ABSTRACT
Oxidative stress is broadly implicated in chronic, inflammatory diseases because it causes protein and lipid damage, cell death, and stimulation of inflammatory signaling. Supplementation of innate antioxidant mechanisms with drugs such as the superoxide dismutase (SOD) mimetic compound 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) is a promising strategy for reducing oxidative stress-driven pathologies. TEMPO is inexpensive to produce and has strong antioxidant activity, but it is limited as a drug due to rapid clearance from the body. It is also challenging to encapsulate into micellar nanoparticles or polymer microparticles, because it is a small, water soluble molecule that does not efficiently load into hydrophobic carrier systems. In this work, we pursued a polymeric form of TEMPO [poly(TEMPO)] to increase its molecular weight with the goal of improving in vivo bioavailability. High density of TEMPO on the poly(TEMPO) backbone limited water solubility and bioactivity of the product, a challenge that was overcome by tuning the density of TEMPO in the polymer by copolymerization with the hydrophilic monomer dimethylacrylamide (DMA). Using this strategy, we formed a series of poly(DMA-co-TEMPO) random copolymers. An optimal composition of 40 mol % TEMPO/60 mol % DMA was identified for water solubility and O2â¢- scavenging in vitro. In an air pouch model of acute local inflammation, the optimized copolymer outperformed both the free drug and a 100% poly(TEMPO) formulation in O2â¢- scavenging, retention, and reduction of TNFα levels. Additionally, the optimized copolymer reduced ROS levels after systemic injection in a footpad model of inflammation. These results demonstrate the benefit of polymerizing TEMPO for in vivo efficacy and could lead to a useful antioxidant polymer formulation for next-generation anti-inflammatory treatments.
Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Cyclic N-Oxides/chemistry , Cyclic N-Oxides/pharmacology , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
Clinical imaging approaches to detect inflammatory biomarkers, such as cyclooxygenase-2 (COX-2), may facilitate the diagnosis and therapy of inflammatory diseases. To this end, we report the discovery of N-[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide chloride salt (fluorocoxib D), a hydrophilic analog of fluorocoxib A. Fluorocoxib D inhibits COX-2 selectively in purified enzyme preparations and cells. It exhibits adequate photophysical properties to enable detection of COX-2 in intact cells, in a mouse model of carrageenan-induced acute footpad inflammation and inflammation in a mouse model of osteoarthritis. COX-2-selectivity was verified either by blocking the enzyme's active site with celecoxib or by molecular imaging with nontargeted 5-carboxy-X-rhodamine dye. These data indicate that fluorocoxib D is an ideal candidate for early detection of inflammatory or neoplastic lesions expressing elevated levels of COX-2.
ABSTRACT
The inherent antioxidant function of poly(propylene sulfide) (PPS) microspheres (MS) was dissected for different reactive oxygen species (ROS), and therapeutic benefits of PPS-MS were explored in models of diabetic peripheral arterial disease (PAD) and mechanically induced post-traumatic osteoarthritis (PTOA). PPS-MS (â¼1 µm diameter) significantly scavenged hydrogen peroxide (H2O2), hypochlorite, and peroxynitrite but not superoxide in vitro in cell-free and cell-based assays. Elevated ROS levels (specifically H2O2) were confirmed in both a mouse model of diabetic PAD and in a mouse model of PTOA, with greater than 5- and 2-fold increases in H2O2, respectively. PPS-MS treatment functionally improved recovery from hind limb ischemia based on â¼15-25% increases in hemoglobin saturation and perfusion in the footpads as well as earlier remodeling of vessels in the proximal limb. In the PTOA model, PPS-MS reduced matrix metalloproteinase (MMP) activity by 30% and mitigated the resultant articular cartilage damage. These results suggest that local delivery of PPS-MS at sites of injury-induced inflammation improves the vascular response to ischemic injury in the setting of chronic hyperglycemia and reduces articular cartilage destruction following joint trauma. These results motivate further exploration of PPS as a stand-alone, locally sustained antioxidant therapy and as a material for microsphere-based, sustained local drug delivery to inflamed tissues at risk of ROS damage.
ABSTRACT
Small-molecule inhibitors of the mTORC2 kinase (torkinibs) have shown efficacy in early clinical trials. However, the torkinibs under study also inhibit the other mTOR-containing complex mTORC1. While mTORC1/mTORC2 combined inhibition may be beneficial in cancer cells, recent reports describe compensatory cell survival upon mTORC1 inhibition due to loss of negative feedback on PI3K, increased autophagy, and increased macropinocytosis. Genetic models suggest that selective mTORC2 inhibition would be effective in breast cancers, but the lack of selective small-molecule inhibitors of mTORC2 have precluded testing of this hypothesis to date. Here we report the engineering of a nanoparticle-based RNAi therapeutic that can effectively silence the mTORC2 obligate cofactor Rictor. Nanoparticle-based Rictor ablation in HER2-amplified breast tumors was achieved following intratumoral and intravenous delivery, decreasing Akt phosphorylation and increasing tumor cell killing. Selective mTORC2 inhibition in vivo, combined with the HER2 inhibitor lapatinib, decreased the growth of HER2-amplified breast cancers to a greater extent than either agent alone, suggesting that mTORC2 promotes lapatinib resistance, but is overcome by mTORC2 inhibition. Importantly, selective mTORC2 inhibition was effective in a triple-negative breast cancer (TNBC) model, decreasing Akt phosphorylation and tumor growth, consistent with our findings that RICTOR mRNA correlates with worse outcome in patients with basal-like TNBC. Together, our results offer preclinical validation of a novel RNAi delivery platform for therapeutic gene ablation in breast cancer, and they show that mTORC2-selective targeting is feasible and efficacious in this disease setting.Significance: This study describes a nanomedicine to effectively inhibit the growth regulatory kinase mTORC2 in a preclinical model of breast cancer, targeting an important pathogenic enzyme in that setting that has been undruggable to date. Cancer Res; 78(7); 1845-58. ©2018 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Lapatinib/pharmacology , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Xenograft Model Antitumor Assays , Animals , Cell Survival/drug effects , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Nanoparticles , RNA, Small Interfering/genetics , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Receptor, ErbB-2/metabolism , Triple Negative Breast Neoplasms/pathologyABSTRACT
Although siRNA-based nanomedicines hold promise for cancer treatment, conventional siRNA-polymer complex (polyplex) nanocarrier systems have poor pharmacokinetics following intravenous delivery, hindering tumor accumulation. Here, we determined the impact of surface chemistry on the in vivo pharmacokinetics and tumor delivery of siRNA polyplexes. A library of diblock polymers was synthesized, all containing the same pH-responsive, endosomolytic polyplex core-forming block but different corona blocks: 5 kDa (benchmark) and 20 kDa linear polyethylene glycol (PEG), 10 kDa and 20 kDa brush-like poly(oligo ethylene glycol), and 10 kDa and 20 kDa zwitterionic phosphorylcholine-based polymers (PMPC). In vitro, it was found that 20 kDa PEG and 20 kDa PMPC had the highest stability in the presence of salt or heparin and were the most effective at blocking protein adsorption. Following intravenous delivery, 20 kDa PEG and PMPC coronas both extended circulation half-lives 5-fold compared to 5 kDa PEG. However, in mouse orthotopic xenograft tumors, zwitterionic PMPC-based polyplexes showed highest in vivo luciferase silencing (>75% knockdown for 10 days with single IV 1 mg/kg dose) and 3-fold higher average tumor cell uptake than 5 kDa PEG polyplexes (20 kDa PEG polyplexes were only 2-fold higher than 5 kDa PEG). These results show that high molecular weight zwitterionic polyplex coronas significantly enhance siRNA polyplex pharmacokinetics without sacrificing polyplex uptake and bioactivity within tumors when compared to traditional PEG architectures.
Subject(s)
Drug Carriers/chemistry , Nanostructures/chemistry , Neoplasms/therapy , Phosphorylcholine/chemistry , Polyethylene Glycols/chemistry , RNA, Small Interfering/administration & dosage , RNAi Therapeutics/methods , Animals , Cell Line, Tumor , Female , Humans , Male , Mice, Nude , Neoplasms/genetics , Polymers/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacokinetics , RNA, Small Interfering/therapeutic use , Surface PropertiesABSTRACT
A rationally-designed library of ternary siRNA polyplexes was developed and screened for gene silencing efficacy in vitro and in vivo with the goal of overcoming both cell-level and systemic delivery barriers. [2-(dimethylamino)ethyl methacrylate] (DMAEMA) was homopolymerized or copolymerized (50mol% each) with butyl methacrylate (BMA) from a reversible addition - fragmentation chain transfer (RAFT) chain transfer agent, with and without pre-conjugation to polyethylene glycol (PEG). Both single block polymers were tested as core-forming units, and both PEGylated, diblock polymers were screened as corona-forming units. Ternary siRNA polyplexes were assembled with varied amounts and ratios of core-forming polymers to PEGylated corona-forming polymers. The impact of polymer composition/ratio, hydrophobe (BMA) placement, and surface PEGylation density was correlated to important outcomes such as polyplex size, stability, pH-dependent membrane disruptive activity, biocompatibility, and gene silencing efficiency. The lead formulation, DB4-PDB12, was optimally PEGylated not only to ensure colloidal stability (no change in size by DLS between 0 and 24h) and neutral surface charge (0.139mV) but also to maintain higher cell uptake (>90% positive cells) than the most densely PEGylated particles. The DB4-PDB12 polyplexes also incorporated BMA in both the polyplex core- and corona-forming polymers, resulting in robust endosomolysis and in vitro siRNA silencing (~85% protein level knockdown) of the model gene luciferase across multiple cell types. Further, the DB4-PDB12 polyplexes exhibited greater stability, increased blood circulation time, reduced renal clearance, increased tumor biodistribution, and greater silencing of luciferase compared to our previously-optimized, binary parent formulation following intravenous (i.v.) delivery. This polyplex library approach enabled concomitant optimization of the composition and ratio of core- and corona-forming polymers (indirectly tuning PEGylation density) and identification of a ternary nanomedicine optimized to overcome important siRNA delivery barriers in vitro and in vivo.
Subject(s)
Methacrylates/chemistry , Polyethylene Glycols/chemistry , RNA, Small Interfering/administration & dosage , Animals , Cell Line , Cell Line, Tumor , Female , Humans , Hydrophobic and Hydrophilic Interactions , Luciferases/genetics , Mice , Mice, Nude , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacokinetics , Tissue DistributionABSTRACT
The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic ß-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to ß-cells in culture and in transplanted tissue models. These technologies allow the study of candidate ß-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating ß-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on ß-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess ß-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.
Subject(s)
Biocompatible Materials , Drug Delivery Systems , Microspheres , Mitogens , Animals , Coculture Techniques , Glycols , Humans , Insulin-Secreting Cells , Islets of Langerhans , Lactic Acid , Mice , Polyglycolic AcidABSTRACT
Self-assembled polymer/porous silicon nanocomposites overcome intracellular and systemic barriers for in vivo application of peptide nucleic acid (PNA) anti-microRNA therapeutics. Porous silicon (PSi) is leveraged as a biodegradable scaffold with high drug-cargo-loading capacity. Functionalization with a diblock polymer improves PSi nanoparticle colloidal stability, in vivo pharmacokinetics, and intracellular bioavailability through endosomal escape, enabling PNA to inhibit miR-122 in vivo.
Subject(s)
MicroRNAs/antagonists & inhibitors , Nanocomposites/chemistry , Peptide Nucleic Acids/administration & dosage , Peptide Nucleic Acids/therapeutic use , Polymers/chemistry , Silicon/chemistry , Animals , Cell Line, Tumor , Colloids/chemistry , Female , Humans , Mice , MicroRNAs/genetics , Peptide Nucleic Acids/pharmacology , Porosity , RNAi TherapeuticsABSTRACT
Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core relative to the analogous polyplexes formed with unmodified siRNA, si-NPs. Hydrophobized siPA loaded into the NPs at a lower charge ratio (N(+):P(-)) relative to unmodified siRNA, and siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (â¼40% at 24 h after one treatment and â¼60% at 48 h after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and tumor gene silencing relative to conventional formulations that are stabilized solely by electrostatic interactions.
Subject(s)
Drug Carriers/chemistry , Gene Silencing , Hydrophobic and Hydrophilic Interactions , Neoplasms/therapy , Palmitic Acid/chemistry , Polyethylene Glycols/chemistry , Polymers/chemistry , RNA, Small Interfering/pharmacokinetics , Animals , Cell Line, Tumor , Female , Humans , Mice, Nude , Reproducibility of Results , Tissue DistributionABSTRACT
Cyclooxygenase-2 (COX-2) is expressed in virtually all solid tumors and its overexpression is a hallmark of inflammation. Thus, it is a potentially powerful biomarker for the early clinical detection of inflammatory disease and human cancers. We report a reactive oxygen species (ROS) responsive micellar nanoparticle, PPS-b-POEGA, that solubilizes the first fluorescent COX-2-selective inhibitor fluorocoxib A (FA) for COX-2 visualization in vivo. Pharmacokinetics and biodistribution of FA-PPS-b-POEGA nanoparticles (FA-NPs) were assessed after a fully-aqueous intravenous (i.v.) administration in wild-type mice and revealed 4-8 h post-injection as an optimal fluorescent imaging window. Carrageenan-induced inflammation in the rat and mouse footpads and 1483 HNSCC tumor xenografts were successfully visualized by FA-NPs with fluorescence up to 10-fold higher than that of normal tissues. The targeted binding of the FA cargo was blocked by pretreatment with the COX-2 inhibitor indomethacin, confirming COX-2-specific binding and local retention of FA at pathological sites. Our collective data indicate that FA-NPs are the first i.v.-ready FA formulation, provide high signal-to-noise in inflamed, premalignant, and malignant tissues, and will uniquely enable clinical translation of the poorly water-soluble FA compound.
Subject(s)
Cyclooxygenase 2/metabolism , Indoles/pharmacology , Inflammation/enzymology , Nanoparticles/chemistry , Neoplasms/enzymology , Rhodamines/pharmacology , Animals , Cell Line, Tumor , Dynamic Light Scattering , Female , Humans , Indoles/administration & dosage , Indoles/pharmacokinetics , Indoles/toxicity , Injections, Intraperitoneal , Mice, Inbred C57BL , Mice, Nude , Molecular Imaging , Nanoparticles/toxicity , Nanoparticles/ultrastructure , Polymers/chemical synthesis , Polymers/chemistry , Rhodamines/administration & dosage , Rhodamines/pharmacokinetics , Rhodamines/toxicity , Tissue Distribution/drug effectsABSTRACT
Osteoarthritis (OA) is a disease characterized by degradation of joints with the development of painful osteophytes in the surrounding tissues. Currently, there are a limited number of treatments for this disease, and many of these only provide temporary, palliative relief. In this review, we discuss particle-based drug delivery systems that can provide targeted and sustained delivery of imaging and therapeutic agents to OA-affected sites. We focus on technologies such as polymeric micelles and nano-/microparticles, liposomes, and dendrimers for their potential treatment and/or diagnosis of OA. Several promising studies are highlighted, motivating the continued development of delivery technologies to improve treatments for OA.
Subject(s)
Delayed-Action Preparations/administration & dosage , Drug Delivery Systems/methods , Osteoarthritis/diagnosis , Osteoarthritis/drug therapy , Animals , Cell-Derived Microparticles , Dendrimers/administration & dosage , Dendrimers/therapeutic use , Humans , Liposomes/administration & dosage , Liposomes/therapeutic use , Micelles , Nanoparticles/administration & dosage , Nanoparticles/therapeutic useABSTRACT
The development of elastomeric, bioresorbable and biocompatible segmented polyurethanes (SPUs) for use in tissue-engineering applications has attracted considerable interest because of the existing need of mechanically tunable scaffolds for regeneration of different tissues, but the incorporation of osteoinductive molecules into SPUs has been limited. In this study, SPUs were synthesized from poly (ε-caprolactone)diol, 4,4'-methylene bis(cyclohexyl isocyanate) using biologically active compounds such as ascorbic acid, L-glutamine, ß-glycerol phosphate, and dexamethasone as chain extenders. Fourier transform infrared spectroscopy (FTIR) revealed the formation of both urethanes and urea linkages while differential scanning calorimetry, dynamic mechanical analysis, X-ray diffraction and mechanical testing showed that these polyurethanes were semi-crystalline polymers exhibiting high deformations. Cytocompatibility studies showed that only SPUs containing ß-glycerol phosphate supported human mesenchymal stem cell adhesion, growth, and osteogenic differentiation, rendering them potentially suitable for bone tissue regeneration, whereas other SPUs failed to support either cell growth or osteogenic differentiation, or both. This study demonstrates that modification of SPUs with osteogenic compounds can lead to new cytocompatible polymers for regenerative medicine applications.
Subject(s)
Biocompatible Materials/chemistry , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Polyurethanes/chemistry , Tissue Scaffolds/chemistry , Bone and Bones/cytology , Bone and Bones/drug effects , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Humans , Materials Testing , Mechanical Phenomena , Mesenchymal Stem Cells/physiology , Osteogenesis/drug effects , Tissue Engineering/instrumentation , Tissue Engineering/methodsABSTRACT
A new microparticle-based delivery system was synthesized from reactive oxygen species (ROS)-responsive poly(propylene sulfide) (PPS) and tested for "on demand" antioxidant therapy. PPS is hydrophobic but undergoes a phase change to become hydrophilic upon oxidation and thus provides a useful platform for ROS-demanded drug release. This platform was tested for delivery of the promising anti-inflammatory and antioxidant therapeutic molecule curcumin, which is currently limited in use in its free form due to poor pharmacokinetic properties. PPS microspheres efficiently encapsulated curcumin through oil-in-water emulsion and provided sustained, on demand release that was modulated in vitro by hydrogen peroxide concentration. The cytocompatible, curcumin-loaded microspheres preferentially targeted and scavenged intracellular ROS in activated macrophages, reduced in vitro cell death in the presence of cytotoxic levels of ROS, and decreased tissue-level ROS in vivo in the diabetic mouse hind limb ischemia model of peripheral arterial disease. Interestingly, due to the ROS scavenging behavior of PPS, the blank microparticles also showed inherent therapeutic properties that were synergistic with the effects of curcumin in these assays. Functionally, local delivery of curcumin-PPS microspheres accelerated recovery from hind limb ischemia in diabetic mice, as demonstrated using non-invasive imaging techniques. This work demonstrates the potential for PPS microspheres as a generalizable vehicle for ROS-demanded drug release and establishes the utility of this platform for improving local curcumin bioavailability for treatment of chronic inflammatory diseases.
Subject(s)
Antioxidants/therapeutic use , Curcumin/therapeutic use , Diabetes Mellitus, Experimental/drug therapy , Microspheres , Peripheral Arterial Disease/drug therapy , Reactive Oxygen Species/metabolism , Animals , Antioxidants/pharmacology , Cell Survival/drug effects , Chemokine CCL2/metabolism , Curcumin/pharmacology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/pathology , Endocytosis/drug effects , Female , Hindlimb/blood supply , Hindlimb/pathology , Hydrogen Peroxide/pharmacology , Interferon-gamma/pharmacology , Intracellular Space/drug effects , Intracellular Space/metabolism , Ischemia/complications , Ischemia/drug therapy , Ischemia/pathology , Kinetics , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Mice , Muscles/blood supply , Muscles/pathology , NIH 3T3 Cells , Oxygen/blood , Particle Size , Perfusion , Peripheral Arterial Disease/complications , Peripheral Arterial Disease/pathology , Polymers/chemical synthesis , Polymers/chemistry , Sulfides/chemical synthesis , Sulfides/chemistryABSTRACT
Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The ß1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete ß1 integrin knockout results in embryonic lethality, studies of ß1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that ß1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of ß1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of ß1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and osteocalcin-Cre lines to generate conditional ß1 integrin deletions, where Cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific ß1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific ß1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of ß1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that ß1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while ß1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic chondryocyte-lineage cells regulate incisor eruption and perinatal bone formation in both intramembranously and endochondrally formed bones in young, rapidly growing mice. In contrast, the osteocalcin-specific ß1 integrin deletion had only minor effects on skeletal phenotype.
Subject(s)
Bone and Bones/pathology , Gene Silencing , Integrases/metabolism , Integrin beta1/metabolism , Osteocalcin/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Twist-Related Protein 1/metabolism , Animals , Biomechanical Phenomena , Bone Density , Bone Development , Bone and Bones/embryology , Bone and Bones/physiopathology , Calcification, Physiologic , Embryo Loss/metabolism , Embryo Loss/pathology , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Female , Femur/abnormalities , Femur/embryology , Femur/physiopathology , Gene Deletion , Incisor/abnormalities , Incisor/embryology , Incisor/metabolism , Male , Mice , Phenotype , Skull/abnormalities , Skull/diagnostic imaging , Skull/embryology , Sp7 Transcription Factor , Stem Cells/metabolism , X-Ray MicrotomographyABSTRACT
Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2ß1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.