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While specific cell signaling pathway inhibitors have yielded great success in oncology, directly triggering cancer cell death is one of the great drug discovery challenges facing biomedical research in the era of precision oncology. Attempts to eradicate cancer cells expressing unique target proteins, such as antibody-drug conjugates (ADCs), T-cell engaging therapies, and radiopharmaceuticals have been successful in the clinic, but they are limited by the number of targets given the inability to target intracellular proteins. More recently, heterobifunctional small molecules such as Proteolysis Targeting Chimera (PROTACs) have paved the way for protein proximity inducing therapeutic modalities.Here, we describe a proof-of-concept study using novel heterobifunctional small molecules calledRegulatedInducedProximityTargetingChimeras or RIPTACs, which elicit a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting cooperative protein:protein interaction (PPI) abrogates the function of the essential protein, thus leading to cell death selectively in cells expressing the target protein. This approach not only opens new target space by leveraging differentially expressed intracellular proteins but also has the advantage of not requiring the target to be a driver of disease. Thus, RIPTACs can address non-target mechanisms of resistance given that cell killing is driven by inactivation of the essential protein.Using the HaloTag7-FKBP model system as a target protein, we describe RIPTACs that incorporate a covalent or non-covalent target ligand connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013 or dinaciclib. We show that these RIPTACs exhibit positive co-operativity, accumulate selectively in cells expressing HaloTag7-FKBP, form stable target:RIPTAC:effector trimers in cells, and induce an anti-proliferative response in target-expressing cells. We propose that RIPTACs are a novel heterobifunctional therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein.

There is an urgent unmet medical need to develop therapeutic options for the ~50% of depression patients suffering from treatment-resistant depression, which is difficult to treat with existing psycho- and pharmaco-therapeutic options. Classical psychedelics, such as the 5HT

This study was to determine to what extent a cryogenic surface treatment technique could improve the wear resistance of a Co-Cr-Mo hip implant material. Dry and cryogenic treatments were used to cr...

Basic science. Investigate the ability of local applicaiton of vancomycin, either in powder form or suspended within poly(lactic-co-glycolic acid) microspheres (MS), to treat infection using a rat spinal model. Surgical site infections (SSIs) are a serious complication after spine surgery and are associated with high morbidity and mortality and often caused my coagulase negative staphylococci. A comprehensive approach to reduce SSIs has been recommended including the use of topical vancomycin. Animal and human studies have shown improved control of infection with local compared to systemic antibiotics. K-wires seeded with methicillin-resistant Staphylococcus epidermidis RP62A (MRSE) were treated with vancomycin powder, carboxymethylcellulose sodium salt (CMC) (microsphere carrier), vancomycin powder, blank MS or vancomycin-loaded MS for 24 or 48 h in vitro after which bacteria were enumerated. In addition, a spinal instrumentation model was developed in rats with a bacterial seeded K-wire implanted into the right side of L4 and L5. Rats underwent no treatment or were treated locally with either vancomycin powder, blank MS or vancomycin-loaded MS. After 8 weeks, the K-wire, bone, soft tissue and wire fastener were cultured and results analyzed. Vancomycin powder and vancomycin-loaded MS resulted in significantly fewer bacteria remaining in vitro than did CMC. Vancomycin powder- treated animals’ cultures were significantly lower than all other groups (P

Introduction Nonhuman animal models have been used extensively to study orthodontic tooth movement (OTM). However, rodent models have disadvantages, including a reported reduction in bone volume during OTM. The purpose of this study was to determine the viability of a skeletal anchorage and the effect of low force (∼3 cN) on interradicular bone volume during OTM. Methods Ninety Sprague-Dawley rats were divided into 5 time points. A miniscrew and a nickel titanium coil spring placed a load of 3 cN (experimental) or 0 cN (sham) on the maxillary first molar in a split-mouth design. Displacement of the first molar and bone volume/total volume (BV/TV) in the interradicular region were quantified. Results The success rate of the miniscrew was 98.9% (89 out of 90). Linear and angular tooth movement increased steadily (mean 0.1 mm/wk, 0.48 mm at 40 days). BV/TV was significantly reduced between the tooth movement and non–tooth movement sides in the 3 cN group: by 13%, 23%, 15%, 23%, and 16% at 3, 7, 14, 28, and 40 days, respectively. Conclusions Our model resulted in efficient OTM without skeletal anchorage failure. BV/TV reduction was lower than in previous reports. This novel validated model is likely to be the basis for future studies.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules which preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.

Recently, biomaterials have been designed to contain redox-sensitive moieties, such as thiols and disulfides, to impart responsive degradation and/or controlled release. However, due to the high sensitivity of cellular redox-based systems which maintain free-radical homeostasis (e.g. glutathione/glutathione disulfide), if these biomaterials modify the cellular redox environment, they may inadvertently affect cellular compatibility and/or oxidative stress defenses. In this work, we hypothesize that the degradation products of a poly(β-amino ester) (PBAE) hydrogel formed with redox sensitive disulfide (cystamine) crosslinking could serve as a supplement to the environmental cellular antioxidant defenses. Upon introduction into a reducing environment, these disulfide-containing hydrogels cleave to present bound-thiol groups, yet remain in the bulk form at up to 66 mol% cystamine of the total amines. By controlling the molar fraction of cystamine, it was apparent that the thiol content varied human umbilical vein endothelial cell (HUVEC) viability IC50 values across an order of magnitude. Further, upon introduction of an enzymatic oxidative stress generator to the cell culture (HX/XO), pre-incubated thiolated hydrogel degradation products conferred cellular and mitochondrial protection from acute oxidative stress, whereas non-reduced disulfide-containing degradation products offered no protection. This polymer may be an advantageous implantable drug delivery system for use in acute oxidative stress prophylaxis and/or chronic oxidative stress cell therapies due to its solid/liquid reversibility in a redox environment, controlled thiolation, high loading capacity through covalent drug-addition, and simple post-synthesis modification which bound-thiols introduce. Statement of Significance In this work, we demonstrate a unique property of disulfide containing degradable biomaterials. By changing the redox state of the degradation products (from oxidized to reduced), it is possible to increase the IC50 of the material by an order of magnitude. This dramatic shift is linked directly to the oxidative stress response of the cells and suggests a possible mechanism by which one can tune the cellular response to degradable biomaterials.

Small molecules that selectively bind to the pseudokinase JH2 domain over the JH1 kinase domain of JAK2 kinase are sought. Virtual screening led to the purchase of 17 compounds among which 9 were found to bind to V617F JAK2 JH2 with affinities of 40 – 300 μM in a fluorogenic assay. Ten analogues were then purchased yielding 9 additional active compounds. Aminoanilinyltriazine 22 was particularly notable as it shows no detectable binding to JAK2 JH1, and it has a 65-μM dissociation constant Kd with V617F JAK2 JH2. A crystal structure for 22 in complex with wild-type JAK2 JH2 was obtained to elucidate the binding mode. Additional de novo design led to the synthesis of 19 analogues of 22 with the most potent being 33n with Kd values of 2–3 μM for WT and V617F JAK2 JH2, and with 16-fold selectivity relative to binding with WT JAK2 JH1.

Large infected bone defects, often resulting from high energy traumas, are difficult to treat due to their variability in complexity and location. Standard treatment for infected bone defects begins with a protocol that includes a series of debridements in conjunction with an extended course of systemic antibiotics. Only after the infection has been eliminated will repair of the defect commence, typically with implantation of autologous bone. To address some of the shortcomings of the standard treatment methods, such as serial procedures, limited grafting material, and the need for a second surgical site for autologous bone, a sequential, dual drug-releasing, moldable, calcium sulfate-based bone graft substitute was developed previously. In the present studies, the effectiveness of the material for treating both the infection with vancomycin and bone defect with simvastatin was evaluated in vivo using a critically sized, infected segmental defect model in rat femurs. Although the infection was not fully eliminated, the local release of vancomycin increased survivorship of infected animals by 464% compared to nontreated controls. Infected animals receiving antimicrobial treatment showed comparable amounts of new bone formation within the defect site when compared to noninfected controls. Incorporating agents capable of disrupting established biofilms into bone graft substitutes may enhance effectiveness in treating a biofilm infection within a bone defect. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1878-1886, 2018.

Janus kinases (JAKs) regulate hematopoiesis via the cytokine-mediated JAK-STAT signaling pathway. JAKs contain tandem C-terminal pseudokinase (JH2) and tyrosine kinase (JH1) domains. The JAK2 pseudokinase domain adopts a protein kinase fold and, despite its pseudokinase designation, binds ATP with micromolar affinity. Recent evidence shows that displacing ATP from the JAK2 JH2 domain alters the hyperactivation state of the oncogenic JAK2 V617F protein while sparing the wild type JAK2 protein. In this study, small molecule binders of JAK2 JH2 were identified via an in vitro screen. Top hits were characterized using biophysical and structural approaches. Development of pseudokinase-selective compounds may offer novel pharmacological opportunities for treating cancers driven by JAK2 V617F and other oncogenic JAK mutants.

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

Previous research from our labs demonstrated the synthesis of polymerized simvastatin by ring-opening polymerization and slow degradation with controlled release of simvastatin in vitro. The objective of the present study was to evaluate the degradation and intramembranous bone-forming potential of simvastatin-containing polyprodrugs in vivo using a rat calvarial onlay model. Poly(ethylene glycol)-block-poly(simvastatin) and poly(ethylene glycol)-block-poly(simvastatin)-ran-poly(glycolide) were compared with simvastatin conventionally encapsulated in poly(lactic-co-glycolic acid) (PLGA) and pure PLGA. The rate of degradation was higher for PLGA with and without simvastatin relative to the simvastatin polyprodrugs. Significant new bone growth at the circumference of poly(ethylene glycol)-block-poly(simvastatin) disks was observed beginning at 4 weeks, whereas severe bone resorption (4 weeks) and bone loss (8 weeks) were observed for PLGA loaded with simvastatin. No significant systemic effects were observed for serum total cholesterol and body weight. Increased expression of osteogenic (BMP-2, Runx2, and ALP), angiogenic (VEGF), and inflammatory cytokines (IL-6 and NF-ĸB) genes was seen with all polymers at the end of 8 weeks. Poly(ethylene glycol)-block-poly(simvastatin), with slow degradation and drug release, controlled inflammation, and significant osteogenic effect, is a candidate for use in bone regeneration applications. Statement of Significance Traditional drug delivery systems, e.g., drug encapsulated in poly(lactic-co-glycolic acid) (PLGA), are typically passive and have limited drug payload. As an alternative, we polymerized the drug simvastatin, which has multiple physiological effects, into macromolecules (“polysimvastatin”) via ring-opening polymerization. We previously demonstrated that the rate of degradation and drug (simvastatin) release can be adjusted by copolymerizing it with other monomers. The present results demonstrate significant new bone growth around polysimvastatin, whereas severe bone loss occurred for PLGA loaded with simvastatin. This degradable biomaterial with biofunctionality integrated into the polymeric backbone is a useful candidate for bone regeneration applications.

The effects of surface roughness and carboxyl functionalization of multi-walled carbon nanotubes (MWCNTs) mixed with collagen coated onto titanium (Ti) substrates on MC3T3-E1 osteoblasts were evaluated. The proliferation, differentiation, and matrix mineralization were investigated using (1) smooth-surfaced Ti discs, (2) Ti discs coated with collagen and MWCNT (Ti-MWCNT), and (3) Ti discs coated with collagen and MWCNT-COOH (Ti-MWCNT-COOH) for applications in orthodontic mini screw implants (MSIs). The coatings were uniform when analyzed using scanning electron microscopy (SEM), and surface roughness was evaluated by surface profilometry that demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 μm) and MWCNT-COOH (0.84 ± 0.01 μm) groups. MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assay was performed after days 1, 3, and 7 to assess proliferation. Alkaline phosphatase (ALP)-specific activity was assessed after day 7 to quantify differentiation. Alizarin red staining was measured after day 28 to quantify matrix mineralization. All data were analyzed with JMP Pro11 software (SAS, USA) with a statistical significance of p < 0.05. Surface profilometry demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 μm) and MWCNT-COOH (0.84 ± 0.01 μm) groups. On day 7, ALP assay showed that MWCNT-COOH (mean ± SD 0.98 ± 0.26 U/μg of protein) enhanced cell differentiation when compared to the uncoated group (p = 0.05). Alizarin red staining after 28 days of cell culture revealed that MWCNT-COOH (mean ± SD 1.5 ± 0.2 OD405) increased (p = 0.03) matrix mineralization when compared to the uncoated group (0.9 ± 0.09 OD405). This study showed that coatings containing MWCNT-COOH (increased hydrophilic surface chemistry) influence osteoblast proliferation, differentiation, and matrix mineralization and should be further studied for applications in orthodontic MSIs.

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.

Composite scaffolds fabricated from biodegradable polymers as drug carriers are used extensively in the field of tissue engineering. Ideal tissue engineering scaffolds must possess a 3D interconnected porous network for tissue ingrowth and construct vascularization. However, the porous interconnected network may reduce the initial mechanical support needed for the scaffold in the implant site. In this study, composite scaffolds consisted of poly(lactic-co-glycolic acid) (PLGA) $\leq$ 250 $\mu$ MS and Simvastatin loaded poly beta amino acid (PBAE) Hydrogel Microspheres (HG MS) $\leq \sim 500 \mu$ were fabricated via a novel device to investigate several new functions: controlled drug release, PBAE HG MS time-dependent degradation exhibiting pore opening resulting in interconnected pattern of porosity development, better initial mechanical support and the true wet-state compressive moduli.

Bone pain is the leading cause of morbidity in patients with metastatic cancer. Systemic administration of zoledronic acid (ZA) decreases skeletally-related events in bone cancer patients but is associated with major side effects. This project investigated two biomaterials, poly(methyl methacrylate) (PMMA) bone cement and poly(lactic-co-glycolic acid) (PLGA), for local ZA delivery. Compressive properties of PMMA samples were tested with increased drug loading, and in vitro ZA release profiles from PMMA cylinders and PLGA films were measured over 8 weeks. The activity of ZA eluted from both materials was evaluated on the RAW 264.7 macrophage cell line. PMMA samples released up to only 17% of incorporated drug, whereas PLGA films released over 95%. A burst profile was observed for PMMA, while ZA release from PLGA exhibited a typical triphasic profile. Drug bioactivity remained above 50% for both materials. Local ZA delivery from these materials may be useful in the treatment of metastatic bone cancer.

Simvastatin was previously converted to a polymeric prodrug with higher drug loading, but the hydrophobic nature of the poly(simvastatin) component of the block copolymer led to slow release of the drug in vitro. In this study, we hypothesized that degradation could be accelerated by chemically modifying the polymer backbone by introducing glycolide and lactide comonomers. Copolymers were formed by ring-opening polymerization using 5 kDa monomethyl ether poly(ethylene glycol) as the microinitiator in presence of triazabicyclodecene catalyst. In addition to simvastatin, modified reaction mixtures contained lactide or glycolide. Incorporation of the less hydrophobic glycolide comonomer led to in vitro degradation of up to two times greater mass loss, release of up to ~7 times more simvastatin, and a 2-3 times increase in compressive modulus compared to the lactide-containing and parent polymers.

Disulfides are used extensively in reversible cross-linking because of the ease of reduction into click-reactive thiols. However, the free-radical scavenging properties upon reduction are often under-considered. The free thiols produced upon reduction of this disulfide material mimic the cellular reducing chemistry (glutathione) that serves as a buffer against acute oxidative stress. A nanoparticle formulation producing biologically relevant concentrations of thiols may not only provide ample chemical conjugation sites, but potentially be useful against severe acute oxidative stress exposure, such as in targeted radioprotection. In this work, we describe the synthesis and characterization of highly thiolated poly (&beta, amino ester) (PBAE) nanoparticles formed from the reduction of bulk disulfide cross-linked PBAE hydrogels. Degradation-tunable PBAE hydrogels were initially synthesized containing up to 26 wt % cystamine, which were reduced into soluble thiolated oligomers and formulated into nanoparticles upon single emulsion. These thiolated nanoparticles were size-stable in phosphate buffered saline consisting of up to 11.0 &plusmn, 1.1 mM (3.7 &plusmn, 0.3 mmol thiol/g, n = 3 M &plusmn, SD), which is an antioxidant concentration within the order of magnitude of cellular glutathione (1&ndash, 10 mM).

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,β) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor–receptor interactions.

A locally injectable system sequentially delivering an antiresorptive drug (clodronate) followed by an osteogenic agent (simvastatin) was hypothesized to improve femoral head microarchitecture, size, and shape compared with untreated or partial treatment groups in an established piglet osteonecrosis model. After 6 weeks, the clodronate+simvastatin treatment resulted in no collapse, microCT measurements and epiphyseal quotients within 10% of control, normal microstructure, and healthy histology. All other groups exhibited collapse, lower epiphyseal quotients and total femoral head volumes (P

Mucin networks are lubricous biofunctional coats formed through the continuous deposition of mucin glycoproteins. Previously, we demonstrated the synthesis of a mucin mimic using biotinylated-filomicelles crosslinked via streptavidin using a layer-by-layer approach. These networks recreate the fibrous nature of mucin and can serve as a drug-releasing network. In this work, the ability to vary the network properties by blending filomicelles with spherical micelles is demonstrated. In addition, the deposition of a dense polymer coating on the mucin network was shown to act as a barrier to control diffusion and improved the structural stability under simulated oral chemical conditions. These biomimetic coatings can be utilized as a delivery system, providing a tunable drug release for oral applications.

Development of drug-delivery devices typically involves characterizing in vitro release performance with the inherent assumption that this will closely approximate in vivo performance. Yet, as delivery devices become more complex, for instance with a sequential drug release pattern, it is important to confirm that in vivo properties correlate with the expected "programming" achieved in vitro. In this work, a systematic comparison between in vitro and in vivo biomaterial erosion and sequential release was performed for a multilayered association polymer system comprising cellulose acetate phthalate and Pluronic F-127. After assessing the materials during incubation in phosphate-buffered saline, devices were implanted supracalvarially in rats. Devices with two different doses and with different erosion rates were harvested at increasing times post-implantation, and the in vivo thickness loss, mass loss, and the drug release profiles were compared with their in vitro counterparts. The sequential release of four different drugs observed in vitro was successfully translated to in vivo conditions. Results suggest, however, that the total erosion time of the devices was longer and that release rates of the four drugs were different, with drugs initially released more quickly and then more slowly in vivo. Many comparative studies of in vitro and in vivo drug release from biodegradable polymers involved a single drug, whereas this research demonstrated that sequential release of four drugs can be maintained following implantation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1302-1310, 2016.

The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 μg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of compound 1 and disrupts the interactions with residues in the pocket. Compound 2 maintains the same conformation in wild-type and mutant structures, in addition to several interactions with the NNRTI binding pocket. Comparison of the six crystal structures will assist in the understanding of compound binding modes and future optimization of the catechol diether series. Fil: Frey, Kathleen M.. University of Yale; Estados Unidos Fil: Puleo, David. University of Yale; Estados Unidos Fil: Spasov, Krasimir A.. University of Yale; Estados Unidos Fil: Bollini, Mariela. University of Yale; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Jorgensen, William L.. University of Yale; Estados Unidos Fil: Anderson, Karen S.. University of Yale; Estados Unidos

The aim of the present work is to investigate the effect of a severe plastic deformation (SPD) process, cryogenic burnishing, on the surface integrity modifications of a Co–Cr–Mo alloy due to the burnishing-induced surface integrity properties. A set of experiments was conducted to investigate the influence of different burnishing parameters on distribution of grain size, phase structure and hardness of the processed material. The results from this work show that the proper selection of burnishing conditions can significant improve the surface integrity of the Co–Cr–Mo alloy due to refined microstructure, high hardness, and favorable phase structure on the surface layer, which could potentially lead to advanced wear performance of such material.

Growth plate fractures account for nearly 18.5% of fractures in children. Depending on the type and severity of the injury, inhibited bone growth or angular deformity caused by bone forming in place of the growth plate can occur. The current treatment involves removal of the bony bar and replacing it with a filler substance, such as a free fat graft. Unfortunately, reformation of the bony bar frequently occurs, preventing the native growth plate from regenerating. The goal of this pilot study was to determine whether biodegradable scaffolds can enhance native growth plate regeneration following a simulated injury that resulted in bony bar formation in the proximal tibial growth plate of New Zealand white rabbits. After removing the bony bar, animals received one of the following treatments: porous poly(lactic-co-glycolic acid) (PLGA) scaffold; PLGA scaffold loaded with insulin-like growth factor I (IGF-I); PLGA scaffold loaded with IGF-I and seeded with autogenous bone marrow cells (BMCs) harvested at the time of implantation; or fat graft (as used clinically). The PLGA scaffold group showed an increased chondrocyte population and a reduced loss of the remaining native growth plate compared to the fat graft group (the control group). An additional increase in chondrocyte density was seen in scaffolds loaded with IGF-I, and even more so when BMCs were seeded on the scaffold. While there was no significant reduction in the angular deformation of the limbs, the PLGA scaffolds increased the amount of cartilage and reduced the amount of bony bar reformation.

Simvastatin was polymerized into copolymers to better control drug loading and release for therapeutic delivery. When using the conventional stannous octoate catalyst in ring-opening polymerization (ROP), reaction temperatures ≥200 °C were required, which promoted uncontrollable and undesirable side reactions. Triazabicyclodecene (TBD), a highly reactive guanidine base organocatalyst, was used as an alternative to polymerize simvastatin. Polymerization was achieved at 150 °C using 5 kDa methyl-terminated poly(ethylene glycol) (mPEG) as the initiator. ROP reactions with 2 kDa or 550 Da mPEG initiators were also successful using TBD at 150 °C instead of stannous octoate, which required a higher reaction temperature. Biodegradability of the poly(simvastatin) copolymer in phosphate-buffered saline was also improved, losing twice as much mass than the copolymer synthesized via stannous octoate. The three copolymers exhibited modified rates of simvastatin release, demonstrating tunablity for drug delivery applications.

Sequential release of drugs aligned with the phases of tissue healing could reduce scarring. To achieve this aim, layered film devices comprising cellulose acetate phthalate (CAP) and Pluronic F-127 (Pluronic) were loaded with ketoprofen, quercetin, and pirfenidone. Citrate plasticizers were added to impart flexibility. Release of two or three drugs in sequence over several days was obtained for all multilayered devices tested. Mechanical analysis showed that elongation increased and modulus decreased with the increasing plasticizer content. Release profiles can be tailored by order of layers, plasticizer concentration, and drug loading, making CAP–Pluronic an appealing system for inhibiting scar tissue formation.

Large infected bone defects, often resulting from high energy traumas, are difficult to treat due to their variability in complexity and location. Standard treatment for infected bone defects begins with a protocol that includes a series of debridements in conjunction with an extended course of systemic antibiotics. Only after the infection has been eliminated will repair of the defect commence, typically with implantation of autologous bone. To address some of the shortcomings of the standard treatment methods, such as serial procedures, limited grafting material, and the need for a second surgical site for autologous bone, a sequential, dual drug-releasing, moldable, calcium sulfate-based bone graft substitute was developed previously. In the present studies, the effectiveness of the material for treating both the infection with vancomycin and bone defect with simvastatin was evaluated in vivo using a critically sized, infected segmental defect model in rat femurs. Although the infection was not fully eliminated, the local release of vancomycin increased survivorship of infected animals by 464% compared to nontreated controls. Infected animals receiving antimicrobial treatment showed comparable amounts of new bone formation within the defect site when compared to noninfected controls. Incorporating agents capable of disrupting established biofilms into bone graft substitutes may enhance effectiveness in treating a biofilm infection within a bone defect. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1878-1886, 2018.

In situ forming poly(lactic-co-glycolic acid) (PLGA) implants have not been strongly considered for bone applications because of their poor mechanical properties. Here, in situ forming scaffolds containing hydroxyapatite micro- and nanoparticles were characterized to determine their mechanical properties, injectability, and microarchitecture. Scaffolds were prepared with various concentrations of hydroxyapatite, as well as poly(β-amino ester) microparticles that facilitate drug delivery. Strength was increased threefold, from 2 to 6 MPa, while compressive modulus was improved sixfold, from 24 to 141 MPa, via the addition of 30% nanohydroxyapatite, which provided greater benefits at equivalent concentrations compared to micro-hydroxyapatite. Scaffolds retained a uniformly porous microarchitecture, and hydroxyapatite particles were distributed evenly throughout the PLGA phase. Injectability, determined by the force required to inject 0.5 mL of material within 60 s, remained clinically acceptable at

Objective To investigate the ability of mucoadhesive films to locally deliver an immune response modifier (imiquimod) to oral mucosa. Study Design After determining the residence time of films in hamster cheek pouches, drug-loaded films and commercially available imiquimod cream were tested for localization of drug in mucosal tissue. The residence time of drug-free films at different locations was also tested in humans. Results Mucoadhesive films delivered imiquimod to the buccal mucosa with no measureable amounts in blood. In contrast, although the cream formulation resulted in higher tissue levels, it also led to significant systemic distribution of imiquimod. In humans, the films resided on tissue for up to 4 hours, increasing in the order of tongue Conclusion Preclinical findings of localized imiquimod delivery in animals and residence in humans support future investigations of the mucoadhesive system in controlled clinical trials for treating oral precancerous lesions.

Biodegradable polyesters are commonly used as drug delivery vehicles, but their role is typically passive, and encapsulation approaches have limited drug payload. An alternative drug delivery method is to polymerize the active agent or its precursor into a degradable polymer. The prodrug simvastatin contains a lactone ring that lends itself to ring-opening polymerization (ROP). Consequently, simvastatin polymerization was initiated with 5 kDa monomethyl ether poly(ethylene glycol) (mPEG) and catalyzed via stannous octoate. Melt condensation reactions produced a 9.5 kDa copolymer with a polydispersity index of 1.1 at 150 °C up to a 75 kDa copolymer with an index of 6.9 at 250 °C. Kinetic analysis revealed first-order propagation rates. Infrared spectroscopy of the copolymer showed carboxylic and methyl ether stretches unique to simvastatin and mPEG, respectively. Slow degradation was demonstrated in neutral and alkaline conditions. Lastly, simvastatin, simvastatin-incorporated molecules, and mPEG were identified as the degradation products released. The present results show the potential of using ROP to polymerize lactone-containing drugs such as simvastatin.

The current standard for treating infected bony defects, such as those caused by periodontal disease, requires multiple time-consuming steps and often multiple procedures to fight the infection and recover lost tissue. Releasing an antibiotic followed by an osteogenic agent from a synthetic bone graft substitute could allow for a streamlined treatment, reducing the need for multiple surgeries and thereby shortening recovery time. Tailorable bilayered calcium sulfate (CS) bone graft substitutes were developed with the ability to sequentially release multiple therapeutic agents. Bilayered composite samples having a shell and core geometry were fabricated with varying amounts (1 or 10 wt%) of metronidazole-loaded poly poly(lactic-co-glycolic acid) (PLGA) particles embedded in the shell and simvastatin directly loaded into either the shell, core, or both. Microcomputed tomography (MicroCT) images showed the overall layered geometry as well as homogenous distribution of PLGA within the shells. Dissolution studies demonstrated that the amount of PLGA particles (i.e., 1 vs. 10 wt%) had a small but significant effect on the erosion rate (3% vs. 3.4% per day). Mechanical testing determined that introducing a layered geometry had a significant effect on the compressive strength, with an average reduction of 35%, but properties were comparable to mandibular trabecular bone. Sustained release of simvastatin directly loaded into CS demonstrated that changing the shell to core volume ratio dictates the duration of drug release from each layer. When loaded together in the shell or in separate layers, sequential release of metronidazole and simvastatin was achieved. By introducing a tunable layered geometry capable of releasing multiple drugs, CS-based bone graft substitutes could be tailored in order to help streamline multiple steps needed to regenerate tissue in infected defects.

Mucin networks are formed in the oral cavity by complexation of glycoproteins with other salivary proteins, yielding a hydrated lubricating barrier. The function of these networks is linked to their structural, chemical, and mechanical properties. Yet, as these properties are interdependent, it is difficult to tease out their relative importance. Here, we demonstrate the ability to recreate the fibrous like network through a series of complementary rinses of polymeric worm-like micelles, resulting in a 3-dimensional (3D) porous network that can be deposited layer-by-layer onto any surface. In this work, stability, structure, and microbial capture capabilities were evaluated as a function of network properties. It was found that network structure alone was sufficient for bacterial capture, even with networks composed of the adhesion-resistant polymer, poly(ethylene glycol). The synthetic networks provide an excellent, yet simple, means of independently characterizing mucin network properties (e.g., surface chemistry, stiffness, and pore size).

Treatment of infected bone defects presents a considerable challenge due to the complications that occur from significant bone damage concomitant with contaminated tissue. These wounds are most often treated in a two-step sequence, where the infection is first eliminated before any attempt to repair the bone is undertaken. In order to combine these two treatment steps into one procedure, a moldable bone grafting material was developed to deliver drugs in a temporally separated manner. This was accomplished by a two-layered calcium sulfate composite consisting of a moldable outer shell containing antibiotic-loaded poly(lactic-co-glycolic acid) microspheres wrapped around a preformed core containing an osteogenic drug. The release of vancomycin from the shell portion began immediately and continued over the course of 6 weeks, while the release of simvastatin from the core was delayed for 12 days before being released over the next 4 weeks. Bioactivity of vancomycin was shown in modified Kirby-Bauer experiments in which whole samples inhibited Staphylococcus aureus (S. aureus) growth for 2 weeks. This two-layered system is capable of delivering antibiotics locally for clinically relevant periods of time and delaying the release of osteogenic drugs to mimic a two-step procedure that has potential for treating infected bone defects.

To help reduce the need for autografts, calcium sulfate (CS)-based bone graft substitutes are being developed to provide a stable platform to aid augmentation while having the ability to release a broad range of bioactive agents. CS has an excellent reputation as a biocompatible and osteoconductive substance, but addition of bioactive agents may further enhance these properties. Samples were produced with either directly loaded small, hydrophobic molecule (i.e., simvastatin), directly loaded hydrophilic protein (i.e., lysozyme), or 1 and 10 wt % of fast-degrading poly(β-amino ester) (PBAE) particles containing protein. Although sustained release of directly loaded simvastatin was achieved, direct loading of small amounts of lysozyme resulted in highly variable release. Direct loading of a larger amount of protein generated a large burst, 65% of total loading, followed by sustained release of protein. Release of lysozyme from 1 wt % of PBAE particles embedded into CS was more controllable than when directly loaded, and for 10 wt % of protein-loaded PBAE particles, a higher burst was followed by sustained release, comparable to the results for the high direct loading. Compression testing determined that incorporation of directly loaded drug or drug-loaded PBAE particles weakened CS. In particular, PBAE particles had a significant effect on the strength of the composites, with a 25 and 80% decrease in strength for 1 and 10 wt % particle loadings, respectively. CS-based composites demonstrated the ability to sustainably release both macromolecules and small molecules, supporting the potential for these materials to release a range of therapeutic agents.

Unsatisfactory corrosion resistance is one of the major disadvantages of magnesium alloys that impede their wide application. Microstructural changes, especially grain sizes, of Mg alloys have significant influence on their corrosion resistance. Cryogenic machining was reported to effectively induce grain refinement on Mg alloys and has a potential to improve their corrosion resistance. It is important to model these changes so that proper machining conditions can be found to enhance the corrosion rate of Mg alloys. In this paper, a preliminary study was conducted to model the microstructural changes of AZ31B Mg alloy during dry and cryogenic machining using the finite element (FE) method and a user subroutine based on the dynamic recrystallization (DRX) mechanism of Mg alloys. Good agreement in terms of grain size and affected layer thickness was found between experimental and predicted results. A numerical study was conducted using this model to investigate the influence of rake angle on microstructural changes after cryogenic machining.

Periodontal disease is highly prevalent, with 90% of the world population affected by either periodontitis or its preceding condition, gingivitis. These conditions are caused by bacterial biofilms on teeth, which stimulate a chronic inflammatory response that leads to loss of alveolar bone and, ultimately, the tooth. Current treatment methods for periodontitis address specific parts of the disease, with no individual treatment serving as a complete therapy. The present research sought to demonstrate development of a multiple drug delivery system for stepwise treatment of different stages of periodontal disease. More specifically, multilayered films were fabricated from an association polymer comprising cellulose acetate phthalate and Pluronic F-127 to achieve sequential release of drugs. The four types of drugs used were metronidazole, ketoprofen, doxycycline, and simvastatin to eliminate infection, inhibit inflammation, prevent tissue destruction, and aid bone regeneration, respectively. Different erosion times and adjustable sequential release profiles were achieved by modifying the number of layers or by inclusion of a slower-eroding polymer layer. Analysis of antibiotic and anti-inflammatory bioactivity showed that drugs released from the devices retained 100% bioactivity. The multilayered CAPP delivery system offers a versatile approach for releasing different drugs based on the pathogenesis of periodontitis and other conditions.

The capacity to quickly regenerate or augment bone lost as a result of resorption is crucial to ensure suitable application of prosthetics for restoring masticatory function. Calcium sulfate hemihydrate (CS)-based bone graft substitute composites containing poly(β-amino ester) (PBAE) biodegradable hydrogel particles were developed to act as a ‘tenting’ barrier to soft tissue infiltration, potentially providing adequate space to enable vertical bone regeneration. CS has long been recognized as an osteoconductive biomaterial with an excellent reputation as a biocompatible substance. Composite samples were fabricated with varying amounts (1 or 10 wt%) and sizes (53–150 or 150–250 µm) of gel particles embedded in CS. The swelling and degradation rates of PBAE gels alone were rapid, resulting in complete degradation in less than 24 hours, an important characteristic to aid in controlled release of drug. MicroCT images revealed a homogeneous distribution of gel particles within the CS matrix. All CS samples degraded via surface erosion, with the amount of gel particles (i.e., 10 wt% gel particles) having only a small, but significant, effect on the dissolution rate (4% vs. 5% per day). Compression testing determined that the amount, but not the size, of gel particles had a significant effect on the overall strength of the composites. As much as a 75% drop in strength was seen with a 10 wt% loading of particles. A pilot study using PBAE particles loaded with the multipotential drug curcumin demonstrated sustained release of drug from CS composites. By adjusting the amount and/or size of the biodegradable gel particles embedded in CS, mechanical strength and degradation rates of the composites, as well as the drug release kinetics, can be tuned to provide sufficient, multi-functional ‘space-making’ bone grafting substitutes.

Biodegradable hydrogels have become a rapidly expanding area of research for biomedical applications. Poly(β-amino ester) (PBAE) biodegradable hydrogels, in particular, have been studied extensively because of the ease of synthesis and range of properties exhibited. In this work, PBAE macromers were created with poly(ethylene glycol) diacrylate (PEGDA), diethylene glycol diacrylate (DEGDA), and isobutylamine. Many methods have been presented that allow control over the resulting hydrogel behavior. Here, properties were varied by combining multiple diacrylate components in the macromer synthesis step (single macromer (SM)) or through the combination of multiple macromers prior to polymerization (double macromer (DM)). The exhibited properties are a result of the hydrophilic/hydrophobic contributions of the different diacrylate molecules to the macromer. The SM systems exhibited linear degradation profiles, the rate of degradation increased as the PEGDA concentration increased. DM systems exhibited a multiphase degradation profile as a result of the combination of macromers with different hydrophilic properties.

Failure of soft tissue implants has been largely attributed to the influence of biomaterial surface properties on the foreign body response, but some implant complications, e.g. macrophage accumulation and necrosis, are still not effectively addressed with surface treatments to minimize deleterious biomaterial effects. We explored an alternative explanation for implant failure, linking biocompatibility with implant micromotion-induced pressure fluctuations at the tissue-biomaterial interface. For this purpose, we used a custom in vitro system to characterize the effects of pressure fluctuations on the activity of macrophages, the predominant cells at a healing implant site. Initially, we quantified superoxide production by HL60-derived macrophage-like cells under several different pressure regimes with means of 5-40 mmHg, amplitudes of 0-15 mmHg and frequencies of 0-1.5 Hz. All pressure regimes tested elicited significantly (p2=0.74; p (Less)

Poly(β-amino ester) (PBAE) biodegradable hydrogel systems have garnered much attention in recent years due to their appealing properties for biomedical applications. These hydrogel systems exhibit properties similar to natural soft tissue, degrade in aqueous environments, and have easily tunable properties that have been well studied and understood. In most cases, tissue engineering scaffolds must possess a three-dimensional interconnected porous network for tissue ingrowth and construct vascularization. Here, PBAE properties were explored and systems were selected to serve as both the pore-forming agent and the outer matrix of a scaffold that exhibits controlled pore opening upon degradation. To our knowledge, this is the first demonstration of a biodegradable hydrogel porogen system entrapped in a degradable hydrogel outer matrix. Scaffolds were prepared, and the degradation, compressive moduli, and porosity were analyzed. An added advantage of a degradable porogen is the potential for controlled drug release, and a model protein was released from the porogen particles to demonstrate this application. Finally, pluripotent cells seeded onto predegraded scaffolds were viable during the first 24 h of exposure, and furthermore, cell tracking confirmed the presence of cells within the pores of the scaffold. Overall, these present studies demonstrate the possibility of using these biodegradable hydrogel porogen-matrix systems as tissue engineering scaffolding materials. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 400–412, 2014.

Biodegradable polymer scaffolds are being extensively investigated for uses in tissue engineering because of their versatility in fabrication methods and range of achievable chemical and mechanical properties. In this study, poly(lactic-co-glycolic acid) (PLGA) was used to make various types of microspheres that were processed into porous scaffolds that possessed a wide range of properties. A heat sintering step was used to fuse microspheres together around porogen particles that were subsequently leached out, allowing for a 10-fold increase in mechanical properties over other PLGA scaffolds. The sintering temperature was based on the glass transition temperature that ranged from 43 to 49°C, which was low enough to enable drug loading. Degradation times were observed to be between 30 and 120 days, with an initial compressive modulus ranging from 10 to 100 MPa, and after 5 days of degradation up to 10 MPa was retained. These scaffolds were designed to allow for cell ingrowth, enable drug loading, and have an adjustable compressive modulus to be applicable for soft or hard tissue implants. This study combined well-established methods, such as double emulsion microspheres, polymer sintering, and salt leaching, to fabricate polymer scaffolds useful for different tissue engineering applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 348–357, 2014.

The objective of this work was to demonstrate the bioactivity of parathyroid hormone (1-34) (PTH) delivered through a single molecule of bisphosphonate to improve tissue/cell interactions. Bifunctional hydrazine-bisphosphonates (HBPs) with varying length and lipophilicity were used as a drug delivery vehicle. PTH was oxidized with periodate treatment to obtain an N-terminal aldehyde that was then conjugated to HBPs. The toxicity and apoptotic properties of HBPs and HBP–PTH conjugates were studied with macrophages (RAW 264.7). It was found that one of the HBPs had significant apoptotic characteristics similar to alendronate, which is a widely prescribed drug in the treatment of osteoporosis. The improved binding affinity of PTH following conjugation to HBP was determined using a hydroxyapatite binding assay. The amount of PTH delivered to bone through HBPs was not affected by the length or lipophilicity of the HBPs. Furthermore, the improved bioactivity of PTH delivered to bone through HBPs, in comparison to adsorbed PTH, was demonstrated by quantifying the cAMP produced by pre-osteoblastic (MC3T3-E1) cells in response to PTH. The delivery of bioactive PTH to bone tissue by HBP conjugation demonstrates the potential use of HBPs in delivering therapeutic macromolecules to bone for the treatment of several skeletal diseases.

Osteoporosis, which is characterized by resorption of bone exceeding formation, remains a significant human health concern, and the impact of this condition will only increase with the “greying” of the worldwide population. This review focuses on current and emerging approaches for delivering therapeutic agents to restore bone remodeling homeostasis. Well-known antiresorptive and anabolic agents such as estrogen, estrogen analogs, bisphosphonates, calcitonin, and parathyroid hormone, along with newer modulators and antibodies, are primarily administered orally, intravenously, or subcutaneously. Although these treatments can be effective, continuing problems include patient non-compliance and adverse systemic or remote-site effects. Controlled drug delivery via polymeric, targeted, and active release systems extends drug half-life by shielding against premature degradation and improves bioavailability, while also providing prolonged, sustained, or intermittent release at therapeutic doses to more effectively treat osteoporosis and associated fracture risk.

Formation of scar tissue may be reduced or prevented if wounds are locally treated with a combination of molecules tuned to the different healing phases, guiding tissue regeneration along a scar free path. To this end, drug delivery devices made of cellulose acetate phthalate and Pluronic F-127 were loaded with either quercetin or pirfenidone and plasticized with either triethyl citrate or tributyl citrate. Quercetin inhibits oxidative stress, and pirfenidone has been shown to reduce production of pro-inflammatory and fibrogenic molecules. The combined effects of drug and plasticizer on erosion, release, and mechanical properties of the drug delivery films were investigated. Triethyl citrate-plasticized films containing quercetin released drug at a slower rate than did tributyl citrate films. Pirfenidone-loaded films released drug at a faster rate than erosion occurred for both types of plasticizers. Higher plasticizer contents of both triethyl citrate and tributyl citrate increased the elongation and decreased the elastic modulus. In contrast, increased pirfenidone loading in both triethyl citrate and tributyl citrate films resulted in a significantly higher modulus, an antiplasticizer effect. Adding pirfenidone significantly decreased elongation for all film types, but quercetin-loaded samples had significantly greater elongation with increasing drug content. Films containing quercetin elongated more than did pirfenidone-loaded films. Quercetin is over 1.5 times larger than pirfenidone, has water solubility over 12 times lower, and has 6 times more bonding sites than pirfenidone. These differences affected how the two drugs interacted with cellulose acetate phthalate and Pluronic F-127 and thereby determined polymer properties. Drug release, erosion, and mechanical properties of association polymer films can be tailored by the characteristics of the drugs and plasticizers included in the system.

Growth plate fracture can lead to retarded growth and unequal limb length, which may have a lifelong effect on a person's physical stature. The goal of this research was to develop an in vivo tissue-engineering approach for the treatment of growth plate injury via localized delivery of insulin-like growth factor I (IGF-I) from cell-free poly(lactic-co-glycolic acid) (PLGA) scaffolds. Mass loss and drug release studies were conducted to study the scaffold degradation and IGF-I release patterns. In vitro cell studies showed that rat bone marrow stromal cells seeded on the porous scaffolds colonized the pores and deposited matrix within the scaffolds. These in vitro evaluations were followed by a proof-of-concept animal study involving implantation of scaffolds in proximal tibial growth plate defects in New Zealand white rabbits. Histological analysis of tissue sections from the in vivo studies showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF-I-releasing scaffolds; in contrast, only bone was formed in empty defects and those treated with IGF-free scaffolds. The present findings show the potential for treating growth plate injury using in vivo tissue engineering techniques.