Education & Certifications
Bachelor of Science, Georgia Institute of Technology, Biomedical Engineering (2015)
Osteogenic ability of rat bone marrow concentrate is at least as efficacious as mesenchymal stem cells in vitro.
Journal of biomedical materials research. Part B, Applied biomaterials
Cell therapy using bone marrow concentrate (BMC) or purified and expanded mesenchymal stem cells (MSCs) has been shown to have a promising osteogenic capacity. However, few studies have directly compared their relative osteogenic ability. The aim of this study was to compare the osteogenic ability of BMC isolated by density gradient centrifugation with bone marrow-derived MSCs in vitro using the cells of 3-month-old Sprague-Dawley rats. The isolated cells were seeded onto 24-well plates (1*105 cells/well) and cultured in control growth media, osteogenic media with dexamethasone, or media without dexamethasone (which simulated the in vivo tissue environment). Alkaline phosphatase activity at week 2, osteocalcin using quantitative real-time polymerase chain reaction at week 4, and Alizarin red staining at week 4 were evaluated. In the osteogenic media with dexamethasone, BMC showed equivalent (osteocalcin) or even greater (Alizarin red staining) osteogenic ability compared to MSCs, suggesting that cross-talk among various cells in the BMC leads to greater osteogenesis. Furthermore, in the osteogenic media without dexamethasone, BMC showed equivalent (osteocalcin) or a trend for greater (Alizarin red staining) bone formation than MSCs alone. Our results suggest that BMC has at least comparable bone regeneration potential to MSCs. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: 00B: 000-000, 2019.
View details for DOI 10.1002/jbm.b.34340
View details for PubMedID 30779478
Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages.
Tissue engineering. Part A
Chronic inflammation-associated bone diseases involve continuous destruction and impaired regeneration of bone. Mesenchymal stem cell (MSC)-based therapy has great potential to modulate inflammatory responses and enhance tissue regeneration. We previously showed that lipopolysaccharide [LPS] plus TNF preconditioned MSCs or genetically modified inflammation-sensing (driven by NFB activation) IL4-secreting MSCs enhanced immunomodulation of macrophages to the more desired tissue repaired M2 type. In the current study, the paracrine regulation of therapeutic MSCs on the pro-inflammatory response and osteogenesis of macrophage-MSC co-cultures (representing endogenous cells) was examined using an in vitro transwell system. In the co-cultures, IL4-secreting MSCs decreased TNF and iNOS expression, and increased Arginase 1 and CD206 expression in the presence of LPS-contaminated polyethylene particles. The preconditioned MSCs decreased TNF and CD206 expression in the bottom MSC-macrophage co-cultures in the presence of contaminated particles. In osteogenesis assays, IL4-secreting MSCs decreased ALP expression, but increased alizarin red staining in the presence of contaminated particles. The preconditioned MSCs increased ALP and osteocalcin expression, and had no significant effect on alizarin red staining. These results suggest that potential treatments using preconditioned MSCs at an earlier stage, or IL4-secreting MSCs at a later stage could enhance bone regeneration in inflammatory conditions including periprosthetic osteolysis.
View details for DOI 10.1089/ten.TEA.2018.0292
View details for PubMedID 30652628
Trained murine mesenchymal stem cells have anti-inflammatory effect on macrophages, but defective regulation on T-cell proliferation.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Mesenchymal stem cell (MSC)-mediated immunomodulation affects both innate and adaptive immune systems. These responses to environmental cues, such as pathogen-associated molecular patterns, damage-associated molecular patterns, or proinflammatory cytokines, are crucial for resolution of inflammation, as well as successful tissue healing and regeneration. We observed that intermittent, repeated exposure of MSCs to LPS induced stronger NF-kappaB activation than singular stimulation. A similar phenomenon, named innate immune memory or trained immunity, has been reported with macrophages. However, the potential regulation of "immune memory" in nonclassic immune cells, such as MSCs, has not been reported. In the current study, we chose IFN-gamma plus TNF-alpha restimulation-induced iNOS expression as a model of MSC activation, because IFN-gamma and TNF-alpha play crucial roles in MSC-mediated immunomodulation. The iNOS expression was enhanced in LPS-trained MSCs, 3 d after a washout period following primary stimulation. LPS-trained MSCs enhanced the anti-inflammatory (arginase 1 and CD206) marker expression, but decreased the proinflammatory marker (TNF-alpha, IL-1beta, iNOS, and IL-6) expression using an MSC-macrophage coculture model. In contrast, LPS-trained MSCs demonstrated a defective regulation on CD4 T-cell proliferation. Mechanistic studies suggested that histone methylation and the JNK pathway are involved in LPS-trained immunomodulation in MSCs. Our results demonstrate differential immunomodulatory effects of trained MSCs on macrophages and T cells. These immunomodulatory consequences are critical, because they will have a major impact on current MSC-based cell therapies.-Lin, T., Pajarinen, J., Kohno, Y., Huang, J.-F., Maruyama, M., Romero-Lopez, M., Nathan, K., Yao, Z., Goodman, S. B. Trained murine mesenchymal stem cells have anti-inflammatory effect on macrophages, but defective regulation on T-cell proliferation.
View details for DOI 10.1096/fj.201801845R
View details for PubMedID 30521384
NFkappaB sensing IL-4 secreting mesenchymal stem cells mitigate the proinflammatory response of macrophages exposed to polyethylene wear particles.
Journal of biomedical materials research. Part A
Total joint replacement is a highly effective treatment for patients with end-stage arthritis. Proinflammatory macrophages (M1) mediate wear particle-associated inflammation and bone loss. Anti-inflammatory macrophages (M2) help resolve tissue damage and favor bone regeneration. Mesenchymal stem cell (MSC)-based therapy mitigates the M1 dominated inflammatory reaction and favorably modulates the bone remodeling process. In the current study, the immunomodulating ability of (1) unmodified MSCs, (2) MSCs preconditioned by NFkappaB stimulating ligands [lipopolysaccharide (LPS) plus TNFalpha], and (3) genetically modified MSCs that secrete IL-4 as a response to NFkappaB activation (NFkappaB-IL4) was compared in a macrophage/MSC co-culture system. Sterile or LPS-contaminated ultra-high molecular weight polyethylene particles were used to induce the proinflammatory responses in the macrophages. Contaminated particles induced M1 marker expression (TNFalpha, IL1beta, and iNOS), while NFkappaB-IL4 MSCs modulated the macrophages from an M1 phenotype into a more favorable M2 phenotype (Arginase 1/Arg 1 and CD206 high). The IL4 secretion by NFkappaB-IL4 MSCs was significantly induced by the contaminated particles. The induction of Arg 1 and CD206 in macrophages via the preconditioned or naive MSCs was negligible when compared with NFkappaB-IL4 MSC. Our findings indicated that NFkappaB-IL4 MSCs have the "on-demand" immunomodulatory ability to mitigate wear particle-associated inflammation with minimal adverse effects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2018.
View details for DOI 10.1002/jbm.a.36504
View details for PubMedID 30084534
Transplanted interleukin-4--secreting mesenchymal stromal cells show extended survival and increased bone mineral density in the murine femur.
BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy has great potential to modulate chronic inflammation and enhance tissue regeneration. Crosstalk between MSC-lineage cells and polarized macrophages is critical for bone formation and remodeling in inflammatory bone diseases. However, the translational application of this interaction is limited by the short-term viability of MSCs after cell transplantation.METHODS: Three types of genetically modified (GM) MSCs were created: (1) luciferase-expressing reporter MSCs; (2) MSCs that secrete interleukin (IL)-4 either constitutively; and (3) MSCs that secrete IL-4 as a response to nuclear factor kappa-light-chain-enhancer of activated B cell (NFkappaB) activation. Cells were injected into the murine distal femoral bone marrow cavity. MSC viability and bone formation were examined in vivo. Cytokine secretion was determined in a femoral explant organ culture model.RESULTS: The reporter MSCs survived up to 4 weeks post-implantation. No difference in the number of viable cells was found between high (2.5 * 106) and low (0.5 * 106) cell-injected groups. Injection of 2.5 * 106 reporter MSCs increased local bone mineral density at 4 weeks post-implantation. Injection of 0.5 * 106 constitutive IL-4 or NFkappaB-sensing IL-4-secreting MSCs increased bone mineral density at 2 weeks post-implantation. In the femoral explant organ culture model, LPS treatment induced IL-4 secretion in the NFkappaB-sensing IL-4-secreting MSC group and IL-10 secretion in all the femur samples. No significant differences in tumor necrosis factor (TNF)alpha and IL-1beta secretion were observed between the MSC-transplanted and control groups in the explant culture.DISCUSSION: Transplanted GM MSCs demonstrated prolonged cell viability when transplanted to a compatible niche within the bone marrow cavity. GM IL-4-secreting MSCs may have great potential to enhance bone regeneration in disorders associated with chronic inflammation.
View details for DOI 10.1016/j.jcyt.2018.06.009
View details for PubMedID 30077567
Mesenchymal stem cell-macrophage crosstalk and bone healing.
Recent research has brought about a clear understanding that successful fracture healing is based on carefully coordinated cross-talk between inflammatory and bone forming cells. In particular, the key role that macrophages play in the recruitment and regulation of the differentiation of mesenchymal stem cells (MSCs) during bone regeneration has been brought to focus. Indeed, animal studies have comprehensively demonstrated that fractures do not heal without the direct involvement of macrophages. Yet the exact mechanisms by which macrophages contribute to bone regeneration remain to be elucidated. Macrophage-derived paracrine signaling molecules such as Oncostatin M, Prostaglandin E2 (PGE2), and Bone Morphogenetic Protein-2 (BMP2) have been shown to play critical roles; however the relative importance of inflammatory (M1) and tissue regenerative (M2) macrophages in guiding MSC differentiation along the osteogenic pathway remains poorly understood. In this review, we summarize the current understanding of the interaction of macrophages and MSCs during bone regeneration, with the emphasis on the role of macrophages in regulating bone formation. The potential implications of aging to this cellular cross-talk are reviewed. Emerging treatment options to improve facture healing by utilizing or targeting MSC-macrophage crosstalk are also discussed.
View details for DOI 10.1016/j.biomaterials.2017.12.025
View details for PubMedID 29329642
- Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis STEM CELL RESEARCH & THERAPY 2017; 8
Inflammation, ageing, and bone regeneration
JOURNAL OF ORTHOPAEDIC TRANSLATION
2017; 10: 28–35
Bone healing involves complex biological pathways and interactions among various cell types and microenvironments. Among them, the monocyte-macrophage-osteoclast line-age and the mesenchymal stem cell-osteoblast lineage are critical, in addition to an initial inflammatory microenvironment. These cellular interactions induce the necessary inflammatory milieu and provide the cells for bone regeneration and immune modulation. Increasing age is accompanied with a rise in the basal state of inflammation, potentially impairing osteogenesis.Translational research has shown multiple interactions between inflammation, ageing, and bone regeneration. This review presents recent, relevant considerations regarding the effects of inflammation and ageing on bone healing.
View details for DOI 10.1016/j.jot.2017.04.002
View details for Web of Science ID 000406877300006
View details for PubMedID 29094003
View details for PubMedCentralID PMC5662134
Mesenchymal stem cells in the aseptic loosening of total joint replacements.
Journal of biomedical materials research. Part A
2017; 105 (4): 1195-1207
Peri-prosthetic osteolysis remains as the main long-term complication of total joint replacement surgery. Research over four decades has established implant wear as the main culprit for chronic inflammation in the peri-implant tissues and macrophages as the key cells mediating the host reaction to implant-derived wear particles. Wear debris activated macrophages secrete inflammatory mediators that stimulate bone resorbing osteoclasts; thus bone loss in the peri-implant tissues is increased. However, the balance of bone turnover is not only dictated by osteoclast-mediated bone resorption but also by the formation of new bone by osteoblasts; under physiological conditions these two processes are tightly coupled. Increasing interest has been placed on the effects of wear debris on the cells of the bone-forming lineage. These cells are derived primarily from multipotent mesenchymal stem cells (MSCs) residing in bone marrow and the walls of the microvasculature. Accumulating evidence indicates that wear debris significantly impairs MSC-to-osteoblast differentiation and subsequent bone formation. In this review, we summarize the current understanding of the effects of biomaterial implant wear debris on MSCs. Emerging treatment options to improve initial implant integration and treat developing osteolytic lesions by utilizing or targeting MSCs are also discussed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1195-1207, 2017.
View details for DOI 10.1002/jbm.a.35978
View details for PubMedID 27977880
Pro-inflammatory M1 macrophages promote osteogenesis by mesenchymal stem cells via the COX-2-prostaglandin E2 pathway.
Journal of orthopaedic research
Bone fractures are among the most common orthopaedic problems that affect individuals of all ages. Immediately after injury, activated macrophages dynamically contribute to and regulate an acute inflammatory response that involves other cells at the injury site, including mesenchymal stem cells (MSCs). These macrophages and MSCs work in concert to modulate bone healing. In this study, we co-cultured undifferentiated M0, pro-inflammatory M1, and anti-inflammatory M2 macrophages with primary murine MSCs in vitro to determine the cross-talk between polarized macrophages and MSCs and their effects on osteogenesis. After 4 weeks of co-culture, MSCs grown with macrophages, especially M1 macrophages, had enhanced bone mineralization compared to MSCs grown alone. The level of bone formation after 4 weeks of culture was closely associated with prostaglandin E2 (PGE2) secretion early in osteogenesis. Treatment with celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, significantly reduced bone mineralization in all co-cultures but most dramatically in the M1-MSC co-culture. We also found that the presence of macrophages reduced the secretion of osteoprotegerin (OPG), the decoy RANKL receptor, suggesting that macrophages may indirectly modulate osteoclast activity in addition to enhancing bone formation. Taken together, these findings suggest that an initial pro-inflammatory phase modulated by M1 macrophages promotes osteogenesis in MSCs via the COX-2-PGE2 pathway. Understanding the complex interactions between macrophages and MSCs provide opportunities to optimize bone healing and other regenerative processes via modulation of the inflammatory response. This study provides one possible biological mechanism for the adverse effects of non-steroidal anti-inflammatory drugs on fracture healing and bone regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
View details for DOI 10.1002/jor.23553
View details for PubMedID 28248001
Establishment of NF-κB sensing and interleukin-4 secreting mesenchymal stromal cells as an "on-demand" drug delivery system to modulate inflammation.
Chronic inflammation is associated with up-regulation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and excessive inflammatory cytokine secretion by M1 macrophages. The anti-inflammatory cytokine interleukin (IL)-4 converts pro-inflammatory M1 macrophages into an anti-inflammatory and tissue-regenerative M2 phenotype, thus reducing inflammation and enhancing tissue regeneration. We have generated NF-κB responsive, or constitutively active IL-4 expression lentiviral vectors transduced into murine bone marrow-derived mesenchymal stromal cells (MSCs). MSCs with a constitutively active IL-4 expression vector produced large quantities of IL-4 continuously, whereas IL-4 secretion was significantly induced by lipopolysaccharide (LPS) in the NF-κB sensing MSCs. In contrast, LPS had no effect on MSCs with IL-4 secretion driven by a constitutively active promoter. We also found that intermittent and continuous LPS treatment displayed distinct NF-κB activation profiles, and this regulation was independent of IL-4 signaling. The supernatant containing IL-4 from the LPS-treated MSCs suppressed M1 marker (inducible nitric oxide synthase [iNOS] and tumor necrosis factor alpha [TNFα]) expression and enhanced M2 marker (Arginase 1, CD206 and IL1 receptor antagonist [IL1Ra]) expression in primary murine macrophages. The IL-4 secretion at the basal, non-LPS induced level was sufficient to suppress TNFα and enhance Arginase 1 at a lower level, but had no significant effects on iNOS, CD206 and IL1Ra expression. Finally, IL-4 secretion at basal or LPS-induced levels significantly suppressed osteogenic differentiation of MSCs. Our findings suggest that the IL-4 secreting MSCs driven by NF-κB sensing or constitutive active promoter have great potential for mitigating the effects of chronic inflammation and promoting earlier tissue regeneration.
View details for DOI 10.1016/j.jcyt.2017.06.008
View details for PubMedID 28739167
Murine Model of Progressive Orthopaedic Wear Particle Induced Chronic Inflammation and Osteolysis.
Tissue engineering. Part C, Methods
Periprosthetic osteolysis and subsequent aseptic loosening of total joint replacements are driven by byproducts of wear released from the implant. Wear particles cause macrophage mediated inflammation that culminates with periprosthetic bone loss. Most current animal models of particle- induced osteolysis are based on the acute inflammatory reaction induced by wear debris, which is distinct from the slowly progressive clinical scenario. To address this limitation we previously developed a murine model of periprosthetic osteolysis that is based on slow continuous delivery of wear particles into the murine distal femur over a period of 4 weeks. The particle delivery was accomplished by using subcutaneously implanted osmotic pumps and tubing, and a hollow titanium rod press-fit into the distal femur. In this study we report a modification of our prior model in which particle delivery is extended to 8 weeks to better mimic the progressive development of periprosthetic osteolysis and to allow the assessment of interventions in a setting where the chronic particle induced osteolysis is already present at the initiation of the treatment. Compared to 4 week samples extending the particle delivery to 8 weeks significantly exacerbated the local bone loss observed with µCT and the amount of both peri-implant F4/80+ macrophages and TRAP+ osteoclasts detected with immunohistochemical and histochemical stainings. Furthermore systemic recruitment of reporter macrophages to peri-implant tissues observed with bioluminescence imaging continued even at the later stages of particle induced inflammation. This modified model system could provide new insights into the mechanisms of chronic inflammatory bone loss and be particularly useful in assessing the efficacy of treatments in a setting that resembles the clinical scenario of developing periprosthetic osteolysis more closely than currently existing model systems.
View details for DOI 10.1089/ten.TEC.2017.0166
View details for PubMedID 28978284
Orthopaedic Wear Particle-induced Bone Loss and Exogenous Macrophage Infiltration is mitigated by Local Infusion of NF-κB Decoy Oligodeoxynucleotide.
Journal of biomedical materials research. Part A
Excessive production of wear particles from total joint replacements (TJRs) induces chronic inflammation, macrophage infiltration, and consequent bone loss (periprosthetic osteolysis). This inflammation and bone remodeling are critically regulated by the transcription factor NF-κB. We previously demonstrated that inhibition of NF-κB signaling by using the decoy oligodeoxynucleotide (ODN) mitigates polyethylene wear particle-induced bone loss using in vitro and in vivo models. However, the mechanisms of NF-κB decoy ODN action, and in particular its impact on systemic macrophage recruitment, remain unknown. In the current study, this systemic macrophage infiltration was examined in our established murine femoral continuous particle infusion model. RAW264.7 murine macrophages expressing a luciferase reporter gene were injected into the systemic circulation. Quantification of bioluminescence showed that NF-κB decoy ODN reduced the homing of these reporter macrophages into the distal femurs exposed to continuous particle delivery. Particle-induced reduction in bone mineral density at the distal diaphysis of the femur was also mitigated by infusion of decoy ODN. Histological staining showed that the decoy ODN infusion decreased osteoclast and macrophage numbers, but had no significant effects on osteoblasts. Local infusion of NF-κB decoy ODN reduced systemic macrophage infiltration and mitigated particle-induced bone loss, thus providing a potential strategy to treat periprosthetic osteolysis. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/jbm.a.36169
View details for PubMedID 28782280
Hydrolysis and sulfation pattern effects on release of bioactive bone morphogenetic protein-2 from heparin-based microparticles
JOURNAL OF MATERIALS CHEMISTRY B
2015; 3 (40): 8001-8009
Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.
View details for DOI 10.1039/c5tb00933b
View details for Web of Science ID 000362663000019
View details for PubMedID 27785363
View details for PubMedCentralID PMC5077163