Philipp Leucht

Wnt Proteins Promote Bone Regeneration

A liposome-encased ligand for the Wnt signaling pathway can accelerate bone regeneration after injury. Some of the best medicines act as facilitators. They tweak the body’s systems so that its own homeostatic mechanisms can more easily return things to normal. One potentially tweakable process is the Wnt signaling pathway, a soluble ligand-receptor system that controls transcription of β-catenin and other genes. This pathway is used over and over again for specification of the body axis and morphogenesis during development and goes awry in cancer. Minear and colleagues noted that Wnt signaling participates in regrowth and healing of broken bones and found a way to augment its action. Their treatment accelerates natural recovery from bone injury. The authors used genetically altered mice to show that Wnt signaling is increased in regions in which bone is healing, which suggests that it participates in the bone regeneration process. By interfering with the action of a Wnt signaling inhibitor, they potentiated this pathway even more, which resulted in more rapid healing of small holes that they made in the tibial bone of the mice. To begin to translate these results to patients, the authors then increased Wnt signaling in these mice with an approach that, unlike genetic manipulation, could be replicated in the clinic. Getting around the fact that Wnt ligands are insoluble in aqueous solution by using a liposome package, the authors applied Wnt-filled liposomes to healing bone and again showed faster healing. The basis of this effect was both the simulation of skeletal progenitor cell proliferation and the promotion of differentiation of these progenitors into osteoblasts, the cellular builder of bone. Wnt packaged in lipid membrane vesicles could have advantages over other treatments that can facilitate bone healing, specifically bone morphogenetic protein, because of the advantageous type of bone that is created. And the fact that the Wnt pathway controls numerous fundamental processes may mean that Wnt-containing vesicles could prove useful in tweaking other systems to help the body more rapidly achieve healing and homeostasis. The Wnt signaling pathway plays a central role in bone development and homeostasis. In most cases, Wnt ligands promote bone growth, which has led to speculation that Wnt factors could be used to stimulate bone healing. We gained insights into the mechanism by which Wnt signaling regulates adult bone repair through the use of the mouse strain Axin2LacZ/LacZ in which the cellular response to Wnt is increased. We found that bone healing after injury is accelerated in Axin2LacZ/LacZ mice, a consequence of more robust proliferation and earlier differentiation of skeletal stem and progenitor cells. In parallel, we devised a biochemical strategy to increase the duration and strength of Wnt signaling at the sites of skeletal injury. Purified Wnt3a was packaged in liposomal vesicles and delivered to skeletal defects, where it stimulated the proliferation of skeletal progenitor cells and accelerated their differentiation into osteoblasts, cells responsible for bone growth. The end result was faster bone regeneration. Because Wnt signaling is conserved in mammalian tissue repair, this protein-based approach may have widespread applications in regenerative medicine.

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

Bone regeneration is regulated by wnt signaling.

Tissue regeneration is increasingly viewed as reactivation of a developmental process that, when misappropriated, can lead to malignant growth. Therefore, understanding the molecular and cellular pathways that govern tissue regeneration provides a glimpse into normal development as well as insights into pathological conditions such as cancer. Herein, we studied the role of Wnt signaling in skeletal tissue regeneration.

Wnt signaling mediates regional specification in the vertebrate face

At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.

Epidemiology of traumatic spine fractures

OBJECTIVES To illustrate the correlations and effects of age, gender and cause of accident on the type of vertebral fracture and fracture distribution, as well as on the likelihood to sustain an associated injury or neurological deficit. DESIGN Retrospective analysis of 562 patients with a traumatic fracture of the spine. Each patient was analysed by reviewing the medical records, the initial radiographs and CT-scans. SETTING Level 1 trauma centre from 01/1996 to 12/2000. RESULTS The most common cause of accident was a high-energy fall (39%), followed by traffic accidents (26.5%). While fall related fractures were evenly distributed over the whole spine, traffic accidents induced significantly more fractures of the cervical and thoracic spine. Sixty-five percent of all cervical spine fractures and 80% of the multisegmental injuries were accompanied by an associated injury. The highest incidence of associated injuries was observed in patients with multilevel fractures (96.5%). Patients with a concomitant injury were more likely to sustain a spinal cord lesion. Sixty-three (11.2%) patients exhibited a complete motor and sensory deficit, 76 (13.5%) an incomplete and 423 (75.3%) no neurological deficit. The highest number of complete motor and sensory neurological deficits was found in cervical spine fractures (19.7%). The majority of patients, 308 (54.8%), sustained a compression fracture, 95 (16.9%) a distraction fracture, and 104 (18.5%) patients experienced a rotational fracture. CONCLUSIONS This study demonstrates correlations between the cause of accident, the type of spinal fracture and the fracture distribution. Using the AO classification, the likelihood to sustain either associated and/or spinal cord injuries, is predictable.

Fgf-9 is required for angiogenesis and osteogenesis in long bone repair

Bone healing requires a complex interaction of growth factors that establishes an environment for efficient bone regeneration. Among these, FGFs have been considered important for intrinsic bone-healing capacity. In this study, we analyzed the role of Fgf-9 in long bone repair. One-millimeter unicortical defects were created in tibias of Fgf-9+/− and wild-type mice. Histomorphometry revealed that half-dose gene of Fgf-9 markedly reduced bone regeneration as compared with wild-type. Both immunohistochemistry and RT-PCR analysis revealed markedly decreased levels of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (Runx2), osteocalcin, Vega-a, and platelet endothelial cell adhesion molecule 1 (PECAM-1) in Fgf-9+/− defects. μCT angiography indicated dramatic impairment of neovascularization in Fgf-9+/− mice as compared with controls. Treatment with FGF-9 protein promoted angiogenesis and successfully rescued the healing capacity of Fgf-9+/− mice. Importantly, although other pro-osteogenic factors [Fgf-2, Fgf-18, and bone morphogenic protein 2 (Bmp-2)] still were present in Fgf-9+/− mice, they could not compensate for the haploinsufficiency of the Fgf-9 gene. Therefore, endogenous Fgf-9 seems to play an important role in long bone repair. Taken together our data suggest a unique role for Fgf-9 in bone healing, presumably by initiating angiogenesis through Vegf-a. Moreover, this study further supports the embryonic phenotype previously observed in the developing limb, thus promoting the concept that healing processes in adult organisms may recapitulate embryonic skeletal development.

Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

Translating insights from development into regenerative medicine: The function of Wnts in bone biology

The Wnt pathway constitutes one of the most attractive candidates for modulating skeletal tissue regeneration based on its functions during skeletal development and homeostasis. Wnts participate in every stage of skeletogenesis, from the self-renewal and proliferation of skeletal stem cells to the specification of osteochondroprogenitor cells and the maturation of chondrocytes and osteoblasts. We propose that the function of Wnts depend upon a skeletogenic cells state of differentiation. In this review we summarize recent data with a focus on the roles of Wnt signaling in mesenchymal stem cell fate, osteoprogenitor cell differentiation, chondrocyte maturation, bone remodeling, and bone regeneration.

Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling

Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh−/− phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh−/− mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro‐osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh−/− calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification. genesis 49:784–796, 2011.

FAK-Mediated mechanotransduction in skeletal regeneration.

The majority of cells are equipped to detect and decipher physical stimuli, and then react to these stimuli in a cell type-specific manner. Ultimately, these cellular behaviors are synchronized to produce a tissue response, but how this is achieved remains enigmatic. Here, we investigated the genetic basis for mechanotransduction using the bone marrow as a model system. We found that physical stimuli produced a pattern of principal strain that precisely corresponded to the site-specific expression of sox9 and runx2, two transcription factors required for the commitment of stem cells to a skeletogenic lineage, and the arrangement and orientation of newly deposited type I collagen fibrils. To gain insights into the genetic basis for skeletal mechanotransduction we conditionally inactivated focal adhesion kinase (FAK), an intracellular component of the integrin signaling pathway. By doing so we abolished the mechanically induced osteogenic response and thus identified a critical genetic component of the molecular machinery required for mechanotransduction. Our data provide a new framework in which to consider how physical forces and molecular signals are synchronized during the program of skeletal regeneration.