Sayuri Yoshizawa

Magnesium ion stimulation of bone marrow stromal cells enhances osteogenic activity, simulating the effect of magnesium alloy degradation

Magnesium alloys are being investigated for load-bearing bone fixation devices due to their initial mechanical strength, modulus similar to native bone, biocompatibility and ability to degrade in vivo. Previous studies have found Mg alloys to support bone regeneration in vivo, but the mechanisms have not been investigated in detail. In this study, we analyzed the effects of Mg(2+) stimulation on intracellular signaling mechanisms of human bone marrow stromal cells (hBMSCs). hBMSCs were cultured in medium containing 0.8, 5, 10, 20 and 100mM MgSO4, either with or without osteogenic induction factors. After 3weeks, mineralization of extracellular matrix (ECM) was analyzed by Alizarin red staining, and gene expression was analyzed by quantitative polymerase chain reaction array. Mineralization of ECM was enhanced at 5 and 10mM MgSO4, and collagen type X mRNA (COL10A1, an ECM protein deposited during bone healing) expression was increased at 10mM MgSO4 both with and without osteogenic factors. We also confirmed the increased production of collagen type X protein by Western blotting. Next, we investigated the mechanisms of intracellular signaling by analyzing the protein production of hypoxia-inducible factor (HIF)-1α and 2α (transcription factors of COL10A1), vascular endothelial growth factor (VEGF) (activated by HIF-2α) and peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α (transcription coactivator of VEGF). We observed that 10mM MgSO4 stimulation enhanced COL10A1 and VEGF expression, possibly via HIF-2α in undifferentiated hBMSCs and via PGC-1α in osteogenic cells. These data suggest possible ECM proteins and transcription factors affected by Mg(2+) that are responsible for the enhanced bone regeneration observed around degradable Mg orthopedic/craniofacial devices.

In vivo study of magnesium plate and screw degradation and bone fracture healing.

Each year, millions of Americans suffer bone fractures, often requiring internal fixation. Current devices, like plates and screws, are made with permanent metals or resorbable polymers. Permanent metals provide strength and biocompatibility, but cause long-term complications and may require removal. Resorbable polymers reduce long-term complications, but are unsuitable for many load-bearing applications. To mitigate complications, degradable magnesium (Mg) alloys are being developed for craniofacial and orthopedic applications. Their combination of strength and degradation make them ideal for bone fixation. Previously, we conducted a pilot study comparing Mg and titanium devices with a rabbit ulna fracture model. We observed Mg device degradation, with uninhibited healing. Interestingly, we observed bone formation around degrading Mg, but not titanium, devices. These results highlighted the potential for these fixation devices. To better assess their efficacy, we conducted a more thorough study assessing 99.9% Mg devices in a similar rabbit ulna fracture model. Device degradation, fracture healing, and bone formation were evaluated using microcomputed tomography, histology and biomechanical tests. We observed device degradation throughout, and calculated a corrosion rate of 0.40±0.04mm/year after 8 weeks. In addition, we observed fracture healing by 8 weeks, and maturation after 16 weeks. In accordance with our pilot study, we observed bone formation surrounding Mg devices, with complete overgrowth by 16 weeks. Bend tests revealed no difference in flexural load of healed ulnae with Mg devices compared to intact ulnae. These data suggest that Mg devices provide stabilization to facilitate healing, while degrading and stimulating new bone formation.

Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes

Significance Periodontal disease (gum disease) is an extremely prevalent inflammatory disease initiated by persistent bacterial insult, leading to the destruction of bone and gingival tissues. Current clinical treatments focus solely on the removal of bacteria. In this study, we put forth a strategy to address the underlying inflammatory imbalance in periodontal disease by harnessing the body’s own sophisticated immunoregulatory mechanisms through the recruitment of regulatory T cells (Tregs). This is accomplished by controllably releasing small quantities (nanogram/kilogram range) of chemokine recognized by Tregs using biodegradable, resorbable polymers with an excellent track record of regulatory approval. Administration of Treg-recruiting treatments to the gingiva of mice and canines reduces clinical scores of disease as well as hard and soft tissue destruction. The hallmark of periodontal disease is the progressive destruction of gingival soft tissue and alveolar bone, which is initiated by inflammation in response to an invasive and persistent bacterial insult. In recent years, it has become apparent that this tissue destruction is associated with a decrease in local regulatory processes, including a decrease of forkhead box P3-expressing regulatory lymphocytes. Accordingly, we developed a controlled release system capable of generating a steady release of a known chemoattractant for regulatory lymphocytes, C-C motif chemokine ligand 22 (CCL22), composed of a degradable polymer with a proven track record of clinical translation, poly(lactic-co-glycolic) acid. We have previously shown that this sustained presentation of CCL22 from a point source effectively recruits regulatory T cells (Tregs) to the site of injection. Following administration of the Treg-recruiting formulation to the gingivae in murine experimental periodontitis, we observed increases in hallmark Treg-associated anti-inflammatory molecules, a decrease of proinflammatory cytokines, and a marked reduction in alveolar bone resorption. Furthermore, application of the Treg-recruiting formulation (fabricated with human CCL22) in ligature-induced periodontitis in beagle dogs leads to reduced clinical measures of inflammation and less alveolar bone loss under severe inflammatory conditions in the presence of a diverse periodontopathogen milieu.

Role of magnesium ions on osteogenic response in bone marrow stromal cells

Abstract Biodegradable magnesium (Mg) alloys have been investigated for craniofacial and orthopedic bone fracture fixation due to their initial mechanical strength and high biocompatibility. Although Mg alloys have been reported to enhance bone regeneration in vivo, and enhanced osteogenic marker expression in human bone marrow stromal cells (hBMSCs) cultured in Mg alloy extract was reported, however, the biological mechanism is not fully understood. Thus, it is important to elucidate which signaling pathway in the hBMSCs are activated by Mg2+ that enhances bone formation. We investigated possible mechanisms underlying effects of Mg2+ on bone regeneration by culturing differentiated and undifferentiated hBMSCs in the presence of culture medium containing 10 mM MgSO4 both with or without osteogenic factors. mRNA expression of osteogenic genes was analyzed using quantitative PCR arrays. Quantitative PCR array data indicated increased mRNA expression of collagen type X and insulin-like growth factor 2, and decreased expression of integrin alpha 3 in the presence of 10 mM MgSO4. Moreover, Western blotting analysis showed enhanced expression of collagen type X, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-2α, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the presence of 10 mM MgSO4. In conclusion, 10 mM of MgSO4 enhanced the production of collagen type X and VEGF by hBMSCs. These results also suggest that Mg2+ released from bone fixation devices may promote bone regeneration by enhancing the production of collagen type X and VEGF of osteogenic cells in bone tissue.

Fracture Healing Using Degradable Magnesium Fixation Plates and Screws

PURPOSE Internal bone fixation devices made with permanent metals are associated with numerous long-term complications and may require removal. We hypothesized that fixation devices made with degradable magnesium alloys could provide an ideal combination of strength and degradation, facilitating fracture fixation and healing while eliminating the need for implant removal surgery. MATERIALS AND METHODS Fixation plates and screws were machined from 99.9% pure magnesium and compared with titanium devices in a rabbit ulnar fracture model. Magnesium device degradation and the effect on fracture healing and bone formation were assessed after 4 weeks. Fracture healing with magnesium device fixation was compared with that of titanium devices using qualitative histologic analysis and quantitative histomorphometry. RESULTS Micro-computed tomography showed device degradation after 4 weeks in vivo. In addition, 2-dimensional micro-computed tomography slices and histologic staining showed that magnesium degradation did not inhibit fracture healing or bone formation. Histomorphology showed no difference in bone-bridging fractures fixed with magnesium and titanium devices. Interestingly, abundant new bone was formed around magnesium devices, suggesting a connection between magnesium degradation and bone formation. CONCLUSION Our results show potential for magnesium fixation devices in a loaded fracture environment. Furthermore, these results suggest that magnesium fixation devices may enhance fracture healing by encouraging localized new bone formation.

IL‐4/CCL22/CCR4 Axis Controls Regulatory T‐Cell Migration That Suppresses Inflammatory Bone Loss in Murine Experimental Periodontitis

Inflammatory bone resorption is a hallmark of periodontitis, and Tregs and Th2 cells are independently associated with disease progression attenuation. In this study, we employed an infection‐triggered inflammatory osteolysis model to investigate the mechanisms underlying Treg and Th2 cell migration and the impact on disease outcome. Aggregatibacter actinomycetemcomitans–infected C57Bl/6 (wild‐type [WT]) mice develop an intense inflammatory reaction and alveolar bone resorption, and Treg and Th2 cell migration is temporally associated with disease progression attenuation. Tregs extracted from the lesions preferentially express CCR4 and CCR8, whereas Th2 cells express CCR3, CCR4, and CCR8. The absence of CCR5 and CCR8 did not significantly impact the migration of Tregs and Th2 cells or affect the disease outcome. CCR4KO mice presented a minor reduction in Th2 cells in parallel with major impairment of Treg migration, which was associated with increased inflammatory bone loss and higher proinflammatory and osteoclastogenic cytokine levels. The blockade of the CCR4 ligand CCL22 in WT mice resulted in an increased inflammatory bone loss phenotype similar to that in the CCR4KO strain. Adoptive transfer of CCR4+ Tregs to the CCR4KO strain revert the increased disease phenotype to WT mice–like levels; also, the in situ production of CCL22 in the lesions is mandatory for Tregs migration and the consequent bone loss arrest. The local release of exogenous CCL22 provided by poly(lactic‐co‐glycolic acid) (PLGA) microparticles promotes migration of Tregs and disease arrest in the absence of endogenous CCL22 in the IL‐4KO strain, characterized by the lack of endogenous CCL22 production, defective migration of Tregs, and exacerbated bone loss. In summary, our results show that the IL‐4/CCL22/CCR4 axis is involved in the migration of Tregs to osteolytic lesion sites, and attenuates development of lesions by inhibiting inflammatory migration and the production of proinflammatory and osteoclastogenic mediators.

Bone marrow skeletal stem/progenitor cell defects in dyskeratosis congenita and telomere biology disorders

Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM-derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.

Strategies to Direct the Enrichment, Expansion, and Recruitment of Regulatory Cells for the Treatment of Disease

Disease and injury perturb the balance of processes associated with inflammation and tissue remodeling, resulting in positive feedback loops, exacerbation of disease and compromised tissue repair. Conversely, under homeostatic healthy conditions, these processes are tightly regulated through the expansion and/or recruitment of specific cell populations, promoting a balanced steady-state. Better understanding of these regulatory processes and recent advances in biomaterials and biotechnology have prompted strategies to utilize cells for the treatment and prevention of disease through regulation of inflammation and promotion of tissue repair. Herein, we describe how cells that regulate these processes can be increased in prevalence at a site of disease or injury. We review several relevant cell therapy approaches as well as new strategies for directing endogenous regulatory cells capable of promoting environmental homeostasis and even the establishment of a pro-regenerative micro-environment. Collectively, these examples may provide a blueprint for next-generation “medicine” that spurs the body’s own cells to action and replaces conventional drugs.

An in vivo model to assess magnesium alloys and their biological effect on human bone marrow stromal cells.

UNLABELLED Magnesium (Mg) alloys have many unique qualities which make them ideal candidates for bone fixation devices, including biocompatibility and degradation in vivo. Despite a rise in Mg alloy production and research, there remains no standardized system to assess their degradation or biological effect on human stem cells in vivo. In this study, we developed a novel in vivo model to assess Mg alloys for craniofacial and orthopedic applications. Our model consists of a collagen sponge seeded with human bone marrow stromal cells (hBMSCs) around a central Mg alloy rod. These scaffolds were implanted subcutaneously in mice and analyzed after eight weeks. Alloy degradation and biological effect were determined by microcomputed tomography (microCT), histological staining, and immunohistochemistry (IHC). MicroCT showed greater volume loss for pure Mg compared to AZ31 after eight weeks in vivo. Histological analysis showed that hBMSCs were retained around the Mg implants after 8 weeks. Furthermore, immunohistochemistry showed the expression of dentin matrix protein 1 and osteopontin around both pure Mg and AZ31 with implanted hBMSCs. In addition, histological sections showed a thin mineral layer around all degrading alloys at the alloy-tissue interface. In conclusion, our data show that degrading pure Mg and AZ31 implants are cytocompatible and do not inhibit the osteogenic property of hBMSCs in vivo. These results demonstrate that this model can be used to efficiently assess the biological effect of corroding Mg alloys in vivo. Importantly, this model may be modified to accommodate additional cell types and clinical applications. STATEMENT OF SIGNIFICANCE Magnesium (Mg) alloys have been investigated as ideal candidates for bone fixation devices due to high biocompatibility and degradation in vivo, and there is a growing need of establishing an efficient in vivo material screening system. In this study, we assessed degradation rate and biological effect of Mg alloys by transplanting Mg alloy rod with human bone marrow stromal cells seeded on collagen sponge subcutaneously in mice. After 8 weeks, samples were analyzed by microcomputed tomography and histological staining. Our data show that degrading Mg alloys are cytocompatible and do not inhibit the osteogenic property of hBMSCs in vivo. These results demonstrate that this model can be used to efficiently assess the biological effect of corroding Mg alloys in vivo.

Finite Element Analysis of Magnesium Alloy Based Bone Fixation Devices

Each year, there are over six million bone fractures in the U.S., over 30% of which require internal fixation devices to stabilize bone fragments during healing [1–2]. Currently, the gold standard materials for these devices are non-degradable titanium alloys. Unfortunately, these devices cause numerous complications and often require a secondary invasive removal surgery [3–4]. To circumvent these issues, resorbable polymeric devices have been developed, but their mechanical limitations render them inadequate for most load bearing applications [5–7].Copyright