Andrew A. Smith

Wnt Signaling and Injury Repair

Wnt signaling is activated by wounding and participates in every subsequent stage of the healing process from the control of inflammation and programmed cell death, to the mobilization of stem cell reservoirs within the wound site. In this review we summarize recent data elucidating the roles that the Wnt pathway plays in the injury repair process. These data provide a foundation for potential Wnt-based therapeutic strategies aimed at stimulating tissue regeneration.

Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing

Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2(LacZ/+) reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2(LacZ/LacZ) mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2(LacZ/LacZ) mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.

Multiscale Analyses of the Bone-implant Interface:

Implants placed with high insertion torque (IT) typically exhibit primary stability, which enables early loading. Whether high IT has a negative impact on peri-implant bone health, however, remains to be determined. The purpose of this study was to ascertain how peri-implant bone responds to strains and stresses created when implants are placed with low and high IT. Titanium micro-implants were inserted into murine femurs with low and high IT using torque values that were scaled to approximate those used to place clinically sized implants. Torque created in peri-implant tissues a distribution and magnitude of strains, which were calculated through finite element modeling. Stiffness tests quantified primary and secondary implant stability. At multiple time points, molecular, cellular, and histomorphometric analyses were performed to quantitatively determine the effect of high and low strains on apoptosis, mineralization, resorption, and collagen matrix deposition in peri-implant bone. Preparation of an osteotomy results in a narrow zone of dead and dying osteocytes in peri-implant bone that is not significantly enlarged in response to implants placed with low IT. Placing implants with high IT more than doubles this zone of dead and dying osteocytes. As a result, peri-implant bone develops micro-fractures, bone resorption is increased, and bone formation is decreased. Using high IT to place an implant creates high interfacial stress and strain that are associated with damage to peri-implant bone and therefore should be avoided to best preserve the viability of this tissue.

Reengineering autologous bone grafts with the stem cell activator WNT3A

Autologous bone grafting represents the standard of care for treating bone defects but this biomaterial is unreliable in older patients. The efficacy of an autograft can be traced back to multipotent stem cells residing within the bone graft. Aging attenuates the viability and function of these stem cells, leading to inconsistent rates of bony union. We show that age-related changes in autograft efficacy are caused by a loss in endogenous Wnt signaling. Blocking this endogenous Wnt signal using Dkk1 abrogates autograft efficacy whereas providing a Wnt signal in the form of liposome-reconstituted WNT3A protein (L-WNT3A) restores bone forming potential to autografts from aged animals. The bioengineered autograft exhibits significantly better survival in the hosting site. Mesenchymal and skeletal stem cell populations in the autograft are activated by L-WNT3A and mitotic activity and osteogenic differentiation are significantly enhanced. In a spinal fusion model, aged autografts treated with L-WNT3A demonstrate superior bone forming capacity compared to the standard of care. Thus, a brief incubation in L-WNT3A reliably improves autologous bone grafting efficacy, which has the potential to significantly improve patient care in the elderly.

Disrupting the intrinsic growth potential of a suture contributes to midfacial hypoplasia

Children with unoperated cleft palates have nearly normal growth of their faces whereas patients who have had early surgical repair often exhibit midfacial hypoplasia. Surgical repair is responsible for the underlying bone growth arrest but the mechanisms responsible for these surgical sequelae are poorly understood. We simulated the effect of cleft palate repair by raising a mucoperiosteal flap in the murine palate. Three-dimensional micro-CT reconstructions of the palate along with histomorphometric measurements, finite element (FE) modeling, immunohistochemical analyses, and quantitative RT-PCR were employed to follow the skeletal healing process. Inflammatory bone resorption was observed during the first few days after denudation, which destroyed the midpalatal suture complex. FE modeling was used to predict and map the distribution of strains and their associated stresses in the area of denudation and the magnitude and location of hydrostatic and distortional strains corresponded to sites of skeletal tissue destruction. Once re-epithelialization was complete and wound contracture subsided, the midpalatal suture complex reformed. Despite this, growth at the midpalatal suture was reduced, which led to palatal constriction and a narrowing of the dental arch. Thus the simple act of raising a flap, here mimicked by denuding the mucoperiosteum, was sufficient to cause significant destruction to the midpalatal suture complex. Although the bone and cartilage growth plates were re-established, mediolateral skeletal growth was nonetheless compromised and the injured palate never reached its full growth potential. These data strongly suggest that disruption of suture complexes, which have intrinsic growth potential, should be avoided during surgical correction of congenital anomalies.

Molecular mechanisms underlying skeletal growth arrest by cutaneous scarring

In pediatric surgeries, cutaneous scarring is frequently accompanied by an arrest in skeletal growth. The molecular mechanisms responsible for this effect are not understood. Here, we investigated the relationship between scar contracture and osteogenesis. An excisional cutaneous wound was made on the tail of neonatal mice. Finite element (FE) modeling of the wound site was used to predict the distribution and magnitude of contractile forces within soft and hard tissues. Morphogenesis of the bony vertebrae was monitored by micro-CT analyses, and vertebral growth plates were interrogated throughout the healing period using assays for cell proliferation, death, differentiation, as well as matrix deposition and remodeling. Wound contracture was grossly evident on post-injury day 7 and accompanying it was a significant shortening in the tail. FE modeling indicated high compressive strains localized to the dorsal portions of the vertebral growth plates and intervertebral disks. These predicted strain distributions corresponded to sites of increased cell death, a cessation in cell proliferation, and a loss in mineralization within the growth plates and IVD. Although cutaneous contracture resolved and skeletal growth rates returned to normal, vertebrae under the cutaneous wound remained significantly shorter than controls. Thus, localized contractile forces generated by scarring led to spatial alterations in cell proliferation, death, and differentiation that inhibited bone growth in a location-dependent manner. Resolution of cutaneous scarring was not accompanied by compensatory bone growth, which left the bony elements permanently truncated. Therefore, targeting early scar reduction is critical to preserving pediatric bone growth after surgery.

The Capacity of Neural Crest-Derived Stem Cells for Ocular Repair

Whether it is due to a particular epigenetic signature, or some other component of an embryonic differentiation program, accumulating evidence indicates that the origins of a stem cell has a profound impact on the potential of a tissue to regenerate and repair. Here, we focus on Müller glia, long considered the stem cells of the retina, and their surprising derivation from the neural crest. Whether the multipotent properties of a subset of Müller glia is associated with their neural crest origin remains a tantalizing possibility.

Author Correction: WNT-activated bone grafts repair osteonecrotic lesions in aged animals

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

WNT-activated bone grafts repair osteonecrotic lesions in aged animals

The Wnt pathway is a new target in bone therapeutic space. WNT proteins are potent stem cell activators and pro-osteogenic agents. Here, we gained insights into the molecular and cellular mechanisms responsible for liposome-reconstituted recombinant human WNT3A protein (L-WNT3A) efficacy to treat osteonecrotic defects. Skeletal injuries were coupled with cryoablation to create non-healing osteonecrotic defects in the diaphysis of the murine long bones. To replicate clinical therapy, osteonecrotic defects were treated with autologous bone graft, which were simulated by using bone graft material from syngeneic ACTB-eGFP-expressing mice. Control osteonecrotic defects received autografts alone; test sites received autografts treated ex vivo with L-WNT3A. In vivo µCT monitored healing over time and immunohistochemistry were used to track the fate of donor cells and assess their capacity to repair osteonecrotic defects according to age and WNT activation status. Collectively, analyses demonstrated that cells from the autograft directly contributed to repair of an osteonecrotic lesion, but this contribution diminished as the age of the donor increased. Pre-treating autografts from aged animals with L-WNT3A restored osteogenic capacity to autografts back to levels observed in autografts from young animals. A WNT therapeutic approach may therefore have utility in the treatment of osteonecrosis, especially in aged patients.

Linking suckling biomechanics to the development of the palate

Skulls are amongst the most informative documents of evolutionary history but a complex geometry, coupled with composite material properties and complicated biomechanics, have made it particularly challenging to identify mechanical principles guiding the skull’s morphogenesis. Despite this challenge, multiple lines of evidence, for example the relationship between masticatory function and the evolution of jaw shape, nonetheless suggest that mechanobiology plays a major role in skull morphogenesis. To begin to tackle this persistent challenge, cellular, molecular and tissue-level analyses of the developing mouse palate were coupled with finite element modeling to demonstrate that patterns of strain created by mammalian-specific oral behaviors produce complementary patterns of chondrogenic gene expression in an initially homogeneous population of cranial neural crest cells. Neural crest cells change from an osteogenic to a chondrogenic fate, leading to the materialization of cartilaginous growth plate-like structures in the palatal midline. These growth plates contribute to lateral expansion of the head but are transient structures; when the strain patterns associated with suckling dissipate at weaning, the growth plates disappear and the palate ossifies. Thus, mechanical cues such as strain appear to co-regulate cell fate specification and ultimately, help drive large-scale morphogenetic changes in head shape.