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INTRODUCTION: Mesenchymal stem and progenitor cells (MSCs), which normally reside in the bone marrow, are critical to bone health and can be recruited to sites of traumatic bone injury, contributing to new bone formation. The ability to control the trafficking of MSCs provides therapeutic potential for improving traumatic bone healing and therapy for genetic bone diseases such as hypophosphatasia. METHODS: In this study, we explored the sphingosine-1-phosphate (S1P) signaling axis as a means to control the mobilization of MSCs into blood and possibly to recruit MSCs enhancing bone growth. RESULTS: Loss of S1P receptor 3 (S1PR3) leads to an increase in circulating CD45-/CD29+/CD90+/Sca1 putative mesenchymal progenitor cells, suggesting that blocking S1PR3 may stimulate MSCs to leave the bone marrow. Antagonism of S1PR3 with the small molecule VPC01091 stimulated acute migration of CD45-/CD29+/CD90+/Sca1+ MSCs into the blood as early as 1.5 hours after treatment. VPC01091 administration also increased ectopic bone formation induced by BMP-2 and significantly increased new bone formation in critically sized rat cranial defects, suggesting that mobilized MSCs may home to injuries to contribute to healing. We also explored the possibility of combining S1P manipulation of endogenous host cell occupancy with exogenous MSC transplantation for potential use in combination therapies. Importantly, reducing niche occupancy of host MSCs with VPC01091 does not impede engraftment of exogenous MSCs. CONCLUSIONS: Our studies suggest that MSC mobilization through S1PR3 antagonism is a promising strategy for endogenous tissue engineering and improving MSC delivery to treat bone diseases.
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Hematopoietic stem and progenitor cells (HSPCs) egress from bone marrow (BM) during homeostasis and at increased rates during stress; however, the mechanisms regulating their trafficking remain incompletely understood. Here we describe a novel role for lipid receptor, sphingosine-1-phosphate receptor 3 (S1PR3), in HSPC residence within the BM niche. HSPCs expressed increased levels of S1PR3 compared to differentiated BM cells. Pharmacological antagonism or knockout (KO) of S1PR3 mobilized HSPCs into blood circulation, suggesting that S1PR3 influences niche localization. S1PR3 antagonism suppressed BM and plasma SDF-1, enabling HSPCs to migrate toward S1P-rich plasma. Mobilization synergized with AMD3100-mediated antagonism of CXCR4, which tethers HSPCs in the niche, and recovered homing deficits of AMD3100-treated grafts. S1PR3 antagonism combined with AMD3100 improved re-engraftment and survival in lethally irradiated recipients. Our studies indicate that S1PR3 and CXCR4 signaling cooperate to maintain HSPCs within the niche under homeostasis. These results highlight an important role for S1PR3 in HSPC niche occupancy and trafficking that can be harnessed for both rapid clinical stem cell mobilization and re-engraftment strategies, as well as the opportunity to design novel therapeutics for control of recruitment, homing, and localization through bioactive lipid signaling. Stem Cells 2017;35:1040-1052.
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Células-Tronco Hematopoéticas/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Adesão Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Ligantes , Lisofosfolipídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Radiação Ionizante , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Nicho de Células-Tronco/efeitos dos fármacosRESUMO
We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (P(VEGF)) or BMP-6 (P(BMP-6)) or both (P(VEGF+BMP-6)) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with P(VEGF+BMP-6) scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the P(VEGF+BMP-6) group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in P(VEGF), P(BMP-6) and P(VEGF+BMP-6) but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with P(VEGF), P(BMP-6), or P(VEGF+BMP-6) but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on P(VEGF+BMP-6) but not on P, P(VEGF), or P(BMP-6). Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.
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Proteína Morfogenética Óssea 6 , Ácido Láctico , Doenças Mandibulares/terapia , Células-Tronco Mesenquimais/metabolismo , Ácido Poliglicólico , Alicerces Teciduais/química , Fator A de Crescimento do Endotélio Vascular , Animais , Proteína Morfogenética Óssea 6/química , Proteína Morfogenética Óssea 6/farmacologia , Humanos , Ácido Láctico/química , Ácido Láctico/farmacologia , Mandíbula/metabolismo , Mandíbula/patologia , Doenças Mandibulares/patologia , Células-Tronco Mesenquimais/patologia , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Endogâmicos F344 , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to locally deliver the immunomodulatory small molecule FTY720 in tibial defects created in rat bone marrow chimeras containing genetically-labeled bone marrow for monitoring cell origin and fate. Donor bone marrow contributed significantly to both myeloid and osteogenic cells in remodeling tissue surrounding allografts. FTY720 coatings altered the phenotype of immune cells two weeks post-injury, which was associated with increased vascularization and bone formation surrounding allografts. Consequently, degradable polymer coating strategies that deliver small molecule growth factors such as FTY720 represent a novel therapeutic strategy for harnessing endogenous bone marrow-derived progenitors and enhancing healing in load-bearing bone defects.
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Transplante Ósseo , Cloridrato de Fingolimode/administração & dosagem , Fatores Imunológicos/administração & dosagem , Aloenxertos , Animais , Células da Medula Óssea/citologia , Osso e Ossos/fisiologia , Linhagem da Célula , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Ácido Láctico/administração & dosagem , Masculino , Mielopoese/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ácido Poliglicólico/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Quimera por Radiação , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Regeneração , Estresse Mecânico , Tíbia/lesões , Tíbia/cirurgia , Microtomografia por Raio-XAssuntos
Desferroxamina/farmacologia , Retalho Miocutâneo/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Reto do Abdome/irrigação sanguínea , Sideróforos/farmacologia , Animais , Fenômenos Biomecânicos , Desferroxamina/administração & dosagem , Esquema de Medicação , Injeções Subcutâneas , Ratos , Reto do Abdome/cirurgia , Sideróforos/administração & dosagemRESUMO
Craniofacial fractures and bony defects are common causes of morbidity and contribute to increasing health care costs. Successful regeneration of bone requires the concomitant processes of osteogenesis and neovascularization. Current methods of repair and reconstruction include rigid fixation, grafting, and free tissue transfer. However, these methods carry innate complications, including plate extrusion, nonunion, graft/flap failure, and donor site morbidity. Recent research efforts have focused on using stem cells and synthetic scaffolds to heal critical-sized bone defects similar to those sustained from traumatic injury or ablative oncologic surgery. Growth factors can be used to augment both osteogenesis and neovascularization across these defects. Many different growth factor delivery techniques and scaffold compositions have been explored yet none have emerged as the universally accepted standard. In this review, we will discuss the recent literature regarding the use of stem cells, growth factors, and synthetic scaffolds as alternative methods of craniofacial fracture repair.
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BACKGROUND: Radiation therapy results in permanent damage to the microvasculature, leading to dermal damage and inelasticity in normal tissues. Deferoxamine is a U.S. Food and Drug Administration-approved iron-chelating medication that has also been shown to increase angiogenesis. The authors hypothesize that the application of deferoxamine will result in increased vascularity and improved tissue elasticity in a rat irradiated transverse rectus abdominis musculocutaneous flap model. METHODS: Fifteen rats underwent a transverse rectus abdominis myocutaneous flap and were randomized to three groups: control, radiation therapy, and radiation therapy plus deferoxamine. The flaps in the radiation therapy and radiation therapy plus deferoxamine groups were irradiated with 35 Gy in a single dose. Four weeks after irradiation, rats in the radiation therapy plus deferoxamine group were treated with deferoxamine. Flaps were imaged with micro-computed tomographic angiography. Flap creep and stress relaxation were assessed using a tensiometer. Hematoxylin and eosin, picrosirius red, and Verhoeff-van Gieson staining was performed. RESULTS: Irradiated flaps demonstrated gross stigmata of cutaneous radiation injury within 4 weeks. Histologically, the epidermis in the radiation therapy flaps was found to be thicker than in the radiation therapy plus deferoxamine and control flaps (p < 0.001). Micro-computed tomographic angiography demonstrated a statistically significant (p < 0.05) increase in vascularity in the radiation therapy plus deferoxamine flaps compared with radiation therapy alone. The creep curve was indicative of increased elasticity in the radiation therapy plus deferoxamine flaps compared with radiation therapy flaps. CONCLUSION: Deferoxamine appears to mitigate radiation-induced hypovascularity and improve tissue elasticity in a rat model of soft-tissue reconstruction.
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Desferroxamina/uso terapêutico , Lesões por Radiação/tratamento farmacológico , Retalhos Cirúrgicos , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Reto do Abdome/transplanteRESUMO
Local delivery of lipid mediators has become a promising new approach for therapeutic angiogenesis and regenerative medicine. In this study, we investigated how gradient stimulation (either abluminal/distal or luminal/proximal) of engineered microvessels with sphingosine 1-phosphate (S1P) receptor-subtype-targeted molecules affects endothelial sprout growth using a microfluidic device. Our studies show that distal stimulation of microvessels with FTY720, an S1P1/3 selective agonist, promotes both arterial and venular sprout growth, whereas proximal stimulation does not. Using novel pharmacological antagonists of S1P receptor subtypes, we further show that S1P3 functionality is necessary for VEGF-induced sprouting, and confirmed these findings ex vivo using a murine aortic ring assay from S1P3-deficient mice. S1P3 agonist stimulation enhanced vascular stability in both cell types via upregulation of the interendothelial junction protein VE-cadherin. Lastly, S1P3 activation under flow promoted endothelial sprouting and branching while decreasing migratory cell fate in the microfluidic device. We used an in vivo murine dorsal skinfold window chamber model to confirm S1P3's role in neovascular branching. Together, these data suggest that a distal transendothelial gradient of S1P1/3-targeted drugs is an effective technique for both enhancing and stabilizing capillary morphogenesis in angiogenic applications.
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Células Endoteliais/metabolismo , Neovascularização Fisiológica , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Antígenos CD , Aorta/citologia , Caderinas , Movimento Celular/efeitos dos fármacos , Difusão , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Endogenous signals originating at the site of injury are involved in the paracrine recruitment, proliferation, and differentiation of circulating progenitor and diverse inflammatory cell types. Here, we investigate a strategy to exploit endogenous cell recruitment mechanisms to regenerate injured bone by local targeting and activation of sphingosine-1-phosphate (S1P) receptors. A mandibular defect model was selected for evaluating regeneration of bone following trauma or congenital disease. The particular challenges of mandibular reconstruction are inherent in the complex anatomy and function of the bone given that the area is highly vascularized and in close proximity to muscle. Nanofibers composed of poly(DL-lactide-co-glycolide) (PLAGA) and polycaprolactone (PCL) were used to delivery FTY720, a targeted agonist of S1P receptors 1 and 3. In vitro culture of bone progenitor cells on drug-loaded constructs significantly enhanced SDF1α mediated chemotaxis of bone marrow mononuclear cells. In vivo results show that local delivery of FTY720 from composite nanofibers enhanced blood vessel ingrowth and increased recruitment of M2 alternatively activated macrophages, leading to significant osseous tissue ingrowth into critical sized defects after 12 weeks of treatment. These results demonstrate that local activation of S1P receptors is a regenerative cue resulting in recruitment of wound healing or anti-inflammatory macrophages and bone healing. Use of such small molecule therapy can provide an alternative to biological factors for the clinical treatment of critical size craniofacial defects.
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Macrófagos/metabolismo , Mandíbula , Nanofibras/química , Receptores de Lisoesfingolipídeo/metabolismo , Cicatrização/fisiologia , Animais , Cloridrato de Fingolimode , Lisofosfolipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Poliésteres/química , Propilenoglicóis/química , Ratos , Esfingosina/análogos & derivados , Esfingosina/químicaRESUMO
Endogenous stem cell recruitment to the site of skeletal injury is key to enhanced osseous remodeling and neovascularization. To this end, this study utilized a novel bone allograft coating of poly(lactic-co-glycolic acid) (PLAGA) to sustain the release of FTY720, a selective agonist for sphingosine 1-phosphate (S1P) receptors, from calvarial allografts. Uncoated allografts, vehicle-coated, low dose FTY720 in PLAGA (1:200 w:w) and high dose FTY720 in PLAGA (1:40) were implanted into critical size calvarial bone defects. The ability of local FTY720 delivery to promote angiogenesis, maximize osteoinductivity and improve allograft incorporation by recruitment of bone progenitor cells from surrounding soft tissues and microcirculation was evaluated. FTY720 bioactivity after encapsulation and release was confirmed with sphingosine kinase 2 assays. HPLC-MS quantified about 50% loaded FTY720 release of the total encapsulated drug (4.5 µg) after 5 days. Following 2 weeks of defect healing, FTY720 delivery led to statistically significant increases in bone volumes compared to controls, with total bone volume increases for uncoated, coated, low FTY720 and high FTY720 of 5.98, 3.38, 7.2 and 8.9 mm(3), respectively. The rate and extent of enhanced bone growth persisted through week 4 but, by week 8, increases in bone formation in FTY720 groups were no longer statistically significant. However, micro-computed tomography (microCT) of contrast enhanced vascular ingrowth (MICROFIL®) and histological analysis showed enhanced integration as well as directed bone growth in both high and low dose FTY720 groups compared to controls.
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Transplante Ósseo , Sistemas de Liberação de Medicamentos/métodos , Osteogênese/efeitos dos fármacos , Propilenoglicóis/administração & dosagem , Propilenoglicóis/farmacologia , Crânio/efeitos dos fármacos , Esfingosina/análogos & derivados , Actinas/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/crescimento & desenvolvimento , Remodelação Óssea/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Feminino , Cloridrato de Fingolimode , Fluorescência , Ácido Láctico/química , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Periósteo/irrigação sanguínea , Periósteo/diagnóstico por imagem , Periósteo/efeitos dos fármacos , Periósteo/crescimento & desenvolvimento , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Sprague-Dawley , Crânio/irrigação sanguínea , Crânio/patologia , Crânio/cirurgia , Esfingosina/administração & dosagem , Esfingosina/farmacologia , Transplante Homólogo , Microtomografia por Raio-XRESUMO
Angiogenesis is crucial during many physiological processes and is influenced by various biochemical and biomechanical factors. Models have proved useful in understanding the mechanisms of angiogenesis and also the characteristics of the capillaries formed as part of the process. We have developed a three-dimensional hybrid, agent-field model where individual cells are modelled as sprout-forming agents in a matrix field. Cell independence, cell-cell communication and stochastic cell response are integral parts of the model. The model simulations incorporate probabilities of an individual cell to transition into one of four stages--quiescence, proliferation, migration and apoptosis. We demonstrate that several features, such as continuous sprouts, cell clustering and branching, that are observed in microfluidic experiments conducted under controlled conditions using few angiogenic factors can be reproduced by this model. We also identify the transition probabilities that result in specific sprout characteristics such as long continuous sprouts and specific branching patterns. Thus, this model can be used to cluster sprout morphology as a function of various influencing factors.