Nancy Troiano

2000 Young Investigator Research Award winner. Evaluation of OP-1 as a graft substitute for intertransverse process lumbar fusion.

Study Design. An established rabbit intertransverse process lumbar fusion model was used to evaluate osteogenic protein (OP)-1 as a potential graft substitute. Objectives. To determine whether OP-1 is effective in producing intertransverse process lumbar fusion in a rabbit model. Summary of Background Data. Autogenous iliac crest bone is the gold standard in grafting material for inducing intertransverse process fusion. However, bone graft substitutes are being considered as supplementary or alternative means to achieve such fusion with less morbidity. Relatively little research has been undertaken to investigate the efficacy of OP-1 in this role. Methods. Single-level intertransverse process lumbar fusions were performed at L5–L6 of 31 New Zealand White rabbits. These were divided into three study groups: autograft, carrier alone, and carrier with OP-1. The animals were killed 5 weeks after surgery. Resultant fusion masses were evaluated by manual palpation, radiography, biomechanical multidirectional flexibility testing, and histology. Results. Seven rabbits (23%) were excluded because of complications. Of the remaining 24 rabbits, 5 (63%) of the 8 in the autograft group had fusion detected by manual palpation, none (0%) of the 8 in the carrier-alone group had fusion, and all 8 (100%) in the OP-1 group had fusion. Radiographs were 55% sensitive and 92% specific for determining fusion. Biomechanical testing results correlated well with those of manual palpation. Histologically, autograft specimens were predominantly fibrocartilage, OP-1 specimens were predominantly maturing bone, and carrier-alone specimens did not show significant bone formation. Conclusions. OP-1 was found to reliably induce solid intertransverse process fusion in a rabbit model at 5 weeks.

Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2

Mice deficient in GATA‐1 or NF‐E2 have a 200–300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6‐fold when cultured with megakaryocytes. A megakaryocyte‐osteoblast interaction plays a role in the increased bone formation in these mice.

Osteogenic protein-1 overcomes the inhibitory effect of nicotine on posterolateral lumbar fusion.

Study Design. An established rabbit posterolateral lumbar fusion model was used to evaluate the ability of osteogenic protein-1 to overcome the inhibitory effect of nicotine. Objective. To determine whether osteogenic protein-1 should be considered as a bone graft alternative for the patient who smokes. Summary of Background Data. Smoking interferes with the success of posterolateral lumbar fusion. This inhibitory effect has been attributed to nicotine and confirmed in a New Zealand white rabbit model. Osteoinductive protein-1 has been shown to induce posterolateral spine fusion reliably in the rabbit model. The effectiveness with which osteogenic protein-1 induces fusion in the presence of nicotine has not been studied previously. Methods. Single-level posterolateral intertransverse process fusions were performed at L5–L6 in 18 New Zealand white rabbits. Either autograft or osteogenic protein-1 was used as grafting material. Nicotine was administered via subcutaneous mini-osmotic pumps. The animals were killed 5 weeks after surgery, and the resulting fusion masses were studied. Results. Three rabbits (17%) were excluded because of complications. By manual palpation, two of the eight nicotine-exposed autograft rabbits (25%) and all of the nicotine-exposed osteogenic protein-1 rabbits (100%) were found to be fused.. These results correlated well with those obtained from biomechanical testing. Histologically, the fusion zones of the nicotine-exposed autograft rabbits were distinctly less mature than the fusion masses of the nicotine-exposed osteogenic protein-1 rabbits. Conclusion. Osteoinductive protein-1 was able to overcome the inhibitory effects of nicotine in a rabbit posterolateral spine fusion model, and to induce bony fusion reliably at 5 weeks.

Effects of calcitonin gene-related peptide on bone turnover in ovariectomized rats

Calcitonin gene-related peptide (CGRP) is a neuropeptide abundantly concentrated in sensory nerve endings innervating bone metaphysis and periosteum, which indicates that it plays a local role in bone metabolism. CGRP-alpha and -beta share structural and functional homology with calcitonin (CT) and have been shown to inhibit bone resorption in vitro and to induce hypocalcemia in vivo. We recently reported that CGRP stimulates the production of the growth factor insulin-like growth factor-I and inhibits that of the cytokine tumor necrosis factor-alpha by osteoblasts, suggesting that CGRP may control bone cell activity. To investigate this possibility, we used ovariectomized (ovx) rats as a high bone turnover model and compared the effects of CGRP to those of CT. ovx young female rats were injected daily starting the day after surgery with either phosphate-buffered saline, CGRP-alpha (1.15 mg/kg per day), or CT (3 micrograms/kg per day) for 28 days. Ovariectomy induced an increase in bone turnover associated with a 60% loss in trabecular bone volume of the proximal tibia. CGRP inhibited bone resorption but not bone formation, and was nevertheless less efficient than CT in preventing bone loss, since CGRP-treated rats had a loss of 46% of cancellous bone, whereas CT-treated rats had a loss of 21%. This suggests that CGRP is either less potent than CT at inhibiting bone resorption or else very rapidly degraded. These data indicate that CGRP can control bone cells through a mechanism that is in part different from that of CT, and further suggest that CGRP may play a local role in bone metabolism.

Use of osmium tetroxide staining with microcomputerized tomography to visualize and quantify bone marrow adipose tissue in vivo.

Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.

Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content

Pigment epithelium‐derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF‐directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator‐activated receptor‐γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control‐treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt‐β‐catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt‐β‐catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.—Gattu, A. K., Swenson, E. S., Iwakiri, Y., Samuel, V. T., Troiano, N., Berry, R., Church, C. D., Rodeheffer, M. S., Carpenter, T. O., Chung, C. Determination of mesenchymal stem cell fate by pigment epithelium‐derived factor (PEDF) results in increased adiposity and reduced bone mineral content. FASEB J. 27, 4384–4394 (2013). www.fasebj.org

Development of a femoral non-union model in the mouse

OBJECTIVES Advancements in our knowledge of fracture healing have occurred in large part by the understanding of this process on a microscopic level. The ability to develop experimental non-union models in animals will assist in the investigation of this problem and are likely to lead to novel treatments. We report on a technique for developing experimental non-unions in mice. METHODS Femoral fractures were created in 48 CD1 mice, 24 mice underwent standard closed femoral fractures, and 24 mice underwent creation of a femoral non-union through an open osteotomy and fracture devascularisation method. All fractures were subsequently rodded. Histological examinations of the fractures were then conducted at eight time points post-operatively. RESULTS The control group showed normal fracture healing with histological evidence of bony fracture bridging by 28 days and mature bony remodelling at 63 days. The non-union group showed delayed fracture healing at all time points and no evidence of bony healing at 63 days. CONCLUSION This is the first report of a reliable method to develop fracture non-union in mice. We believe this technique will be critical to further the investigation of fracture non-union in normal mice and provides the great advantage of using the plethora of transgenic and knockout mouse models to analyse non-union at the cell and molecular level.

Increased bone volume and correction of HYP mouse hypophosphatemia in the Klotho/HYP mouse.

Inactivating mutations of PHEX cause X-linked hypophosphatemia and result in increased circulating fibroblast growth factor 23 (FGF23). FGF23 action is dependent upon Klotho, which converts FGF receptor 1 into an FGF23-specific receptor. Disruption of Klotho results in a complex bone phenotype and hyperphosphatemia, the converse phenotype of X-linked hypophosphatemia. We examined effects of disrupting both Klotho and PHEX by creating a double-knockout (Klotho/HYP) mouse. The combined disruption corrected the hypophosphatemia in HYP mice, indicating that Klotho is epistatic to PHEX. FGF23 levels remained elevated in all groups except wild-type, indicating that Klotho is necessary for FGF23-dependent phosphaturic activity. 1,25-Dihydroxyvitamin D levels, reduced in HYP mice, were comparably elevated in Klotho and Klotho/HYP mice, demonstrating that Klotho is necessary for FGF23s effect on vitamin D metabolism. Serum PTH levels were reduced in both Klotho and Klotho/HYP mice. Moreover, the Klotho null phenotype persisted in Klotho/HYP, maintaining the runty phenotype and decreased life span of Klotho null mice. Notably, microcomputed tomography analysis demonstrated greater trabecular bone volume fraction in Klotho/HYP mice than that in all other groups (Klotho/HYP, 56.2 +/- 6.3%; Klotho, 32.5 +/- 10.3%; HYP, 8.6 +/- 7.7%; and wild type, 21.4 +/- 3.4%; P < 0.004). Histomorphometric analysis confirmed the markedly increased trabecular bone density in Klotho/HYP mice and the well-established increase in osteoid volume in HYP mice. These observations suggest that with addition of Klotho loss of function, the overabundant osteoid typically produced in HYP mice (but fails to mineralize) is produced and mineralized in the double knockout, resulting in markedly enhanced trabecular bone density.

Effects of Ethanol Post-Fixation on the Histological, Histochemical, and Immunohistochemical Analysis of Murine Bone Specimens

Abstract Proper histological and histomorphometric assessment of bones requires acute visualization of cellular detail. Because fat is soluble in ethanol, adding an ethanol post-fixation step can help remove excess fat in tissue specimens, thus enhancing cellular visualization. However, the effects of adding this additional ethanol post-fixation step on bone-specific histochemical and immunohistochemical protocols have not yet been elucidated. Here, we describe the differences between using or not using an additional ethanol post-fixation step on six types of histochemical and immunohistochemical protocols. We assessed the effects in undecalcified tibiae sections routinely stained with hematoxylin and eosin, toluidine blue, and Von Kossa and found that addition of ethanol post-fixation enhanced specimen visualization for each of these stains. We examined the of ethanol post-fixation on enzymatic staining and found that alkaline phosphatase activity was equally preserved, whereas acid phosphatase activity was visualized more readily in specimens that underwent this treatment. Finally, the ethanol post-fixation improved antigen preservation in osteocalcin immunohistochemical labeling. (The J Histotechnol 27: 15, 2004) Submitted: July 8, 2003; Accepted: October 31, 2003

LIM kinase 1 deficient mice have reduced bone mass

The cytoskeleton determines cell shape and is involved in cell motility. It also plays a role in differentiation and in modulating specialized cellular functions. LIM kinase 1 (LIMK1) participates in cytoskeletal remodeling by phosphorylating and inactivating the actin-severing protein, cofilin. Severing F-actin to release G-actin monomers is required for actin cytoskeletal remodeling. Although less well established, LIMK1 may also influence the cell cycle and modulate metalloproteinase activity. Since the role of LIMK1 in bone cell biology has not been reported, the skeletal phenotype of LIMK1(-/-) mice was examined. LIMK1(-/-) mice had significantly reduced trabecular bone mass when analyzed by microCT (p<0.01). Histomorphometric analyses demonstrated a 31% reduction in the number of osteoblasts (p=0.0003) and a 23% reduction in osteoid surface (p=0.0005). The number of osteoclasts was no different in control and knock out animals. Consistent with the in vivo findings in osteoblasts, the number of osteoblast colony forming units in LIMK1(-/-) bone marrow was reduced by nearly 50%. Further, osteoblasts isolated from LIMK1(-/-) mice showed significantly reduced rates of mineralization in vitro. Osteoclasts from LIMK1(-/-) mice evidenced more rapid cytoskeletal remodeling in response to treatment with CSF1. In keeping with this latter finding, basal levels of phospho-cofilin were reduced in LIMK1(-/-) osteoclasts. LIMK1(-/-) osteoclasts also resorbed dentine slices to a greater extent in vitro and were more active in a pit assay. These data support the hypothesis that LIMK1 is required for normal osteoblast differentiation. In addition, its absence leads to increased cytoskeletal remodeling and bone resorption in osteoclasts.