Elsevier

Biomaterials

Volume 288, September 2022, 121692
Biomaterials

Murine macrophages or their secretome delivered in alginate dressings enhance impaired wound healing in diabetic mice

https://doi.org/10.1016/j.biomaterials.2022.121692Get rights and content

Abstract

Diabetic foot ulceration is a devastating diabetic complication with unmet needs. We explored the efficacy of calcium-crosslinked alginate dressings in topically delivering primary macrophages and their secretome to diabetic wounds. The alginate bandages had a microporous structure that enabled even cell loading with prolonged cell survival and egress following wound placement. In vitro experiments showed that we could successfully differentiate and polarize primary murine bone marrow derived monocytes into M0, M1, M2a and M2c defined states with distinct gene expression, surface protein and secretome profiles. The primary macrophages were delivered in the bandages, migrated within the wounds and were still present for as long as 16 days post-injury. In wounds of db/db mice, treatment with all macrophage subtypes and their secretome, when compared to control, accelerated wound healing. Bulk RNA sequencing analysis and multiplex protein quantification of wound lysates revealed that M2c macrophages conditioned media had the most impact in wound healing affecting processes like neurogenesis, while M1 conditioned media promoted keratinization and epidermal differentiation. Collectively, our results indicate that alginate dressings can serve as a delivery platform for topical treatment of diabetic wounds and that conditioned media from distinctly polarized macrophages is equally or more effective than their parental cells in advancing wound healing and could therefore be a promising and technically advantageous alternative to cell therapy.

Section snippets

CRediT author statement

Conceptualization: G.T, D.M., A.V.; Formal analysis: G.T, S.R, X.K, S.M., M.N.; Funding acquisition: D.M., A.V.; Investigation: G.T, S.R, S.L., Z.L., A.L, K.K, P.W.; Methodology: G.T., S.R., S.L.; Supervision: I.V., M.N., D.M., A.V.; Validation: G.T., S.R.; Visualization: G.T., S.R.; Writing - original draft: G.T.; Writing - review & editing: G.T., M.N., D. M., and A.V.

Alginate bandage fabrication and optimization of cell seeding

Alginate bandages with micron-sized pores were fabricated by a process of lyophilization, calcium crosslinking, and a second lyophilization step to yield dry, off-the-shelf available materials (Fig. 1A). Fabricated bandages demonstrated consistent porosity across their cross-section (Fig. 1B), with continuous pores running throughout the volume (Fig. 1C). The calcium crosslinking and second lyophilization did not markedly alter the structure nor porosity resulting from the initial bandage

Discussion

In the present study, we first showed that alginate dressings with a microporous structure enable cell loading, and are an appropriate delivery platform for macrophages and their conditioned media. A microporous structure was desirable, in order to enable cell loading throughout the dressing volume, and subsequent cellular egress into wounds after placement. These dressings further enabled cell proliferation and maintenance of viability. In the lyophilized state, these dressings can be readily

Animals

All animals were housed and treated in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental procedures were approved by the IACUC at Beth Israel Deaconess Medical Center. Male C57BL/6 J (stock # 000664), CAG:mRFP1 [B6. Cg-Tg(CAG-mRFP1)1F1Hadj/J, stock # 005884)] and db/db [B6·BKS(D)-Leprdb/J, stock # 000697] mice were obtained from Jackson Laboratories and were acclimated to the animal facility for at least 1 week before

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Institutes of Health Grant DP3DK108224 (D.M. and A.V.). A.V. received funding from the National Rongxiang Xu Foundation. G.T. received a George and Marie Vergottis Foundation Postdoctoral Fellowship.

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    These authors contributed equally to this work as co-senior authors.

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