Faculty Bibliography

BACKGROUND: Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity. METHODS: Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]). RESULTS: Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls. CONCLUSIONS: Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals. CLINICAL RELEVANCE: We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.

OBJECTIVE: To compare clinical and radiological outcome between acetabular fractures with marginal impaction that were treated with either cancellous bone graft (CBG) or tricalcium phosphate cement (TPC) as bone void filler. DESIGN: Retrospective study. PATIENTS: Forty-three patients with acetabular fractures with marginal impaction. INTERVENTION: Eighteen patients received cancellous bone graft and 25 patients received tricalcium phosphate cement as bone void filler. MAIN OUTCOME MEASUREMENT: Clinical outcome was assessed using the Merle d'Aubigne score and Short-form-36. Radiographs were evaluated for postoperative reduction, arthritis grade and development of heterotopic ossification. RESULTS: Forty-three patients met the inclusion criteria. There was no significant difference in the demographics, laterality, fracture type, associated injuries, surgical approach and postoperative quality of reduction between the groups. At final follow-up, a significantly higher number of patients in the cancellous bone graft group exhibited signs of moderate to severe post-traumatic arthritis (CBG: 6 (33%) vs. TPC: 4 (20%), p=0.007) and required a total hip arthroplasty (CBG: 4 (22.2%) vs. TPC: 1 (5%), p=0.08). There was no significant difference between the two groups in the SF-36 score and the modified Merle d'Aubigne score. CONCLUSION: Patients with acetabular fractures with marginal impaction treated with tricalcium phosphate cement exhibit a significantly lower incidence of post-traumatic arthritis when compared to patients treated with cancellous bone graft. LEVEL OF EVIDENCE: III.

The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

PURPOSE: Muller glia respond to retinal injury by a reactive gliosis, but only rarely do mammalian glial cells re-enter the cell cycle and generate new neurons. In the nonmammalian retina, however, Muller glia act as stem/progenitor cells. Here, we tested the function of Wnt signaling in the postinjury retina, focusing on its ability to influence mammalian Muller cell dedifferentiation, proliferation, and neurogenesis. METHODS: A 532 nm frequency doubled neodymium-doped yttrium aluminum garnet (ND:YAG) laser was used to create light burns on the retina of Axin2(LacZ/+) Wnt reporter mice. At various time points after injury, retinas were analyzed for evidence of Wnt signaling as well as glial cell response, proliferation, and apoptosis. Laser injuries also were created in Axin2(LacZ/LacZ) mice, and the effect of potentiated Wnt signaling on retinal repair was assessed. RESULTS: A subpopulation of mammalian Muller cells are Wnt responsive and, when Wnt signaling is increased, these cells showed enhanced proliferation in response to injury. In an environment of heightened Wnt signaling, caused by the loss of the Wnt negative regulator Axin2, Muller cells proliferated after injury and adopted the expression patterns of retinal progenitor cells (RPCs). The Wnt-responsive Muller cells also exhibited long-term survival and, in some cases, expressed the rod photoreceptor marker, rhodopsin. CONCLUSIONS: The Wnt pathway is activated by retinal injury, and prolonging the endogenous Wnt signal causes a subset of Muller cells to proliferate and dedifferentiate into RPCs. These data raised the possibility that transient amplification of Wnt signaling after retinal damage may unlock the latent regenerative capacity long speculated to reside in mammalian neural tissues.

Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Colalpha1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Colalpha1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Colalpha1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.

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.

The strength of a Wnt-based strategy for tissue regeneration lies in the central role that Wnts play in healing. Tissue injury triggers local Wnt activation at the site of damage, and this Wnt signal is required for the repair and/or regeneration of almost all tissues including bone, neural tissues, myocardium, and epidermis. We developed a biologically based approach to create a transient elevation in Wnt signaling in peri-implant tissues, and in doing so, accelerated bone formation around the implant. Our subsequent molecular and cellular analyses provide mechanistic insights into the basis for this pro-osteogenic effect. Given the essential role of Wnt signaling in bone formation, this protein-based approach may have widespread application in implant osseointegration.

Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed that Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, and Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgehog (Shh-N) significantly increased in vitro osteogenic differentiation in mouse ASCs, by all markers examined (*p < 0.01). Concomitantly, Shh-N abrogated adipogenic differentiation, by all markers examined (*p < 0.01). Conversely, blockade of endogenous Hedgehog signaling, with the Hedgehog antagonist cyclopamine, enhanced adipogenesis at the expense of osteogenesis. We next translated these results to a mouse model of appendicular skeletal regeneration. Using quantitative real-time polymerase chain reaction and in situ hybridization, we found that skeletal injury (a monocortical 1 mm defect in the tibia) results in a localized increase in Hedgehog signaling. Moreover, grafting of ASCs treated with Shh-N resulted in significantly increased bone regeneration within the defect site. In conclusion, Hedgehog signaling enhances the osteogenic differentiation of mouse ASCs, at the expense of adipogenesis. These data suggest that Hedgehog signaling directs the lineage differentiation of mesodermal stem cells and represents a promising strategy for skeletal tissue regeneration.

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. muCT 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.

Bone morphogenetic proteins (BMPs) participate in multiple stages of the fetal skeletogenic program from promoting cell condensation to regulating chondrogenesis and bone formation through endochondral ossification. Here, we show that these pleiotropic functions are recapitulated when recombinant BMPs are used to augment skeletal tissue repair. In addition to their well-documented ability to stimulate chondrogenesis in a skeletal injury, we show that recombinant BMPs (rBMPs) simultaneously suppress the differentiation of skeletal progenitor cells in the endosteum and bone marrow cavity to an osteoblast lineage. Both the prochondrogenic and antiosteogenic effects are achieved because rBMP inhibits endogenous beta-catenin-dependent Wnt signaling. In the injured periosteum, this repression of Wnt activity results in sox9 upregulation; consequently, cells in the injured periosteum adopt a chondrogenic fate. In the injured endosteum, rBMP also inhibits Wnt signaling, which results in the runx2 and collagen type I downregulation; consequently, cells in this region fail to differentiate into osteoblasts. In muscle surrounding the skeletal injury site, rBMP treatment induces Smad phosphorylation followed by exuberant cell proliferation, an increase in alkaline phosphatase activity, and chondrogenic differentiation. Thus different populations of adult skeletal progenitor cells interpret the same rBMP stimulus in unique ways, and these responses mirror the pleiotropic effects of BMPs during fetal skeletogenesis. These mechanistic insights may be particularly useful for optimizing the reparative potential of rBMPs while simultaneously minimizing their adverse outcomes.