Notch-Wnt signal crosstalk regulates proliferation and differentiation of osteoprogenitor cells during intramembranous bone healing.

Adult bone regeneration is orchestrated by the precise actions of osteoprogenitor cells (OPCs). However, the mechanisms by which OPC proliferation and differentiation are linked and thereby regulated are yet to be defined. Here, we present evidence that during intramembranous bone formation OPC proliferation is controlled by Notch signaling, while differentiation is initiated by activation of canonical Wnt signaling. The temporospatial separation of Notch and Wnt signal activation during the early stages of bone regeneration suggests crosstalk between the two pathways. In vitro and in vivo manipulation of the two essential pathways demonstrate that Wnt activation leads to initiation of osteogenic differentiation and at the same time inhibits Notch signaling, which results in termination of the proliferative phase. Here, we establish canonical Wnt signaling as a key regulator that facilitates the crosstalk between OPC proliferation and differentiation during intramembranous, primary bone healing.

Temporary inhibition of the plasminogen activator inhibits periosteal chondrogenesis and promotes periosteal osteogenesis during appendicular bone fracture healing.

INTRODUCTION: Aminocaproic acid is approved as an anti-fibrinolytic for use in joint replacement and spinal fusion surgeries to limit perioperative blood loss. Previous animal studies have demonstrated a pro-osteogenic effect of aminocaproic acid in spine fusion models. Here, we tested if aminocaproic acid enhances appendicular bone healing and we sought to uncover the effect of aminocaproic acid on osteoprogenitor cells (OPCs) during bone regeneration. METHODS: We employed a well-established murine femur fracture model in adult C57BL/6J mice after receiving two peri-operative injections of aminocaproic acid. Routine histological assays, biomechanical testing and micro-CT analyses were utilized to assess callus volume, and strength, progenitor cell proliferation, differentiation, and remodeling in vivo. Two disparate ectopic transplantation models were used to study the effect of the growth factor milieu within the early fracture hematoma on osteoprogenitor cell fate decisions. RESULTS: Aminocaproic acid treated femur fractures healed with a significantly smaller cartilaginous callus, and this effect was also observed in the ectopic transplantation assays. We hypothesized that aminocaproic acid treatment resulted in a stabilization of the early fracture hematoma, leading to a change in the growth factor milieu created by the early hematoma. Gene and protein expression analysis confirmed that aminocaproic acid treatment resulted in an increase in Wnt and BMP signaling and a decrease in TGF-ß-signaling, resulting in a shift from chondrogenic to osteogenic differentiation in this model of endochondral bone formation. CONCLUSION: These experiments demonstrate for the first time that inhibition of the plasminogen activator during fracture healing using aminocaproic acid leads to a change in cell fate decision of periosteal osteoprogenitor cells, with a predominance of osteogenic differentiation, resulting in a larger and stronger bony callus. These findings may offer a promising new use of aminocaproic acid, which is already FDA-approved and offers a very safe risk profile.

The use of ultra-low-dose CT scans for the evaluation of limb fractures: is the reduced effective dose using ct in orthopaedic injury (REDUCTION) protocol effective?

AIMS: To evaluate whether an ultra-low-dose CT protocol can diagnose selected limb fractures as well as conventional CT (C-CT). PATIENTS AND METHODS: We prospectively studied 40 consecutive patients with a limb fracture in whom a CT scan was indicated. These were scanned using an ultra-low-dose CT Reduced Effective Dose Using Computed Tomography In Orthopaedic Injury (REDUCTION) protocol. Studies from 16 selected cases were compared with 16 C-CT scans matched for age, gender and type of fracture. Studies were assessed for diagnosis and image quality. Descriptive and reliability statistics were calculated. The total effective radiation dose for each scanned site was compared. RESULTS: The mean estimated effective dose (ED) for the REDUCTION protocol was 0.03 milliSieverts (mSv) and 0.43 mSv (p < 0.005) for C-CT. The sensitivity (Sn), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the REDUCTION protocol to detect fractures were 0.98, 0.89, 0.98 and 0.89 respectively when two occult fractures were excluded. Inter- and intra-observer reliability for diagnosis using the REDUCTION protocol (κ = 0.75, κ = 0.71) were similar to those of C-CT (κ = 0.85, κ = 0.82). Using the REDUCTION protocol, 3D CT reconstructions were equivalent in quality and diagnostic information to those generated by C-CT (κ = 0.87, κ = 0.94). CONCLUSION: With a near 14-fold reduction in estimated ED compared with C-CT, the REDUCTION protocol reduces the amount of CT radiation substantially without significant diagnostic decay. It produces images that appear to be comparable with those of C-CT for evaluating fractures of the limbs. Cite this article: Bone Joint J 2016;98-B:1668-73.

Primary cilia act as mechanosensors during bone healing around an implant.

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.

Controlling the in vivo activity of Wnt liposomes.

Liposomes offer a method of delivering small molecules, nucleic acids, and proteins to sites within the body. Typically, bioactive materials are encapsulated within the liposomal aqueous core and liposomal phase transition is elicited by pH or temperature changes. We developed a new class of liposomes for the in vivo delivery of lipid-modified proteins. First, we show that the inclusion of a chromophore into the liposomal or vesosomal membrane renders these lipid vesicles extremely sensitive to very small (muJ) changes in energy. Next, we demonstrate that the lipid-modified Wnt protein is not encapsulated within a liposome but rather is tethered to the exoliposomal surface in an active configuration. When applied to intact skin, chromophore-modified liposomes do not penetrate past the corneal layer of the epidermis, but remain localized to the site of application. Injury to the epidermis allows rapid penetration of liposomes into the dermis, which suggests that mild forms of dermabrasion will greatly enhance transdermal delivery of liposome-packaged molecules. Finally, we demonstrate that topical application of Wnt3a liposomes rapidly stimulates proliferation of cells in the corneal layer, resulting in a thicker, more fibrillous epidermis.

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 cell's 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.

Bone voyage: an expedition into the molecular and cellular parameters affecting bone graft fate.

The demand for bone grafts in orthopaedic and craniofacial surgery is steadily increasing. Estimations suggest that about 500,000 are performed annually in the United States that include bone grafting as a component of the surgery, and the majority of these surgeries employ autografts. This perspective focuses on the biological events that occur during osseointegration of such bone grafts. Here, three key factors of graft osseointegration--the embryonic origin, the inclusion of skeletal progenitor cells, and the integrity of the recipient site--are discussed. Altogether, they form the foundation for survival of the bone graft and eventually for a positive clinical outcome of the procedure.