said poly(ethylene glycol) microgel particles with azide surface functionalities comprise 4-arm 10 kDa PEG-N.sub.3.

12. The microgel of claim 1, wherein said network comprises particles with a size range between 10.sup.2 nm and 10.sup.4 m.

13. The microgel of claim 1, wherein said network comprises particles further comprise an adhesion ligand comprising a clickable reactive group.

14. The hydrogel network of claim 3, wherein said poly(ethylene glycol) microgel particles further comprise an azide-labeled adhesion ligand.

15. A method, comprising: a) providing, i) a first group of microgel particles with a first surface functionality, ii) a second group of microgel particles with a second surface functionality, wherein said first and second surface functionalities are complementary clickable reactive surface groups, iii) a population of cells, and b) mixing said cells with said first and second microgel particles, and c) centrifuging said mixture to spontaneously form a hydrogel network encapsulating said population of cells.

16. The method of claim 15, wherein said first group of microgel particles comprises poly(ethylene glycol) microgel particles with dibenzocylcoctyne surface functionalities and said second group of microgel particles comprises poly(ethylene glycol) microgel particles with azide surface functionalities.

17. The method of claim 15, wherein said particles are composed of materials selected from the group consisting of poly(ethylene glycol), hyaluronic acid, gelatin, alginate, poly(vinyl alcohol), and polypeptides.

18. The method of claim 15, wherein said groups react via click reactions.

19. The method of claim 18, wherein said click reactions are selected from the group consisting of thiol-ene chemistry, Michael type additions, copper-click azide alkyne chemistries, strain-promoted alkyne-azide cycloadditions chemistry, and Diels Alder type reactions.

20. The method of claim 15, wherein said network is porous.

Description

DESCRIPTION OF THE FIGURES

[0051] The accompanying figures, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The figures are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention.

[0052] FIG. 1 shows an inverse suspension polymerization of microgel building blocks. Scheme for microgel formation. 8arm 20 kDa PEG-DBCO, 4arm 10 kDa PEG-N.sub.3, and azide labeled RGD were combined in PBS and dispersed in cyclohexane. Solutions were exposed to either continuous vortexing or sonication, while polymerizations were performed off-stoichiometry to yield microgels with excess DBCO or N.sub.3 surface functionalities.

[0053] FIG. 2A-C shows the size characterization of vortexed and sonicated microgels. FIG. 2A shows fluorescently labeled (Alexa Fluor 594) microgels were imaged and sized using a custom Matlab script (scale bar 100 m). FIG. 2B shows the size distribution of 10.sup.2 m gels with varying stoichiometry of reactive groups. Microgels exhibited a heterogeneous population for both excess DBCO and N.sub.3 gels, with average diameters of 120 m and 130 m, respectively. FIG. 2C the size distribution of 10.sup.1 m gels with varied excess surface functionalities. 10.sup.1 m microgels had average diameters of 16 m and 15 m for the excess DBCO and excess N.sub.3 cases, respectively.

[0054] FIG. 3A-C shows the Assembly of microgels into microporous scaffolds. FIG. 3A shows both the 10.sup.2 m and 10.sup.1 m particles, equal volumes of microgels containing excess DBCO or N.sub.3 groups were combined and centrifuged to form a percolated network. Interactions between particles bearing surface DBCO and N.sub.3 results in triazole formation and a covalently connected microgel network FIG. 3B shows the macroscopic scaffold formed from fluorescently labeled 10.sup.2 m particles (scale bar=10 mm). FIG. 3C shows the values for the average compressive moduli of networks formed from 10.sup.2 m and 10.sup.1 m particles. Compressive modulus was calculated from the slope of the stress-strain curve, yielding networks of 2.1 kPa and 3.3 kPa for the 10.sup.2 m and 10.sup.1 m particles, respectively.

[0055] FIG. 4A-C shows the pore analysis of microgel-assembled networks. FIG. 4A shows the three-dimensional images of scaffolds with a fluorescently labeled high molecular weight dextran solution to demonstrate the interconnectivity of pores within the scaffold. FIG. 4B shows the Matlab analysis of pores within microgel-assembled networks (10.sup.2 m particles shown, scale bar 100 m). FIG. 4C shows the void fraction analysis of microgel-assembled networks. Void space was consistent among networks, with 29% and 12% negative space in the 10.sup.2 m and 10.sup.1 m networks, respectively.

[0056] FIG. 5A-C shows the analysis of pore dimensions in assembled networks. FIG. 5A shows the analysis of pore area within 10 m and 10.sup.1 m scaffolds. 75% of pores in 10.sup.2 m particle gels were less than 3300 m, while the corresponding percentage of pores in 10.sup.1 m gels were below 60 m. FIG. 5B shows the analysis of the major axis of pores within assembled networks. The majority (75%) of pores within 10.sup.2 m networks had a major axis length smaller than 120 m. Networks containing 10.sup.1 m particles had much smaller pores, with the same percentage of pores being less than 14 m in length. FIG. 5C shows the aspect ratio of pores within microgel scaffolds. Pores in both networks had heterogeneous distributions of pore shapes, with networks consisting of 10.sup.2 m particles having an average aspect ratio of 2.1. Networks containing 10.sup.1 m microgels had a slightly smaller average aspect ratio of 1.9.

[0057] FIG. 6A-C shows Cell encapsulation within microporous assembled scaffolds. FIG. 6A shows representative images of cells encapsulated within 10.sup.2 m (left) and 10.sup.1 m (right) microgel networks. hMSCs are stained with calcein AM (green, live cells) and ethidium homodimer (red, dead cells) prior to imaging. Insert: Higher magnification image demonstrating cell morphology and interaction with microgels (gray). Scale bar denotes 10 m. FIG. 6B shows cell staining for cytoskeletal morphology. Cells were stained with DAPI (nuclei, blue) and phalloidin (actin, green). Scale bars in inserts denote 10.sup.0 m. Insert: Higher magnification image showing actin structure. Scale bar denotes 10.sup.0 m. FIG. 6C shows quantification of cell viability and morphology. Viability (top) in both networks was similar, with approximately 95% of cells viable after 4 days in culture. However, cells in the 10.sup.1 m gel network were significantly more circular (bottom) than those encapsulated in 10.sup.2 M networks (**** denotes p<0.0001).

[0058] FIG. 7A-C shows In situ gelation data for SPAAC hydrogels at microgel conditions using FIG. 7A shows 11 mM excess DBCO and FIG. 7B shows 11 mM excess N.sub.3. Each formulation was mixed for 10-15 seconds prior to the rheological measurement. Both graphs represent a single measurement for clarity, but each condition was repeated three times with similar results. Shear modulus (G) is shown in black while loss modulus (G) is shown in gray. FIG. 7C shows Bulk gels were also tested after 24 hours in PBS to assess the swollen compressive modulus. Observed moduli differences between the two conditions is very likely a result in the differing functionalities of the two PEG macromers (8 arm PEG-DBCO vs. 4 arm PEG-N3), which have different contributions to cross-linking density.

[0059] FIG. 8 shows Matlab code for analyzing particle size.

[0060] FIG. 9 shows Matlab code for analyzing pore size and dimensions.

[0061] FIG. 10 shows images of microgel assembled networks and unformed networks from homogenous particle solutions. FIG. 10 Top: Solutions (post-centrifugation) containing equal densities of a mixture of DBCO and N.sub.3 particles (left), only DBCO functionalized particles (middle), and only N.sub.3 functionalized particles for 10.sup.2 m (left group) and 10.sup.1 m (right group) cases. FIG. 10 Bottom: After multiple tube inversions a network was only observed in the heterogeneous particle mixture.

DETAILED DESCRIPTION OF THE INVENTION

[0062] This invention is in the field of medicinal chemistry. The present invention provides cell-laden hydrogels and hydrogel assemblies thereof for use in tissue engineering. The present invention provides methods of producing various hydrogels and hydrogel assemblies and pharmaceutical compositions thereof. The present invention provides for a microgel comprising an encapsulated population of live primary human cells in a hydrogel comprising a polymeric network.

[0063] Hydrogels are a versatile class of polymeric networks that have been utilized for a wide variety of cell culture and tissue repair applications. These highly water-swollen polymer networks have served as a platform to support cell growth in vitro, and to improve viability and engraftment in cell transplantation therapies. Hydrogels can also be spatially confined as microscopic shapes (microgels) and used as structural components or delivery vehicles to affect cell function. Microgels have found numerous uses in cell culture, including as drug delivery vehicles, structural or bioactive components of bulk hydrogels, or cellular aggregates, and as 3D cell culture platforms.

[0064] More recently, however, microgels have been utilized as the building blocks for cell culture scaffolds. In this way, hydrogel scaffolds are produced via bottom-up fabrication, by assembling microgels into a fully percolated network. While traditional bulk networks have a porosity (mesh size) on the order of nanometers, microgel-assembled networks contain an inherent porosity on the size scale of tens to hundreds of microns, allowing for rapid cell proliferation, infiltration, and motility. While top-down approaches to creating porous scaffolds, that is pore formation by degrading part of a bulk network, have been traditionally used, bottom-up network fabrication can aid in the recreation of complex tissue architectures. Bottom-up approaches have already been used to create heterogeneous tissue mimics, and the inclusion of multiple building blocks could allow for numerous cues to be rapidly incorporated into a porous cell culture scaffold.

[0065] Previously, these building blocks have been assembled through physical entanglements or via external cross-linking agents. Herein a method to fabricate cell-laden microporous hydrogels by the co-assembly of reactive hydrogel particles with primary cells without the need for external cross-linking moieties is presented. Macromolecular poly(ethylene glycol) monomers end functionalized with azide and alkyne moieties were synthesized and reacted using an inverse suspension polymerization method to generate microgels with average diameters either on the order of 100 m or 10 m. Particles were synthesized with excess alkyne or azide functional groups, and then introduced into a cell suspension. Upon centrifugation, the particle-cell composite formed a macroscopic, but microporous, hydrogel via a spontaneous azide alkyne cycloaddition bio-click reaction (SPAAC). Introduction of a cell-adhesive ligand into the microsphere formulation allowed for cell-particle interactions, cell spreading and process extension. The ability to co-assemble functionalized particles with cells to create cell-laden scaffolds can lend itself to numerous tissue engineering applications.

[0066] While microporous scaffolds are increasingly used for regenerative medicine and tissue repair applications, the most common techniques to fabricate these scaffolds use templating or top-down fabrication approaches. Cytocompatible bottom-up assembly methods afford the opportunity to assemble microporous systems in the presence of cells and create complex polymer-cell composite systems in situ. Here, microgel building blocks with clickable surface groups are synthesized for the bottom-up fabrication of porous cell laden scaffolds. The facile nature of assembly allows for human mesenchymal stem cells to be incorporated throughout the porous scaffold. Particles are designed with mean diameters of 10 and 100 m, and assembled to create varied microenvironments. The resulting pore sizes and their distribution significantly alter cell morphology and cytoskeletal formation. This microgel-based system provides numerous tunable properties that can be used to control multiple aspects of cellular growth and development, as well as providing the ability to recapitulate various biological interfaces.

1. Introduction

[0067] Hydrogels are a versatile class of polymeric networks that have been utilized for a wide variety of cell culture and tissue repair applications. These highly water-swollen polymer networks have served as a platform to support cell growth in vitro [1, 2], and to improve viability and engraftment in cell transplantation therapies [3, 4]. Additionally, hydrogels can be modified to mimic many important facets of the native extracellular matrix or selectively functionalized to present specific chemical [5-7] or mechanical [8-10] cues to trigger desired cellular responses. Hydrogels can also be spatially confined as microscopic shapes (microgels) and functionalized with specific moieties to affect cell function. Microgels have found numerous uses in cell culture, including as drug delivery vehicles [11], structural or bioactive components of hydrogels [12, 13], or cellular aggregates [14, 15], and as 3D cell culture platforms [16-18].

[0068] More recently, however, microgels have been utilized as the building blocks for cell culture scaffolds. In this way, hydrogel scaffolds are produced via bottom-up fabrication, by assembling microgels into a fully percolated network. While traditional bulk networks have a porosity (mesh size) on the order of nanometers, microgel-assembled networks contain an inherent porosity on the size scale of tens to hundreds of microns, allowing for rapid cell proliferation, infiltration, and motility [19, 20]. Top-down approaches to creating porous scaffolds, that is, pore formation by degrading part of a bulk network, have been traditionally used, and have been successful at creating biocompatible scaffolds with highly tunable pore characteristics [21, 22]. While top down approaches have proven useful in numerous applications, bottom-up network fabrication can aid in the recreation of complex tissue architectures. Bottom-up approaches have already been used to create heterogeneous tissue mimics [23], and the inclusion of multiple building blocks could allow for several cues to be rapidly incorporated into a porous cell culture scaffold. Elbert and colleagues have successfully demonstrated this concept, by creating microgel assembled scaffolds with three types of microgel components, functioning as structural supports, porogens, or drug delivery systems [24]. Similarly, microgels have been functionalized with cell adhesive peptides allowing for increased cell invasion to facilitate more rapid wound healing [19]. Finally, the Zhang group has explored sub-micron sized gels for similar purposes, demonstrating how particle size and interconnectivity within porous scaffolds can be utilized to control cell growth [25].

[0069] Thus, these scaffolds provide a wealth of opportunities to investigate cell interactions with both chemical and mechanical cues. Fabricating a scaffold with surface features on the size scale of a cell allows for the examination of how micro-structured environments (for example, trabecular bone or alveoli) affect cellular function and growth. The ability to co-assemble cells with microgel building blocks allows for facile creation of these environments in vitro, as well as providing numerous opportunities for cell transplantation and wound healing. Finally, these microgel building blocks can be functionalized with active moieties to impart specific cues to cells contained within the assembled network. Thus, a bottom-up type fabrication, which is presented herein, may be used, not only to create porous scaffolds, but to permit the assembly of heterogeneous particles and cells that can create varied complex culture environments.

[0070] In one embodiment, the present invention contemplates a method to fabricate cell-laden microporous hydrogels by the co-assembly of reactive hydrogel particles with primary cells. Macromolecular poly(ethylene glycol) monomers end functionalized with azide and alkyne moieties were synthesized and reacted using an inverse suspension polymerization method to generate microgels with average diameters either on the order of 100 m or 10 m. Particles were synthesized with excess alkyne or azide functional groups, and then introduced into a cell suspension. Upon centrifugation, the particle-cell composite formed a macroscopic, but microporous, hydrogel via a strain-promoted azide alkyne cycloaddition (SPAAC) with a cell density of 310.sup.6 cells mL.sup.1. Introduction of a fibronectin-derived adhesive ligand, GRGDS, into the microsphere formulation allowed for cell-particle interactions, cell spreading and process extension. The described microgel networks may have widespread applications in both in vitro cell culture and regenerative medicine.

2. Results

2.1 Microgel Synthesis

[0071] Microgels containing excess DBCO (dibenzocylcooctyne) or azide groups were formed using an inverse suspension polymerization method (FIG. 1). Briefly, aqueous precursor solutions, containing PEG-DBCO (8-arm, 20 kDa), PEG-N.sub.3 (4-arm, 10 kDa), an azide functionalized fluorophore (AlexaFluor-594-N.sub.3), and an azide functionalized adhesive peptide (N.sub.3-GRGDS) were prepared with 11 mM excess of either functional group. Immediately after the addition of the limiting group (PEG-DBCO or PEG-N.sub.3), the aqueous solution was transferred to a continuous phase of hexane, Span-80, and Tween-20. In order to control microgel size, solutions were exposed to either low shear (vortexing) or high shear (sonication) for 5 minutes, which is sufficient time for modulus evolution (FIG. 7A-C). Particles were subsequently washed with hexanes (3), isopropanol (3), and transitioned to PBS.

[0072] Next, the size distribution of the microgels for each formulation was categorized using a custom Matlab script (FIG. 2A-C, FIG. 8). Microgels formed using low shear (hereafter referred to as 10.sup.2 m microgels) had a broad distribution, ranging between 30 m to 350 m, with mean particle diameters of 120 m and 130 m for gels containing excess DBCO and azide groups, respectively (FIG. 2B). Microgels formed using high shear (hereafter referred to as 10.sup.1 m microgels) were an order of magnitude smaller, with average particle sizes of 16 m and 15 m for DBCO and azide gels, respectively (FIG. 2C). Microgel mechanical properties were approximated by measuring the moduli of bulk gels at identical cross-linking densities. Gels with excess DBCO functional groups were 15 kPa in compressive modulus, while excess azide gels had a compressive modulus of 8.7 kPa (FIG. 7C).

2.2 Assembly of Microgels into Microporous Scaffolds and Characterization

[0073] To assemble the PEG microgels into a microporous, covalently linked material, a microgel suspension was prepared containing equal densities of both DBCO and N.sub.3 functionalized microgels. In order to increase the number of particle interactions and covalent cross-linking between microgels, solutions were concentrated via centrifuged for scaffold formation (FIG. 3A-C). While solutions containing both DBCO excess and N.sub.3 excess microgels formed networks in both the 10.sup.2 m and 10.sup.1 m particle cases (FIG. 3B and FIG. 3C), solutions containing homogenous particles (only DBCO or N.sub.3 surface reactive groups) at identical densities exposed to the same centrifugation did not form full networks (FIG. 10). The mechanical properties of the assembled gels were assessed by measuring their compressive moduli. Networks composed of 10.sup.2 m or 10.sup.1 m microgels formed networks with compressive moduli of approximately 2.1 kPa and 3.3 kPa, respectively (calculated via the slope of the linear stress-strain plot) (FIG. 3C). The bulk scaffold had a lower modulus (approximately 3-7 times lower) than the compressive moduli of the microgels.

[0074] Next was the categorization of the overall porosity, pore connectivity, morphology, and size distribution. Each hydrogel was first incubated in a high molecular weight fluorescein-labeled dextran solution to visualize the interconnectedness of the void space. The 250 kDa dextran readily diffused throughout the open pore structures, but did not penetrate the microgels themselves on the time scale of the experiment (FIG. 4A-C). Three-dimensional renderings of the scaffold demonstrate a continuous network of pores in both the 10.sup.2 m and 10.sup.1 m gel conditions. To quantify the overall porosity of the scaffolds, as well as the distribution and size of the pores, the overall void fraction and selected morphological characteristics of the pores were calculated using a custom Matlab script (FIG. 9). In brief, Z stack images were collected using a laser scanning confocal microscope of fluorescently labeled microgel assembled networks and analyzed at 12 m intervals over 400 m. A custom Matlab script was used to analyze the porous region in each image, identifying individual contiguous pores (FIG. 4B), and measure each pore's area, as well as major and minor axes lengths. The void space was calculated for each slice (area of pores/total area), averaged across the entire scaffold, and reported as the overall void fraction for each microgel type. The 10.sup.2 m networks had a void fraction of 293%, while the 10.sup.1 m gels had a lower void fraction of 122% (FIG. 4C).

[0075] While the void space distribution was relatively non-disperse across the gels, the pore areas within the gel were relatively heterogeneous. The 10.sup.1 m networks contained a higher number of pores than did the 10.sup.2 m networks, while the average pore size was much larger in 10.sup.2 m networks compared to 10.sup.1 m networks (FIG. 5A-C). In the case of the 10.sup.2 m system, 75% of the pores were less than 3300 m.sup.2 in area, while the corresponding amount of pores were less than 60 m.sup.2 in area in the 10.sup.1 m system (FIG. 5A). For reference, these sizes correspond to circles with diameters of approximately 65 m and 9 m, respectively. However, as the pores in this gel were irregularly shaped and typically acircular, the major axes lengths were measured (FIG. 5B), and the aspect ratio (major: minor) of the pores in each gel type is reported (FIG. 5C) were assessed to better categorize the dimensions of the void space in the gels. The majority (75%) of pores in the 10.sup.2 m and 10.sup.1 m gels had major axes at or below 120 m and 14.0 m, respectively (FIG. 5B). Despite the difference in average pore size, the shape of pores (as determined by aspect ratio) was very similar in both networks (FIG. 5B). In both cases, the average aspect ratio was approximately 2, indicating elongated, acircular pore shapes (FIG. 5C).

2.3 Co-Assembly of Cells and Microgels

[0076] To assess the ability to co-assemble primary cells and microgels during the scaffold fabrication process, bone marrow derived mesenchymal stem cells (hMSCs) were selected as a model system. Specifically, hMSCs were suspended in the microgel formulations prior to centrifugation, and encapsulated at approximately 310.sup.6 cells mL.sup.1. Upon assembly, the cell-laden microporous scaffolds were cultured for 96 hours, and overall cell viability, cytoskeletal structure, and cell morphology were assessed (FIG. 6A-C). Cells showed high survival in both cases, with approximately 95% viability observed in both the 10.sup.2 m and 10.sup.1 m networks (FIG. 6C). In the 10.sup.2 m networks, hMSCs spread robustly, stretching between multiple microgels or spreading across the surface of a single microgel (FIG. 6A insert, left). Similarly, the cells in the 10.sup.1 m networks also adopted a spread morphology, with an increase in thin, fibular-like projections from cells (FIG. 6A insert, right). Next cellular morphology was categorized by assessing cytoskeletal structure. Cells in the 10 m.sup.2 microgel networks spread and exhibited visible actin fibers (FIG. 6B, left), while those in the 10.sup.1 m microgel network had only small protrusions into the matrix, with diffuse actin staining FIG. 6B, right). Finally, cell shape was quantified by analyzing area:perimeter.sup.2 ratio in ImageJ, with cells in the 10.sup.1 m microgel networks significantly more circular than those in the 10.sup.2 m microgel networks (FIG. 6C).

3. Discussion

[0077] In one embodiment, the present invention contemplates a self-assembling microgel network was introduced for cell culture applications. This approach allows for the simple assembly of microgel components into a porous network without the need for porogens or post fabrication processing. The described SPAAC chemistry was used for microgel synthesis, as well as system assembly, without the addition of external initiators or cross-linking agents (FIG. 3A-C). Furthermore, stable networks did not form when only microgels containing a single surface functionality were centrifuged (FIG. 10), indicating that the networks form via covalent interactions rather than simple physical entanglements. Finally, the scaffolds demonstrated that they could be assembled in the presence of living cells at several densities, allowing for facile encapsulation and high cellular viability (FIG. 6A). By varying particle size, and thus network porosity and pore size, cell shape could be controlled, varying from a fibroblast type morphology in 10.sup.2 m microgel networks to a more rounded morphology with thin protrusions in the 10.sup.1 m microgel networks (FIG. 6C). This resulted in variations in cytoskeletal organization, with cells in larger pore sizes being able to form visible actin fibers, while those encapsulated in smaller pores showing only diffuse actin (FIG. 6B).

[0078] In one embodiment, the present invention contemplates modification of the microgels with the adhesive ligand RGD to allow for rapid cell spreading within the network. These cell-microgel interactions can be advantageous for network formation and engraftment in in vivo studies. Furthermore, the ability to functionalize microgels with bioactive moieties can be extended to numerous applications. In particular, functionalizing microgels with specific differentiation factors or using microgels as drug depots could allow for a new class of cell instructive materials. Microgel scaffolds have already been synthesized with deliverable growth factors [24, 26], and the fabrication of scaffolds with multiple chemical cues could allow for the creation of complex culture environments.

[0079] Beyond the inclusion of chemical cues, many material properties of this system can also be tuned. While nanoporous networks can achieve similar, or even higher void fractions [27, 28], as the current invention microgel assembled system, it has been demonstrated how particle size, and thus network pore size, affect cell growth. Both morphology and actin structure were significantly different between the 10.sup.2 m and 10.sup.1 m microgel networks. In the former, fibroblast morphology was observed and defined cytoskeletal actin fibers, while in the latter case cells were more contracted with thin protrusions into the network and diffuse cytosolic actin (FIG. 6A-C). This is similar to reported differences observed in cell morphology and actin structure as culture dimensionality and network elasticity is varied [29, 30]. It is possible that the smaller pore size in 10.sup.1 m microgel networks restricts cell spreading to thin processes, limiting cytoskeletal organization.

[0080] One advantage of this material system is the ability to manipulate the bulk versus local mechanical properties. It has been observed that a local microenvironmental stiffness greater than that of the macroscopic scaffolds, with microgels approximately 3-7 times the moduli of the bulk material. This stands to reason, as one may expect the significant percentage of void space to detract from the compressive modulus of the assembled networks. It is also possible that the dispersity of microgel sizes play an important role in determining both porosity and scaffold mechanical properties, as it is likely that this dispersity allows for tighter microgel packing than monodisperse samples. More importantly, this tunability can be highly advantageous, as one can recapitulate stiffer, yet porous, cellular environments, which may have broad reaching implications for regenerative medicine for different tissue environments (e.g., trabecular bone). Furthermore, as porous hydrogels allow for more rapid cell infiltration [31], ECM deposition [32], and a mitigated immune response [19], this system could aid in cell transplantation therapies. Thus, a microgel-assembled networks present a highly manipulatable system that could allow for a better understanding of cell function in native tissues, such as the bone marrow stem cell niche, and for in vivo tissue regeneration.

[0081] Finally, while this initial formulation relied on microgels only differing in their size and surface functionality (e.g., to allow for intraparticle cross-linking), this formulation could be readily adapted to include a more heterogeneous particle distribution to recapitulate aspects of native tissues. As material stiffness affects cellular function and fate [8, 9, 21, 33], microgels with varied stiffness, or even chemical moieties, might be included within the formulation to probe cell growth fate decisions in unique ways. While homogenous particles can be incorporated ubiquitously within the formulation, they could also be segregated to produce sub-regions within the material. Based on the nature of assembly, gradients or even distinct zones of functionalized microgels could be created via a layer-by-layer deposition method during centrifugation. These scaffolds could then be utilized to control specific cellular responses to recapitulate complex tissue interfaces both in vitro and in vivo [34]. This approach may be highly beneficial for recapitulating complex tissue interfaces (e.g., an osteochondral interface), where cell growth and fate decisions must be precisely regulated.

4. Conclusion

[0082] In one embodiment of the present invention, a bottom-up assembled microgel network suitable for cellular encapsulation has been designed. Clickable microgels of varied size were formed without the need for device fabrication, with varied surface functionalities allowing for spontaneous network formation. This system proved a useful cell culture platform, allowing for facile encapsulation, as well as controllable morphology, of mesenchymal stem cells. These porous scaffolds offer a high degree of tunability over both mechanical and chemical properties, and can be used to recapitulate highly varied or complex tissue environments. Collectively, the bio-click functionalized microgels and their cytocompatible assembly processes offer a unique platform to study and direct cell growth, interactions and function for both in vitro and in vivo applications.

5. Experimental Section

Monomer Synthesis

[0083] Eight-arm poly(ethylene glycol) (PEG) amine (M.sub.n20000 Da) was end functionalized with dibenzylcyclooctyne (DBCO) using standard HATU chemistry. Briefly, dibenzocyclooctyne acid (Click Chem Tools, USA), HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) coupling agent (1:1 ratio with DBCO, Sigma Aldrich, USA), and di-isopropylethylamine (DIPEA) (2:1 ratio with DBCO, Sigma Aldrich, USA) were dissolved in N,N-dimethylformamide (peptide synthesis grade) and combined with PEG amine (1:15 ratio to DBCO). The reaction was run overnight at room temperature, subsequently concentrated under reduced pressure by rotoevaporation, and precipitated in diethyl ether at 4 C. The resulting product was resuspended in DI H.sub.2O, dialyzed for 72 hours, and lyophilized. The extent of end-group functionalization was confirmed by .sup.1H NMR (Bruker AV-III) to be approximately 85%; .sup.1H NMR (400 MHz, CDCl.sub.3, ): 7.70 (d, J=7.5 Hz, 1H), 7.45 (m, 8H), 5.18 (d, J=13.8 Hz, 1H), 3.84 (q, J=4.0 Hz, 2H), 3.72 (s, 1H), 3.67 (m, PEG), 3.36 (q, J=5.3 Hz, 2H).

[0084] Four-arm poly(ethylene glycol) (PEG) azide (M.sub.n10000 Da) was synthesized as previously reported [35]. Briefly, 4-arm poly(ethylene glycol) was dissolved in dichloromethane (DCM) and pyridine (Sigma Aldrich) and cooled to 0 C. Methanesulfonyl chloride (20 fold excess to PEG) (Sigma Aldrich) dissolved in DCM was then added dropwise, and allowed to react overnight. The product was washed with aqueous sodium bicarbonate, dried with MgSO.sub.4, and precipitated in diethyl ether (Fisher). The mesylate activated PEG was then dissolved in anhydrous DMF along with sodium azide (5 fold excess to PEG) (Sigma Aldrich) and stirred overnight at 80 C. under argon. The product was filtered, concentrated under reduced pressure, resuspended in DI H.sub.2O, dialyzed for 72 hours, and lyophilized. The extent of end group functionalization was confirmed by .sup.1H NMR (Bruker AV-III) to be >98%; .sup.1H NMR (400 MHz, CDCl.sub.3, ): 3.65 (m, PEG), 3.41 (m, 2H).

[0085] The fibronectin derived adhesive peptide GRGDS (RGD) was synthesized on a Protein Technologies Tribute Peptide Synthesizer using standard Fmoc chemistry and a Rink Amide MBHA resin (Chempep Inc, USA). Azide functionalized RGD was synthesized by coupling 4-azidobutanoic acid to the free N-terminus using standard HATU chemistry. The peptide was purified using reverse phase High Pressure Liquid Chromatography (HPLC, Waters Corporation, mobile phase: water and acetonitrile) on an XSELECT CSH C18 column and confirmed via mass spectrometry using a Voyager DE-STR MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight).

Microgel Synthesis

[0086] Off-stoichiometry monomer solutions were prepared by combining PEG-DBCO, PEG-N.sub.3, and RGD-N.sub.3 in Phosphate Buffered Saline (PBS) (total volume 50 L). Solutions were made on ice with either excess DBCO or N.sub.3 groups at a concentration of 11 mM. In the case of DBCO excess gels, 8-arm 20 kDa PEG-DBCO (3 mM), 4-arm 10 kDa PEG-N.sub.3 (3 mM) and RGD-N.sub.3 (1 mM) were used, while for N.sub.3 excess gels PEG-DBCO (2 mM), PEG-N.sub.3 (6.5 mM), and RGD-N.sub.3 (1 mM) were used in the formulation. Each macromer solution was rapidly mixed and transferred to a continuous phase consisting of hexane with Span-80 (2.25% v/v) and Tween-20 (0.75% v/v) (20:1 ratio of continuous phase: aqueous phase). Solutions were then exposed to shear stress in the form of vortex mixing or bath sonication for 5 minutes to allow for complete polymerization (based on in situ gelation data (FIG. 7A and FIG. 7B)). Microgels were concentrated via ultracentrifugation (18,000 rcf, room temperature, 10 minutes) and washed with hexane (3), isopropyl alcohol (3), and PBS (1), followed by resuspension in 1 mL of PBS. After isopropyl alcohol washes, all gels were maintained in sterile conditions.

Microgel Size Categorization

[0087] In situ gelation data (FIG. 7A and FIG. 7B) was obtained by pipetting 20 L of monomer solution (for either DBCO-excess or N.sub.3-excess conditions) (cooled on ice) between the bottom plate and an 8 mm flat tool on a shear rheometer (TA DH-R3). Time sweeps were performed at 1% strain and 1 rad s.sup.1 for 300 seconds to observe full modulus evolution.

[0088] In order to assess the swollen moduli of microgels, 30 L of monomer solutions (for either DBCO-excess or N.sub.3-excess conditions) were pipetted into a 5 mm mold and allowed to swell overnight in PBS after formation. Swollen gels were then tested using an MTS Synergie 100. Tested gels were approximately 2 mm in height and 5.3 mm in diameter. Gels were exposed to compression up to 15% strain at a rate of 0.5 mm min.sup.1, and the compressive modulus was taken to be the slope of the reported stress-strain curve (linear region). Reported values are taken from four separate gels for each condition (DBCO-excess or N.sub.3-excess) (FIG. 7C).

Assembly of Microgels into Macroscopic, Porous Scaffolds

[0089] Microgel-based scaffolds were assembled by co-centrifuging equal quantities of DBCO-excess and N.sub.3-excess microgels together in 15 mL conical tubes. Particle densities of 810.sup.5 and 510.sup.7 particles/mL were used for 10.sup.2 m and 10.sup.1 m particle networks, respectively. The resulting microgel suspension was mixed and centrifuged (room temperature, 1,000 rcf for 10 minutes, and 3,000 rcf for 3 minutes) to form the covalently-linked microgel-assembled scaffold. The microporous gel assembly was carefully removed from the conical tube and left to equilibrate overnight in PBS, with both gels reaching final swollen volumes of 180 L.

Microgel Size Categorization

[0090] For imaging purposes, AlexaFluor594-N.sub.3 (Life Technologies) was included in the DBCO-excess and N.sub.3-excess formulations in the aqueous phase at 40 M. After washing and transitioning to PBS, the washed microgels were diluted and placed between a glass slide and a cover slip. Gels were imaged on a Zeiss LSM710 scanning confocal microscope with a 10 objective. Images were analyzed using a custom Matlab script (FIG. 8) to quantify particle sizes and distributions, analyzing at least 200 microgels (in total) originating from three separate syntheses. PDI values are calculated by (*d.sup.1).sup.2, where is the standard deviation and d is the average particle diameter. Particle size distributions were fit to Gaussian curves using Graph Pad Prism software.

Assembly of Microgels into Macroscopic, Porous Scaffolds

[0091] Microgel-based scaffolds were assembled by co-centrifuging equal quantities of DBCO-excess and N.sub.3-excess microgels together in 15 mL conical tubes. In both conditions, DBCO-excess and N.sub.3-excess gels were mixed in 2 mL of PBS to reach final particle densities of 810.sup.5 and 910.sup.7 particles mL.sup.1 for 10.sup.2 m and 10.sup.1 m particle networks, respectively. These densities correspond to equal microgel volumes (50 L starting monomer volume). The resulting microgel suspension was mixed and centrifuged (room temperature, 1,000 rcf for 10 minutes, and 3,000 rcf for 3 minutes) to form the covalently-linked microgel-assembled scaffold. Centrifugation speeds were chosen to ensure network formation, without limiting viability in subsequent cell studies. The microporous gel assembly was carefully removed from the conical tube and left to equilibrate overnight in PBS. Gel volumes were determined by displacement, with both 10.sup.2 and 10.sup.1 m gel networks having average volumes of 160 L (pre-swollen) and 180 (final swollen volume) (average measurements of at least 3 gels per condition).

Characterization of the Microporous Gel Assembly

[0092] After equilibration in PBS, the porosity of the scaffolds containing the fluorescently labeled particles was assessed using quantitative image analysis techniques. First, images were collected on a Zeiss LSM710 scanning confocal microscope using a 10 objective; z-stacks were taken every 3-4 m. Next, gels were incubated with high molecular weight fluorescein labeled dextran (250 kDa, Sigma Aldrich) to assess pore interconnectivity. The high molecular weight prevents dextran diffusion into the microgels, while still allowing for transport through the pores. The images were analyzed using a custom Matlab code (FIG. 9) to quantify the dimensions and size of each pore, as well as the overall scaffold porosity of the scaffolds. In brief, slices were taken every 12 m, converted to binary, and thresholded to identify individual contiguous pores. In both cases, 400 m stacks were imaged from three separate microgel-assembled scaffolds. Over 1500 identified pores were then categorized for each case (10.sup.2 m or 10.sup.1 m scaffolds). The area, major and minor axes lengths for each pore were then identified and averaged across each condition.

[0093] The macroscopic mechanical properties of the reacted microgel assemblies were tested using an MTS Synergie 100. Tested microporous hydrogels were conical, approximately 7.55 mm in height and 9 mm in diameter at the base. The microporous hydrogels were exposed to compression up to 15% strain at a rate of 0.5 mm min.sup.1, and the compressive modulus was taken to be the slope of the reported stress-strain curve (linear region). Reported compressive moduli are taken from five gels from each condition (10.sup.2 m and 10.sup.1 m gel networks).

Cell Culture

[0094] Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates (Lonza Biosciences) as previously reported [36]. Bone marrow samples were plated on 10 mm tissue culture polystyrene plates (Corning, USA) and cultured in growth media (low-glucose DMEM (1 g L.sup.1) with 10% (v/v) fetal bovine serum, penicillin (50 U mL.sup.1), streptomycin (50 g amphotericin B (500 ng mL.sup.1), and basic fibroblast growth factor (bFGF) (1 ng mL.sup.1)) for 72 hours at 5% CO, and 37 C. Media was aspirated to remove non-adherent cells; adherent cells were expanded in growth media, trypsinized, and frozen until use in experiments. Prior to encapsulation, cells were similarly plated, expanded (not exceeding 80% confluency), and trypsinized, with all experiments using cells at passage three.

Assembly of Microgel-Cell Composite Scaffolds and Cell Categorization

[0095] Cell-laden microporous networks were then fabricated by co-assembling hMSCs with both DBCO-excess and N.sub.3-excess microgels. hMSCs were mixed with DBCO-excess and N.sub.3-excess gels and centrifuged at high speed (room temperature, 1,000 rcf for 10 minutes, and 3,000 rcf for 3 minutes) to form cell-laden gels at 310.sup.6 cells mL.sup.1. The resulting 180 L gels were placed in wells containing hMSC experimental media (growth media without bFGF). Then cell viability and morphology was quantified to assess the potential of the network as a cell culture scaffold. Cell viability was quantified at 96 hours after encapsulation via calcein (0.5 M, green, live) and ethidium homodimer (1 M, red, dead) staining. Cell morphology was characterized by measuring the aspect ratio and circularity of each cell, where circularity is defined as 4n*Area*Perimeter.sup.2. Four separate gels for each condition (10.sup.2 m or 10.sup.1 m microgel networks) were analyzed with at least 100 cells from each gel (totaling 500 cells per condition). Cell circularity was averaged for each gel, and statistical analysis was then performed using an unpaired t-test with Welch's correction (to account for differing standard deviations) using Graph Pad Prism software. Finally, all images used are maximum intensity projections of 300-400 m stacks taken with a 10 objective.

[0096] Cytoskeletal morphology of cells within microporous scaffolds was also assessed after 96 hours in culture. Microgel networks were fixed in 10% formalin (Sigma Aldrich) for 30 minutes, permeabilized in 0.1% Triton (100, Sigma Aldrich) in PBS for 1 hour, blocked with 1% bovine serum albumin solution, and stained with DAPI (300 nM, Life Technologies) and rhodamine-phalloidin (22 nM) overnight. All images are maximum intensity projections of 200-300 m stacks taken with a 20 objective.

[0097] Thus, specific compositions and methods of synthesis and assembly of clickable microgels into cell-laden porous scaffolds have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Moreover, in interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms comprises and comprising should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

[0098] Although the invention has been described with reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all applications, patents, and publications cited above, and of the corresponding application are hereby incorporated by reference.

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