MiMHC 5th Annual Musculoskeletal Health Symposium

Skeleton is one of body’s largest organs and osteocytes (OT) make up to 95% of all skeletal cells. Peroxisome proliferator-activated receptor gamma (PPARG) is a transcription factor known for regulating energy metabolism and insulin sensitivity; however, its role in regulation of osteocyte function is largely unknown. In this study, we report that PPARG is abundantly expressed in osteocytes, as compared to endosteal osteoblasts, and is transcriptionally essential for sclerostin production which is a major regulator of bone formation by osteoblasts. Mice lacking PPARG primarily in osteocytes (γOTKO) have increased bone mass and decreased marrow fat, and there is an excellent correlation of sclerostin protein level in osteocytes with the level of PPARG deletion. This is paralleled by increased WNT signaling and bone-forming activity of endosteal osteoblasts. Moreover, osteocyte conditioned medium depleted of sclerostin decreases adipogenesis in recipient bone marrow stromal cells consistent with decreased marrow adiposity in γOTKO mice. Examination of 8 kb sequence upstream of Sost gene transcription start site (TSS), using position weighted matrix prediction for transcription factors binding sites (JASPAR), revealed multiple PPARG binding elements (PPREs). Two of them, with highest scores for consensus PPARG binding sequence, were chosen for further analysis. Chromatin immunoprecipitation assays (ChIP) showed that PPARG binds to both PPREs. Interestingly, pharmacologic modulation of PPARG activity with full agonist rosiglitazone increases PPARG binding to both PPREs and increases sclerostin mRNA and protein expression. Consistently, γOTKO mice are at least partially resistant to the negative effects of rosiglitazone on bone. These findings indicate that transcriptional activities of PPARG are essential for sclerostin expression in osteocytes and support consideration of targeting PPARG activities with selective modulators to treat osteoporosis.

Baroi, Sudipta <sudipta.baroi@rockets.utoledo.edu>

1Department of Orthopedic Surgery, University of Toledo, College of Medicine and Life Sciences, Toledo, OH.

2Department of Physiology and Pharmacology, 3Center for Diabetes and Endocrine Research, University of Toledo, College of Medicine and Life Sciences, Toledo, OH. 4The Scripps Research Institute, Jupiter FL

Monoclonal gammopathy of undetermined significance (MGUS) is a precursor condition for multiple myeloma (MM). It is characterized by the presence of serum monoclonal antibody derived from clonal plasma cells (PC). Despite similar oncogenes to MM, MGUS PCs are non-proliferative and the mechanisms that trigger disease progression remain unclear. Because oncogene activation is a stressor known to cause senescence-induced growth arrest, we hypothesized that MGUS clonal PCs are senescent. Thus, we have employed the KaLwRij mouse model of MGUS to assess senescence markers in KaLwRij PCs and the response of these cells to senolytic therapy (dasatinib+quercetin, D+Q). Age and sex-matched KaLwRij mice (10-12 mo) were treated (tx) with D+Q (5, 50mg/kg) or placebo for 3 days, biweekly for 2.5 months. The related C57BL/6 mice served as controls. Blood samples were collected biweekly to measure serum antibody (Ab) content. Following euthanasia, bone marrow PCs were enriched via magnetic sorting, followed by staining for senescence-associated βGal activity (SA-BGal) and further isolation/analysis of PCs by fluorescence-activated cell sorting. As observed in Figure 1, female KaLwRij mice exhibited significantly reduced total PC numbers compared to C57BL/6, with increased percentage (%) SA-βGal+ PCs. The % of SA-βgal+ PCs was significantly decreased in female D+Q tx KaLwRij mice, and these mice exhibited a rescue in total PC number. Male D+Q tx KaLwRij mice showed no differences in these parameters compared to placebo. RT-qPCR revealed increased senescence gene expression (Bcl2, Bmi, Fas, Irf4, Nfkb1, Prdm1, and Relb) with reduced proliferation marker, Mki67, in SA-BGal+ PCs. D+Q tx significantly improved the expression of PC markers Cd40, Slamf7, and Xbp1, with trends for improved Sdc1 and Cd40Ig in SA-BGalNeg PCs. Serum Ab content served as a functional readout of MGUS. While individual Ab isotype levels were variable, total Ab content significantly increased with time in placebo mice. This increase was not observed in D+Q tx female KaLwRij mice. The increased Ab content was driven by increased IgG1, and D+Q tx female KaLwRij mice exhibited a significant decrease in IgG1 percent change compared to placebo. Overall, these results are consistent with senescence in clonal PCs. Recent findings suggest senescent cells can exhibit increased genomic instability. Thus, further evaluation of PC senescence phenotypes in MGUS may lead to the identification of biomarkers to predict risk of progression to MM.

Password (if required): 315668

Borges, Gabriel Alvares <galvares@umich.edu>

1 University of Michigan, Ann Arbor, MI, USA. 2 University of Oxford, Oxford, UK. 3 University of Connecticut, Storrs, CT, USA. 4 Mayo Clinic, Rochester, MN, USA.

INTRODUCTION: Increased risk of skeletal fractures due to bone mass loss is a major public health problem resulting in significant morbidity and mortality, particularly in the case of hip fractures. Current clinical methods based on two-dimensional measures of bone mineral density (areal BMD or aBMD) are often unable to identify individuals at risk of fracture. The underlying hypothesis of this study was that combinations of femur structural traits are different for those femurs that suffer a fragility fracture within the proximal region of the femur and those that sustain a fracture in either the subtrochanteric or midshaft region of the femur, resulting in an “atypical femur fracture”.

STUDY GOALS: The objective of this study was to determine the effects of varying combinations of structural traits, material properties, and loading conditions on femur stress response and the location of stress response variation using a validated parametric finite element model.

METHODS: Computed tomography (CT) image data for forty-seven intact cadaver specimens (39 males, 64.3 +/- 8.6 years; 8 females, 67.1 +/- 12.7 years) was previously obtained (University of Virginia Center for Applied Biomechanics, Charlottesville, VA). CT data were filtered and segmented to develop surfaces describing the outer cortical bone surface and the transition surface from cortical bone to trabecular bone and marrow space for the right femur. Spatial distributions of bone mineral density (BMD) within the femurs were determined from CT data calibrated to relate image intensity to BMD. Statistical shape and trait modelling of the femur was used to describe variability in the structural organization of the set of femurs in an efficient manner. The resulting description of structural variability was converted to a parametric finite element model and exploited to investigate combinations of factors leading to increased stress under single-leg stance and fall-type loading, whether in the proximal region or in the subtrochanteric and midshaft region.

RESULTS: Under single leg stance, stress was dependent on interactions between femur structure, material property, and loading variables. Proximal femur stress variation was largely dependent on interaction between femur structure and loading conditions. Subtrochanteric femur stress was also dependent on interaction between bone structure, yield properties, and loading direction. Under fall loading, variation in trabecular bone yield stress was primarily responsible for an increase in yielded elements in both cortical and trabecular bone. Trabecular bone stress was also affected by variation in femur structure, although interaction with other factors, rather than main effects, was responsible. Subtrochanteric bone yield was dependent on cortical elastic modulus, yield strength for trabecular and cortical bone, and structural weighting factors.

DISCUSSION: In combination with parameters describing loading condition and material property variation, variation in structural organization is associated with regional increases in maximum principal stress and the percentage of bone expected to damage, and these increases are likely associated with increased fracture risk. The results of this study indicate that there are multiple pathways and combinations of descriptor variation that may result in increased fracture risk. Further, these pathways can lead to fracture in any region of the femur under both overload conditions, such as with sideways fall loading, and stance loading, which due to the repetitive nature may lead to the accumulation of fatigue damage within the bone and further impair bone condition and increased susceptibility to fracture.

Bredbenner, Todd <todd.bredbenner@uccs.edu>

Mechanical and Aerospace Engineering, University of Colorado Colorado Springs

Study Goals: Osteoporotic hip fractures remain a major public health problem. Low areal bone mineral density (aBMD) measured by Dual-energy X-ray absorptiometry (DXA) is used to clinically identify those at increased risk of fracturing. The majority of fractures, however, occur in individuals outside the range considered to be at risk, which indicates the association between aBMD and fracture risk varies among individuals. By better understanding the inter-individual differences in how bone structure influences aBMD, we can begin to better predict who is at increased risk of fracturing. The goal of this study was to determine the association between the amount of cortical and trabecular bone from NanoCT with bone parameters from DXA and to test if this association differs between narrow and wide proximal femurs.

Methods: Femoral neck (FN) area, bone mineral content (BMC), and aBMD were measured using DXA for 22 cadaveric proximal femurs (white women, 42-89+ years old). High resolution 3D volumes of the proximal femurs were obtained using a NanoCT system at 27um voxel size. Using ORS Dragonfly, FN volumes of interest (VOI) corresponding to the DXA ROI were extracted from the NanoCT scans. Validated convolutional neural networks were used to segment the bone from background and the cortical bone from the trabecular bone. A custom plugin was used to count the number of cortical, trabecular, and total bone voxels in the direction of the 2D projection of the DXA scans. Samples were sorted based on FN area from the DXA scans into narrow and wide subgroups and linear regression analysis was performed.

Results: Linear regression analysis revealed strong correlations between the NanoCT and DXA FN regions for area (R2=0.91, p<0.0001), BMC (R2=0.91, p<0.0001), and aBMD (R2=0.89, p<0.0001). There was a strong association between BMC and the total number of bone voxels which did not differ between narrow (R2 =0.87, p<0.0001) and wide (R2 =0.93, p<0.0001) subgroups (ANCOVA: slope, p=0.199; y-int, p=0.559). The association between BMC and the number of cortical vs. trabecular voxels, however, differed between subgroups. While both subgroups showed significant associations between BMC and the number of cortical bone voxels (R2 =0.88, p<0.0001 and R2 =0.54, p=0.010, respectively), the slope was significantly greater for narrow bones (ANCOVA, slope p=0.002). In contrast, the wide (R2=0.88, p<0.0001) but not narrow (R2=0.008, p=0.79) subgroup showed a significant association between BMC and the number of trabecular voxels (ANCOVA, slope, p=0.001).

Discussion/Conclusion: The strong correlation between the NanoCT and DXA regions for area, BMC, and aBMD indicates successful alignment of the NanoCT and DXA VOIs. When separated into the narrow and wide subgroups, both demonstrated a strong correlation between BMC and the total number of bone voxels, which was expected because X-ray attenuation is based largely on the presence of bone. Importantly, the subgroup analysis revealed that for narrow bones, lower BMC values reflect lower amounts of cortical bone. Alternatively, for wide bones, lower BMC values reflect proportionally lower amounts of both cortical and trabecular bone. Therefore, this study demonstrates that FN BMC values from DXA reflect different proportions of cortical and trabecular tissues in narrow and wide proximal femurs. This outcome motivates future studies investigating how the relationship between changes in BMC and changes in bone strength differ between narrow and wide subgroups.

Casden, Michael

University of Michigan, Department of Orthopaedic Surgery

University of Michigan Medical School; University of Michigan School of Public Health

On average, males have larger muscle mass than females, while females have greater muscular endurance than males. Understanding how sex contributes to the phenotypic variation in skeletal muscle provides a better understanding of muscle biology. In the FUSION Tissue Biopsy Study, we used single nuclei isolation and RNA-sequencing to quantify gene expression in skeletal muscle biopsies from 287 living Finnish donors (Age 60.1±0.9, BMI 27.6±0.5, 42.9% female). We clustered nuclei into 13 cell types, including Type 1, 2a, and 2x muscle fibers.

We tested for differences in muscle composition by sex using Negative Binomial Regression with DESeq2. To test for differential gene expression by sex within each cell type, we used linear regression adjusting for age and technical covariates. Genes on the X and Y chromosomes were most strongly differentially expressed between men and women. We found the highest percentage of genes differentially expressed by sex in Type 2a fibers (15.3%). We used gene set enrichment analysis to determine the biological processes with genes that are enriched for higher expression in males or in females. Across all muscle fiber types, genes involved in mitochondria and energy metabolism are enriched for higher expression in males. Genes involved in signal transduction, development, and cell adhesion are enriched for higher expression in females. Gene set analyses for differential expression by sex were highly concordant across Type 1, 2a, and 2x fibers.

In skeletal muscle, muscle composition and gene expression within muscle cell types differ between the sexes. Further analysis of these differences could aid in identifying general and cell-type-specific gene regulation underlying phenotypic differences in skeletal muscle by sex.

Ciotlos, Dan Liviu

University of Michigan, Department of Biostatistics

Osteoclasts (OCs) are multinucleated bone-resorbing cells derived from a heterogeneous pool of myeloid progenitors. Recent reports show that similar to other myeloid cells (macrophages, MΦ; dendritic cells, DC) OCs have the ability to phagocytose, process, and present antigen to activate specific T cell responses in the bone microenvironment. The ability of OCs to activate T cell responses has important implications for inflammatory bone pathologies, such as rheumatoid arthritis, osteomyelitis, and periodontitis, in which OCs and T cells both play crucial roles. The majority of functional analyses of OC antigen uptake, presentation, and activation of T cells have been performed on plastic tissue culture plates. Adherence to the bone surface affects osteoclast activities via activation of integrin signaling and matrix stiffness. In order to determine how these factors affect the ability of osteoclasts to act as antigen presenting cells, and thus better assess how bone resorbing osteoclasts impact disease pathology in vivo, we performed a direct comparison of antigen uptake by MΦ, DC, and MΦ or DC-derived osteoclasts cultured on plastic, glass, or bovine bone chips. In each condition, cells were incubated with fluorescent (FL)-ovalbumin (OVA) and assessed for antigen uptake and expression of MHC class II and CD80 by IHC. Similar to previous studies, we found that DC cultures exhibited significantly improved FL-OVA uptake on all substrates (p<0.005), including on bone, in which mononucleated cells within the DC-OC cultures showed improved FL-OVA uptake compared to mononucleated cells within BMM-OC cultures. DC uptake of FL-OVA was greatest on plastic, with lesser uptake on glass and bone. In contrast to mononucleated cells, BMM-derived OCs exhibited comparable FL-OVA uptake to DC-derived OCs on plastic and glass (p>0.05). However, when plated on bone uptake of FL-OVA by osteoclasts was attenuated. These findings demonstrate that both adherence to bone, and matrix stiffness, reduce OC ability to uptake and present antigens.

Durdan, Margaret

University of Michigan

Cellular and Molecular Biology, Program in Biomedical Sciences, Biointerfaces Institute, University of Michigan Medical School

Study Goals/Purpose Disuse osteopenia develops in individuals with decrease lower limb use especially after injury necessitating non-weightbearing, hospitalization or both. However, the nature of this change is not well characterized and, therefore, cannot be appropriately addressed. Conventional CT produces a reliable evaluation of bone quality as Hounsfield Units (HU) and offers the opportunity to longitudinally study bone density in activity-altered patients. The purpose of this study is to estimate the degree of osteopenia in non-weightbearing hospitalized patients and to identify patient related factors associated with this loss.

Methods Initial screening criteria included patients who presented to the emergency department between January 1, 2010 to August 1, 2020; baseline lower extremity orthopaedic trauma evaluated by CT. Further criteria included CT imaging capturing the ipsilateral hip at the time of admission; a second CT scan, for any indication (eg, small bowel obstruction, diverticulitis, etc) that include that same hip; strict non-weightbearing in between scans. Exclusion criteria included change in weightbearing status and CT imaging changes that would not allow eBMD analysis (eg, intervention). Estimated BMD (eBMD) was measured at baseline and and at the secondary time point via HU approximation 12mm inferior to superior aspect of femoral neck on axial CT. The change in eBMD was calculated as a proxy for disuse osteopenia and the relationship between relative bone loss (as a nominal rate of osteopenia, eBMD loss/time) and patient specific factors was explored.

Results Of 200,000 patients who received CT evaluation including their hip during the targeted timeframe, seventeen patients met our inclusion and exclusion criteria. There was an average of 7.3 days (median = 6 days; mode = 4 days; interquartile range: 4 – 8.5 days) between CT scans. The average rate of disuse osteopenia in the cohort was a net average decrease of 7.5 ± 5.8 HU/day. To place in a standardized clinical context, this change is estimated to correlate linearly with respect to change in DEXA BMD T-Score and may be clinically relevant when HU approach values <100. Associated patient factors that may further precipitate osteopenia whilst non-weightbearing are smoking, female gender, and diabetes.

Conclusions We report novel, preliminary data estimating the rate of disuse osteopenia in admitted, non-weightbearing orthopaedic trauma patients. This data underscores an important consideration all physicians should make when non-weightbearing status is being considered or required after injury or admission.

Eichman, Jack

Department of Orthopaedic Surgery, University of Michigan, Ann Arbor, MI, USA

Skeletal muscle’s remarkable regenerative capacity is mainly attributed to resident stem cells, termed satellite cells. Recently, it has been shown that some interstitial cells exhibit myogenic capacity and may contribute to myofibers in vivo. However, muscle interstitial stroma contains heterogenous populations and their in vivo myogenic capacity is not fully understood. Here, we identify a population of muscle interstitial cells that express the evolutionarily conserved transcription factors of the Hox11 paralogous group. Hox genes are crucial for regional patterning of musculoskeletal elements during embryonic development, and regionalized Hox expression is maintained throughout postnatal and adult stages. In bone marrow stroma, endosteum, and periosteum, previous work has shown that Hox11-expressing cells function as skeletal stem cells and play major roles in skeletal homeostasis and fracture repair. Although regionally-restricted Hox11-expressing muscle interstitial cells have been observed during embryogenesis, their continued expression and potential contribution to differentiated cell types remains unexplored. Using a Hoxa11CreERT2/+; ROSALSL-tdTomato/+ lineage reporter, we are investigating the contribution of Hox11-expressing cells to skeletal muscle at adult stages. Surprisingly, Hox11-expressing interstitial cells progressively contribute to zeugopod-attached myofibers (radius/ulna or tibia/fibula). Hox11-expressing interstitial cells do not overlap with Pax7-expressing satellite cells. Further, lineage-labeled cells do not traverse through satellite cells to contribute to myofibers; Pax7-expressing cells are not lineage labeled by Hoxa11CreERT2. Hox11 lineage contribution to muscle is independent of Hox function; loss of Hox11 function does not affect the lineage labeling of interstitial cells to muscle fibers. Together, these data support a satellite cell-independent lineage of interstitial cells that contribute to adult myofibers. Zoom Passcode: 981034

Flynn, Corey <cflynn9@wisc.edu>

University of Wisconsin-Madison

Bone loss caused by trauma, neoplasia, congenital defects or periodontal disease is a major cause of disability and human suffering. Bone regeneration procedures are necessary for treating non-union fractures and other refractory bone defects. Skeletal progenitor/stem cell:extracellular matrix (ECM) interactions are critical for bone regeneration. Integrins are the most‐studied bone ECM receptors and scaffolds containing integrin‐binding regions of the ECM have been used for bone regeneration. However, little is known about the involvement of other ECM receptors in bone. This project will focus on discoidin domain receptor 2 (DDR2), an understudied but important collagen receptor that preferentially binds fibrillar collagens and modulates integrin activity. Ddr2 loss-of-function mutations in humans and mice cause severe craniofacial and skeletal defects including altered cranial shape, dwarfing, reduced trabecular and cortical bone and alveolar bone/periodontal defects. In this study, we examine the role of DDR2 in bone regeneration and explore DDR2-based therapeutics as a new class of bone regeneration agents. DDR2 in bone regeneration. Calvarial subcritical-size defects and tibial fractures were generated in wildtype and Ddr2 deficient mice. For calvarial defects, complete bridging, seen in WT controls at 4 weeks post-surgery, was not observed in Ddr2 deficient mice. Quantitation of defect bone area by CT revealed a 50% reduction in new bone volume in Ddr2 deficient mice. For tibial fractures, WT mice showed union of fractures 6 weeks post-fracture while union was reduced in Ddr2 deficient mice. This was manifested as a significant reduction in mRUST (radiographic union scoring tibial fracture) score. Mutant mice also exhibited a 35% reduction in callous volume. Ddr2 expression during calvarial and tibial bone regeneration was also defined using Ddr2-LacZ knock-in mice. Ddr2 expression, which was restricted to periosteal surfaces of uninjured calvarial bone and tibia, was first detected in select regions of the fracture site 2 days post fracture and expanded during the healing process. Lineage tracing of Ddr2-expressing cells was examined using Ddr2creERT, Ai14 TdTomato mice. Ddr2 positive cells and their progeny appeared within the three days after injury and TdTomato-labeled populations greatly expanded throughout the fracture callus three weeks post-surgery indicating that most new bone cells were derived from Ddr2-expressing cells. To examine the requirement for DDR2 in MSC-mediated bone formation, cells were isolated from marrow of wildtype and Ddr2-deficient mice and subcutaneously implanted into wild type hosts. Ectopic bone formation was measured after 4 weeks and found to be reduced by 50% in Ddr2-deficient MSCs versus wild type control cells. This shows that the osteogenic activity of MSCs requires DDRS. DDR2-activating peptides promote osteoblast differentiation. Both β1 integrins and DDR2 bind fibrillar collagens, with each receptor recognizing a distinct sequence. Specific triple helical peptides binding DDR2 (GVMGFO) and 1 integrin (GFOGER) were synthesized. GVMGFO peptide strongly stimulated MSC osteoblast differentiation as measured by induction of mineralization and osteoblast marker mRNAs. GFOGER peptide was also stimulatory, but to a lesser extent than the DDR2 peptide. Most interestingly, the combination of both peptides synergistically enhanced osteoblast differentiation. Analysis of downstream signals after peptide activation indicated possible cross-talk between these two peptides via activation of DDR2 and integrin signals. Development of scaffolds containing DDR2 and integrin-activating peptides may provide a new route for promoting bone regeneraton. This study shows DDR2 is required for bone regeneration in two well-accepted bone defect models. DDR2-expressing MSCs and their progeny are involved in the bone healing process. Activation of DDR2 with specific peptides enhances osteoblast differentiation which may provide a novel tissue engineering strategy for bone regeneration.

Ge, Chunxi

University of Michigan, School of Dentistry, Department of Periodontics and Oral Medicine

University of Michigan, Orthopaedics Research Laboratory

Chondrocytes in the resting zone of the postnatal growth plate are characterized by their slow-cycling nature, which include a small population of skeletal stem cells that continue to form columnar chondrocytes and differentiate into osteoblasts and bone marrow stromal cells over time. Slow-cycling chondrocytes express parathyroid hormone-related protein (PTHrP), which constitutes the PTHrP-indian hedgehog (Ihh) negative feedback loop essential for maintaining the growth plate. However, the molecular mechanisms underlying how resting chondrocytes differentiate into proliferating, columnar chondrocytes and beyond remain unknown. Here, we utilized a genetic pulse-chase approach to isolate slow cycling chondrocytes (LRCs) from the postnatal growth plate resting zone using a chondrocyte-specific doxycycline-controllable Tet-Off system that regulates expression of histone-linked GFP. We crossed this model with a type II Collagen, chondrocyte-specific, lineage reporter mouse (hereafter Col2a1-creERtdTomato) to isolate double-positive growth plate chondrocytes that maintain low levels of mitotic capability, marked by H2B-GFP, following a 10-week chase period. Growth plate chondrocytes were isolated by microdissection and a series of collagenase washes, leaving behind the remaining ossified portions of the epiphysis. Fluorescence-activated cell sorting was utilized to isolate H2B-GFPhigh; Col2a1-creERtdTomato double-positive LRCs and to discriminate between H2B-GFPlow; Col2a1-creERtdTomato non-LRCs. An RNA-sequencing transcriptomic analysis of LRCs identified significant enrichment of Wnt inhibitors (e.g. Sfrp1/5) and resting zone chondrocyte markers (e.g. Gas1 and PTHrP) in these cells. Conversely, non-LRCs were significantly enriched for markers of further differentiated pre-hypertrophic and hypertrophic chondrocytes (e.g. Ihh and Col10a1, respectively). To define the functional roles of Wnt signaling on LRCs and their differentiation into columnar chondrocytes, we activated Wnt/ß-catenin signaling in PTHrP+ resting chondrocytes by conditionally inducing haploinsufficiency of Adenomatous polyposis coli (Apc), which is a critical component of the ß-catenin degradation complex, using a PTHrP-creER line and an Apc-floxed allele in combination with a tdTomato reporter allele allowing cell-lineage analyses. Animals were injected with tamoxifen at postnatal day 6 (P6) to induce Cre recombination and chased until P12, 21, 36 and 96. PTHrP+ resting chondrocytes and the numbers of short (composed of <10 cells) and long (composed of >10 cells) columnar chondrocytes were quantified to assess PTHrP+ skeletal stem cell formation and short/long-term self-renewal capability of these cells in a Wnt activated environment. By P12 and P21, Apc-haploinsufficient PTHrP+ resting chondrocytes were significantly reduced. Moreover, at P21 the number of PTHrP+ short, columnar, chondrocytes was significantly reduced in mutants. Interestingly, after 3 months of chase at P96, Apc-haploinsufficient PTHrP+ resting chondrocytes established long columnar chondrocytes less effectively than their controls. These data indicate that Wnt/ß-catenin activation impairs initial formation and short and long-term self-renewal of PTHrP+ skeletal stem cells in the postnatal growth plate. Taken together, these findings reveal that slow-cycling chondrocytes are maintained in a canonical Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating self-renewal and differentiation of PTHrP+ skeletal stem cells of the postnatal growth plate.

Hallett, Shawn

University of Michigan School of Dentistry

Department of Orthodontics and Pediatric Dentistry

INTRODUCTION & OBJECTIVES: There is growing interest in the potential biological role of bone marrow fat on skeletal development. Examining this relationship is particularly important for pediatric populations with bone fragility, as they often have excess bone marrow fat, which may exacerbate their bone fragility as they age. However, the field is limited as the available methods for human pediatrics often rely on tissue distribution (e.g., in vivo imaging) rather than biological interactions. Therefore, the objective of this study was to develop a new methodology to directly investigate the biological interaction of the bone marrow environment and bone development in children. To this end, we examined the effect of pediatric bone marrow-derived secretome on osteoblast cells across various conditions to identify an optimal method for future studies in the field. METHODS: Bone marrow samples from children aged 8-17 with cerebral palsy (CP (n = 2M, 4F)) and adolescent idiopathic scoliosis (AIS (n = 3M, 7F)) were collected during routine surgery. Single cell suspensions were obtained by passing the cells sequentially through a serological pipette, a syringe fitted with a 21G needle, and a 70-micron cell strainer (needle preparatory method, n = 11) or by bypassing the needle step (filter preparatory method, n = 7). Bone marrow cells either underwent red blood cell lysis or did not and were subsequently immediately plated at different densities (10x106, 25x106, 50x106, and 500x106 cells/mL) in basal culture media, where they released secretome. Equal volumes of this secretome were collected at 1, 3, and 7 days. Bone marrow secretomes were co-cultured with ST2 cells, a murine marrow derived mesenchymal stem cell line with osteogenic and adipogenic potential. MTT assays were then performed on the cultured cells, and resulting optical density (OD) values were examined relative to a control condition (ST2 cells grown in MSC growth medium) to determine the effect of the secretome on ST2 cells at different cell doses and stages of secretome development. OD values are a rough marker of cell number, and differences in OD values can be due to differences in cell survival, proliferation, and metabolism. RESULTS: The filter preparatory method showed a nearly 10-fold higher total cell yield compared to the needle method (1153±417 vs. 168±179 million cells, respectively; p<0.0001), indicating a better approach to harvesting marrow. Data also show that secretome has a negative effect on OD values, and this effect is more pronounced at higher cell densities and later stages of secretome development. For each case prepared using the filter method, secretomes developed with the 500 million cell dose elicited the most substantial reduction in MTT OD values compared to the control condition. Relative OD values for the 500 million cell dose were 0.66±0.16, 0.53±0.14 and 0.49±0.09 at days 1, 3, and 7 respectively, showing an increasing effect with further development of the secretome. Interestingly, RBC-depleted marrow did not differ significantly from whole marrow in its effects on OD values at 1 day collection, but had a less negative effect on OD values at 3 and 7 days. CONCLUSION: Our results show that bone marrow secretome derived from children with CP and AIS is biologically active and that secretome-ST2 cell interactions are influenced by time, cell dose, and presence of RBCs. The filter preparatory method should be followed in future research as it resulted in a substantially higher cell yield. Importantly, we found that secretome has a cell dose-and-time-dependent negative effect on ST2 cell OD values. Furthermore, at later timepoints, the biological potency of whole marrow-derived secretomes is stronger than those derived from RBC-depleted marrow. More work is needed to determine if cell proliferation, apoptosis, or differences in mitochondrial activity are responsible for the observed effect of secretome on osteoblasts.

Kannikeswaran, Sanjana P

University of Michigan, Dept. of Physical Medicine & Rehabilitation & University of Michigan, Dept. of Orthopaedic Surgery

Synthetic hydrogels represent an exciting avenue in the field of regenerative biomaterials given their injectability, orthogonally tunable mechanical properties, and potential for modular inclusion of cellular cues. Separately, recent advances in soluble factor release technology have demonstrated microparticle-mediated, sustained release of growth factors. A composite hydrogel incorporating both of these components could robustly mediate tendon healing with a single injection. Therefore, we sought to assess whether 3D migration of cells involved in tendon healing can be enhanced via combined mechanical, topographical, and microparticle-delivered soluble cues. Tendon stem/progenitor cells (TSPCs) and mesenchymal stem cells (MSCs) were isolated and formed into spheroids using established methods. A droplet-based microfluidic device generated hybrid dextran vinyl sulfone (DexVS) / heparin methacrylate microgels, which were loaded with platelet derived growth factor (PDGF)-BB. Composite hydrogels were fabricated by electrospinning DexVS fibers and then co-encapsulating them with spheroids in a DexVS bulk; a cell-degradable peptide crosslinker was added at 12.5 mM and 20.0 mM to achieve bulk stiffnesses of ~0.5 and 2.0 kPa. PDGF-BB enhanced 3D tendon MSC migration in DexVS and showed that fiber density can synergistically increase migration but can potentially hinder outgrowth above 2.5 vol%. Both TSPCs and tendon MSCs exhibited increased spheroid outgrowth in response to microgel-delivered PDGF-BB. Moreover, we found that these trends persist at a higher bulk matrix stiffness, albeit with an attenuated response. These studies suggest that modular, composite DexVS hydrogels meet fundamental criteria for an injectable musculoskeletal scaffold: (1) mechanical durability via fiber reinforcement over a range of bulk moduli permissive to migration and (2) programmed regulation of the soluble milieu following a single administration. Thus, this biomaterial approach could likely be generalized to a range of connective tissue injuries including skin, ligaments, and bone.

Kent, Bobby <rnkent@umich.edu>

University of Michigan

University of Illinois Chicago

Study goals: Post-traumatic osteoarthritis (PTOA) develops following joint injury and involves mechanical, inflammatory, and structural degeneration of the joint. The Wnt signaling pathway has recently gained attention in PTOA, however little is known about the underlying molecular or cellular mechanisms. Here we sought to identify the effectors and targets of Wnt signaling in a mouse model of PTOA, with a focus on the role of the secreted Wnt agonist, R-spondin 2, in the synovium. Methods: To induce joint injury and PTOA, mice were subjected to non-invasive anterior cruciate ligament (ACL) rupture, in which maximal synovitis occurs by day 7 and PTOA is established by day 28 (experiments were IACUC-approved). We utilized immunofluorescence, ELISA and RNA-seq approaches to assess expression and spatial distribution of R-spondin 2, in addition to flow cytometry to characterize R-spondin 2-expressing cell types in synovium. Cell culture and gene expression assays were used to test mechanistic crosstalk between synovial fibroblasts and macrophages. Results: RNA-seq of synovium revealed that Wnt pathway genes, including Rspo2, were induced following injury (Fig 1A-B). Flow cytometry showed that R-spondin 2 expression in the synovium was confined to non-hematopoietic (CD45-) and non-endothelial (CD31-) cells, indicative of synovial fibroblasts (Fig 1C). We also observed elevated RSPO2 protein secreted into synovial fluid 28d post-injury (Fig 1D). Intra-articular RSPO2 injection resulted in synovial lining hyperplasia (Fig 1E), suggesting an inflammatory component to the effects of R-spondin 2. Treatment of synovial fibroblasts with RSPO2 activated Wnt and inflammatory gene expression (Fig 1F). Given that macrophages are another synovial cell type, we sought to test whether inflammatory factors derived from RSPO2-treated fibroblasts could affect macrophage polarization. Conditioned media from RSPO2-treated fibroblasts was applied to bone marrow macrophages, and an M1-M2 score was generated based on expression of a pro- (M1) and anti-inflammatory (M2) gene panel (Fig 1G). These results suggest that RSPO2-activated fibroblasts can polarize macrophages towards a more inflammatory phenotype. Discussion/Conclusion: Our results show that the Wnt pathway agonist R-spondin 2 is induced in synovium following joint injury, and that synovial fibroblasts are the primary source. Elevated secreted R-spondin 2 protein in synovial fluid suggests that this protein can act in a paracrine manner to activate Wnt signaling throughout the synovium and joint space during PTOA. R-spondin 2 delivery induced mild synovial hyperplasia, indicating possible inflammatory effects of pathological synovial Wnt signaling, that we hypothesize constitutes a fibroblast-macrophage signaling axis.

Knights, Alexander <aknights@umich.edu>

University of Michigan Orthopaedic Research Laboratories

In 2014, NIH issued guidelines to ensure the inclusion of both sexes in preclinical research [1]. However, the success of these policies is mostly unknown. A recent study found that less than 50% of biomechanical studies had balanced sex distribution in their samples [2]. We opted to investigate a more specific field in our current study: finite element (FE) analysis in hip arthroplasty. Hip fracture is nearly twice as common in women compared to men in the US [3], as are total hip arthroplasty (THA) surgeries. A common method for comparing the viability of THA implants is FE modeling because of accessibility, relatively low-cost, and subject-specific insights when modeled accordingly. In the current study, we asked if and how FE modeling has been systematically used for comparing sex differences in FE modeling of THA implants.

Lamia, Syeda Noor E

(1) Department of Orthopaedic Surgery, University of Michigan, Ann Arbor, Michigan (2) Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan (3) Department of Biomedical Engineering, University of Texas, Austin, Texas

Study Goals: Traumatic or surgical loss of a critical muscle volume (known as volumetric muscle loss, or VML) is responsible for over 90% of muscle conditions that lead to long-term disability and presents a considerable financial burden. Unfortunately, regenerative strategies have so far failed to recover more than 16% of functional deficits. While persistent inflammation has been shown to overwhelm the endogenous repair mechanisms of muscle stem cells (MuSCs), inadequate understanding of which signaling networks drive the fibrotic phenotype preclude the development of successful regenerative therapies. Thus, we sought to characterize the cellular and molecular mechanisms driving fibrotic degeneration of skeletal muscle after VML by comparing the healing trajectories between muscle loss injuries that regenerate to those that result in fibrosis.

Methods: We administered bilateral full thickness 2mm (5% of muscle mass, healing) or 3mm (15% of muscle mass, non-healing) punch defects to the rectus femoris of 3-4-month-old wild type mice as previously published. To probe cell responses to healing and non-healing defects, we performed droplet-based single cell RNA sequencing (scRNA-Seq) on mononucleated cells at 0-, 7-, 14-, and 28-days post injury. Seurat and NicheNet were used to analyze the data and predict dysregulated cell-cell communication networks, respectively. Flow cytometry was performed to validate time-courses of cell infiltration, immunohistochemistry was used to co-localize cells predicted to be communicating, in vivo cell transplants and small molecule inhibition were used to validate predicted signaling networks, myoblast culture in neutrophil-conditioned media was used to assess the impact of the neutrophil secretome on fusion, and histological and muscle function tests were performed to evaluate improvements in regeneration.

Results: We observed and validated that non-healing VML injuries result in persistent infiltration of inflammatory cells, including neutrophils, which exert lasting consequences on myogenic capacity of resident MuSCs. Moreover, we identified and characterized an inter-cellular communication circuit between neutrophils and cytolytic natural killer (NK) cells, which combat neutrophil accumulation via a chemokine ligand 5 (CCL5) - chemokine receptor type 1 (CCR1) axis. Small molecule inhibition of CCR1 exacerbated neutrophil accumulation in degenerative defects, while NK transplants significantly reduced neutrophil populations and enhanced healing. Finally, prediction of inter-cellular communication networks from scRNA-Seq data suggested elevated transforming growth factor beta 1 (TGFb) levels contribute to neutrophil persistence and confer MuSC fusion impairments. In accordance, small molecule inhibition of TGFb1 signaling in vivo reduced neutrophil abundance and improved tissue morphology and specific maximal tetanic force at late timepoints following non-healing VML injuries.

Discussion: These findings enhance our understanding of cellular communication dynamics governing muscle healing outcomes. We envision this work not only provides a valuable resource for further exploration into mechanisms driving VML-induced fibrosis, but also helps elucidate drivers of fibrosis and chronic inflammation in other pathologies, including cirrhosis, myocardial infarction, and pulmonary fibrosis.

Larouch, Jacqueline Adelaide

University of Michigan, Department of Biomedical Engineering

GOAL: Demonstrate that bone ridge enlargement in Fgf9null embryos is driven by muscle-to-bone signaling.

METHODS All experiments were approved by University of Delaware IACUC. Fgf9WT/Ex; Prrx1Cre-positive dams were bred with Fgf9flx/flx males to generate Fgf9null global mutants and Fgf9WT/Ex (WT) neonates. For muscle-specific knockouts, Fgf9flx/WT; Acta1-Cre mice were crossed with Fgf9flx/flx mice and dams were given doxycycline chow throughout gestation and weaning. Fgf9null embryos were harvested at postnatal day 0 (P0) and quantitative measurements of the humeri and DT area were performed using whole-mount staining. For bulk RNA-sequencing experiments, skeletal muscle and humeri were carefully micro-dissected from E18.5 embryos (n=3/genotype) and RNA was isolated from each tissue using cold tissue pulverization (Retsch MM400) and an RNeasy kit (Qiagen). Total RNA from muscle and bone were sequenced in Illumina HiSeq 2000 (Paired-end 2*50bp; threshold length=30bp; Phred score=28) following library preparation (NEBNext® UltraTM RNA Library Prep kit, Illumina). RNA quality was assessed before and after sequencing (using fragment analysis and FastQC, respectively) and mapped to the mouse reference genome Grcm38 (mm10), and quantified (HT-Seq). Sample pool was uploaded to DAVID Bioinformatics Resources 6.8 and run against a Mus musculus background. GOTERM libraries were examined, and terms were ranked based on Benjamini number (DAVID p value). Genes in terms of interest were plotted using Prism (GraphPad). MicroCT was used to obtain 3D renderings of the left humerus of 8-week old mice to visualize topographical data on DT volume. MicroCT scans were performed using a Scanco µCT 35 (X-ray energy: 60kV; X-ray intensity: 95mA; integration time: 1100msec; voxel size: 10.6 cubic microns; rotation step: 0.4º). All raw scans were reconstructed and analyzed using Dragonfly software (Object Research Systems). DT bone volume was selected based on voxel value and filled in using bone analysis to obtain total volume. Statistical analyses were performed using Prism. Student’s t-tests were used for DT size.

RESULTS Fgf9null neonates had larger DTs (Fig 1A,B) than WT neonates at P0. There were 805 differentially expressed genes that correlated with Mitochondria (Fig 1C; GO:0006631, GO:0008152, GO:0055114, GO:0005747, GO:0016491, GO:0070469, GO:0005743), Innervation (Fig 1D; GO:0007411, GO:0048485), Wnt (Fig 1E; GO:0016055, GO:0005109), and Interleukin (Fig 1F; GO:0005152) terms. At 8-weeks, Fgf9cKO had larger DTs (Fig 1G-H) than WT mice.

DISCUSSION Our findings elucidate the role that Fgf9 plays during the development and growth of the DT. This bone ridge regulating role of Fgf9 seems to be driven by muscle as the majority of gene expression difference between Fgf9null and WT embryos was in muscle. Additionally, muscle-specific knockouts of Fgf9 had larger DTs than WT mice.

Leek, Connor

University of Michigan

University of Delaware

Incidences of low-trauma fractures among osteopenic women may be related to changes in bone quality. In this blinded, prospective-controlled study, compositional and heterogeneity contributors of bone quality to fracture risk were examined. We hypothesize that Raman spectroscopy can differentiate between osteopenic women with one or more fractures (cases) from women without fractures (controls). This study involved the Raman spectroscopic analysis of cortical and cancellous bone composition using iliac crest biopsies obtained from 59-cases and 59-controls, matched for age (62.0 ± 7.5 and 61.7 ± 7.3 years, respectively, p = 0.38) and hip bone mineral density (BMD, 0.827 ± 0.083 and 0.823 ± 0.072 g/cm3, respectively, p = 0.57). Based on aggregate univariate case-control and odds ratio based logistic regression analyses, we discovered two Raman ratiometric parameters that were predictive of past fracture risk. Specifically, 1244/1268 and 1044/959 cm−1 ratios, were identified as the most differential aspects of bone quality in cortical cases with odds ratios of 0.617 (0.406–0.938 95% CI, p = 0.024) and 1.656 (1.083–2.534 95% CI, p = 0.020), respectively. Both 1244/1268 and 1044/959 cm−1 ratios exhibited moderate sensitivity (59.3–64.4%) but low specificity (49.2–52.5%). These results suggest that the organization of mineralized collagen fibrils were significantly altered in cortical cases compared to controls. In contrast, compositional and heterogeneity parameters related to mineral/matrix ratios, B-type carbonate substitutions, and mineral crystallinity, were not significantly different between cases and controls. In conclusion, a key outcome of this study is the significant odds ratios obtained for two Raman parameters (1244/1268 and 1044/959 cm−1 ratios), which from a diagnostic perspective, may assist in the screening of osteopenic women with suspected low-trauma fractures. One important implication of these findings includes considering the possibility that changes in the organization of collagen compositional structure plays a far greater role in postmenopausal women with osteopenic fractures.

Zoom Passcode: 381557

Mandair, Gurjit Singh

School of Dentistry, University of Michigan

Introduction/Study Goals: Soft tissue repair and reconstruction commonly utilize the Krackow stitch and commercially designed looped whipstitch techniques, and both have been biomechanically evaluated. Perpendicular multi-planar fixation may improve the biomechanical properties compared to the commonly used techniques, as has been demonstrated with fracture fixation. The purpose of this study was to compare the elongation, yield load, ultimate failure, stiffness, and mode of failure of the traditional Krackow stitch, standard looped whipstitch, and a novel, multi-planar perpendicular looped whipstitch technique. The hypothesis was that the multi-planar perpendicular technique would demonstrate superior biomechanical properties over the standard techniques.

Methods: Thirty tibialis anterior cadaveric tendons were randomly assigned into 3 groups of 10. Three suturing techniques: the Krackow stitch (KS), standard commercial looped whipstitch (WS), and a novel, multi-planar perpendicular looped whipstitch (MP) were performed. The MP stitch was performed with orthogonal throws starting right-to-left, then front-to-back, left-to-right, and back-to-front. Each technique used 4 passes of Number 2 FiberWire spaced 5mm apart and ending 10mm from the end of the tendon. Tendons were secured to a custom clamp and the other end was sutured. Tendons were pre-loaded to 5N, pre-tensioned to 50N at 100mm/min for 3 cycles, returned to 5N for 1 minute, cycled from 5N to 100N at 200mm/min for 100 cycles, and then loaded to failure at 20mm/min. Elongation measurements were recorded as displacement after pre-tensioning and cycling, and measured across the suture-tendon interface, and from the base of the suture-tendon interface to markings on the suture limbs (construct elongation). One-way analyses of variance were performed, with Bonferroni post hoc analysis when appropriate.

Results: There were no differences in cross-sectional area or stiffness among the 3 techniques (p>.05). The ultimate load for WS (183.33±57.44N) was lower compared to both MP (270.76±39.36N) and KS (298.90±25.94N) (p≤.001). The yield load for KS was greater than WS (186.32±32.66 vs 147.60±31.93, p=0.019). There was less total construct elongation for KS (5.96±0.86) compared to WS (20.54±8.24) and MP (24.27±6.16) for total displacement measured from pre-tensioning to the end of cycling (p<.001). All 3 techniques were noted to have a decrease in length at the suture-tendon interface during testing, termed compression. There was more compression at the suture-tendon interface for WS compared to KS (p=.006). All 10 KS and 6 of 10 MP failed by suture breakage whereas all 10 WS failed by suture pull-out through the tendon.

Discussion/Conclusion: The Krackow stitch maximizes strength, while minimizing suture pull through, construct elongation, or graft compression. If using the looped commercial whipstitch for ease of use, the multi-planar perpendicular technique offers improved biomechanical properties over the standard technique and should be utilized.

Passcode: 220080

Muscatelli, Stefano

1University of Michigan Health System, Ann Arbor, MI 2Eastern Kentucky University, Richmond, KY 3Ann Arbor VA Healthcare System, Ann Arbor, MI 4Stanford Health Care, Palo Alto, CA

Study goals: The jawbone is a unique structure and plays an important role in mastication. Given the fact that the jawbone is remodeled faster than other skeletal bones, bone cells in the jawbone may respond differently to local and systemic cues to regulate bone remodeling. Bone morphogenetic protein (BMP) signaling plays critical roles in skeletal development and bone homeostasis. Our previous studies showed that loss of function of Bmpr1a, which encodes the type 1A receptor for BMPs, in osteoblasts results in increased trabecular bone mass in long bones due to an imbalance between bone resorption and formation. Here, we investigated the impacts of BMP signaling through BMPR1A in osteoblasts on the mandibular alveolar bone homeostasis to understand distinctive features of jawbone remodeling.

Methods: We conditionally deleted Bmpr1a in Osx-expressing cells to assess the function of BMPR1A in osteoblasts. Tibiae and mandibles were collected to perform micro-CT, histological and gene expression analyses at 12-week of age.

Results: As mentioned, bone mass in the long bones of the Bmpr1a cKO mice were higher than control mice, however, that in the mandibular alveolar bones were lower in the cKO mice (Fig. 1A). The number of TRAP-positive cells and expression of Trap, Ctsk and Mmp9 in the long bones were higher in the cKO mice, while those in the mandibular alveolar bones were lower in the cKO mice. Interestingly, expression of Rankl was higher in the cKO mandibles contrasting to its lower expression in the long bones (Fig. 1B), indicating a possible reason for higher osteoclast activities in the jawbone.

Conclusion: In sum, the Bmpr1a cKO mice display opposite bone phenotypes in long bones and jawbones. Loss of BMP signaling in osteoblasts leads to reduced bone resorption in long bones, resulting in more bone mass, while loss of BMP signaling promotes bone resorption in jawbones, resulting in lower bone mass (Fig. 2). These results emphasize that BMP signaling plays distinct roles in maintaining bone mass, depending on anatomical locations of bones.

Omi, Maiko

University of Michigan School of Dentistry, Department of Biologic and Materials Sciences & Prosthodontics

Goals: Bone marrow adipose tissue (BMAT), located inside the bone marrow cavity, is unique from white adipose tissue (WAT). Unlike WAT depots, BMAT expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unknown, our prior data suggests there may be an intermediary role for increased circulating glucocorticoids. Therefore, we designed a series of experiments to test the hypothesis that glucocorticoids regulate BMAT volume and functions following CR. Methods: To test this hypothesis, we generated a novel mouse, the Flippase-Activated Cre (FAC) model, which has Cre recombinase exclusively expressed in bone marrow adipocyte (BMAd), to selectively eliminate the glucocorticoid receptor (GR) (gene name nr3c1) from bone marrow adipocytes (BMAds) whilst maintaining GR expression in other WAT depots. Mice lacking GR in bone marrow adipocytes (FAC-nr3c1) and control mice (WT) (male, 30-42 weeks old) were placed on a CR diet with 30% caloric reduction or fed ad libitum for six weeks. Results: Both WT and FAC-nr3c1 mice showed increased circulating glucocorticoid concentrations following CR. As expected, CR caused a decrease in weight of WAT depots, liver and spleen. Ct analysis of tibiae showed no significant differences in trabecular and cortical bone in WT or FAC-nr3c1 mice. Histological sections demonstrated that BMAT was increased in both the femur and tibia of mice on CR compared to WT mice fed ad lib. However, osmium tetroxide labeling combined with Ct analysis to quantify BMAd volume showed no difference in BMAT accumulation between WT or FAC-nr3c1 mice. Quantification of adipocyte size demonstrated a small shift towards larger adipocytes within the marrow of FAC-nr3c1 mice compared to WT controls. An additional cohort of female WT or FAC-nr3c1 mice (22-32 weeks old) were placed on a CR diet with 30% caloric reduction or fed ad libitum for six weeks and showed similar results to the male cohort previously described. A complete blood count on the female cohort demonstrated no differences in red blood cells, white blood cells, platelets, hemoglobin or hematocrit levels between WT or FAC-nr3c1 mice. Conclusions: Although CR caused elevated circulating glucocorticoids, deletion of GR in BMAT did not alter glucocorticoid concentrations, adipose tissue, spleen, or liver mass, or bone volumes or mineral density at baseline or following CR. In addition, BMAT volume of WT and FAC-nr3c1 mice was similar, as assessed in H&E-stained bone sections or by osmium tetroxide labeling. In conclusion, our data suggests that eliminating GR from BMAd does not impact BMAT accumulation during CR. We are currently investigating whether, in the absence of GR, BMAd upregulate another receptor capable of recognizing glucocorticoids, the mineralocorticoid receptor (gene name nr3c2), or if CR alters local glucocorticoid levels within the marrow by altering the levels of the two enzymes responsible for controlling glucocorticoid activation, 11-βHSD1 and 11-βHSD2.

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Schill, Rebecca Lynn

University of Michigan

Study Goals: Osteogenesis Imperfecta (OI) is a genetic dysplasia characterized by low bone density. A novel strategy for OI treatment is Sclerostin Antibody (SclAb), which induces a strong anabolic response to increase bone mass. Recent studies have shown that systemic delivery of SclAb leads to acceleration of the peri-implant bone formation rate and increases pull-out strength of implants on normally loaded and unloaded long bones. If similar findings were to be observed in the maxilla of OI bone, they might lead to a new strategy to improve the craniofacial peri-implant osseointegration in OI. Clinically, dental implants among OI patients are usually avoided based on the assumption that poor bone quality inherent to OI may have detrimental effects on maxillary peri-implant osseointegration. We propose that systemic delivery of SclAb will improve the peri-implant osseointegration of both bone types, maxilla and tibia, in an OI mouse model. Methods: 3 month-old female WT and Brtl/+ mice were randomly assigned to the following 4 experimental groups 1) SclAb-implant-WT, 2) SclAb- implant-Brtl/+; 3) Vehicle-implant-WT, 4) Vehicle-implant-Brtl/+. Under general anesthesia, titanium implants were placed in the maxilla and tibia, concomitantly and randomly the mice received subcutaneous SclAb (SclAb VI, Amgen) treatment or vehicle injection (PBS) at 25 mg/kg, 2X/week, for 5 weeks. The peri-implant bone formation was assessed with microCT (xRadia) imaging at 7µm voxel size and analyzed with deep learning segmentation (Dragonfly). Results: SclAb significantly increases this BV/TV in Brtl/+ (20%). Tibial peri-implant osseointegration is more robust compared to the maxilla in both genotypes. SclAb increases the bone-implant contact (BIC) in Brtl/+ 60% (tibia) and 40% (maxilla) vs non-treated Brtl/+ (Figure 2). Discussion/Conclusion: The Brtl/+ mouse recapitulates the craniofacial features of the OI patient. Several investigators use the mouse tibia as a model for oral implant osseointegration, assuming both sites--maxillae and long-bones--have the same healing patterns. However, we found more robust peri-implant osseointegration in OI long bones compared to the maxilla. These findings are similar to results observed in CD1 mice (Mouraret et al, 2014) and as reported previously, the tibia has more marrow space containing abundant numbers of osteoprogenitor cells and stem cell niche signals, while maxillae have little or no marrow space (Leucht et al, 2007). Therefore, the results from an implant placed in OI mouse long-bones cannot be extrapolated to the maxilla. Despite the low bone density in Brtl/+ long bones, the tibial implant was able to induce peri-implant osseointegration with increased BIC. In contrast, non-treated Brtl/+ maxilla has a decreased BIC. SclAb is able to increase the peri-implant bone formation in Brtl and WT, increasing the BIC. Our findings suggest the combination of SclAb/implant could work as a potential clinical alternative to address patients’ edentulism.

zoom passcode: 757956

Sung, Hsiao

University of Michigan

Radboudumc

Study Goals/Purpose Disuse osteopenia develops in individuals with decrease lower limb use especially after injury necessitating non-weightbearing, hospitalization or both. However, the nature of this change is not well characterized and, therefore, cannot be appropriately addressed. Conventional CT produces a reliable evaluation of bone quality as Hounsfield Units (HU) and offers the opportunity to longitudinally study bone density in activity-altered patients. The purpose of this study is to estimate the degree of osteopenia in non-weightbearing hospitalized patients and to identify patient related factors associated with this loss.

Methods Initial screening criteria included patients who presented to the emergency department between January 1, 2010 to August 1, 2020; baseline lower extremity orthopaedic trauma evaluated by CT. Further criteria included CT imaging capturing the ipsilateral hip at the time of admission; a second CT scan, for any indication (eg, small bowel obstruction, diverticulitis, etc) that include that same hip; strict non-weightbearing in between scans. Exclusion criteria included change in weightbearing status and CT imaging changes that would not allow eBMD analysis (eg, intervention). Estimated BMD (eBMD) was measured at baseline and and at the secondary time point via HU approximation 12mm inferior to superior aspect of femoral neck on axial CT. The change in eBMD was calculated as a proxy for disuse osteopenia and the relationship between relative bone loss (as a nominal rate of osteopenia, eBMD loss/time) and patient specific factors was explored.

Results Of 200,000 patients who received CT evaluation including their hip during the targeted timeframe, seventeen patients met our inclusion and exclusion criteria. There was an average of 7.3 days (median = 6 days; mode = 4 days; interquartile range: 4 – 8.5 days) between CT scans. The average rate of disuse osteopenia in the cohort was a net average decrease of 7.5 ± 5.8 HU/day. To place in a standardized clinical context, this change is estimated to correlate linearly with respect to change in DEXA BMD T-Score and may be clinically relevant when HU approach values <100. Associated patient factors that may further precipitate osteopenia whilst non-weightbearing are smoking, female gender, and diabetes.

Conclusions We report novel, preliminary data estimating the rate of disuse osteopenia in admitted, non-weightbearing orthopaedic trauma patients. This data underscores an important consideration all physicians should make when non-weightbearing status is being considered or required after injury or admission.

Walley, Kempland C.

Department of Orthopaedic Surgery, University of Michigan, Ann Arbor, MI, USA

STUDY GOALS: Skeletal muscle tissue engineering technologies have the potential to treat volumetric muscle loss (VML) by growing exogenous muscle tissue. However, there has been limited success in engineering human cell-sourced skeletal muscle with structure and function comparable to native adult human muscle. The mitogenic protein human epidermal growth factor (hEGF) is of interest due to its ability to enhance proliferation and sarcomeric structure formation in myogenic cell cultures. In this study, we used our scaffold-free tissue-engineered skeletal muscle units (SMUs) to examine the effects of hEGF on the structure and function of human cell-sourced engineered skeletal muscle.

METHODS: Using our established SMU fabrication process, human muscle cell isolates were exposed to media treated with 7.5 nM hEGF at three different time spans during the 21 day cell culture period: 0 to 6 days post-seeding (hEGF-treated Muscle Growth Media (MGM) Only), 7 to 21 days post-seeding (hEGF-treated Muscle Differentiation Media (MDM) Only), and 0 to 21 days post-seeding (hEGF-treated MGM+MDM). Control cell cultures were fed standard MGM and MDM (no hEGF treatment). During the fabrication process, light microscopy was used to examine proliferation and differentiation of myogenic cells in monolayer. 14 days post-seeding, the cell monolayers were manually delaminated and pinned into 3 cm long 3D cylindrical SMUs. After SMU formation, the 3-D constructs underwent maximum tetanic isometric force (Fo) measurements to evaluate contractile function and immunohistochemical staining to examine SMU composition and cross-sectional area (CSA). Differences between experimental groups were assessed using a one-way ANOVA with Tukey’s multiple comparisons test. Significance was established at p < 0.05. Numerical results are listed as mean ± standard error of the mean.

RESULTS: hEGF administration significantly impacted myogenesis (n=15 per experimental group, p < 0.01), increasing myotube diameter in hEGF-treated MGM only and hEGF-treated MDM only cell cultures and increasing myotube density in hEGF-treated MGM+MDM cultures. SMUs exposed to hEGF-treated MDM and hEGF-treated MGM+MDM exhibited greater whole SMU CSAs and more organized sarcomeric structure. The exposure of myogenic cell cultures to hEGF during any timespan also led to a significant increase in SMU myosin heavy chain content (n=5 per experimental group, p = 0.002). Mean myosin heavy chain CSAs for hEGF-treated MGM and MGM+MDM SMUs were over two times greater than the untreated control mean CSA of 0.070± 0.01 mm2. Average isometric tetanic forces were 2.6 ± 1.3 μN, 10.4 ± 1.2 μN, 54.2 ± 6.7 μN, and 90.4 ± 7.6 μN for control, hEGF-treated MGM only, hEGF-treated MDM only, and hEGF-treated MGM+MDM groups respectively (n=15 per experimental group). When compared to control, SMUs in hEGF-treated MDM and hEGF-treated MGM+MDM groups showed a 20x and 35x increase in average Fo respectively (p < 0.0001 for both).

DISCUSSION: Our data suggests that hEGF starting time point and duration of treatment play a significant role in SMU contractile function. The structural development induced by hEGF in 2D monolayers and 3D SMUs contributed to greater SMU force production, most notably in hEGF-treated MGM+MDM SMUs. Using our scaffold-free tissue engineering model, we demonstrated that the supplementation of hEGF to human primary myogenic cell cultures improved tissue engineered skeletal muscle structural and functional characteristics with the most promising advancements occurring during sustained administration of 7.5 nM hEGF during myogenic cell proliferation, differentiation, and maturation phases. Our results suggest that hEGF serves as a critical growth factor in advancing human skeletal muscle engineered tissue models and developing skeletal muscle with adult phenotypes. Since this study utilized freshly isolated muscle progenitor cells from human skeletal muscle, further research needs to be conducted to determine whether hEGF has a similar impact on different skeletal muscle cell lines and cells sourced from other species. Passcode for Video Chat: 616443

Wroblewski, Olga Maria

University of Michigan (Ann Arbor), Department of Biomedical Engineering, Department of Molecular and Integrative Physiology, Department of Plastic Surgery

Study Goals: Exposure to environmental teratogenic pollutant leads to severe birth defects. However, the biological events underlying these developmental abnormalities remain undefined. Here, we report a molecular link between an environmental stress response pathway and key developmental signaling pathway during craniofacial development. Methods: We constitutively activated type I BMP receptor, Acvr1 or Bmpr1a, in neural crest cells using P0-Cre. Immunostaining was used to determine changes of AhR signaling, cell proliferation and cell death in facial primordia. Pregnant females were treated with activator or inhibitor of AhR signaling to identify its involvement in facial defects caused in enhanced BMP signaling. Results: We found mutant mice with constitutively activated Acvr1 in neural crest cells (ca-ACVR1) displayed severe craniofacial skeletal defects, including facial cleft, and maxilla and mandible hypoplasia. We identified that these defects were associated with an upregulation of the signaling pathway mediated by Aryl hydrocarbon receptor (AhR), a receptor to environmental toxicants. Activation of AhR signaling in ca-ACVR1 mutants further suppressed growth and migration of facial mesenchymal cells. Accordingly, inhibiting AhR activity rescued growth defects of these cells and the facial skeleton formation of ca-ACVR1 mutants, revealing an interaction between BMP and AhR signaling. Importantly, using another transgenic mouse line with constitutively activated type I BMP receptor Bmpr1a in neural crest cells (ca-BMPR1A), which do not show facial clefts without treatment, we identified that suboptimal activation of AhR signaling caused facial clefts and skeleton defects in ca-BMPR1A mutants but not in control littermates, mimicking the phenotype of ca-ACVR1 mutants. Conclusion: Together, our findings demonstrate that BMP signaling functions as a risk factor for AhR-mediated teratogenesis during craniofacial development.

Yang, Jingwen

Department of Biologic and Materials Sciences & Prosthodontics, School of Dentistry, University of Michigan

The temporomandibular joint (TMJ) articulates the mandible with the skull and is the most frequently used joint in the body. TMJ plays essential roles in chewing, drinking, suckling, licking, swallowing, breathing, speaking, and yawning. Because of its critical life-sustaining role, proper TMJ function is essential. The temporomandibular joint disorders (TMJD) are a set of disorders characterized by pain, dysfunction and structural changes in the masticatory muscles and in the TMJ. It is estimated that 2-16% of the population suffer from TMJD, with a 4.6% prevalence in the United States. In the past decades, advances have been put to understand the pathophysiology of muscle pains in TMJD and the osteoarthritis in TMJ. However, up to today, the actual etiology of TMJD still remains to be elusive. This is partially due to most of the available rodent models develop TMJD through invasive procedures and lack of models that naturally develop TMJD.

EVC2 is one the causative genes of Ellis van Creveld syndrome (OMIM#225500). Previous studies from us and others demonstrated that EVC2 intracellularly locate to the bottom of primary cilium. Evc2 loss of function leads to compromised response to Hedgehog ligand. Strikingly, in Evc2 mutant mice, we observed abnormally developed cartilage at the posterior part of the TMJ at postnatal day 21 (P21). The same type of abnormalities in TMJ structure can be traced back to as early as P8 during development, but not in P2. Studies using mice with conditionally deleted Evc2 suggest that Evc2 plays a critical role in chondrocyte progenitor cells rather than in chondrocyte cells to ensure a correctly developed TMJ. Additionally, associated with abnormal TMJ structure, we observed abnormal TMJ functions in Evc2 mutant mice. We observed decreased chewing rate, decreased vertical displacement, increased chewing variation and decreased biting rate in Evc2 mutant mice. Through tracking jaw movement, we observed that mutant mice have difficulty in opening jaw during chewing. Finally, we observed spontaneous pain in Evc2 mutant mice.

Since spontaneous pain, decreased biting force, decreased chewing rate, decreased vertical displacement and increased chewing variation are typical signs in TMJD, our studies demonstrate that Evc2 mutant mice are a novel model for studies to understand the pathophysiological mechanism leading to TMJD.

Zhang, Honghao

Department of Biologic & Materials Sciences & Prosthodontics, School of Dentistry, University of Michigan

Department of Molecular, Cellular and Developmental Biology, University of Michigan; Department of Anatomy, Heritage College of Osteopathic Medicine, Ohio University