Impacts of Type V Collage Haploinsufficiency on the Structural Integrity and Immunoresponses of Dermal Wound Healing

Introduction: Pathological wounds are a prevalent healthcare problem, affecting more than 40 million Americans [1]. Imbalanced healing often results in fibrotic scar formation, marked by excessive deposition and aberrant assembly of collagen I fibers. Type V collagen, a minor fibril-forming collagen, is a key regulator of the initial fibrillogenesis of collagen I during embryonic development [2], and could be an important player in wound healing [3]. For patients with classic Ehlers-Danlos syndrome (cEDS), a genetic disorder caused by the mutation of human COL5A1 gene, major symptoms include fragile skin, delayed wound healing and healed scar tissue [4, 5]. Understanding how collagen V contributes to skin tissue integrity and immune response from wound healing will provide new insights for developing effective regenerative strategies and improved care for cEDS patients. This study aims to elucidate how collagen V regulates the tissue structural integrity and immune responses during wound healing by studying the collagen V heterozygous mice, Col5a1+/−, an established model of cEDS [6].

Materials and

Methods: Two 6-mm diameter circular full-thickness dermal punch wounds were created on the lower dorsal skin of Col5a1+/− and wild-type (WT) mice at 7 weeks of age and harvested at different post-wound days (PWD) [7]. We applied Picrosirius Red staining and polarized light microscope imaging (PLM) (Olympus BX51-P) to assess the collagen fiber structure of intact skin. CT-Fire was utilized to analyze the fiber length and width of PLM images [8]. Scanning electron microscopy (SEM) was applied at 20k× magnification to image the fibril nanostructure on 10-µm thick cross-sections of intact skin and scar tissue at PWD35. ImageJ was used to measure the fiber diameter of SEM images. PWD35 skin samples were harvested and stamped out into dumbbell-shaped pieces for tensile tests of maximum stress to failure and Young’s modulus for both the intact skin and scar tissue (Instron 5542) [9]. Flow cytometry (BD Fortessa or Symphony) was performed to assess PWD14 macrophage activities after re-epithelialization and results were analyzed using FlowJo software [10]. Unpaired two-sample student’s t-test was applied to determine the effects of genotype at α = 0.05.

Results, Conclusions, and Discussions: This study highlights the crucial role of collagen V in mediating the integrity of scar tissue and immunoresponses during dermal wound healing. Studying the Col5a1+/− mice, we have previously shown that reduction of collagen V led to delayed wound healing and substantially increased scar tissue size using the dermal wound punch model [3]. Here, we further demonstrated that the reduction of collagen V led to an increase in collagen I fiber size, as marked by wider and longer fibers than their WT counterparts (Fig. 1b,c). At the nanoscale, for both genotypes, the scar tissue showed thinner, densely packed and more aligned collagen fibrils compared to the intact dermis, illustrating the aberrant assembly of collagen during wound healing (Fig. 2a, arrowheads). In comparison to the WT, Col5a1+/− mice exhibited thicker collagen fibrils for both intact skin and scar tissue, supporting that reduction of collagen V led to disrupted collagen fibrillogenesis in both normal matrix assembly and aggravated scar formation (Fig. 2b). In alignment with these structural defects, we further demonstrated that Col5a1+/− tissues demonstrated lower linear modulus and maximum stress to failure under tensile test compared to the WT counterparts, for both intact skin and scar tissue (Fig. 3b,c). Together, these results illustrated reduction of collagen V impaired the structural integrity and mechanical functions of skin tissue during dermal wound healing. In addition, at PWD14, Col5a1+/− mice retained a higher percentage of macrophages in the wound cell population (Fig. 4a), as well as a higher proportion of CD301b and CD206 positive macrophages (Fig. 4b) compared to the WT. These results evidenced elevated activities with dermal dendritic cells and M2 type macrophages [11, 12], which likely results from disrupted matrix remodeling with the reduction of collagen V.

This study highlights that collagen V is a crucial regulator of the structural integrity, mechanical function as well as inflammation-driven macrophage activities during dermal wound healing. Modulating the activities of collagen V could serve as a potential therapeutic target for enhancing wound regeneration and improving the care for cEDS patients.

Acknowledgements (Optional): This work is supported by NSF CAREER CMMI-1751898 as well as the NIH Grant P30AR069619 to the Penn Center for Musculoskeletal Disorders (Biomechanics Core).

References: [1] Birk+ 1990. [2] Wenstrup+ 2004. [3] DeNigris+ 2016. [4] Mak+ 2016. [5] Malfait+ 2010. [7] Wenstrup+ 2006. [8] Jorgensen+ 2020. [9] Bermudez+ 2011. [10] O’Brien+ 2022. [11] Kim+ 2018. [12] Xu+ 2020.