Hyaluronic Acid and Emergent Tissue Mechanics Orchestrate Digit Tip Regeneration

Background

If we suffer the loss of the tip of our finger, it will grow back. However, we lack the ability to regenerate any more than this, for example, if we lose an arm then it doesn’t come back. As well as being an interesting developmental biology problem, a better understanding of the mechanisms controlling regeneration could also enable better therapeutic treatment of wounds (Simkin et al., 2015; Storer & Miller, 2021).

Humans are not completely lacking in regenerative ability, for example foetal wounds heal/regenerate without scarring (Moore et al., 2018). Unlike foetal wounds, adult injuries heal through deposition of extracellular matrix (ECM) components in a process called fibrosis, eventually leading to scar formation (Wynn & Ramalingam, 2012). In these examples, hyaluronic acid (HA) is key in the foetal wound repair, whilst fibrotic wounds typically get much stiffer (Mast et al., 1992; Ziol et al., 2005). The ECM substrate can therefore play a vital role in the regenerative capability of a wound, and changes in the mechanical environment affect tissue repair and regeneration (Kuehlmann et al., 2020; Rosińczuk et al., 2016). Nevertheless, by comparison to molecular and cellular signals, mechanical cues from the ECM are an understudied area which has been of increasing interest.

In this first preprint from the Storer lab, Mui and colleagues delve into the importance of the ECM in wound regeneration in mice, revealing that soft substrates and HA deposition promote regeneration.

Key findings

Summary

The team used a mouse model of regeneration where digits were amputated to generate regenerative (blastema) or non-regenerative wounds. They first compared the two wound types and found blastemas to be softer and have higher levels of HA deposition compared to non-regenerative wounds which were stiffer, fibrotic and had more collagen. They then experimentally challenged the system by preventing HA synthesis and showed HA levels can determine wound stiffness and regenerative capability, and that HA levels and substrate stiffness also affect BMP signalling. Finally, they showed HAPLN1 is mechanosensitive and can stabilise HA, with overexpression restoring regenerative capacity.

Blastemas have a softer ECM environment compared to non-regenerative wounds

First, the team applied second harmonic generation microscopy (SHGM) and atomic force microscopy (AFM) to image Blastema and non-regenerative wounds (Fig. 1). SHGM is label free non-invasive and especially good for labelling collagen and muscle structures (Aghigh et al., 2023; Chen et al., 2012). Whilst AFM uses a cantilever to scan over the tissue at a set pressure and measure the substrate stiffness.

Using these novel techniques on blastemas and non-regenerative wounds, the authors demonstrated that the blastema has less collagen deposition and is softer than the non-regenerative wounds.

Figure 1: Second harmonic generation microscopy from figure 1 of Mui et al (2024) showing greater collagen deposition in non-regenerative (NR) than regenerative (R) wounds. Image made available under a CC-BY-NC-ND 4.0 International license.

The composition of fibroblast populations differs between Blastema and non-regenerative wounds

Fibroblasts are the principal cell type in connective tissue and have functional heterogeneity within their population (Ganier et al., 2022; Lendahl et al., 2022). Here, Mui and colleagues performed single cell sequencing and identified fibroblasts based on expression of PDGFRA (Fig. 2). They found that two populations (fibroblast 1 and 2) deposit collagen and contribute to a stiff environment. However, in the blastema, an osteo-lineage (OL) fibroblast population synthesises hyaluronic acid (HA), Hyaluronan and Proteglycan Link Protein 1 (HAPLN1), and aggrecan (ACAN). This OL population is three times more prevalent in Blastema than non-regenerative wounds and the synthesis of HA, HAPLN1 and ACAN form a distinct ECM niche.

Figure 2: Uniform Manifold Approximation and Projection (UMAP) plots from figure 1 of Mui et al (2024) showing different fibroblast populations detected in single cell RNA sequencing of non-regenerative and regenerative (blastema) wounds. The same cells were coloured for the data set they originated from highlighting the prevalence of different fibroblast subtypes in the two wound conditions. Image made available under a CC-BY-NC-ND 4.0 International license.

A balance of hyaluronic acid and collagen determines wound stiffness and regenerative capability

4-methylumbelliferone (4-MU) is widely used to inhibit HA synthesis and is also an approved therapeutic (as hymecromone) in humans (Nagy et al., 2015). The team provided 4-MU in the mouse diet, followed by regenerative digit amputations. The 4-MU diet prevented digit regeneration as 4-MU digits were smaller in length and area, as well as bone volume, surface area and length, compared to control digits (Fig. 3). 4-MU digit wounds had more collagen and fibrotic ECM and were stiffer, more closely resembling a non-regenerative wound. There were also fewer OL fibroblasts, suggesting these are sensitive to the levels of HA. This led the team to propose a model where the balance of HA and collagen can determine the blastema/wound stiffness and fluid properties, with low stiffness and high fluidity leading to a regenerative blastema.

Figure 3: Gross images of control and 4-MU digits showed reduced nail length/area 14- and 28-days post amputation in 4-MU treated mice. Similarly, microcomputed tomography analysis of bones 28 days after amputation showed that bone volume and length was reduced in 4-MU treated mice. Adapted from figure 3 of Mui et al (2024). Image made available under a CC-BY-NC-ND 4.0 International license.

The defect in bone regeneration further inspired the team to look at the bone morphogenic protein (BMP) pathway. Immunofluorescence and Western blots were used to confirm that the effector of BMP signalling, pSmad1/5/8, was reduced in 4-MU digits compared to control, and that culturing fibroblasts on soft hydrogels led to the highest levels of pSmad1/5/8 (Fig. 4). This prompted the team to propose that BMP signalling can be affected by HA levels and that substrate stiffness can further tune the BMP signalling pathway.

Figure 4: Immunofluorescent labelling of pSmad1/5/8 and qPCR analysis of its target gene Id1 from figure 5 of Mui et al (2024). Fibroblasts were cultured on soft or stiff hydrogels and treated with BMP-7. Increases in pSmad1/5/8 fluorescence and Id1 expression on the soft hydrogels showed BMP signalling can be tuned by substrate stiffness. Image made available under a CC-BY-NC-ND 4.0 International license.

Overexpressing HAPLN1 in stiff contexts can promote HA deposition and wound regeneration

Finally, the team asked if a soft substrate could promote the production of a regenerative ECM from fibroblasts. They first cultured fibroblasts on stiff and soft hydrogels and induced HA synthesis. Fibroblasts grown on soft gels were found to have increased expression of Hapln1 and had dense aggregates of HA and HAPLN1 together on the cell surface (Fig. 5). HAPLN1 and HA were also observed to colocalise together in uninjured digits, with less HAPLN1/HA in P2 (non-regenerative) and P3 (regenerative) regions. Therefore, HAPLN1 is mechanosensitive and able to retain or stabilise HA, suggesting soft substrates upregulate HAPLN1, which promotes HA retention, further promoting soft ECM and creating a positive feedback loop.

Figure 5: Immunofluorescence images of fibroblasts grown on stiff and soft hydrogels from figure 5 of Mui et al (2024). HA binding protein (HABP) and HAPLN1 levels increased in fibroblasts cultured on the soft substrate. Image made available under a CC-BY-NC-ND 4.0 International license.

To further test this, the team finished by overexpressing HAPLN1 (Hapln1^OE) in fibroblasts. On stiff substrates Hapln1^OE fibroblasts deposited more HA and reduced collagen fibrillogenesis compared to control cells. Furthermore, transplanting Hapln1^OE fibroblasts into non-regenerative wounds (from immune-compromised mice, to avoid rejection) promoted HA accumulation, reduced fibrosis, enhanced bone repair and increased the presence of OL cells.

What I like about the preprint/why I think this new work is important

I was initially attracted to this preprint due to my interest in how mechanical cues affect cell responses and found it to be a tour de force in studying ECM composition and stiffness in wound healing. I really appreciated the combination of many different techniques to build a compelling narrative about the role of different cell types regulating HA networks in regenerative wounds and the influence stiffness has on this process. Finally, out of curiosity about the plethora of creams and gels in pharmacies promoting the properties of HA, I was interested to dig deeper into some primary literature and see what truth there is in the hype around HA.

Future directions and questions for the authors

1. How is the initial regenerative or non-regenerative response to a wound determined? For example, are there differences in the stiffnesses or fibroblast populations of “prewounded” digits that determine the wound response?
2. Can you comment further on what mechanisms individual cells use to transduce the mechanical cues from collagen/HA networks to transcriptional responses? Could these also be therapeutically targeted?
3. How might infiltration of wound sites by cells from the immune system affect ECM remodelling? And vice versa?

References

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Tags: ecm, fibroblast, hyaluronic acid, mechanosensing, regeneration

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