[Show abstract][Hide abstract] ABSTRACT: Beyond the glamorous and futuristic notion of building new organs articulated by two generations of academic bioengineers lies the reality of the business of regenerative medicine. How do we reconcile the relentless innovation of promising new biomaterials created with "free" grant money, with the creation of products that are safe, effective, scalable, and affordable, return value to investors, show clinically relevant outcomes, and have a viable business model? In this chapter, we (1) discuss the general principles of creating a clinical biomaterial, (2) identify potential business models used for commercializing biomaterials, and (3) present a case study in building successful businesses from a single technology by licensing specific fields of use.
[Show abstract][Hide abstract] ABSTRACT: Titanium (Ti) is a widely used material for surgical implants; total joint replacements (TJRs), screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA) and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.
[Show abstract][Hide abstract] ABSTRACT: Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units. These studies tested the ability of CMHA to stabilize the formation of calcium phosphate nanoparticles and evaluated their potential to target therapy resistant, CD44(+)/CD24(-/low) human breast cancer cells (BT-474EMT). CMHA stabilized particles (nCaP(CMHA)) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaP(CMHA)CDDP. nCaP(CMHA)CDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a -43 mV zeta potential. nCaP(CMHA)CDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaP(CMHA)CDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaP(CMHA-AF488) was taken up by CD44(+)/CD24(-) BT-474EMT breast cancer cells within 18 hours. nCaP(CMHA)CDDP was as cytotoxic as free CDDP against the BT-474EMT cells. Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaP(CMHA)CDDP. This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.
Full-text · Article · Oct 2015 · International Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Background:
Cathelicidin (LL-37) is an endogenous innate immune peptide that is elevated in patients with chronic rhinosinusitis (CRS). The role of LL-37 in olfactory epithelium (OE) inflammation remains unknown. We hypothesized that: (1) LL-37 topically delivered would elicit profound OE inflammation; and (2) LL-37 induced inflammation is associated with increased infiltration of neutrophils and mast cells.
To test our hypothesis we challenged C57BL/6 mice intranasally with increasing concentrations of LL-37. At 24 hours tissues were examined histologically and scored for inflammatory cell infiltrate, edema, and secretory hyperplasia. In separate experiments, fluorescently conjugated LL-37 was instilled and tissues were examined at 0.5 and 24 hours. To test our last hypothesis, we performed tissue myeloperoxidase (MPO) assays for neutrophil activity and immunohistochemistry for tryptase to determine the mean number of mast cells per mm(2) .
LL-37 caused increased inflammatory cell infiltrate, edema, and secretory cell hyperplasia of the sinonasal mucosa, with higher LL-37 concentrations yielding significantly more inflammatory changes (p < 0.01). Fluorescent LL-37 demonstrated global sinonasal epithelial binding and tissue distribution. Further, higher concentrations of LL-37 led to significantly greater MPO levels with dose-dependent increases in mast cell infiltration (p < 0.01).
LL-37 has dramatic inflammatory effects in the OE mucosa that is dose-dependent. The observed inflammatory changes in the olfactory mucosa were associated with the infiltration of both neutrophils and mast cells. Our biologic model represents a new model to further investigate the role of LL-37 in OE inflammation.
No preview · Article · Sep 2015 · International Forum of Allergy and Rhinology
[Show abstract][Hide abstract] ABSTRACT: While approximately 30 % US adults are suffer from moderate to
severe form of periodontitis, the available treatment options are far
too limited. This chronic disease occurs due to a disruption of
homeostasis between the microorganisms residing in the oral cavity
and the host immune system. GM-0111 is a modified glycosaminoglycan
and it has demonstrated anti-inflammatory effects on various
inflammatory diesease models molecular targets and possibly offer
alternative therapeutic choices for treating periodontitis. We investigated
whether GM-0111, a modified GAG, could block molecular
events important for the development of periodontitis and the
resulting bone loss. First, we tested GM-0111 on RANKL-induced
osteoclast formation and bone resorption in cultured mouse pre-osteoclasts.
Next, we tested whether GM-0111 could block proinflammatory
TLR2 and TLR4 signaling pathways in mouse macrophage
RAW 264.7 cells and heterologously expressed HEK293 cells.
Lastly, we investigated the antibacterial effects of GM-0111 on
Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.
GM-0111 blocked RANKL-induced osteoclast formation even at
300 ng/mL and it also reduced the resulting bone resorption. The antiosteoclastic
effects of GM-0111 were independent of RANKL to
RANK signaling. We also found that GM-0111 selectively targets
TLR2 (IC50 of 1–10 ng/mL) than TLR4 (IC50[100 lg/mL). By
blocking TLR-mediated inflammatory pathways and inhibiting key
process leading bone loss, we suggest that GM-0111 can provide a
therapeutic potential to treat periodontitis.
[Show abstract][Hide abstract] ABSTRACT: Acute Lung Injury (ALI) is a life-threatening, diffuse heterogeneous lung injury characterized by acute onset, pulmonary edema and respiratory failure. Lipopolysaccharide (LPS) is a common cause of both direct and indirect lung injury and when administered to a mouse induces a lung phenotype exhibiting some of the clinical characteristics of human ALI. Here, we report that LPS inhalation in mice results in increased bronchoalveolar lavage fluid (BALF) levels of Autotaxin (ATX, Enpp2), a lysophospholipase D largely responsible for the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) in biological fluids and chronically inflamed sites. In agreement, gradual increases were also detected in BALF LPA levels, following inflammation and pulmonary edema. However, genetic or pharmacologic targeting of ATX had minor effects in ALI severity, suggesting no major involvement of the ATX/LPA axis in acute inflammation. Moreover, systemic, chronic exposure to increased ATX/LPA levels was shown to predispose to and/or to promote acute inflammation and ALI unlike chronic inflammatory pathophysiological situations, further suggesting a differential involvement of the ATX/LPA axis in acute versus chronic pulmonary inflammation.
[Show abstract][Hide abstract] ABSTRACT: Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term 'stem cell therapy' is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call "design for optimal functionality." Third, we describe an alternative approach to sECM design and implementation, called "design for simplicity," in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ. We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.
Preview · Article · Jan 2015 · Expert Opinion on Biological Therapy
[Show abstract][Hide abstract] ABSTRACT: Significance: Hyaluronic acid (HA, or hyaluronan) is a ubiquitous naturally occurring polysaccharide that plays a role in virtually all tissues in vertebrate organisms. HA-based hydrogels have wound-healing properties, support cell delivery, and can deliver drugs locally. Recent Advances: A few HA hydrogels can be customized for composition, physical form, and biomechanical properties. No clinically approved HA hydrogel allows for in vivo crosslinking on administration, has a tunable gelation time to meet wound-healing needs, or enables drug delivery. Recently, a thiolated carboxymethyl HA (CMHA-S) was developed to produce crosslinked hydrogels, sponges, and thin films. CMHA-S can be crosslinked with a thiol-reactive crosslinker or by oxidative disulfide bond formation to form hydrogels. By controlled crosslinking, the shape and form of this material can be manipulated. These hydrogels can be subsequently lyophilized to form sponges or air-dried to form thin films. CMHA-S films, liquids, and gels have been shown to be effective in vivo for treating various injuries and wounds in the eye in veterinary use, and are in clinical development for human use. Critical Issues: Better clinical therapies are needed to treat ophthalmic injuries. Corneal wounds can be treated using this HA-based crosslinked hydrogel. CMHA-S biomaterials can help heal ocular surface defects, can be formed into a film to deliver drugs for local ocular drug delivery, and could deliver autologous limbal stem cells to treat extreme ocular surface damage associated with limbal stem cell deficiencies. Future Directions: This CMHA-S hydrogel increases the options that could be available for improved ocular wound care, healing, and regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. Although the 5-year survival rate of localized disease approaches 75%, the prognosis of metastatic and/or therapy-resistant disease remains dismal despite the wide use of aggressive therapeutic strategies. We previously reported that high expression of glutathione S-transferase M4 (GSTM4) in primary tumors correlates with poor patient outcomes. GSTM4 is required for oncogenic transformation and mediates resistance to chemotherapeutic drugs in Ewing sarcoma cells. Here, we performed RNA-sequencing analyses of Ewing sarcoma cells and combined our results with publicly-available datasets to demonstrate that GSTM4 is a major GST specifically expressed in Ewing sarcoma. Pharmacological inhibition of GSTM4 activity using a pan GST inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), significantly limited cellular proliferation and oncogenic transformation of Ewing sarcoma cells. Moreover, combined use of NBDHEX and etoposide synergistically increased cytotoxicity, suggesting a role for GSTM4 as an inhibitor of apoptosis. Mechanistic studies revealed that GSTM4 limits apoptosis owing to its ability to interact with Apoptosis Signal-regulating Kinase 1 (ASK1) and inhibit signaling via the c-Jun N-terminal Kinase axis. To exploit our observation that GSTM4 expression is specifically up-regulated in Ewing sarcoma, we tested the effect of a GSTM4-activated anti-cancer agent, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K, on tumor growth and survival. We found that JS-K robustly decreased Ewing sarcoma cell viability and xenograft tumor growth, and improved overall survival of xenograft mice. Our data suggest that GSTM4 is a novel therapeutic target for the treatment of high GSTM4-expressing Ewing sarcoma. Strategies that combine standard chemotherapy with agents that inhibit GSTM4, or that are activated by GSTM4, or that block GSTM4/ASK1 interaction
Full-text · Article · Aug 2014 · Frontiers in Pediatrics
[Show abstract][Hide abstract] ABSTRACT: A new water-soluble fluorescent perylene sensor was synthesized and compared to the previous non-aqueous sensors. The new sensor, MSI-1-9, exhibits sensitive and selective detection of mercuric ion (Hg2+) directly in aqueous solution through fluorescence quenching. The detection was not affected by the coexistence of other common divalent metal ions. MSI-1-9 possesses the necessary criteria for use in affordable, real-time measurement of Hg2+ in environmental water samples, permitting its incorporation into a portable mercury detection kit.
No preview · Article · Jul 2014 · Sensors and Actuators B Chemical
[Show abstract][Hide abstract] ABSTRACT: Autotaxin (ATX), an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2) that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.
[Show abstract][Hide abstract] ABSTRACT: Interstitial cystitis (IC), often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG) layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1) the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2) the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3) the physiological and molecular markers that correlate with the severity of the inflammation, and (4) the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.
[Show abstract][Hide abstract] ABSTRACT: Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Rationale: Bioactive lipid mediators, derived from membrane lipid precursors, are released into the airway and airspace where they bind high affinity cognate receptors and may mediate asthma pathogenesis. Lysophosphatidic acid (LPA), a bioactive lipid mediator generated by the enzymatic activity of extracellular autotaxin (ATX), binds LPA receptors resulting in an array of biological actions on cell proliferation, migration, survival, differentiation and motility, and therefore could mediate asthma pathogenesis. Objectives: To define a role for the ATX-LPA pathway in human asthma pathogenesis and a murine model of allergic lung inflammation. Methods: We investigated the profiles of LPA molecular species and the level of ATX exoenzyme in BAL fluids of human asthmatics subjected to sub-segmental broncho-provocation with allergen. We interrogated the role of the ATX-LPA pathway in allergic lung inflammation using a murine allergic asthma model in ATX-LPA pathway-specific genetically modified mice. Measurements and Main Results: Sub-segmental broncho-provocation with allergen in mild asthmatics resulted in a remarkable increase in BAL fluid levels of LPA enriched in polyunsaturated 22:5 and 22:6 fatty acids in association with increased concentrations of ATX protein. Using a triple allergen mouse asthma model, we showed that ATX over-expressing transgenic mice had a more severe asthmatic phenotype, whereas blocking ATX activity and knockdown of the LPA2 receptor in mice produced a marked attenuation of Th2 cytokines and allergic lung inflammation. Conclusions: The ATX-LPA pathway plays a critical role in the pathogenesis of asthma. These preclinical data indicate that targeting the ATX-LPA pathway could be an effective anti-asthma treatment strategy.
Full-text · Article · Sep 2013 · American Journal of Respiratory and Critical Care Medicine