Local microarchitecture affects mechanical properties of deposited extracellular matrix for osteonal regeneration

Authors

M. Pilia, T. Guda, B. Pollot, V. Aguero, M. Appleford

Abstract

Multiple biomimetic approaches have been attempted to accelerate the regeneration of functional bone tissue. While most synthetic scaffolds are designed to mimic the architecture of trabecular bone, in the current study, cortical bone-like extracellular matrix was regenerated in vitro within organized structures. Biphasic calcium phosphate (BCaP) and hydroxyapatite (HAp) scaffolds were developed with longitudinal microchannels (250 μm diameter) that resembled native osteons in cortical bone. BCaP and HAp scaffolds had a compressive strength of 7.61 ± 1.42 and 9.98 ± 0.61 MPa respectively. The constructs were investigated in vitro to evaluate the organization and stiffness of the extracellular matrix (ECM) formed by human fetal osteoblasts (HFObs) cultured inside the microchannels. The ECM deposited on the BCaP scaffolds was found to have a higher micro-hardness (h) (1.93 ± 0.40GPa) than the ECM formed within the HAp microchannels (h = 0.80 ± 0.20GPa) (p < 0.05) or native bone (h = 0.47-0.74GPa). ECM deposition within the microchannels resembled osteoid organization and showed a significant increase in both osteoid area and thickness after 24 days (p < 0.001). These observations indicate that controlled microarchitecture; and specifically cylindrical microchannels; plays a fundamental role in stimulating the appropriate cellular response aimed at recreating organized, cortical bone-like matrix. These findings open the door for researchers to develop a new generation of cortical bone scaffolds that can restore strong, organized bone.

Link to Article

http://dx.doi.org/10.1016/j.msec.2013.10.018

The Inhibition of Subchondral Bone Lesions Significantly Reversed the Weight-Bearing Deficit and the Overexpression of CGRP in...

Title

The Inhibition of Subchondral Bone Lesions Significantly Reversed the Weight-Bearing Deficit and the Overexpression of CGRP in DRG Neurons, GFAP and Iba-1 in the Spinal Dorsal Horn in the Monosodium Iodoacetate Induced Model of Osteoarthritis Pain

Authors

Degang Yu, Fengxiang Liu, Ming Liu, Xin Zhao, Xiaoqing Wang, Yang Li, Yuanqing Mao, Zhenan Zhu

Abstract

Background

Chronic pain is the most prominent and disabling symptom of osteoarthritis (OA). Clinical data suggest that subchondral bone lesions contribute to the occurrence of joint pain. The present study investigated the effect of the inhibition of subchondral bone lesions on joint pain.

Methods

Osteoarthritic pain was induced by an injection of monosodium iodoacetate (MIA) into the rat knee joint. Zoledronic acid (ZOL), a third generation of bisphosphonate, was used to inhibit subchondral bone lesions. Joint histomorphology was evaluated using X-ray micro computed tomography scanning and hematoxylin-eosin staining. The activity of osteoclast in subchondral bone was evaluated using tartrate-resistant acid phosphatase staining. Joint pain was evaluated using weight-bearing asymmetry, the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG), and spinal glial activation status using glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) immunofluorescence. Afferent neurons in the DRGs that innervated the joints were identified using retrograde fluorogold labeling.

Results

MIA injections induced significant histomorphological alterations and joint pain. The inhibition of subchondral bone lesions by ZOL significantly reduced the MIA-induced weight-bearing deficit and overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn at 3 and 6 weeks after MIA injection; however, joint swelling and synovial reaction were unaffected.

Conclusions

The inhibition of subchondral bone lesions alleviated joint pain. Subchondral bone lesions should be a key target in the management of osteoarthritic joint pain.

Link to Article

http://dx.doi.org/10.1371/journal.pone.0077824

Allogeneic Mesenchymal Progenitor Cells for Posterolateral Lumbar Spine Fusion in Sheep

Authors

Donna L. Wheeler, PhD, Joseph M. Lane, MD, Howard B. Seim III, DVM, Dipl. ACVS, Christian Puttlitz, PhD, Silviu Itescu, MD, A. Simon Turner, B.V.Sc, M.S, Dipl. ACVS

Abstract

Background

Osteoconductive porous ceramic bone graft materials supplemented with mesenchymal precursor cells (MPC) derived from autologous bone marrow aspirates have been shown to stimulate successful interbody and posterolateral spine fusion in preclinical models. Recent advances in immunomagnetic cell sorting have enabled purification and isolation of pleuripotent stem cells from marrow aspirates and have expanded stem cell technology to allogeneic cell sources. Allogeneic MPC technology combined with appropriate synthetic biomaterial carriers could provide both the osteogenic and osteoconductive components needed for successful posterolateral spine fusion without the need for autologous bone harvest or expensive recombinant protein technology.

Purpose

To determine the safety and efficacy of a hydroxyapatite:tricalcium phosphate graft material supplemented with allogeneic mesenchymal precursor cells in posterolateral lumbar spine fusion using an ovine model.

Study Design

Skeletally mature ewes underwent single-level instrumented posterolateral lumbar spine fusion using either autograft (AG), hydroxyapatite:tricalcium phosphate carrier (CP), or CP supplemented with allogeneic mesenchymal progenitor cells (MPCs). Three doses of MPCs were evaluated: 25 X 106 cells (low dose, LD), 75 X 106 cells (mid dose, MD), and 225 X 106 cell (high dose, HD). Animals survived for either 4 or 9 months.

Methods

Plain radiographs were acquired and scored for bridging bone at regular intervals during healing to monitor fusion development. Hematology, coagulation and serum chemistry were monitored at regular interval throughout the study to monitor animal health. After necropsy, computed tomography, high resolution radiography, biomechanical testing, organ pathology, bone histopathology, and bone histomorphometry were conducted to monitor the safety and ascertain the efficacy of MPC treatment.

Results

MPC treatment in this spine fusion model resulted in no observed adverse systemic or local tissue responses. Radiographically, fusion scores for MPC treated animals were uniformly higher compared to those treated with carrier alone (CP) after 3 months and continued the same trend throughout 9 month of healing. Quantitative computed tomography (qCT) confirmed better connectivity of the fusion for MPC treatment groups compared to CP. Biomechanical analyses were not able to differentiate between treatment groups. Histomorphometry results confirmed radiographic and qCT results; cell-supplemented treatment groups and autograft had equivalent amounts of bone within the fusion mass and less bony fusion tissue was found within the fusion mass in specimens from the CP treatment group. No conclusive effects of cell dose of fusion efficacy were noted.

Conclusions

Adult allogeneic mesenchymal precursor cells delivered via a hydroxyapatite:tricalcium phosphate carrier were both safe and efficacious in this ovine spine fusion model. Results from this preclinical study support that allogeneic mesenchymal precursor cells produced fusion efficacy similar to that achieved using iliac crest autograft, thereby providing a safe and viable option to achieve successful posterolateral spine fusion.

Link to Article

http://dx.doi.org/10.1016/j.spinee.2013.09.048

The interaction of force and repetition on musculoskeletal and neural tissue responses and sensorimotor behavior in a rat model of work-related musculoskeletal disorders

Authors

Mary F Barbe, Sean Gallagher, Vicky S Massicotte, Michael Tytell, Steven N Popoff, and Ann E Barr-Gillespie

Abstract

Background

We examined the relationship of musculoskeletal risk factors underlying force and repetition on tissue responses in an operant rat model of repetitive reaching and pulling, and if force x repetition interactions were present, indicative of a fatigue failure process. We examined exposure-dependent changes in biochemical, morphological and sensorimotor responses occurring with repeated performance of a handle-pulling task for 12 weeks at one of four repetition and force levels: 1) low repetition with low force, 2) high repetition with low force, 3) low repetition with high force, and 4) high repetition with high force (HRHF).

Methods

Rats underwent initial training for 4–6 weeks, and then performed one of the tasks for 12 weeks, 2 hours/day, 3 days/week. Reflexive grip strength and sensitivity to touch were assayed as functional outcomes. Flexor digitorum muscles and tendons, forelimb bones, and serum were assayed using ELISA for indicators of inflammation, tissue stress and repair, and bone turnover. Histomorphometry was used to assay macrophage infiltration of tissues, spinal cord substance P changes, and tissue adaptative or degradative changes. MicroCT was used to assay bones for changes in bone quality.

Results

Several force x repetition interactions were observed for: muscle IL-1alpha and bone IL-1beta; serum TNFalpha, IL-1alpha, and IL-1beta; muscle HSP72, a tissue stress and repair protein; histomorphological evidence of tendon and cartilage degradation; serum biomarkers of bone degradation (CTXI) and bone formation (osteocalcin); and morphological evidence of bone adaptation versus resorption. In most cases, performance of the HRHF task induced the greatest tissue degenerative changes, while performance of moderate level tasks induced bone adaptation and a suggestion of muscle adaptation. Both high force tasks induced median nerve macrophage infiltration, spinal cord sensitization (increased substance P), grip strength declines and forepaw mechanical allodynia by task week 12.

Conclusions

Although not consistent in all tissues, we found several significant interactions between the critical musculoskeletal risk factors of force and repetition, consistent with a fatigue failure process in musculoskeletal tissues. Prolonged performance of HRHF tasks exhibited significantly increased risk for musculoskeletal disorders, while performance of moderate level tasks exhibited adaptation to task demands.

Link to Article

http://dx.doi.org/10.1186/1471-2474-14-303

Role of TGF-β in a Mouse Model of High Turnover Renal Osteodystrophy

Authors

Shiguang Liu, Wenping Song, Joseph H Boulanger, Wen Tang, Yves Sabbagh, Brian Kelley, Russell Gotschall, Susan Ryan, Lucy Phillips, Katie Malley, Xiaohong Cao, Tai-He Xia, Gehua Zhen, Xu Cao, Hong Ling, Paul C Dechow, Teresita M Bellido, Steven R Ledbetter, Susan C Schiavi

Abstract

Altered bone turnover is a key pathologic feature of chronic kidney disease-mineral and bone disorder (CKD-MBD). Expression of TGF-β1, a known regulator of bone turnover, is increased in bone biopsies from individuals with CKD. Similarly, TGF-β1 mRNA and downstream signaling is increased in bones from jck mice, a model of high-turnover renal osteodystropy. A neutralizing anti-TGF-β antibody (1D11) was used to explore TGF-βs role in renal osteodystrophy. 1D11 administration to jck significantly attenuated elevated serum osteocalcin and type I collagen C-telopeptides. Histomorphometric analysis indicated that 1D11 administration increased bone volume and suppressed the elevated bone turnover in a dose-dependent manner. These effects were associated with reductions in osteoblast and osteoclast surface areas. µCT confirmed the observed increase in trabecular bone volume and demonstrated improvements in trabecular architecture and increased cortical thickness. 1D11 administration was associated with significant reductions in expression of osteoblast marker genes (Runx2, alkaline phosphatase, osteocalcin) and the osteoclast marker gene, Trap5. Importantly, in this model, 1D11 did not improve kidney function or reduce serum PTH levels indicating that 1D11 effects on bone are independent of changes in renal or parathyroid function. 1D11 also significantly attenuated high turnover bone disease in the adenine-induced uremic rat model. Antibody administration was associated with a reduction in pSMAD2/SMAD2 in bone but not bone marrow as assessed by quantitative immunoblot analysis. Immunostaining revealed pSMAD staining in osteoblasts and osteocytes but not osteoclasts, suggesting 1D11 effects on osteoclasts may be indirect. Immunoblot and whole genome mRNA expression analysis confirmed our previous observation that repression of Wnt/β catenin expression in bone is correlated with increased osteoclast activity in jck mice and bone biopsies from CKD patients. Furthermore, our data suggests that elevated TGF-β may contribute to the pathogenesis of high turnover disease partially through inhibition of β-catenin signaling.

Link to Article

http://dx.doi.org/10.1002/jbmr.2120

NF1 is a critical regulator of muscle development and metabolism

Authors

Kate Sullivan, Jad El-Hoss, Kate G. R. Quinlan, Nikita Deo, Fleur Garton, Jane T. C. Seto, Marie Gdalevitch, Nigel Turner, Gregory J. Cooney, Mateusz Kolanczyk, Kathryn N. North, David G. Little and Aaron Schindeler

Abstract

There is emerging evidence for reduced muscle function in children with Neurofibromatosis type 1 (NF1). We have examined three murine models featuring NF1 deficiency in muscle to study the effect on muscle function as well as any underlying pathophysiology. The Nf1+/- mouse exhibited no differences in overall weight, lean tissue mass, fiber size, muscle weakness as measured by grip strength, or muscle atrophy-recovery with limb disuse, although this model lacks many other characteristic features of the human disease. Next, muscle-specific knockout mice (Nf1muscle−/-) were generated and they exhibited a failure to thrive leading to neonatal lethality. Intramyocellular lipid accumulations were observed by electron microscopy (EM) and Oil Red O staining. More mature muscle specimens lacking Nf1 expression taken from the limb-specific Nf1Prx1−/- conditional knockout line showed a 10-fold increase in muscle triglyceride content. Enzyme assays revealed a significant increase in the activities of oxidative metabolism enzymes in the Nf1Prx1−/- mice. Western analyses showed increases in the expression of Fatty Acid Synthase (FAS) and the hormone Leptin, as well as decreased expression of a number of fatty acid transporters in this mouse line. These data support the hypothesis that NF1 is essential for normal muscle function and survival and are the first to suggest a direct link between NF1 and mitochondrial fatty acid metabolism.

Link to Article

http://dx.doi.org/10.1093/hmg/ddt515