The Sonic hedgehog (Shh) gene is the key to repairing large bone injuries


A USC Stem Cell study in npj Regenerative Medicine presents intriguing evidence that large bone injuries might trigger a repair strategy in adults that recapitulates elements of skeletal formation in utero.

Key to this repair strategy is a gene with a fittingly heroic name: Sonic hedgehog.

In the study, first author Maxwell Serowoky, a Ph.D. student in the USC Stem Cell laboratory of Francesca Mariani, and his colleagues took a close look at how mice are able to regrow large sections of missing rib – an ability they share with humans, and one of the most impressive examples of bone regeneration in mammals.

To their surprise, the scientists observed an increase in the activity of Sonic hedgehog (Shh), which plays an important role in skeletal formation in embryos, but hasn’t previously been linked to injury repair in adults.

In their experiments, Shh appeared to play a necessary role in healing the central region of large sections of missing ribs, but not in closing small-scale fractures.

“Our evidence suggests that large-scale bone regeneration requires the redeployment of an embryonic developmental program involving Shh, whereas small injuries heal through a distinct repair program that does not mirror development,” said Mariani, the study’s corresponding author and an associate professor of stem cell biology and regenerative medicine at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at the Keck School of Medicine of USC.

Serowoky added: “It’s still a fascinating mystery which factors or conditions result in Shh activity following large, but not small bone injuries.”

In mice, Shh activity increased briefly after a large rib injury, and then quickly returned to normal levels within 5 days. Although transient, this increase in Shh was a prerequisite for successfully building a callus, which is an initial scaffold that bridges a fracture or injury but then converts to bone and regenerates the missing section of rib. Mice genetically modified to lack Shh couldn’t successfully form calluses or heal their ribs.

In contrast, mice that had Shh at the time of injury, but were genetically altered to lose Shh after a 5-day healing period, were able to repair their ribs normally. A related gene known as Smoothened (Smo) was also required only during the first 5 days of the healing process.

The researchers expected that the source of Shh would be from specific progenitor cells that the group had previously shown to be essential for healing large injuries and that reside in the periosteum, which is the sheath of tissue surrounding each rib. Instead, they discovered that the source of Shh was an unexpected population of stem cell-like cells, known as mesenchymal cells.

When these mesenchymal cells increased their Shh activity, this seemed to serve as a signal to summon a separate population of stem cell-like bone marrow cells to the injury site to assist in the healing process.

“Our discovery may inform future therapeutic strategies for situations where patients are missing large sections of bone following high energy injuries such as traffic accidents or combat wounds, or after cancer-related bone resections,” said co-author Jay R. Lieberman, chair and professor of orthopaedic surgery at the Keck School.

Additional co-authors for this USC study include Stephanie Kuwahara and Shuwan Liu from the Department of Stem Cell Biology and Regenerative Medicine, and Venus Vakhshori from the Department of Orthopaedic Surgery.

The fracture healing process consists of four overlapping phases, namely, inflammation, proliferation, callus formation, and bone remodeling. Immediately following fracture, the injury initiates an inflammatory response that is necessary to promote healing.

The response induces the development of a hematoma, which consists of cells from both peripheral blood vessels and bone marrow. The hematoma coagulates between and around the fracture site and within the bone marrow, providing a template for callus formation [1].

Vascularization supplies mesenchymal stem cells (MSCs), which differentiate into chondrocytes or osteoblasts simultaneously with cartilage tissue development (proliferation phase) [2,3]. The cartilage matrix begins to form at the fractured bone gap during the callus formation phase.

Meanwhile, intramembranous ossification occurs internal to the periosteum adjacent to the fracture line and forms the bone matrix [4]. MSCs directly differentiate into osteoblasts at the fracture site along the proximal and distal edges of fractured bone during intramembranous ossification.

After cartilage tissue maturation, new bone formation is initiated as the cartilage tissue is resorbed and vascularization is induced to replace the cartilage tissue with bone. It has also been reported that primary bone formation is initiated peripheral to the newly formed cartilage region at the fractured bone site [5].

The bone remodeling phase recapitulates embryonic bone development with a combination of cellular proliferation and differentiation, increasing the cellular volume and matrix deposition [1]. Finally, remodeling of the hard callus into a lamellar bone structure occurs (bone remodeling phase).

The biological process occurring during bone fracture healing is regulated by several signaling molecules. Hedgehog (HH) proteins are among the signaling molecules required for endochondral bone formation during embryonic development, and they regulate bone homeostasis by controlling MSC proliferation [6,7]. HH signaling is also involved in the regulation of MSC proliferation in adult tissues. Aberrant activation of HH pathways has been linked to multiple types of human cancer [7].

These pathways are also activated during intramembranous and endochondral ossification in the fracture healing process, but it is not clear if they are involved in the healing process [5]. HH signaling pathways play critical roles in developmental processes and in the postnatal homeostasis of many tissues, including bone and cartilage. The HH family of intercellular signaling proteins plays important roles in regulating the development of many tissues and organs.

Their name is derived from the observation of a hedgehog-like appearance in Drosophila embryos with genetic mutations that block their action. Three types of HH proteins have been reported in mammals, namely Sonic HH (Shh), Indian HH (Ihh), and Desert HH (Dhh). Ihh is up-regulated during the initial stage of fracture repair, and it regulates differentiation indirectly by controlling cartilage development at the fracture site. Ihh regulates osteoblast differentiation indirectly by controlling cartilage development [8].

In general, Shh acts in the early stages of development to regulate patterning and growth [9]. Recently, several studies reported that Shh might be related to fracture healing [10,11]. Following the inactivation of HH signaling, the activity of Smo is inhibited by a receptor known as Patched (Ptch).

Binding of the HH ligand Ptch relieves the inhibition of Smo, and activated Smo blocks the proteolysis of Gli proteins in the cytoplasm and promotes their dissociation from suppressor of fused (SuFu). Following dissociation from SuFu, activated Gli proteins translocate into the nucleus and promote the expression of Hh target genes, including Gli1 [9,12]. Gli1 positivity has been identified as a marker for MSCs [13]. Another study uncovered that Gli1 is involved in osteoblast differentiation [14]. However, it is unclear that whether Shh proteins are involved in fracture healing. In this study, we demonstrated that Shh protein and the related proteins Smo and Gli1 were involved in osteoblast differentiation at the fracture healing site via immunohistochemical analysis.

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More information: A murine model of large-scale bone regeneration reveals a selective requirement for Sonic Hedgehog, npj Regenerative Medicine (2022). DOI: 10.1038/s41536-022-00225-8.


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