Vertebral Bone Formation Research Articles

Induction of osteoblast apoptosis stimulates macrophage efferocytosis and paradoxical bone formation

Apoptosis is crucial for tissue homeostasis and organ development. In bone, apoptosis is recognized to be a main fate of osteoblasts, yet the relevance of this process remains underexplored. Using our murine model with inducible Caspase 9, the enzyme that initiates intrinsic apoptosis, we triggered apoptosis in a proportion of mature osteocalcin (OCN+) osteoblasts and investigated the impact on postnatal bone development. Osteoblast apoptosis stimulated efferocytosis by osteal macrophages. A five-week stimulation of OCN+ osteoblast apoptosis in 3-week-old male and female mice significantly enhanced vertebral bone formation while increasing osteoblast precursors. A similar treatment regimen to stimulate osterix+ cell apoptosis had no impact on bone volume or density. The vertebral bone accrual following stimulation of OCN+ osteoblast apoptosis did not translate in improved mechanical strength due to disruption of the lacunocanalicular network. The observed bone phenotype was not influenced by changes in osteoclasts but was associated with stimulation of macrophage efferocytosis and vasculature formation. Phenotyping of efferocytic macrophages revealed a unique transcriptomic signature and expression of factors including VEGFA. To examine whether macrophages participated in the osteoblast precursor increase following osteoblast apoptosis, macrophage depletion models were employed. Depletion of macrophages via clodronate-liposomes and the CD169-diphtheria toxin receptor mouse model resulted in marked reduction in leptin receptor+ and osterix+ osteoblast precursors. Collectively, this work demonstrates the significance of osteoblast turnover via apoptosis and efferocytosis in postnatal bone formation. Importantly, it exposes the potential of targeting this mechanism to promote bone anabolism in the clinical setting.

A vertebral skeletal stem cell lineage driving metastasis.

Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases1-4. The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis.

Lumbar vertebral canal stenosis due to marked bone overgrowth after routine hemilaminectomy in a dog

BackgroundBone overgrowth after decompressive surgery for lumbar stenosis resulting in recurrence of neurological signs has not been reported in veterinary literature. However, there are few cases described in human medicine.Case presentationA 13-month-old entire female dog, a crossbreed between a Springer Spaniel and a Border Collie, weighing 24 kg, was referred with a 5-day history of progressive spastic paraplegia, indicative of a T3-L3 myelopathy. Magnetic resonance (MR) imaging revealed a right-sided L2-L3 compressive extradural lesion, compatible with epidural haemorrhage, which was confirmed by histopathology. The lesion was approached via right-sided L2-L3 hemilaminectomy and was successfully removed. One-year postoperatively the dog re-presented with pelvic limb ataxia. MR and computed tomography (CT) images demonstrated excessive vertebral bone formation affecting the right articular processes, ventral aspect of the spinous process of L2-L3, and contiguous vertebral laminae, causing spinal cord compression. Revision surgery was performed, and histopathology revealed normal or reactive osseous tissue with a possible chondroid metaplasia and endochondral ossification, failing to identify a definitive reason for the bone overgrowth. Nine-month postoperatively, imaging studies showed a similar vertebral overgrowth, resulting in minimal spinal cord compression. The patient remained stable with mild proprioceptive ataxia up until the last follow-up 18 months post-revision surgery.ConclusionThis is the first report in the veterinary literature of bone overgrowth after lumbar hemilaminectomy which resulted in neurological deficits and required a revision decompressive surgery.

Loss-of-Function of p21-Activated Kinase 2 Links BMP Signaling to Neural Tube Patterning Defects.

Closure of the neural tube represents a highly complex and coordinated process, the failure of which constitutes common birth defects. The serine/threonine kinase p21-activated kinase 2 (PAK2) is a critical regulator of cytoskeleton dynamics; however, its role in the neurulation and pathogenesis of neural tube defects (NTDs) remains unclear. Here, the results show that Pak2-/- mouse embryos fail to develop dorsolateral hinge points (DLHPs) and exhibit craniorachischisis, a severe phenotype of NTDs. Pak2 knockout activates BMP signaling that involves in vertebrate bone formation. Single-cell transcriptomes reveal abnormal differentiation trajectories and transcriptional events in Pak2-/- mouse embryos during neural tube development. Two nonsynonymous and one recurrent splice-site mutations in the PAK2 gene are identified in five human NTD fetuses, which exhibit attenuated PAK2 expression and upregulated BMP signaling in the brain. Mechanistically, PAK2 regulates Smad9 phosphorylation to inhibit BMP signaling and ultimately induce DLHP formation. Depletion of pak2a in zebrafish induces defects in the neural tube, which are partially rescued by the overexpression of wild-type, but not mutant PAK2. The findings demonstrate the conserved role of PAK2 in neurulation in multiple vertebrate species, highlighting the molecular pathogenesis of PAK2 mutations in NTDs.

Follow-up MRI appearance of the surgical site in dogs treated for thoracolumbar intervertebral disc herniation and showing ongoing or recurrent neurological symptoms.

Reherniation and reoperation rates of 4.5%-36% are reported in canine patients treated for intervertebral disc herniation (IVDH). Decision-making for surgical reintervention can prove challenging, especially since common postoperative changes are poorly described on MRI. The purpose of this single-center, retrospective, descriptive study was to describe the MRI characteristics ofthe surgical site in dogs treated for thoracolumbar IVDH and presenting for ongoing or recurrent neurological signs. Twenty-one patients were included for a total of 42 MRI studies. Chondrodystrophic breeds, specifically Dachshunds, were overrepresented. Mean number of days between surgery and second MRI was335(range 2-1367). Metallic susceptibility artifacts were seen in seven of 21cases(33%), but these were limited in extent, spanning on average 1.3 vertebral bodies. In 11 cases, spinal cord compression suspected to be clinically significant was found at the surgical site; the extradural compressive material consisted of intervertebral disc material only, or a combination of intervertebral disc material and hematoma or inflammatory changes in 10 cases, and a displaced articular process and fibrous tissue in one case. The latter is a newly described complication of mini-hemilaminectomies. Paravertebral soft tissue changes and vertebral new bone formation varied according to the postoperative stage at which the patients were imaged. The results of this study supported the use of MRI as a diagnostic modality for spinal imaging following IVDH surgery, and showed that the presence of extradural disc material at a spinal surgical site is common along with various vertebral and paravertebral changes.

Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAP-expressing cells in mice

Pannexin1 (Panx1) is a hemichannel-forming protein that participates in the communication of cells with the extracellular space. To characterize the role of osteoclastic Panx1 on bone, Panx1fl/fl;TRAP-Cre (Panx1ΔOc) mice were generated, and compared to Panx1fl/fl littermates at 6 weeks of age. Total and femoral BMD was ~20% lower in females and males whereas spinal BMD was lower only in female Panx1ΔOc mice. μCT analyses showed that cortical bone of the femoral mid-diaphysis was not altered in Panx1ΔOc mice. In contrast, cancellous bone in the distal femur and lumbar vertebra was significantly decreased in both female and male Panx1ΔOc mice compared to Panx1fl/fl controls and was associated with higher osteoclast activity in female Panx1ΔOc mice, with no changes in the males. On the other hand, vertebral bone formation was decreased for both sexes, resulting from lower mineral apposition rate in the females and lower mineralizing surface in the males. Consistent with an osteoclastic effect in female Panx1ΔOc mice, osteoclast differentiation with RANKL/M-CSF and osteoclast bone resorbing activity in vitro were higher in female, but not male, Panx1ΔOc mice, compared to Panx1fl/fl littermates. Surprisingly, although Panx1 expression was normal in bone marrow stromal-derived osteoblasts from male and female Panx1ΔOc mice, mineral deposition by male (but not female) Panx1ΔOc osteoblasts was lower than controls, and it was reduced in male Panx1fl/fl osteoblasts when conditioned media prepared from male Panx1ΔOc osteoclast cultures was added to the cell culture media. Thus, deletion of Panx1 in TRAP-expressing cells in female mice leads to low bone mass primarily through a cell autonomous effect in osteoclast activity. In contrast, our evidence suggests that changes in the osteoclast secretome drive reduced osteoblast function in male Panx1ΔOc mice, resulting in low bone mass.

Effects of lithium administration on vertebral bone disease in mucopolysaccharidosis I dogs.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease characterized by deficient activity of the enzyme alpha-L-iduronidase, leading to abnormal accumulation of heparan and dermatan sulfate glycosaminoglycans in cells and tissues. Patients commonly exhibit progressive skeletal abnormalities, in part due to failures of endochondral ossification during postnatal growth. Previously, using the naturally-occurring canine model, we showed that bone and cartilage cells in MPS I exhibit elevated lysosomal storage from an early age and that animals subsequently exhibit significantly diminished vertebral trabecular bone formation. Wnts are critical regulators of endochondral ossification that depend on glycosaminoglycans for signaling. The objective of this study was to examine whether lithium, a glycogen synthase kinase-3 inhibitor and stimulator of Wnt/beta-catenin signaling, administered during postnatal growth could attenuate progression of vertebral trabecular bone disease in MPS I. MPS I dogs were treated orally with therapeutic levels of lithium carbonate from 14days to 6months-of-age. Untreated heterozygous and MPS I dogs served as controls. Serum was collected at 3 and 6months for assessment of bone turnover markers. At the study end point, thoracic vertebrae were excised and assessed using microcomputed tomography and histology. Lithium-treated animals exhibited significantly improved trabecular spacing, number and connectivity density, and serum bone-specific alkaline phosphatase levels compared to untreated animals. Growth plates from lithium-treated animals exhibited increased numbers of hypertrophic chondrocytes relative to both untreated MPS I and heterozygous animals. These findings suggest that bone and cartilage cells in MPS I are still capable of responding to exogenous osteogenic signals even in the presence of significant lysosomal storage, and that targeted osteogenic therapies may represent a promising approach for attenuating bone disease progression in MPS I.

Progression of vertebral bone disease in mucopolysaccharidosis VII dogs from birth to skeletal maturity

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, leading to accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans. Patients with MPS VII exhibit progressive spinal deformity, which decreases quality of life. Previously, we demonstrated that MPS VII dogs exhibit impaired initiation of secondary ossification in the vertebrae and long bones. The objective of this study was to build on these findings and comprehensively characterize how vertebral bone disease manifests progressively in MPS VII dogs throughout postnatal growth. Vertebrae were collected postmortem from MPS VII and healthy control dogs at seven ages ranging from 9 to 365 days. Microcomputed tomography and histology were used to characterize bone properties in primary and secondary ossification centers. Serum was analyzed for bone turnover biomarkers. Results demonstrated that not only was secondary ossification delayed in MPS VII vertebrae, but that it progressed aberrantly and was markedly diminished even at 365 days-of-age. Within primary ossification centers, bone volume fraction and bone mineral density were significantly lower in MPS VII at 180 and 365 days-of-age. MPS VII growth plates exhibited significantly lower proliferative and hypertrophic zone cellularity at 90 days-of-age, while serum bone-specific alkaline phosphatase (BAP) was significantly lower in MPS VII dogs at 180 days-of-age. Overall, these findings establish that vertebral bone formation is significantly diminished in MPS VII dogs in both primary and secondary ossification centers during postnatal growth.

Effects of systemically administered abaloparatide, an osteoanabolic PTHrP analog, as an adjuvant therapy for spinal fusion in rats.

BackgroundAbaloparatide is a parathyroid hormone receptor agonist that increases bone formation and reduces vertebral and nonvertebral fracture risk in women with postmenopausal osteoporosis. Animal studies indicate abaloparatide stimulates vertebral bone formation and enhances bony bridging and biomechanical stability of fracture calluses.AimsThe current study is evaluating the potential utility for abaloparatide as an adjunct therapy for spinal fusions.Material and MethodsThe effects of 14 or 28 days of daily subcutaneous injections of abaloparatide (20 μg/kg/d) or vehicle were evaluated in 32 male Sprague‐Dawley rats starting 1 day after noninstrumented posterolateral fusion (PLF) with bone autograft. Fusion mass microarchitecture was analyzed by micro‐computed tomography (micro‐CT) and serum markers of bone formation and bone resorption were evaluated. Motion segments were scored in a blinded manner as fused or unfused by postmortem radiography and manual palpation.ResultsAbaloparatide‐treated rats showed higher bone formation (serum osteocalcin) at day 14 and 28 compared with vehicle controls, without increases in the bone resorption marker serum TRACP‐5b. Micro‐CT showed greater trabecular number in fusion masses from the abaloparatide group vs vehicle controls at day 14. Manual palpation and radiography indicated no fusions in either group at day 14, whereas 25% of vehicle‐treated rats and 50% of abaloparatide‐treated rats had bilateral fusion at day 28.Discussion and ConclusionIn summary, this rat PLF model showed that abaloparatide treatment was associated with higher levels of the bone formation marker osteocalcin, improved fusion mass architecture, and a non‐ significant 2‐fold higher fusion rate compared with vehicle.

Multigenerational Impacts of Benzo[a]pyrene on Bone Modeling and Remodeling in Medaka (Oryzias latipes).

Ancestral benzo[a]pyrene (BaP) (1 μg/L, 21 days) exposure has previously been shown to cause skeletal deformities in medaka (Oryzias latipes) larvae in the F1-F3 generation. However, when and how this deformity is induced during bone development remain to be elucidated. The col10a1:nlGFP/osx:mCherry double transgenic medaka model was employed to determine the temporal and spatial changes of col10a1:nlGFP- positive osteochondral progenitor cells (OPCs) and osx:mCherry-positive premature osteoblasts (POBs) [8 days postfertilization (dpf)-31 dpf] in combination with changes in bone mineralization at the tissue level. Ancestral BaP exposure delayed the development of col10a1:nlGFP- and osx:mCherry-positive osteoblasts and reduced the abundance of col10a1:nlGFP-positive osteoblast progenitors and col10a1:nlGFP/osx:mCherry double-positive premature osteoblasts during critical windows of early vertebral bone formation, associated with reduced bone mineralization in embryos (14 dpf) and larvae (31 dpf), compressed vertebral segments in larvae (31 dpf), and reduced bone thickness in adult male medaka (6 months old) of the F1-F3 generations. Both Col10a1:nlGFP and osx:mCherry were identified as potential targets of epigenetic modifications underlying the transgenerational inheritance of BaP bone toxicity. The present study provides novel knowledge of the underlying mechanisms of transgenerational toxicity of BaP at the cellular level.

Notochord vacuoles absorb compressive bone growth during zebrafish spine formation.

The vertebral column or spine assembles around the notochord rod which contains a core made of large vacuolated cells. Each vacuolated cell possesses a single fluid-filled vacuole, and loss or fragmentation of these vacuoles in zebrafish leads to spine kinking. Here, we identified a mutation in the kinase gene dstyk that causes fragmentation of notochord vacuoles and a severe congenital scoliosis-like phenotype in zebrafish. Live imaging revealed that Dstyk regulates fusion of membranes with the vacuole. We find that localized disruption of notochord vacuoles causes vertebral malformation and curving of the spine axis at those sites. Accordingly, in dstyk mutants the spine curves increasingly over time as vertebral bone formation compresses the notochord asymmetrically, causing vertebral malformations and kinking of the axis. Together, our data show that notochord vacuoles function as a hydrostatic scaffold that guides symmetrical growth of vertebrae and spine formation.

HIF-1α metabolically controls collagen synthesis and modification in chondrocytes.

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

Reversal of loss of bone mass in old mice treated with mefloquine.

Aging is accompanied by imbalanced bone remodeling, elevated osteocyte apoptosis, and decreased bone mass and mechanical properties; and improved pharmacologic approaches to counteract bone deterioration with aging are needed. We examined herein the effect of mefloquine, a drug used to treat malaria and systemic lupus erythematosus and shown to ameliorate bone loss in glucocorticoid-treated patients, on bone mass and mechanical properties in young and old mice. Young 3.5-month-old and old 21-month-old female C57BL/6 mice received daily injections of 5 mg/kg/day mefloquine for 14 days. Aging resulted in the expected changes in bone volume and mechanical properties. In old mice mefloquine administration reversed the lower vertebral cancellous bone volume and bone formation; and had modest effects on cortical bone volume, thickness, and moment of inertia. Mefloquine administration did not change the levels of the circulating bone formation markers P1NP or alkaline phosphatase, whereas levels of the resorption marker CTX showed trends towards increase with mefloquine treatment. In addition, and as expected, aging bones exhibited an accumulation of active caspase3-expressing osteocytes and higher expression of apoptosis-related genes compared to young mice, which were not altered by mefloquine administration at either age. In young animals, mefloquine induced higher periosteal bone formation, but lower endocortical bone formation. Further, osteoclast numbers were higher on the endocortical bone surface and circulating CTX levels were increased, in mefloquine- compared to vehicle-treated young mice. Consistent with this, addition of mefloquine to bone marrow cells isolated from young mice led to increased osteoclastic gene expression and a tendency towards increased osteoclast numbers in vitro. Taken together our findings identify the age and bone-site specific skeletal effects of mefloquine. Further, our results highlight a beneficial effect of mefloquine administration on vertebral cancellous bone mass in old animals, raising the possibility of using this pharmacologic inhibitor to preserve skeletal health with aging.

Decreased osteoprogenitor proliferation precedes attenuation of cancellous bone formation in ovariectomized rats treated with sclerostin antibody

Sclerostin antibody (Scl-Ab) stimulates bone formation, which with long-term treatment, attenuates over time. The cellular and molecular mechanisms responsible for the attenuation of bone formation are not well understood, but in aged ovariectomized (OVX) rats, the reduction in vertebral cancellous bone formation is preceded by a reduction in osteoprogenitor (OP) number and significant induction of signaling pathways known to suppress mitogenesis and cell cycle progression in the osteocyte (OCy) (Taylor et al., 2016). To determine if the reduction in OP number is associated with a decrease in proliferation, aged OVX rats were administered vehicle or Scl-Ab for 9 or 29 days and implanted with continuous-delivery 5-bromo-2′-deoxyuridine (BrdU) mini-osmotic pumps 5 days prior to necropsy. The total number of BrdU-labeled osteoblasts (OB) was quantified in vertebral cancellous bone to indirectly assess the effects of Scl-Ab treatment on OP proliferation at the time of activation of modeling-based bone formation at day 9 and at the time of maximal mineralizing surface, initial decrease in OP number, and transcriptional changes in the OCy at day 29. Compared with vehicle, Scl-Ab resulted in an increase in the total number of BrdU-positive OB (+260%) at day 9 that decreased with continued treatment (+50%) at day 29. These differences in proliferation occurred at time points when the increase in total OB number was significant and similar in magnitude. These findings suggest that reduced OP proliferation contributes to the decrease in OP numbers, an effect that would limit the OB pool and contribute to the attenuation of bone formation that occurs with long-term Scl-Ab treatment.

A vertebrate specific and essential role for sp7/osterix in osteogenesis revealed by gene knock-out in the teleost medaka

osterix (osx; sp7) encodes a zinc-finger transcription factor that controls osteoblast differentiation in mammals. Although identified in all vertebrate lineages, its role in non-mammalian bone formation remains elusive. Here, we show that an osx mutation in medaka results in severe bone defects and larval lethality. Pre-osteoblasts fail to differentiate leading to severe intramembranous and perichondral ossification defects. The notochord sheath mineralizes normally, supporting the idea of an osteoblast-independent mechanism for teleost vertebral centra formation. This study establishes a key role for Osx for bone formation in a non-mammalian species, and reveals conserved and non-conserved features in vertebrate bone formation.

MiR-29a Participated in Nacre Formation and Immune Response by Targeting Y2R in Pinctada martensii.

miR-29a is a conserved miRNA that participates in bone formation and immune response in vertebrates. miR-29a of Pinctada martensii (Pm-miR-29a) was identified in the previous research though deep sequencing. In this report, the precise sequence of mature Pm-miR-29a was validated using miRNA rapid amplification of cDNA ends (miR-RACE) technology. The precursor sequence of Pm-miR-29a was predicted to have 87 bp. Stem loop qRT-PCR analysis showed that Pm-miR-29a was easily detected in all the tissues, although expressions in the mantle and gill were low. The microstructure showed the disrupted growth of the nacre after Pm-miR-29a over-expression, which was induced by mimic injection into P. martensii. Results of the target analysis indicated that neuropeptide Y receptor type 2 (Y2R) was the potential target of Pm-miR-29a. Meanwhile, Pm-miR-29a mimics could obviously inhibit the relative luciferase activity of the reporter containing 3′ UTR (Untranslated Regions) of the Y2R gene. Furthermore, the expression of Y2R was downregulated whereas expressions of interleukin 17 (IL-17) and nuclear factor κB (NF-κB) were upregulated after Pm-miR-29a over-expression in the mantle and gill, thereby suggesting that Pm-miR-29a could activate the immune response of the pearl oyster. Results showed that Pm-miR-29a was involved in nacre formation and immune response by regulating Y2R in pearl oyster P. martensii.

Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.

A single CT-guided percutaneous intraosseous injection of thermosensitive simvastatin/poloxamer 407 hydrogel enhances vertebral bone formation in ovariectomized minipigs.

The ultimate goal of osteoporosis treatment is prevention of fragile fracture. Local treatment targeting specific bone may decrease the incidence of osteoporotic fractures. We developed an injectable, thermosensitive simvastatin/poloxamer 407 hydrogel; a single CT-guided percutaneous intraosseous injection augmented vertebrae in ovariectomized minipigs. The greatest hazard associated with osteoporosis is local fragility fractures. An adjunct, local treatment might be helpful to decrease the incidence of osteoporotic fracture. Studies have found that simvastatin stimulates bone formation, but the skeletal bioavailability of orally administered is low. Directly delivering simvastatin to the specific bone that is prone to fractures may reinforce the target bone and reduce the incidence of fragility fractures. We developed an injectable, thermosensitive simvastatin/poloxamer 407 hydrogel, conducted scanning electron microscopy, rheological, and drug release analyses to evaluate the delivery system; injected it into the lumbar vertebrae of ovariectomized minipigs via minimally invasive CT-guided percutaneous vertebral injection. Three months later, BMD, microstructures, mineral apposition rates, and strength were determined by DXA, micro-CT, histology, and biomechanical test; expression of VEGF, BMP2, and osteocalcin were analyzed by immunohistochemistry and Western blots. Poloxamer 407 is an effective controlled delivery system for intraosseous-injected simvastatin. A single injection of the simvastatin/poloxamer 407 hydrogel significantly increased BMD, bone microstructure, and strength; the bone volume fraction and trabecular thickness increased nearly 150%, bone strength almost doubled compared with controls (all P < 0.01); and induced higher expression of VEGF, BMP2, and osteocalcin. CT-guided percutaneous vertebral injection of a single simvastatin/poloxamer 407 thermosensitive hydrogel promotes bone formation in ovariectomized minipigs. The underlying mechanism appears to involve the higher expression of VEGF and BMP-2.

On the pathway of mineral deposition in larval zebrafish caudal fin bone

A poorly understood aspect of bone biomineralization concerns the mechanisms whereby ions are sequestered from the environment, concentrated, and deposited in the extracellular matrix. In this study, we follow mineral deposition in the caudal fin of the zebrafish larva in vivo. Using fluorescence and cryo-SEM-microscopy, in combination with Raman and XRF spectroscopy, we detect the presence of intracellular mineral particles located between bones, and in close association with blood vessels. Calcium-rich particles are also located away from the mineralized bone, and these are also in close association with blood vessels. These observations challenge the view that mineral formation is restricted to osteoblast cells juxtaposed to bone, or to the extracellular matrix. Our results, derived from observations performed in living animals, contribute a new perspective to the comprehensive mechanism of bone formation in vertebrates, from the blood to the bone. More broadly, these findings may shed light on bone mineralization processes in other vertebrates, including humans.

Failed vertebral bone formation in mucopolysaccharidosis VII dogs is associated with impaired chondrocyte hypertrophic differentiation

Failed vertebral bone formation in mucopolysaccharidosis VII dogs is associated with impaired chondrocyte hypertrophic differentiation