Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks

Abstract

Injectable hydrogels can provide a scaffold for in situ tissue regrowth and regeneration, yet gel degradation before tissue reformation limits the gels' ability to provide physical support. Here, we show that this shortcoming can be circumvented through an injectable, interconnected microporous gel scaffold assembled from annealed microgel building blocks whose chemical and physical properties can be tailored by microfluidic fabrication. In vitro, cells incorporated during scaffold formation proliferated and formed extensive three-dimensional networks within 48 h. In vivo, the scaffolds facilitated cell migration that resulted in rapid cutaneous-tissue regeneration and tissue-structure formation within five days. The combination of microporosity and injectability of these annealed gel scaffolds should enable novel routes to tissue regeneration and formation in vivo.

Extended Data Figure 1

Microfluidic generation of building block μgels using a water-in-oil segmentation approach. A: Scheme of the microfluidic channel design used, with two aqueous inlets and two oil inlets. The collection well lies at the channel outlet (right side of scheme). B: In the droplet segmentation region, mineral oil with 0.25% Span 80 pinches and segments PEG pre-gel, and downstream a 5% Span 80 solution in mineral oil mixes and prevents downstream coalescence of μgels before complete gelation. Droplet’s internal contents mix during incubation in the bifurcation region and exit from the microchannel to the collection well. C High Peclet number (Pe > 10) prevents mixing of PEG and crosslinker (x-link) upstream of the segmentation region. D: Building block mixtures are injectable (e.g. 25 Gauge syringe) and can be molded to any shape (here shown after conforming to a star-shaped laser cut acrylic mold).

Extended Data Figure 2

Control over the μgel building block parameters and the resultant MAP scaffolds. A: Gelation kinetics of the pre-polymer and crosslinker solution were altered by tuning solution pH and gelation temperature. The gelation environment chosen for this study was pH 8.25 and 37°C. Tuning both B: the weight % of PEG and C: the r-ratio of free crosslinker ends (-SH) to vinyl groups (-VS) on the PEG backbone allows control over the storage modulus of the resultant gels. D: Degradation kinetics of MAP and non-porous (equal volumes) gels in vitro. MAP gels degrade faster than non-porous due to higher surface area to volume ratios and faster transport through the microporous gel. Degradation was carried out using 1:1000 TriplE, resulting in higher protease concentrations than in a wound bed and faster degradation kinetics. E: SEM images of a MAP scaffold annealed with FXIIIa. F: SEM images of μgel building blocks without FXIIIa. Green highlights indicate μgel building blocks.

Extended Data Figure 3

MAP scaffolds promote proliferation and network formation in vitro. A: representative images of HDF, AhMSC, and BMhMSC cell lines grown in non-porous PEG hydrogels of the same chemical composition as the MAP scaffolds. B: Representative images of the same cell lines grown in the MAP scaffolds.

Extended Data Figure 4

A: Maximum Intensity projections (MIPs) of the representative images from Extended Data Fig. 3a. B: MIPs of the MAP scaffolds after 6 days in vitro are shown for comparison.

Extended Data Figure 5

Scheme for image analysis of DAPI stained in vitro MAP gels for proliferation studies. A: Volumetric data within the scaffold is captured using a Nikon Ti eclipse confocal. Each volume is 317 × 317 × 150 um³ (L × W × H), and consists of 55 slices. B: Slices are combined into 11 groups, each containing 5 slices. The maximum intensity projection (MIP) of those five slices is projected into 1 2D image. C: These MIP images are then fed through an automated image analysis script written in MATLAB that filters the image, converts it to black and white, and counts the number of nuclei present in each section that is in focus, indicating that its centroid resides within the original five slices for that MIP. The counts for each MIP are summed for each gel, and this is presented as a total number of cells (plotted in figure 3d).

Extended Data Figure 6

Scheme for technique used to analyze and quantitate the immune response of adjacent tissue to injected MAP scaffolds and non-porous bilateral controls 5 days post-injection. A: Each image is a 3×3 stitched image using a 40× water immersion objective (Nikon). These images are decomposed into their three distinct channels, where red represents injected gel, blue shows DAPI stained nuclei, and green indicated the presence of the CD11b antigen on cell surfaces, a marker of activated leukocytes. The red channel is used to find the edge of the gel-tissue interface, and this line is expanded 75μm both into the surrounding tissue and into the gel itself. B: These lines are then imposed on the green and blue channels, used as cropping margins. C: The resultant images are merged and counted, where data is presented as the fraction of DAPI nuclei associated with CD11b⁺ signal. Three non-adjacent sections from each tissue block were used to count cells for both MAP scaffolds and non-porous controls.

Extended Data Figure 7

The MAP scaffold promotes tissue-like structure formation in vivo. A: Examples of normal hair follicles in non-wound areas of CLR:SKH1-Hr^hr mice. B: Two examples of structure formation within the MAP scaffold 5 days post injection. The early stages of large invaginations from the growing epidermis (top panel) and formation of structures (bottom panel).

Extended Data Figure 8

Large-scale tissue structures formed after treatment with the MAP scaffold in vivo. A: Further examples of vascular formation within the MAP scaffold after 5 days in vivo, as depicted via the juxtaposition of PECAM-1⁺ cells with PDGFRβ⁺ cells, as well as co-staining of NG2 and PDGFRβ on cells within the MAP scaffold. B: Stitched images of cross-sections of the wound beds treated with MAP after 5 days in vivo. These sections have been completely covered with sheets positive for both Keratin 5 and Keratin 14. C: Examples of normal tissue epithelial structure in the SKH1-Hr^hr mouse. Stratified epithelium is shown, where basilar layers express high levels of Keratin 5 and CD49f, where as more apical layers continue to express Keratin -14, but loose Keratin 5 expression. This same pattern is seen in tissue growing within the wounds treated with MAP (figure 5).

Figure 1

Microfluidic generation of microsphere hydrogel building blocks for the creation of Microporous Annealed Particle (MAP) scaffolds. A–B: Scheme illustrating μgel formation using a microfluidic water-in-oil emulsion system. A pre-gel and crosslinker solution are segmented into monodisperse droplets followed by in-droplet mixing and crosslinking via Michael-addition. μgels are purified into an aqueous solution and annealed using FXIIIa into a microporous scaffold, either in the presence of cells or as a pure scaffold. C: Fluorescent images showing purified μgel building blocks and a subsequent cell-laden MAP scaffold. D–E: MAP scaffolds are moldable to macro-scale shapes, and can be injected to form complex shapes that are maintained after annealing. A process that can be performed in the presence of live cells.

Figure 2

High precision fabrication of μgel building blocks allows creation of defined MAP scaffolds. A: The operational regime for microfluidic μgel generation has a large dynamic range, spanning almost an order of magnitude in size while maintaining tight control at each condition, with CVs < 6% in all cases. B: Hydrogel building blocks swell in buffer after aqueous extraction from the oil phase. The swelling ratio (Q_v) is predictable and determined by polymer network characteristics. In our chosen formulation, Q_v=4.5. C: Representative images of μgel droplets in flow after generation. D: Rheological characterization of the MAP scaffold. Without the addition of FXIIIa the μgel building blocks display some gel-like characteristics, however the onset of annealing results in significantly increased macro-scale mechanical moduli. E: Different building block sizes allow for deterministic control over resultant micro-porous network characteristics, presented here as median pore sizes +/− SD. F: Single confocal slices of MAP scaffolds created using different building block sizes. All data presented as average +/−- SD unless otherwise stated. All experiments performed in triplicate.

Figure 3

MAP scaffolds facilitate 3D cellular network formation and proliferation in vitro. A: Schematic illustrating how to read images of 3D cell growth and network formation presented in C. B: Cell survival 24 hours post annealing is greater than 93% across three cell lines representing different human tissue types. HDF: Human dermal fibroblasts, AhMSC: Adipose-derived human mesenchymal stem cells, BMhMSC: Bone marrow-derived human mesenchymal stem cells. C: Fluorescent images demonstrating the formation of 3D cellular networks during six days of culture in MAP scaffolds in vitro as well as non-porous gels after 6 days. (350 Pa: bulk modulus identical to MAP, 600 Pa: microscale modulus matched to individual μgels) D: Cells proliferate within the MAP scaffold while forming interconnected networks. HDF and AhMSC cells proliferate quickly within the scaffolds with doubling times of ~1.5 and ~2 days, respectively. BMhMSC cells proliferate significantly slower, with a calculated doubling time of ~12 days. These are analogous to previously observed normal growth phenotypes for these lines. All data presented as average +/− SD. All experiments performed in triplicate.

Figure 4

MAP scaffolds promote fast wound closure in SKH1-Hr^hr and Balb/c epidermal mouse models. A: H&E staining of tissue sections indicate seamless integration of the injected MAP scaffold as well as the non-porous control 24 hours post injection in SKH1-Hr^hr mice. B: Quantification of wound closure over a 5 day period shows statistically significant wound closure rates for MAP scaffolds when compared to non-porous bilateral controls (N = 5). C: Representative images of would closure during a 5 day in vivo wound healing model in SKH1-Hr^hr mice. D: Representative images of wound closure during 7 day in vivo Balb/c experiments. E: Quantification of wound closure data form Balb/c in vivo wound healing. After 7 days in vivo, the MAP scaffolds promote significantly faster wound healing than the no treatment control, the non-porous PEG gel, and the MAP gels lacking the K and Q peptides. Porous gels created ex vivo to precisely match the wound shape using the canonical, porogen-based, casting method showed appreciable wound healing rates, comparable to the MAP scaffolds, but lacking injectability (N≥5). F: Traces of wound bed closure during 7 days in vivo for each treatment category. All data are presented as average +/− SEM. Statistical significance performed using standard two-tailed t-test (*: p<0.05; **p<0.01).

Figure 5

MAP scaffolds allow for faster tissue regeneration compared to non-porous controls in vivo. A: Matching wound closure data (Figure 4), the MAP scaffolds also allow for significant re-epithelialization 5 days post injection. By comparison, the non-porous constructs show very little to no re-epithelialization by day 5. Importantly, in addition to stratified expression of keratin-5, keratin-14, and CD49f above the gel, we also observe large-scale tissue structures within the construct. Keratin-5 staining of the basement epithelial layer outline developing hair follicles and sebaceous glands within the MAP scaffold after 5 days. Non-porous controls are devoid of similar complex multicellular structures. B–C?: MAP scaffolds contain large networks of cells staining positive for the endothelial marker, PECAM-1, juxtapositioned with cells expressing NG2 and PDGFR-β (a pericyte phenotype), indicative of developing vasculature.

Figure 6

MAP scaffolds elicit a significantly lower immune response than non-porous hydrogels in vivo. A: Quantification of both total cellular infiltration into the constructs and immune response in the surrounding tissue 24 hours post injection. Inflammation is measured using a paired test for each mouse, where the fraction is the number of inflammatory cells for each construct relative to its bilateral control. C: Quantification of immune response 5 days after injection, as measured by the fraction of total cells expressing CD11b. MAP scaffolds elicit a significantly lower response of CD11b⁺ cells as compared to non-porous controls, both inside the construct and in the surrounding tissue. D: Representative images of tissue sections from 5 days after injection for MAP scaffolds and non-porous controls. All data presented as average +/− SD. Statistical significance performed using standard two-tailed t-test (*: p<0.05; **p<0.01; ****p<0.001).