Dietary intervention rescues a bone porosity phenotype in a murine model of Neurofibromatosis Type 1 (NF1).

Neurofibromatosis type 1 (NF1) is a complex genetic disorder that affects a range of tissues including muscle and bone. Recent preclinical and clinical studies have shown that Nf1 deficiency in muscle causes metabolic changes resulting in intramyocellular lipid accumulation and muscle weakness. These can be subsequently rescued by dietary interventions aimed at modulating lipid availability and metabolism. It was speculated that the modified diet may rescue defects in cortical bone as NF1 deficiency has been reported to affect genes involved with lipid metabolism. Bone specimens were analyzed from wild type control mice as well as Nf1Prx1-/- (limb-targeted Nf1 knockout mice) fed standard chow versus a range of modified chows hypothesized to influence lipid metabolism. Mice were fed from 4 weeks to 12 weeks of age. MicroCT analysis was performed on the cortical bone to examine standard parameters (bone volume, tissue mineral density, cortical thickness) and specific porosity measures (closed pores corresponding to osteocyte lacunae, and larger open pores). Nf1Prx1-/- bones were found to have inferior bone properties to wild type bones, with a 4-fold increase in the porosity attributed to open pores. These measures were rescued by dietary interventions including a L-carnitine + medium-chain fatty acid supplemented chow previously shown to improve muscle histology function. Histological staining visualized these changes in bone porosity. These data support the concept that lipid metabolism may have a mechanistic impact on bone porosity and quality in NF1.

Targeted postnatal knockout of Sclerostin using a bone-targeted adeno-associated viral vector increases bone anabolism and decreases canalicular density.

PURPOSE: The creation of murine gene knockout models to study bone gene functions often requires the resource intensive crossbreeding of Cre transgenic and gene-floxed strains. The developmental versus postnatal roles of genes can be difficult to discern in such models. For example, embryonic deletion of the Sclerostin (Sost) gene establishes a high-bone mass phenotype in neonatal mice that may impact on future bone growth. To generate a postnatal skeletal knockout of Sost in adult mice, this study used a single injection of a bone-targeted recombinant adeno-associated virus (rAAV) vector. METHODS: 8-week-old Sostflox/flox mice were injected with saline (control) or a single injection containing 5 × 1011 vg AAV8-Sp7-Cre vector. Ai9 fluorescent Cre reporter mice were dosed in parallel to confirm targeting efficiency. After 6 weeks, detailed bone analysis was performed via microCT, biomechanical testing, and bone histology on vertebral and long bone specimens. RESULTS: The AAV8-Sp7-Cre vector induced widespread persistent recombination in the bone compartment. Regional microCT analyses revealed significant increases in bone with vector treatment. In the L3 vertebrae, Sostflox/flox:AAV-Cre showed a 22 % increase in bone volume and 21 % in trabecular bone fraction compared to controls; this translated to a 17 % increase in compressive strength. In the tibiae, Sostflox/flox:AAV-Cre led to small but statistically significant increases in cortical bone volume and thickness. These were consistent with a 25 % increase in mineral apposition rate, but this did not translate into increased four-point bending strength. Ploton silver nitrate stain on histological sections revealed an unexpected increase in canalicular density associated with Sost ablation. CONCLUSION: This report demonstrates a proof-of-concept that the AAV8-Sp7-Cre vector can efficiently produce postnatal skeletal knockout mice using gene-floxed strains. This technology has the potential for broad utility in the bone field with existing conditional lines. These data also confirm an important postnatal role for Sost in regulating bone homeostasis, consistent with prior studies using neutralizing Sclerostin antibodies, and highlights a novel role of Sost in canalicular remodeling.

Combination treatment with growth hormone and zoledronic acid in a mouse model of Osteogenesis imperfecta.

INTRODUCTION: Osteogenesis imperfecta (OI) or brittle bone disease is a genetic disorder that results in bone fragility. Bisphosphonates such as zoledronic acid (ZA) are used clinically to increase bone mass and reduce fracture risk. Human growth hormone (hGH) has been used to promote long bone growth and forestall short stature in children with OI. The potential for hGH to improve bone quality, particularly in combination with ZA has not been robustly studied. METHODS: A preclinical study was performed using n = 80 mice split evenly by genotype (WT, Col1a2+/G610C). Groups of n = 10 were treated with +/-ZA and +/-hGH in a factorial design for each genotype. Outcome measures included bone length, isolated muscle mass, bone parameters assessed by microCT analysis, dynamic histomorphometry, and biomechanical testing. RESULTS: Treatment with hGH alone led to an increase in femur length in WT but not OI mice, however bone length was increased in both genotypes with the combination of hGH/ZA. MicroCT showed that hGH/ZA treatment increased cortical BV in both WT (+15%) and OI mice (+14.3%); hGH/ZA were also found to be synergistic in promoting cortical thickness in OI bone. ZA was found to have a considerably greater positive impact on trabecular bone than hGH. ZA was found to suppress bone turnover, and this was rescued by hGH treatment in terms of cortical periosteal perimeter, but not by dynamic bone remodeling. Statistically significant improvements in long bone by microCT did not translate into improvements in mechanical strength in a 4-point bending test, nor did vertebral strength improve in L4 compression testing in WT/OI bone. DISCUSSION/CONCLUSION: These data support hGH/ZA combination as a treatment for short stature, however the improvements granted by hGH alone and in combination with ZA on bone quality are modest. Increased periosteal perimeter does show promise in improving bone strength in OI, however a longer treatment time may be required to see effects on bone strength through mechanical testing.

Curative Cell and Gene Therapy for Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) describes a series of genetic bone fragility disorders that can have a substantive impact on patient quality of life. The multidisciplinary approach to management of children and adults with OI primarily involves the administration of antiresorptive medication, allied health (physiotherapy and occupational therapy), and orthopedic surgery. However, advances in gene editing technology and gene therapy vectors bring with them the promise of gene-targeted interventions to provide an enduring or perhaps permanent cure for OI. This review describes emergent technologies for cell- and gene-targeted therapies, major hurdles to their implementation, and the prospects of their future success with a focus on bone disorders. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Characterization of the Developing Lacunocanalicular Network During Fracture Repair.

Fracture repair is a normal physiological response to bone injury. During the process of bony callus formation, a lacunocanalicular network (LCN) is formed de novo that evolves with callus remodeling. Our aim was the longitudinal assessment of the development and evolution of the LCN during fracture repair. To this end, 45 adult wild-type C57BL/6 mice underwent closed tibial fracture surgery. Fractured and intact contralateral tibias were harvested after 2, 3, and 6 weeks of bone healing (n = 15/group). High-resolution micro-computed tomography (µCT) and deconvolution microscopy (DV) approaches were applied to quantify lacunar number density from the calluses and intact bone. On histological sections, Goldner's trichrome staining was used to assess lacunar occupancy, fluorescein isothiocyanate staining to visualize the canalicular network, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining to examine osteocyte apoptosis. Analysis of µCT scans showed progressive decreases in mean lacuna volume over time (-27% 2-3 weeks; -13% 3-6 weeks). Lacunar number density increased considerably between 2 and 3 weeks (+156%). Correlation analysis was performed, showing a positive linear relationship between canalicular number density and trabecular thickness (R 2 = 0.56, p < 0.001) and an inverse relationship between mean lacuna volume and trabecular thickness (R 2 = 0.57, p < 0.001). Histology showed increases in canalicular number density over time (+22% 2-3 weeks, +51% 3-6 weeks). Lacunar occupancy in new bone of the callus was high (>90%), but the old cortical bone within the fracture site appeared necrotic as it underwent resorption. In conclusion, our data shows a progressive increase in the complexity of the LCN over time during fracture healing and demonstrates that this network is initiated during the early stages of repair. Further studies are needed to address the functional importance of osteocytes in bone healing, particularly in detecting and translating the effects of micromotion in the fracture. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Reprogramming of human fibroblasts into osteoblasts by insulin-like growth factor-binding protein 7.

The induced pluripotent stem cell (iPSC) is a promising cell source for tissue regeneration. However, the therapeutic value of iPSC technology is limited due to the complexity of induction protocols and potential risks of teratoma formation. A trans-differentiation approach employing natural factors may allow better control over reprogramming and improved safety. We report here a novel approach to drive trans-differentiation of human fibroblasts into functional osteoblasts using insulin-like growth factor binding protein 7 (IGFBP7). We initially determined that media conditioned by human osteoblasts can induce reprogramming of human fibroblasts to functional osteoblasts. Proteomic analysis identified IGFBP7 as being significantly elevated in media conditioned with osteoblasts compared with those with fibroblasts. Recombinant IGFBP7 induced a phenotypic switch from fibroblasts to osteoblasts. The switch was associated with senescence and dependent on autocrine IL-6 signaling. Our study supports a novel strategy for regenerating bone by using IGFBP7 to trans-differentiate fibroblasts to osteoblasts.

Increased anabolic bone response in Dkk1 KO mice following tibial compressive loading.

A viable Dkk1 knockout (KO) mouse strain in which embryonic lethality is rescued by developmental Wnt3 heterozygosity (Dkk1-/-:Wnt3+/-) exhibits increased bone formation and a high bone mass phenotype. We hypothesized that in vivo mechanical loading would further augment the bone formation response in Dkk1 KO mice, comparable to results from Sost KO mice. A cyclic loading protocol was applied to Dkk1 KO mice, wild type mice (WT; Dkk1+/+:Wnt3+/+), and Wnt3 heterozygote (Wnt3+/-; Dkk1+/+:Wnt3+/-) controls. The left tibiae of 10-week-old female mice were dynamically loaded in vivo with 7N maximum compressive force 5 days/week for 2 weeks. Dkk1 KO bones were significantly stiffer, and so an additional group of Dkk1 KO received 12N maximum compressive force to achieve an equivalent +1200µÎµ strain at the mid-diaphysis. MicroCT and bone histomorphometry analyses were subsequently performed. All groups responded to tibial loading with increased mid-diaphyseal bone volume. The largest effect size was in the Dkk1 KO -12N group. Thus, Dkk1 KO animals had enhanced sensitivity to mechanical loading. Increases in cortical bone volume reflected increased periosteal bone formation. Bone volume and formation were not altered between WT and Wnt3+/- controls. These data support the concept that agonists of Wnt/ß-catenin signaling can act synergistically with load-bearing exercise. Notably, Sost expression decreased with loading in Dkk1 KO and WT mice, independent of genotype. These data suggest that a compensatory downregulation of Sost in Dkk1 KO mice is not likely the primary mechanism for the augmented response to mechanical load.

Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton.

A panel of 18 recombinant adeno-associated virus (rAAV) variants, both natural and engineered, constitutively expressing Cre recombinase under the cytomegalovirus early enhancer/chicken ß actin (CAG) promoter, were screened for their ability to transduce bone in Ai9 fluorescent reporter mice. Transgenic Cre-induced tdTomato expression served as a measure of transduction efficiency and alkaline phosphatase (AP) activity as an osteoblastic marker. Single injections of AAV8, AAV9, and AAV-DJ into midshaft tibial fractures yielded robust tdTomato expression in the callus. Next, the bone cell-specific promoters Sp7 and Col2.3 were tested to restrict Cre expression in an alternate model of systemic delivery by intravenous injection. Although CAG promoter constructs packaged into AAV8 produced high levels of tdTomato in the bone, liver, heart, spleen, and kidney, bone-specific promoter constructs restricted Cre expression to osseous tissues. AAV variants were further tested in vitro in a human osteoblast cell line (hFOB1.19), measuring GFP reporter expression by flow cytometry after 72 h. AAV2, AAV5, and AAV-DJ showed the highest transduction efficiency. In summary, we produced AAV vectors for selective and high-efficiency in vivo gene delivery to murine bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion postnatally in floxed mouse models.

Bone Marrow Transplantation for Treatment of the Col1a2+/G610C Osteogenesis Imperfecta Mouse Model.

Bone marrow transplantation (BMT) of healthy donor cells has been postulated as a strategy for treating osteogenesis imperfecta (OI) and other bone fragility disorders. The effect of engraftment by tail vein injection and/or marrow ablation by 6 Gy whole body irradiation were tested in Col1a2+/G610C (OI) mice as a model of mild-moderate OI. Dual-emission X-ray absorptiometry, microCT, and 4-point bending were used to measure bone volume (BV), bone mineral density (BMD), and biomechanical strength. BV, BMD, and mechanical strength were reduced in OI mice compared to wild type (WT) controls. BMT with and without irradiation yielded no difference in BV and BMD outcomes for both OI and WT mice, at 3 weeks. Transplantation of OI cells into OI mice to test for paracrine effects of BMT also showed no difference with non-transplanted OI mice. In a parallel cell tracking study, donor marrow was taken from transgenic mice constitutively expressing tdTomato and transplanted into WT mice. Lineage tracking demonstrated that irradiation considerably enhanced engraftment of tdTomato+ cells. However, tdTomato+ cells predominantly expressed TRAP and not AP, indicating engrafted donor cells were chiefly from the hematopoietic lineages. These data show that whole marrow transplantation fails to rescue the bone phenotype of Col1a2+/G610C (OI) mice and that osteopoietic engraftment is not significantly enhanced by irradiation. These findings are highly relevant to modern approaches focused on the gene repair of patient cells ex vivo and their subsequent reintroduction into the osteopoietic compartment via the circulation.

Homozygous Dkk1 Knockout Mice Exhibit High Bone Mass Phenotype Due to Increased Bone Formation.

Wnt antagonist Dkk1 is a negative regulator of bone formation and Dkk1 +/- heterozygous mice display a high bone mass phenotype. Complete loss of Dkk1 function disrupts embryonic head development. Homozygous Dkk1 -/- mice that were heterozygous for Wnt3 loss of function mutation (termed Dkk1 KO) are viable and allowed studying the effects of homozygous inactivation of Dkk1 on bone formation. Dkk1 KO mice showed a high bone mass phenotype exceeding that of heterozygous mice as well as a high incidence of polydactyly and kinky tails. Whole body bone density was increased in the Dkk1 KO mice as shown by longitudinal dual-energy X-ray absorptiometry. MicroCT analysis of the distal femur revealed up to 3-fold increases in trabecular bone volume and up to 2-fold increases in the vertebrae, compared to wild type controls. Cortical bone was increased in both the tibiae and vertebrae, which correlated with increased strength in tibial 4-point bending and vertebral compression tests. Dynamic histomorphometry identified increased bone formation as the mechanism underlying the high bone mass phenotype in Dkk1 KO mice, with no changes in bone resorption. Mice featuring only Wnt3 heterozygosity showed no evident bone phenotype. Our findings highlight a critical role for Dkk1 in the regulation of bone formation and a gene dose-dependent response to loss of DKK1 function. Targeting Dkk1 to enhance bone formation offers therapeutic potential for osteoporosis.