Faculty Bibliography

Introduction With an ever-increasing number of bone fractures, physicians are constantly faced with patients with delayed unions or non-unions. Aminocaproic acid has been approved and used in joint replacement and spinal fusion surgeries as an anti-fibrinolytic to limit perioperative blood loss (1-3). The efficacy and safety of aminocaproic acid has been studied extensively, and it has been shown to have a very safe and favorable risk profile (2,3). Cuellar et al. showed that aminocaproic acid significantly enhanced spine fusion in an animal model (4). We investigated aminocaproic acid as a pro-osteogenic agent and test whether it can be used as an adjunct to enhance bone regeneration after long-bone fracture in contrast to axial bone. Methods In order to investigate the osteoinductive capacity of aminocaproic acid, we performed an in vitro analysis with mouse marrow mesenchymal progenitor cells cultured with 150, 200, 250 ug/ml aminocaproic acid. The gene expression of osteogenic markers was assessed by quantitative RT-PCR and cell proliferation was measured by BrdU assay. For the in vivo study, we employed two well-established mouse models using 12 week-old male C57BL/6J mice to evaluate intramembranous healing (1 mm monocortical tibia defect) and endochondral healing (femur fracture). Immediately after euthanasia, the tibiae and femurs were dissected, fixed in paraformaldehyde then scanned at high resolution with a Bruker SkyScan microCT. Ten mum resolution images were used to assess bone volume, bone volume/total volume, trabecular thickness, cortical thickness and overall regenerate volume. Histology was completed using Movat's pentachrome and aniline blue to detect cartilaginous and osseous tissues. Histomorphometric analyses were completed as follows: the stained slides were photographed using a Leica digital imaging system (5X objective). The digital images were imported into Adobe Photoshop CC 2015 and the region of interest was set to 106 x 106 pixels. The number of aniline blue/alcian blue stained pixels was determined using the magic wand tool (tolerance setting; 60, histogram pixel setting; cache level 1) by a single blinded investigator, and confirmed by a second independent investigator. Two-tailed Student's t-tests were used to determine significant differences between data sets that are normally distributed. For non-normally distributed data sets, Mann-Whitney U test was used. Significance was attained at p < 0.05 and all statistical analyses were performed with Graphpad Prism software (GraphPad Software, San Diego, California). Results In vitro data showed no significant difference in cell proliferation of mouse bone marrow cells treated at varying concentrations of aminocaproic acid. Osteogenic gene expression, specifically alkaline phosphatase, osterix and osteocalcin, were all significantly decreased at all concentrations of aminocaproic acid treatment while collagen I gene expression was significantly decreased only at higher concentrations (Figure 1). When looking at the femur fracture model (endochondral healing) a significant difference in cartilage volume was noted both on day 14 (p=0.007) and day 21 (p=0.045) (Figure 2). Contrasting the cartilage findings, bone volume on histomorphometric analysis in the femur fracture model was significantly increased in the treatment group compared to the control group on both day 14, p= 0.059 and day 21, p=0.032 (Figure 3). No difference was detected in bone volume in micro CT analysis at either day 14 or day 21 for the femur fracture model. A significant difference in early fracture healing was observed in the tibial defect (intramembranous) model at day 7 in the control group compared to the treatment group in both histomorphometric analyses (p=0.038) and microCT data, where bone volume was reduced in the treated group at day 7 compared to controls (p<0.05). Discussion: Aminocaproic acid is an important hemostasis pharmacologic used to decrease blood loss in joint replacements and spinal fusions but its effects on bone remodeling are not completely understood. We sought to analyze the differences in the effect of aminocaproic acid on the appendicular skeleton versus its effects on the axial skeleton. Our results show a decrease in early bone formation and osteogenic gene expression but no difference in cellular proliferation, which suggests that aminocaproic acid interferes with osteogenic differentiation. Additionally the decrease in cartilage volume in the femur fracture model could indicate that aminocaproic acid interferes with chondrogenesis, which is essential to endochondral ossification and should be further investigated. (Figure Presented)

INTRODUCTION: All tissues are affected by aging, but diseases that weaken the skeleton constitute the most prevalent chronic impairment in the United States. Although skeletal diseases and conditions are seldom fatal, they can significantly compromise function and diminish quality of life. Perhaps most importantly, age-related changes in skeletal health can be traced back to a decline in both the number and function of osteoprogenitor cells (OPCs). However, the cause for the decline in both the number and function of OPCs is not well understood. Chronic inflammation in the elderly (inflamm-aging) is thought to be a major contributor to this decline in the regenerative capacity of many tissues, including the skeleton. In contrast to a well-balanced inflammatory response after trauma, which is crucial for successful bone repair, chronic unbalanced elevation of pro-inflammatory cytokines has been shown to inhibit regeneration in a variety of tissues. We hypothesize that inflamm-aging is the major cause for the decline in OPC number and dysfunction in elderly patients and that this decline in OPC number and dysfunction can be halted by treatment with an anti-inflammatory drug. METHODS: Young, 12 week-old and aged, 52 week-old C57BL/6J were used following the IACUC guidelines at our institution. Aged animals were randomly distributed into a no-treatment (n=5) and a treatment group (n=5). Animals in the no-treatment group received regular drinking water, while animals in the treatment group received sodium salicylate water (12mg/day) for 8 weeks. The inflammatory status of young and aged untreated and treated mice was assessed using a multiplex platform screening for multiple pro- and anti-inflammatory cytokines, and utilizing qRT-PCR for IL-1, IL-6, NF-kappaB, TNF-alpha. FACS analysis using the LepR as a marker for osteogenic precursor cells was employed to identify the effect of chronic low-level inflammation on progenitor cell number. In addition, bMSCs were harvested from femurs and tibia from young and aged untreated and treated mice and cultured in growth media and osteogenic media. Cell proliferation and osteogenic differentiation (qRT-PCR for col 1, runx2, osx and oc, and alizarin red and alkaline phosphatase staining) were assessed in vitro. Results are presented in the form of mean +/- standard deviation, with N equal to the number of samples analyzed. Two-tailed Student's t-tests were used to determine significant differences between data sets that are normally distributed. For non-normally distributed data sets, Mann-Whitney U test was used. Significance was attained at p < 0.05 and all statistical analyses were performed with Graphpad Prism software (GraphPad Software, San Diego, California). RESULTS: First, we set out to identify age-related chronic inflammation in mice. We analyzed blood by multiplex analysis and tibial and femoral bone marrow by qRT-PCR for pro-inflammatory markers. Both analyses revealed an increase in pro-inflammatory and a decrease in anti-inflammatory cytokines in aged animals, confirming the presence of inflamm-aging (Fig. 1A). Next, we aimed at understanding how aging effects osteoprogenitor cell number using flow cytometry. We harvested cells from young and aged mice, removed red blood cells, and then stained with antibodies to CD31, CD45, Ter-119 and LepR. Cell sorting was performed and CD31^-CD45^-Ter^-119^-LepR+ cells were isolated and quantified. Flow cytometry analysis revealed that 0.38% of cells from 12 week old mice were LepR⁺ osteoprogenitor cells, confirming the findings published by Zhou et al. Analysis of the cells from 52 week-old mice revealed a significant decrease in number to 0.017% of bone marrow cells (Fig. 1B). Next, we had to establish that an 8-week course of sodium salicylate successfully represses chronic inflammation. In response to NSAID treatment, the expression level of IL-10 significantly increased above the level of the juvenile animals, while the NF-kappaB, TNFalpha and Cox-2 levels returned to baseline (Fig. 1A). This experiment served as a proof-of-principle that mice exhibit inflamm-aging and that this inflammatory state can be suppressed by administration of an NSAID. Having established that our dosing of the systemic NSAID suppresses chronic ageinduced inflammation, we assessed whether OPC frequency changes as a result of suppressed inflamm-aging. Bone marrow from 3 month-old, 12 month-old and 12 monthold NSAID-treated animals was subjected to flow cytometry. OPC frequency declined with aging, however, after an 8 week course of NSAID treatment, we noticed a two-fold increase in LepR⁺ OPCs within the bone marrow (Fig. 1B). We then aimed at testing whether NSAID treatment of aged mice resulted in a restoration of the osteogenic potential of this OPC population. Quantitative RT-PCR of the bone marrow of sodium salicylate-treated mice showed an increase in osteogenic gene expression (osx, oc and alkaline phosphatase) compared to untreated aged mice. The expression level of osx reached that of young, 3 month-old mice, while oc and ALP expression levels were significantly higher than those of juvenile animals (Fig. 1C). In order to further characterize this increase in osteogenic potential, we harvested MSCs from young and aged treated and untreated animals, plated them in vitro and then subjected them to osteogenic differentiation media. Mineralization assays and expression analysis of osx, oc and ALP showed decreased osteogenesis of aged cells, while treatment with sodium salicylate recovered this decline and resulted in restoration of the osteogenic potential (Fig. 1D). DISCUSSION: These experiments demonstrate for the first time that age-related chronic inflammation is responsible for the decreased proliferative and osteogenic potential of aged OPCs and that this process is reversible by anti-inflammatory treatment. The findings from this study may have a profound translational impact: If we could restore the regenerative potential of the aged skeleton by treating age-related inflammation, then theoretically, we may have a tool at hand to improve the healing process of osteoporotic fracture patients. (Figure Presented)