BackgroundBone 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.MethodsIn 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.ResultsComparison 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.ConclusionsThe 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.

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.

Objective The angiotensin II type 1 receptor (AT1R) can be activated under conditions of mechanical stretch in some cellular systems. Whether this activity influences signaling within the abdominal aorta to promote to abdominal aortic aneurysm (AAA) development remains unknown. We evaluated the hypothesis that mechanical AT1R activation can occur under conditions of hypertension (HTN) and contribute to AAA formation. Methods BPH/2 mice, which demonstrate spontaneous neurogenic, low-renin HTN, and normotensive BPN/3 mice underwent AAA induction via the calcium chloride model, with or without an osmotic minipump delivering 30 mg/kg/d of the AT1R blocker Losartan. Systolic blood pressure (SBP) was measured at baseline and weekly via a tail cuff. The aortic diameter (AoD) was measured at baseline and terminal surgery at 21 days by digital microscopy. Aortic tissue was harvested for immunoblotting (phosphorylated extracellular signal-regulated kinase-1 and -2 [pERK1/2] to ERK1/2 ratio) and expressed as the fold-change from the BPN/3 control mice. Aortic vascular smooth muscle cells (VSMCs) underwent stretch with or without Losartan (1 μM) treatment to assess the mechanical stimulation of ERK1/2 activity. Statistical analysis of the blood pressure, AoD, and VSMC ERK1/2 activity was performed using analysis of variance. However, the data distribution was determined to be log-normal (Shapiro-Wilk test) for ERK1/2 activity. Therefore, it was logarithmically transformed before analysis of variance. Results At baseline, the SBP was elevated in the BPH/2 mice relative to the BPN/3 mice ( P < .05). Losartan treatment significantly reduced the SBP in both mouse strains ( P < .05). AAA induction did not affect the SBP. At 21 days after induction, the percentage of increase in the AoD from baseline was significantly greater in the BPH/2 mice than in the BPN/3 mice (101.28% ± 4.19% vs 75.59% ± 1.67% above baseline; P < .05). Losartan treatment significantly attenuated AAA growth in both BPH/2 and BPN/3 mice (33.88% ± 2.97% and 43.96% ± 3.05% above baseline, respectively; P < .05). ERK1/2 activity was increased approximately fivefold in the BPH/2 control mice relative to the BPN/3 control mice ( P < .05). In the BPH/2 and BPN/3 mice with AAA, ERK1/2 activity was significantly increased relative to the respective baseline control ( P < .05) and effectively reduced by concomitant Losartan therapy ( P < .05). Biaxial stretch of the VSMCs in the absence of angiotensin II demonstrated increased ERK1/2 activation ( P < .05 vs static control), which was significantly inhibited by Losartan. Conclusions In BPH/2 mice with spontaneous neurogenic, low-renin HTN, AAA growth was amplified compared with the normotensive control a...

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Introduction Elevated interleukin-6 (IL-6) plasma levels have been associated with abdominal aortic aneurysm (AAA), but whether this cytokine plays a causative role in the degenerative remodeling or represents an effect from the inflammatory cascades initiated by infiltrating leukocytes remained unclear. This project aims to demonstrate that within the aortic wall, signaling from IL-6 through the STAT3 transcription factor is necessary for infiltration of proteolytically-active macrophages and development of small AAA. Methods Following measurement of baseline infrarenal aortic diameter (AoD, digital microscopy), C57Bl/6 and IL-6 knockout (IL-6KO) mice underwent AAA induction by application of peri-adventitial CaCl2 (0.5 M) +/− implantation of an osmotic mini-pump delivering IL-6 (4.36 µg/kg/day over 21 days). At the terminal procedure, AoDs were measured by digital microscopy and aortas harvested for immunoblot (pSTAT3/STAT3), matrix metalloproteinase (MMP) quantification, or flow cytometric analysis of macrophage content. Plasma was collected for cytokine analysis. Results IL-6 infusion significantly increased the plasma IL-6 levels in C57Bl/6 and IL-6KO animals. The C57Bl/6 + CaCl2 group developed AAA (AoD >50% above baseline) but IL-6KO + CaCl2 did not. In the IL-6KO + IL-6+CaCl2 group, AAA developed to match that of C57Bl/6 + CaCl2 mice. STAT3 activity was significantly increased in animals with advanced stages of dilation (>40% from baseline), compared to those with ectasia (≤25%). Although cytokine profiles did not support T-cells or neutrophils as being active contributors in this stage of aortic remodeling, changes in the profile of elaborated MMPs suggested macrophage activity with a trend toward alternatively activated pathways. Flow cytometry confirmed significantly increased macrophage abundance specifically in animals with upregulated STAT3 activity and advanced aortic dilation. Conclusion In this murine model of AAA, progressive dilation to development of true AAA was only accomplished when IL-6 signaling upregulated STAT3 activity to effect accumulation of proteolytically-active macrophages. This pathway warrants further investigation to identify potential therapeutic avenues to abrogate growth of small AAA.