TLR4 knockout promoted fracture healing, reduced the number of osteoclasts, increased bone callus volume (BV) and callus mineralized volume fraction (BV/TV%) (P < 0.05), increased the maximum torque and torsional stiffness of callus (P < 0.05), reduced TNF-α, IL-1β and IL-6 expression (P < 0.01), and increased the expression levels of β-catenin, Wnt4, Wnt5B, PCNA and BMP-2 (P < 0.01).
At 24 weeks, tissue mineral density, the ratio of bone volume to total volume, and volumetric bone mineral density of the callus were higher in the BMP-2 group than in control animals.
Repetitive brief ischemia increased the callus area at 2 weeks and boosted the synthesis of BMP-2, VEGF, TGF-<i>β</i>, and ALP in the fracture region at 2 weeks from tissue stains.
Specific components of the BMP-2 pathway were analyzed in fracture callus on days 3, 7, 14, and 21 after fracture via western immunoassays and enzyme-linked immunosorbent assay.
The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1osx (-/-) mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol-based delivery method.
Using MSCs from BMP-2-Lac Z mice genetically modified using a bacterial artificial chromosome system to be beta-gal reporters for bone morphogenic protein 2 (BMP-2) expression, we found that MSCs contributed to the callus initiation by expressing BMP-2.
Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus.
In vivo bending stiffness measurements during fracture healing as well as ex vivo torsional stiffness measurements demonstrated stiffer callus tissue after treatment with Ad.BMP-2.
Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses.
We concluded that continuous PTH<sub>1-34</sub> infusion resulted in a delayed fracture healing process due to delayed callus cell maturation but ultimately increased biomechanical properties.
Histological analysis revealed that administration of PTH1-34 increased the size of both the total callus and cartilaginous callus at 14 days after the surgery in ACH mice.
Conclusions PTH can promote fracture healing and callus hardness in ovariectomized mice by increasing callus formation and reconstructing trabecular bone via a PLC-independent pathway.
In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1-34) can increase callus formation and mechanical strength.
We hypothesized that fracture fixation with a smaller pin would permit greater interfragmentary strain resulting in increased total amount of vascular endothelial growth factor within the callus and greater angiogenesis compared to fixation with a larger pin.
Real-time and Fluorescence- Activated Cell Sorting (FACS) results demonstrated that standard Thymoquinone and callus extracts down-regulated the VEGF-A gene expression, and all three induced apoptosis in the AGS cell line.
Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus.
Compared to the control, ADSC<sup>bFGF</sup> treatment increased VEGF expression at the periosteal region of the callus, remodeling of collagen into mineralized callus and bone strength.
Based on our data, local administration of VEGF in the callus to stimulate revascularization, or transplantation of stem cells to enhance bone turnover represent potentially feasible approaches to improve outcomes in clinical practice.
Furthermore, qRT-PCR analysis showed a 1-fold upregulation in mRNA levels of transforming growth factor beta and roughly 6-fold increases in vascular endothelial growth factor mRNA expression in calluses from the tanshinol groups.
Further, the Cu<sup>2+</sup> content and Runx2 level in the callus were determined, and local mechanical test of the fracture was performed to assess the healing quality.