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

Journal name:
Nature Materials
Volume:
14,
Pages:
737–744
Year published:
DOI:
doi:10.1038/nmat4294
Received
Accepted
Published online

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.

At a glance

Figures

  1. Microfluidic generation of microsphere hydrogel building blocks for the creation of microporous annealed particle (MAP) scaffolds.
    Figure 1: Microfluidic generation of microsphere hydrogel building blocks for the creation of microporous annealed particle (MAP) scaffolds.

    a, Scheme illustrating microgel 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. b, microgels 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 microgel building blocks (left) and a subsequent cell-laden MAP scaffold (right). d, MAP scaffolds are mouldable to macroscale shapes, and can be injected to form complex shapes that are maintained after annealing. e, This process can be performed in the presence of live cells.

  2. High-precision fabrication of microgel building blocks allows the creation of defined MAP scaffolds.
    Figure 2: High-precision fabrication of microgel building blocks allows the creation of defined MAP scaffolds.

    a, The operational regime for microfluidic microgel 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 (Qv) is predictable and determined by polymer network characteristics. In our chosen formulation, Qv = 4.5. c, Representative images of microgel droplets in flow after generation. d, Rheological characterization of the MAP scaffold. Without the addition of FXIIIa the microgel building blocks exhibit some gel-like characteristics; however, the onset of annealing results in significantly increased macroscale mechanical moduli. e, Different building-block sizes allow deterministic control over resultant microporous network characteristics, presented here as median pore sizes ± s.d. f, Single confocal slices of MAP scaffolds created using different building-block sizes. All data presented as average ± s.d. unless otherwise stated. All experiments performed in triplicate.

  3. MAP scaffolds facilitate 3D cellular network formation and proliferation in vitro.
    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 h 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 six days. (350 Pa, bulk modulus identical to MAP; 600 Pa, microscale modulus matched to individual microgels). 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 more slowly, 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 ± s.d. All experiments performed in triplicate.

  4. MAP scaffolds promote fast wound closure in SKH1-Hrhr and Balb/c epidermal mouse models.
    Figure 4: MAP scaffolds promote fast wound closure in SKH1-Hrhr 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 h post injection in SKH1-Hrhr mice. b, Quantification of wound closure over a five-day period shows statistically significant wound-closure rates for MAP scaffolds when compared with non-porous bilateral controls (N = 6). Statistical significance performed using standard two-tailed t-test (p < 0.05). c, Representative images of wound closure during a five-day in vivo wound-healing model in SKH1-Hrhr mice. d, Representative images of wound closure during seven-day in vivo Balb/c experiments. e, Quantification of wound-closure data from Balb/c in vivo wound healing. After seven 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 = 5–7). f, Traces of wound-bed closure during seven days in vivo for each treatment category. All data are presented as average ± s.e.m. Statistical significance performed using one-way ANOVA with a Dunnett post hoc multiple comparison test (p < 0.05; p < 0.01).

  5. MAP scaffolds allow faster tissue regeneration compared with non-porous controls in vivo.
    Figure 5: MAP scaffolds allow faster tissue regeneration compared with non-porous controls in vivo.

    a, Matching wound-closure data (Fig. 4), the MAP scaffolds also allow significant re-epithelialization five 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 five days. Non-porous controls are devoid of similar complex multicellular structures. b, Non-porous gels show some PECAM-1 positive cells ingrowth, however, there is no positive staining for supporting vascular cells (NG2). 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.

  6. MAP scaffolds elicit a significantly lower immune response than non-porous hydrogels in vivo.
    Figure 6: MAP scaffolds elicit a significantly lower immune response than non-porous hydrogels in vivo.

    a,b, Quantification of total cellular infiltration into the constructs (a) and immune response in the surrounding tissue 24 h post injection (b). 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 five 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 with non-porous controls, both inside the construct and in the surrounding tissue (N = 6 for ac). d, Representative images of tissue sections from five days after injection for MAP scaffolds and non-porous controls. All data presented as average ± s.d. Statistical significance performed using standard two-tailed t-test (p < 0.05; p < 0.01; p < 0.001).

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Author information

  1. These authors contributed equally to this work.

    • Donald R. Griffin &
    • Westbrook M. Weaver

Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, California 90095, USA

    • Donald R. Griffin
  2. Department of Bioengineering, University of California, Los Angeles, Los Angeles, California 90095, USA

    • Westbrook M. Weaver
  3. Division of Dermatology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA

    • Philip O. Scumpia
  4. Department of Bioengineering, California NanoSystems Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California 90095, USA

    • Dino Di Carlo
  5. Department of Chemical and Biomolecular Engineering, California NanoSystems Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California 90095, USA

    • Tatiana Segura

Contributions

D.R.G. and W.M.W. contributed equally to this manuscript, both in conceptual design, troubleshooting, experimental execution and manuscript writing. P.O.S. performed Day 1 immunological analysis and in vivo interpretation. D.D.C. and T.S. contributed equally to overseeing experimental design and interpretation.

Competing financial interests

The authors have a financial interest in Tempo Therapeutics, which aims to commercialize MAP technology.

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