Gloria Gutierrez

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic beta subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.

Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents.

A synthetic peptide corresponding to the first 34 amino acids of the parathyroid hormone-related protein (PTH-rP) produced by a human tumor associated with hypercalcemia was examined for skeletal and renal effects on calcium metabolism in vivo and in vitro. These effects were compared with those of human parathyroid hormone (1-34), hPTH (1-34). Equal doses of PTH-rP(1-34) and hPTH(1-34) produced equivalent stimulation of adenylate cyclase in vitro in bone cells and kidney cells and tubules. Subcutaneous injection of PTH-rP(1-34) in mice caused a significant dose-related increase in blood ionized calcium similar to that seen with hPTH(1-34) at equivalent doses. Repeated injections of equal doses of both peptides caused sustained hypercalcemia which was significantly greater in PTH-rP(1-34)-treated mice, although each induced comparable increases in histomorphometric indices of osteoclastic bone resorption. PTH-rP(1-34) and hPTH(1-34) also caused similar increases in bone resorption when incubated with fetal rat long bones in organ culture. Infusion of either peptide into thyroparathyroidectomized rats suppressed urinary calcium excretion and increased urinary excretion of cyclic AMP. PTH-rP appears to have similar effects to those of PTH on the skeleton, the kidney, and overall calcium homeostasis.

Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref‐1

Dlk‐1/Pref‐1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref‐1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells.

Cytokines and Bone Remodeling

Publisher Summary Bone is continuously remodeled in normal individuals and this is achieved via a finely regulated balance between the processes of bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. This bone remodeling is regulated, in part, by local factors including cytokines generated in the bone microenvironment. The purpose of this chapter is to summarize what is currently known about the role of cytokines and their receptors in bone remodeling. Recent advances in molecular biological techniques have meant that most of the biological activities ascribed to cytokines have now been associated with specific molecules, and their receptors identified and molecularly cloned. Several cytokines and their cognate receptors have been shown to be expressed by bone cells, marrow cells, or accessory cells in the bone microenvironment. Moreover, studies using knockout and transgenic mice have increased the understanding of the complex signal transduction mechanisms utilized by cytokines and are opening up new and exciting areas of study. Cytokines tend to be pleiotropic and multifactorial, and may have overlapping and seemingly redundant biological effects. Some of this redundancy is apparent in the receptor mechanisms and signal transduction pathways used by groups of cytokines. Classic examples that illustrate this vividly are the various cytokines belonging to the interleukin (IL)-6 family, such as IL-6, leukemia inhibitory factor, oncostatin-M, and IL-11, which utilize a common signal transduction protein known as gp130. These cytokines bind to distinct membrane-associated receptors, which form hetero- or homo-dimers upon binding to the ligand. Further, there is now a body of data derived from in vivo studies in animals which show that over- or under-production of certain cytokines cause profound effects on bone. These fundamental observations have the potential of not only increasing the understanding of the pathophysiology of osteoporosis, but also leading to new and better forms of therapy using these molecules as targets for drug discovery programs.

Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography

Although fracture healing is frequently studied in pre-clinical models of long bone fractures using rodents, there is a dearth of objective quantitative techniques to assess successful healing. Biomechanical testing is possibly the most quantitative and relevant to a successful clinical outcome, but it is a destructive technique providing little insight into the cellular mechanisms associated with healing. The advent of X-ray computed tomography (CT) has provided the opportunity to quantitatively and non-destructively assess bone structure and density, but it is unknown how measurements derived using this technology relate to successful healing. To examine possible relationships, we used a pre-clinical model to test for statistically significant correlations between quantitative characteristics of the callus by micro-CT (microCT) and the bending strength, stiffness, and energy-to-failure of the callus as assessed by three-point bending of excised bones. A closed, transverse fracture was generated in the mid-shaft of rat femurs by impact loading. Shortly thereafter, the rats received a one-time, local injection of either the vehicle or one of four doses of lovastatin. Following sacrifice after 4 weeks of healing, fractured femurs were extracted for microCT analysis and then three-point bending. Setting the region of interest to be 3.2 mm above and below the fracture line, we acquired standard and new microCT-derived measurements. The mineralized callus volume and the mineral density of the callus correlated positively with callus strength (rxy = -0.315, p = 0.016 and rxy = 0.444, p<0.0005, respectively) and stiffness (rxy = -0.271, p = 0.040 and rxy = 0.325, p = 0.013, respectively), but the fraction of the callus that mineralized and the moment of inertia of the callus did not. This fraction did correlate with energy-to-failure (rxy = -0.343, p = 0.0085). Of the microCT-derived measurements, quantifying defects within the outer bridging cortices of the callus produced the strongest correlation with both callus strength (rxy = 0.557, p<0.0001) and stiffness (rxy = 0.468, p = 0.0002). By both reducing structural defects and increasing mineralization, lovastatin appears to increase the callus strength.

Transdermal Lovastatin Enhances Fracture Repair in Rats

Statins have been shown to stimulate BMP2 transcription and bone formation. This raises the possibility that they could be useful for enhancing rates of fracture repair. Observational studies in patients treated with oral statins for lipid‐lowering have been controversial. The likely reason for their inconsistent effects is that the statin concentration reaching the periphery was too low after oral administration to produce a reproducible biologic effect. Thus, we examined the effects of lovastatin (LV) given transdermally in a well‐described preclinical model of fracture repair. Effects on the healing fracture callus were assessed by biomechanical strength, radiographs, and quantitative morphology. LV was administered transdermally (TD) for 5 days after fracture in several doses (0.1–5 mg/kg/d) and compared with vehicle‐treated control rats and rats treated with LV by oral gavage (PO) at 5–25 mg/kg/d for 5 days from the day of fracture. Radiological evaluation of bones treated with TD LV showed enhanced fracture repair at 2 and 6 wk. BMD in the callus area at 6 wk was also increased in the TD group compared with vehicle‐treated controls (p < 0.05). The force required to break TD‐treated bones (0.1 mg/kg/d for 5 days) was 42% greater than vehicle‐treated controls (p < 0.02), and there was a 90% increase in stiffness (p < 0.01). PO LV at much higher doses (10 and 25 mg/kg/d) showed increased stiffness but no change in other biomechanical properties. By histological examination, a significant increase was also observed in the size of the callus, surrounding proliferating cell nuclear antigen–positive cells, and osteoblast and osteoclast number in TD‐treated rats compared with controls at day 8 after fracture (n = 6). In summary, we found that TD LV in low doses accelerates fracture healing, whereas 10‐fold the lipid‐lowering dose was required to produce any effect when it was administered orally. These studies provide valuable information on the potential of statins and TD delivery as a new and effective therapeutic modality in fracture repair.

Local low-dose lovastatin delivery improves the bone-healing defect caused by Nf1 loss of function in osteoblasts.

Postfracture tibial nonunion (pseudoarthrosis) leads to lifelong disability in patients with neurofibromatosis type I (NF1), a disorder caused by mutations in the NF1 gene. To determine the contribution of NF1 in bone healing, we assessed bone healing in the Nf1  ob−/− conditional mouse model lacking Nf1 specifically in osteoblasts. A closed distal tibia fracture protocol and a longitudinal study design were used. During the 21‐ to 28‐day postfracture period, callus volume, as expected, decreased in wild‐type but not in Nf1  ob−/− mice, suggesting delayed healing. At these two time points, bone volume (BV/TV) and volumetric bone mineral density (vBMD) measured by 3D micro–computed tomography were decreased in Nf1  ob−/− callus‐bridging cortices and trabecular compartments compared with wild‐type controls. Histomorphometric analyses revealed the presence of cartilaginous remnants, a high amount of osteoid, and increased osteoclast surfaces in Nf1  ob−/− calluses 21 days after fracture, which was accompanied by increased expression of osteopontin, Rankl, and Tgfβ. Callus strength measured by three‐point bending 28 days after fracture was reduced in Nf1  ob−/− versus wild‐type calluses. Importantly, from a clinical point of view, this defect of callus maturation and strength could be ameliorated by local delivery of low‐dose lovastatin microparticles, which successfully decreased osteoid volume and cartilaginous remnant number and increased callus BV/TV and strength in mutant mice. These results thus indicate that the dysfunctions caused by loss of Nf1 in osteoblasts impair callus maturation and weaken callus mechanical properties and suggest that local delivery of low‐dose lovastatin may improve bone healing in NF1 patients.

Approaches to treating NF1 tibial pseudarthrosis: Consensus from the children's tumor foundation NF1 bone abnormalities consortium

Background: Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with various skeletal abnormalities occurring as part of a complex phenotype. Tibial dysplasia, which typically presents as anterolateral bowing of the leg with subsequent fracture and nonunion (pseudarthrosis), is a serious but infrequent osseous manifestation of NF1. Over the past several years, results from clinical and experimental studies have advanced our knowledge of the role of NF1 in bone. On the basis of current knowledge, we propose a number of concepts to consider as a theoretical approach to the optimal management of tibial pseudarthrosis. Methods: A literature review for both clinical treatment and preclinical models for tibial dysplasia in NF1 was performed. Concepts were discussed and developed by experts who participated in the Children’s Tumor Foundation sponsored International Bone Abnormalities Consortium meeting in 2011. Results: Concepts for a theoretical approach to treating tibial pseudarthrosis include: bone fixation appropriate to achieve stability in any given case; debridement of the “fibrous pseudarthrosis tissue” between the bone segments associated with the pseudarthrosis; creating a healthy vascular bed for bone repair; promoting osteogenesis; controlling overactive bone resorption (catabolism); prevention of recurrence of the “fibrous pseudarthrosis tissue”; and achievement of long-term bone health to prevent recurrence. Conclusions: Clinical trials are needed to assess effectiveness of the wide variation of surgical and pharmacologic approaches currently in practice for the treatment of tibial pseudarthrosis in NF1. Level of Evidence: Level V, expert opinion.

Local injection of lovastatin in biodegradable polyurethane scaffolds enhances bone regeneration in a critical‐sized segmental defect in rat femora

Statins, a class of naturally‐occurring compounds that inhibit HMG‐CoA reductase, are known to increase endogenous bone morphogenetic protein‐2 (BMP‐2) expression. Local administration of statins has been shown to stimulate fracture repair in in vivo animal experiments. However, the ability of statins to heal more challenging critical‐sized defects at the mid‐diaphyseal region in long bones has not been investigated. In this study, we examined the potential of injectable lovastatin microparticles combined with biodegradable polyurethane (PUR) scaffolds in preclinical animal models: metaphyseal small plug defects and diaphyseal segmental bone defects in rat femora. Sustained release of lovastatin from the lovastatin microparticles was achieved over 14 days. The released lovastatin was bioactive, as evidenced by its ability to stimulate BMP‐2 gene expression in osteoblastic cells. Micro‐computed tomography (CT) and histological examinations showed that lovastatin microparticles, injected into PUR scaffolds implanted in femoral plug defects, enhanced new bone formation. Furthermore, bi‐weekly multiple injections of lovastatin microparticles into PUR scaffolds implanted in critical‐sized femoral segmental defects resulted in increased new bone formation compared to the vehicle control. In addition, bridging of the defect with newly formed bone was observed in four of nine defects in the lovastatin microparticle treatment group, whereas none of the defects in the vehicle group showed bridging. These observations suggest that local delivery of lovastatin combined with PUR scaffold can be an effective approach for treatment of orthopaedic bone defects and that multiple injections of lovastatin may be useful for large defects. Copyright

Design and assessment of a wrapped cylindrical Ca-P AZ31 Mg alloy for critical-size ulna defect repair†

Recently, magnesium has been investigated as a promising bioresorbable orthopedic biomaterial. Its mechanical properties are very similar to natural bone, making it appropriate for load-bearing orthopedic fracture repair applications. However, significant hurdles remain regarding the design of practical implants and methods to control degradation and enhance biocompatibility. Although attempts have been made to hinder magnesiums rapid corrosion via alloying and coating, these studies have used solid monoliths. In an effort to reduce the amount of alloy used for implantation in a shape that mimics cortical bone shape, this study used a thin sheet of Mg AZ31 which was rolled into hollow cylindrical scaffolds. The scaffold was coated with different amounts of Ca-P; this implant demonstrated slowed corrosion in simulated body fluid (SBF) as well as enhanced biocompatibility for mesenchymal stem cells (MSC). In vivo implantation of magnesium alloy scaffold adjacent to the rat femur showed significant biointegration with further deposition of complex Mg-Ca phosphates/carbonates typical of natural bone. Finally, the implant was placed in a critical-size ulna defect in live rabbits, which lead to radiographic union and partial restoration of biomechanical strength in the defect. This study demonstrated that a thin sheet of coated Mg alloy that was spirally wrapped wound be a promising orthopedic biomaterial for bone repair.