Sfrp4 expression in thyroxine treated calvarial cells.

AIMS: Evidence suggests alterations of thyroid hormone levels can disrupt normal bone development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of exogenous thyroid hormone. MAIN METHODS: Here we aimed to model these interactions in vitro using primary and isotype cell lines to determine if thyroid hormone drives increased Sfrp4 expression in cells relevant to craniofacial development. Transcriptional profiling, bioinformatics interrogation, protein and function analyses were used. KEY FINDINGS: Affymetrix transcriptional profiling found Sfrp4 overexpression in primary cranial suture derived cells stimulated with thyroxine in vitro. Interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4 in murine calvarial pre-osteoblasts. Functional analysis after thyroxine hormone stimulation for alkaline phosphatase activity shows that pre-osteoblasts increase alkaline phosphatase activity compared to other cell types, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre-osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity. SIGNIFICANCE: Taken together, these results suggest SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. These data corroborate clinical findings indicating a potential for SFRP4 as a diagnostic or therapeutic target for hyperostotic craniofacial disorders.

Bi-Criteria Radio Spectrum Sharing With Subspace-Based Pareto Tracing.

Radio spectrum is a scarce resource. To meet demands, new wireless technologies must operate in shared spectrum over unlicensed bands (coexist). We consider coexistence of Long-Term Evolution (LTE) License-Assisted Access (LAA) with incumbent Wi-Fi systems. Our scenario consists of multiple LAA and Wi-Fi links sharing an unlicensed band; we aim to simultaneously optimize performance of both coexistence systems. To do this, we present a technique to continuously estimate the Pareto frontier of parameter sets (traces) which approximately maximize all convex combinations of network throughputs over network parameters. We use a dimensionality reduction approach known as active subspaces to determine that this near-optimal parameter set is primarily composed of two physically relevant parameters. A choice of two-dimensional subspace enables visualizations augmenting explainability and the reduced-dimension convex problem results in approximations which dominate random grid search.

rhBMP2 alone does not induce macrophage polarization towards an increased inflammatory response.

Once thought to have revolutionized therapeutic intervention in surgery, Recombinant Human Bone Morphogenic Protein 2 (rhBMP2) is now in its second decade of sustained controversy over the side effects associated with its use. Side effects associated with clinical use of rhBMP2 (Infuse, Medtronic Inc) include a marked inflammatory response, pain, therapeutic failures, ectopic bone, tissue degradation, and death. What is missing, despite the depth of literature on the subject, is a direct interrogation of rhBMP2, specifically for inflammation. Here we set out to determine if rhBMP2 alters traditional macrophage markers associated with pro-inflammatory responses, and pro-reparative responses to injury. Based on our previous work, we hypothesized there would be no direct effect of the peptide on macrophage polarization. Here we utilized commercially available murine macrophages, RAW 264.7, and treated these cells with rhBMP2 in standard growth media or macrophage polarizing media (M1 and M2) at several doses of the peptide. Our readouts were cell viability, apoptosis, gene expression of M1 and M2 markers, and ELISA for M1 marker iNOS, and M2 marker Arg1. Our data give very little evidence to support an alteration in macrophage phenotype by rhBMP2 alone, or alteration of the phenotype when cultured in enriched M1 or M2 media. These results further suggest that other factors associated with the clinical use of Infuse, likely supraphysiological rhBMP2 doses and off label usage, are more likely the culprit for poor outcomes. This further reinforces the utility of rhBMP2 and other peptides in tissue engineering therapies when conditions are tightly controlled.

Sub-clinical dose of bone morphogenetic protein-2 does not precipitate rampant, sustained inflammatory response in bone wound healing.

Large bone injuries, defects, and chronic wounds present a major problem for medicine. Several therapeutic strategies are used clinically to precipitate bone including a combination therapy delivering osteoinductive bone morphogenetic protein 2 (rhBMP-2) via an osteoconductive scaffold (absorbable collagen sponge [ACS], i.e., INFUSE). Adverse side effects reportedly associated with rhBMP2 administration include rampant inflammation and clinical failures. Although acute inflammation is necessary for proper healing in bone, inflammatory cascade dysregulation can result in sustained tissue damage and poor healing. We hypothesized that a subclinical dose of rhBMP2 modeled in the murine calvarial defect would not precipitate alterations to inflammatory markers during acute phases of bone wound healing. We utilized the 5 mm critical size calvarial defect in C57BL6 wild-type mice which were subsequently treated with ACS and a subclinical dose of rhBMP2 shown to be optimal for healing. Three and 7-day postoperative time points were used to assess the role that rhBMP-2 plays in modulating inflammation vs. ACS alone by cytokine array and histological interrogation. Data revealed that rhBMP-2 delivery resulted in substantial modulation of several markers associated with inflammation, most of which decreased to levels similar to control by the 7-day time point. Additionally, while rhBMP-2 administration increased macrophage response, this peptide had a little noticeable effect on traditional markers of macrophage polarization (M1-iNOS, M2-Arg1). These results suggest that rhBMP-2 delivered at a lower dose does not precipitate rampant inflammation. Thus, an assessment of dosing for rhBMP-2 therapies may lead to better healing outcomes and less surgical failure.

Optimizing bone wound healing using BMP2 with absorbable collagen sponge and Talymed nanofiber scaffold.

BACKGROUND: Bone is a highly vascularized and resilient organ with innate healing abilities, however some bone injuries overwhelm these attributes and require intervention, such as bone tissue engineering strategies. Combining biomaterials and growth factors, such as bone morphogenetic protein 2 (BMP2), is one of the most commonly used tissue engineering strategies. However, use of BMP2 has been correlated with negative clinical outcomes including aberrant inflammatory response, poor quality bone, and ectopic bone. METHODS: In the present study, a novel poly-n-acetyl glucosamine (pGlcNAc, trade name Talymed) scaffold was utilized in addition to the commonly used acellular collagen sponge (ACS) BMP2 delivery system in a murine calvarial defect model to investigate whether the innate properties of Talymed can reduce the noted negative bone phenotypes associated with BMP2 treatment. RESULTS: Comparison of murine calvarial defect healing between ACS with and without Talymed revealed that there was no measurable healing benefit for the combined treatment. Healing was most effective utilizing the traditional acellular collagen sponge with a reduced dose of BMP2. CONCLUSIONS: The results of this investigation lead to the conclusion that excessive dosing of BMP2 may be responsible for the negative clinical side effects observed with this bone tissue engineering strategy. Rather than augmenting the currently used ACS BMP2 bone wound healing strategy with an additional anti-inflammatory scaffold, reducing the dose of BMP2 used in the traditional delivery system results in optimal healing without the published negative side effects of BMP2 treatment.

Selective serotonin re-uptake inhibitor sertraline inhibits bone healing in a calvarial defect model.

Bone wound healing is a highly dynamic and precisely controlled process through which damaged bone undergoes repair and complete regeneration. External factors can alter this process, leading to delayed or failed bone wound healing. The findings of recent studies suggest that the use of selective serotonin reuptake inhibitors (SSRIs) can reduce bone mass, precipitate osteoporotic fractures and increase the rate of dental implant failure. With 10% of Americans prescribed antidepressants, the potential of SSRIs to impair bone healing may adversely affect millions of patients' ability to heal after sustaining trauma. Here, we investigate the effect of the SSRI sertraline on bone healing through pre-treatment with (10 mg·kg-1 sertraline in drinking water, n = 26) or without (control, n = 30) SSRI followed by the creation of a 5-mm calvarial defect. Animals were randomized into three surgical groups: (a) empty/sham, (b) implanted with a DermaMatrix scaffold soak-loaded with sterile PBS or (c) DermaMatrix soak-loaded with 542.5 ng BMP2. SSRI exposure continued until sacrifice in the exposed groups at 4 weeks after surgery. Sertraline exposure resulted in decreased bone healing with significant decreases in trabecular thickness, trabecular number and osteoclast dysfunction while significantly increasing mature collagen fiber formation. These findings indicate that sertraline exposure can impair bone wound healing through disruption of bone repair and regeneration while promoting or defaulting to scar formation within the defect site.

Testing a novel nanofibre scaffold for utility in bone tissue regeneration.

Many variables serve to alter the process of bone remodelling and diminish regeneration including the size and nature of the wound bed and health status of the individual. To overcome these inhibitory factors, tissue-engineered osteoconductive scaffolds paired with various growth factors have been utilized clinically. However, many limitations still remain, for example, bone morphogenetic protein 2 (BMP2) can lead to rampant inflammation, ectopic bone formation, and graft failure. Here, we studied the ability for a nanofiber scaffold (Talymed) to accelerate BMP2 growth factor-induced bone healing compared with the traditional absorbable collagen sponge (ACS) delivery system. One hundred fifty-five adult wild type mice were arranged in 16 groups by time, 4 and 8 weeks, and treatment, ACS or Talymed, loaded with control, low, medium, or high dosages of BMP2. Skulls were subjected to microCT, biomechanical, and histological analysis to assess bone regeneration. The use of Talymed within the defect site was found to decrease the bone volume, bone formation rate, and alkaline phosphatase activity compared with ACS/BMP2 combinations. Interestingly, though Talymed regenerated less bone, the regenerate was found to have a greater hardness value than that of bone within the ACS groups. However, the difference in bone hardness between scaffolds was not detectable by 8 weeks. Based on these results, we found that the nanofiber scaffold generated a better quality of bone regenerate at 4 weeks but, due to the lack of overall bone formation and the inhibition of normal remodelling processes, was not as efficacious as the current clinical standard ACS/BMP2 therapy.

Involvement of calvarial stem cells in healing: A regional analysis of large cranial defects.

Large craniofacial defects present a substantial clinical challenge that often requires the use of osteoconductive matrices and osteoinductive cues (i.e., bone morphogenetic proteins [BMP2]) to augment healing. While these methods have improved clinical outcomes, a better understanding of how the osteogenic fronts surrounding the defect, the underlying dura mater, and the cranial suture area contribute to healing may lead to more targeted therapies to enhance bone regeneration. We hypothesized that healing within a large bone defect will be precipitated from cells within the remaining or available suture mesenchyme abutting the edges of a murine critical sized defect. To investigate this hypothesis, 39 adult, wild-type mice were randomly arranged into groups (9 or 10 per group) by time (4 and 8 weeks) and treatment (control, acellular collagen sponge alone, or acellular collagen sponge loaded with a clinically relevant scaled dosage of BMP2). The skulls were then subjected to microcomputed tomography and histological analysis to assess bone regeneration in regions of interest within the defect area. A regional assessment of healing indicated that BMP2 drives greater healing than control and that healing emanates from the surgical margin, particularly from the margin associated with undisrupted suture mesenchyme. Though BMP2 treatment drove an increase in cell presence within the healing defect, there was no regional orientation of craniofacial stem cells or vascularity. Overall, these data reinforce that osteoconductive matrices in conjunction with osteoinductive peptides result in better healing of large calvarial defects. This healing is characterized as emanating from the surgical margin where there is an abundant supply of vasculature and progenitor cells.