Mitchell B. Schaffler

Sustained release of multiple growth factors from injectable polymeric system as a novel therapeutic approach towards angiogenesis.

PurposeThe aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion.MethodsThree groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF165 (VEGF), or alginate containing VEGF165 combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points.ResultsIn the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles.ConclusionsSequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.

Osteocyte apoptosis is required for production of osteoclastogenic signals following bone fatigue in vivo.

Osteocyte apoptosis is spatially, temporally and functionally linked to the removal and replacement of microdamage in the bone. Recently we showed that microdamage elicits distinct responses in two populations of osteocytes near the injury site. Osteocytes directly adjacent to microdamage undergo apoptosis, whereas there is a second group of osteocytes located adjacent to the apoptotic population that upregulate expression of osteoclastogenic signaling molecules. In this study we used the pan-caspase inhibitor QVD to test the hypothesis that osteocyte apoptosis is an obligatory step in the production of key osteoclastogenic signals by in situ osteocytes in fatigue-damaged bone. We found, based on real-time PCR and immunohistochemistry assays, that expression of the apoptosis marker caspase-3 as well osteoclastogenic proteins RANKL and VEGF were increased following fatigue, while expression of the RANKL antagonist OPG decreased. However, when apoptosis was inhibited using QVD, these changes in gene expression were completely blocked. This dependence on apoptosis for neighboring non-apoptotic cells to produce signals that promote tissue remodeling also occurs in response to focal ischemic injury in the brain and heart, indicating that osteoclastic bone remodeling follows a common paradigm for localized tissue repair.

Matrix metalloproteinase-3 in articular cartilage is upregulated by joint immobilization and suppressed by passive joint motion

Both underloading and overloading of joints can lead to articular cartilage degradation, a process mediated in part by matrix metalloproteinases (MMPs). Here we examine the effects of reduced loading of rat hindlimbs on articular cartilage expression of MMP-3, which not only digests matrix components but also activates other proteolytic enzymes. We show that hindlimb immobilization resulted in elevated MMP-3 mRNA expression at 6h that was sustained throughout the 21day immobilization period. MMP-3 upregulation was higher in the medial condyle than the lateral, and was greatest in the superficial cartilage zone, followed by middle and deep zones. These areas also showed decreases in safranin O staining, consistent with reduced cartilage proteoglycan content, as early as 7days after immobilization. One hour of daily moderate mechanical loading, applied as passive joint motion, reduced the MMP-3 and ADAMTS-5 increases that resulted from immobilization, and also prevented changes in safranin O staining. Intra-articular injections of an MMP-3 inhibitor, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), dampened the catabolic effects of a 7day immobilization period, indicating a likely requirement for MMP-3 in the regulation of proteoglycan levels through ADAMTS-5. These results suggest that biomechanical forces have the potential to combat cartilage destruction and can be critical in developing effective therapeutic strategies.

Reductions in serum IGF-1 during aging impair health span.

In lower or simple species, such as worms and flies, disruption of the insulin‐like growth factor (IGF)‐1 and the insulin signaling pathways has been shown to increase lifespan. In rodents, however, growth hormone (GH) regulates IGF‐1 levels in serum and tissues and can modulate lifespan via/or independent of IGF‐1. Rodent models, where the GH/IGF‐1 axis was ablated congenitally, show increased lifespan. However, in contrast to rodents where serum IGF‐1 levels are high throughout life, in humans, serum IGF‐1 peaks during puberty and declines thereafter during aging. Thus, animal models with congenital disruption of the GH/IGF‐1 axis are unable to clearly distinguish between developmental and age‐related effects of GH/IGF‐1 on health. To overcome this caveat, we developed an inducible liver IGF‐1‐deficient (iLID) mouse that allows temporal control of serum IGF‐1. Deletion of liver Igf ‐1 gene at one year of age reduced serum IGF‐1 by 70% and dramatically impaired health span of the iLID mice. Reductions in serum IGF‐1 were coupled with increased GH levels and increased basal STAT5B phosphorylation in livers of iLID mice. These changes were associated with increased liver weight, increased liver inflammation, increased oxidative stress in liver and muscle, and increased incidence of hepatic tumors. Lastly, despite elevations in serum GH, low levels of serum IGF‐1 from 1 year of age compromised skeletal integrity and accelerated bone loss. We conclude that an intact GH/IGF‐1 axis is essential to maintain health span and that elevated GH, even late in life, associates with increased pathology.

Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor

States of growth hormone (GH) resistance, such those observed in Laron dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF‐1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF‐1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH‐resistant (GHR knockout [GHRKO]) mouse model with mice expressing the hepatic Igf‐1 transgene (HIT) to generate the GHRKO‐HIT mouse model. In GHRKO‐HIT mice, serum IGF‐1 levels were restored via transgenic expression of Igf‐1, allowing us to study how endocrine IGF‐1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH‐resistant state, normalization of serum IGF‐1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF‐1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf‐1 gene expression, blunted GH‐mediated effects on the skeleton that are independent of serum or tissue IGF‐1, and poor delivery of IGF‐1 to the tissues. These findings are consistent with clinical data showing that IGF‐I replacement therapy in patients with Laron syndrome does not achieve full skeletal growth.

High Resolution Micro Arthrography of Hard and Soft Tissues in a Murine Model

OBJECTIVEnRecent developments on high resolution micro computed tomography (μCT) allow imaging of soft tissues in small animal joints. Nevertheless, μCT images cannot distinguish soft tissues from synovial fluid due to their similar mass density, limiting the 3D assessment of soft tissues volume and thickness. This study aimed to evaluate a lead chromate contrast agent for μCΤ arthrography of rat knee joints ex vivo.nnnDESIGNnIntact tibiofemoral rat joints were injected with the contrast agent at different concentrations and imaged using a μCT at 2.7 μm isotropic voxel size. Cartilage thickness was measured using an automated procedure, validated against histological measurements, and analyzed as a function of μCT image resolution. Changes in hard and soft tissues were also analyzed in tibiofemoral joints 4 weeks after surgical destabilization of the medial meniscus (DMM).nnnRESULTSnThe contrast agent diffused well throughout the whole knee cavity without penetrating the tissues, therefore providing high contrast at the boundaries between soft tissues and synovial fluid space. Thickness analysis of cartilage demonstrated a high similarity between histology and μ-arthrography approaches (R(2) = 0.90). Four weeks after surgical DMM, the development of osteophytes (Oph) and cartilage ulcerations was recognizable with μCT, as well as a slight increase in trabecular bone porosity, and decrease in trabecular thickness.nnnCONCLUSIONSnA lead chromate-based contrast agent allowed discriminating the synovial fluid from soft tissues of intact knee joints, and thus made possible both qualitative and quantitative assessment of hard and soft tissues in both intact and DMM tibiofemoral joints using high resolution μCT.

Low levels of plasma IGF-1 inhibit intracortical bone remodeling during aging.

Studies linking insulin-like growth factor-1 (IGF-1) to age-related bone loss in humans have been reported but remain only correlative. In this investigation, we characterized the bone phenotype of aged WT C57BL/6J male mice in comparison to that of C57BL/6J mice with reduced serum IGF-1 levels arising from an igfals gene deletion (ALS knockout (ALSKO)). During the aging process, WT mice showed an increase in fat mass and decrease lean mass while ALSKO mice had stable lean and fat mass values. Skeletal analyses of femora from WT mice revealed an expansion of the marrow area and a significant accumulation of intracortical porosity associated with increased intracortical remodeling. In contrast, ALSKO mice showed only small age-related declines in the amount of cortical bone tissue and minimal intracortical porosity, at 2xa0years of age. Accordingly, mechanical tests of femora from 2-year-old WT mice revealed reduced stiffness and maximal load when compared to bones from ALSKO mice. We show here that lifelong reductions in serum IGF-1 compromise skeletal size in development leading to slender bones; they are also associated with decreased intracortical bone remodeling and preservation of bone strength during aging.

Development and Validation of a Motion and Loading System for a Rat Knee Joint In Vivo

The influence of biomechanical stimuli on modulating cartilage homeostasis is well recognized. However, many aspects of cellular mechanotransduction in cartilage remain unknown. We developed a computer-controlled joint motion and loading system (JMLS) to study the biological response of cartilage under well-characterized mechanical loading environments. The JMLS was capable of controlling (i) angular displacement, (ii) motion frequency, (iii) magnitude of the axial compressive load applied to the moving joint, and it featured real-time monitoring. The accuracy and repeatability of angular position measurements, the kinematic misalignment error as well as the repositioning error of the JMLS were evaluated. The effectiveness of the JMLS in implementing well-defined loading protocols such as moderate Passive Motion Loading (PML) and increased Compressive Motion Loading (CML) were tested. The JMLS demonstrated remarkable accuracy and reliability for the measurement and kinematics tests. Moreover, the effectiveness test demonstrated the ability of the JMLS to produce an effective stimulus via PML that led to the suppression of the catabolic effects of immobilization. Interestingly, the biological response of the CML group was catabolic and exhibited a pattern similar to that observed in the immobilization group. This novel non-invasive system may be useful for joint biomechanics studies that require different treatment conditions of load and motion in vivo.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF‐1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF‐1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)‐1‐mediated Ghr knockout (DMP‐GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP‐GHRKO did not affect linear growth but compromised overall bone accrual. DMP‐GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte‐like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase‐2 and IGF‐1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.—Liu, Z., Kennedy, O. D., Cardoso, L., Basta‐Pljakic, J., Partridge, N. C., Schaffler, M. B., Rosen, C. J., Yakar, S. DMP‐1‐mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH. FASEB J. 30, 635‐652 (2016). www.fasebj.org