Exogenous PTH 1-34 Attenuates Impaired Fracture Healing in Endogenous PTH Deficiency Mice via Activating Indian Hedgehog Signaling Pathway and Accelerating Endochondral Ossification

Fracture healing is a complicated, long-term, and multistage repair process. Intermittent administration of parathyroid hormone (PTH) has been proven effective on intramembranous and endochondral bone formation during the fracture healing process, however, the mechanism is unclear. In this study, we investigated the role of exogenous PTH and endogenous PTH deficiency in bone fracture healing and explored the mechanism by using PTH knockout (PTH-/-) mice and ATDC5 cells. In a mouse femur fracture model, endogenous PTH deficiency could delay endochondral ossification whereas exogenous PTH promotes accumulation of endochondral bone, accelerates cartilaginous callus conversion to bony callus, enhances maturity of bony callus, and attenuates impaired fracture healing resulting from endogenous PTH deficiency. In fracture callus tissue, endogenous PTH deficiency could inhibit chondrocyte proliferation and differentiation whereas exogenous PTH could activate the IHH signaling pathway to accelerate endochondral ossification and rescue impaired fracture healing resulting from endogenous PTH deficiency. In vitro, exogenous PTH promotes cell proliferation by activating IHH signaling pathway on ATDC5 cells. In mechanistic studies, by using ChIP and luciferase reporter assays, we showed that PTH could phosphorylate CREB, and subsequently bind to the promoter of IHH, causing the activation of IHH gene expression. Therefore, results from this study support the concept that exogenous PTH 1-34 attenuates impaired fracture healing in endogenous PTH deficiency mice via activating the IHH pathway and accelerating endochondral ossification. Hence, the investigation of the mechanism underlying the effects of PTH treatment on fracture repair might guide the exploration of effective therapeutic targets for fracture.

Novel Fracture Site Targeting Drug Improves Fracture Healing with Pain Relief: Preclinical Evaluation of MAK123

Non-union bone fracture occurs in 5-10% of fracture injuries. Interventions include surgery with local implantation of autograft, allograft, demineralized bone matrix, and/or bone morphogenetic proteins. These types of fracture injuries are also accompanied by acute and chronic pain states. In most instances, opioids are provided to injured patients during and after surgery. With the opioid crisis, identifying new analgesic therapies that could reduce or eliminate opioid use, while also improving bone healing is important. Here we show the ability of a novel compound, MAK123, to both enhance bone healing and reduce pain behavior in a surgically induced femoral fracture mouse model. Briefly, 20 male C57BL/6 mice underwent a surgically induced femoral fracture and then were treated with 0, 2, 6, or 20 mg/kg, 3X/week for the 3 week study duration. Weekly X-rays were used to examine healing progression. Prior to euthanasia, mice underwent behavioral testing to measure evoked pain behaviors. Upon euthanasia, ex vivo µCT imaging and analysis was completed to assess fracture callus size and composition. While all doses of MAK123 tested resulted in improved healing, the 6mg/kg dose resulted in accelerated bone healing and a significant increase in mineralized callus volume (p<0.05). Similarly, while all doses of MAK123 reduced evoked responses to tactile stimulus as demonstrated by increased paw withdrawal thresholds, 6 mg/kg of MAK123 resulted in a more robust and significant improvement (p<0.05). We postulate that optimization of the dosing schedule/concentration could further improve both bone healing and behavioral measures thought to represent pain in rodents. That said, these promising pre-clinical data warrant further evaluation as MAK123 may prove to be a unique tool for orthopaedic surgery usage whereby it could both improve bone healing and reduce clinical pain, improving overall patient outcomes.

Estrogen Receptor α Signaling in Osteoblasts is Required for Mechanotransduction in Bone Fracture Healing

Biomechanical stimulation by whole-body low-magnitude high-frequency vibration (LMHFV) has demonstrated to provoke anabolic effects on bone metabolism in both non-osteoporotic and osteoporotic animals and humans. However, preclinical studies reported that vibration improved fracture healing and bone formation in osteoporotic, ovariectomized (OVX) mice representing an estrogen-deficient hormonal status, but impaired bone regeneration in skeletally healthy non-OVX mice. These effects were abolished in general estrogen receptor α (ERα)-knockout (KO) mice. However, it remains to be elucidated which cell types in the fracture callus are targeted by LMHFV during bone healing. To answer this question, we generated osteoblast lineage-specific ERα-KO mice that were subjected to ovariectomy, femur osteotomy and subsequent vibration. We found that the ERα specifically on osteoblastic lineage cells facilitated the vibration-induced effects on fracture healing, because in osteoblast lineage-specific ERα-KO (ERαfl/fl; Runx2Cre) mice the negative effects in non-OVX mice were abolished, whereas the positive effects of vibration in OVX mice were reversed. To gain greater mechanistic insights, the influence of vibration on murine and human osteogenic cells was investigated in vitro by whole genome array analysis and qPCR. The results suggested that particularly canonical WNT and Cox2/PGE2 signaling is involved in the mechanotransduction of LMHFV under estrogen-deficient conditions. In conclusion, our study demonstrates a critical role of the osteoblast lineage-specific ERα in LMHFV-induced effects on fracture healing and provides further insights into the molecular mechanism behind these effects.

Apolipoprotein E impairs aged bone fracture healing

Bone fracture healing and osteoblast differentiation are impaired with advanced age. Using a combination of parabiosis and proteomic models, we identified apolipoprotein E (ApoE) to be an aging factor in bone regeneration. Circulating levels of ApoE increased with age in patients and in mice. ApoE impaired bone fracture healing by decreasing bone deposition in the fracture callus which subsequently decreased the mechanical strength of healed tissue. Osteoblasts serve as the sole bone forming cells within the body. In tissue culture models, ApoE treatment decreased osteoblast differentiation and activity which led to decreased matrix formation and mineralization. This inhibition of osteoblast differentiation relied on down-regulation of the Wnt/β-catenin pathway. In mouse models, aged bone repair was rejuvenated when we lowered circulating ApoE levels using a hepatotropic AAV-siRNA model – serving as a proof of concept that ApoE can be targeted to improve bone repair in an older population. While promising, knockdown of circulating ApoE in such a fashion is likely not translatable to patient care. Thus, current work in our laboratory is focused on developing treatment strategies that temporally decrease circulating ApoE levels and consequently improve bone healing after acute injury and/or surgical orthopedic procedure in the geriatric population.

Sarcomatoid Lesions of Head and Neck Region: A Diagnostic Dilemma

One challenging feature of head and neck pathology is that a dizzying array of sarcomatoid lesions occurs here ranging all the way from reactive to malignant and very aggressive. This makes accurate diagnosis critical. These lesions are quite diverse with great clinical and biological heterogeneity. Some are malignant while many others are benign or simply reactive in nature. For example; at mucosal sites, a well known lesion is spindle cell carcinoma (SpCC), which are overtly malignant, and the differential diagnosis then includes a number of different malignant spindle cell lesions. However, there are several benign or even non-neoplastic lesions that can sometimes be difficult to discern from SpCC, e.g. Nodular fasciitis, Proliferative myositis, Cellular schwannoma, Benign fibrous histiocytoma, Carcino sarcoma, Sarcomatoid melanoma. Fracture callus, etc. Aim of Study: There is a diagnostic challenge to the oral pathologists to differentiate dizzying array of sarcoma like lesions from other similar microscopic simulates ranging all the way from reactive to malignant and very aggressive. This article aims to review the sarcomatoid lesions of the head and neck region with emphasis on differential diagnosis histologically and immunohistochemicaly.

Modulation of fracture healing by the transient accumulation of senescent cells

Senescent cells have detrimental effects across tissues with aging but may have beneficial effects on tissue repair, specifically on skin wound healing. However, the potential role of senescent cells in fracture healing has not been defined. Here, we performed an in silico analysis of public mRNAseq data and found that senescence and senescence-associated secretory phenotype (SASP) markers increased during fracture healing. We next directly established that the expression of senescence biomarkers increased markedly during murine fracture healing. We also identified cells in the fracture callus that displayed hallmarks of senescence, including distension of satellite heterochromatin and telomeric DNA damage; the specific identity of these cells, however, requires further characterization. Then, using a genetic mouse model (Cdkn2aLUC) containing a Cdkn2aInk4a-driven luciferase reporter, we demonstrated transient in vivo senescent cell accumulation during callus formation. Finally, we intermittently treated young adult mice following fracture with drugs that selectively eliminate senescent cells ('senolytics', Dasatinib plus Quercetin), and showed that this regimen both decreased senescence and SASP markers in the fracture callus and significantly accelerated the time course of fracture healing. Our findings thus demonstrate that senescent cells accumulate transiently in the murine fracture callus and, in contrast to the skin, their clearance does not impair but rather improves fracture healing.

Study on the Mechanism of Qigu Capsule in Upregulating NF-κB/HIF-1α Pathway to Improve the Quality of Bone Callus in Mice at Different Stages of Osteoporotic Fracture Healing

Objective. The present study intends to investigate the effects and underlying molecular mechanism of Qigu Capsule (QG) on fracture healing in mice with osteoporosis. Methods. Ten-week-old female C57BL/6 mice were ovariectomized and three weeks later were evaluated for successful modeling. Then, all mice were prepared into models of transverse fracture in the right middle femoral shaft. Mice were treated daily using a gavage with normal saline (the NS group), Qigu Capsule (the QG group), or alendronate (the ALN group) postoperatively. Fracture callus tissues were collected and analyzed by X-ray, micro-CT, western blot (WB), and transmission electron microscope (TEM) on postoperation Day 14 (POD14), POD28, and POD42. Results. (1) X-ray results showed that on POD14, the QG group had the fracture healing score significantly higher than the NS and ALN groups, and on POD28, it had the fracture healing score higher than the NS group, suggesting that QG could promote fracture healing. (2) Micro-CT results showed that on POD14, the QG group had tissue bone density (TMD) significantly higher than the NS and ALN groups, and on POD28 and POD42, it had bone volume fraction, trabecular number, and TMD significantly higher than the NS group. (3) WB results showed that, compared with the NS group, the QG group had significantly increased expression of nuclear factor kappa-B (NF-κB), hypoxia-inducible factor-1α (HIF-1α), bone alkaline phosphatase (BALP), runt-related transcription factor 2 (Runx2), bone Gla protein (BGP) and collagen Iα1 (COLIα1) on POD14, significantly increased expression of NF-κB, HIF-1α, BALP and COLIα1 on POD28, and significantly increased expression of NF-κB, HIF-1α, and Runx2 on POD42. (4) TEM scanning results showed that, compared with the NS and ALN groups, the QG group had significantly increased numbers of autophagic vacuoles (AVs) in osteocytes on POD14, POD28, and POD42. Conclusion. QG could accelerate osteoporotic fracture healing by promoting bone formation and osteocyte autophagy, possibly through upregulating the NF-κB/HIF-1α signaling pathway.

Differences in Fracture Healing Between Female and Male C57BL/6J Mice

BackgroundMice are increasingly used in fracture healing research because of the opportunity to use transgenic animals. While both, male and female mice are employed, there is no consensus in the literature whether fracture healing differs between both sexes. Therefore, the aim of the present study was to analyze diaphyseal fracture healing in female and male C57BL/6J mice, a commonly used mouse strain in bone research.MethodsFor that purpose, 12-week-old Female (17–20 g) and Male mice (22–26 g) received a standardized femur midshaft osteotomy stabilized by an external fixator. Mice were euthanized 10 and 21 days after fracture and bone healing was analyzed by biomechanical testing, μCT, histology, immunohistochemistry and qPCR.ResultsTen days after fracture, Male mice displayed significantly more cartilage but less fibrous tissue in the fracture callus compared to Female mice, whereas the amount of bone did not differ. At day 21, Male mice showed a significantly larger fracture callus compared to Female mice. The relative amount of bone in the fracture callus did not significantly differ between both sexes, whereas its tissue mineral density was significantly higher in Male mice on day 21, indicating more mature bone and slightly more rapid fracture healing. These results were confirmed by a significantly greater absolute bending stiffness of the fractured femurs of Male mice on day 21. On the molecular level, Male mice displayed increased active β-catenin expression in the fracture callus, whereas estrogen receptor α (ERα) expression was lower.ConclusionThese results suggest that Male mice display more rapid fracture healing with more prominent cartilaginous callus formation. This might be due to the higher weight of Male mice, resulting in increased mechanical loading of the fracture. Furthermore, Male mice displayed significantly greater activation of osteoanabolic Wnt/β-catenin signaling, which might also contribute to more rapid bone regeneration.